The basic helix-loop-helix transcription factor OsBLR1 regulates leaf angle in rice via brassinosteroid signalling.

The OsMAPK6-OsWRKY72 module positively regulates rice leaf angle through brassinosteroid signals.

Brassinosteroids (BRs) are steroid hormones that are essential for the development of plants. A tight control of BR homeostasis is vital for modulating their impact on growth responses. Although it is recognized that the rapid adaptation of de novo synthesis has a key role in adjusting required BR levels, our knowledge of the mechanisms governing feedback control is limited. In this study, we identify the transcription factor CESTA as a regulator of BR biosynthesis. ces-D was isolated in a screen of Arabidopsis mutants by BR over-accumulation phenotypes. Loss-of-function analysis and the use of a dominant repressor version revealed functional overlap among CESTA and its homologues and confirmed the role of CESTA in the positive control of BR-biosynthetic gene expression. We provide evidence that CESTA interacts with its homologue BEE1 and can directly bind to a G-box motif in the promoter of the BR biosynthesis gene CPD. Moreover, we show that CESTA subnuclear localization is BR regulated and discuss a model, in which CESTA interplays with BEE1 to control BR biosynthesis and other BR responses.

WRKY transcription factors orchestrate diverse responses to environmental stimuli in plants. Although considerable research has been conducted on the stress resistance mechanisms associated with WRKYs, little is known about the roles of WRKYs in regulating plant growth and development. Here, we identified OsWRKY36, a key regulator of rice (Oryza sativa) leaf angle and plant architecture, from a rice oswrky mutant library. OsWRKY36 is strongly expressed in the leaf lamina joint and promotes cell growth and expansion in adaxial parenchyma cells, leading to an increased leaf angle. A mechanistic investigation revealed that the E3 ubiquitin ligase OsPUB24, a negative component of brassinosteroid (BR) signaling, interacts with OsWRKY36 and facilitates its ubiquitination and degradation. Genetic evidence indicated that OsWRKY36 acts downstream of OsPUB24 to regulate the BR-induced increase in leaf angle. Furthermore, OsWRKY36 physically interacts with DWARF AND LOW-TILLERING (DLT), a positive regulator of BR, and cooperatively activates the expression of the BR-responsive gene OsBZR4. BR signaling stabilizes OsWRKY36 by inhibiting OsPUB24, allowing accumulated OsWRKY36 to interact with DLT and drive OsBZR4 expression, thereby positively regulating leaf inclination. Collectively, our study elucidates the mechanism underlying the regulation of leaf inclination and plant architecture by the BR-OsPUB24-OsWRKY36 and DLT-OsWRKY36-OsBZR4 modules. These findings not only advance the understanding of BR-mediated growth regulation but also offer a potential strategy for optimizing crop architecture through modulation of BR signaling.

Changes in plant architecture, such as leaf size, leaf shape, leaf angle, plant height, and floral organs, have been major factors in improving the yield of cereal crops. Moreover, changes in grain size and weight can also increase yield. Therefore, screens for additional factors affecting plant architecture and grain morphology may enable additional improvements in yield. Among the basic Helix-Loop-Helix (bHLH) transcription factors in rice (Oryza sativa), we found an enhancer-trap T-DNA insertion mutant of OsbHLH079 (termed osbhlh079-D). The osbhlh079-D mutant showed a wide leaf angle phenotype and produced long grains, similar to the phenotypes of mutants with increased brassinosteroid (BR) levels or enhanced BR signaling. Reverse transcription-quantitative PCR analysis showed that BR signaling-associated genes are largely upregulated in osbhlh079-D, but BR biosynthesis-associated genes are not upregulated, compared with its parental japonica cultivar ‘Dongjin’. Consistent with this, osbhlh079-D was hypersensitive to BR treatment. Scanning electron microscopy revealed that the expansion of cell size in the adaxial side of the lamina joint was responsible for the increase in leaf angle in osbhlh079-D. The expression of cell-elongation-associated genes encoding expansins and xyloglucan endotransglycosylases/hydrolases increased in the lamina joints of leaves in osbhlh079-D. The regulatory function of OsbHLH079 was further confirmed by analyzing 35S::OsbHLH079 overexpression and 35S::RNAi-OsbHLH079 gene silencing lines. The 35S::OsbHLH079 plants showed similar phenotypes to osbhlh079-D, and the 35S::RNAi-OsbHLH079 plants displayed opposite phenotypes to osbhlh079-D. Taking these observations together, we propose that OsbHLH079 functions as a positive regulator of BR signaling in rice.

Leaf angle is an important trait in plants. Here, we demonstrate that the leucine-rich repeat receptor-like kinase OsSLA1 plays an important role in leaf angle regulation in rice (Oryza sativa). OsSLA1 mutant plants exhibited a small leaf angle phenotype due to changes of adaxial cells in the lamina joint. GUS staining revealed that OsSLA1 was highly expressed in adaxial cells of the lamina joint. The OsSLA1 mutant plants were insensitive to exogenous epibrassinolide (eBL) and showed upregulated expression of DWARF and CPD, but downregulated expression of BU1, BUL1, and ILI1, indicating that brassinosteroid (BR) signal transduction was blocked. Fluorescence microscopy showed that OsSLA1 was localized to the plasma membrane and nearby periplasmic vesicles. Further study showed that OsSLA1 interacts with OsBRI1 and OsBAK1 via its intracellular domain and promotes the interaction between OsBRI1 and OsBAK1. In addition, phosphorylation experiments revealed that OsSLA1 does not possess kinase activity, but that it can be phosphorylated by OsBRI1 in vitro. Knockout of OsSLA1 in the context of d61 caused exacerbation of the mutant phenotype. These results demonstrate that OsSLA1 regulates leaf angle formation via positive regulation of BR signaling by enhancing the interaction of OsBRI1 with OsBAK1.

Atypical basic helix-loop-helix (bHLH) transcription factors, which lack the basic region for DNA binding, are important elements of brassinosteroid (BR) signaling. Recently, our systematic characterization of the rice (Oryza sativa) INCREASED LEAF INCLINATION (ILI) subfamily of atypical bHLHs revealed their indispensable roles in BR-mediated growth and development. Here, we reported the isolation of two additional rice ILI-interacting atypical bHLHs, ATBS1-INTERACTING FACTOR 1 (OsAIF1)/OsbHLH176 and OsAIF2/OsbHLH178. Genetic and cytological analyses of the OsAIFs knockout mutants and overexpression lines revealed that OsAIF1 and OsAIF2 negatively regulate rice leaf inclination and grain size in a synergistic and redundant manner. Compared to the wild-type, osaif knockout mutants exhibited hypersensitivity to BR, while OsAIF1 and OsAIF2 overexpression lines showed greatly reduced sensitivity or complete insensitivity to BR, indicating that these two OsAIFs act as major negative regulators of rice BR signaling. As ILI-interacting negative atypical HLHs, OsAIF1 and OsAIF2 genetically counteracted the positive ILI subfamily of atypical HLHs. Moreover, OsAIF1 and OsAIF2 physically interacted with and antagonized OsbHLH92, a positive regulator of BR signaling, thereby modulating rice development and gene transcription. These findings suggested that the atypical HLHs (ILIs and OsAIF1/OsAIF2) and the bHLH (OsbHLH92) transcription factors form a triantagonistic cascade in rice, counteracting each other to fine-tune leaf angle and grain size through BR signaling. Our results provide insights into the mechanisms balancing BR signaling and growth in rice.

MYB42 inhibits hypocotyl cell elongation by coordinating brassinosteroid homeostasis and signalling in Arabidopsis thaliana.

The GSK3/SHAGGY-like kinase plays critical roles in plant development and response to stress, but its specific function remains largely unknown in wheat (Triticum aestivum L.). In this study, we investigated the function of TaGSK3, a GSK3/SHAGGY-like kinase, in wheat development and response to stress. Our findings demonstrated that TaGSK3 mutants had significant effects on wheat seedling development and brassinosteroid (BR) signalling. Quadruple and quintuple mutants showed amplified BR signalling, promoting seedling development, while a sextuple mutant displayed severe developmental defects but still responded to exogenous BR signals, indicating redundancy and non-BR-related functions of TaGSK3. A gain-of-function mutation in TaGSK3-3D disrupted BR signalling, resulting in compact and dwarf plant architecture. Notably, this mutation conferred significant drought and heat stress resistance of wheat, and enhanced heat tolerance independent of BR signalling, unlike knock-down mutants. Further research revealed that this mutation maintains a higher relative water content by regulating stomatal-mediated water loss and maintains a lower ROS level to reduces cell damage, enabling better growth under stress. Our study provides comprehensive insights into the role of TaGSK3 in wheat development, stress response, and BR signal transduction, offering potential for modifying TaGSK3 to improve agronomic traits and enhance stress resistance in wheat.

Defects in brassinosteroid (BR) biosynthetic or signaling genes result in dwarfed plants, whereas overexpression of these genes increases overall stature. An Arabidopsis elongated-D (elg-D) mutant shares phenotypic similarities with BR overexpression lines, suggesting its implication in BR pathways. Here, we determine how elg-D affects BR signaling. Since elg-D rescued dwarfism in bri1-5 plants, a BR receptor mutant, but not in BR-insensitive bin2/dwf12-1D plants, elg-D appears to act between bri1-5 and bin2/dwf12-1D in BR signaling. We found that elg-D had an increased response to epi-brassinolide (epi-BL); that the BES1 transcription factor was shifted toward the dephosphorylated form in elg-D; that the expression of a BR responsive gene, SAUR-AC1, was upregulated in elg-D; and that transcription of BR biosynthetic genes, DWF4 and CPD, was downregulated by feedback inhibition. Thus, endogenous levels of CS and BL as well as biosynthetic intermediates were reduced by the elg-D mutation, whereas basal levels of BR signaling were elevated. Map-based cloning and sequencing revealed that elg-D is allelic to the BR co-receptor protein, BAK1, and has an Asp(122) to Asn substitution in the third repeat of the extracellular leucine-rich repeat (LRR) domain. In agreement with the finding that BAK1/ELG is involved in the perception of pathogen-associated molecular patterns (PAMPs), the bak1/elg-D plants exhibited increased Pseudomonas syringae growth. Therefore, bak1/elg-D promotes Arabidopsis growth by stimulating BR signaling at the expense of its readiness to respond to biotic stress factors. The BAK1/ELG BR co-receptor thus plays an important role in BR signaling that is mediated by its LRR domain.

Ammonium protects rice against rice stripe virus by activating HDA703/OsBZR1-mediated BR signaling

Brassinosteroids (BRs) are essential growth-promoting hormones that regulate many aspects of plant growth and development. Two leucine-rich repeat receptor-like kinases (LRR-RLKs) are involved in BR perception and signal transduction: brassinosteroid insensitive 1 (BRI1), which is the BR receptor, and its coreceptor BRI1-associated kinase 1 (BAK1). Both proteins are classified as serine/threonine protein kinases, but here we report that recombinant cytoplasmic domains of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases. With BRI1, Tyr-831 and Tyr-956 are identified as autophosphorylation sites in vitro and in vivo. Interestingly, Tyr-956 in kinase subdomain V is essential for activity, because the Y956F mutant is catalytically inactive and thus this site cannot be simply manipulated by mutagenesis. In contrast, Tyr-831 in the juxtamembrane domain is not essential for kinase activity but plays an important role in BR signaling in vivo, because expression of BRI1(Y831F)-Flag in transgenic bri1-5 plants results in plants with larger leaves (but altered leaf shape) and early flowering relative to plants expressing wild-type BRI1-Flag. Acidic substitutions of Tyr-831 restored normal leaf size (but not shape) and normal flowering time. This is an example where a specific tyrosine residue has been shown to play an important role in vivo in plant receptor kinase function. Interestingly, 6 additional LRR-RLKs (of the 23 tested) were also found to autophosphorylate on tyrosine in addition to serine and threonine, suggesting that tyrosine signaling should be considered with other plant receptor kinases as well.

The brassinosteroid (BR) class of steroid hormones regulates plant development and physiology. The BR signal is transduced by a receptor kinase-mediated signal transduction pathway, which is distinct from animal steroid signalling systems. Recent studies have fully connected the BR signal transduction chain and have identified thousands of BR target genes, linking BR signalling to numerous cellular processes. Molecular links between BR and several other signalling pathways have also been identified. Here, we provide an overview of the highly integrated BR signalling network and explain how this steroid hormone functions as a master regulator of plant growth, development and metabolism.

While BRASSINOSTEROID (BR) SIGNALING KINASEs (BSKs) are known to interact with the BR receptor BR-INSENSITIVE 1 (BRI1), the extent of their genetic redundancy and essential contribution to BR signaling remain unclear. This study aimed to systematically investigate the function of BSK family members and uncover novel developmental outputs controlled by BR signaling via BSKs mediated pathway in Arabidopsis. We generated an undecuple bsk knockout/knockdown mutant (bsk-u) in Arabidopsis. Comprehensive phenotypic observation, molecular analyses, and genetic complementation were performed to characterize the mutant. Subsequently, the role of the LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD16) in mediating BR-suppressed adventitious root (AR) development was further examined using genetic and physiological approaches. The bsk-u mutant exhibited severe BR-insensitive phenotypes, including dwarfism, impaired BZR1 dephosphorylation, and attenuated transcriptional responses to BR. We identified LBD16 as a key BZR1 target repressed by BR signaling. BR application inhibited AR formation in wild-type explants, whereas BR-deficient mutants produced more ARs. Overexpression of LBD16 partially alleviated BR-mediated suppression of AR development. This study establishes that BSKs are indispensable for the BR signaling pathway in Arabidopsis. Furthermore, it reveals a novel role for BR in negatively regulating AR development through transcriptional repression of LBD16, expanding our understanding of BR-mediated developmental control.

Brassinosteroids (BRs) regulate of maize (Zea mays L.) growth, but the underlying molecular mechanism remains unclear. In this study, we used a multi-disciplinary approach to determine how BRs regulate maize morphology and physiology during development. Treatment with the BRs promoted primary root the elongation and growth during germination, and the early development of lateral roots. BRs treatment during the middle growth stage increased the levels of various stress resistance factors, and enhanced resistance to lodging, likely by protecting the plant against stem rot and sheath rot. BRs had no significant effect on plant height during late growth, but it increased leaf angle and photosynthetic efficiency, as well as yield and quality traits. Our findings increase our understanding of the regulatory effects of BR on maize root growth and development and the mechanism by which BR improves disease resistance, which could further the potential for using BR to improve maize yield.

Ovule initiation is a key step that strongly influences ovule number and seed yield. Notably, mutants with enhanced brassinosteroid (BR) and cytokinin (CK) signaling produce more ovules and have a higher seed number per silique (SNS) than wild-type plants. Here, we crossed BR- and CK-related mutants to test whether these phytohormones function together in ovule initiation. We determined that simultaneously enhancing BR and CK contents led to higher ovule and seed numbers than enhancing BR or CK separately, and BR and CK enhanced each other. Further, the BR-response transcription factor BZR1 directly interacted with the CK-response transcription factor ARABIDOPSIS RESPONSE REGULATOR1 (ARR1). Treatments with BR or BR plus CK strengthened this interaction and subsequent ARR1 targeting and induction of downstream genes to promote ovule initiation. Enhanced CK signaling partially rescued the reduced SNS phenotype of BR-deficient/insensitive mutants whereas enhanced BR signaling failed to rescue the low SNS of CK-deficient mutants, suggesting that BR regulates ovule initiation and SNS through CK-mediated and -independent pathways. Our study thus reveals that interaction between BR and CK promotes ovule initiation and increases seed number, providing important clues for increasing the seed yield of dicot crops.