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.

LMA or vivipary? Wheat grain can germinate precociously during grain maturation under the cool conditions used to induce late maturity alpha-amylase (LMA).

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.

Seed dormancy loss from dry after-ripening is associated with increasing gibberellin hormone levels in Arabidopsis thaliana.

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.

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.

Leaf temperature impacts canopy water use efficiency independent of changes in leaf level water use efficiency.

Canopy water use efficiency (above-ground biomass over lifetime water loss, WUEcanopy) can influence yield in wheat and other crops. Breeding for WUEcanopy is difficult because it is influenced by many component traits. For example, intrinsic water use efficiency (WUEi), the ratio of net carbon assimilation (Anet) over stomatal conductance, contributes to WUEcanopy 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 (VPDleaf). Alternatively, measurements of instantaneous leaf water use efficiency (WUEleaf) are defined as Anet 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 VPDleaf that impact transpiration but not Anet. This alters WUEleaf and in turn affects WUEcanopy. To test this relationship, WUEcanopy was measured in conjunction with WUEi, WUEcanopy, 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 WUEcanopy likely because of the effect of canopy temperature on VPDleaf driving T. However, deviations between WUEi, WUEleaf, and WUEcanopy were present because measurements made at the leaf level do not account for variations in leaf temperature. This uncoupled the relationship of measured WUEleaf and WUEi from WUEcanopy and emphasizes the importance of canopy temperature on carbon uptake and transpired water loss.

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.

Exome sequencing of bulked segregants identified a novel TaMKK3-A allele linked to the wheat ERA8 ABA-hypersensitive germination phenotype.

KEY MESSAGE: Using bulked segregant analysis of exome sequence, we fine-mapped the ABA-hypersensitive mutant ERA8 in a wheat backcross population to the TaMKK3-A locus of chromosome 4A. 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-A660S allele as Zak, whereas Louise carries the PHS-tolerant MKK3-A-C660R 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.

Genome-Wide Association Mapping for Tolerance to Preharvest Sprouting and Low Falling Numbers in Wheat.

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.

Positive and negative regulation of seed germination by the Arabidopsis GA hormone receptors, GID1a, b, and c.

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 germinate 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 suggested that there may be some specialization of GID1 gene function during germination because GID1b mRNA expression was more highly induced by after-ripening, whereas GID1a and GID1c mRNA levels were more highly induced by cold stratification. 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 germination. Further experiments showed that GID1b can negatively regulate dark germination. 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.

Transcriptional mechanisms associated with seed dormancy and dormancy loss in the gibberellin-insensitive sly1-2 mutant of Arabidopsis thaliana.

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 overaccumulation 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. Approximately half of the transcriptome changes with after-ripening appear to depend on SLY1-triggered 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.

Biology in the Dry Seed: Transcriptome Changes Associated with Dry Seed Dormancy and Dormancy Loss in the Arabidopsis GA-Insensitive sleepy1-2 Mutant.

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.

Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.).

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 genome-specific 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.

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.

Registration of Zak ERA8 Soft White Spring Wheat Germplasm with Enhanced Response to ABA and Increased Seed Dormancy.

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 ERA8was tested under experimental number 60.1.27.10. The ERA8mutation 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.

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.

Increased ABA sensitivity results in higher seed dormancy in soft white spring wheat cultivar 'Zak'.

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.

Wheat ABA-insensitive mutants result in reduced grain dormancy.

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. ScABI 1, 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 insensi-tivity 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.