Class II knotted-like homeodomain protein SlKN5 with BEL1-like homeodomain proteins suppresses fruit greening in tomato fruit.

Knotted1-like homeodomain (KNOX) proteins are essential in regulating plant organ differentiation. Land plants, including tomato (Solanum lycopersicum), have two classes of the KNOX protein family, namely, class I (KNOX I) and class II KNOX (KNOX II). While tomato KNOX I proteins are known to stimulate chloroplast development in fruit, affecting fruit coloration, the role of KNOX II proteins in this context remains unclear. In this study, we employ CRISPR/Cas9 to generate knockout mutants of the KNOX II member, SlKN5. These mutants display increased leaf complexity, a phenotype commonly associated with reduced KNOX II activity, as well as enhanced accumulation of chloroplasts and chlorophylls in smaller cells within young, unripe fruit. RNA-seq data analyses indicate that SlKN5 suppresses the transcriptions of genes involved in chloroplast biogenesis, chlorophyll biosynthesis, and gibberellin catabolism. Furthermore, protein-protein interaction assays reveal that SlKN5 physically interacts with three transcriptional repressors from the BLH1-clade of BEL1-like homeodomain (BLH) protein family, SlBLH4, SlBLH5, and SlBLH7, with SlBLH7 showing the strongest interaction. CRISPR/Cas9-mediated knockout of these SlBLH genes confirmed their overlapping roles in suppressing chloroplast biogenesis, chlorophyll biosynthesis, and lycopene cyclization. Transient assays further demonstrate that the SlKN5-SlBLH7 interaction enhances binding capacity to regulatory regions of key chloroplast- and chlorophyll-related genes, including SlAPRR2-like1, SlCAB-1C, and SlGUN4. Collectively, our findings elucidate that the KNOX II SlKN5-SlBLH regulatory modules serve to inhibit fruit greening and subsequently promote lycopene accumulation, thereby fine-tuning the color transition from immature green fruit to mature red fruit.

Transcription Factors behind MYB98 Regulation: What Does the Discovery of SaeM Suggest?

MYB98 is master regulator of the molecular network involved in pollen tube attraction. Until recently, it was unclear how this gene exhibits exclusively synergid cell-specific expression in ovule. Our recent study has established that a 16-bp-long SaeM element is crucial for its synergid cell-specific expression in ovule, and an 84-bp-long fragment harboring SaeM is sufficient to drive the process. In this study, we have developed a workflow to predict functional roles of potential transcription factors (TFs) putatively binding to the promoter region, taking MYB98 promoter as a test subject. After sequential assessment of co-expression pattern, network analysis, and potential master regulator identification, we have proposed a multi-TF model for MYB98 regulation. Our study suggests that ANL2, GT-1, and their respective homologs could be direct regulators of MYB98 and indicates that TCP15, TCP16, FRS9, and HB34 are likely master regulators of the majority of the TFs involved in its regulation. Comprehensive studies in the future are expected to offer more insights into such propositions. Developed workflow can be used while designing similar regulome-related studies for any other species and genes.

Application of ethanol alleviates heat damage to leaf growth and yield in tomato.

Chemical priming has emerged as a promising area in agricultural research. Our previous studies have demonstrated that pretreatment with a low concentration of ethanol enhances abiotic stress tolerance in Arabidopsis and cassava. Here, we show that ethanol treatment induces heat stress tolerance in tomato (Solanum lycopersicon L.) plants. Seedlings of the tomato cultivar 'Micro-Tom' were pretreated with ethanol solution and then subjected to heat stress. The survival rates of the ethanol-pretreated plants were significantly higher than those of the water-treated control plants. Similarly, the fruit numbers of the ethanol-pretreated plants were greater than those of the water-treated ones. Transcriptome analysis identified sets of genes that were differentially expressed in shoots and roots of seedlings and in mature green fruits of ethanol-pretreated plants compared with those in water-treated plants. Gene ontology analysis using these genes showed that stress-related gene ontology terms were found in the set of ethanol-induced genes. Metabolome analysis revealed that the contents of a wide range of metabolites differed between water- and ethanol-treated samples. They included sugars such as trehalose, sucrose, glucose, and fructose. From our results, we speculate that ethanol-induced heat stress tolerance in tomato is mainly the result of increased expression of stress-related genes encoding late embryogenesis abundant (LEA) proteins, reactive oxygen species (ROS) elimination enzymes, and activated gluconeogenesis. Our results will be useful for establishing ethanol-based chemical priming technology to reduce heat stress damage in crops, especially in Solanaceae.

BIL9 Promotes Both Plant Growth via BR Signaling and Drought Stress Resistance by Binding with the Transcription Factor HDG11.

Drought stress is a major threat leading to global plant and crop losses in the context of the climate change crisis. Brassinosteroids (BRs) are plant steroid hormones, and the BR signaling mechanism in plant development has been well elucidated. Nevertheless, the specific mechanisms of BR signaling in drought stress are still unclear. Here, we identify a novel Arabidopsis gene, BRZ INSENSITIVE LONG HYPOCOTYL 9 (BIL9), which promotes plant growth via BR signaling. Overexpression of BIL9 enhances drought and mannitol stress resistance and increases the expression of drought-responsive genes. BIL9 protein is induced by dehydration and interacts with the HD-Zip IV transcription factor HOMEODOMAIN GLABROUS 11 (HDG11), which is known to promote plant resistance to drought stress, in vitro and in vivo. BIL9 enhanced the transcriptional activity of HDG11 for drought-stress-resistant genes. BIL9 is a novel BR signaling factor that enhances both plant growth and plant drought resistance.

Jasmonate activates secondary cell wall biosynthesis through MYC2-MYB46 module.

Formation of secondary cell wall (SCW) is tightly regulated spatiotemporally by various developmental and environmental signals. Successful fine-tuning of the trade-off between SCW biosynthesis and stress responses requires a better understanding of how plant growth is regulated under environmental stress conditions. However, the current understanding of the interplay between environmental signaling and SCW formation is limited. The lipid-derived plant hormone jasmonate (JA) and its derivatives are important signaling components involved in various physiological processes including plant growth, development, and abiotic/biotic stress responses. Recent studies suggest that JA is involved in SCW formation but the signaling pathway has not been studied for how JA regulates SCW formation. We tested this hypothesis using the transcription factor MYB46, a master switch for SCW biosynthesis, and JA treatments. Both the transcript and protein levels of MYB46, a master switch for SCW formation, were significantly increased by JA treatment, resulting in the upregulation of SCW biosynthesis. We then show that this JA-induced upregulation of MYB46 is mediated by MYC2, a central regulator of JA signaling, which binds to the promoter of MYB46. We conclude that this MYC2-MYB46 module is a key component of the plant response to JA in SCW formation.

The AT-hook protein AHL29 promotes Bacillus subtilis colonization by suppressing SWEET2-mediated sugar retrieval in Arabidopsis roots.

Beneficial Bacillus subtilis (BS) symbiosis could combat root pathogenesis, but it relies on root-secreted sugars. Understanding the molecular control of sugar flux during colonization would benefit biocontrol applications. The SWEET (Sugar Will Eventually Be Exported Transporter) uniporter regulates microbe-induced sugar secretion from roots; thus, its homologs may modulate sugar distribution upon BS colonization. Quantitative polymerase chain reaction revealed that gene transcripts of SWEET2, but not SWEET16 and 17, were significantly induced in seedling roots after 12 h of BS inoculation. Particularly, SWEET2-ß-glucuronidase fusion proteins accumulated in the apical mature zone where BS abundantly colonized. Yet, enhanced BS colonization in sweet2 mutant roots suggested a specific role for SWEET2 to constrain BS propagation, probably by limiting hexose secretion. By employing yeast one-hybrid screening and ectopic expression in Arabidopsis protoplasts, the transcription factor AHL29 was identified to function as a repressor of SWEET2 expression through the AT-hook motif. Repression occurred despite immunity signals. Additionally, enhanced SWEET2 expression and reduced colonies were specifically detected in roots of BS-colonized ahl29 mutant. Taken together, we propose that BS colonization may activate repression of AHL29 on SWEET2 transcription that would be enhanced by immunity signals, thereby maintaining adequate sugar secretion for a beneficial Bacillus association.

The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice.

CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated OsLCYß genome-edited lines generated by RNPs. The calli and regenerated shoot of the OsLCYß mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.

NAC domain transcription factors VNI2 and ATAF2 form protein complexes and regulate leaf senescence.

The NAM, ATAF1/2, and CUC2 (NAC) domain transcription factor VND-INTERACTING2 (VNI2) negatively regulates xylem vessel formation by interacting with another NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), a master regulator of xylem vessel formation. Here, we screened interacting proteins with VNI2 using yeast two-hybrid assay and isolated two NAC domain transcription factors, Arabidopsis thaliana ACTIVATION FACTOR 2 (ATAF2) and NAC DOMAIN CONTAINING PROTEIN 102 (ANAC102). A transient gene expression assay showed that ATAF2 upregulates the expression of genes involved in leaf senescence, and VNI2 effectively inhibits the transcriptional activation activity of ATAF2. vni2 mutants accelerate leaf senescence, whereas ataf2 mutants delay leaf senescence. In addition, the accelerated leaf senescence phenotype of the vni2 mutant is recovered by simultaneous mutation of ATAF2. Our findings strongly suggest that VNI2 interacts with and inhibits ATAF2, resulting in negatively regulating leaf senescence.

Stone cell formation in the pedicel of pears and apples.

MAIN CONCLUSION: For the first time, stone cells in pear and apple pedicel were studied. The lignification of the pedicel outer part was correlated with flesh, and the secondary cell wall biosynthesis genes were activated. Fruit pedicels act as bridges between the fruit and the shoot. They have secondary thickened cell walls that presumably function in mechanical support, water and nutrient transport. Stone cells are cells with a secondary cell wall thickening. In pears, yet not in apples, the stone cells affect the flesh texture. There have been few reports on stone cell formation in pear and apple pedicels; therefore, we studied these cells for the first time. The apple pedicel had few stone cells in the cortex. The formation of stone cells in pear continued until seven weeks after flowering (WAF), and the density was significantly higher than in apple. The stone cell formation degree (SFD) of pear was 3.6-7.1 times higher than that of apple. Total lignin and lignin non-condensed structure (G and S units) content in the pear pedicle outer part was 1.5-2.7 times higher than that of the apple at harvest. The SFD of the pedicel outer part had a positive correlation with the G and S units content of the flesh. The total lignin and G and S units content between flesh and the pedicel outer part were positively correlated. Correlation analysis revealed a positive relationship between fruit and pedicel formation of the stone cells. The WGCNA showed that NST3 was linked to NAC028, MYB46, CESA, POD, LAC, and VSR6. These genes were highly expressed in the outer part of the pear pedicel, while they were suppressed in that issue of the apple at 4 WAF.

A bHLH heterodimer regulates germ cell differentiation in land plant gametophytes.

Land plants are a monophyletic group of photosynthetic eukaryotes that diverged from streptophyte algae about 470 million years ago. During both the alternating haploid and diploid stages of the life cycle, land plants form multicellular bodies.1,2,3,4 The haploid multicellular body (gametophyte) produces progenitor cells that give rise to gametes and the reproductive organs.5,6,7,8 In the liverwort Marchantia polymorpha, differentiation of the initial cells of gamete-producing organs (gametangia) from the gametophyte is regulated by MpBONOBO (MpBNB), a member of the basic helix-loop-helix (bHLH) transcription factor subfamily VIIIa. In Arabidopsis thaliana, specification of generative cells in developing male gametophytes (pollen) requires redundant action of BNB1 and BNB2.9 Subfamily XI bHLHs, such as LOTUS JAPONICUS ROOTHAIRLESS LIKE1 (LRL1)/DEFECTIVE REGION OF POLLEN1 (DROP1) and LRL2/DROP2 in A. thaliana and the single LRL/DROP protein MpLRL in M. polymorpha, are the evolutionarily conserved regulators of rooting system development.10 Although the role of LRL1/DROP1 and LRL2/DROP2 in gametogenesis remains unclear, their loss leads to the formation of abnormal pollen devoid of sperm cells.11 Here, we show that BNBs and LRL/DROPs co-localize to gametophytic cell nuclei and form heterodimers. LRL1/DROP1 and LRL2/DROP2 act redundantly to regulate BNB expression for generative cell specification in A. thaliana after asymmetric division of the haploid microspore. MpLRL is required for differentiation of MpBNB-expressing gametangium initial cells in M. polymorpha gametophytes. Our findings suggest that broadly expressed LRL/DROP stabilizes BNB expression, leading to the formation of an evolutionarily conserved bHLH heterodimer, which regulates germ cell differentiation in the haploid gametophyte of land plants.

Heterologous gene expression system for the production of hydrolyzable tannin intermediates in herbaceous model plants.

Aluminum toxicity is the main factor limiting the elongation of plant roots in acidic soil. The tree species Eucalyptus camaldulensis is considerably more resistant to aluminum than herbaceous model plants and crops. Hydrolyzable tannins (HTs) accumulating in E. camaldulensis roots can bind and detoxify the aluminum taken up by the roots. However, in herbaceous model plants, HTs do not accumulate and the genes involved in the HT biosynthetic pathway are largely unknown. The aim of this study was to establish a method for reconstituting the HT biosynthetic pathway in the HT non-accumulating model plant Nicotiana benthamiana. Four E. camaldulensis enzymes were transiently expressed in N. benthamiana leaves via Agrobacterium tumefaciens-mediated transformation. These enzymes included dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDH2 and EcDQD/SDH3), which catalyze the synthesis of gallic acid, the first intermediate of the HT biosynthetic pathway that branches off from the shikimate pathway. The others were UDP-glycosyltransferases (UGT84A25 and UGT84A26), which catalyze the conversion of gallic acid to ß-glucogallin, the second intermediate. The co-expression of the EcDQD/SDHs in transgenic N. benthamiana leaf regions promoted the synthesis of gallic acid. Moreover, the co-expression of the UGT84As in addition to the EcDQD/SDHs resulted in the biosynthesis of ß-glucogallin, the universal metabolic precursor of HTs. Thus, we successfully reconstituted a portion of the HT biosynthetic pathway in HT non-accumulating N. benthamiana plants. This heterologous gene expression system will be useful for co-expressing candidate genes involved in downstream reactions in the HT biosynthetic pathway and for clarifying their in planta functions.

Role of autophagy-related proteins ATG8f and ATG8h in the maintenance of autophagic activity in Arabidopsis roots under phosphate starvation.

Nutrient starvation-induced autophagy is a conserved process in eukaryotes. Plants defective in autophagy show hypersensitivity to carbon and nitrogen limitation. However, the role of autophagy in plant phosphate (Pi) starvation response is relatively less explored. Among the core autophagy-related (ATG) genes, ATG8 encodes a ubiquitin-like protein involved in autophagosome formation and selective cargo recruitment. The Arabidopsis thaliana ATG8 genes, AtATG8f and AtATG8h, are notably induced in roots under low Pi. In this study, we show that such upregulation correlates with their promoter activities and can be suppressed in the phosphate response 1 (phr1) mutant. Yeast one-hybrid analysis failed to attest the binding of the AtPHR1 transcription factor to the promoter regions of AtATG8f and AtATG8h. Dual luciferase reporter assays in Arabidopsis mesophyll protoplasts also indicated that AtPHR1 could not transactivate the expression of both genes. Loss of AtATG8f and AtATG8h leads to decreased root microsomal-enriched ATG8 but increased ATG8 lipidation. Moreover, atg8f/atg8h mutants exhibit reduced autophagic flux estimated by the vacuolar degradation of ATG8 in the Pi-limited root but maintain normal cellular Pi homeostasis with reduced number of lateral roots. While the expression patterns of AtATG8f and AtATG8h overlap in the root stele, AtATG8f is more strongly expressed in the root apex and root hair and remarkably at sites where lateral root primordia develop. We hypothesize that Pi starvation-induction of AtATG8f and AtATG8h may not directly contribute to Pi recycling but rely on a second wave of transcriptional activation triggered by PHR1 that fine-tunes cell type-specific autophagic activity.

Nicotiana benthamiana XYLEM CYSTEINE PROTEASE genes facilitate tracheary element formation in interfamily grafting.

Grafting is a plant propagation technique widely used in agriculture. A recent discovery of the capability of interfamily grafting in Nicotiana has expanded the potential combinations of grafting. In this study, we showed that xylem connection is essential for the achievement of interfamily grafting and investigated the molecular basis of xylem formation at the graft junction. Transcriptome and gene network analyses revealed gene modules for tracheary element (TE) formation during grafting that include genes associated with xylem cell differentiation and immune response. The reliability of the drawn network was validated by examining the role of the Nicotiana benthamiana XYLEM CYSTEINE PROTEASE (NbXCP) genes in TE formation during interfamily grafting. Promoter activities of NbXCP1 and NbXCP2 genes were found in differentiating TE cells in the stem and callus tissues at the graft junction. Analysis of a Nbxcp1;Nbxcp2 loss-of-function mutant indicated that NbXCPs control the timing of de novo TE formation at the graft junction. Moreover, grafts of the NbXCP1 overexpressor increased the scion growth rate as well as the fruit size. Thus, we identified gene modules for TE formation at the graft boundary and demonstrated potential ways to enhance Nicotiana interfamily grafting.

Discovery of a cis-regulatory element SaeM involved in dynamic regulation of synergid-specific MYB98.

MYB98 is a key regulator of the genetic network behind pollen tube attraction toward the female gametophyte. MYB98 is specifically expressed in the synergid cells (SCs), a female gametophyte component cells specialized for pollen tube attraction. However, it had not been clear how exactly MYB98 achieves this specific expression pattern. In the current study, we have determined that a normal SC-specific expression of MYB98 is dependent on a 16-bp-long cis-regulatory element, CATTTACACATTAAAA, freshly named as the "S ynergid-specific A ctivation E lement of M YB98" (SaeM). An 84 bp fragment harboring SaeM in the middle was sufficient to drive exclusively SC-specific expression. The element was present in a significantly large proportion of SC-specific gene promoters and in the promoter of MYB98 homologous genes in the Brassicaceae (pMYB98s). Significance of such family-wide SaeM-like element conservation in exclusive SC-specific expression was confirmed by the Arabidopsis-like activation feature of Brassica oleracea-derived pMYB98 and absence of such feature of pMYB98 derived from a non-Brassicaceae member Prunus persica. Additionally, the yeast-one-hybrid assay showed that the SaeM can be recognized by ANTHOCYANINLESS2 (ANL2) and DAP-seq data further suggested for additional three ANL2 homologs targeting the similar cis-element. Overall, our study has concluded that SaeM plays a crucial role in driving exclusively SC-specific expression of MYB98 and strongly suggests for the involvement of ANL2 and its homologs in its dynamic regulation in planta. Future study on the transcription factors is expected to shed more light on the mechanism behind the process.

Contribution of vasculature to stem integrity in Arabidopsis thaliana.

In plants, coordinated growth is important for organ mechanical integrity because cells remain contiguous through their walls. So far, defects in inflorescence stem integrity in Arabidopsis thaliana have mainly been related to epidermal defects. Although these observations suggest a growth-limiting function at the stem cortex, deeper layers of the stem could also contribute to stem integrity. The nac secondary cell wall thickening promoting factor1 (nst1) nst3 double-mutant background is characterized by weaker vascular bundles without cracks. By screening for the cracking phenotype in this background, we identified a regulator of stem cracking, the transcription factor INDETERMINATE DOMAIN9 (IDD9). Stem cracking was not caused by vascular bundle breakage in plants that expressed a dominant repressor version of IDD9. Instead, cracking emerged from increased cell expansion in non-lignified interfascicular fiber cells that stretched the epidermis. This phenotype could be enhanced through CLAVATA3-dependent cell proliferation. Collectively, our results demonstrate that stem integrity relies on three additive mechanical components: the epidermis, which resists inner cell growth; cell proliferation in inner tissues; and growth heterogeneity associated with vascular bundle distribution in deep tissues.

Transcriptome analyses uncover reliance of endosperm gene expression on Arabidopsis embryonic development.

Endosperm-embryo development in flowering plants is regulated coordinately by signal exchange during seed development. However, such a reciprocal control mechanism has not been clearly identified. In this study, we identified an endosperm-specific gene, LBD35, expressed in an embryonic development-dependent manner, by a comparative transcriptome and cytological analyses of double-fertilized and single-fertilized seeds prepared by using the kokopelli mutant, which frequently induces single fertilization events. Transcriptome analysis using LBD35 as a marker of the central cell fertilization event identified that 141 genes, including 31 genes for small cysteine-rich peptides, are expressed in a double fertilization-dependent manner. Our results reveal possible embryonic signals that regulate endosperm gene expression and provide a practicable method to identify genes involved in the communication during endosperm-embryo development.

Elongation of Siliques Without Pollination 3 Regulates Nutrient Flow Necessary for Embryogenesis.

Apomixis, defined as the transfer of maternal germplasm to offspring without fertilization, enables the fixation of F1-useful traits, providing advantages in crop breeding. However, most apomictic plants require pollination to produce the endosperm. The endosperm is essential for embryogenesis, and its development is suppressed until fertilization. We show that the expression of a chimeric repressor of the Elongation of Siliques without Pollination 3 (ESP3) gene (Pro35S:ESP3-SRDX) induces ovule enlargement without fertilization in Arabidopsis thaliana. The ESP3 gene encodes a protein similar to the flowering Wageningen homeodomain transcription factor containing a StAR-related lipid transfer domain. However, ESP3 lacks the homeobox-encoding region. Genes related to the cell cycle and sugar metabolism were upregulated in unfertilized Pro35S:ESP3-SRDX ovules similar to those in fertilized seeds, while those related to autophagy were downregulated similar to those in fertilized seeds. Unfertilized Pro35S:ESP3-SRDX ovules partially nourished embryos when only the egg was fertilized, accumulating hexoses without central cell proliferation. ESP3 may regulate nutrient flow during seed development, and ESP3-SRDX could be a useful tool for complete apomixis that does not require pseudo-fertilization.

Transcription factors KNAT3 and KNAT4 are essential for integument and ovule formation in Arabidopsis.

Integuments form important protective cell layers surrounding the developing ovules in gymno- and angiosperms. Although several genes have been shown to influence the development of integuments, the transcriptional regulatory mechanism is still poorly understood. In this work, we report that the Class II KNOTTED1-LIKE HOMEOBOX (KNOX II) transcription factors KNOTTED1-LIKE HOMEBOX GENE 3 (KNAT3) and KNAT4 regulate integument development in Arabidopsis (Arabidopsis thaliana). KNAT3 and KNAT4 were co-expressed in inflorescences and especially in young developing ovules. The loss-of-function double mutant knat3 knat4 showed an infertility phenotype, in which both inner and outer integuments of the ovule are arrested at an early stage and form an amorphous structure as in the bell1 (bel1) mutant. The expression of chimeric KNAT3- and KNAT4-EAR motif repression domain (SRDX repressors) resulted in severe seed abortion. Protein-protein interaction assays demonstrated that KNAT3 and KNAT4 interact with each other and also with INNER NO OUTER (INO), a key transcription factor required for the outer integument formation. Transcriptome analysis showed that the expression of genes related with integument development is influenced in the knat3 knat4 mutant. The knat3 knat4 mutant also had a lower indole-3-acetic acid (IAA) content, and some auxin signaling pathway genes were downregulated. Moreover, transactivation analysis indicated that KNAT3/4 and INO activate the auxin signaling gene IAA INDUCIBLE 14 (IAA14). Taken together, our study identified KNAT3 and KNAT4 as key factors in integument development in Arabidopsis.

Brassinosteroid-induced gene repression requires specific and tight promoter binding of BIL1/BZR1 via DNA shape readout.

BRZ-INSENSITIVE-LONG 1 (BIL1)/BRASSINAZOLE-RESISTANT 1 (BZR1) and its homologues are plant-specific transcription factors that convert the signalling of the phytohormones brassinosteroids (BRs) to transcriptional responses, thus controlling various physiological processes in plants. Although BIL1/BZR1 upregulates some BR-responsive genes and downregulates others, the molecular mechanism underlying the dual roles of BIL1/BZR1 is still poorly understood. Here we show that BR-responsive transcriptional repression by BIL1/BZR1 requires the tight binding of BIL1/BZR1 alone to the 10 bp elements of DNA fragments containing the known 6 bp core-binding motifs at the centre. Furthermore, biochemical and structural evidence demonstrates that the selectivity for two nucleobases flanking the core motifs is realized by the DNA shape readout of BIL1/BZR1 without direct recognition of the nucleobases. These results elucidate the molecular and structural basis of transcriptional repression by BIL1/BZR1 and contribute to further understanding of the dual roles of BIL1/BZR1 in BR-responsive gene regulation.