GSK3 phosphorylates and regulates the Green Revolution protein Rht-B1b to reduce plant height in wheat.

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.

Variation of TaMyb10 and their function on grain color and pre-harvest sprouting resistance of wheat.

Pre-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.

BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth.

The BRASSINAZOLE-RESISTANT 1 (BZR1) transcription factor family plays an essential role in plant brassinosteroid (BR) signaling, but the signaling mechanism through which BZR1 and its homologs cooperate with certain coactivators to facilitate transcription of target genes remains incompletely understood. In this study, we used an efficient protein interaction screening system to identify blue-light inhibitor of cryptochromes 1 (BIC1) as a new BZR1-interacting protein in Arabidopsis thaliana. We show that BIC1 positively regulates BR signaling and acts as a transcriptional coactivator for BZR1-dependent activation of BR-responsive genes. Simultaneously, BIC1 interacts with the transcription factor PIF4 to synergistically and interdependently activate expression of downstream genes including PIF4 itself, and to promote plant growth. Chromatin immunoprecipitation assays demonstrate that BIC1 and BZR1/PIF4 interdependently associate with the promoters of common target genes. In addition, we show that the interaction between BIC1 and BZR1 is evolutionally conserved in the model monocot plant Triticum aestivum (bread wheat). Together, our results reveal mechanistic details of BR signaling mediated by a transcriptional activation module BIC1/BZR1/PIF4 and thus provide new insights into the molecular mechanisms underlying the integration of BR and light signaling in plants.

Plant-specific BLISTER interacts with kinase BIN2 and BRASSINAZOLE RESISTANT1 during skotomorphogenesis.

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.

The PGS1 basic helix-loop-helix protein regulates Fl3 to impact seed growth and grain yield in cereals.

Plant 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.

Photoexcited phytochrome B interacts with brassinazole resistant 1 to repress brassinosteroid signaling in Arabidopsis.

Photoreceptor phytochrome B (phyB) mediates a variety of light responses in plants. To further elucidate the molecular mechanisms of phyB-regulated hypocotyl elongation, we performed firefly luciferase complementation imaging (LCI) screening for phyB-interacting transcription factors (TFs). LCI assays showed that phyB possibly interacts with brassinazoleresistant 1 (BZR1), BZR2, AUXIN RESPONSE FACTOR 6 (ARF6), and several WRKY DNA-binding TFs in a red light-dependent manner. Furthermore, biochemical assays demonstrated that photoexcited phyB specifically interacts with non-phosphorylated BZR1, the physiologically active form of a master TF in brassinosteroid (BR) signaling, and this interaction can be competitively interfered by phytochrome-interacting factor 4. Furthermore, we showed that phyB can directly interact with the DNA-binding domain of BZR1 and affect the enrichment of BZR1 on the chromatin of target genes. Moreover, our genetic evidence and RNA-seq analysis demonstrated that phyB negatively regulates BR signaling. Together, we revealed that photoexcited phyB directly interacts with the TF BZR1 to repress BR signaling in Arabidopsis.

TaCOLD1 defines a new regulator of plant height in bread wheat.

Plant height is among the most important agronomic traits that influence crop yield. However, in addition to the Rht-1 alleles, the molecular basis of plant height in bread wheat remains largely unclear. Based on wheat gene expression profiling analysis, we identify a light-regulated gene from bread wheat, designated as TaCOLD1, whose encoding protein is homologous to cold sensor COLD1 in rice. We show that TaCOLD1 protein is localized to the endoplasmic reticulum (ER) and plasma membrane. Phenotypic analyses show that overexpression of a mutated form of TaCOLD1 (M187K) in bread wheat cultivar Kenong199 (Rht-B1b) background resulted in an obvious reduction in plant height. Further, we demonstrate that the hydrophilic loop of TaCOLD1 (residues 178-296) can interact with TaGα-7A (the α subunit of heterotrimeric G protein) protein but not TaGα-1B, and the mutation (M187K) in TaCOLD1 remarkably enhances its interaction with TaGα-7A. Physical interaction analyses show that the C-terminal region of TaGα-7A, which is lacking in the TaGα-1B protein, is necessary for its interaction with TaCOLD1. Intriguingly, the C-terminal region of TaGα-7A is also physically associated with the TaDEP1 protein (an atypical Gγ subunit). Significantly, we discover that TaCOLD1 and mTaCOLD1 (M187K) can interfere with the physical association between TaGα-7A and TaDEP1. Together, this study reveals that TaCOLD1 acts as a novel regulator of plant height through interfering with the formation of heterotrimeric G protein complex in bread wheat and is a valuable target for the engineering of wheat plant architecture.

Functional regulation of Q by microRNA172 and transcriptional co-repressor TOPLESS in controlling bread wheat spikelet density.

Bread wheat (Triticum aestivum) spike architecture is an important agronomic trait. The Q gene plays a key role in the domestication of bread wheat spike architecture. However, the regulatory mechanisms of Q expression and transcriptional activity remain largely unknown. In this study, we show that overexpression of bread wheat tae-miR172 caused a speltoid-like spike phenotype, reminiscent of that in wheat plants with the q gene. The reduction in Q transcript levels in the tae-miR172 overexpression transgenic bread wheat lines suggests that the Q expression can be suppressed by tae-miR172 in bread wheat. Indeed, our RACE analyses confirmed that the Q mRNA is targeted by tae-miR172 for cleavage. According to our analyses, the Q protein is localized in nucleus and confers transcriptional repression activity. Meanwhile, the Q protein could physically interact with the bread wheat transcriptional co-repressor TOPLESS (TaTPL). Specifically, the N-terminal ethylene-responsive element binding factor-associated amphiphilic repression (EAR) (LDLNVE) motif but not the C-terminal EAR (LDLDLR) motif of Q protein mediates its interaction with the CTLH motif of TaTPL. Moreover, we show that the N-terminal EAR motif of Q protein is also essentially required for the transcriptional repression activity of Q protein. Taken together, we reveal the functional regulation of Q protein by tae-miR172 and transcriptional co-repressor TaTPL in controlling the bread wheat spike architecture.

The trxG protein ULT1 regulates Arabidopsis organ size by interacting with TCP14/15 to antagonize the LIM peptidase DA1 for H3K4me3 on target genes.

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.

BIN2 phosphorylates the Thr280 of CO to restrict its function in promoting Arabidopsis flowering.

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.

Identification of candidate gene for the defective kernel phenotype using bulked segregant RNA and exome capture sequencing methods 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.

Wheat male-sterile 2 reduces ROS levels to inhibit anther development by deactivating ROS modulator 1.

Ms2 is an important dominant male-sterile gene in wheat, but the biochemical function of Ms2 and the mechanism by which it causes male sterility remain elusive. Here, we report the molecular basis underlying Ms2-induced male sterility in wheat. We found that activated Ms2 specifically reduces the reactive oxygen species (ROS) signals in anthers and thereby induces termination of wheat anther development at an early stage. Furthermore, our results indicate that Ms2 is localized in mitochondria, where it physically interacts with a wheat homolog of ROS modulator 1 (TaRomo1). Romo1 positively regulates the ROS levels in humans but has never been studied in plants. We found that single amino acid substitutions in the Ms2 protein that rescue the ms2 male-sterile phenotype abolish the interaction between Ms2 and TaRomo1. Significantly, Ms2 promotes the transition of TaRomo1 proteins from active monomers to inactive oligomers. Taken together, our findings unravel the molecular basis of Ms2-induced male sterility and reveal a regulatory mechanism in which ROS act as essential signals guiding the anther development program in wheat.

MIR156-Targeted SPL9 Is Phosphorylated by SnRK2s and Interacts With ABI5 to Enhance ABA Responses in Arabidopsis.

The miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors play key roles in regulating plant development, but little is known about their function in abscisic acid (ABA) signaling. Here, we report that the miR156-targeted SPLs enhance ABA responses and contribute to the inhibition of pre-harvest sprouting. We find that SPL9 directly activates the expression of ABA responsive genes through binding to their promoters. SPL9 was further shown to physically interact with ABSCISIC ACID INSENSITIVE 5 (ABI5), a master transcription factor in ABA signaling, thus promoting its association with the promoters of ABA responsive genes. Furthermore, we reveal that the protein kinases SnRK2s interact with and phosphorylate SPL9, which is essential for its role in the activation of ABA responses. Together, our results disclose a SnRK2s-SPLs-ABI5 regulatory module in ABA signaling in Arabidopsis.

The blue light receptor CRY1 interacts with GID1 and DELLA proteins to repress gibberellin signaling and plant growth.

Improvements in plant architecture, such as reduced plant height under high-density planting, are important for agricultural production. Light and gibberellin (GA) are essential external and internal cues that affect plant architecture. In this study, we characterize the direct interaction of distinct receptors that link light and GA signaling in Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum L.). We show that the light receptor CRY1 represses GA signaling through interaction with all five DELLA proteins and promotion of RGA protein accumulation in Arabidopsis. Genetic analysis shows that CRY1-mediated growth repression is achieved by means of the DELLA proteins. Interestingly, we find that CRY1 also directly interacts with the GA receptor GID1 to competitively inhibit the GID1-GAI interaction. We also show that overexpression of TaCRY1a reduces plant height and coleoptile growth in wheat and that TaCRY1a interacts with both TaGID1 and Rht1 to competitively attenuate the TaGID1-Rht1 interaction. Based on these findings, we propose that the photoreceptor CRY1 competitively inhibits the GID1-DELLA interaction, thereby stabilizing DELLA proteins and enhancing their repression of plant growth.

The Blue-Light Receptor CRY1 Interacts with BZR1 and BIN2 to Modulate the Phosphorylation and Nuclear Function of BZR1 in Repressing BR Signaling in Arabidopsis.

The blue-light receptor cryptochrome 1 (CRY1) primarily mediates blue-light inhibition of hypocotyl elongation in Arabidopsis. However, the underlying mechanisms remain largely elusive. We report here that CRY1 inhibits hypocotyl elongation by repressing brassinosteroid (BR) signaling. A genetic interaction assay reveals the negative regulatory effect of CRY1 on the function of BZR1, a core transcription factor in the BR signaling pathway. We demonstrated that CRY1 interacts with the DNA-binding domain of BZR1 to interfere with the DNA-binding ability of BZR1, and represses its transcriptional activity. Furthermore, we found that CRY1 promotes the phosphorylation of BZR1 and inhibits the nuclear accumulation of BZR1. Interestingly, we discovered that CRY1 interacts with the GSK3-like kinase BIN2 and enhances the interaction of BIN2 and BZR1 in a light-dependent manner. Our findings revealed that CRY1 negatively regulates the function of BZR1 through at least two mechanisms: interfering with the DNA-binding ability of BZR1 and promoting the phosphorylation of BZR1. Therefore, we uncover a novel CRY1-BIN2-BZR1 regulatory module that mediates crosstalk between blue light and BR signaling to coordinate plant growth in Arabidopsis.