Preharvest Sprouting in Quinoa: A New Screening Method Adapted to Panicles and GWAS Components.

The introduction of quinoa into new growing regions and environments is of interest to farmers, consumers, and stakeholders around the world. Many plant breeding programs have already started to adapt quinoa to the environmental and agronomic conditions of their local fields. Formal quinoa breeding efforts in Washington State started in 2010, led by Professor Kevin Murphy out of Washington State University. Preharvest sprouting appeared as the primary obstacle to increased production in the coastal regions of the Pacific Northwest. Preharvest sprouting (PHS) is the undesirable sprouting of seeds that occurs before harvest, is triggered by rain or humid conditions, and is responsible for yield losses and lower nutrition in cereal grains. PHS has been extensively studied in wheat, barley, and rice, but there are limited reports for quinoa, partly because it has only recently emerged as a problem. This study aimed to better understand PHS in quinoa by adapting a PHS screening method commonly used in cereals. This involved carrying out panicle-wetting tests and developing a scoring scale specific for panicles to quantify sprouting. Assessment of the trait was performed in a diversity panel (N = 336), and the resulting phenotypes were used to create PHS tolerance rankings and undertake a GWAS analysis (n = 279). Our findings indicate that PHS occurred at varying degrees across a subset of the quinoa germplasm tested and that it is possible to access PHS tolerance from natural sources. Ultimately, these genotypes can be used as parental lines in future breeding programs aiming to incorporate tolerance to PHS.

Evaluating relationships between seed morphological traits and seed dormancy in Chenopodium quinoa Willd.

Introduction: Quinoa is a high-value, nutritious crop that performs well in variable environments, marginal soils, and in diverse crop rotations. Quinoa's many attributes make it an ideal crop for supporting human health in global communities and economies. To date, quinoa research has largely focused on traits in adult plants important for enhancing plant phenotypic plasticity, abiotic stress, disease resistance, and yield. Fewer studies have evaluated quinoa seed dormancy and suggest that most modern quinoa varieties have weak or no seed dormancy, and a narrow window of seed viability post-harvest. In other crops, diminished seed dormancy is a major risk factor for preharvest sprouting (PHS; germination on the panicle due to rain prior to harvest) and may also pose a similar risk for quinoa. Methods: This study (1) developed a dormancy screening assay to characterize seed dormancy strength in a large collection of quinoa varieties, (2) investigated if morphological variables including seed coat color, seed coat thickness, seed shape including eccentricity which evaluates the roundness or flatness of a seed, and other agronomic traits like crude protein content and seed moisture, contribute to quinoa seed dormancy, and (3) evaluated the use of a phenetic modeling approach to explore relationships between seed morphology and seed dormancy. Results: Dormancy screening indicated seed dormancy ranges in quinoa varieties from none to strong dormancy. Further, phenetic modeling approaches indicate that seed coat thickness and eccentricity are important morphological variables that impact quinoa seed dormancy strength. Conclusions: While dormancy screening and phenetic modeling approaches do not provide a direct solution to preventing PHS in quinoa, they do provide new tools for identifying dormant varieties as well as morphological variables contributing to seed dormancy.

Development of Novel Monoclonal Antibodies to Wheat Alpha-Amylases Associated with Grain Quality Problems That Are Increasing with Climate Change.

Accurate, rapid testing platforms are essential for early detection and mitigation of late maturity 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.

GA signaling expands: The plant UBX domain-containing protein 1 is a binding partner for the GA receptor.

The plant Ubiquitin Regulatory X (UBX) domain-containing protein 1 (PUX1) functions as a negative regulator of gibberellin (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.

As the number falls, alternatives to the Hagberg-Perten falling number method: A review.

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 additional 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.

Late-maturity α-amylase (LMA): exploring the underlying mechanisms and end-use quality effects in wheat.

MAIN CONCLUSION: A comprehensive understanding of LMA from the underlying molecular aspects to the end-use quality effects will greatly benefit the global wheat industry and those whose livelihoods depend upon it. Late-maturity α-amylase (LMA) leads to the expression and protein accumulation of high pI α-amylases during late grain development. This α-amylase is maintained through harvest and leads to an unacceptable low falling number (FN), the wheat industry's standard measure for predicting end-use quality. Unfortunately, low FN leads to significant financial losses for growers. As a result, wheat researchers are working to understand and eliminate LMA from wheat breeding programs, with research aims that include unraveling the genetic, biochemical, and physiological mechanisms that lead to LMA expression. In addition, cereal chemists and quality scientists are working to determine if and how LMA-affected grain impacts end-use quality. This review is a comprehensive overview of studies focused on LMA and includes open questions and future directions.

Seed Dormancy and Preharvest Sprouting in Quinoa (Chenopodium quinoa Willd.).

Quinoa (Chenopodium quinoa Willd.) is a culturally significant staple food source that has been grown for thousands of years in South America. Due to its natural drought and salinity tolerance, quinoa has emerged as an agronomically important crop for production in marginal soils, in highly variable climates, and as part of diverse crop rotations. Primary areas of quinoa research have focused on improving resistance to abiotic stresses and disease, improving yields, and increasing nutrition. However, an evolving issue impacting quinoa seed end-use quality is preharvest sprouting (PHS), which is when seeds with little to no dormancy experience a rain event prior to harvest and sprout on the panicle. Far less is understood about the mechanisms that regulate quinoa seed dormancy and seed viability. This review will cover topics including seed dormancy, orthodox and unorthodox dormancy programs, desiccation sensitivity, environmental and hormonal mechanisms that regulate seed dormancy, and breeding and non-breeding strategies for enhancing resistance to PHS in quinoa.

GA signaling is essential for the embryo-to-seedling transition during Arabidopsis seed germination, a ghost story.

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.

Population structure and genetic diversity of Bromus tectorum within the small grain production region of the Pacific Northwest.

Bromus tectorum L. is an invasive winter annual grass naturalized across the United States. Numerous studies have investigated B. tectorum population structure and genetics in the context of B. tectorum as an ecological invader of natural areas and rangeland. Despite the wealth of information regarding B. tectorum, previous studies have not focused on, or made comparisons to, B. tectorum as it persists in individual agroecosystems. The objectives of this study were to assess the genetic diversity and structure, the occurrence of generalist and specialist genotypes, and the influence of climate on distribution of B. tectorum sourced exclusively from within small grain production regions of the Pacific Northwest. Genetic diversity of B. tectorum sourced from agronomic fields was found to be similar to what has been observed from other land use histories. Six distinct genetic clusters of B. tectorum were identified, with no evidence to indicate that any of the genetic clusters were better adapted to a particular geographical area or climate within the region. Given the apparent random spatial distribution of B. tectorum genetic clusters at the spatial scale of this analysis, unique genotypes may be well mixed within region, similar to what was reported for other inbreeding weedy grass species.

Loss of Arabidopsis thaliana Seed Dormancy is Associated with Increased Accumulation of the GID1 GA Hormone Receptors.

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 (GIBBERELLIN-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 probably 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 and the 26S proteasome. 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 roles of the GA receptors GID1a, GID1b, and GID1c in sly1-independent GA signaling.

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 in sly1 mutants where GA cannot target DELLA proteins for destruction, 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.

Lifting della repression of Arabidopsis seed germination by nonproteolytic gibberellin signaling.

DELLA repression of Arabidopsis (Arabidopsis thaliana) seed germination can be lifted either through DELLA proteolysis by the ubiquitin-proteasome pathway or through proteolysis-independent gibberellin (GA) hormone signaling. GA binding to the GIBBERELLIN-INSENSITIVE DWARF1 (GID1) GA receptors stimulates GID1-GA-DELLA complex formation, which in turn triggers DELLA protein ubiquitination and proteolysis via the SCF(SLY1) E3 ubiquitin ligase and 26S proteasome. Although DELLA cannot be destroyed in the sleepy1-2 (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 abscisic acid (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 messenger RNA levels. Higher levels of GID1 overexpression are associated with more efficient sly1 germination and increased GID1-GA-DELLA complex formation, suggesting that GID1 down-regulates 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.