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2023

• Shrestha, S. L., Garland‐Campbell, K. A., Steber, C. M., Pan, W. L., & Hulbert, S. H. (2023). Association of canopy temperature with agronomic traits in spring wheat inbred populations. Euphytica, 219(7), 1–14. (Show/Hide Abstract)
  Abstract: Canopy temperature (CT) is considered a reliable proxy for stomatal conductance. Low CT values of plant canopies under water-limited conditions are associated with high transpiration indicating plants’ drought tolerance. Many U.S. Pacific Northwest (PNW) adapted wheat (Triticum aestivum L.) cultivars lack stress-adaptive traits resulting in poor performance in drought environments. This study aims to identify the stress-adaptive traits by evaluating the CT in spring wheat populations across different soil moisture conditions in the PNW. An infrared thermometer was used to estimate the CT in two families of recombinant inbred lines, 'Alpowa' × 'Express' (AE population) and 'Hollis' × 'Drysdale' (HD population), in rainfed and irrigated environments of the dryland PNW in 2011 to 2013. Higher reductions in grain yield up to 170%, spike length up to 25%, and spikelets spike⁻¹ up to 19% were observed in a rainfed environment compared to the reductions in an irrigated environment. A significant variation in CT was observed in both AE and HD populations. With 1 °C increase in CT at the anthesis stage, grain yield was lowered up to 38 g m⁻². Low CT was associated with high grain yield and agronomic traits in both wheat populations (r =  − 0.18 to − 0.55, P ≤ 0.05). The highest association between CT and grain yield was observed at anthesis (r =  − 0.47) and milking (r =  − 0.38) stages (P ≤ 0.001). Our results show that screening for low CT during terminal wheat growth stage is an effective strategy for improving the selection of new drought-tolerant wheat varieties in the PNW.
• Hauvermale, A. L., Parveen, R. S., Harris, T. J., Tuttle, K. M., Mikhaylenko, G., Nair, S., McCubbin, A. G., Pumphrey, M. O., & Steber, C. M. (2023). Streamlined alpha-amylase assays for wheat preharvest sprouting and late maturity alpha-amylase detection. Agrosystems, Geosciences & Environment, 6(7), 1–10. (Show/Hide Abstract)
  Abstract: Late maturity alpha-amylase (LMA) and preharvest sprouting (PHS) lead to elevated alpha-amylase in wheat (Triticum aestivum L.) grain. Risk of poor end-product quality due to elevated alpha-amylase is detected in the wheat industry using the Hagberg–Perten falling number (FN) method. In breeding programs, selection for PHS and LMA tolerance requires higher throughput methods requiring a smaller sample size than the 7 g required for the FN method. Specifically, LMA can only be screened only using detection of alpha-amylase activity or protein after cold treatment of individual wheat spikes at a specific stage of grain development resulting in very small samples (≤1 g). This study developed and evaluated a high through-put 96-well method for the Phadebas alpha-amylase enzyme assay for small wheat grain samples and compared this method to FN and the Megazyme Alpha-Amylase SD (Sprout Damage) Assay Kit performed on the automated Awareness Technology ChemWell-T Analyzer. In parallel, the efficacy of low-cost small-scale milling methods was evaluated relative to traditional larger scale mills. The Phadebas enzyme activity was highly reproducible and showed a strong correlation to the SD enzyme assay and FN method regardless of which mill was used to process the grain. The SD assay offers simpler standardization and calculation of enzyme activity, whereas the Phadebas assay offers higher sensitivity and lower expense. Both the 96-well Phadebas and automated Megazyme SD assays are suitable for alpha-amylase detection from small samples, and the use of low-cost coffee grinders to process small samples did not significantly impact assay performance.
• Nelson, S. K., Kanno, Y. Seo, M., & Steber, C. M. (2023). Seed dormancy loss from dry after-ripening is associated with increasing gibberellin hormone levels in Arabidopsis thaliana. Frontiers in Plant Science, 14, 1–18. (Show/Hide Abstract)
  Abstract: Introduction: The seeds of many plants are dormant and unable to germinate at maturity, but gain the ability to germinate through after-ripening during dry storage. The hormone abscisic acid (ABA) stimulates seed dormancy, whereas gibberellin A (GA) stimulates dormancy loss and germination. Methods: To determine whether dry after-ripening alters the potential to accumulate ABA and GA, hormone levels were measured during an after-ripening time course in dry and imbibing ungerminated seeds of wildtype Landsberg erecta (Ler) and of the highly dormant GA-insensitive mutant sleepy1-2 (sly1-2). Results: The elevated sly1-2 dormancy was associated with lower rather than higher ABA levels. Ler germination increased with 2-4 weeks of after-ripening whereas sly1-2 required 21 months to after-ripen. Increasing germination capacity with after-ripening was associated with increasing GA₄ levels in imbibing sly1-2 and wild-type Ler seeds. During the same 12 hr imbibition period, after-ripening also resulted in increased ABA levels. Discussion: The decreased ABA levels with after-ripening in other studies occurred later in imbibition, just before germination. This suggests a model where GA acts first, stimulating germination before ABA levels decline, and ABA acts as the final checkpoint preventing germination until processes essential to survival, like DNA repair and activation of respiration, are completed. Overexpression of the GA receptor GID1b (GA INSENSITIVE DWARF1b) was associated with increased germination of sly1-2 but decreased germination of wildtype Ler. This reduction of Ler germination was not associated with increased ABA levels. Apparently, GID1b is a positive regulator of germination in one context, but a negative regulator in the other.
• Peery, S. R., Carle, S. W., Wysock, M., Pumphrey, M. O., & Steber, C. M. (2023). LMA or vivipary? Wheat grain can germinate precociously during grain maturation under the cool conditions used to induce late maturity alpha-amylase (LMA). Frontiers in Plant Science, 14, 1–18. (Show/Hide Abstract)
  Abstract: Introduction: This study found that wheat (Triticum aestivum) grain can germinate precociously during the maturation phase of grain development, a phenomenon called vivipary that was associated with alpha-amylase induction. Farmers receive severe discounts for grain with low falling number (FN), an indicator that grain contains sufficiently elevated levels of the starch-digesting enzyme alpha-amylase to pose a risk to end-product quality. High grain alpha- amylase can result from: preharvest sprouting (PHS)/germination when mature wheat is rained on before harvest, or from late maturity alpha-amylase (LMA) when grain experiences cool temperatures during the soft dough stage of grain maturation (Zadoks growth stage 85). An initial LMA-induction experiment found that low FN was associated with premature visible germination, suggesting that cool and humid conditions caused vivipary. Methods: To examine whether LMA and vivipary are related, controlled environment experiments examined the conditions that induce vivipary, whether LMA could be induced without vivipary, and whether the pattern of alpha-amylase expression during vivipary better resembled PHS or LMA. Results: Vivipary was induced in the soft to hard dough stages of grain development (Zadok’s stages 83-87) both on agar and after misting of the mother plant. This premature germination was associated with elevated alpha-amylase activity. Vivipary was more strongly induced under the cooler conditions used for LMA-induction (18°C day/7.5°C night) than warmer conditions (25°C day/18°C night). Cool temperatures could induce LMA with little or no visible germination when low humidity was maintained, and susceptibility to vivipary was not always associated with LMA susceptibility in a panel of 8 varieties. Mature grain preharvest sprouting results in much higher alpha-amylase levels at the embryo-end of the kernel. In contrast, vivipary resulted in a more even distribution of alpha-amylase that was reminiscent of LMA. Discussion: Vivipary can occur in susceptible varieties under moist, cool conditions, and the resulting alpha-amylase activity may result in low FN problems when a farm experiences cool, rainy conditions before the crop is mature. While there are genotypic differences in LMA and vivipary susceptibility, overlapping mechanisms are likely involved since they are similarly controlled by temperature and growth stage, and result in similar patterns of alpha-amylase expression.
• Hauvermale, A. L., Matzke, C., Bohaliga, G., Pumphrey, M.O., Steber, C.M., & McCubbin, A.G. (2023). Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change. Plants, 12(22):3798, 1–18. (Show/Hide Abstract)
  Abstract: Accurate, rapid testing platforms are essential for early detection and mitigation of late ma- turity α-amylase (LMA) and preharvest sprouting (PHS) in wheat. These conditions are characterized by elevated α-amylase levels and negatively impact flour quality, resulting in substantial economic losses. The Hagberg–Perten Falling Number (FN) method is the industry standard for measuring α-amylase activity in wheatmeal. However, FN does not directly detect α-amylase and has major limitations. Developing α-amylase immunoassays would potentially enable early, accurate detection regardless of testing environment. With this goal, we assessed an expression of α-amylase isoforms during seed development. Transcripts of three of the four isoforms were detected in developing and mature grain. These were cloned and used to develop E. coli expression lines expressing single isoforms. After assessing amino acid conservation between isoforms, we identified peptide sequences specific to a single isoform (TaAMY1) or that were conserved in all isoforms, to develop monoclonal antibodies with targeted specificities. Three monoclonal antibodies were developed, anti-TaAMY1-A, anti-TaAMY1-B, and anti-TaAMY1-C. All three detected endogenous α-amylase(s). Anti-TaAMY1-A was specific for TaAMY1, whereas anti-TaAMY1-C detected TaAMY1, 2, and 4. Thus, confirming that they possessed the intended specificities. All three antibodies were shown to be compatible for use with immuno-pulldown and immuno-assay applications.
• Chen, C.-P. J., Hu, Y., Li, X., Morris, C. F., Delwiche, S., Carter, A. H., Steber, C., & Zhang, Z. (2023). An independent validation reveals the potential to predict Hagberg–Perten falling number using spectrometers. The Plant Phenome Journal, 6, 1–15. (Show/Hide Abstract)
  Abstract: The Hagberg–Perten falling number (HFN) method is the international standard used to evaluate the damage to wheat (Triticum aestivum) grain quality due to preharvest sprouting (PHS) and late maturity alpha-amylase (LMA). However, the HFN test requires specialized laboratory facilities and is time consuming. Spectrometers were known as a potential tool for quick HFN assessment, but none of the studies have val- idated the assessment results across different datasets. In this study, an independent validation was conducted using independent samples and spectral instruments. The calibration set had 462 grain samples of 92 varieties grown at 24 locations in 2019 and examined using a near-infrared spectrometer. In the validation set, 19 varieties collected from 10 locations in 2 years that experienced either PHS or LMA were scanned with a hyperspectral camera. The association between spectra and HFN was modeled by partial least square regression. As a result, the independent validation correlation accuracy was r = 0.72 and a mean absolute error of 56 s. Furthermore, this study showed a cost-effective alternative using only 10 spectral bands to predict HFN, and it achieved better performance than the full spectrum of the hyperspectral system. In conclusion, this is the first study that showed the potential that wheat HFN could be predicted on an independent dataset measured by a different instrument. The result suggested that spectrometers can potentially serve as a faster alternative for plant breeders to develop varieties resistant to PHS and LMA, and for growers to screen damaged grains in transportation processes.

2022

• Hu, Y., Sjoberg, S. M., Chen, C., Hauvermale, A. L., Morris, C. F., Delwiche, S. R., Cannon, A. E., Steber, C. M., & Zhang, Z. (2022). As the number falls, alternatives to the Hagberg–Perten falling number method: A review. Comprehensive Reviews in Food Science and Food Safety , 1–13. (Show/Hide Abstract)
  Abstract: This review examines the application, limitations, and potential alternatives to the Hagberg–Perten falling number (FN) method used in the global wheat industry for detecting the risk of poor end-product quality mainly due to starch degradation by the enzyme α-amylase. By viscometry, the FN test indirectly detects the presence of α-amylase, the primary enzyme that digests starch. Elevated α-amylase results in low FN and damages wheat product quality resulting in cakes that fall, and sticky bread and noodles. Low FN can occur from preharvest sprouting (PHS) and late maturity α-amylase (LMA). Moist or rainy conditions before harvest cause PHS on the mother plant. Continuously cool or fluctuating temperatures during the grain filling stage cause LMA. Due to the expression of addi- tional hydrolytic enzymes, PHS has a stronger negative impact than LMA. Wheat grain with low FN/high α-amylase results in serious losses for farmers, traders, millers, and bakers worldwide. Although blending of low FN grain with sound wheat may be used as a means of moving affected grain through the marketplace, care must be taken to avoid grain lots from falling below contract-specified FN. A large amount of sound wheat can be ruined if mixed with a small amount of sprouted wheat. The FN method is widely employed to detect α-amylase after harvest. However, it has several limitations, including sampling variability, high cost, labor intensiveness, the destructive nature of the test, and an inability to differentiate between LMA and PHS. Faster, cheaper, and more accurate alternatives could improve breeding for resistance to PHS and LMA and could preserve the value of wheat grain by avoiding inadvertent mixing of high- and low- FN grain by enabling testing at more stages of the value stream including at harvest, delivery, transport, storage, and milling. Alternatives to the FN method explored here include the Rapid Visco Analyzer, enzyme assays, immunoassays, near-infrared spectroscopy, and hyperspectral imaging.
• Garland-Campbell, K., Bellinger, B. S., Carter, A. H., Chen, X., DeMacon, P., Engle, D., Hagerty, C. H., Kiszonas, A., Klarquist, E., Murray, T., Morris, C., Neely, C., Odubiyi, S., Rashad, A., See, D., Steber, C., & Wen, N. (2022). Registration of 'Cameo' soft white winter club wheat. Journal of Plant Registrations, 16, 585–596. (Show/Hide Abstract)
  Abstract: Soft white club winter wheat (Triticum aestivum L. ssp. compactum) is an important component of soft white wheat production in the Pacific Northwest of the United States. Most of the current club wheat production is in the <350 mm annual precipitation zone in central Washington, but there is interest in club wheat in the Palouse region of the United States (the counties of Whitman and Garfield in Washington and in Latah County in Idaho). Growers are continuing to grow the older club wheat cultivars 'Cara' and 'Coda', and there is a need for a new winter club wheat targeted to this region. ‘Cameo’ club wheat (Reg. no. CV-1192, PI 699960), tested as ARS09X492-6CBW, with awned spikes and soft white kernels, was developed using the bulk-pedigree breeding method from the cross ARSC96059- 2/IL01-11934//ARSC96059-2-0-16. Cameo has better agronomic performance than other club wheat cultivars in trials on the Palouse, better stripe rust resistance than the club wheat 'ARS Crescent', tolerance to several major biotic and abiotic stressors, consistent good grain volume weight, mid-season maturity and moderate height, excellent club wheat quality, and tolerance to low falling numbers. Cameo is not as competitive for grain yield in the traditional club wheat growing area in central Washington but is well suited to increasing the acreage of club wheat in the Palouse region of Idaho and Washington.
• Hauvermale, A. L., Cárdenas, J. J., Bednarek, S. Y., & Steber, C. M. (2022). GA signaling expands: The plant UBX domain-containing protein 1 is a binding partner for the GA receptor. Plant Physiology, 190(4), 2651–2670. (Show/Hide Abstract)
  Abstract: The plant Ubiquitin Regulatory X (UBX) domain-containing protein 1 (PUX1) functions as a negative regulator of gibberel- lin (GA) signaling. GAs are plant hormones that stimulate seed germination, the transition to flowering, and cell elongation and division. Loss of Arabidopsis (Arabidopsis thaliana) PUX1 resulted in a ''GA-overdose'' phenotype including early flowering, increased stem and root elongation, and partial resistance to the GA-biosynthesis inhibitor paclobutrazol during seed germination and root elongation. Furthermore, GA application failed to stimulate further stem elongation or flowering onset suggesting that elongation and flowering response to GA had reached its maximum. GA hormone partially repressed PUX1 protein accumulation, and PUX1 showed a GA-independent interaction with the GA receptor GA-INSENSITIVE DWARF-1 (GID1). This suggests that PUX1 is GA regulated and/or regulates elements of the GA signaling pathway. Consistent with PUX1 function as a negative regulator of GA signaling, the pux1 mutant caused increased GID1 expression and decreased accumulation of the DELLA REPRESSOR OF GA1-3, RGA. PUX1 is a negative regulator of the hexameric AAA + ATPase CDC48, a protein that functions in diverse cellular processes including unfolding proteins in preparation for proteasomal degradation, cell division, and expansion. PUX1 binding to GID1 required the UBX domain, a binding motif necessary for CDC48 interaction. Moreover, PUX1 overexpression in cell culture not only stimulated the disassembly of CDC48 hexamer but also resulted in co-fractionation of GID1, PUX1, and CDC48 subunits in velocity sedimentation assays. Based on our results, we propose that PUX1 and CDC48 are additional factors that need to be incorporated into our understanding of GA signaling.
• Steber, C. M. (2022, November). Rising to the challenge of falling numbers. Wheat Life, 65(10), 44–45.

2021

• Liu, C., Tuttle, K., Garland Campbell, K., Pumphrey, M., & Steber, C. (2021). Investigating conditions that induce late maturity alpha-amylase (LMA) using Northwestern US spring wheat (Triticum aestivum L.). Seed Science Research, 1–9. https://doi.org/10.1017/S0960258521000052 (Show/Hide Abstract)
  Abstract: The wheat industry rejects grain with unacceptably high α-amylase enzyme levels due to the risk of poor end product quality. There are two main causes of elevated grain α-amylase: (1) preharvest sprouting in response to rain before harvest and (2) late maturity α-amylase (LMA) induction in response to a cool temperature shock during late grain development. LMA induction was detected in a panel of 24 Northwestern US spring wheat lines. Thus, this problem previously described in Australian and U.K. varieties also exists in U.S. varieties. Because LMA induction results were highly variable using published methods, a characterization of LMA-inducing conditions was conducted in an LMA-susceptible soft white spring wheat line, WA8124. Problems with elevated α-amylase in untreated controls were reduced by raising the temperature, 25°C day/18°C night versus 20°C day/10°C night. LMA induction was not improved by colder temperatures (15°C day/4°C night) versus moderately cold temperatures (18°C day/7.5°C night or 10°C day/10°C night). While previous studies observed LMA induction by heat stress, it failed to induce LMA in WA8124. Thus, not all LMA-susceptible cultivars respond to heat. The timing of LMA susceptibility varied between two cultivars and within a single cultivar grown at slightly different temperatures. Thus, variability in LMA induction likely results from variability in the timing of the grain developmental stage during which cold shock induces LMA. Thus, it was concluded that the visual inspection of grain is needed to correctly identify LMA-sensitive spikes at the soft dough stage of grain development (Zadok's stage 85).
• Liu C., Parveen R. S., Revolinski S. R., Garland Campbell K. A., Pumphrey M. O., & Steber C. M. (2021). The genetics of late maturity alpha-amylase (LMA) in North American spring wheat (Triticum aestivum L.). Seed Science Research, 1–10. https://doi.org/10.1017/S0960258521000064 (Show/Hide Abstract)
  Abstract: Genetic susceptibility to late maturity alpha-amylase (LMA) in wheat (Triticum aestivum L.) results in increased alpha-amylase activity in mature grain when cool conditions occur during late grain maturation. Farmers are forced to sell wheat grain with elevated alpha-amylase at a discount because it has an increased risk of poor end-product quality. This problem can result from either LMA or preharvest sprouting, grain germination on the mother plant when rain occurs before harvest. Whereas preharvest sprouting is a well-understood problem, little is known about the risk LMA poses to North American wheat crops. To examine this, LMA susceptibility was characterized in a panel of 251 North American hard spring wheat lines, representing ten geographical areas. It appears that there is substantial LMA susceptibility in North American wheat since only 27% of the lines showed reproducible LMA resistance following cold-induction experiments. A preliminary genome-wide association study detected six significant marker-trait associations. LMA in North American wheat may result from genetic mechanisms similar to those previously observed in Australian and International Maize and Wheat Improvement Center (CIMMYT) germplasm since two of the detected QTLs, QLMA.wsu.7B and QLMA.wsu.6B, co-localized with previously reported loci. The Reduced height (Rht) loci also influenced LMA. Elevated alpha-amylase levels were significantly associated with the presence of both wild-type and tall height, rht-B1a and rht-D1a, loci in both cold-treated and untreated samples.
• Sexton, T. M., Steber, C. M., & Cousins, A. B. (2021). Leaf temperature impacts canopy water use efficiency independent of changes in leaf level water use efficiency. Journal of Plant Physiology, 25, 1–10. (Show/Hide Abstract)
  Abstract: Canopy water use efficiency (above-ground biomass over lifetime water loss, WUE_canopy) can influence yield in wheat and other crops. Breeding for WUE_canopy is difficult because it is influenced by many component traits. For example, intrinsic water use efficiency (WUE_i), the ratio of net carbon assimilation (A_net) over stomatal conductance, contributes to WUE_canopy and can be estimated from carbon isotope discrimination (Δ). However, Δ is not sensitive to differences in the water vapor pressure deficit between the air and leaf (VPD_leaf). Alternatively, measurements of instantaneous leaf water use efficiency (WUE_leaf) are defined as A_net over transpiration and can be determined with gas exchange, but the dynamic nature of field conditions are not represented. Specifically, fluctuations in canopy temperature lead to changes in VPD_leaf that impact transpiration but not A_net. This alters WUE_leaf and in turn affects WUE_canopy. To test this relationship, WUE_canopy was measured in conjunction with WUE_i, WUE_canopy, and canopy temperature under well-watered and water-limited conditions in two drought- tolerant wheat cultivars that differ in canopy architecture. In this experiment, boundary layer conductance was low and significant changes in leaf temperature occurred between cultivars and treatments that correlated with WUE_canopy likely because of the effect of canopy temperature on VPD_leaf driving T. However, deviations between WUE_i, WUE_leaf, and WUE_canopy were present because measurements made at the leaf level do not ac­count for variations in leaf temperature. This uncoupled the relationship of measured WUE and WUE_i from WUE_canopy and emphasizes the importance of canopy temperature on carbon uptake and transpired water loss.
• Horgan, A. M., Garland-Campbell, K. A., Carter, A. H., & Steber, C. M. (2021). Seedling elongation responses to gibberellin seed treatments in wheat. Agrosystems, Geosciences & Environment, 4, 1–13. (Show/Hide Abstract)
  Abstract: The wheat (Triticum aestivum L.) Reduced height (Rht) alleles are widely used to prevent lodging through semi-dwarfism. Seedling elongation and coleoptile length can be significantly decreased by some of these alleles, leading to reduced soil emergence after deep sowing in certain semi-arid environments. Application of the elongation-promoting hormone gibberellin A₃ (GA₃) as a seed treatment has been used as an alternative to improve seedling emergence. Seedling responses to GA₃ seed treatment were investigated under controlled conditions in a collection of varieties differing for Rht dwarfing alleles and the ability to emerge from deep planting. Data between treated and untreated seed were collected on overall coleoptile length and emergence from deep planting in simulated pot studies. Gibberellin-sensitive varieties, carrying either no dwarfing gene or the Rht8 dwarfing gene, responded to the GA₃ seed treatment with increased coleoptile and subcrown internode elongation. Comparison of near-isogenic lines carrying no dwarfing allele or the GA-insensitive Rht-B1b and/or Rht-D1b semi-dwarfing alleles showed that GA insensitivity was associated with reduced seedling response to GA₃ seed treatment. However, there was variation in seedling elongation and GA₃ response in a collection of GA-insensitive varieties. Thus, it cannot be assumed that all Rht-B1b and Rht-D1b varieties will fail to respond to GA₃ seed treatment. Interestingly, some better emerging GA-insensitive varieties had longer coleoptiles after treatment, suggesting that selection for better emergence in semi-arid regions of the U.S. Pacific Northwest may have led to responses in GA₃ application independent of the dwarfing gene used.

2020

• Sjoberg, S., Carter, A. H., Steber, C. M., & Garland-Campbell, K. A. (2020). Unravelling Complex Traits in Wheat: Approaches for Analyzing Genotype × Environment Interactions in a Multi-environment Study of Falling Numbers. Crop Science, 60, 3013–3026. (Show/Hide Abstract)
  Abstract: Multi-environment trials provide useful information about highly variable, complex plant traits like yield and quality, but are difficult to analyze due to their frequently unbalanced nature, with genotypes and locations varying from year to year. Our objectives were to use multiple approaches, including joint regression and principal components analysis, to characterize patterns in the genotype by environment interactions across an unbalanced three-year multi-environment wheat variety trial dataset, examining falling numbers (FN) test results in wheat. The FN test measures the decrease in flour gelling capacity resulting from starch digestion by the enzyme α-amylase. Low FN/high α-amylase grain is discounted because it is associated with poor end-use quality. Low FN can be caused by susceptibility either to preharvest sprouting when it rains before harvest or to late maturity α-amylase induction by temperature fluctuations during grain maturation. The most effective and visually intuitive approaches for selecting varieties with high FN across variable environments was a combination of joint regression, such as Finlay-Wilkinson and Eberhart and Russell, with biplot methods such as the additive main effects and multiplicative interaction model (AMMI) and the genotype main effects and genotype × environment interaction model (GGE). These results identified stable lines for FN resistance and provide a means to analyze unbalanced, multi-environment data from breeding and variety trials.
• Ghimire, B., Hulbert, S., Steber, C. M., Garland-Campbell, K., & Sanguinet, K. A. (2020). Characterization of root traits for improvement of spring wheat in the Pacific Northwest. Agronomy Journal, 112, 228–240. (Show/Hide Abstract)
  Abstract: Understanding the genetic basis of root traits and overall root physiology provides essential information on a largely untapped resource for crop improvement as roots are instrumental for the uptake of water and nutrients. However, breeding for improved root traits is challenging due to laborious and time-consuming root phenotyping in soil. Our studies sought to uncover spatiotemporal root growth dynamics of mature plant root systems in five spring wheat (Triticum aestivum L.) cultivars - Louise, Alpowa, Hollis, Drysdale, and Dharwar Dry - and a facultative spring landrace, AUS28451 using the in situ minirhizotron technique. The two-year greenhouse study revealed that the root system grows rapidly after early node elongation to gain maximum size during anthesis after which root growth slows and transitions to senescence. We were able to detect quantifiable differences among wheat cultivars in root traits in both 5-day old seedlings and root systems at anthesis. Furthermore, the positive correlation of the observed root traits with grain yield and the consistency in root traits observed using minirhizotrons and through extraction of young and mature root systems has reinforced the experimental results. A negative correlation was found between root number, area, and length and root diameter. We found that the spring wheat cultivars - AUS28451, Dharwar Dry, and Alpowa - had increased root number, area, and length, but also increased time to heading. The results from this study can be further leveraged to screen breeding lines for root traits of interest as well as assess the heritability of root traits for dryland farming in the inland Pacific Northwest.
• Shrestha, S. L., Garland-Campbell, K. A., Steber, C. M., & Hulbert, S. H. (2020). Carbon isotope discrimination association with yield and test weight in Pacific Northwest-adapted spring and winter wheat. Agrosystems, Geosciences & Environment, 3(1), e20052, (15 pages). (Show/Hide Abstract)
  Abstract: Due to considerable influence of environment on yield, breeding for drought tolerance could benefit from focusing on selection of more heritable physiological traits, such as carbon isotope discrimination (as measured by delta, Δ) for indirectly assessing water use efficiency (WUE) of wheat (Triticum aestivum L.). Spring and winter wheat cultivars were assayed for ∆, and these values were used to determine the relationships with performance in over 13 environments in the U.S. Pacific Northwest. The correlation coefficients of Δ values between the wheat cultivars grown in different environments ranged from 0.11 to 0.73 for both spring and winter wheat. There was significant genotypic variation for Δ in soft spring and hard winter wheat but not in hard spring and soft winter wheat. The Δ values were poor indicators of yield for this set of wheat cultivars in most environments, although low values (better WUE) were sometimes correlated with yield. A population of 165 hard spring wheat recombinant inbred lines derived from a cross between two hard spring wheat varieties that differed in Δ was also screened in low-rainfall dryland and irrigated environments. High yields in this recombinant inbred population were weakly correlated with high Δ values or low WUE in most environments.
• Hauvermale, A. L., & Steber, C. M. (2020). GA signaling is essential for the embryo-to-seedling transition during Arabidopsis seed germination, a ghost story. Plant Signaling & Behavior, 15(1), e1705028 (9 pages). (Show/Hide Abstract)
  Abstract: The plant hormone gibberellin (GA) stimulates developmental transitions including seed germination, flowering, and the transition from juvenile to adult growth stage. This study provided evidence that GA and the GA receptor GID1 (GA-INSENSITIVE DWARF1) are also needed for the embryo-to-seedling transition in Arabidopsis. The ga1-3 GA biosynthesis mutant fails to germinate unless GA is applied, whereas the gid1abc triple mutant fails to germinate because it cannot perceive endogenous or applied GA. Overexpression of the GID1a, GID1b, and GID1c GA receptors rescued the germination of a small percentage of ga1-3 seeds without GA application, and this rescue was improved by dormancy-breaking treatments, after-ripening and cold stratification. While GID1 overexpression stimulated ga1-3 seed germination, this germination was aberrant suggesting incomplete rescue of the germination process. Cotyledons emerged before the radicle, and the resulting “ghost” seedlings failed to develop a primary root, lost green coloration, and eventually died. The development of ga1-3 seedlings overexpressing GID1 was rescued by pre-germinative but not post-germinative GA application. Since the gid1abc mutant also exhibited a ghost phenotype after germination was rescued by cutting the seed coat, we concluded that both GA and GID1 are needed for the embryo-to-seedling transition prior to emergence from the seed coat.
• Martinez, S. A., Shorinola, O., Conselman, S., See, D., Skinner, D. Z., Uauy, C., & Steber, C. M. (2020). Exome sequencing of bulked segregants identified a novel TaMKK3‐A allele linked to the wheat ERA8 ABA‐hypersensitive germination phenotype. Theoretical and Applied Genetics, 133, 719–736. (Show/Hide Abstract)
  Abstract: Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest. The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft white wheat 'Zak'. ERA8 was mapped to chromosome 4A in a Zak/'ZakERA8' backcross population using bulked segregant analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb region containing 70 genes. In the backcross population, the ERA8 ABA-hypersensitive phenotype was strongly linked to a missense mutation in TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat. The map position of ERA8 was confirmed in an 'Otis'/ZakERA8 but not in a 'Louise'/ZakERA8 mapping population. This is likely because Otis carries the same natural PHS susceptible MKK3-A-A660^S allele as Zak, whereas Louise carries the PHS- tolerant MKK3-A-C660^R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results from the MKK3-A-G1093A mutation.
• Sjoberg, S. M., Carter, A. H., Steber, C. M., & Garland-Campbell, K. A. (2020) Application of the factor analytic model to assess wheat falling number performance and stability in multi‐environment trials. Crop Science, doi:10.1002/csc2.20293. (Show/Hide Abstract)
  Abstract: A factor analytic model was used to characterize data generated with the Hagberg‐Perten Falling Number (FN) method, a measure of wheat quality influenced by genotype‐by‐environment interactions. The FN method detects starch degradation due to the presence of the enzyme alpha‐amylase in wheat grain such that a low FN indicates high alpha‐amylase activity and high risk of poor end‐product quality. Because farmers receive severe discounts for low FN, FN data has been collected over multiple years for the Washington State University multilocation variety trials to help farmers and breeders identify lower risk varieties. Analysis of this data to objectively rank varieties is challenging because the dataset is unbalanced and because FN is subject to complex genotype‐by‐environment interactions. Low FN can result from environmental differences at multiple stages in grain development because there are two major causes of alpha‐amylase accumulation in grain, late‐maturity alpha‐amylase (LMA) and preharvest sprouting (PHS). A five‐factor analytic model extracted explicit measures of overall performance and of stability in variable environments from historical falling number data from the multilocation trial, providing a basis for breeding and planting decisions. Whereas a linear model explained 70.3% of the variation, the five‐factor analytic model accounted for 92.5% of variation in the data. Examination of factor loadings enabled us to separate environments and genotype response to either PHS or LMA, specifically. This is the first application of a factor analytic model to evaluate the end‐use quality trait, FN, providing a method to rank varieties for grower decisions and for breeder selections.

2019

• Steber, C. M., & Garland Campbell, K. A. (2019, April). Rising optimism fuels falling numbers research. Wheat Life, 62(4), 40–52.
• Liu, C. (2019). Late maturity alpha-amylase in north American spring wheat (Triticum aestivum L.). (Master's thesis). Washington State University. (Show/Hide Abstract)
  Abstract: Late maturity alpha-amylase (LMA) is considered a genetic defect in wheat (Triticum aestivum L.), resulting in the induction of alpha-amylase enzyme in response to a low or high temperature shock during late grain filling. The wheat industry uses the Hagberg-Perten Falling Number (FN) method to detect loss of gelling capacity due to the presence of alpha-amylase in meal or flour. Low FN is associated with higher risk of poor end-product quality, such as cakes that fall and sticky bread or noodles. To improve selection for LMA resistance, LMA testing methods were optimized and then used to characterize the LMA susceptibility in North American wheat. Preliminary LMA testing results were highly variable in cold-treated and in untreated controls. Warmer (25°C day/ 18°C night) and drier (˜55-65% relative humidity) conditions reduced alpha- amylase levels in untreated controls. Colder LMA-induction experiments did not result in stronger or more consistent LMA-induction in spring wheat variety, WA8124. The most significant cause of LMA phenotypic variability appeared to be variability in the developmental window during which a 7-day low temperature shock triggered LMA. This window varied with environmental conditions prior to grain development and by genotype, such that WA8124 induced LMA at 20-24 days past anthesis (dpa) whereas Australian ‘Kennedy’ induced at 25-29 dpa. Only 21% of the 256 North American breeding lines characterized showed LMA resistance, suggesting that improved selection for LMA resistance is needed. In this panel, the tall rht-B1a rht-D1a genotype was associated with higher LMA in both cold-treated and untreated experiments, suggesting the presence of a constitutive LMA phenotype that did not require cold treatment. However, some rht-B1a rht-D1a lines required cold induction whereas some semi-dwarf lines had constitutive LMA, suggesting that the constitutive LMA phenotype is genetically complex. A preliminary genome-wide association study identified six significant marker-trait associations on chromosomes 2B, 3A, 3B, 5A, 7B, and 7D. The QLMA.wsu.7B locus detected in this study co-localized a QTL detected in four previous studies of Australian and CIMMYT germplasm. Future work will determine if these molecular markers are effective in selecting LMA resistance in U.S. wheat.

2018

• Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2018, January). Hunting for genes: falling numbers project seeks to reduce risk by breeding for genetic resistance. Wheat Life, 61(1), 50–51.
• Martinez, S. A., Godoy, J., Huang, M., Zhang, Z., Carter, A. H., Garland Campbell, K. A., & Steber, C. M. (2018). Genome-Wide Association Mapping for Tolerance to Preharvest Sprouting and Low Falling Numbers in Wheat. Frontiers in Plant Science, 9, 1–16, https://doi.org/10.3389/fpls.2018.00141. (Show/Hide Abstract)
  Abstract: Preharvest sprouting (PHS), the germination of grain on the mother plant under cool and wet conditions, is a recurring problem for wheat farmers worldwide. α-amylase enzyme produced during PHS degrades starch resulting in baked good with poor end-use quality. The Hagberg-Perten Falling Number (FN) test is used to measure this problem in the wheat industry, and determines how much a farmer’s wheat is discounted for PHS damage. PHS tolerance is associated with higher grain dormancy. Thus, breeding programs use germination-based assays such as the spike-wetting test to measure PHS susceptibility. Association mapping identified loci associated with PHS tolerance in U.S. Pacific Northwest germplasm based both on FN and on spike-wetting test data. The study was performed using a panel of 469 white winter wheat cultivars and elite breeding lines grown in six Washington state environments, and genotyped for 15,229 polymorphic markers using the 90k SNP Illumina iSelect array. Marker-trait associations were identified using the FarmCPU R package. Principal component analysis was directly and a kinship matrix was indirectly used to account for population structure. Nine loci were associated with FN and 34 loci associated with PHS based on sprouting scores. None of the QFN.wsu loci were detected in multiple environments, whereas six of the 34 QPHS.wsu loci were detected in two of the five environments. There was no overlap between the QTN detected based on FN and PHS, and there was little correlation between the two traits. However, both traits appear to be PHS-related since 19 of the 34 QPHS.wsu loci and four of the nine QFN.wsu loci co-localized with previously published dormancy and PHS QTL. Identification of these loci will lead to a better understanding of the genetic architecture of PHS and will help with the future development of genomic selection models.
• Martinez, S. A., Thompson, A. L., Wen, N., Murphy, L., Sanguinet, K. A., Steber, C. M., & Garland Campbell, K. A. (2018). Registration of the Louise/Alpowa Wheat Recombinant Inbred Line Mapping Population. Journal of Plant Registrations, doi:10.3198/jpr2017.08.0053crmp. (Show/Hide Abstract)
  Abstract: A mapping population was developed from the cross of soft white spring wheat (Triticum aestivum L.) cultivars 'Louise' and 'Alpowa' for use in investigating the genetic architecture of drought tolerance in the US Pacific Northwest. The Louise/Alpowa (Reg. No. MP-8, NSL 520824 MAP) recombinant inbred line mapping population was developed through single seed descent from the F₂ generation to the F₅ generation. The population consists of 141 F_5:6 recombinant inbred lines, of which 132 were used to construct the genetic linkage map. The 32 linkages groups included 882 single nucleotide polymorphism markers and one simple sequence repeat marker spanning 18 of 21 chromosomes. The Louise/Alpowa population was characterized for variation in agronomic traits, phenology, and end-use quality traits. This population will be used for identification and introgression of multiple loci providing resistance to environmental stress such as drought, stripe rust, and high temperatures.
• Aramrak, A., Lawrence, N. C., Demacon, V. L., Carter, A. H., Kidwell, K. K., Burke, I. C., & Steber, C. M. (2018). Isolation of Mutations Conferring Increased Glyphosate Resistance in Spring Wheat. Crop Science, 58, 84–97. (Show/Hide Abstract)
  Abstract: A mutation breeding approach was used to explore the feasibility of isolating glyphosate- resistant (GR) wheat (Triticum aestivum L.) lines. Although transgenic GR wheat cultivars were developed, they were never introduced due to lack of consumer acceptance and concern over management of volunteer wheat in rotation. Large-scale screening experiments recovered ethyl methanesulfonate mutants able to resist 360 to 480 g acid equivalent (ae) ha^-1 glyphosate in four spring wheat cultivars, 'Hollis', 'Louise', 'Macon', and 'Tara2002', indicating that it is possible to recover resistance in a wide range of genetic backgrounds (glyphosate is typically applied at 840 g ae ha^-1 in transgenic crops). Glyphosate rates of 420 to 530 g ae ha^-1 were sufficient to kill the susceptible wild-type parents. Seven GR mutants were characterized: GRH9-5, GRH9-8, GRL1, GRL33, GRL65, GRM14, and GRT20. Glyphosate resistance was examined at the whole-plant level in dose-response experiments. Three mutant lines – GRL33, GRH9-5, and GRT20 – exhibited resistance based on a significant increase in the dose required to retard growth compared with the corresponding susceptible wild type. According to F₂ segregation analysis, GRL1, GRL65, and GRT20 segregated as a single dominant gene, whereas GRL33, GRH9-5, and GRH9-8 appeared to be either a single semidominant or polygenic trait. Although GRL1 was associated with an amino acid substitution (L239F) in TaEPSPS-7D1, no nucleotide changes were observed in the coding regions of wheat 5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS) gene in GRL33 and GRH9-8. Results suggest that glyphosate resistance can result from multiple genetic mechanisms in wheat.
• Ge, W., & Steber, C. M. (2018). Positive and negative regulation of seed germination by the Arabidopsis GA hormone receptors, GID1a, b, and c. Plant Direct, 2(9), 1–11. (Show/Hide Abstract)
  Abstract: Epistasis analysis of gid1 single and double mutants revealed that GID1c is a key positive regulator of seed germination, whereas the GID1b receptor can negatively regulate germination in dormant seeds and in the dark. The GID1 GA receptors were expected to positively regulate germination because the plant hormone gibberellin (GA) is required for seed germination in Arabidopsis thaliana. The three GA hormone receptors, GID1a, GID1b, and GID1c, positively regulate GA responses via GA/GID1‐ stimulated destruction of DELLA (Asp‐Glu‐Leu‐Leu‐Ala) repressors of GA responses. The fact that the gid1abc triple mutant but not gid1 double mutants fail to germi- nate indicates that all three GA receptors can positively regulate non‐dormant seed germination in the light. It was known that the gid1abc triple mutant fails to lose dormancy through the dormancy breaking treatments of cold stratification (moist chilling of seeds) and dry after‐ripening (a period of dry storage). Previous work sug- gested that there may be some specialization of GID1 gene function during germina- tion because GID1b mRNA expression was more highly induced by after‐ripening, whereas GID1a and GID1c mRNA levels were more highly induced by cold stratifica- tion. In light‐germinated dormant seeds, the gid1b mutation can partly rescue the germination efficiency of gid1a but not of gid1c seeds. Thus, GID1b can function as an upstream negative regulator GID1c, a positive regulator of dormant seed germina- tion. Further experiments showed that GID1b can negatively regulate dark germina- tion. Wild‐type Arabidopsis seeds do not germinate well in the dark. The gid1b and gid1ab double mutants germinated much more efficiently than wild type, gid1c, or gid1ac mutants in the dark. The observation that the gid1ab double mutant also shows increased dark germination suggests that GID1b, and to some extent GID1a, can act as upstream negative regulators of GID1c. Since the gid1abc triple mutant failed to germinate in the dark, it appears that GID1c is a key downstream positive regulator of dark germination. This genetic analysis indicates that the three GID1 receptors have partially specialized functions in GA signaling.
• Delwiche, S. R., Higginbotham, R. W., & Steber, C. M.(2018). Falling number of soft white wheat by near-infrared spectroscopy: A challenge revisited. Cereal Chemistry, 95(3), 469–477. (Show/Hide Abstract)
  Abstract: Background and objectives: Wheat Hagberg falling number (FN) is a long- standing quality test that, by means of measuring the viscosity of a heated water-meal or water-flour mixture, characterizes the activity of endogenous α-amylase, the enzyme primarily responsible for starch hydrolysis. The accuracy, time requirement, and cost of this test have come under heightened scrutiny, particularly in seasons when weather conditions have been favorable to preharvest sprouting or late maturity amylase. Near-infrared (NIR) spectroscopy, an analytical approach routinely used in the grain industry to measure contents of protein and moisture, was reexamined as a possible alternative to the FN procedure. Findings: Partial least squares (PLS) regression quantitative models developed on a genetically diverse set of Washington grown white wheat demonstrated low accuracy, with standard errors of performance ranging from 40 to 77 s. Alternatively, linear discriminant analysis and PLS discriminant analysis (PLSDA) qualitative models, developed and tested using a FN cutoff (pass/fail) value, also demonstrated low accuracy, with the best model correctly identifying 67% and 71% of the samples, respectively, above and below a threshold value established as the median value of FN in a calibration set of several hundred samples. Conclusions: Replacement of the FN test with one based on NIR spectroscopy on either whole grain or ground meal for making decisions on segregating wheat lots according to α-amylase activity is not recommended. Significance and novelty: Because NIR spectroscopy is not sufficiently accurate to quantitatively model FN or differentiate low from high FN grain, viscometry procedures for starch integrity, such as FN, will continue their use in grain commerce.

2017

• Nelson, S. K., Ariizumi, T., & Steber, C. M. (2017). Biology in the Dry Seed: Transcriptome Changes Associated with Dry Seed Dormancy and Dormancy Loss in the Arabidopsis GA-Insensitive sleepy1-2 Mutant. Frontiers in Plant Science, 8, 1–21, https://doi.org/10.3389/fpls.2017.02158. (Show/Hide Abstract)
  Abstract: Plant embryos can survive years in a desiccated, quiescent state within seeds. In many species, seeds are dormant and unable to germinate at maturity. They acquire the capacity to germinate through a period of dry storage called after-ripening (AR), a biological process that occurs at 5–15% moisture when most metabolic processes cease. Because stored transcripts are among the first proteins translated upon water uptake, they likely impact germination potential. Transcriptome changes associated with the increased seed dormancy of the GA-insensitive sly1-2 mutant, and with dormancy loss through long sly1-2 after-ripening (19 months) were characterized in dry seeds. The SLY1 gene was needed for proper down-regulation of translation-associated genes in mature dry seeds, and for AR up-regulation of these genes in germinating seeds. Thus, sly1-2 seed dormancy may result partly from failure to properly regulate protein translation, and partly from observed differences in transcription factor mRNA levels. Two positive regulators of seed dormancy, DELLA GAI (GA-INSENSITIVE) and the histone deacetylase HDA6/SIL1 (MODIFIERS OF SILENCING1) were strongly AR-down- regulated. These transcriptional changes appeared to be functionally relevant since loss of GAI function and application of a histone deacetylase inhibitor led to decreased sly1-2 seed dormancy. Thus, after-ripening may increase germination potential over time by reducing dormancy-promoting stored transcript levels. Differences in transcript accumulation with after-ripening correlated to differences in transcript stability, such that stable mRNAs appeared AR-up-regulated, and unstable transcripts AR-down-regulated. Thus, relative transcript levels may change with dry after-ripening partly as a consequence of differences in mRNA turnover.
• Nelson, S. K, & Steber, C. M. (2017). Transcriptional mechanisms associated with seed dormancy and dormancy loss in the gibberellin-insensitive sly1-2 mutant of Arabidopsis thaliana. PLoS ONE 12(6), 1–32, https://doi.org/10.1371/journal.pone.0179143. (Show/Hide Abstract)
  Abstract: While widespread transcriptome changes were previously observed with seed dormancy loss, this study specifically characterized transcriptional changes associated with the increased seed dormancy and dormancy loss of the gibberellin (GA) hormone-insensitive sleepy1-2 (sly1-2) mutant. The SLY1 gene encodes the F-box subunit of an SCF E3 ubiquitin ligase needed for GA-triggered proteolysis of DELLA repressors of seed germination. DELLA over- accumulation in sly1-2 seeds leads to increased dormancy that can be rescued without DELLA protein destruction either by overexpression of the GA receptor, GA-INSENSITIVE DWARF1b (GID1b-OE) (74% germination) or by extended dry after-ripening (11 months, 51% germination). After-ripening of sly1 resulted in different transcriptional changes in early versus late Phase II of germination that were consistent with the processes known to occur. Approxi- mately half of the transcriptome changes with after-ripening appear to depend on SLY1-trig- gered DELLA proteolysis. Given that many of these SLY1/GA-dependent changes are genes involved in protein translation, it appears that GA signaling increases germination capacity in part by activating translation. While sly1-2 after-ripening was associated with transcript-level changes in 4594 genes over two imbibition timepoints, rescue of sly1-2 germination by GID1b- OE was associated with changes in only 23 genes. Thus, a big change in sly1-2 germination phenotype can occur with relatively little change in the global pattern of gene expression during the process of germination. Most GID1b-OE-responsive transcripts showed similar changes with after-ripening in early Phase II of imbibition, but opposite changes with after-ripening by late Phase II. This suggests that GID1b-OE stimulates germination early in imbibition, but may later trigger negative feedback regulation.
• Steber, C. M., & Campbell, K. G. (2017, November). All hands on deck: USDA-ARS, WSU scientists tackle falling numbers. Wheat Life, 60(10), 40–41.
• Steber, C. M. (2017, March-April). Avoiding problems in wheat with low Falling Numbers. Crops & Soils Magazine, (50)2, 22–25.

2016

• Nelson, S. K, & Steber, C. M. (2016). Gibberellin hormone signal perception: down-regulating DELLA repressors of plant growth and development. In P. Hedden & S. G. Thomas (Eds), Annual Plant Reviews, Volume 49, The Gibberellins (pp. 153–187). Chichester, UK: Wiley Blackwell. (Show/Hide Abstract)
  Abstract: The gibberellin (GA) hormone signal is perceived by a receptor with homology to hormone-sensitive lipases, GID1 (GA-INSENSITIVE DWARF1). This leads to GA-stimulated responses, including stem elongation, seed germination and the transition to flowering. GA-binding enables GID1 to interact with and block the function of the DELLA repressors of GA responses. DELLA repression can be blocked both by proteolytic and non-proteolytic mechanisms triggered by the formation of a GID1-GA-DELLA complex. DELLA is down-regulated by the SLEEPY1/GID2 F-box proteins via the ubiquitin-proteasome pathway, and can be regulated by other post-translational modifications. This chapter reviews the structural requirements for GA-binding by GID1 and for GID1-GA-DELLA protein complex formation, and reviews the current understanding of the mechanisms regulating DELLA repressors.
• Steber, C. M. (2016). Managing the risk of low falling numbers in wheat. Washington State University Extension, FS242E, 1–6.
• Martinez, S. A., Tuttle K. M., Takebayashi, Y., Seo, M., Campbell, K. G., & Steber, C. M. (2016). The wheat ABA hypersensitive ERA8 mutant is associated with increased preharvest sprouting tolerance and altered hormone accumulation. Euphytica, 212(2), 229–245. (Show/Hide Abstract)
  Abstract: Wheat preharvest sprouting (PHS) is the germination of mature grain on the mother plant when rain occurs before harvest. Higher abscisic acid (ABA) hormone levels and sensitivity are associated with higher seed dormancy and PHS tolerance. Consistent with this, the ABA hypersensitive ENHANCED RESPONSE TO ABA8 (ERA8) mutant resulted in increased dormancy and PHS tolerance in soft white spring wheat 'Zak'. ERA8 seeds were initially less responsive to germination-rescue by the hormone gibberellin (GA). ERA8 gained GA and lost ABA sensitivity more slowly than wild-type during dormancy loss through after-ripening and cold imbibition. This study examined if increased ABA sensitivity in ERA8 likely resulted from increased ABA signaling or increased ABA hormone levels. Zak ERA8 had higher initial grain dormancy although endogenous embryo ABA levels were similar in Zak ERA8 and wild-type, suggesting that increased dormancy was due to increased ABA signaling rather than increased ABA accumulation. ABA levels declined with Zak ERA8 after-ripening, suggesting that ABA turnover is not defective. Elevated ERA8 dormancy was also associated with increased embryonic jasmonic acid-Ile and aleurone indole-3-acetic acid (IAA) levels. The possible implication that other plant hormones may influence wheat seed dormancy and germination are discussed.
• Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2016, December). Get out your hard hats, the numbers are falling. Wheat Life, 59(11), 47–49.
• Steber, C. M., Kidwell, K. K., Demacon, V. L., & Okubara, P. A. (2016). MUTATION BREEDING FOR RESISTANCE TO FUNGAL DISEASE AND FOR DROUGHT TOLERANCE. United States Patent No.: 9,491,916. (Show/Hide Abstract)
  Abstract: Particular aspects provide novel mutant plants and plant parts thereof, derived via mutagenesis, having disease resistance and other useful traits. Particular embodiments provide a wheat genotype 'RRR Scarlet' ('Scarlet-Rz1'), plants and seeds thereof, methods for producing a plant comprising crossing 'Scarlet-Rz1' plants with another wheat plant, hybrid wheat seeds and plants produced by crossing 'Scarlet-Rz1' plants with another line or plant, and creation of variants by mutagenesis or transformation of 'Scarlet-Rz1'. Additional aspects provide methods for producing other varieties or breeding lines derived from 'Scarlet-Rz1' and to varieties or breeding lines produced thereby. Further aspects provide for mutant plants and plant parts thereof that are resistant and/or tolerant to plant root fungal pathogens such as Rhizoctonia and Pythium. Additional embodiments provide mutant plants and plant parts thereof that exhibit stress tolerance and/or resistance. Yet further aspects provide mutant plants and plant parts thereof that are drought resistant or tolerant.

2015

• Tuttle, K. M., Martinez, S. A., Schramm, E. C., Takebayashi, Y., Seo, M., & Steber, C. M. (2015). Grain dormancy loss is associated with changes in ABA and GA sensitivity and hormone accumulation in bread wheat, Triticum aestivum (L.). Seed Science Research, 25(2), 179–193. (Show/Hide Abstract)
  Abstract: Knowledge about the hormonal control of grain dormancy and dormancy loss is essential in wheat, because low grain dormancy at maturity is associated with the problem of pre-harvest sprouting (PHS) when cool and rainy conditions occur before harvest. Low GA (gibberellin A) hormone sensitivity and high ABA (abscisic acid) sensitivity were associated with higher wheat grain dormancy and PHS tolerance. Grains of two PHS-tolerant cultivars were very dormant at maturity, and insensitive to GA stimulation of germination. More PHS-susceptible cultivars were less sensitive to ABA inhibition of germination, and were either more GA sensitive or germinated efficiently without GA at maturity. As grain dormancy was lost through dry afterripening or cold imbibition, grains first gained GA sensitivity and then lost ABA sensitivity. These changes in GA and ABA sensitivity can serve as landmarks defining stages of dormancy loss that cannot be discerned without hormone treatment. These dormancy stages can be used to compare different cultivars, seed lots and studies. Previous work showed that wheat afterripening is associated with decreasing ABA levels in imbibing seeds. Wheat grain dormancy loss through cold imbibition also led to decreased endogenous ABA levels, suggesting that reduced ABA signalling is a general mechanism triggering dormancy loss.
• Hauvermale, A. L., Tuttle, K. M., Takebayashi, Y., Seo, M., and Steber, C. M. (2015). Loss of Arabidopsis thaliana seed dormancy is associated with increased accumulation of the GID1 GA hormone receptors. Plant and Cell Physiology, 56(9), 1773–1785. (Show/Hide Abstract)
  Abstract: Dormancy prevents seeds from germinating under favorable conditions until they have experienced dormancy-breaking conditions, such as after-ripening through a period of dry storage or cold imbibition. Abscisic acid (ABA) hormone signaling establishes and maintains seed dormancy, whereas gibberellin (GA) signaling stimulates germination. ABA levels decrease and GA levels increase with after-ripening and cold stratification. However, increasing GA sensitivity may also be critical to dormancy loss since increasing seed GA levels are detectable only with long periods of after-ripening and imbibition. After-ripening and cold stratification act additively to enhance GA hormone sensitivity in ga1-3 seeds that cannot synthesize GA. Since the overexpression of the GA receptor GID1 (GA-INSENSITIVE DWARF1) enhanced this dormancy loss, and because gid1a gid1b gid1c triple mutants show decreased germination, the effects of dormancy–breaking treatments on GID1 mRNA and protein accumulation were examined. Partial after-ripening resulted in increased GID1b, but not GID1a or GID1c mRNA levels. Cold imbibition stimulated the accumulation of all three GID1 transcripts, but resulted in no increase in GA sensitivity during ga1-3 seed germination unless seeds were also partially after-ripened. This is likely because after-ripening was needed to enhance GID1 protein accumulation, independently of transcript abundance. The rise in GID1b transcript with after-ripening was not associated with decreased ABA levels, suggesting there is ABA-independent GID1b regulation by after-ripening. GA and the DELLA RGL2 repressor of GA responses differentially regulated the three GID1 transcripts. Moreover, DELLA RGL2 appeared to switch between positive and negative regulation of GID1 expression in response to dormancy breaking treatments. The rise in GID1b transcript with after-ripening was not associated with decreased ABA levels, suggesting that after-ripening regulates GID1 independently of ABA.
• Aramrak, A., Kidwell, K. K., Steber, C. M., & Burke, I. C. (2015). Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.). BMC Genomics, 16(844), 1–14. (Show/Hide Abstract)
  Abstract:
  Background: 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the sixth and penultimate enzyme in the shikimate biosynthesis pathway, and is the target of the herbicide glyphosate. The EPSPS genes of allohexaploid wheat (Triticum aestivum, AABBDD) have not been well characterized. Herein, the three homoeologous copies of the allohexaploid wheat EPSPS gene were cloned and characterized.
  Methods: Genomic and coding DNA sequences of EPSPS from the three related genomes of allohexaploid wheat were isolated using PCR and inverse PCR approaches from soft white spring ''Louise''. Development of genomespecific primers allowed the mapping and expression analysis of TaEPSPS-7A1, TaEPSPS-7D1, and TaEPSPS-4A1 on chromosomes 7A, 7D, and 4A, respectively. Sequence alignments of cDNA sequences from wheat and wheat relatives served as a basis for phylogenetic analysis.
  Results: The three genomic copies of wheat EPSPS differed by insertion/deletion and single nucleotide polymorphisms (SNPs), largely in intron sequences. RT-PCR analysis and cDNA cloning revealed that EPSPS is expressed from all three genomic copies. However, TaEPSPS-4A1 is expressed at much lower levels than TaEPSPS-7A1 and TaEPSPS-7D1 in wheat seedlings. Phylogenetic analysis of 1190-bp cDNA clones from wheat and wheat relatives revealed that: 1) TaEPSPS-7A1 is most similar to EPSPS from the tetraploid AB genome donor, T. turgidum (99.7% identity); 2) TaEPSPS-7D1 most resembles EPSPS from the diploid D genome donor, Aegilops tauschii (100% identity); and 3) TaEPSPS-4A1 resembles EPSPS from the diploid B genome relative, Ae. speltoides (97.7% identity). Thus, EPSPS sequences in allohexaploid wheat are preserved from the most two recent ancestors. The wheat EPSPS genes are more closely related to Lolium multiflorum and Brachypodium distachyon than to Oryza sativa (rice).
  Conclusions: The three related EPSPS homoeologues of wheat exhibited conservation of the exon/intron structure and of coding region sequence, but contained significant sequence variation within intron regions. The genome-specific primers developed will enable future characterization of natural and induced variation in EPSPS sequence and expression. This can be useful in investigating new causes of glyphosate herbicide resistance.

2014

• Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2014, July). Falling numbers: research strategies to stay out of the red. Wheat Life, 57, 56–59.
• Hauvermale, A. L., Ariizumi, T., & Steber, C. M. (2014). The roles of the GA receptors GID1a, GID1b, and GID1c in sly1-independent GA signaling. Plant Signaling & Behavior 9:e28030. (Show/Hide Abstract)
  Abstract: Gibberellin (GA) hormone signaling occurs through proteolytic and non-proteolytic mechanisms. GA binding to the GA receptor GID1 (GA-INSENSITIVE DWARF1) enables GID1 to bind negative regulators of GA responses called DELLA proteins. In proteolytic GA signaling, the SLEEPY1 (SLY1) F-box protein targets DELLA proteins in the GID1-GA-DELLA complex for destruction through the ubiquitin-proteasome pathway. Non-proteolytic GA signaling occurs in sly1 mutants when GA cannot target DELLA proteins for destruction, and requires GA and GID1 gene function. Based on comparison of gid1 multiple mutants to sly1 gid1 mutants, GID1a is the primary GA receptor stimulating stem elongation in proteolytic and non-proteolytic signaling, and stimulating fertility in proteolytic GA signaling. GID1b plays the primary role in fertility, and a secondary role in elongation during non-proteolytic GA signaling. The stronger role of GID1b in non-proteolytic GA signaling may result from the fact that GID1b has higher affinity for DELLA protein than GID1a and GID1c.
• Kidwell, K. K., Steber, C. M., Demacon, V. L., Shelton, G. B., & Burke, A. B. (2014). GLYPHOSATE-TOLERANT WHEAT GENOTYPES. United States Patent No.: 8,637,738. (Show/Hide Abstract)
  Abstract: The present invention provides methods for producing glyphosate-tolerant wheat genotypes by mutagenesis, glyphosate wheat plants produced by such methods, and related compositions and methods.
• Martinez, S. A., Schramm, E. C., Harris, T. J., Kidwell, K. K., Garland-Campbell, K., & Steber, C. M. (2014). Registration of Zak ERA8 Soft White Spring Wheat Germplasm with Enhanced Response to ABA and Increased Seed Dormancy. Journal of Plant Registrations, 8(2), 217–220. (Show/Hide Abstract)
  Zak ERA8 (ENHANCED RESPONSE to ABA8) (Reg. No. GP-966, PI 669443) is a unique line derived from soft white spring wheat (Triticum aestivum L.) cultivar Zak that has increased seed dormancy but after-ripens within 10 to 16 wk. The goal in developing this germplasm was to use increased seed dormancy to improve tolerance to preharvest sprouting, a problem that can cause severe economic losses. This germplasm was developed by USDA–ARS, Pullman, WA, in collaboration with Washington State University. Zak ERA8 was tested under experimental number 60.1.27.10. The ERA8 mutation was generated by chemical mutagenesis followed by selection for the inability to germinate on abscisic acid (ABA) concentrations too low to inhibit wild-type Zak seed germination. The semidominant Zak ERA8 line has been backcrossed twice to wild-type Zak. Following the first backcross, Zak ERA8 showed similar morphological and grain quality traits to the original Zak cultivar.

2013

• Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2013, August/September). Preventing those falling number blues. Wheat Life, 56, 67–69.
• Ariizumi, T., Hauvermale, A. L., Nelson, S. K., Hanada, A., Yamaguchi, S., & Steber, C. M. (2013). Lifting DELLA repression of Arabidopsis seed germination by non-proteolytic GA signaling. Plant Physiology, 162(4), 2125–2139. (Show/Hide Abstract)
  Abstract: DELLA repression of Arabidopsis thaliana seed germination can be lifted either through DELLA proteolysis by the ubiquitin-proteasome pathway or through proteolysis-independent GA hormone signaling. GA-binding to the GID1 (GIBBERELLIN-INSENSITIVE DWARF1) GA receptors stimulates GID1-GA-DELLA complex formation which in turn triggers DELLA protein ubiquitination and proteolysis via the SCF^SLY1 E3 ubiqutin ligase and 26S proteasome. Although DELLA cannot be destroyed in the sly1-2 F-box mutant, long dry after-ripening and GID1 overexpression can relieve the strong sly1-2 seed dormancy phenotype. It appears that sly1-2 seed dormancy results from ABA signaling downstream of DELLA since dormant sly1-2 seeds accumulate high levels of ABA hormone and loss of ABA sensitivity rescues sly1-2 seed germination. DELLA positively regulates the expression of XERICO, an inducer of ABA biosynthesis. GID1b overexpression rescues sly1-2 germination through proteolysis-independent DELLA down-regulation associated with increased expression of GA-inducible genes and decreased ABA accumulation, apparently as a result of decreased XERICO mRNA levels. Higher levels of GID1 overexpression are associated with more efficient sly1 germination and increased GID1-GA-DELLA complex formation, suggesting that GID1 downregulates DELLA through protein binding. After-ripening results in increased GA accumulation and GID1a-dependent GA signaling, suggesting that after-ripening triggers GA-stimulated GID1-GA-DELLA protein complex formation which in turn blocks DELLA transcriptional activation of the XERICO inhibitor of seed germination.
• Schramm, E. C., Nelson, S. K., Kidwell, K. K., & Steber, C. M. (2013). Increased ABA sensitivity results in higher seed dormancy in soft white spring wheat cultivar ‘Zak’. Theoretical and Applied Genetics, 126(3), 791–803. (Show/Hide Abstract)
  Abstract: As a strategy to increase the seed dormancy of soft white wheat, mutants with increased sensitivity to the plant hormone abscisic acid (ABA) were identified in mutagenized grain of soft white spring wheat “Zak”. Lack of seed dormancy is correlated with increased susceptibility to preharvest sprouting in wheat, especially those cultivars with white kernels. ABA induces seed dormancy during embryo maturation and inhibits the germination of mature grain. Three mutant lines called Zak ERA8, Zak ERA19A, and Zak ERA19B (Zak ENHANCED RESPONSE to ABA) were recovered based on failure to germinate on 5 μM ABA. All three mutants resulted in increased ABA sensitivity over a wide range of concentrations such that a phenotype can be detected at very low ABA concentrations. Wheat loses sensitivity to ABA inhibition of germination with extended periods of dry after-ripening. All three mutants recovered required more time to after-ripen sufficiently to germinate in the absence of ABA and to lose sensitivity to 5 μM ABA. However, an increase in ABA sensitivity could be detected after as long as 3 years of after-ripening using high ABA concentrations. The Zak ERA8 line showed the strongest phenotype and segregated as a single semi-dominant mutation. This mutation resulted in no obvious decrease in yield and is a good candidate gene for breeding preharvest sprouting tolerance.

2012

• Schramm, E. C., Nelson, S. K., & Steber, C. M. (2012). Wheat ABA-insensitive mutants result in reduced grain dormancy. Euphytica, 188(1), 35–49. (Show/Hide Abstract)
  Abstract: This paper describes the isolation of wheat mutants in the hard red spring Scarlet resulting in reduced sensitivity to the plant hormone abscisic acid (ABA) during seed germination. ABA induces seed dormancy during embryo maturation and inhibits the germination of mature seeds. Wheat sensitivity to ABA gradually decreases with dry after-ripening. Scarlet grain normally fails to germinate when fully dormant, shows ABA sensitive germination when partially after-ripened, and becomes ABA insensitive when after-ripened for 8–12 months. Scarlet ABA-insensitive (ScABI) mutants were isolated based on the ability to germinate on 5 μM ABA after only 3 weeks of after-ripening, a condition under which Scarlet would fail to germinate. Six independent seed-specific mutants were recovered. ScABI1, ScABI2, ScABI3 and ScABI4 are able to germinate more efficiently than Scarlet at up to 25 μM ABA. The two strongest ABA insensitive lines, ScABI3 and ScABI4, both proved to be partly dominant suggesting that they result from gain-of-function mutations. The ScABI1, ScABI2, ScABI3, ScABI4, and ScABI5 mutants after-ripen more rapidly than Scarlet. Thus, ABA insensitivity is associated with decreased grain dormancy in Scarlet wheat. This suggests that ABA sensitivity is an important factor controlling grain dormancy in wheat, a trait that impacts seedling emergence and pre-harvest sprouting resistance.
• Hauvermale, A. L., Ariizumi, T., & Steber, C. M. (2012). Gibberellin signaling: A theme and variations on DELLA repression. Plant Physiology, 160(1), 83–92.

2011

• Ariizumi, T., Lawrence, P. K., & Steber, C. M. (2011). The role of two F-box proteins, SLEEPY1 and SNEEZY, in arabidopsis gibberellin signaling. Plant Physiology, 155(2), 765–775. (Show/Hide Abstract)
  Abstract: The SLEEPY1 (SLY1) F-box gene is a positive regulator of gibberellin (GA) signaling in Arabidopsis (Arabidopsis thaliana). Loss of SLY1 results in GA-insensitive phenotypes including dwarfism, reduced fertility, delayed flowering, and increased seed dormancy. These sly1 phenotypes are partially rescued by overexpression of the SLY1 homolog SNEEZY (SNE)/SLY2, suggesting that SNE can functionally replace SLY1. GA responses are repressed by DELLA family proteins. GA relieves DELLA repression when the SCF^SLY1 (for Skp1, Cullin, F-box) E3 ubiquitin ligase ubiquitinates DELLA protein, thereby targeting it for proteolysis. Coimmunoprecipitation experiments using constitutively expressed 35S:hemagglutinin (HA)-SLY1 and 35S:HA-SNE translational fusions in the sly1-10 background suggest that SNE can function similarly to SLY1 in GA signaling. Like HA-SLY1, HA-SNE interacted with the CULLIN1 subunit of the SCF complex, and this interaction required the F-box domain. Like HA-SLY1, HA-SNE coimmunoprecipitated with the DELLA REPRESSOR OF GA1-3 (RGA), and this interaction required the SLY1 or SNE carboxyl-terminal domain. Whereas HA-SLY1 overexpression resulted in a decrease in both DELLA RGA and RGA-LIKE2 (RGL2) protein levels, HA-SNE caused a decrease in DELLA RGA but not in RGL2 levels. This suggests that one reason HA-SLY1 is able to effect a stronger rescue of sly1-10 phenotypes than HA-SNE is because SLY1 regulates a broader spectrum of DELLA proteins. The FLAG-SLY1 fusion protein was found to coimmunoprecipitate with the GA receptor HA-GA-INSENSITIVE DWARF1b (GID1b), supporting the model that SLY1 regulates DELLA through interaction with the DELLA-GA-GID1 complex.
• Ariizumi, T., & Steber, C. M. (2011). Mutations in the F-box gene SNEEZY result in decreased Arabidopsis GA signaling. Plant Signaling & Behavior, 6(6), 831–833. (Show/Hide Abstract)
  Abstract: We previously reported that the SLEEPY1 (SLY1) homolog, F-box gene SNEEZY/SLEEPY2 (SNE/SLY2), can partly replace SLY1 in gibberellin (GA) hormone signaling through interaction with DELLAs RGA and GAI. To determine whether SNE normally functions in GA signaling, we characterized the phenotypes of two T-DNA alleles, sne-t2 and sne-t3. These mutations result in no apparent vegetative phenotypes, but do result in increased ABA sensitivity in seed germination. Double mutants sly1-t2 sne-t2 and sly1-t2 sne-t3 result in a significant decrease in plant fertility and final plant height compared to sly1-t2. The fact that sne mutations have an additive effect with sly1 suggests that SNE normally functions as a redundant positive regulator of GA signaling.
• Nirmala, J., Drader, T., Lawrence, P. K., Yin, C., Hulbert, S., Steber, C. M., Steffenson, B. J., Szabo, L.J., von Wettstein, D., & Kleinhof, A. (2011). Concerted action of two avirulent spore effectors activates Reaction to Puccinia graminis 1 (Rpg1)-mediated cereal stem rust resistance. Proc. Natl. Acad. Sci. U.S.A., 108(35), 14676–14681. (Show/Hide Abstract)
  Abstract: The barley stem rust resistance gene Reaction to Puccinia graminis 1 (Rpg1), encoding a receptor-like kinase, confers durable resistance to the stem rust pathogen Puccinia graminis f. sp. tritici. The fungal urediniospores form adhesion structures with the leaf epidermal cells within 1 h of inoculation, followed by hyphae and haustorium formation. The RPG1 protein is constitutively expressed and not phosphorylated. On inoculation with avirulent urediniospores, it is phosphorylated in vivo within 5 min and subsequently degraded. Application of arginine-glycine-aspartic acid peptide loops prevented the formation of adhesion structures for spore attachment, the phosphorylation of RPG1, and germination of the viable spores. Arginine-glycine-aspartic acid affinity chromatography of proteins from the ungerminated avirulent rust spores led to the purification and identification of a protein with fibronectin type III and breast cancer type 1 susceptibility protein domains and a vacuolar protein sorting-associated protein 9 with a coupling of ubiquitin to endoplasmic reticulum degradation domain. Both proteins are required to induce in vivo phosphorylation and degradation of RPG1. Combined application of both proteins caused hypersensitive reaction on the stem rust-resistant cultivar Morex but not on the susceptible cultivar Steptoe. Expression studies indicated that mRNA of both genes are present in ungerminated urediniospores and are constitutively transcribed in sporelings, infected leaves, and haustoria in the investigated avirulent races. Evidence is presented that RPG1, in yeast, interacts with the two protein effectors from the urediniospores that activate cooperatively the stem rust resistance protein RPG1 long before haustoria formation.
• Steber, C. M., & Skinner, D. (2011, April). It takes a village: (of ARS scientists working together to develop new wheat varieties). Wheat Life, 54, 58–61.

2010

• Schramm, E. C., Abellera, J. C., Strader, L. C., Campbell, K. G., & Steber, C. M. (2010). Isolation of ABA-responsive mutants in allohexaploid bread wheat ( Triticum aestivum L.): Drawing connections to grain dormancy, preharvest sprouting, and drought tolerance. Plant Science, 179(6), 620–629. (Show/Hide Abstract)
  Abstract: This paper describes the isolation of Wheat ABA-responsive mutants (Warm) in the Chinese Spring background of allohexaploid Triticum aestivum L. Because the hormone abscisic acid (ABA) is required for the induction of seed dormancy, stomatal closure, and drought, cold and salt tolerance, ABA-hypersensitive wheat mutants were expected to show increased seed dormancy and decreased leaf transpiration in drying soils. Lack of wheat grain dormancy is associated with a propensity for preharvest sprouting (PHS), the germination of seed on the mother plant when moist conditions persist before harvest. PHS tolerance correlates with higher seed dormancy resulting from red grain color, higher ABA accumulation and sensitivity. Wheat grain loses dormancy and sensitivity to ABA inhibition of seed germination with afterripening. Warm lines maintained higher ABA sensitivity when partially after-ripened. The Warm1 and Warm4 mutants showed the strongest and most reproducible increase in ABA sensitivity, accompanied by a requirement for more prolonged after-ripening to break dormancy. Warm2, Warm3, Warm5, and Warm6 showed normal germination without ABA but increased sensitivity to inhibition of germination by applied ABA. The Warm4 mutant showed decreased whole plant transpiration in drying soil, consistent with the role of ABA in inhibiting vegetative leaf transpiration.
• Schramm, E. C. (2010). Isolation of ABA-response mutants in allohexaploid bread wheat (Triticum aestivum L.): Drawing connections to grain dormancy, preharvest sprouting, and drought tolerance (Doctoral dissertation). Washington State University. (Show/Hide Abstract)
  Abstract: This dissertation describes the characterization of seed dormancy and drought tolerance phenotypes of abscisic acid (ABA) response mutants in allohexaploid wheat (Triticum aestivum L.). The plant hormone ABA is required for the induction of seed dormancy, stomatal closure in drying soils, and drought tolerance. Preharvest sprouting (PHS), the germination of seed on the mother plant, occurs when cool moist conditions persist before harvest. PHS resistance is correlated with seed dormancy resulting from red grain color, higher ABA accumulation or sensitivity. ABA only inhibits the germination of imbibing dormant wheat grain, not of afterripened grain. ABA hypersensitive lines were isolated based on the phenotype of maintaining higher ABA sensitivity when partially after-ripened. ABA hypersensitive phenotypes were stronger and persisted longer in the soft white spring 'Zak' compared to the hard red springs Chinese Spring and 'Scarlet'. ZakERA (Zak Enhanced Response to ABA) 0 and 19A mutants showed increased dormancy associated with a reproducible increase in ABA sensitivity in germination. These mutants required more time to after-ripen than Zak wild type (WT), and still showed ABA sensitivity after 3 years after-ripening. Warm1 (Wheat ABA-responsive mutant) and Warm4 showed the strongest ABA-hypersensitive germination phenotype among Chinese Spring mutants, and were accompanied by decreased germination in the absence of ABA. Although these mutants required more time to after-ripen than WT, they lost ABA sensitivity within 3-12 months. ScERA (Scarlet Enhanced Response to ABA) 10A, 68A, 10C, and 4A showed the strongest ABA-hypersensitive germination phenotype. ScERA 4A is unique in having enhanced ABA sensitivity without any apparent increase in seed dormancy. ABA insensitive mutants in Scarlet, ScABI (Scarlet ABA-Insensitive) 2A, 2C, 1B, and 1C, were insensitive to ABA inhibition of germination with little or no after-ripening. ABA mutants are also expected to show altered water relations. All of the ScERA or ScABI mutants tested so far appear to have only seed germination phenotypes. Consistent with the role of ABA in downregulating transpiration in drying soils, Warm3, Warm4, and ZakERA 19A are associated with decreased leaf transpiration. In Warm4 and ZakERA 19A this decrease in transpiration is associated with higher canopy temperatures under field conditions.
• Steber, C. M. (2010, June). Solutions that don't fall from the sky: New tools will aid future farmers. Wheat Life, 53, 44–47.

2009

• Okubara, P. A., Steber, C. M., Demacon, V. L., Walter, N. L., Paulitz, T. C., & Kidwell, K. K. (2009). Scarlet-Rz1, an EMS-generated hexaploid wheat with tolerance to the soilborne necrotrophic pathogens Rhizoctonia solani AG-8 and R. oryzae. Theoretical and Applied Genetics, 119(2), 293–303. (Show/Hide Abstract)
  Abstract: The necrotrophic root pathogens Rhizoctonia solani AG-8 and R. oryzae cause Rhizoctonia root rot and damping-off, yield-limiting diseases that pose barriers to the adoption of conservation tillage in wheat production systems. Existing control practices are only partially effective, and natural genetic resistance to Rhizoctonia has not been identified in wheat or its close relatives. We report the first genetic resistance/tolerance to R. solani AG-8 and R. oryzae in wheat (Triticum aestivum L. em Thell) germplasm 'Scarlet-Rz1'. Scarlet-Rz1 was derived from the allohexaploid spring wheat cultivar Scarlet using EMS mutagenesis. Tolerant seedlings displayed substantial root and shoot growth after 14 days in the presence of 100–400 propagules per gram soil of R. solani AG-8 and R. oryzae in greenhouse assays. BC₂F₄ individuals of Scarlet-Rz1 showed a high and consistent degree of tolerance. Seedling tolerance was transmissible and appeared to be dominant or co-dominant. Scarlet-Rz1 is a promising genetic resource for developing Rhizoctonia-tolerant wheat cultivars because the tolerance trait immediately can be deployed into wheat breeding germplasm through cross-hybridization, thereby avoiding difficulties with transfer from secondary or tertiary relatives as well as constraints associated with genetically modified plants. Our findings also demonstrate the utility of chemical mutagenesis for generating tolerance to necrotrophic pathogens in allohexaploid wheat.
• McGuire, P. E., Loar, S., Steber, C. M., & Zale, J. (2009). Floral transformation of wheat. In H. D. Jones & P. R. Shewry (Eds), Transgenic Wheat, Barley and Oats: Production and Characterization Protocols (Methods in Molecular Biology) (pp. 105–113). Clifton, NJ: Humana Press. (Show/Hide Abstract)
  Abstract: A method is described for the floral transformation of wheat using a protocol similar to the floral dip of Arabidopsis. This method does not employ tissue culture of dissected embryos, but instead pre-anthesis spikes with clipped florets at the early, mid to late uninucleate microspore stage are dipped in Agrobacterium infiltration media harboring a vector carrying anthocyanin reporters and the NPTII selectable marker. T1 seeds are examined for color changes induced in the embryo by the anthocyanin reporters. Putatively transformed seeds are germinated and the seedlings are screened for the presence of the NPTII gene based on resistance to paromomycin spray and assayed with NPTII ELISAs. Genomic DNA of putative transformants is digested and analyzed on Southern blots for copy number to determine whether the T-DNA has integrated into the nucleus and to show the number of insertions. The nonoptimized transformation efficiencies range from 0.3 to 0.6% (number of transformants/number of florets dipped) but the efficiencies are higher in terms of the number of transformants produced/number of seeds set ranging from 0.9 to 10%. Research is underway to maximize seed set and optimize the protocol by testing different Agrobacterium strains, visual reporters, vectors, and surfactants.
• Zale, J. M., Agarwal, S., Loar, S., & Steber, C. M. (2009). Evidence for stable transformation of wheat by floral dip in agrobacterium tumefaciens. Plant Cell Reports, 28(6), 903–913. (Show/Hide Abstract)
  Abstract: Hexaploid wheat, one of the world’s most important staple crops, remains a challenge for genetic transformation. We are developing a floral transformation protocol for wheat that does not require tissue culture. This paper presents three transformants in the hard red germplasm line Crocus that have been characterized thoroughly at the molecular level over three to six generations. Wheat spikes at the early boot stage, i.e. the early, mid or late uninucleate microspore stages, were immersed in an infiltration medium of strain C58C1 harboring pDs(Hyg)35S, or strain AGL1 harboring pBECKSred. pDs(Hyg)35S contains the NPTII and hph selectable markers, and transformants were detected using paromomycin spray at the whole plant level, NPTII ELISAs, or selection on medium with hygromycin. Strain AGL1, harboring pBECKSred, which contains the maize anthocyanin regulators, Lc and C1, and the NPTII gene, was also used to produce a Crocus transformant. T1 and T2 seeds with red embryos were selected; T1 and T2 plants were screened by sequential tests for paromomycin resistance and NPTII ELISAs. The transformants were low copy number and showed Mendelian segregation in the T2. Stable transmission of the transgenes over several generations has been demonstrated using Southern analysis. Gene expression in advanced progeny was shown using Reverse Transcriptase-PCR and ELISA assays for NPTII protein expression. This protocol has the potential to reduce the time and expense required for wheat transformation.
• Steber, C. M. (2009, March). Up-and-coming wheat research revealed Down Under. Wheat Life, 52, 40–41.

2008

• Ariizumi, T., Murase, K., Sun, T.-p., & Steber, C. M. (2008). Proteolysis-independent downregulation of DELLA repression in Arabidopsis by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1. The Plant Cell, 20(9), 2447–2459. (Show/Hide Abstract)
  Abstract: This article presents evidence that DELLA repression of gibberellin (GA) signaling is relieved both by proteolysis-dependent and -independent pathways in Arabidopsis thaliana. DELLA proteins are negative regulators of GA responses, including seed germination, stem elongation, and fertility. GA stimulates GA responses by causing DELLA repressor degradation via the ubiquitin-proteasome pathway. DELLA degradation requires GA biosynthesis, three functionally redundant GA receptors GIBBERELLIN INSENSITIVE DWARF1 (GID1a, b, and c), and the SLEEPY1 (SLY1) F-box subunit of an SCF E3 ubiquitin ligase. The sly1 mutants accumulate more DELLA proteins but display less severe dwarf and germination phenotypes than the GA biosynthesis mutant ga1-3 or the gid1abc triple mutant. Interestingly, GID1 overexpression rescued the sly1 dwarf and infertility phenotypes without decreasing the accumulation of the DELLA protein REPRESSOR OF ga1-3. GID1 rescue of sly1 mutants was dependent on the level of GID1 protein, GA, and the presence of a functional DELLA motif. Since DELLA shows increasing interaction with GID1 with increasing GA levels, it appears that GA-bound GID1 can block DELLA repressor activity by direct protein–protein interaction with the DELLA domain. Thus, a SLY1-independent mechanism for GA signaling may function without DELLA degradation.
• Finkelstein, R. R., Reeves, W., Ariizumi, T., & Steber, C. M. (2008). Molecular aspects of seed dormancy. Annual Review of Plant Biology, 59, 387–415. (Show/Hide Abstract)
  Abstract: Seed dormancy provides a mechanism for plants to delay germination until conditions are optimal for survival of the next generation. Dormancy release is regulated by a combination of environmental and endogenous signals with both synergistic and competing effects. Molecular studies of dormancy have correlated changes in transcriptomes, proteomes, and hormone levels with dormancy states ranging from deep primary or secondary dormancy to varying degrees of release. The balance of abscisic acid (ABA):gibberellin (GA) levels and sensitivity is a major, but not the sole, regulator of dormancy status. ABA promotes dormancy induction and maintenance, whereas GA promotes progression from release through germination; environmental signals regulate this balance by modifying the expression of biosynthetic and catabolic enzymes. Mediators of environmental and hormonal response include both positive and negative regulators, many of which are feedback-regulated to enhance or attenuate the response. The net result is a slightly heterogeneous response, thereby providing more temporal options for successful germination.
• Schramm, E. C., Abellera, J. C., Strader, L. C., Campbell, K. G., & Steber, C. M. (2008). Can ABA signaling be used to develop drought tolerant wheat? In R. Appels, R. Eastwood, E. Lagudah, P. Langridge, M. Mackay, L. MacIntyre & P. Sharp (Eds.) 11th International Wheat Genetics Symposium 2008 Proceedings – Volume 1 (pp. 116–118). Sydney, Australia: Sydney University Press. (Show/Hide Abstract)
  Abstract: Wheat yield and quality can be compromised by drought stress and preharvest sprouting (PHS) on the mother plant. It is believed that problems with PHS are due to lack of grain dormancy partly resulting from lack of ABA sensitivity. The long term goal is to use wheat mutants with increased sensitivity to ABA to increase grain dormancy and drought tolerance. ABA is required to set up grain dormancy and embryo desiccation tolerance during embryo maturation, and stimulate storage of nutrients. ABA also inhibits germination of mature seeds and stimulates stomatal closure in response to drought stress. A screen for wheat mutants with altered response to ABA in seed germination has been used as a first step to isolate wheat plants with increased ABA sensitivity. Mutants have been characterized for changes in ABA dose-response. In addition, ABA hypersensitive grain germination appears to correspond to reduced vegetative transpiration under drought stress and decreased carbon isotope discrimination.
• Kidwell, K. K., Steber, C. M., Demacon, V. L., Shelton, G. B., & Burke, A. B. (2008). GLYPHOSATE-TOLERANT WHEAT GENOTYPES. United States Patent Application. (Show/Hide Abstract)
  Abstract: The present invention provides methods for producing glyphosate-tolerant wheat genotypes by mutagenesis, glyphosate wheat plants produced by such methods, and related compositions and methods.

2007

• Ariizumi, T., & Steber, C. M. (2007). Seed germination of GA-insensitive sleepy1 mutants does not require RGL2 protein disappearance in Arabidopsis. The Plant Cell, 19(3), 791–804. (Show/Hide Abstract)
  Abstract: We explore the roles of gibberellin (GA) signaling genes SLEEPY1 (SLY1) and RGA-LIKE2 (RGL2) in regulation of seed germination in Arabidopsis thaliana, a plant in which the hormone GA is required for seed germination. Seed germination failure in the GA biosynthesis mutant ga1-3 is rescued by GA and by mutations in the DELLA gene RGL2, suggesting that RGL2 represses seed germination. RGL2 protein disappears before wild-type seed germination, consistent with the model that GA stimulates germination by causing the SCF^SLY1 E3 ubiquitin ligase complex to trigger ubiquitination and destruction of RGL2. Unlike ga1-3, the GA-insensitive sly1 mutants show variable seed dormancy. Seed lots with high seed dormancy after-ripened slowly, with stronger alleles requiring more time. We expected that if RGL2 negatively controls seed germination, sly1 mutant seeds that germinate well should accumulate lower RGL2 levels than those failing to germinate. Surprisingly, RGL2 accumulated at high levels even in after-ripened sly1 mutant seeds with 100% germination, suggesting that RGL2 disappearance is not a prerequisite for seed germination in the sly1 background. Without GA, several GA-induced genes show increased accumulation in sly1 seeds compared with ga1-3. It is possible that the RGL2 repressor of seed germination is inactivated by after-ripening of sly1 mutant seeds.
• Steber, C. M. (2007). De-repression of seed germination by GA signaling. In K. J. Bradford, H. Nonogaki, (Eds.) Annual Plant Reviews Volume 27: Seed Development, Dormancy and Germination (pp. 248–263). Oxford, UK: Blackwell Publishing Ltd.

2006

• Ariizumi, T., & Steber, C. M. (2006). Essay 20.2: Ubiquitin becomes ubiquitous in GA signaling. In L. Taiz & E. Zeigler (Eds.) Plant Physiology (Fourth ed.). Sunderland, MA: Sinauer Associates, Inc.
• Abellera, J. C. (2006). ABA Responsive when Afterripened (ARA) mutants increase seed dormancy, ABA sensitivity, and improve water relations: towards wheat varieties with improved preharvest sprouting and drought tolerance. (Master's thesis). Washington State University. (Show/Hide Abstract)
  Abstract: This thesis explores the hypothesis that mutants isolated based on ABA (abscisic acid) hypersensitive seed germination can be used to obtain drought tolerance and resistance to preharvest sprouting through increased seed dormancy in allohexaploid wheat. The plant hormone ABA is required for establishment of dormancy during seed development, stimulates stomatal closure in response to drought stress and induces expression of genes that promote osmotic adjustment. Arabidopsis mutants isolated for ABA hypersensitive seed germination have increased seed dormancy and drought tolerance resulting from increased sensitivity of stomates to ABA. Based on this result, wheat mutants showing increased sensitivity to ABA in germination of afterripened grain have been isolated and tested for improved vegetative drought tolerance. A total of 25 mutants were isolated from fastneutron mutagenized Chinese Spring, whereas four and 27 mutants were isolated from EMS mutagenized cv. Zak and cv. Scarlet, respectively. Of the 25 Chinese Spring mutants, 4 demonstrated a clear and reproducible hypersensitive response to ABA in dose-response germination assays, whereas 2 lines showed embryo dormancy. The remaining mutants showed inconsistent phenotypes over seven retest experiments suggesting variable expressivity. Genetic analysis of Chinese Spring mutants showed that two are the result of a single dominant mutation, whereas three others may be single semi-dominant mutations. Mutants with a vegetative ABA-hypersensitive phenotype should close their stomates earlier in response to drought stress and have slower transpiration. Drought tolerance was evaluated by comparing the mutant rate of soil moisture loss during drought stress, stomatal conductance, and carbon isotope discrimination (Δ¹³C) to wild-type. Of eight Chinese Spring mutants tested, four showed a slower transpiration rate under drought stress, and one of the four showed reduced stomatal conductance compared to wild-type. The four mutants with ABA-hypersensitive seed germination were the same four mutants that showed lower rate of soil moisture loss under drought stress. Five of 19 mutants appeared to have reduced Δ¹³C relative to wild type, suggesting that they may have improved transpiration efficiency.
• Steber, C. M., Kidwell, K. K., Demacon, V. L., & Okubara, P. A. (2006). MUTATION BREEDING FOR RESISTANCE TO DISEASE AND OTHER USEFUL TRAITS. United States Patent Application. (Show/Hide Abstract)
  Abstract: Particular aspects provide novel mutant plants and plant parts thereof, derived via mutagensis, having disease resistance and other useful traits. Particular embodiments provide a wheat genotye 'RRR Scarlet' ('Scarlet-Rz1'), plants and seeds thereof, methods for producing a plant comprising crossing 'Scarlet-Rz1' plants with another wheat plant, hybrid wheat seeds and plants produced by crossing 'Scarlet-Rz1' plants with another line or plant, and creation of variants by mutagenesis or transformation of 'Scarlet-Rz1'. Additional aspects provide methods for producing other varieties or breeding lines derived from 'Scarlet-Rz1' and to varieties or breeding lines produced thereby. Further aspects provide for mutant plants and plant parts thereof that are resistant and/or tolerant to plant root fungal pathogens such as Rhizoctonia and Pythium. Additional embodiments provide mutant plants and plant parts thereof that exhibit stress tolerance and/or resistance. Yet further aspects provide mutant plants and plant parts thereof that are drought resistant or tolerant.

2005

• Thomas, S. G., Rieu, I., & Steber, C. M. (2005). Gibberellin metabolism and signaling. In G. Litwack (Ed.) Plant Hormones, Volume 72 (Vitamins & Hormones) (pp. 228–338). San Diego & London: Elsevier Academic Press. (Show/Hide Abstract)
  Abstract: Gibberellins (GAs) are a family of plant hormones controlling many aspects of plant growth and development including stem elongation, germination, and the transition from vegetative growth to flowering. Cloning of the genes encoding GA biosynthetic and inactivating enzymes has led to numerous insights into the developmental regulation of GA hormone accumulation that is subject to both positive and negative feedback regulation. Genetic and biochemical analysis of GA‐signaling genes has revealed that posttranslational regulation of DELLA protein accumulation is a key control point in GA response. The highly conserved DELLA proteins are a family of negative regulators of GA signaling that appear subject to GA‐stimulated degradation through the ubiquitin‐26S proteasome pathway. This review discusses the regulation of GA hormone accumulation and signaling in the context of its role in plant growth and development.
• Nelson, S. (2005). Isolating drought tolerant wheat using germination screens for increased ABA hormone sensitivity (Honors thesis). Washington State University.

2004

• Dill, A., Thomas, S. G., Hu, J., Steber, C. M., & Sun, T.-p. (2004). The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation. The Plant Cell, 16(6), 1392–1405. (Show/Hide Abstract)
  Abstract: The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants. In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA). SLEEPY1 (SLY1) encodes an F-box–containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA. These findings suggested that SLY1 recruits RGA to the SCF^SLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome. In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response. By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain. The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCF^SLY1 targets both RGA and GAI for degradation. The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation. However, we found that this DELLA domain is not required for protein–protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins. We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants. This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins.
• Strader, L. C., Ritchie, S., Soule, J. D., McGinnis, K. M., & Steber, C. M. (2004). Recessive-interfering mutations in the gibberellin signaling gene SLEEPY1 are rescued by overexpression of its homologue, SNEEZY. Proc. Natl. Acad. Sci. U.S.A., 101(34), 12771–12776. (Show/Hide Abstract)
  Abstract: This article reports the genetic interaction of two F-box genes, SLEEPY1 (SLY1) and SNEEZY (SNE), in Arabidopsis thaliana gibberellin (GA) signaling. The SLY1 gene encodes an F-box subunit of a Skp1–cullin–F-box (SCF) E3 ubiquitin ligase complex that positively regulates GA signaling. The sly1-2 and sly1-10 mutants have recessive, GA-insensitive phenotypes including delayed germination, dwarfism, reduced fertility, and overaccumulation of the DELLA proteins RGA (Repressor of ga1–3), GAI (GA-Insensitive), and RGL2 (RGA-Like 2). The DELLA domain proteins are putative transcription factors that negatively regulate GA signaling. The requirement for SLY1 in GA-stimulated disappearance of DELLA proteins suggests that GA targets DELLA proteins for destruction via SCF^SLY1-mediated ubiquitylation. Overexpression of SLY1 in sly1-2 and sly1-10 plants rescues the recessive GA-insensitive phenotype of these mutants. Surprisingly, antisense expression of SLY1 also suppresses these mutants. This result caused us to hypothesize that the SLY1 homologue SNE can functionally replace SLY1 in the absence of the recessive interfering sly1-2 or sly1-10 genes. This hypothesis was supported because overexpression of SNE in sly1-10 rescues the dwarf phenotype. In addition to rescuing the sly1-10 dwarf phenotype, SNE overexpression also restored normal RGA protein levels, suggesting that the SNE F-box protein can replace SLY1 in the GA-induced proteolysis of RGA. If the C-terminal truncation in the sly1-2 and sly1-10 alleles interferes with SNE rescue, we reasoned that overexpression of sly1-2 might interfere with wild-type SLY1 function. Indeed, overexpression of sly1-2 in wild-type Ler (Landsberg erecta) yields dwarf plants.
• Zale, J. M., Borchardt-Wier, H., Kidwell, K. K., & Steber, C. M. (2004). Callus induction and plant regeneration from mature embryos of a diverse set of wheat genotypes. Plant Cell, Tissue and Organ Culture, 76(3), 277–281. (Show/Hide Abstract)
  Abstract: This paper compared the behavior of a diverse set of wheat genotypes in their tissue culture response. Significant differences were detected in plant regeneration, culture efficiency, and regeneration capacity when mature embryos of 47 wheat cultivars, breeding lines, and the common wheat progenitors, Triticum monococcum, T.tauschii, and Aegilops speltoides were compared. Although not currently used in wheat tissue culture, mature embryo-derived callus of cv. 'Zak' (SWS), 'Scarlet' (HRS), 'Tara' (SWS), 'Jagger' (HRW), 'UC 1036' (HRS), and 'Kyle' durum showed better or comparable plant regeneration than commonly cultured cultivars 'Fielder' and 'Bobwhite'. Of the three diploid wheat progenitors tested, Ae. speltoides regenerated the most plants. In one replicated experiment, callus induction was correlated with culture efficiency (r = 0.42; p = 0.002) and regeneration capacity (r = 0.39; p = 0.002), and in a second larger screen, callus induction correlated with the total number of plants regenerated (r = 0.6; p = 0.001). Immature and mature embryos of 'Bobwhite' and 'Crocus' were compared for callus induction and plant regeneration. Immature embryos were superior explants in terms of plant regeneration. However, sufficient numbers of plants can be regenerated from mature embryos saving on growth facility resources and time required for the collection of immature embryos.
• Strader, L. C. (2004). Hormonal control of seed dormancy and germination: drawing connections between Arabidopsis thaliana L. and Triticum aestivum L. (Doctoral dissertation). Washington State University.
• Strader, L. C., Zale, J., & Steber, C. M. (2004). Sivb 2003 congress symposium proceeding: Mutation- and transposon-based approaches for the identification of genes for pre-harvest sprouting in wheat. In Vitro Cellular & Developmental Biology - Plant, 40(3), 256–259. (Show/Hide Abstract)
  Abstract: This article reviews techniques for gene identification and cloning in allohexaploid bread wheat (Triticum aestivum L.). Gene identification and cloning in wheat are complicated by the large size and high redundancy of the genome. Both classical mutagenesis and transposon tagging are important tools for the study of grain dormancy and plant hormone signaling in wheat. While classical mutagenesis can be used to identify wheat mutants with altered hormone sensitivity, it can be difficult to clone the corresponding genes. We review the techniques available for gene identification in wheat, and propose that transposon-based activation tagging will be an important tool for wheat genetics.

2003

• McGinnis, K. M., Thomas, S. G., Soule, J. D., Strader, L. C., Zale, J. M., Sun, T.-p., & Steber, C. M. (2003). The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase. The Plant Cell, 15(5), 1120–1130. (Show/Hide Abstract)
  Abstract: The Arabidopsis SLY1 (SLEEPY1) gene positively regulates gibberellin (GA) signaling. Positional cloning of SLY1 revealed that it encodes a putative F-box protein. This result suggests that SLY1 is the F-box subunit of an SCF E3 ubiquitin ligase that regulates GA responses. The DELLA domain protein RGA (repressor of ga1-3) is a repressor of GA response that appears to undergo GA-stimulated protein degradation. RGA is a potential substrate of SLY1, because sly1 mutations cause a significant increase in RGA protein accumulation even after GA treatment. This result suggests SCF^SLY1-targeted degradation of RGA through the 26S proteasome pathway. Further support for this model is provided by the observation that an rga null allele partially suppresses the sly1-10 mutant phenotype. The predicted SLY1 amino acid sequence is highly conserved among plants, indicating a key role in GA response.
• Itoh, H., Matsuoka, M., & Steber, C. M. (2003). A role for the ubiquitin-26S-proteasome pathway in gibberellin signaling. Trends in Plant Science, 8(10), 492–497. (Show/Hide Abstract)
  Abstract: The gibberellin (GA) signaling pathway, like auxin and jasmonate signaling, uses the ubiquitin–proteasome pathway to control expression through protein degradation. A conserved F-box protein of an SCF E3 ubiquitin ligase is a positive regulator of GA signaling in Arabidopsis and rice. GA apparently stimulates stem elongation by causing this SCF complex to regulate negatively a family of negative regulators of GA response (the DELLA family of putative transcription factors). The DELLA family members AtRGA or (Repressor of ga1-3) and OsSLR1 (SLENDER RICE1) proteins both appear to be subject to GA-induced proteolysis. The need to have the F-box genes AtSLY1 and OsGID2 for this proteolysis suggests that GA causes proteolysis of AtRGA/OsSLR1 via the SCF^{AtSLY1/OsGID2} ubiquitin ligase.
• Akhunov, E. D., Goodyear, A. W., Geng, S., Qi, L.-L., Echalier, B., Gill, B. S., Miftahudin, Gustafson, J. P., Lazo, G., Chao, S., Anderson, O. D., Linkiewicz, A. M., Dubcovsky, J., La Rota, M., Sorrells, M. E., Zhang, D., Nguyen, H. T., Kalavacharla, V., Hossain, K., Kianian, S. F., Peng, J., Lapitan, N. L.V., Gonzalez-Hernandez, J. L., Anderson, J. A., Choi, D.-W., Close, T. J., Dilbirligi, M., Gill, K. S., Walker-Simmons, M. K., Steber, C. M., McGuire, P. E., Qualset, C. O. & Dvorak, J. (2003). The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms. Genome Research, 13(5), 753–763. (Show/Hide Abstract)
  Abstract: Genes detected by wheat expressed sequence tags (ESTs) were mapped into chromosome bins delineated by breakpoints of 159 overlapping deletions. These data were used to assess the organizational and evolutionary aspects of wheat genomes. Relative gene density and recombination rate increased with the relative distance of a bin from the centromere. Single-gene loci present once in the wheat genomes were found predominantly in the proximal, low-recombination regions, while multigene loci tended to be more frequent in distal, high-recombination regions. One-quarter of all gene motifs within wheat genomes were represented by two or more duplicated loci (paralogous sets). For 40 such sets, ancestral loci and loci derived from them by duplication were identified. Loci derived by duplication were most frequently located in distal, high-recombination chromosome regions whereas ancestral loci were most frequently located proximal to them. It is suggested that recombination has played a central role in the evolution of wheat genome structure and that gradients of recombination rates along chromosome arms promote more rapid rates of genome evolution in distal, high-recombination regions than in proximal, low-recombination regions.
• Sorrells, M. E., La Rota, M., Bermudez-Kandianis, C. E., Greene, R. A., Kantety, R., Munkvold, J. D., Miftahudin, Mahmoud, A., Ma, X., Gustafson, P. J., Qi, L.-L., Echalier, B., Gill, B. S., Matthews, D. E., Lazo, G. R., Chao, S., Anderson, O. D., Edwards, H., Linkiewicz, A. M., Dubcovsky, J., Akhunov, E. D., Dvorak, J., Zhang, D., Nguyen, H. T., Peng, J., Lapitan, N. L., Gonzalez-Hernandez, J. L., Anderson, J. A., Hossain, K., Kalavacharla, V., Kianian, S. F., Choi, D. W., Close, T. J., Dilbirligi, M., Gill, K. .S, Steber, C. M., Walker-Simmons, M. K., McGuire, P. E., & Qualset, C. O. (2003). Comparative DNA sequence analysis of wheat and rice genomes. Genome Research, 13(8), 1818–1827. (Show/Hide Abstract)
  Abstract: The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to crop species has revolutionized molecular genetics and crop improvement strategies. This study compared 4485 expressed sequence tags (ESTs) that were physically mapped in wheat chromosome bins, to the public rice genome sequence data from 2251 ordered BAC/PAC clones using BLAST. A rice genome view of homologous wheat genome locations based on comparative sequence analysis revealed numerous chromosomal rearrangements that will significantly complicate the use of rice as a model for cross-species transfer of information in nonconserved regions.

2002

• Zale, J. M., & Steber, C. M. (2002). Transposon-related sequences in the Triticeae. Cereal Research Communications, 30(3-4), 237–244. (Show/Hide Abstract)
  Abstract: The purpose of this work was to determine whether sequences homologous to maize transposable elements exist in wheat and its relatives. Genomic Southern blots of Triticeae members were used to detect copy number of homologues to the major open reading frames of the maize Ac and En transposases, and MudrB, a regulator of transposition. The number of similar sequences was the least with Ac, intermediate with Mu, and the greatest with En. Maize transposons at the nucleotide and amino acid level were used in EST and genomic database searches. Nucleotide identity between maize and wheat was 58% for Ac, 59% for Mu, and 61% for En. Protein homologues were found for Ac and Mu in wheat and barley, and for En in barley.

2001

• Steber, C. M., & McCourt, P. (2001). A role for Brassinosteroids in germination in Arabidopsis. Plant Physiology, 125(2), 763–769. (Show/Hide Abstract)
  Abstract: This paper presents evidence that plant brassinosteroid (BR) hormones play a role in promoting germination. It has long been recognized that seed dormancy and germination are regulated by the plant hormones abscisic acid (ABA) and gibberellin (GA). These two hormones act antagonistically with each other. ABA induces seed dormancy in maturing embryos and inhibits germination of seeds. GA breaks seed dormancy and promotes germination. Severe mutations in GA biosynthetic genes in Arabidopsis, such as ga1-3, result in a requirement for GA application to germinate. Whereas previous work has shown that BRs play a critical role in controlling cell elongation, cell division, and skotomorphogenesis, no germination phenotypes have been reported in BR mutants. We show that BR rescues the germination phenotype of severe GA biosynthetic mutants and of the GA-insensitive mutant sleepy1. This result shows that BR stimulates germination and raises the possibility that BR is needed for normal germination. If true, we would expect to detect a germination phenotype in BR mutants. We found that BR mutants exhibit a germination phenotype in the presence of ABA. Germination of both the BR biosynthetic mutant det2-1 and the BR-insensitive mutant bri1-1 is more strongly inhibited by ABA than is germination of wild type. Thus, the BR signal is needed to overcome inhibition of germination by ABA. Taken together, these results point to a role for BRs in stimulating germination.

1998

• Steber, C. M., Cooney, S. E., & McCourt, P. (1998). Isolation of the GA-response mutant sly1 as a suppressor of ABI1-1 in Arabidopsis thaliana. Genetics, 149(2), 509–521. (Show/Hide Abstract)
  Abstract: Seed dormancy and germination in higher plants are partially controlled by the plant hormones abscisic acid (ABA) and gibberellic acid (GA). ABA establishes dormancy during embryo maturation, whereas GA breaks dormancy and induces germination. Previous attempts to identify GA response genes were confounded because GA mutants are not expected to germinate and, unlike GA auxotrophs, should fail to be rescued by exogenous GA. Here, we describe a screen for suppressors of the ABA-insensitive mutant ABI1-1 that enriches for GA auxotrophs and GA-insensitive mutants. The vast majority (76%) of the suppressors of ABI1-1 strongly resemble GA auxotrophs in that they are severely dwarfed and have dark green foliage and flowers with underdeveloped petals and stamen. Three isolates were alleles of the GA auxotroph ga1. The remaining severe dwarves were not rescued by GA and belong to a single complementation group that we designate sly1 (Sleepy 1). The alleles of sly1 identified are the first recessive GAinsensitive mutations to reflect the full spectrum of GA-associated phenotypes, including the failure to germinate in the absence of the ABI1-1 lesion. Thus, we postulate that SLY1 is a key factor in GA reception.

Wheat

Falling numbers, preharvest sprouting (PHS), and drought tolerance

• Shrestha, S. L., Garland‐Campbell, K. A., Steber, C. M., Pan, W. L., & Hulbert, S. H. (2023). Association of canopy temperature with agronomic traits in spring wheat inbred populations. Euphytica, 219(7), 1–14. (Show/Hide Abstract)
  Abstract: Canopy temperature (CT) is considered a reliable proxy for stomatal conductance. Low CT values of plant canopies under water-limited conditions are associated with high transpiration indicating plants’ drought tolerance. Many U.S. Pacific Northwest (PNW) adapted wheat (Triticum aestivum L.) cultivars lack stress-adaptive traits resulting in poor performance in drought environments. This study aims to identify the stress-adaptive traits by evaluating the CT in spring wheat populations across different soil moisture conditions in the PNW. An infrared thermometer was used to estimate the CT in two families of recombinant inbred lines, 'Alpowa' × 'Express' (AE population) and 'Hollis' × 'Drysdale' (HD population), in rainfed and irrigated environments of the dryland PNW in 2011 to 2013. Higher reductions in grain yield up to 170%, spike length up to 25%, and spikelets spike⁻¹ up to 19% were observed in a rainfed environment compared to the reductions in an irrigated environment. A significant variation in CT was observed in both AE and HD populations. With 1 °C increase in CT at the anthesis stage, grain yield was lowered up to 38 g m⁻². Low CT was associated with high grain yield and agronomic traits in both wheat populations (r =  − 0.18 to − 0.55, P ≤ 0.05). The highest association between CT and grain yield was observed at anthesis (r =  − 0.47) and milking (r =  − 0.38) stages (P ≤ 0.001). Our results show that screening for low CT during terminal wheat growth stage is an effective strategy for improving the selection of new drought-tolerant wheat varieties in the PNW.
• Hauvermale, A. L., Parveen, R. S., Harris, T. J., Tuttle, K. M., Mikhaylenko, G., Nair, S., McCubbin, A. G., Pumphrey, M. O., & Steber, C. M. (2023). Streamlined alpha-amylase assays for wheat preharvest sprouting and late maturity alpha-amylase detection. Agrosystems, Geosciences & Environment, 6(7), 1–10. (Show/Hide Abstract)
  Abstract: Late maturity alpha-amylase (LMA) and preharvest sprouting (PHS) lead to elevated alpha-amylase in wheat (Triticum aestivum L.) grain. Risk of poor end-product quality due to elevated alpha-amylase is detected in the wheat industry using the Hagberg–Perten falling number (FN) method. In breeding programs, selection for PHS and LMA tolerance requires higher throughput methods requiring a smaller sample size than the 7 g required for the FN method. Specifically, LMA can only be screened only using detection of alpha-amylase activity or protein after cold treatment of individual wheat spikes at a specific stage of grain development resulting in very small samples (≤1 g). This study developed and evaluated a high through-put 96-well method for the Phadebas alpha-amylase enzyme assay for small wheat grain samples and compared this method to FN and the Megazyme Alpha-Amylase SD (Sprout Damage) Assay Kit performed on the automated Awareness Technology ChemWell-T Analyzer. In parallel, the efficacy of low-cost small-scale milling methods was evaluated relative to traditional larger scale mills. The Phadebas enzyme activity was highly reproducible and showed a strong correlation to the SD enzyme assay and FN method regardless of which mill was used to process the grain. The SD assay offers simpler standardization and calculation of enzyme activity, whereas the Phadebas assay offers higher sensitivity and lower expense. Both the 96-well Phadebas and automated Megazyme SD assays are suitable for alpha-amylase detection from small samples, and the use of low-cost coffee grinders to process small samples did not significantly impact assay performance.
• Peery, S. R., Carle, S. W., Wysock, M., Pumphrey, M. O., & Steber, C. M. (2023). LMA or vivipary? Wheat grain can germinate precociously during grain maturation under the cool conditions used to induce late maturity alpha-amylase (LMA). Frontiers in Plant Science, 14, 1–18. (Show/Hide Abstract)
  Abstract: Introduction: This study found that wheat (Triticum aestivum) grain can germinate precociously during the maturation phase of grain development, a phenomenon called vivipary that was associated with alpha-amylase induction. Farmers receive severe discounts for grain with low falling number (FN), an indicator that grain contains sufficiently elevated levels of the starch-digesting enzyme alpha-amylase to pose a risk to end-product quality. High grain alpha- amylase can result from: preharvest sprouting (PHS)/germination when mature wheat is rained on before harvest, or from late maturity alpha-amylase (LMA) when grain experiences cool temperatures during the soft dough stage of grain maturation (Zadoks growth stage 85). An initial LMA-induction experiment found that low FN was associated with premature visible germination, suggesting that cool and humid conditions caused vivipary. Methods: To examine whether LMA and vivipary are related, controlled environment experiments examined the conditions that induce vivipary, whether LMA could be induced without vivipary, and whether the pattern of alpha-amylase expression during vivipary better resembled PHS or LMA. Results: Vivipary was induced in the soft to hard dough stages of grain development (Zadok’s stages 83-87) both on agar and after misting of the mother plant. This premature germination was associated with elevated alpha-amylase activity. Vivipary was more strongly induced under the cooler conditions used for LMA-induction (18°C day/7.5°C night) than warmer conditions (25°C day/18°C night). Cool temperatures could induce LMA with little or no visible germination when low humidity was maintained, and susceptibility to vivipary was not always associated with LMA susceptibility in a panel of 8 varieties. Mature grain preharvest sprouting results in much higher alpha-amylase levels at the embryo-end of the kernel. In contrast, vivipary resulted in a more even distribution of alpha-amylase that was reminiscent of LMA. Discussion: Vivipary can occur in susceptible varieties under moist, cool conditions, and the resulting alpha-amylase activity may result in low FN problems when a farm experiences cool, rainy conditions before the crop is mature. While there are genotypic differences in LMA and vivipary susceptibility, overlapping mechanisms are likely involved since they are similarly controlled by temperature and growth stage, and result in similar patterns of alpha-amylase expression.
• Hauvermale, A. L., Matzke, C., Bohaliga, G., Pumphrey, M.O., Steber, C.M., & McCubbin, A.G. (2023). Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change. Plants, 12(22):3798, 1–18. (Show/Hide Abstract)
  Abstract: Accurate, rapid testing platforms are essential for early detection and mitigation of late ma- turity α-amylase (LMA) and preharvest sprouting (PHS) in wheat. These conditions are characterized by elevated α-amylase levels and negatively impact flour quality, resulting in substantial economic losses. The Hagberg–Perten Falling Number (FN) method is the industry standard for measuring α-amylase activity in wheatmeal. However, FN does not directly detect α-amylase and has major limitations. Developing α-amylase immunoassays would potentially enable early, accurate detection regardless of testing environment. With this goal, we assessed an expression of α-amylase isoforms during seed development. Transcripts of three of the four isoforms were detected in developing and mature grain. These were cloned and used to develop E. coli expression lines expressing single isoforms. After assessing amino acid conservation between isoforms, we identified peptide sequences specific to a single isoform (TaAMY1) or that were conserved in all isoforms, to develop monoclonal antibodies with targeted specificities. Three monoclonal antibodies were developed, anti-TaAMY1-A, anti-TaAMY1-B, and anti-TaAMY1-C. All three detected endogenous α-amylase(s). Anti-TaAMY1-A was specific for TaAMY1, whereas anti-TaAMY1-C detected TaAMY1, 2, and 4. Thus, confirming that they possessed the intended specificities. All three antibodies were shown to be compatible for use with immuno-pulldown and immuno-assay applications.
• Chen, C.-P. J., Hu, Y., Li, X., Morris, C. F., Delwiche, S., Carter, A. H., Steber, C., & Zhang, Z. (2023). An independent validation reveals the potential to predict Hagberg–Perten falling number using spectrometers. The Plant Phenome Journal, 6, 1–15. (Show/Hide Abstract)
  Abstract: The Hagberg–Perten falling number (HFN) method is the international standard used to evaluate the damage to wheat (Triticum aestivum) grain quality due to preharvest sprouting (PHS) and late maturity alpha-amylase (LMA). However, the HFN test requires specialized laboratory facilities and is time consuming. Spectrometers were known as a potential tool for quick HFN assessment, but none of the studies have val- idated the assessment results across different datasets. In this study, an independent validation was conducted using independent samples and spectral instruments. The calibration set had 462 grain samples of 92 varieties grown at 24 locations in 2019 and examined using a near-infrared spectrometer. In the validation set, 19 varieties collected from 10 locations in 2 years that experienced either PHS or LMA were scanned with a hyperspectral camera. The association between spectra and HFN was modeled by partial least square regression. As a result, the independent validation correlation accuracy was r = 0.72 and a mean absolute error of 56 s. Furthermore, this study showed a cost-effective alternative using only 10 spectral bands to predict HFN, and it achieved better performance than the full spectrum of the hyperspectral system. In conclusion, this is the first study that showed the potential that wheat HFN could be predicted on an independent dataset measured by a different instrument. The result suggested that spectrometers can potentially serve as a faster alternative for plant breeders to develop varieties resistant to PHS and LMA, and for growers to screen damaged grains in transportation processes.
• Steber, C. M. (2022, November). Rising to the challenge of falling numbers. Wheat Life, 65(10), 44–45.
• Hu, Y., Sjoberg, S. M., Chen, C., Hauvermale, A. L., Morris, C. F., Delwiche, S. R., Cannon, A. E., Steber, C. M., & Zhang, Z. (2022). As the number falls, alternatives to the Hagberg–Perten falling number method: A review. Comprehensive Reviews in Food Science and Food Safety , 1–13. (Show/Hide Abstract)
  Abstract: This review examines the application, limitations, and potential alternatives to the Hagberg–Perten falling number (FN) method used in the global wheat industry for detecting the risk of poor end-product quality mainly due to starch degradation by the enzyme α-amylase. By viscometry, the FN test indirectly detects the presence of α-amylase, the primary enzyme that digests starch. Elevated α-amylase results in low FN and damages wheat product quality resulting in cakes that fall, and sticky bread and noodles. Low FN can occur from preharvest sprouting (PHS) and late maturity α-amylase (LMA). Moist or rainy conditions before harvest cause PHS on the mother plant. Continuously cool or fluctuating temperatures during the grain filling stage cause LMA. Due to the expression of addi- tional hydrolytic enzymes, PHS has a stronger negative impact than LMA. Wheat grain with low FN/high α-amylase results in serious losses for farmers, traders, millers, and bakers worldwide. Although blending of low FN grain with sound wheat may be used as a means of moving affected grain through the marketplace, care must be taken to avoid grain lots from falling below contract-specified FN. A large amount of sound wheat can be ruined if mixed with a small amount of sprouted wheat. The FN method is widely employed to detect α-amylase after harvest. However, it has several limitations, including sampling variability, high cost, labor intensiveness, the destructive nature of the test, and an inability to differentiate between LMA and PHS. Faster, cheaper, and more accurate alternatives could improve breeding for resistance to PHS and LMA and could preserve the value of wheat grain by avoiding inadvertent mixing of high- and low- FN grain by enabling testing at more stages of the value stream including at harvest, delivery, transport, storage, and milling. Alternatives to the FN method explored here include the Rapid Visco Analyzer, enzyme assays, immunoassays, near-infrared spectroscopy, and hyperspectral imaging.
• Liu, C., Tuttle, K., Garland Campbell, K., Pumphrey, M., & Steber, C. (2021). Investigating conditions that induce late maturity alpha-amylase (LMA) using Northwestern US spring wheat (Triticum aestivum L.). Seed Science Research, 1–9. https://doi.org/10.1017/S0960258521000052 (Show/Hide Abstract)
  Abstract: The wheat industry rejects grain with unacceptably high α-amylase enzyme levels due to the risk of poor end product quality. There are two main causes of elevated grain α-amylase: (1) preharvest sprouting in response to rain before harvest and (2) late maturity α-amylase (LMA) induction in response to a cool temperature shock during late grain development. LMA induction was detected in a panel of 24 Northwestern US spring wheat lines. Thus, this problem previously described in Australian and U.K. varieties also exists in U.S. varieties. Because LMA induction results were highly variable using published methods, a characterization of LMA-inducing conditions was conducted in an LMA-susceptible soft white spring wheat line, WA8124. Problems with elevated α-amylase in untreated controls were reduced by raising the temperature, 25°C day/18°C night versus 20°C day/10°C night. LMA induction was not improved by colder temperatures (15°C day/4°C night) versus moderately cold temperatures (18°C day/7.5°C night or 10°C day/10°C night). While previous studies observed LMA induction by heat stress, it failed to induce LMA in WA8124. Thus, not all LMA-susceptible cultivars respond to heat. The timing of LMA susceptibility varied between two cultivars and within a single cultivar grown at slightly different temperatures. Thus, variability in LMA induction likely results from variability in the timing of the grain developmental stage during which cold shock induces LMA. Thus, it was concluded that the visual inspection of grain is needed to correctly identify LMA-sensitive spikes at the soft dough stage of grain development (Zadok's stage 85).
• Liu C., Parveen R. S., Revolinski S. R., Garland Campbell K. A., Pumphrey M. O., & Steber C. M. (2021). The genetics of late maturity alpha-amylase (LMA) in North American spring wheat (Triticum aestivum L.). Seed Science Research, 1–10. https://doi.org/10.1017/S0960258521000064 (Show/Hide Abstract)
  Abstract: Genetic susceptibility to late maturity alpha-amylase (LMA) in wheat (Triticum aestivum L.) results in increased alpha-amylase activity in mature grain when cool conditions occur during late grain maturation. Farmers are forced to sell wheat grain with elevated alpha-amylase at a discount because it has an increased risk of poor end-product quality. This problem can result from either LMA or preharvest sprouting, grain germination on the mother plant when rain occurs before harvest. Whereas preharvest sprouting is a well-understood problem, little is known about the risk LMA poses to North American wheat crops. To examine this, LMA susceptibility was characterized in a panel of 251 North American hard spring wheat lines, representing ten geographical areas. It appears that there is substantial LMA susceptibility in North American wheat since only 27% of the lines showed reproducible LMA resistance following cold-induction experiments. A preliminary genome-wide association study detected six significant marker-trait associations. LMA in North American wheat may result from genetic mechanisms similar to those previously observed in Australian and International Maize and Wheat Improvement Center (CIMMYT) germplasm since two of the detected QTLs, QLMA.wsu.7B and QLMA.wsu.6B, co-localized with previously reported loci. The Reduced height (Rht) loci also influenced LMA. Elevated alpha-amylase levels were significantly associated with the presence of both wild-type and tall height, rht-B1a and rht-D1a, loci in both cold-treated and untreated samples.
• Sexton, T. M., Steber, C. M., & Cousins, A. B. (2021). Leaf temperature impacts canopy water use efficiency independent of changes in leaf level water use efficiency. Journal of Plant Physiology, 25, 1–10. (Show/Hide Abstract)
  Abstract: Canopy water use efficiency (above-ground biomass over lifetime water loss, WUE_canopy) can influence yield in wheat and other crops. Breeding for WUE_canopy is difficult because it is influenced by many component traits. For example, intrinsic water use efficiency (WUE_i), the ratio of net carbon assimilation (A_net) over stomatal conductance, contributes to WUE_canopy and can be estimated from carbon isotope discrimination (Δ). However, Δ is not sensitive to differences in the water vapor pressure deficit between the air and leaf (VPD_leaf). Alternatively, measurements of instantaneous leaf water use efficiency (WUE_leaf) are defined as A_net over transpiration and can be determined with gas exchange, but the dynamic nature of field conditions are not represented. Specifically, fluctuations in canopy temperature lead to changes in VPD_leaf that impact transpiration but not A_net. This alters WUE_leaf and in turn affects WUE_canopy. To test this relationship, WUE_canopy was measured in conjunction with WUE_i, WUE_canopy, and canopy temperature under well-watered and water-limited conditions in two drought- tolerant wheat cultivars that differ in canopy architecture. In this experiment, boundary layer conductance was low and significant changes in leaf temperature occurred between cultivars and treatments that correlated with WUE_canopy likely because of the effect of canopy temperature on VPD_leaf driving T. However, deviations between WUE_i, WUE_leaf, and WUE_canopy were present because measurements made at the leaf level do not ac­count for variations in leaf temperature. This uncoupled the relationship of measured WUE and WUE_i from WUE_canopy and emphasizes the importance of canopy temperature on carbon uptake and transpired water loss.
• Horgan, A. M., Garland-Campbell, K. A., Carter, A. H., & Steber, C. M. (2021). Seedling elongation responses to gibberellin seed treatments in wheat. Agrosystems, Geosciences & Environment, 4, 1–13. (Show/Hide Abstract)
  Abstract: The wheat (Triticum aestivum L.) Reduced height (Rht) alleles are widely used to prevent lodging through semi-dwarfism. Seedling elongation and coleoptile length can be significantly decreased by some of these alleles, leading to reduced soil emergence after deep sowing in certain semi-arid environments. Application of the elongation-promoting hormone gibberellin A₃ (GA₃) as a seed treatment has been used as an alternative to improve seedling emergence. Seedling responses to GA₃ seed treatment were investigated under controlled conditions in a collection of varieties differing for Rht dwarfing alleles and the ability to emerge from deep planting. Data between treated and untreated seed were collected on overall coleoptile length and emergence from deep planting in simulated pot studies. Gibberellin-sensitive varieties, carrying either no dwarfing gene or the Rht8 dwarfing gene, responded to the GA₃ seed treatment with increased coleoptile and subcrown internode elongation. Comparison of near-isogenic lines carrying no dwarfing allele or the GA-insensitive Rht-B1b and/or Rht-D1b semi-dwarfing alleles showed that GA insensitivity was associated with reduced seedling response to GA₃ seed treatment. However, there was variation in seedling elongation and GA₃ response in a collection of GA-insensitive varieties. Thus, it cannot be assumed that all Rht-B1b and Rht-D1b varieties will fail to respond to GA₃ seed treatment. Interestingly, some better emerging GA-insensitive varieties had longer coleoptiles after treatment, suggesting that selection for better emergence in semi-arid regions of the U.S. Pacific Northwest may have led to responses in GA₃ application independent of the dwarfing gene used.
• Sjoberg, S., Carter, A. H., Steber, C. M., & Garland-Campbell, K. A. (2020). Unravelling Complex Traits in Wheat: Approaches for Analyzing Genotype × Environment Interactions in a Multi-environment Study of Falling Numbers. Crop Science, 60, 3013–3026. (Show/Hide Abstract)
  Abstract: Multi-environment trials provide useful information about highly variable, complex plant traits like yield and quality, but are difficult to analyze due to their frequently unbalanced nature, with genotypes and locations varying from year to year. Our objectives were to use multiple approaches, including joint regression and principal components analysis, to characterize patterns in the genotype by environment interactions across an unbalanced three-year multi-environment wheat variety trial dataset, examining falling numbers (FN) test results in wheat. The FN test measures the decrease in flour gelling capacity resulting from starch digestion by the enzyme α-amylase. Low FN/high α-amylase grain is discounted because it is associated with poor end-use quality. Low FN can be caused by susceptibility either to preharvest sprouting when it rains before harvest or to late maturity α-amylase induction by temperature fluctuations during grain maturation. The most effective and visually intuitive approaches for selecting varieties with high FN across variable environments was a combination of joint regression, such as Finlay-Wilkinson and Eberhart and Russell, with biplot methods such as the additive main effects and multiplicative interaction model (AMMI) and the genotype main effects and genotype × environment interaction model (GGE). These results identified stable lines for FN resistance and provide a means to analyze unbalanced, multi-environment data from breeding and variety trials.
• Shrestha, S. L., Garland-Campbell, K. A., Steber, C. M., & Hulbert, S. H. (2020). Carbon isotope discrimination association with yield and test weight in Pacific Northwest-adapted spring and winter wheat. Agrosystems, Geosciences & Environment, 3(1), e20052, (15 pages). (Show/Hide Abstract)
  Abstract: Due to considerable influence of environment on yield, breeding for drought tolerance could benefit from focusing on selection of more heritable physiological traits, such as carbon isotope discrimination (as measured by delta, Δ) for indirectly assessing water use efficiency (WUE) of wheat (Triticum aestivum L.). Spring and winter wheat cultivars were assayed for ∆, and these values were used to determine the relationships with performance in over 13 environments in the U.S. Pacific Northwest. The correlation coefficients of Δ values between the wheat cultivars grown in different environments ranged from 0.11 to 0.73 for both spring and winter wheat. There was significant genotypic variation for Δ in soft spring and hard winter wheat but not in hard spring and soft winter wheat. The Δ values were poor indicators of yield for this set of wheat cultivars in most environments, although low values (better WUE) were sometimes correlated with yield. A population of 165 hard spring wheat recombinant inbred lines derived from a cross between two hard spring wheat varieties that differed in Δ was also screened in low-rainfall dryland and irrigated environments. High yields in this recombinant inbred population were weakly correlated with high Δ values or low WUE in most environments.
• Martinez, S. A., Shorinola, O., Conselman, S., See, D., Skinner, D. Z., Uauy, C., & Steber, C. M. (2020). Exome sequencing of bulked segregants identified a novel TaMKK3‐A allele linked to the wheat ERA8 ABA‐hypersensitive germination phenotype. Theoretical and Applied Genetics, 133, 719–736. (Show/Hide Abstract)
  Abstract: Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest. The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft white wheat 'Zak'. ERA8 was mapped to chromosome 4A in a Zak/'ZakERA8' backcross population using bulked segregant analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb region containing 70 genes. In the backcross population, the ERA8 ABA-hypersensitive phenotype was strongly linked to a missense mutation in TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat. The map position of ERA8 was confirmed in an 'Otis'/ZakERA8 but not in a 'Louise'/ZakERA8 mapping population. This is likely because Otis carries the same natural PHS susceptible MKK3-A-A660^S allele as Zak, whereas Louise carries the PHS- tolerant MKK3-A-C660^R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results from the MKK3-A-G1093A mutation.
• Sjoberg, S. M., Carter, A. H., Steber, C. M., & Garland-Campbell, K. A. (2020) Application of the factor analytic model to assess wheat falling number performance and stability in multi‐environment trials. Crop Science, doi:10.1002/csc2.20293. (Show/Hide Abstract)
  Abstract: A factor analytic model was used to characterize data generated with the Hagberg‐Perten Falling Number (FN) method, a measure of wheat quality influenced by genotype‐by‐environment interactions. The FN method detects starch degradation due to the presence of the enzyme alpha‐amylase in wheat grain such that a low FN indicates high alpha‐amylase activity and high risk of poor end‐product quality. Because farmers receive severe discounts for low FN, FN data has been collected over multiple years for the Washington State University multilocation variety trials to help farmers and breeders identify lower risk varieties. Analysis of this data to objectively rank varieties is challenging because the dataset is unbalanced and because FN is subject to complex genotype‐by‐environment interactions. Low FN can result from environmental differences at multiple stages in grain development because there are two major causes of alpha‐amylase accumulation in grain, late‐maturity alpha‐amylase (LMA) and preharvest sprouting (PHS). A five‐factor analytic model extracted explicit measures of overall performance and of stability in variable environments from historical falling number data from the multilocation trial, providing a basis for breeding and planting decisions. Whereas a linear model explained 70.3% of the variation, the five‐factor analytic model accounted for 92.5% of variation in the data. Examination of factor loadings enabled us to separate environments and genotype response to either PHS or LMA, specifically. This is the first application of a factor analytic model to evaluate the end‐use quality trait, FN, providing a method to rank varieties for grower decisions and for breeder selections.
• Steber, C. M., & Garland Campbell, K. A. (2019, April). Rising optimism fuels falling numbers research. Wheat Life, 62(4), 40–52.
• Liu, C. (2019). Late maturity alpha-amylase in north American spring wheat (Triticum aestivum L.). (Master's thesis). Washington State University. (Show/Hide Abstract)
  Abstract: Late maturity alpha-amylase (LMA) is considered a genetic defect in wheat (Triticum aestivum L.), resulting in the induction of alpha-amylase enzyme in response to a low or high temperature shock during late grain filling. The wheat industry uses the Hagberg-Perten Falling Number (FN) method to detect loss of gelling capacity due to the presence of alpha-amylase in meal or flour. Low FN is associated with higher risk of poor end-product quality, such as cakes that fall and sticky bread or noodles. To improve selection for LMA resistance, LMA testing methods were optimized and then used to characterize the LMA susceptibility in North American wheat. Preliminary LMA testing results were highly variable in cold-treated and in untreated controls. Warmer (25°C day/ 18°C night) and drier (˜55-65% relative humidity) conditions reduced alpha- amylase levels in untreated controls. Colder LMA-induction experiments did not result in stronger or more consistent LMA-induction in spring wheat variety, WA8124. The most significant cause of LMA phenotypic variability appeared to be variability in the developmental window during which a 7-day low temperature shock triggered LMA. This window varied with environmental conditions prior to grain development and by genotype, such that WA8124 induced LMA at 20-24 days past anthesis (dpa) whereas Australian ‘Kennedy’ induced at 25-29 dpa. Only 21% of the 256 North American breeding lines characterized showed LMA resistance, suggesting that improved selection for LMA resistance is needed. In this panel, the tall rht-B1a rht-D1a genotype was associated with higher LMA in both cold-treated and untreated experiments, suggesting the presence of a constitutive LMA phenotype that did not require cold treatment. However, some rht-B1a rht-D1a lines required cold induction whereas some semi-dwarf lines had constitutive LMA, suggesting that the constitutive LMA phenotype is genetically complex. A preliminary genome-wide association study identified six significant marker-trait associations on chromosomes 2B, 3A, 3B, 5A, 7B, and 7D. The QLMA.wsu.7B locus detected in this study co-localized a QTL detected in four previous studies of Australian and CIMMYT germplasm. Future work will determine if these molecular markers are effective in selecting LMA resistance in U.S. wheat.
• Delwiche, S. R., Higginbotham, R. W., & Steber, C. M.(2018). Falling number of soft white wheat by near-infrared spectroscopy: A challenge revisited. Cereal Chemistry, 95(3), 469–477. (Show/Hide Abstract)
  Abstract: Background and objectives: Wheat Hagberg falling number (FN) is a long- standing quality test that, by means of measuring the viscosity of a heated water-meal or water-flour mixture, characterizes the activity of endogenous α-amylase, the enzyme primarily responsible for starch hydrolysis. The accuracy, time requirement, and cost of this test have come under heightened scrutiny, particularly in seasons when weather conditions have been favorable to preharvest sprouting or late maturity amylase. Near-infrared (NIR) spectroscopy, an analytical approach routinely used in the grain industry to measure contents of protein and moisture, was reexamined as a possible alternative to the FN procedure. Findings: Partial least squares (PLS) regression quantitative models developed on a genetically diverse set of Washington grown white wheat demonstrated low accuracy, with standard errors of performance ranging from 40 to 77 s. Alternatively, linear discriminant analysis and PLS discriminant analysis (PLSDA) qualitative models, developed and tested using a FN cutoff (pass/fail) value, also demonstrated low accuracy, with the best model correctly identifying 67% and 71% of the samples, respectively, above and below a threshold value established as the median value of FN in a calibration set of several hundred samples. Conclusions: Replacement of the FN test with one based on NIR spectroscopy on either whole grain or ground meal for making decisions on segregating wheat lots according to α-amylase activity is not recommended. Significance and novelty: Because NIR spectroscopy is not sufficiently accurate to quantitatively model FN or differentiate low from high FN grain, viscometry procedures for starch integrity, such as FN, will continue their use in grain commerce.
• Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2018, January). Hunting for genes: falling numbers project seeks to reduce risk by breeding for genetic resistance. Wheat Life, 61(1), 50–51.
• Martinez, S. A., Godoy, J., Huang, M., Zhang, Z., Carter, A. H., Garland Campbell, K. A., & Steber, C. M. (2018). Genome-Wide Association Mapping for Tolerance to Preharvest Sprouting and Low Falling Numbers in Wheat. Frontiers in Plant Science, 9, 1–16, https://doi.org/10.3389/fpls.2018.00141. (Show/Hide Abstract)
  Abstract: Preharvest sprouting (PHS), the germination of grain on the mother plant under cool and wet conditions, is a recurring problem for wheat farmers worldwide. α-amylase enzyme produced during PHS degrades starch resulting in baked good with poor end-use quality. The Hagberg-Perten Falling Number (FN) test is used to measure this problem in the wheat industry, and determines how much a farmer’s wheat is discounted for PHS damage. PHS tolerance is associated with higher grain dormancy. Thus, breeding programs use germination-based assays such as the spike-wetting test to measure PHS susceptibility. Association mapping identified loci associated with PHS tolerance in U.S. Pacific Northwest germplasm based both on FN and on spike-wetting test data. The study was performed using a panel of 469 white winter wheat cultivars and elite breeding lines grown in six Washington state environments, and genotyped for 15,229 polymorphic markers using the 90k SNP Illumina iSelect array. Marker-trait associations were identified using the FarmCPU R package. Principal component analysis was directly and a kinship matrix was indirectly used to account for population structure. Nine loci were associated with FN and 34 loci associated with PHS based on sprouting scores. None of the QFN.wsu loci were detected in multiple environments, whereas six of the 34 QPHS.wsu loci were detected in two of the five environments. There was no overlap between the QTN detected based on FN and PHS, and there was little correlation between the two traits. However, both traits appear to be PHS-related since 19 of the 34 QPHS.wsu loci and four of the nine QFN.wsu loci co-localized with previously published dormancy and PHS QTL. Identification of these loci will lead to a better understanding of the genetic architecture of PHS and will help with the future development of genomic selection models.
• Martinez, S. A., Thompson, A. L., Wen, N., Murphy, L., Sanguinet, K. A., Steber, C. M., & Garland Campbell, K. A. (2018). Registration of the Louise/Alpowa Wheat Recombinant Inbred Line Mapping Population. Journal of Plant Registrations, doi:10.3198/jpr2017.08.0053crmp. (Show/Hide Abstract)
  Abstract: A mapping population was developed from the cross of soft white spring wheat (Triticum aestivum L.) cultivars 'Louise' and 'Alpowa' for use in investigating the genetic architecture of drought tolerance in the US Pacific Northwest. The Louise/Alpowa (Reg. No. MP-8, NSL 520824 MAP) recombinant inbred line mapping population was developed through single seed descent from the F₂ generation to the F₅ generation. The population consists of 141 F_5:6 recombinant inbred lines, of which 132 were used to construct the genetic linkage map. The 32 linkages groups included 882 single nucleotide polymorphism markers and one simple sequence repeat marker spanning 18 of 21 chromosomes. The Louise/Alpowa population was characterized for variation in agronomic traits, phenology, and end-use quality traits. This population will be used for identification and introgression of multiple loci providing resistance to environmental stress such as drought, stripe rust, and high temperatures.
• Steber, C. M., & Campbell, K. G. (2017, November). All hands on deck: USDA-ARS, WSU scientists tackle falling numbers. Wheat Life, 60(10), 40–41.
• Steber, C. M. (2017, March-April). Avoiding problems in wheat with low Falling Numbers. Crops & Soils Magazine, (50)2, 22–25.
• Martinez, S. A., Tuttle K. M., Takebayashi, Y., Seo, M., Campbell, K. G., & Steber, C. M. (2016). The wheat ABA hypersensitive ERA8 mutant is associated with increased preharvest sprouting tolerance and altered hormone accumulation. Euphytica, 212(2), 229–245. (Show/Hide Abstract)
  Abstract: Wheat preharvest sprouting (PHS) is the germination of mature grain on the mother plant when rain occurs before harvest. Higher abscisic acid (ABA) hormone levels and sensitivity are associated with higher seed dormancy and PHS tolerance. Consistent with this, the ABA hypersensitive ENHANCED RESPONSE TO ABA8 (ERA8) mutant resulted in increased dormancy and PHS tolerance in soft white spring wheat 'Zak'. ERA8 seeds were initially less responsive to germination-rescue by the hormone gibberellin (GA). ERA8 gained GA and lost ABA sensitivity more slowly than wild-type during dormancy loss through after-ripening and cold imbibition. This study examined if increased ABA sensitivity in ERA8 likely resulted from increased ABA signaling or increased ABA hormone levels. Zak ERA8 had higher initial grain dormancy although endogenous embryo ABA levels were similar in Zak ERA8 and wild-type, suggesting that increased dormancy was due to increased ABA signaling rather than increased ABA accumulation. ABA levels declined with Zak ERA8 after-ripening, suggesting that ABA turnover is not defective. Elevated ERA8 dormancy was also associated with increased embryonic jasmonic acid-Ile and aleurone indole-3-acetic acid (IAA) levels. The possible implication that other plant hormones may influence wheat seed dormancy and germination are discussed.
• Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2016, December). Get out your hard hats, the numbers are falling. Wheat Life, 59(11), 47–49.
• Steber, C. M. (2016). Managing the risk of low falling numbers in wheat. Washington State University Extension, FS242E, 1–6.
• Tuttle, K. M., Martinez, S. A., Schramm, E. C., Takebayashi, Y., Seo, M., & Steber, C. M. (2015). Grain dormancy loss is associated with changes in ABA and GA sensitivity and hormone accumulation in bread wheat, Triticum aestivum (L.). Seed Science Research, 25(2), 179–193. (Show/Hide Abstract)
  Abstract: Knowledge about the hormonal control of grain dormancy and dormancy loss is essential in wheat, because low grain dormancy at maturity is associated with the problem of pre-harvest sprouting (PHS) when cool and rainy conditions occur before harvest. Low GA (gibberellin A) hormone sensitivity and high ABA (abscisic acid) sensitivity were associated with higher wheat grain dormancy and PHS tolerance. Grains of two PHS-tolerant cultivars were very dormant at maturity, and insensitive to GA stimulation of germination. More PHS-susceptible cultivars were less sensitive to ABA inhibition of germination, and were either more GA sensitive or germinated efficiently without GA at maturity. As grain dormancy was lost through dry afterripening or cold imbibition, grains first gained GA sensitivity and then lost ABA sensitivity. These changes in GA and ABA sensitivity can serve as landmarks defining stages of dormancy loss that cannot be discerned without hormone treatment. These dormancy stages can be used to compare different cultivars, seed lots and studies. Previous work showed that wheat afterripening is associated with decreasing ABA levels in imbibing seeds. Wheat grain dormancy loss through cold imbibition also led to decreased endogenous ABA levels, suggesting that reduced ABA signalling is a general mechanism triggering dormancy loss.
• Martinez, S. A., Schramm, E. C., Harris, T. J., Kidwell, K. K., Garland-Campbell, K., & Steber, C. M. (2014). Registration of Zak ERA8 Soft White Spring Wheat Germplasm with Enhanced Response to ABA and Increased Seed Dormancy. Journal of Plant Registrations, 8(2), 217–220. (Show/Hide Abstract)
  Zak ERA8 (ENHANCED RESPONSE to ABA8) (Reg. No. GP-966, PI 669443) is a unique line derived from soft white spring wheat (Triticum aestivum L.) cultivar Zak that has increased seed dormancy but after-ripens within 10 to 16 wk. The goal in developing this germplasm was to use increased seed dormancy to improve tolerance to preharvest sprouting, a problem that can cause severe economic losses. This germplasm was developed by USDA–ARS, Pullman, WA, in collaboration with Washington State University. Zak ERA8 was tested under experimental number 60.1.27.10. The ERA8 mutation was generated by chemical mutagenesis followed by selection for the inability to germinate on abscisic acid (ABA) concentrations too low to inhibit wild-type Zak seed germination. The semidominant Zak ERA8 line has been backcrossed twice to wild-type Zak. Following the first backcross, Zak ERA8 showed similar morphological and grain quality traits to the original Zak cultivar.
• Schramm, E. C., Nelson, S. K., Kidwell, K. K., & Steber, C. M. (2013). Increased ABA sensitivity results in higher seed dormancy in soft white spring wheat cultivar ‘Zak’. Theoretical and Applied Genetics, 126(3), 791–803. (Show/Hide Abstract)
  Abstract: As a strategy to increase the seed dormancy of soft white wheat, mutants with increased sensitivity to the plant hormone abscisic acid (ABA) were identified in mutagenized grain of soft white spring wheat “Zak”. Lack of seed dormancy is correlated with increased susceptibility to preharvest sprouting in wheat, especially those cultivars with white kernels. ABA induces seed dormancy during embryo maturation and inhibits the germination of mature grain. Three mutant lines called Zak ERA8, Zak ERA19A, and Zak ERA19B (Zak ENHANCED RESPONSE to ABA) were recovered based on failure to germinate on 5 μM ABA. All three mutants resulted in increased ABA sensitivity over a wide range of concentrations such that a phenotype can be detected at very low ABA concentrations. Wheat loses sensitivity to ABA inhibition of germination with extended periods of dry after-ripening. All three mutants recovered required more time to after-ripen sufficiently to germinate in the absence of ABA and to lose sensitivity to 5 μM ABA. However, an increase in ABA sensitivity could be detected after as long as 3 years of after-ripening using high ABA concentrations. The Zak ERA8 line showed the strongest phenotype and segregated as a single semi-dominant mutation. This mutation resulted in no obvious decrease in yield and is a good candidate gene for breeding preharvest sprouting tolerance.
• Schramm, E. C., Nelson, S. K., & Steber, C. M. (2012). Wheat ABA-insensitive mutants result in reduced grain dormancy. Euphytica, 188(1), 35–49. (Show/Hide Abstract)
  Abstract: This paper describes the isolation of wheat mutants in the hard red spring Scarlet resulting in reduced sensitivity to the plant hormone abscisic acid (ABA) during seed germination. ABA induces seed dormancy during embryo maturation and inhibits the germination of mature seeds. Wheat sensitivity to ABA gradually decreases with dry after-ripening. Scarlet grain normally fails to germinate when fully dormant, shows ABA sensitive germination when partially after-ripened, and becomes ABA insensitive when after-ripened for 8–12 months. Scarlet ABA-insensitive (ScABI) mutants were isolated based on the ability to germinate on 5 μM ABA after only 3 weeks of after-ripening, a condition under which Scarlet would fail to germinate. Six independent seed-specific mutants were recovered. ScABI1, ScABI2, ScABI3 and ScABI4 are able to germinate more efficiently than Scarlet at up to 25 μM ABA. The two strongest ABA insensitive lines, ScABI3 and ScABI4, both proved to be partly dominant suggesting that they result from gain-of-function mutations. The ScABI1, ScABI2, ScABI3, ScABI4, and ScABI5 mutants after-ripen more rapidly than Scarlet. Thus, ABA insensitivity is associated with decreased grain dormancy in Scarlet wheat. This suggests that ABA sensitivity is an important factor controlling grain dormancy in wheat, a trait that impacts seedling emergence and pre-harvest sprouting resistance.
• Schramm, E. C., Abellera, J. C., Strader, L. C., Campbell, K. G., & Steber, C. M. (2010). Isolation of ABA-responsive mutants in allohexaploid bread wheat ( Triticum aestivum L.): Drawing connections to grain dormancy, preharvest sprouting, and drought tolerance. Plant Science, 179(6), 620–629. (Show/Hide Abstract)
  Abstract: This paper describes the isolation of Wheat ABA-responsive mutants (Warm) in the Chinese Spring background of allohexaploid Triticum aestivum L. Because the hormone abscisic acid (ABA) is required for the induction of seed dormancy, stomatal closure, and drought, cold and salt tolerance, ABA-hypersensitive wheat mutants were expected to show increased seed dormancy and decreased leaf transpiration in drying soils. Lack of wheat grain dormancy is associated with a propensity for preharvest sprouting (PHS), the germination of seed on the mother plant when moist conditions persist before harvest. PHS tolerance correlates with higher seed dormancy resulting from red grain color, higher ABA accumulation and sensitivity. Wheat grain loses dormancy and sensitivity to ABA inhibition of seed germination with afterripening. Warm lines maintained higher ABA sensitivity when partially after-ripened. The Warm1 and Warm4 mutants showed the strongest and most reproducible increase in ABA sensitivity, accompanied by a requirement for more prolonged after-ripening to break dormancy. Warm2, Warm3, Warm5, and Warm6 showed normal germination without ABA but increased sensitivity to inhibition of germination by applied ABA. The Warm4 mutant showed decreased whole plant transpiration in drying soil, consistent with the role of ABA in inhibiting vegetative leaf transpiration.
• Schramm, E. C. (2010). Isolation of ABA-response mutants in allohexaploid bread wheat (Triticum aestivum L.): Drawing connections to grain dormancy, preharvest sprouting, and drought tolerance (Doctoral dissertation). Washington State University. (Show/Hide Abstract)
  Abstract: This dissertation describes the characterization of seed dormancy and drought tolerance phenotypes of abscisic acid (ABA) response mutants in allohexaploid wheat (Triticum aestivum L.). The plant hormone ABA is required for the induction of seed dormancy, stomatal closure in drying soils, and drought tolerance. Preharvest sprouting (PHS), the germination of seed on the mother plant, occurs when cool moist conditions persist before harvest. PHS resistance is correlated with seed dormancy resulting from red grain color, higher ABA accumulation or sensitivity. ABA only inhibits the germination of imbibing dormant wheat grain, not of afterripened grain. ABA hypersensitive lines were isolated based on the phenotype of maintaining higher ABA sensitivity when partially after-ripened. ABA hypersensitive phenotypes were stronger and persisted longer in the soft white spring 'Zak' compared to the hard red springs Chinese Spring and 'Scarlet'. ZakERA (Zak Enhanced Response to ABA) 0 and 19A mutants showed increased dormancy associated with a reproducible increase in ABA sensitivity in germination. These mutants required more time to after-ripen than Zak wild type (WT), and still showed ABA sensitivity after 3 years after-ripening. Warm1 (Wheat ABA-responsive mutant) and Warm4 showed the strongest ABA-hypersensitive germination phenotype among Chinese Spring mutants, and were accompanied by decreased germination in the absence of ABA. Although these mutants required more time to after-ripen than WT, they lost ABA sensitivity within 3-12 months. ScERA (Scarlet Enhanced Response to ABA) 10A, 68A, 10C, and 4A showed the strongest ABA-hypersensitive germination phenotype. ScERA 4A is unique in having enhanced ABA sensitivity without any apparent increase in seed dormancy. ABA insensitive mutants in Scarlet, ScABI (Scarlet ABA-Insensitive) 2A, 2C, 1B, and 1C, were insensitive to ABA inhibition of germination with little or no after-ripening. ABA mutants are also expected to show altered water relations. All of the ScERA or ScABI mutants tested so far appear to have only seed germination phenotypes. Consistent with the role of ABA in downregulating transpiration in drying soils, Warm3, Warm4, and ZakERA 19A are associated with decreased leaf transpiration. In Warm4 and ZakERA 19A this decrease in transpiration is associated with higher canopy temperatures under field conditions.
• Schramm, E. C., Abellera, J. C., Strader, L. C., Campbell, K. G., & Steber, C. M. (2008). Can ABA signaling be used to develop drought tolerant wheat? In R. Appels, R. Eastwood, E. Lagudah, P. Langridge, M. Mackay, L. MacIntyre & P. Sharp (Eds.) 11th International Wheat Genetics Symposium 2008 Proceedings – Volume 1 (pp. 116–118). Sydney, Australia: Sydney University Press. (Show/Hide Abstract)
  Abstract: Wheat yield and quality can be compromised by drought stress and preharvest sprouting (PHS) on the mother plant. It is believed that problems with PHS are due to lack of grain dormancy partly resulting from lack of ABA sensitivity. The long term goal is to use wheat mutants with increased sensitivity to ABA to increase grain dormancy and drought tolerance. ABA is required to set up grain dormancy and embryo desiccation tolerance during embryo maturation, and stimulate storage of nutrients. ABA also inhibits germination of mature seeds and stimulates stomatal closure in response to drought stress. A screen for wheat mutants with altered response to ABA in seed germination has been used as a first step to isolate wheat plants with increased ABA sensitivity. Mutants have been characterized for changes in ABA dose-response. In addition, ABA hypersensitive grain germination appears to correspond to reduced vegetative transpiration under drought stress and decreased carbon isotope discrimination.
• Abellera, J. C. (2006). ABA Responsive when Afterripened (ARA) mutants increase seed dormancy, ABA sensitivity, and improve water relations: towards wheat varieties with improved preharvest sprouting and drought tolerance. (Master's thesis). Washington State University. (Show/Hide Abstract)
  Abstract: This thesis explores the hypothesis that mutants isolated based on ABA (abscisic acid) hypersensitive seed germination can be used to obtain drought tolerance and resistance to preharvest sprouting through increased seed dormancy in allohexaploid wheat. The plant hormone ABA is required for establishment of dormancy during seed development, stimulates stomatal closure in response to drought stress and induces expression of genes that promote osmotic adjustment. Arabidopsis mutants isolated for ABA hypersensitive seed germination have increased seed dormancy and drought tolerance resulting from increased sensitivity of stomates to ABA. Based on this result, wheat mutants showing increased sensitivity to ABA in germination of afterripened grain have been isolated and tested for improved vegetative drought tolerance. A total of 25 mutants were isolated from fastneutron mutagenized Chinese Spring, whereas four and 27 mutants were isolated from EMS mutagenized cv. Zak and cv. Scarlet, respectively. Of the 25 Chinese Spring mutants, 4 demonstrated a clear and reproducible hypersensitive response to ABA in dose-response germination assays, whereas 2 lines showed embryo dormancy. The remaining mutants showed inconsistent phenotypes over seven retest experiments suggesting variable expressivity. Genetic analysis of Chinese Spring mutants showed that two are the result of a single dominant mutation, whereas three others may be single semi-dominant mutations. Mutants with a vegetative ABA-hypersensitive phenotype should close their stomates earlier in response to drought stress and have slower transpiration. Drought tolerance was evaluated by comparing the mutant rate of soil moisture loss during drought stress, stomatal conductance, and carbon isotope discrimination (Δ¹³C) to wild-type. Of eight Chinese Spring mutants tested, four showed a slower transpiration rate under drought stress, and one of the four showed reduced stomatal conductance compared to wild-type. The four mutants with ABA-hypersensitive seed germination were the same four mutants that showed lower rate of soil moisture loss under drought stress. Five of 19 mutants appeared to have reduced Δ¹³C relative to wild type, suggesting that they may have improved transpiration efficiency.
• Nelson, S. (2005). Isolating drought tolerant wheat using germination screens for increased ABA hormone sensitivity (Honors thesis). Washington State University.
• Strader, L. C., Zale, J., & Steber, C. M. (2004). Sivb 2003 congress symposium proceeding: Mutation- and transposon-based approaches for the identification of genes for pre-harvest sprouting in wheat. In Vitro Cellular & Developmental Biology - Plant, 40(3), 256–259. (Show/Hide Abstract)
  Abstract: This article reviews techniques for gene identification and cloning in allohexaploid bread wheat (Triticum aestivum L.). Gene identification and cloning in wheat are complicated by the large size and high redundancy of the genome. Both classical mutagenesis and transposon tagging are important tools for the study of grain dormancy and plant hormone signaling in wheat. While classical mutagenesis can be used to identify wheat mutants with altered hormone sensitivity, it can be difficult to clone the corresponding genes. We review the techniques available for gene identification in wheat, and propose that transposon-based activation tagging will be an important tool for wheat genetics.

Rhizoctonia

• Okubara, P. A., Steber, C. M., Demacon, V. L., Walter, N. L., Paulitz, T. C., & Kidwell, K. K. (2009). Scarlet-Rz1, an EMS-generated hexaploid wheat with tolerance to the soilborne necrotrophic pathogens Rhizoctonia solani AG-8 and R. oryzae. Theoretical and Applied Genetics, 119(2), 293–303. (Show/Hide Abstract)
  Abstract: The necrotrophic root pathogens Rhizoctonia solani AG-8 and R. oryzae cause Rhizoctonia root rot and damping-off, yield-limiting diseases that pose barriers to the adoption of conservation tillage in wheat production systems. Existing control practices are only partially effective, and natural genetic resistance to Rhizoctonia has not been identified in wheat or its close relatives. We report the first genetic resistance/tolerance to R. solani AG-8 and R. oryzae in wheat (Triticum aestivum L. em Thell) germplasm 'Scarlet-Rz1'. Scarlet-Rz1 was derived from the allohexaploid spring wheat cultivar Scarlet using EMS mutagenesis. Tolerant seedlings displayed substantial root and shoot growth after 14 days in the presence of 100–400 propagules per gram soil of R. solani AG-8 and R. oryzae in greenhouse assays. BC₂F₄ individuals of Scarlet-Rz1 showed a high and consistent degree of tolerance. Seedling tolerance was transmissible and appeared to be dominant or co-dominant. Scarlet-Rz1 is a promising genetic resource for developing Rhizoctonia-tolerant wheat cultivars because the tolerance trait immediately can be deployed into wheat breeding germplasm through cross-hybridization, thereby avoiding difficulties with transfer from secondary or tertiary relatives as well as constraints associated with genetically modified plants. Our findings also demonstrate the utility of chemical mutagenesis for generating tolerance to necrotrophic pathogens in allohexaploid wheat.

Tissue Culture

• Zale, J. M., Borchardt-Wier, H., Kidwell, K. K., & Steber, C. M. (2004). Callus induction and plant regeneration from mature embryos of a diverse set of wheat genotypes. Plant Cell, Tissue and Organ Culture, 76(3), 277–281. (Show/Hide Abstract)
  Abstract: This paper compared the behavior of a diverse set of wheat genotypes in their tissue culture response. Significant differences were detected in plant regeneration, culture efficiency, and regeneration capacity when mature embryos of 47 wheat cultivars, breeding lines, and the common wheat progenitors, Triticum monococcum, T.tauschii, and Aegilops speltoides were compared. Although not currently used in wheat tissue culture, mature embryo-derived callus of cv. 'Zak' (SWS), 'Scarlet' (HRS), 'Tara' (SWS), 'Jagger' (HRW), 'UC 1036' (HRS), and 'Kyle' durum showed better or comparable plant regeneration than commonly cultured cultivars 'Fielder' and 'Bobwhite'. Of the three diploid wheat progenitors tested, Ae. speltoides regenerated the most plants. In one replicated experiment, callus induction was correlated with culture efficiency (r = 0.42; p = 0.002) and regeneration capacity (r = 0.39; p = 0.002), and in a second larger screen, callus induction correlated with the total number of plants regenerated (r = 0.6; p = 0.001). Immature and mature embryos of 'Bobwhite' and 'Crocus' were compared for callus induction and plant regeneration. Immature embryos were superior explants in terms of plant regeneration. However, sufficient numbers of plants can be regenerated from mature embryos saving on growth facility resources and time required for the collection of immature embryos.

Floral Dip Transformation

• McGuire, P. E., Loar, S., Steber, C. M., & Zale, J. (2009). Floral transformation of wheat. In H. D. Jones & P. R. Shewry (Eds), Transgenic Wheat, Barley and Oats: Production and Characterization Protocols (Methods in Molecular Biology) (pp. 105–113). Clifton, NJ: Humana Press. (Show/Hide Abstract)
  Abstract: A method is described for the floral transformation of wheat using a protocol similar to the floral dip of Arabidopsis. This method does not employ tissue culture of dissected embryos, but instead pre-anthesis spikes with clipped florets at the early, mid to late uninucleate microspore stage are dipped in Agrobacterium infiltration media harboring a vector carrying anthocyanin reporters and the NPTII selectable marker. T1 seeds are examined for color changes induced in the embryo by the anthocyanin reporters. Putatively transformed seeds are germinated and the seedlings are screened for the presence of the NPTII gene based on resistance to paromomycin spray and assayed with NPTII ELISAs. Genomic DNA of putative transformants is digested and analyzed on Southern blots for copy number to determine whether the T-DNA has integrated into the nucleus and to show the number of insertions. The nonoptimized transformation efficiencies range from 0.3 to 0.6% (number of transformants/number of florets dipped) but the efficiencies are higher in terms of the number of transformants produced/number of seeds set ranging from 0.9 to 10%. Research is underway to maximize seed set and optimize the protocol by testing different Agrobacterium strains, visual reporters, vectors, and surfactants.
• Zale, J. M., Agarwal, S., Loar, S., & Steber, C. M. (2009). Evidence for stable transformation of wheat by floral dip in agrobacterium tumefaciens. Plant Cell Reports, 28(6), 903–913. (Show/Hide Abstract)
  Abstract: Hexaploid wheat, one of the world’s most important staple crops, remains a challenge for genetic transformation. We are developing a floral transformation protocol for wheat that does not require tissue culture. This paper presents three transformants in the hard red germplasm line Crocus that have been characterized thoroughly at the molecular level over three to six generations. Wheat spikes at the early boot stage, i.e. the early, mid or late uninucleate microspore stages, were immersed in an infiltration medium of strain C58C1 harboring pDs(Hyg)35S, or strain AGL1 harboring pBECKSred. pDs(Hyg)35S contains the NPTII and hph selectable markers, and transformants were detected using paromomycin spray at the whole plant level, NPTII ELISAs, or selection on medium with hygromycin. Strain AGL1, harboring pBECKSred, which contains the maize anthocyanin regulators, Lc and C1, and the NPTII gene, was also used to produce a Crocus transformant. T1 and T2 seeds with red embryos were selected; T1 and T2 plants were screened by sequential tests for paromomycin resistance and NPTII ELISAs. The transformants were low copy number and showed Mendelian segregation in the T2. Stable transmission of the transgenes over several generations has been demonstrated using Southern analysis. Gene expression in advanced progeny was shown using Reverse Transcriptase-PCR and ELISA assays for NPTII protein expression. This protocol has the potential to reduce the time and expense required for wheat transformation.

Genomics

• Martinez, S. A., Godoy, J., Huang, M., Zhang, Z., Carter, A. H., Garland Campbell, K. A., & Steber, C. M. (2018). Genome-Wide Association Mapping for Tolerance to Preharvest Sprouting and Low Falling Numbers in Wheat. Frontiers in Plant Science, 9, 1–16, https://doi.org/10.3389/fpls.2018.00141. (Show/Hide Abstract)
  Abstract: Preharvest sprouting (PHS), the germination of grain on the mother plant under cool and wet conditions, is a recurring problem for wheat farmers worldwide. α-amylase enzyme produced during PHS degrades starch resulting in baked good with poor end-use quality. The Hagberg-Perten Falling Number (FN) test is used to measure this problem in the wheat industry, and determines how much a farmer’s wheat is discounted for PHS damage. PHS tolerance is associated with higher grain dormancy. Thus, breeding programs use germination-based assays such as the spike-wetting test to measure PHS susceptibility. Association mapping identified loci associated with PHS tolerance in U.S. Pacific Northwest germplasm based both on FN and on spike-wetting test data. The study was performed using a panel of 469 white winter wheat cultivars and elite breeding lines grown in six Washington state environments, and genotyped for 15,229 polymorphic markers using the 90k SNP Illumina iSelect array. Marker-trait associations were identified using the FarmCPU R package. Principal component analysis was directly and a kinship matrix was indirectly used to account for population structure. Nine loci were associated with FN and 34 loci associated with PHS based on sprouting scores. None of the QFN.wsu loci were detected in multiple environments, whereas six of the 34 QPHS.wsu loci were detected in two of the five environments. There was no overlap between the QTN detected based on FN and PHS, and there was little correlation between the two traits. However, both traits appear to be PHS-related since 19 of the 34 QPHS.wsu loci and four of the nine QFN.wsu loci co-localized with previously published dormancy and PHS QTL. Identification of these loci will lead to a better understanding of the genetic architecture of PHS and will help with the future development of genomic selection models.
• Akhunov, E. D., Goodyear, A. W., Geng, S., Qi, L.-L., Echalier, B., Gill, B. S., Miftahudin, Gustafson, J. P., Lazo, G., Chao, S., Anderson, O. D., Linkiewicz, A. M., Dubcovsky, J., La Rota, M., Sorrells, M. E., Zhang, D., Nguyen, H. T., Kalavacharla, V., Hossain, K., Kianian, S. F., Peng, J., Lapitan, N. L.V., Gonzalez-Hernandez, J. L., Anderson, J. A., Choi, D.-W., Close, T. J., Dilbirligi, M., Gill, K. S., Walker-Simmons, M. K., Steber, C. M., McGuire, P. E., Qualset, C. O. & Dvorak, J. (2003). The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms. Genome Research, 13(5), 753–763. (Show/Hide Abstract)
  Abstract: Genes detected by wheat expressed sequence tags (ESTs) were mapped into chromosome bins delineated by breakpoints of 159 overlapping deletions. These data were used to assess the organizational and evolutionary aspects of wheat genomes. Relative gene density and recombination rate increased with the relative distance of a bin from the centromere. Single-gene loci present once in the wheat genomes were found predominantly in the proximal, low-recombination regions, while multigene loci tended to be more frequent in distal, high-recombination regions. One-quarter of all gene motifs within wheat genomes were represented by two or more duplicated loci (paralogous sets). For 40 such sets, ancestral loci and loci derived from them by duplication were identified. Loci derived by duplication were most frequently located in distal, high-recombination chromosome regions whereas ancestral loci were most frequently located proximal to them. It is suggested that recombination has played a central role in the evolution of wheat genome structure and that gradients of recombination rates along chromosome arms promote more rapid rates of genome evolution in distal, high-recombination regions than in proximal, low-recombination regions.
• Sorrells, M. E., La Rota, M., Bermudez-Kandianis, C. E., Greene, R. A., Kantety, R., Munkvold, J. D., Miftahudin, Mahmoud, A., Ma, X., Gustafson, P. J., Qi, L.-L., Echalier, B., Gill, B. S., Matthews, D. E., Lazo, G. R., Chao, S., Anderson, O. D., Edwards, H., Linkiewicz, A. M., Dubcovsky, J., Akhunov, E. D., Dvorak, J., Zhang, D., Nguyen, H. T., Peng, J., Lapitan, N. L., Gonzalez-Hernandez, J. L., Anderson, J. A., Hossain, K., Kalavacharla, V., Kianian, S. F., Choi, D. W., Close, T. J., Dilbirligi, M., Gill, K. .S, Steber, C. M., Walker-Simmons, M. K., McGuire, P. E., & Qualset, C. O. (2003). Comparative DNA sequence analysis of wheat and rice genomes. Genome Research, 13(8), 1818–1827. (Show/Hide Abstract)
  Abstract: The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to crop species has revolutionized molecular genetics and crop improvement strategies. This study compared 4485 expressed sequence tags (ESTs) that were physically mapped in wheat chromosome bins, to the public rice genome sequence data from 2251 ordered BAC/PAC clones using BLAST. A rice genome view of homologous wheat genome locations based on comparative sequence analysis revealed numerous chromosomal rearrangements that will significantly complicate the use of rice as a model for cross-species transfer of information in nonconserved regions.

Transposons

• Zale, J. M., & Steber, C. M. (2002). Transposon-related sequences in the Triticeae. Cereal Research Communications, 30(3-4), 237–244. (Show/Hide Abstract)
  Abstract: The purpose of this work was to determine whether sequences homologous to maize transposable elements exist in wheat and its relatives. Genomic Southern blots of Triticeae members were used to detect copy number of homologues to the major open reading frames of the maize Ac and En transposases, and MudrB, a regulator of transposition. The number of similar sequences was the least with Ac, intermediate with Mu, and the greatest with En. Maize transposons at the nucleotide and amino acid level were used in EST and genomic database searches. Nucleotide identity between maize and wheat was 58% for Ac, 59% for Mu, and 61% for En. Protein homologues were found for Ac and Mu in wheat and barley, and for En in barley.

Wheat Life Magazine

• Steber, C. M. (2022, November). Rising to the challenge of falling numbers. Wheat Life, 65(10), 44–45.
• Steber, C. M., & Garland Campbell, K. A. (2019, April). Rising optimism fuels falling numbers research. Wheat Life, 62(4), 40–52.
• Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2018, January). Hunting for genes: falling numbers project seeks to reduce risk by breeding for genetic resistance. Wheat Life, 61(1), 50–51.
• Steber, C. M., & Campbell, K. G. (2017, November). All hands on deck: USDA-ARS, WSU scientists tackle falling numbers. Wheat Life, 60(10), 40–41.
• Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2016, December). Get out your hard hats, the numbers are falling. Wheat Life, 59(11), 47–49.
• Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2014, July). Falling numbers: research strategies to stay out of the red. Wheat Life, 57, 56–59.
• Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2013, August/September). Preventing those falling number blues. Wheat Life, 56, 67–69.
• Steber, C. M., & Skinner, D. (2011, April). It takes a village: (of ARS scientists working together to develop new wheat varieties). Wheat Life, 54, 58–61.
• Steber, C. M. (2010, June). Solutions that don't fall from the sky: New tools will aid future farmers. Wheat Life, 53, 44–47.
• Steber, C. M. (2009, March). Up-and-coming wheat research revealed Down Under. Wheat Life, 52, 40–41.

Arabidopsis

GA and ABA - seed germination, plant height, and flowering

• Nelson, S. K., Kanno, Y. Seo, M., & Steber, C. M. (2023). Seed dormancy loss from dry after-ripening is associated with increasing gibberellin hormone levels in Arabidopsis thaliana. Frontiers in Plant Science, 14, 1–18. (Show/Hide Abstract)
  Abstract: Introduction: The seeds of many plants are dormant and unable to germinate at maturity, but gain the ability to germinate through after-ripening during dry storage. The hormone abscisic acid (ABA) stimulates seed dormancy, whereas gibberellin A (GA) stimulates dormancy loss and germination. Methods: To determine whether dry after-ripening alters the potential to accumulate ABA and GA, hormone levels were measured during an after-ripening time course in dry and imbibing ungerminated seeds of wildtype Landsberg erecta (Ler) and of the highly dormant GA-insensitive mutant sleepy1-2 (sly1-2). Results: The elevated sly1-2 dormancy was associated with lower rather than higher ABA levels. Ler germination increased with 2-4 weeks of after-ripening whereas sly1-2 required 21 months to after-ripen. Increasing germination capacity with after-ripening was associated with increasing GA₄ levels in imbibing sly1-2 and wild-type Ler seeds. During the same 12 hr imbibition period, after-ripening also resulted in increased ABA levels. Discussion: The decreased ABA levels with after-ripening in other studies occurred later in imbibition, just before germination. This suggests a model where GA acts first, stimulating germination before ABA levels decline, and ABA acts as the final checkpoint preventing germination until processes essential to survival, like DNA repair and activation of respiration, are completed. Overexpression of the GA receptor GID1b (GA INSENSITIVE DWARF1b) was associated with increased germination of sly1-2 but decreased germination of wildtype Ler. This reduction of Ler germination was not associated with increased ABA levels. Apparently, GID1b is a positive regulator of germination in one context, but a negative regulator in the other.
• Hauvermale, A. L., Cárdenas, J. J., Bednarek, S. Y., & Steber, C. M. (2022). GA signaling expands: The plant UBX domain-containing protein 1 is a binding partner for the GA receptor. Plant Physiology, 190(4), 2651–2670. (Show/Hide Abstract)
  Abstract: The plant Ubiquitin Regulatory X (UBX) domain-containing protein 1 (PUX1) functions as a negative regulator of gibberel- lin (GA) signaling. GAs are plant hormones that stimulate seed germination, the transition to flowering, and cell elongation and division. Loss of Arabidopsis (Arabidopsis thaliana) PUX1 resulted in a ''GA-overdose'' phenotype including early flowering, increased stem and root elongation, and partial resistance to the GA-biosynthesis inhibitor paclobutrazol during seed germination and root elongation. Furthermore, GA application failed to stimulate further stem elongation or flowering onset suggesting that elongation and flowering response to GA had reached its maximum. GA hormone partially repressed PUX1 protein accumulation, and PUX1 showed a GA-independent interaction with the GA receptor GA-INSENSITIVE DWARF-1 (GID1). This suggests that PUX1 is GA regulated and/or regulates elements of the GA signaling pathway. Consistent with PUX1 function as a negative regulator of GA signaling, the pux1 mutant caused increased GID1 expression and decreased accumulation of the DELLA REPRESSOR OF GA1-3, RGA. PUX1 is a negative regulator of the hexameric AAA + ATPase CDC48, a protein that functions in diverse cellular processes including unfolding proteins in preparation for proteasomal degradation, cell division, and expansion. PUX1 binding to GID1 required the UBX domain, a binding motif necessary for CDC48 interaction. Moreover, PUX1 overexpression in cell culture not only stimulated the disassembly of CDC48 hexamer but also resulted in co-fractionation of GID1, PUX1, and CDC48 subunits in velocity sedimentation assays. Based on our results, we propose that PUX1 and CDC48 are additional factors that need to be incorporated into our understanding of GA signaling.
• Hauvermale, A. L., & Steber, C. M. (2020). GA signaling is essential for the embryo-to-seedling transition during Arabidopsis seed germination, a ghost story. Plant Signaling & Behavior, 15(1), e1705028 (9 pages). (Show/Hide Abstract)
  Abstract: The plant hormone gibberellin (GA) stimulates developmental transitions including seed germination, flowering, and the transition from juvenile to adult growth stage. This study provided evidence that GA and the GA receptor GID1 (GA-INSENSITIVE DWARF1) are also needed for the embryo-to-seedling transition in Arabidopsis. The ga1-3 GA biosynthesis mutant fails to germinate unless GA is applied, whereas the gid1abc triple mutant fails to germinate because it cannot perceive endogenous or applied GA. Overexpression of the GID1a, GID1b, and GID1c GA receptors rescued the germination of a small percentage of ga1-3 seeds without GA application, and this rescue was improved by dormancy-breaking treatments, after-ripening and cold stratification. While GID1 overexpression stimulated ga1-3 seed germination, this germination was aberrant suggesting incomplete rescue of the germination process. Cotyledons emerged before the radicle, and the resulting “ghost” seedlings failed to develop a primary root, lost green coloration, and eventually died. The development of ga1-3 seedlings overexpressing GID1 was rescued by pre-germinative but not post-germinative GA application. Since the gid1abc mutant also exhibited a ghost phenotype after germination was rescued by cutting the seed coat, we concluded that both GA and GID1 are needed for the embryo-to-seedling transition prior to emergence from the seed coat.
• Ge, W., & Steber, C. M. (2018). Positive and negative regulation of seed germination by the Arabidopsis GA hormone receptors, GID1a, b, and c. Plant Direct, 2(9), 1–11. (Show/Hide Abstract)
  Abstract: Epistasis analysis of gid1 single and double mutants revealed that GID1c is a key positive regulator of seed germination, whereas the GID1b receptor can negatively regulate germination in dormant seeds and in the dark. The GID1 GA receptors were expected to positively regulate germination because the plant hormone gibberellin (GA) is required for seed germination in Arabidopsis thaliana. The three GA hormone receptors, GID1a, GID1b, and GID1c, positively regulate GA responses via GA/GID1‐ stimulated destruction of DELLA (Asp‐Glu‐Leu‐Leu‐Ala) repressors of GA responses. The fact that the gid1abc triple mutant but not gid1 double mutants fail to germi- nate indicates that all three GA receptors can positively regulate non‐dormant seed germination in the light. It was known that the gid1abc triple mutant fails to lose dormancy through the dormancy breaking treatments of cold stratification (moist chilling of seeds) and dry after‐ripening (a period of dry storage). Previous work sug- gested that there may be some specialization of GID1 gene function during germina- tion because GID1b mRNA expression was more highly induced by after‐ripening, whereas GID1a and GID1c mRNA levels were more highly induced by cold stratifica- tion. In light‐germinated dormant seeds, the gid1b mutation can partly rescue the germination efficiency of gid1a but not of gid1c seeds. Thus, GID1b can function as an upstream negative regulator GID1c, a positive regulator of dormant seed germina- tion. Further experiments showed that GID1b can negatively regulate dark germina- tion. Wild‐type Arabidopsis seeds do not germinate well in the dark. The gid1b and gid1ab double mutants germinated much more efficiently than wild type, gid1c, or gid1ac mutants in the dark. The observation that the gid1ab double mutant also shows increased dark germination suggests that GID1b, and to some extent GID1a, can act as upstream negative regulators of GID1c. Since the gid1abc triple mutant failed to germinate in the dark, it appears that GID1c is a key downstream positive regulator of dark germination. This genetic analysis indicates that the three GID1 receptors have partially specialized functions in GA signaling.
• Nelson, S. K., Ariizumi, T., & Steber, C. M. (2017). Biology in the Dry Seed: Transcriptome Changes Associated with Dry Seed Dormancy and Dormancy Loss in the Arabidopsis GA-Insensitive sleepy1-2 Mutant. Frontiers in Plant Science, 8, 1–21, https://doi.org/10.3389/fpls.2017.02158. (Show/Hide Abstract)
  Abstract: Plant embryos can survive years in a desiccated, quiescent state within seeds. In many species, seeds are dormant and unable to germinate at maturity. They acquire the capacity to germinate through a period of dry storage called after-ripening (AR), a biological process that occurs at 5–15% moisture when most metabolic processes cease. Because stored transcripts are among the first proteins translated upon water uptake, they likely impact germination potential. Transcriptome changes associated with the increased seed dormancy of the GA-insensitive sly1-2 mutant, and with dormancy loss through long sly1-2 after-ripening (19 months) were characterized in dry seeds. The SLY1 gene was needed for proper down-regulation of translation-associated genes in mature dry seeds, and for AR up-regulation of these genes in germinating seeds. Thus, sly1-2 seed dormancy may result partly from failure to properly regulate protein translation, and partly from observed differences in transcription factor mRNA levels. Two positive regulators of seed dormancy, DELLA GAI (GA-INSENSITIVE) and the histone deacetylase HDA6/SIL1 (MODIFIERS OF SILENCING1) were strongly AR-down- regulated. These transcriptional changes appeared to be functionally relevant since loss of GAI function and application of a histone deacetylase inhibitor led to decreased sly1-2 seed dormancy. Thus, after-ripening may increase germination potential over time by reducing dormancy-promoting stored transcript levels. Differences in transcript accumulation with after-ripening correlated to differences in transcript stability, such that stable mRNAs appeared AR-up-regulated, and unstable transcripts AR-down-regulated. Thus, relative transcript levels may change with dry after-ripening partly as a consequence of differences in mRNA turnover.
• Nelson, S. K, & Steber, C. M. (2017). Transcriptional mechanisms associated with seed dormancy and dormancy loss in the gibberellin-insensitive sly1-2 mutant of Arabidopsis thaliana. PLoS ONE 12(6), 1–32, https://doi.org/10.1371/journal.pone.0179143. (Show/Hide Abstract)
  Abstract: While widespread transcriptome changes were previously observed with seed dormancy loss, this study specifically characterized transcriptional changes associated with the increased seed dormancy and dormancy loss of the gibberellin (GA) hormone-insensitive sleepy1-2 (sly1-2) mutant. The SLY1 gene encodes the F-box subunit of an SCF E3 ubiquitin ligase needed for GA-triggered proteolysis of DELLA repressors of seed germination. DELLA over- accumulation in sly1-2 seeds leads to increased dormancy that can be rescued without DELLA protein destruction either by overexpression of the GA receptor, GA-INSENSITIVE DWARF1b (GID1b-OE) (74% germination) or by extended dry after-ripening (11 months, 51% germination). After-ripening of sly1 resulted in different transcriptional changes in early versus late Phase II of germination that were consistent with the processes known to occur. Approxi- mately half of the transcriptome changes with after-ripening appear to depend on SLY1-trig- gered DELLA proteolysis. Given that many of these SLY1/GA-dependent changes are genes involved in protein translation, it appears that GA signaling increases germination capacity in part by activating translation. While sly1-2 after-ripening was associated with transcript-level changes in 4594 genes over two imbibition timepoints, rescue of sly1-2 germination by GID1b- OE was associated with changes in only 23 genes. Thus, a big change in sly1-2 germination phenotype can occur with relatively little change in the global pattern of gene expression during the process of germination. Most GID1b-OE-responsive transcripts showed similar changes with after-ripening in early Phase II of imbibition, but opposite changes with after-ripening by late Phase II. This suggests that GID1b-OE stimulates germination early in imbibition, but may later trigger negative feedback regulation.
• Nelson, S. K, & Steber, C. M. (2016). Gibberellin hormone signal perception: down-regulating DELLA repressors of plant growth and development. In P. Hedden & S. G. Thomas (Eds), Annual Plant Reviews, Volume 49, The Gibberellins (pp. 153–187). Chichester, UK: Wiley Blackwell. (Show/Hide Abstract)
  Abstract: The gibberellin (GA) hormone signal is perceived by a receptor with homology to hormone-sensitive lipases, GID1 (GA-INSENSITIVE DWARF1). This leads to GA-stimulated responses, including stem elongation, seed germination and the transition to flowering. GA-binding enables GID1 to interact with and block the function of the DELLA repressors of GA responses. DELLA repression can be blocked both by proteolytic and non-proteolytic mechanisms triggered by the formation of a GID1-GA-DELLA complex. DELLA is down-regulated by the SLEEPY1/GID2 F-box proteins via the ubiquitin-proteasome pathway, and can be regulated by other post-translational modifications. This chapter reviews the structural requirements for GA-binding by GID1 and for GID1-GA-DELLA protein complex formation, and reviews the current understanding of the mechanisms regulating DELLA repressors.
• Hauvermale, A. L., Tuttle, K. M., Takebayashi, Y., Seo, M., and Steber, C. M. (2015). Loss of Arabidopsis thaliana seed dormancy is associated with increased accumulation of the GID1 GA hormone receptors. Plant and Cell Physiology, 56(9), 1773–1785. (Show/Hide Abstract)
  Abstract: Dormancy prevents seeds from germinating under favorable conditions until they have experienced dormancy-breaking conditions, such as after-ripening through a period of dry storage or cold imbibition. Abscisic acid (ABA) hormone signaling establishes and maintains seed dormancy, whereas gibberellin (GA) signaling stimulates germination. ABA levels decrease and GA levels increase with after-ripening and cold stratification. However, increasing GA sensitivity may also be critical to dormancy loss since increasing seed GA levels are detectable only with long periods of after-ripening and imbibition. After-ripening and cold stratification act additively to enhance GA hormone sensitivity in ga1-3 seeds that cannot synthesize GA. Since the overexpression of the GA receptor GID1 (GA-INSENSITIVE DWARF1) enhanced this dormancy loss, and because gid1a gid1b gid1c triple mutants show decreased germination, the effects of dormancy–breaking treatments on GID1 mRNA and protein accumulation were examined. Partial after-ripening resulted in increased GID1b, but not GID1a or GID1c mRNA levels. Cold imbibition stimulated the accumulation of all three GID1 transcripts, but resulted in no increase in GA sensitivity during ga1-3 seed germination unless seeds were also partially after-ripened. This is likely because after-ripening was needed to enhance GID1 protein accumulation, independently of transcript abundance. The rise in GID1b transcript with after-ripening was not associated with decreased ABA levels, suggesting there is ABA-independent GID1b regulation by after-ripening. GA and the DELLA RGL2 repressor of GA responses differentially regulated the three GID1 transcripts. Moreover, DELLA RGL2 appeared to switch between positive and negative regulation of GID1 expression in response to dormancy breaking treatments. The rise in GID1b transcript with after-ripening was not associated with decreased ABA levels, suggesting that after-ripening regulates GID1 independently of ABA.
• Hauvermale, A. L., Ariizumi, T., & Steber, C. M. (2014). The roles of the GA receptors GID1a, GID1b, and GID1c in sly1-independent GA signaling. Plant Signaling & Behavior 9:e28030. (Show/Hide Abstract)
  Abstract: Gibberellin (GA) hormone signaling occurs through proteolytic and non-proteolytic mechanisms. GA binding to the GA receptor GID1 (GA-INSENSITIVE DWARF1) enables GID1 to bind negative regulators of GA responses called DELLA proteins. In proteolytic GA signaling, the SLEEPY1 (SLY1) F-box protein targets DELLA proteins in the GID1-GA-DELLA complex for destruction through the ubiquitin-proteasome pathway. Non-proteolytic GA signaling occurs in sly1 mutants when GA cannot target DELLA proteins for destruction, and requires GA and GID1 gene function. Based on comparison of gid1 multiple mutants to sly1 gid1 mutants, GID1a is the primary GA receptor stimulating stem elongation in proteolytic and non-proteolytic signaling, and stimulating fertility in proteolytic GA signaling. GID1b plays the primary role in fertility, and a secondary role in elongation during non-proteolytic GA signaling. The stronger role of GID1b in non-proteolytic GA signaling may result from the fact that GID1b has higher affinity for DELLA protein than GID1a and GID1c.
• Ariizumi, T., Hauvermale, A. L., Nelson, S. K., Hanada, A., Yamaguchi, S., & Steber, C. M. (2013). Lifting DELLA repression of Arabidopsis seed germination by non-proteolytic GA signaling. Plant Physiology, 162(4), 2125–2139. (Show/Hide Abstract)
  Abstract: DELLA repression of Arabidopsis thaliana seed germination can be lifted either through DELLA proteolysis by the ubiquitin-proteasome pathway or through proteolysis-independent GA hormone signaling. GA-binding to the GID1 (GIBBERELLIN-INSENSITIVE DWARF1) GA receptors stimulates GID1-GA-DELLA complex formation which in turn triggers DELLA protein ubiquitination and proteolysis via the SCF^SLY1 E3 ubiqutin ligase and 26S proteasome. Although DELLA cannot be destroyed in the sly1-2 F-box mutant, long dry after-ripening and GID1 overexpression can relieve the strong sly1-2 seed dormancy phenotype. It appears that sly1-2 seed dormancy results from ABA signaling downstream of DELLA since dormant sly1-2 seeds accumulate high levels of ABA hormone and loss of ABA sensitivity rescues sly1-2 seed germination. DELLA positively regulates the expression of XERICO, an inducer of ABA biosynthesis. GID1b overexpression rescues sly1-2 germination through proteolysis-independent DELLA down-regulation associated with increased expression of GA-inducible genes and decreased ABA accumulation, apparently as a result of decreased XERICO mRNA levels. Higher levels of GID1 overexpression are associated with more efficient sly1 germination and increased GID1-GA-DELLA complex formation, suggesting that GID1 downregulates DELLA through protein binding. After-ripening results in increased GA accumulation and GID1a-dependent GA signaling, suggesting that after-ripening triggers GA-stimulated GID1-GA-DELLA protein complex formation which in turn blocks DELLA transcriptional activation of the XERICO inhibitor of seed germination.
• Ariizumi, T., Lawrence, P. K., & Steber, C. M. (2011). The role of two F-box proteins, SLEEPY1 and SNEEZY, in arabidopsis gibberellin signaling. Plant Physiology, 155(2), 765–775. (Show/Hide Abstract)
  Abstract: The SLEEPY1 (SLY1) F-box gene is a positive regulator of gibberellin (GA) signaling in Arabidopsis (Arabidopsis thaliana). Loss of SLY1 results in GA-insensitive phenotypes including dwarfism, reduced fertility, delayed flowering, and increased seed dormancy. These sly1 phenotypes are partially rescued by overexpression of the SLY1 homolog SNEEZY (SNE)/SLY2, suggesting that SNE can functionally replace SLY1. GA responses are repressed by DELLA family proteins. GA relieves DELLA repression when the SCF^SLY1 (for Skp1, Cullin, F-box) E3 ubiquitin ligase ubiquitinates DELLA protein, thereby targeting it for proteolysis. Coimmunoprecipitation experiments using constitutively expressed 35S:hemagglutinin (HA)-SLY1 and 35S:HA-SNE translational fusions in the sly1-10 background suggest that SNE can function similarly to SLY1 in GA signaling. Like HA-SLY1, HA-SNE interacted with the CULLIN1 subunit of the SCF complex, and this interaction required the F-box domain. Like HA-SLY1, HA-SNE coimmunoprecipitated with the DELLA REPRESSOR OF GA1-3 (RGA), and this interaction required the SLY1 or SNE carboxyl-terminal domain. Whereas HA-SLY1 overexpression resulted in a decrease in both DELLA RGA and RGA-LIKE2 (RGL2) protein levels, HA-SNE caused a decrease in DELLA RGA but not in RGL2 levels. This suggests that one reason HA-SLY1 is able to effect a stronger rescue of sly1-10 phenotypes than HA-SNE is because SLY1 regulates a broader spectrum of DELLA proteins. The FLAG-SLY1 fusion protein was found to coimmunoprecipitate with the GA receptor HA-GA-INSENSITIVE DWARF1b (GID1b), supporting the model that SLY1 regulates DELLA through interaction with the DELLA-GA-GID1 complex.
• Ariizumi, T., & Steber, C. M. (2011). Mutations in the F-box gene SNEEZY result in decreased Arabidopsis GA signaling. Plant Signaling & Behavior, 6(6), 831–833. (Show/Hide Abstract)
  Abstract: We previously reported that the SLEEPY1 (SLY1) homolog, F-box gene SNEEZY/SLEEPY2 (SNE/SLY2), can partly replace SLY1 in gibberellin (GA) hormone signaling through interaction with DELLAs RGA and GAI. To determine whether SNE normally functions in GA signaling, we characterized the phenotypes of two T-DNA alleles, sne-t2 and sne-t3. These mutations result in no apparent vegetative phenotypes, but do result in increased ABA sensitivity in seed germination. Double mutants sly1-t2 sne-t2 and sly1-t2 sne-t3 result in a significant decrease in plant fertility and final plant height compared to sly1-t2. The fact that sne mutations have an additive effect with sly1 suggests that SNE normally functions as a redundant positive regulator of GA signaling.
• Ariizumi, T., Murase, K., Sun, T.-p., & Steber, C. M. (2008). Proteolysis-independent downregulation of DELLA repression in Arabidopsis by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1. The Plant Cell, 20(9), 2447–2459. (Show/Hide Abstract)
  Abstract: This article presents evidence that DELLA repression of gibberellin (GA) signaling is relieved both by proteolysis-dependent and -independent pathways in Arabidopsis thaliana. DELLA proteins are negative regulators of GA responses, including seed germination, stem elongation, and fertility. GA stimulates GA responses by causing DELLA repressor degradation via the ubiquitin-proteasome pathway. DELLA degradation requires GA biosynthesis, three functionally redundant GA receptors GIBBERELLIN INSENSITIVE DWARF1 (GID1a, b, and c), and the SLEEPY1 (SLY1) F-box subunit of an SCF E3 ubiquitin ligase. The sly1 mutants accumulate more DELLA proteins but display less severe dwarf and germination phenotypes than the GA biosynthesis mutant ga1-3 or the gid1abc triple mutant. Interestingly, GID1 overexpression rescued the sly1 dwarf and infertility phenotypes without decreasing the accumulation of the DELLA protein REPRESSOR OF ga1-3. GID1 rescue of sly1 mutants was dependent on the level of GID1 protein, GA, and the presence of a functional DELLA motif. Since DELLA shows increasing interaction with GID1 with increasing GA levels, it appears that GA-bound GID1 can block DELLA repressor activity by direct protein–protein interaction with the DELLA domain. Thus, a SLY1-independent mechanism for GA signaling may function without DELLA degradation.
• Ariizumi, T., & Steber, C. M. (2007). Seed germination of GA-insensitive sleepy1 mutants does not require RGL2 protein disappearance in Arabidopsis. The Plant Cell, 19(3), 791–804. (Show/Hide Abstract)
  Abstract: We explore the roles of gibberellin (GA) signaling genes SLEEPY1 (SLY1) and RGA-LIKE2 (RGL2) in regulation of seed germination in Arabidopsis thaliana, a plant in which the hormone GA is required for seed germination. Seed germination failure in the GA biosynthesis mutant ga1-3 is rescued by GA and by mutations in the DELLA gene RGL2, suggesting that RGL2 represses seed germination. RGL2 protein disappears before wild-type seed germination, consistent with the model that GA stimulates germination by causing the SCF^SLY1 E3 ubiquitin ligase complex to trigger ubiquitination and destruction of RGL2. Unlike ga1-3, the GA-insensitive sly1 mutants show variable seed dormancy. Seed lots with high seed dormancy after-ripened slowly, with stronger alleles requiring more time. We expected that if RGL2 negatively controls seed germination, sly1 mutant seeds that germinate well should accumulate lower RGL2 levels than those failing to germinate. Surprisingly, RGL2 accumulated at high levels even in after-ripened sly1 mutant seeds with 100% germination, suggesting that RGL2 disappearance is not a prerequisite for seed germination in the sly1 background. Without GA, several GA-induced genes show increased accumulation in sly1 seeds compared with ga1-3. It is possible that the RGL2 repressor of seed germination is inactivated by after-ripening of sly1 mutant seeds.
• Dill, A., Thomas, S. G., Hu, J., Steber, C. M., & Sun, T.-p. (2004). The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation. The Plant Cell, 16(6), 1392–1405. (Show/Hide Abstract)
  Abstract: The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants. In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA). SLEEPY1 (SLY1) encodes an F-box–containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA. These findings suggested that SLY1 recruits RGA to the SCF^SLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome. In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response. By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain. The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCF^SLY1 targets both RGA and GAI for degradation. The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation. However, we found that this DELLA domain is not required for protein–protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins. We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants. This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins.
• Strader, L. C., Ritchie, S., Soule, J. D., McGinnis, K. M., & Steber, C. M. (2004). Recessive-interfering mutations in the gibberellin signaling gene SLEEPY1 are rescued by overexpression of its homologue, SNEEZY. Proc. Natl. Acad. Sci. U.S.A., 101(34), 12771–12776. (Show/Hide Abstract)
  Abstract: This article reports the genetic interaction of two F-box genes, SLEEPY1 (SLY1) and SNEEZY (SNE), in Arabidopsis thaliana gibberellin (GA) signaling. The SLY1 gene encodes an F-box subunit of a Skp1–cullin–F-box (SCF) E3 ubiquitin ligase complex that positively regulates GA signaling. The sly1-2 and sly1-10 mutants have recessive, GA-insensitive phenotypes including delayed germination, dwarfism, reduced fertility, and overaccumulation of the DELLA proteins RGA (Repressor of ga1–3), GAI (GA-Insensitive), and RGL2 (RGA-Like 2). The DELLA domain proteins are putative transcription factors that negatively regulate GA signaling. The requirement for SLY1 in GA-stimulated disappearance of DELLA proteins suggests that GA targets DELLA proteins for destruction via SCF^SLY1-mediated ubiquitylation. Overexpression of SLY1 in sly1-2 and sly1-10 plants rescues the recessive GA-insensitive phenotype of these mutants. Surprisingly, antisense expression of SLY1 also suppresses these mutants. This result caused us to hypothesize that the SLY1 homologue SNE can functionally replace SLY1 in the absence of the recessive interfering sly1-2 or sly1-10 genes. This hypothesis was supported because overexpression of SNE in sly1-10 rescues the dwarf phenotype. In addition to rescuing the sly1-10 dwarf phenotype, SNE overexpression also restored normal RGA protein levels, suggesting that the SNE F-box protein can replace SLY1 in the GA-induced proteolysis of RGA. If the C-terminal truncation in the sly1-2 and sly1-10 alleles interferes with SNE rescue, we reasoned that overexpression of sly1-2 might interfere with wild-type SLY1 function. Indeed, overexpression of sly1-2 in wild-type Ler (Landsberg erecta) yields dwarf plants.
• Strader, L. C. (2004). Hormonal control of seed dormancy and germination: drawing connections between Arabidopsis thaliana L. and Triticum aestivum L. (Doctoral dissertation). Washington State University.
• McGinnis, K. M., Thomas, S. G., Soule, J. D., Strader, L. C., Zale, J. M., Sun, T.-p., & Steber, C. M. (2003). The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase. The Plant Cell, 15(5), 1120–1130. (Show/Hide Abstract)
  Abstract: The Arabidopsis SLY1 (SLEEPY1) gene positively regulates gibberellin (GA) signaling. Positional cloning of SLY1 revealed that it encodes a putative F-box protein. This result suggests that SLY1 is the F-box subunit of an SCF E3 ubiquitin ligase that regulates GA responses. The DELLA domain protein RGA (repressor of ga1-3) is a repressor of GA response that appears to undergo GA-stimulated protein degradation. RGA is a potential substrate of SLY1, because sly1 mutations cause a significant increase in RGA protein accumulation even after GA treatment. This result suggests SCF^SLY1-targeted degradation of RGA through the 26S proteasome pathway. Further support for this model is provided by the observation that an rga null allele partially suppresses the sly1-10 mutant phenotype. The predicted SLY1 amino acid sequence is highly conserved among plants, indicating a key role in GA response.
• Steber, C. M., & McCourt, P. (2001). A role for Brassinosteroids in germination in Arabidopsis. Plant Physiology, 125(2), 763–769. (Show/Hide Abstract)
  Abstract: This paper presents evidence that plant brassinosteroid (BR) hormones play a role in promoting germination. It has long been recognized that seed dormancy and germination are regulated by the plant hormones abscisic acid (ABA) and gibberellin (GA). These two hormones act antagonistically with each other. ABA induces seed dormancy in maturing embryos and inhibits germination of seeds. GA breaks seed dormancy and promotes germination. Severe mutations in GA biosynthetic genes in Arabidopsis, such as ga1-3, result in a requirement for GA application to germinate. Whereas previous work has shown that BRs play a critical role in controlling cell elongation, cell division, and skotomorphogenesis, no germination phenotypes have been reported in BR mutants. We show that BR rescues the germination phenotype of severe GA biosynthetic mutants and of the GA-insensitive mutant sleepy1. This result shows that BR stimulates germination and raises the possibility that BR is needed for normal germination. If true, we would expect to detect a germination phenotype in BR mutants. We found that BR mutants exhibit a germination phenotype in the presence of ABA. Germination of both the BR biosynthetic mutant det2-1 and the BR-insensitive mutant bri1-1 is more strongly inhibited by ABA than is germination of wild type. Thus, the BR signal is needed to overcome inhibition of germination by ABA. Taken together, these results point to a role for BRs in stimulating germination.
• Steber, C. M., Cooney, S. E., & McCourt, P. (1998). Isolation of the GA-response mutant sly1 as a suppressor of ABI1-1 in Arabidopsis thaliana. Genetics, 149(2), 509–521. (Show/Hide Abstract)
  Abstract: Seed dormancy and germination in higher plants are partially controlled by the plant hormones abscisic acid (ABA) and gibberellic acid (GA). ABA establishes dormancy during embryo maturation, whereas GA breaks dormancy and induces germination. Previous attempts to identify GA response genes were confounded because GA mutants are not expected to germinate and, unlike GA auxotrophs, should fail to be rescued by exogenous GA. Here, we describe a screen for suppressors of the ABA-insensitive mutant ABI1-1 that enriches for GA auxotrophs and GA-insensitive mutants. The vast majority (76%) of the suppressors of ABI1-1 strongly resemble GA auxotrophs in that they are severely dwarfed and have dark green foliage and flowers with underdeveloped petals and stamen. Three isolates were alleles of the GA auxotroph ga1. The remaining severe dwarves were not rescued by GA and belong to a single complementation group that we designate sly1 (Sleepy 1). The alleles of sly1 identified are the first recessive GAinsensitive mutations to reflect the full spectrum of GA-associated phenotypes, including the failure to germinate in the absence of the ABI1-1 lesion. Thus, we postulate that SLY1 is a key factor in GA reception.

Brassinosteroids

• Steber, C. M., & McCourt, P. (2001). A role for Brassinosteroids in germination in Arabidopsis. Plant Physiology, 125(2), 763–769. (Show/Hide Abstract)
  Abstract: This paper presents evidence that plant brassinosteroid (BR) hormones play a role in promoting germination. It has long been recognized that seed dormancy and germination are regulated by the plant hormones abscisic acid (ABA) and gibberellin (GA). These two hormones act antagonistically with each other. ABA induces seed dormancy in maturing embryos and inhibits germination of seeds. GA breaks seed dormancy and promotes germination. Severe mutations in GA biosynthetic genes in Arabidopsis, such as ga1-3, result in a requirement for GA application to germinate. Whereas previous work has shown that BRs play a critical role in controlling cell elongation, cell division, and skotomorphogenesis, no germination phenotypes have been reported in BR mutants. We show that BR rescues the germination phenotype of severe GA biosynthetic mutants and of the GA-insensitive mutant sleepy1. This result shows that BR stimulates germination and raises the possibility that BR is needed for normal germination. If true, we would expect to detect a germination phenotype in BR mutants. We found that BR mutants exhibit a germination phenotype in the presence of ABA. Germination of both the BR biosynthetic mutant det2-1 and the BR-insensitive mutant bri1-1 is more strongly inhibited by ABA than is germination of wild type. Thus, the BR signal is needed to overcome inhibition of germination by ABA. Taken together, these results point to a role for BRs in stimulating germination.

Review Articles

• Hu, Y., Sjoberg, S. M., Chen, C., Hauvermale, A. L., Morris, C. F., Delwiche, S. R., Cannon, A. E., Steber, C. M., & Zhang, Z. (2022). As the number falls, alternatives to the Hagberg–Perten falling number method: A review. Comprehensive Reviews in Food Science and Food Safety , 1–13. (Show/Hide Abstract)
  Abstract: This review examines the application, limitations, and potential alternatives to the Hagberg–Perten falling number (FN) method used in the global wheat industry for detecting the risk of poor end-product quality mainly due to starch degradation by the enzyme α-amylase. By viscometry, the FN test indirectly detects the presence of α-amylase, the primary enzyme that digests starch. Elevated α-amylase results in low FN and damages wheat product quality resulting in cakes that fall, and sticky bread and noodles. Low FN can occur from preharvest sprouting (PHS) and late maturity α-amylase (LMA). Moist or rainy conditions before harvest cause PHS on the mother plant. Continuously cool or fluctuating temperatures during the grain filling stage cause LMA. Due to the expression of addi- tional hydrolytic enzymes, PHS has a stronger negative impact than LMA. Wheat grain with low FN/high α-amylase results in serious losses for farmers, traders, millers, and bakers worldwide. Although blending of low FN grain with sound wheat may be used as a means of moving affected grain through the marketplace, care must be taken to avoid grain lots from falling below contract-specified FN. A large amount of sound wheat can be ruined if mixed with a small amount of sprouted wheat. The FN method is widely employed to detect α-amylase after harvest. However, it has several limitations, including sampling variability, high cost, labor intensiveness, the destructive nature of the test, and an inability to differentiate between LMA and PHS. Faster, cheaper, and more accurate alternatives could improve breeding for resistance to PHS and LMA and could preserve the value of wheat grain by avoiding inadvertent mixing of high- and low- FN grain by enabling testing at more stages of the value stream including at harvest, delivery, transport, storage, and milling. Alternatives to the FN method explored here include the Rapid Visco Analyzer, enzyme assays, immunoassays, near-infrared spectroscopy, and hyperspectral imaging.
• Nelson, S. K, & Steber, C. M. (2016). Gibberellin hormone signal perception: down-regulating DELLA repressors of plant growth and development. In P. Hedden & S. G. Thomas (Eds), Annual Plant Reviews, Volume 49, The Gibberellins (pp. 153–187). Chichester, UK: Wiley Blackwell. (Show/Hide Abstract)
  Abstract: The gibberellin (GA) hormone signal is perceived by a receptor with homology to hormone-sensitive lipases, GID1 (GA-INSENSITIVE DWARF1). This leads to GA-stimulated responses, including stem elongation, seed germination and the transition to flowering. GA-binding enables GID1 to interact with and block the function of the DELLA repressors of GA responses. DELLA repression can be blocked both by proteolytic and non-proteolytic mechanisms triggered by the formation of a GID1-GA-DELLA complex. DELLA is down-regulated by the SLEEPY1/GID2 F-box proteins via the ubiquitin-proteasome pathway, and can be regulated by other post-translational modifications. This chapter reviews the structural requirements for GA-binding by GID1 and for GID1-GA-DELLA protein complex formation, and reviews the current understanding of the mechanisms regulating DELLA repressors.
• Hauvermale, A. L., Ariizumi, T., & Steber, C. M. (2012). Gibberellin signaling: A theme and variations on DELLA repression. Plant Physiology, 160(1), 83–92.
• Finkelstein, R. R., Reeves, W., Ariizumi, T., & Steber, C. M. (2008). Molecular aspects of seed dormancy. Annual Review of Plant Biology, 59, 387–415. (Show/Hide Abstract)
  Abstract: Seed dormancy provides a mechanism for plants to delay germination until conditions are optimal for survival of the next generation. Dormancy release is regulated by a combination of environmental and endogenous signals with both synergistic and competing effects. Molecular studies of dormancy have correlated changes in transcriptomes, proteomes, and hormone levels with dormancy states ranging from deep primary or secondary dormancy to varying degrees of release. The balance of abscisic acid (ABA):gibberellin (GA) levels and sensitivity is a major, but not the sole, regulator of dormancy status. ABA promotes dormancy induction and maintenance, whereas GA promotes progression from release through germination; environmental signals regulate this balance by modifying the expression of biosynthetic and catabolic enzymes. Mediators of environmental and hormonal response include both positive and negative regulators, many of which are feedback-regulated to enhance or attenuate the response. The net result is a slightly heterogeneous response, thereby providing more temporal options for successful germination.
• Steber, C. M. (2007). De-repression of seed germination by GA signaling. In K. J. Bradford, H. Nonogaki, (Eds.) Annual Plant Reviews Volume 27: Seed Development, Dormancy and Germination (pp. 248–263). Oxford, UK: Blackwell Publishing Ltd.
• Ariizumi, T., & Steber, C. M. (2006). Essay 20.2: Ubiquitin becomes ubiquitous in GA signaling. In L. Taiz & E. Zeigler (Eds.) Plant Physiology (Fourth ed.). Sunderland, MA: Sinauer Associates, Inc.
• Thomas, S. G., Rieu, I., & Steber, C. M. (2005). Gibberellin metabolism and signaling. In G. Litwack (Ed.) Plant Hormones, Volume 72 (Vitamins & Hormones) (pp. 228–338). San Diego & London: Elsevier Academic Press. (Show/Hide Abstract)
  Abstract: Gibberellins (GAs) are a family of plant hormones controlling many aspects of plant growth and development including stem elongation, germination, and the transition from vegetative growth to flowering. Cloning of the genes encoding GA biosynthetic and inactivating enzymes has led to numerous insights into the developmental regulation of GA hormone accumulation that is subject to both positive and negative feedback regulation. Genetic and biochemical analysis of GA‐signaling genes has revealed that posttranslational regulation of DELLA protein accumulation is a key control point in GA response. The highly conserved DELLA proteins are a family of negative regulators of GA signaling that appear subject to GA‐stimulated degradation through the ubiquitin‐26S proteasome pathway. This review discusses the regulation of GA hormone accumulation and signaling in the context of its role in plant growth and development.
• Itoh, H., Matsuoka, M., & Steber, C. M. (2003). A role for the ubiquitin-26S-proteasome pathway in gibberellin signaling. Trends in Plant Science, 8(10), 492–497. (Show/Hide Abstract)
  Abstract: The gibberellin (GA) signaling pathway, like auxin and jasmonate signaling, uses the ubiquitin–proteasome pathway to control expression through protein degradation. A conserved F-box protein of an SCF E3 ubiquitin ligase is a positive regulator of GA signaling in Arabidopsis and rice. GA apparently stimulates stem elongation by causing this SCF complex to regulate negatively a family of negative regulators of GA response (the DELLA family of putative transcription factors). The DELLA family members AtRGA or (Repressor of ga1-3) and OsSLR1 (SLENDER RICE1) proteins both appear to be subject to GA-induced proteolysis. The need to have the F-box genes AtSLY1 and OsGID2 for this proteolysis suggests that GA causes proteolysis of AtRGA/OsSLR1 via the SCF^{AtSLY1/OsGID2} ubiquitin ligase.

Barley

• Nirmala, J., Drader, T., Lawrence, P. K., Yin, C., Hulbert, S., Steber, C. M., Steffenson, B. J., Szabo, L.J., von Wettstein, D., & Kleinhof, A. (2011). Concerted action of two avirulent spore effectors activates Reaction to Puccinia graminis 1 (Rpg1)-mediated cereal stem rust resistance. Proc. Natl. Acad. Sci. U.S.A., 108(35), 14676–14681. (Show/Hide Abstract)
  Abstract: The barley stem rust resistance gene Reaction to Puccinia graminis 1 (Rpg1), encoding a receptor-like kinase, confers durable resistance to the stem rust pathogen Puccinia graminis f. sp. tritici. The fungal urediniospores form adhesion structures with the leaf epidermal cells within 1 h of inoculation, followed by hyphae and haustorium formation. The RPG1 protein is constitutively expressed and not phosphorylated. On inoculation with avirulent urediniospores, it is phosphorylated in vivo within 5 min and subsequently degraded. Application of arginine-glycine-aspartic acid peptide loops prevented the formation of adhesion structures for spore attachment, the phosphorylation of RPG1, and germination of the viable spores. Arginine-glycine-aspartic acid affinity chromatography of proteins from the ungerminated avirulent rust spores led to the purification and identification of a protein with fibronectin type III and breast cancer type 1 susceptibility protein domains and a vacuolar protein sorting-associated protein 9 with a coupling of ubiquitin to endoplasmic reticulum degradation domain. Both proteins are required to induce in vivo phosphorylation and degradation of RPG1. Combined application of both proteins caused hypersensitive reaction on the stem rust-resistant cultivar Morex but not on the susceptible cultivar Steptoe. Expression studies indicated that mRNA of both genes are present in ungerminated urediniospores and are constitutively transcribed in sporelings, infected leaves, and haustoria in the investigated avirulent races. Evidence is presented that RPG1, in yeast, interacts with the two protein effectors from the urediniospores that activate cooperatively the stem rust resistance protein RPG1 long before haustoria formation.

Patent Applications

• Kidwell, K. K., Steber, C. M., Demacon, V. L., Shelton, G. B., & Burke, A. B. (2008). GLYPHOSATE-TOLERANT WHEAT GENOTYPES. United States Patent Application. (Show/Hide Abstract)
  Abstract: The present invention provides methods for producing glyphosate-tolerant wheat genotypes by mutagenesis, glyphosate wheat plants produced by such methods, and related compositions and methods.
• Steber, C. M., Kidwell, K. K., Demacon, V. L., & Okubara, P. A. (2006). MUTATION BREEDING FOR RESISTANCE TO DISEASE AND OTHER USEFUL TRAITS. United States Patent Application. (Show/Hide Abstract)
  Abstract: Particular aspects provide novel mutant plants and plant parts thereof, derived via mutagensis, having disease resistance and other useful traits. Particular embodiments provide a wheat genotye 'RRR Scarlet' ('Scarlet-Rz1'), plants and seeds thereof, methods for producing a plant comprising crossing 'Scarlet-Rz1' plants with another wheat plant, hybrid wheat seeds and plants produced by crossing 'Scarlet-Rz1' plants with another line or plant, and creation of variants by mutagenesis or transformation of 'Scarlet-Rz1'. Additional aspects provide methods for producing other varieties or breeding lines derived from 'Scarlet-Rz1' and to varieties or breeding lines produced thereby. Further aspects provide for mutant plants and plant parts thereof that are resistant and/or tolerant to plant root fungal pathogens such as Rhizoctonia and Pythium. Additional embodiments provide mutant plants and plant parts thereof that exhibit stress tolerance and/or resistance. Yet further aspects provide mutant plants and plant parts thereof that are drought resistant or tolerant.

Patents

• Steber, C. M., Kidwell, K. K., Demacon, V. L., & Okubara, P. A. (2016). MUTATION BREEDING FOR RESISTANCE TO FUNGAL DISEASE AND FOR DROUGHT TOLERANCE. United States Patent No.: 9,491,916. (Show/Hide Abstract)
  Abstract: Particular aspects provide novel mutant plants and plant parts thereof, derived via mutagenesis, having disease resistance and other useful traits. Particular embodiments provide a wheat genotype 'RRR Scarlet' ('Scarlet-Rz1'), plants and seeds thereof, methods for producing a plant comprising crossing 'Scarlet-Rz1' plants with another wheat plant, hybrid wheat seeds and plants produced by crossing 'Scarlet-Rz1' plants with another line or plant, and creation of variants by mutagenesis or transformation of 'Scarlet-Rz1'. Additional aspects provide methods for producing other varieties or breeding lines derived from 'Scarlet-Rz1' and to varieties or breeding lines produced thereby. Further aspects provide for mutant plants and plant parts thereof that are resistant and/or tolerant to plant root fungal pathogens such as Rhizoctonia and Pythium. Additional embodiments provide mutant plants and plant parts thereof that exhibit stress tolerance and/or resistance. Yet further aspects provide mutant plants and plant parts thereof that are drought resistant or tolerant.
• Kidwell, K. K., Steber, C. M., Demacon, V. L., Shelton, G. B., & Burke, A. B. (2014). GLYPHOSATE-TOLERANT WHEAT GENOTYPES. United States Patent No.: 8,637,738. (Show/Hide Abstract)
  Abstract: The present invention provides methods for producing glyphosate-tolerant wheat genotypes by mutagenesis, glyphosate wheat plants produced by such methods, and related compositions and methods.