Flavonoids contribute to seed development, and flavonols can modulate polar auxin transport. We previously showed that overexpressing the wheat bHLH factor TaPGS1 increases grain size. Here, the metabolomic and transcriptomic profiling of TaPGS1 overexpression lines revealed enrichment of flavonol-related metabolites, most notably kaempferol, together with coordinated increases in flavonoid-pathway gene expression, including FLS and upstream nodes. Auxin localization indicated local auxin buildup in developing grains, and exogenous kaempferol reproduced a delay in endosperm cellularization. Promoter-reporter assays showed that TaPGS1 activates representative flavonoid-pathway promoters. These findings support a model in which TaPGS1 enhances the flavonoid pathway and is associated with altered auxin distribution, leading to delayed cellularization and increased endosperm cell number, which together contribute to grain enlargement in wheat.

GLYCOGEN SYNTHASE KINASE 3 (GSK3), a negative regulator of brassinosteroid signaling, phosphorylates and stabilizes the seed dormancy protein DOG1L4, which in turn activates ABSCISIC ACID INSENSITIVE 5 (ABI5) to enhance seed dormancy. GSK3 also phosphorylates ABI5. This dual-target mechanism suggests a strategy for controlling seed dormancy and germination in crops.

Preharvest sprouting (PHS) is the germination of mature grains on the mother plant before harvest, which negatively impacts both the yield and baking/brewing quality of wheat (Triticum aestivum). PHS resistance is a complex quantitative trait influenced by numerous genes. Although gene pyramiding has effectively increased resistance, the combined effects of multiple genes have not been systematically analyzed. We shortlisted 35 germination-related genes through homology searches and transcript profiling, then mined 16 public wheat genomes to locate functional single nucleotide polymorphisms and insertion/deletions. Kompetitive allele-specific PCR assays were designed for 69 non-synonymous candidate variants, and 11 reliably genotyped a panel of 113 cultivars and landraces. Four known functional markers were also verified, giving a 15-marker set. Every assay was significantly associated with germination percentage (%G, p < 0.05), and favorable alleles acted additively: varieties carrying 10 or more resistant alleles germinated at less than 20%. The core haplotype TaOCO1_D_121 / TaPHS1_3A_222 (T:C) was especially stable, with mean %G falling from below 50% in F4 progeny to below 20% in F6. Key three-locus combinations including TaOCO1_D_121/TaDFR_B_-282/TaMKK3_A_660 (T:C:G) and TaOCO1_D_121/ TaDFR_B_282 /TaDOG1_A_740 (T:C:C) reduced %G by up to 80% relative to the population mean. Using extreme-phenotype subsets, the 20 lowest %G and 20 highest %G lines per population from two biparental recombinant inbred line populations (Huaqixiaomai & times; Yanfeng 168 and Huaqixiaomai & times; Chuanmai 23), we found that the tested marker combinations identify low-germination lines across genetic backgrounds. These results clarify the genetic architecture of PHS resistance and provide a practical marker toolkit for pyramiding alleles in wheat breeding.

Tryptophan decarboxylase (TDC) plays an important role in plant hormonal balance and secondary metabolite synthesis by catalyzing the conversion of tryptophan to tryptamine. Prior research has indicated that AevTDC from Aegilops variabilis, a relative of wheat (Triticum aestivum L.), enhances wheat resistance to the pathogen cereal cyst nematode by influencing salicylic acid and flavonoid pathways. Expanding on these findings, this study demonstrates that AevTDC promotes both serotonin and flavonoid synthesis, resulting in altered wheat grain color. Furthermore, we observed that AevTDC overexpression induces the expression of TaMYB310, which is closely associated with flavonoid biosynthesis. TaMYB310 activates the expression of CHS (encoding chalcone synthase) and FLS (encoding flavonol synthase) by directly binding to the MYB binding site (MBS), thereby promoting flavonoid biosynthesis. Additionally, overexpression of AevTDC reduced wheat seed sensitivity to abscisic acid (ABA), providing an explanation for the accelerated seed germination. In summary, this study reveals the critical role of AevTDC in regulating wheat metabolism, emphasizing its importance in promoting serotonin and flavonoid synthesis, altering grain color, and accelerating seed germination. These findings offer insights into plant metabolic regulation mechanisms and their applications in agricultural improvement.

Starch biosynthesis is a complex process that relies on the coordinated action of multiple enzymes. Resistant starch is not digested in the small intestine, thus preventing a rapid rise in the glycemic index. Starch synthase 2a(SS2a) is a key enzyme in amylopectin biosynthesis that has significant effects on starch structure and properties. In this study, we identified an ss2a null mutant(M3-1413) with a single base mutation from an ethyl methane sulfonate(EMS)-mutagenized population of barley. The mutation was located at the 3' end of the first intron of the RNA splicing receptor(AG) site, and resulted in abnormal RNA splicing and two abnormal transcripts of ss2a, which caused the inactivation of the SS2a gene. The starch structure and properties were significantly altered in the mutant, with M3-1413 containing lower total starch and higher amylose and resistant starch levels. This study sheds light on the effect of barley ss2a null mutations on starch properties and will help to guide new applications of barley starch in the development of nutritious food products.

Flavonols play a crucial role in seed development by regulating multiple physiological processes, including seed coat pigmentation, dormancy, fertilization, and endosperm formation. Notably, flavonols influence the polar transport of auxin, thereby affecting seed growth dynamics. In our previous work, we found that overexpression of the wheat bHLH transcription factor TaPGS1 results in increased grain size; however, the underlying mechanism remained unclear. In this study, metabolomic and transcriptomic analyses of TaPGS1 overexpressing wheat lines revealed enhanced flavonol accumulation and upregulation of key flavonol biosynthetic genes. Further investigations suggested that flavonol accumulation in the seed coat may disrupt auxin transport, leading to localized auxin buildup, delayed endosperm cellularization, and an increase in endosperm cell number. These changes collectively contribute to grain enlargement. Our findings uncover a TaPGS1 flavonol regulatory module that links auxin distribution to endosperm development and seed size control in wheat. ### Competing Interest Statement The authors have declared no competing interest.

Plant organ size is an important agronomic trait that greatly determines plant yield. However, despite its central importance, the genetic and molecular mechanisms underlying organ size control remain elusive. Here, we report the trithorax group protein ULTRAPETALA1 (ULT1) interacts with TCP14/15 transcription factors (TFs) by antagonizing the LIM-peptidase DA1 to regulate organ size in Arabidopsis. Loss of ULT1 function significantly increases rosette leaf, petal, silique and seed size, while overexpression of ULT1 results in reduced organ size. ULT1 associates with TCP14 and TCP15 to co-regulate cell size by affecting cellular endoreduplication. Transcriptome analysis reveals that ULT1 and TCP14/15 regulate common target genes involved in endoreduplication and leaf development. ULT1 can be recruited by TCP14/15 to promote H3K4 trimethylation (H3K4me3) at target genes, thereby activating their expression to determine final cell sizes. Furthermore, we found that ULT1 influences the interaction of DA1 and TCP14/15 and antagonizes the effect of DA1’s degradation on TCP14/TCP15. Collectively, our findings uncover a novel epigenetic mechanism underlying the regulation of organ size in Arabidopsis.

SUMMARYPre‐harvest sprouting (PHS) is a significant threat to global food security due to its association with losses in both yield and quality. Among the genes involved in PHS resistance in wheat, PHS‐3D (TaMyb10‐D) plays a crucial role. Here, we characterized the sequence variations of TaMyb10 genes in 416 bread wheat and 302 Aegilops tauschii accessions. Within TaMyb10‐A sequences, we identified a deletion ranging from 214 to 305 bp in the signal and amino acid coding region, present in 61.3% of the accessions. Similarly, 79.3% of the TaMyb10‐B sequences within the third exon region exhibited a 19 bp deletion. Additionally, 40.8% of the accessions lacked the 2.4 Mb fragment (in/del mutations) on Chr3D, where TaMyb10‐D/PHS‐3D was located. Interestingly, the geographical distribution of accessions showed little correlation with the divergence of TaMyb10. TaMyb10‐A‐IIIDele, TaMyb10‐B‐IVDele, and TaMyb10‐D‐VDele genotypes were prevalent in wheat populations across continents. Despite their structural variations, the five distinct protein types exhibited comparable ability to bind the promoters of downstream genes in the flavonoid and ABA pathways, such as CHS, DFR, and NCED. Furthermore, the combination of TaMyb10 homologs was significantly associated with grain color and germination percentages. Accessions exclusively harboring TaMyb10‐D displayed red seed color and reduced germination percentages, indicating the predominant role of TaMyb10‐D compared to TaMyb10‐A and TaMyb10‐B. This comprehensive investigation enhances our understanding of the structural variations and functional divergence of TaMyb10, providing valuable insights and resources for improving PHS resistance in wheat.

Wheat domestication and subsequent genetic improvement have yielded cultivated species with larger seeds compared to wild ancestors. Increasing thousand kernel weight (TKW) remains a crucial goal in many wheat breeding programs. To identify genomic regions influencing TKW across diverse genetic populations, we performed a comprehensive meta-analysis of quantitative trait loci (MQTL), integrating 993 initial QTL from 120 independent mapping studies over recent decades. We refined 242 loci into 66 MQTL, with an average confidence interval (CI) 3.06 times smaller than that of the original QTL. In these 66 MQTL regions, a total of 4,913 candidate genes related to TKW were identified, involved in ubiquitination, phytohormones, G-proteins, photosynthesis, and microRNAs. Expression analysis of the candidate genes showed that 95 were specific to grain and might potentially affect TKW at different seed development stages. These findings enhance our understanding of the genetic factors associated with TKW in wheat, providing reliable MQTL and potential candidate genes for genetic improvement of this trait.

Chuanmai104 (CM104), an elite wheat (Triticum aestivum L.) variety that currently produces the highest yield per unit area in southwestern China, plays a critical role in wheat production. The high quality and stability of grain traits are important factors that ensure the high, stable yields of CM104 in different production areas. In this study, six grain traits of CM104 sampled from 19 environments in five provinces of China during 2018–2022 were evaluated. The traits comprised thousand-kernel weight, grain length, grain width, grain length–width ratio, grain circumference, and grain surface area. Fifteen quantitative trait loci (QTLs) associated with the grain traits were identified based on a recombinant inbred lines (F9–10) population derived from the cross between CM104 and the landrace Baimaomai (BMM), nine and six QTLs derived from CM104 and BMM, respectively. Three mainly pleiotropic QTLs derived from CM104, namely QTL10 (grain circumference, grain surface area), QTL11 (grain length, grain circumference), and QTL12 (grain length, grain circumference), were expressed significantly and stably in multiple environments, and explained 3.34–5.06

The grain-filling stage is highly sensitive to heat, impacting wheat yield and quality. This study uncovered Chuanmai104's grain spatial transcriptome atlas under heat stress, identifying over 120 specifically expressed genes crucial for cell sorting and subsequent spatial sequencing across twelve cell types. The cell differentiation trajectory of endosperm indicated high expression of genes for cell development and programmed cell death at early stages, and the subsequent expressed genes enable grains accumulate nutrients. At 15 DAP, recovery grains under heat stress showed significant gene expression changes, especially in the nucellar projection, endosperm near the scutellum, and embryo. Heat stress reduced starch and ABA synthesis gene expression, shortening grain-filling time, reducing grain weight by 28.9%, and raising germination rates from 7.9% to 52.0%. Spatial transcriptomics revealed unexpected shifts in the major expression domains for 189 genes due to heat stress, featuring three major spatial shifts: AL-SCU, ENC-PR, and EXC-NP.

In plants, the basic leucine zipper (bZIP) family of transcription factors is known for its large size and diversity. Many studies have shown that bZIP transcription factors play an indispensable role in the growth and development of plants; however, there are few reports about the regulation of starch content in grain. To understand the genetic members of the bZIP family, using newly available wheat genome data, we compared our identification of 181 Triticum aestivum bZIP ( TabZIP ) genes to those reported in earlier studies. Some duplicate genes and incorrect annotations in previous studies were supplemented and corrected. Through phylogenetic analysis, transcriptome data, quantitative reverse transcription PCR (qRT-PCR), a dual-luciferase reporter (DLR), and subcellular localization analysis were used to identify transcription factors that may be involved in grain starch synthesis. We divided genes into 13 known groups and five unknown groups by phylogenetic analysis. All of the bZIP genes exhibited a minimum of one bZIP motif in their motif distribution and gene structure. Spatial and temporal expression patterns of bZIP family members during various stages of plant growth vary, as suggested by transcriptome data, and several genes were specifically expressed during grain development. As per the expression data obtained via qRT-PCR, over 10 TabZIP genes showed similarity with starch synthesis in wheat. The in-vitro binding activity of TabZIP68 to the promoter of TaWaxy was demonstrated by a DLR assay. Expression level of TabZIP68 was affected by different plant hormones treated with developing grains. Given its potential involvement in starch synthesis, the TabZIP68 gene presents itself as a strong candidate for further investigation.

Although elevated ambient temperature causes many effects on plant growth and development, the mechanisms of plant high-ambient temperature sensing remain unknown. In this study, we show that GLYCOGEN SYNTHASE KINASE 3s (GSK3s) negatively regulate high-ambient temperature response and oligomerize upon high-temperature treatment. We demonstrate that GSK3 kinase BIN2 specifically interacts with the high-temperature sensor phytochrome B (phyB) but not the high-temperature sensor EARLY FLOWER 3 (ELF3) to phosphorylate and promote phyB photobody formation. Furthermore, we show that phosphorylation of phyB by GSK3s promotes its interaction with ELF3. Subsequently, we find that ELF3 recruits the phyB photobody facilitator HEMERA (HMR) to promote its association with phyB. Taken together, our data reveal a mechanism that GSK3s promote the phyB-ELF3-HMR complex formation in regulating plant thermomorphogenesis.

Plant organ size is an important agronomic trait that makes a significant contribution to plant yield. Despite its central importance, the genetic and molecular mechanisms underlying organ size control remain to be fully clarified. Here, we report that the trithorax group protein ULTRAPETALA1 (ULT1) interacts with the TEOSINTE BRANCHED1/CYCLOIDEA/PCF14/15 (TCP14/15) transcription factors by antagonizing the LIN-11, ISL-1, and MEC-3 (LIM) peptidase DA1, thereby regulating organ size in Arabidopsis. Loss of ULT1 function significantly increases rosette leaf, petal, silique, and seed size, whereas overexpression of ULT1 results in reduced organ size. ULT1 associates with TCP14 and TCP15 to co-regulate cell size by affecting cellular endoreduplication. Transcriptome analysis revealed that ULT1 and TCP14/15 regulate common target genes involved in endoreduplication and leaf development. ULT1 can be recruited by TCP14/15 to promote lysine 4 of histone H3 trimethylation at target genes, activating their expression to determine final cell size. Furthermore, we found that ULT1 influences the interaction of DA1 and TCP14/15 and antagonizes the effect of DA1 on TCP14/15 degradation. Collectively, our findings reveal a novel epigenetic mechanism underlying the regulation of organ size in Arabidopsis.

CONSTANS (CO) is a central regulator of floral initiation in response to photoperiod. In this study, we show that the GSK3 kinase BIN2 physically interacts with CO and the gain-of-function mutant bin2-1 displays late flowering phenotype through down-regulation of FT transcription. Genetic analyses show that BIN2 genetically acts upstream of CO in regulating flowering time. Further, we illustrate that BIN2 phosphorylates the Thr280 residue of CO. Importantly, the BIN2 phosphorylation of Thr280 residue restricts the function of CO in promoting flowering through affecting its DNA-binding activity. Moreover, we reveal that the N-terminal part of CO harboring the B-Box domain mediates the interaction of both CO-CO and BIN2-CO. We find that BIN2 inhibits the formation of CO dimer/oligomer. Taken together, this study reveals that BIN2 regulates flowering time through phosphorylating the Thr280 of CO and inhibiting the CO-CO interaction in Arabidopsis.

The utilization of stabilized DELLA proteins Rht-B1b and Rht-D1b was crucial for increasing wheat (Triticum aestivum) productivity during the Green Revolution. However, the underlying mechanisms remain to be clarified. Here, we cloned a gain-of-function allele of the GSK3/SHAGGY-like kinase-encoding gene GSK3 by characterizing a dwarf wheat mutant. Furthermore, we determined that GSK3 interacts with and phosphorylates the Green Revolution protein Rht-B1b to promote it to reduce plant height in wheat. Specifically, phosphorylation by GSK3 may enhance the activity and stability of Rht-B1b, allowing it to inhibit the activities of its target transcription factors. Taken together, we reveal a positive regulatory mechanism for the Green Revolution protein Rht-B1b by GSK3, which might have contributed to the Green Revolution in wheat.

Wheat is a significant source of protein and starch worldwide. The defective kernel (Dek) mutant AK-3537, displaying a large hollow area in the endosperm and shrunken grain, was obtained through ethyl methane sulfonate (EMS) treatment of the wheat cultivar Aikang 58 (AK58). The mode of inheritance of the AK-3537 grain Dek phenotype was determined to be recessive with a specific statistical significance level. We used bulked segregant RNA-seq (BSR-seq), BSA-based exome capture sequencing (BSE-seq), and the ΔSNP-index algorithm to identify candidate regions for the grain Dek phenotype. Two major candidate regions, DCR1 (Dek candidate region 1) and DCR2, were identified on chromosome 7A between 279.98 and 287.93 Mb and 565.34 and 568.59 Mb, respectively. Based on transcriptome analysis and previous reports, we designed KASP genotyping assays based on SNP variations in the candidate regions and speculated that the candidate gene is TraesCS7A03G0625900 (HMGS-7A), which encodes a 3-hydroxy-3-methylglutaryl-CoA synthase. One SNP variation located at position 1,049 in the coding sequence (G>A) causes an amino acid change from Gly to Asp. The research suggests that functional changes in HMGS-7A may affect the expression of key enzyme genes involved in wheat starch syntheses, such as GBSSII and SSIIIa.

Brassinosteroids play an essential role in promoting skotomorphogenesis, yet the underlying mechanisms remain unknown. Here we report that a plant-specific BLISTER (BLI) protein functions as a positive regulator of both BR signaling and skotomorphogenesis in Arabidopsis (Arabidopsis thaliana). We found that the glycogen synthase kinase 3 (GSK3)-like kinase BRASSINOSTEROID INSENSITIVE2 interacts with and phosphorylates BLI at 4 phosphorylation sites (Ser70, Ser146, Thr256, and Ser267) for degradation; in turn, BR inhibits degradation of BLI. Specifically, BLI cooperates with the BRASSINAZOLE RESISTANT1 (BZR1) transcription factor to facilitate the transcriptional activation of BR-responsive genes. Genetic analyses indicated that BLI is essentially required for BZR1-mediated hypocotyl elongation in the dark. Intriguingly, we reveal that BLI and BZR1 orchestrate the transcriptional expression of gibberellin (GA) biosynthetic genes to promote the production of bioactive GAs. Our results demonstrate that BLI acts as an essential regulator of Arabidopsis skotomorphogenesis by promoting BR signaling and GA biosynthesis.

Promoting seed germination after short episodes of heat stress during the wheat grain filling stage is a serious problem that results in pre-harvest sprouting. The plant hormones abscisic acid (ABA), gibberellins (GAs), and ethylene (ETH) are well known to be involved in germination control. However, the genes associated with the metabolism and responsiveness of these hormones to heat stress during wheat grain filling are not well understood. Transcriptomic analysis was carried out to explore the mechanisms controlling seed germination under five days (15-20 days after flowering, DAF) of heat stress (20, 24, 28, and 32 degrees C) in wheat grains at 15-30 DAF using comparative RNA sequencing. A dataset of 2073 differentially regulated genes was used to help elucidate the molecular mechanisms that respond to heat stress and affect seed germination in wheat. Some genes related to ABA, GA, and ETH biosynthesis, transport, and signaling had significantly different expression levels under heat stress. Among these genes, the transcriptional alterations of plant hormone-related genes, such as NCED9, AAO3, CYP707A2, GA20ox, and SAM1 uncovered here, provide a foundation for identifying key players involved in determining seed dormancy and germination. The expression levels of many germination-related genes did not linearly increase with increasing temperature. In this study, 28 degrees C is a threshold of temperature tolerance during the grain filling stage. Heat stress, especially extremely high temperature (>28 degrees C), represses ABA-related gene expression and promotes seed germination.

SummaryPlant transcription factors (TFs), such as basic helix‐loop‐helix (bHLH) and AT‐rich zinc‐binding proteins (PLATZ), play critical roles in regulating the expression of developmental genes in cereals. We identified the bHLH protein TaPGS1 (T. aestivum Positive Regulator of Grain Size 1) specifically expressed in the seeds at 5–20 days post‐anthesis in wheat. TaPGS1 was ectopically overexpressed (OE) in wheat and rice, leading to increased grain weight (up to 13.81% in wheat and 18.55% in rice lines) and grain size. Carbohydrate and total protein levels also increased. Scanning electron microscopy results indicated that the starch granules in the endosperm of TaPGS1 OE wheat and rice lines were smaller and tightly embedded in a proteinaceous matrix. Furthermore, TaPGS1 was bound directly to the E‐box motif at the promoter of the PLATZ TF genes TaFl3 and OsFl3 and positively regulated their expression in wheat and rice. In rice, the OsFl3 CRISPR/Cas9 knockout lines showed reduced average thousand‐grain weight, grain width, and grain length in rice. Our results reveal that TaPGS1 functions as a valuable trait‐associated gene for improving cereal grain yield.