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In Arabidopsis thaliana, proanthocyanidins (PAs) accumulate in the innermost cell layer of the seed coat (i.e. endothelium, chalaza and micropyle). The expression of the biosynthetic genes involved relies on the transcriptional activity of R2R3‐MYB and basic helix‐loop‐helix (bHLH) proteins which form ternary complexes (‘MBW’) with TRANSPARENT TESTA GLABRA1 (TTG1) (WD repeat protein). The identification of the direct targets and the determination of the nature and spatio‐temporal activity of these MBW complexes are essential steps towards a comprehensive understanding of the transcriptional mechanisms that control flavonoid biosynthesis. In this study, various molecular, genetic and biochemical approaches were used. Here, we have demonstrated that, of the 12 studied genes of the pathway, only dihydroflavonol‐4‐reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), BANYULS (BAN), TRANSPARENT TESTA 19 (TT19), TT12 and H⁺‐ATPase isoform 10 (AHA10) are direct targets of the MBW complexes. Interestingly, although the TT2–TT8–TTG1 complex plays the major role in developing seeds, three additional MBW complexes (i.e. MYB5–TT8–TTG1, TT2–EGL3–TTG1 and TT2–GL3–TTG1) were also shown to be involved, in a tissue‐specific manner. Finally, a minimal promoter was identified for each of the target genes of the MBW complexes. Altogether, by answering fundamental questions and by demonstrating or invalidating previously made hypotheses, this study provides a new and comprehensive view of the transcriptional regulatory mechanisms controlling PA and anthocyanin biosynthesis in Arabidopsis.

• Nonphotochemical quenching (NPQ), an intricate photoprotective process, plays fundamental roles in maintaining plant fitness. The PsbS protein is essential for the rapid induction of NPQ, and acts in a dose-dependent manner in leaves. However, little information is known on the transcriptional control of PsbS in land plants. • Here we demonstrated that the expression of OsPsbS1 is directly upregulated by OsbZIP72 while repressed by OsMYBS2 in rice. We identified a new cis-element GACAGGTG in japonica OsPsbS1 promoter, to which OsbZIP72 could strongly bind and activate the expression of OsPsbS1. The new cis-element CTAATC confers specific binding for OsMYBS2 in japonica OsPsbS1 promoter. • OsbZIP72 can be activated by SAPK1, and acts depending on the abscisic acid (ABA) signalling pathway. GF14A protein affects the repression activity of OsMYBS2 by regulating its nucleocytoplasmic shuttling, and Ser53 is necessary for OsMYBS2 to be retained in the cytoplasm. The inducibility of OsPsbS1 transcription under high light conditions in OsbZIP72 knockout lines was greatly impaired, while the repression of OsPsbS1 transcription under a low light environment in OsMYBS2 knockout lines was significantly alleviated. • These results reveal cross-talk among NPQ processes, the ABA signalling pathway and abiotic stress signalling. The elaborate mechanisms may help enhance photoprotection and improve photosynthesis in rice.

Summary The GT‐1 cis‐element widely exists in many plant gene promoters. However, the molecular mechanism that underlies the response of the GT‐1 cis‐element to abiotic and biotic stresses remains elusive in rice. We previously isolated a rice short‐chain peptide‐encoding gene, Os2H16, and demonstrated that it plays important roles in both disease resistance and drought tolerance. Here, we conducted a promoter assay of Os2H16 and identified GT‐1 as an important cis‐element that mediates Os2H16 expression in response to pathogen attack and osmotic stress. Using the repeated GT‐1 as bait, we characterized an abscisic acid, stress and ripening 2 (ASR2) protein from yeast‐one hybridization screening. Sequence alignments showed that the carboxy‐terminal domain of OsASR2 containing residues 80–138 was the DNA‐binding domain. Furthermore, we identified that OsASR2 was specifically bound to GT‐1 and activated the expression of the target gene Os2H16, as well as GFP driven by the chimeric promoter of 2 × GT‐1‐35S mini construct. Additionally, the expression of OsASR2 was elevated by pathogens and osmotic stress challenges. Overexpression of OsASR2 enhanced the resistance against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani, and tolerance to drought in rice. These results suggest that the interaction between OsASR2 and GT‐1 plays an important role in the crosstalk of the response of rice to biotic and abiotic stresses.

Summary Advancement of DNA‐synthesis technologies has greatly facilitated the development of synthetic biology tools. However, high‐complexity DNA sequences containing tandems of short repeats are still notoriously difficult to produce synthetically, with commercial DNA synthesis companies usually rejecting orders that exceed specific sequence complexity thresholds. To overcome this limitation, we developed a simple, single‐tube reaction method that enables the generation of DNA sequences containing multiple repetitive elements. Our strategy involves commercial synthesis and PCR amplification of padded sequences that contain the repeats of interest, along with random intervening sequence stuffers that include type IIS restriction enzyme sites. GoldenBraid molecular cloning technology is then employed to remove the stuffers, rejoin the repeats together in a predefined order, and subclone the tandem(s) in a vector using a single‐tube digestion–ligation reaction. In our hands, this new approach is much simpler, more versatile and efficient than previously developed solutions to this problem. As a proof of concept, two different phytohormone‐responsive, synthetic, repetitive proximal promoters were generated and tested in planta in the context of transcriptional reporters. Analysis of transgenic lines carrying the synthetic ethylene‐responsive promoter 10x2EBS‐S10 fused to the GUS reporter gene uncovered several developmentally regulated ethylene response maxima, indicating the utility of this reporter for monitoring the involvement of ethylene in a variety of physiologically relevant processes. These encouraging results suggest that this reporter system can be leveraged to investigate the ethylene response to biotic and abiotic factors with high spatial and temporal resolution. A simple method to build repetitive DNA fragments that are not amenable to direct commercial DNA synthesis was developed and employed to generate a new synthetic ethylene‐responsive promoter, 10x2EBS‐S10.

MADS-box genes are major domestication genes involved in a plethora of developmental processes. They might be ideally suited for future targeted breeding efforts. Abstract MADS-box genes are key regulators of virtually every aspect of plant reproductive development. They play especially prominent roles in flowering time control, inflorescence architecture, floral organ identity determination, and seed development. The developmental and evolutionary importance of MADS-box genes is widely acknowledged. However, their role during flowering plant domestication is less well recognized. Here, we provide an overview illustrating that MADS-box genes have been important targets of selection during crop domestication and improvement. Numerous examples from a diversity of crop plants show that various developmental processes have been shaped by allelic variations in MADS-box genes. We propose that new genomic and genome editing resources provide an excellent starting point for further harnessing the potential of MADS-box genes to improve a variety of reproductive traits in crops. We also suggest that the biophysics of MADS-domain protein-protein and protein-DNA interactions, which is becoming increasingly well characterized, makes them especially suited to exploit coding sequence variations for targeted breeding approaches.

Transcription factors (TFs) regulate gene expression through binding to cis- regulatory specific sequences in the promoters of their target genes. In contrast to the genetic code, the transcriptional regulatory code is far from being deciphered and is determined by sequence specificity of TFs, combinatorial cooperation between TFs and chromatin competence. Here we addressed one of these determinants by characterizing the target sequence specificity of 63 plant TFs representing 25 families, using protein-binding microarrays. Remarkably, almost half of these TFs recognized secondary motifs, which in some cases were completely unrelated to the primary element. Analyses of coregulated genes and transcriptomic data from TFs mutants showed the functional significance of over 80% of all identified sequences and of at least one target sequence per TF. Moreover, combining the target sequence information with coexpression analysis we could predict the function of a TF as activator or repressor through a particular DNA sequence. Our data support the correlation between cis- regulatory elements and the sequence determined in vitro using the protein-binding microarray and provides a framework to explore regulatory networks in plants.

Grain weight is a major determinant of grain yield. GS5 is a positive regulator of grain size such that grain width, filling, and weight are correlated with its expression level. Previous work suggested that polymorphisms of GS5 in the promoter region might be responsible for the variation in grain size. In this study, two single nucleotide polymorphisms (SNPs) between the wide-grain allele GS5-1 and the narrow-grain allele GS5-2 in the upstream region of the gene that were responsible for the differential expression in developing young panicles were identified. These two polymorphs altered the responses of the GS5 alleles to abscisic acid (ABA) treatments, resulting in higher expression of GS5-1 than of GS5-2 in developing young panicles. It was also shown that SNPs in light-responsive elements of the promoter altered the response to light induction, leading to higher expression of GS5-2 than GS5-1 in leaves. Enhanced expression of GS5 competitively inhibits the interaction between OsBAK1-7 and OsMSBP1 by occupying the extracellular leucine-rich repeat (LRR) domain of OsBAK1-7, thus preventing OsBAK1-7 from endocytosis caused by interacting with OsMSBP1, providing an explanation for the positive association between grain size and GS5 expression. These results advanced our understanding of the molecular mechanism by which GS5 controls grain size.

The WRKY transcription factor (TF) gene family expanded greatly in the evolutionary process from green algae to flowering plants through whole genome, segmental, and tandem duplications. Genomic sequences from diverse plant species provide valuable information about the origin and evolution of WRKY domain-containing proteins. Accumulating data indicate that WRKY TFs bind W-box and/or other cis -elements, revealing the specificity of cis -element recognition based on the structure of the WRKY protein as well as the nucleotides adjacent to the core sequences. Physical interactions among WRKY proteins or between WRKY proteins and other regulatory factors afford important insights into the regulation of this TF family. WRKY proteins are essential players in the kinase signaling network. The interaction of plant resistance (R) proteins with WRKY TFs and the existence of unusual chimeric R-WRKY proteins suggest diversity in signaling pathways for rapid immune responses. In this review, we focus mainly on progress in understanding the function of WRKY TFs in response to biotic stresses and focus on their multiple roles in Arabidopsis and rice plants.

Mammalian sex chromosomes are highly conserved, and sex is determined by SRY on the Y chromosome. Two exceptional rodent groups in which some species lack a Y chromosome and Sry offer insights into how novel sex genes can arise and replace Sry, leading to sex chromosome turnover. However, intensive study over three decades has failed to reveal the identity of novel sex genes in either of these lineages. We here report our discovery of a male-specific duplication of an enhancer of Sox9 in the Amami spiny rat Tokudaia osimensis, in which males and females have only a single X chromosome (XO/XO) and the Y chromosome and Sry are completely lost. We performed a comprehensive survey to detect sex-specific genomic regions in the spiny rat. Sex-related genomic differences were limited to a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on an autosome. Hi-C analysis using male spiny rat cells showed the duplicated region has potential chromatin interaction with Sox9. The duplicated unit harbored a 1,262-bp element homologous to mouse enhancer 14 (Enh14), a candidate Sox9 enhancer that is functionally redundant in mice. Transgenic reporter mice showed that the spiny rat Enh14 can function as an embryonic testis enhancer in mice. Embryonic gonads of XX mice in which Enh14 was replaced by the duplicated spiny rat Enh14 showed increased Sox9 expression and decreased Foxl2 expression. We propose that male-specific duplication of this Sox9 enhancer substituted for Sry function, defining a novel Y chromosome in the spiny rat.