BnaGSK3-BnaIDD16 module negatively regulates branch angle in Brassica napus.

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

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.

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.

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.

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.

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

Leaf angle is a key factor in plant architecture and crop yield. Brassinosteroids (BRs) regulate many developmental processes, especially the leaf angle in monocots. However, the BR signalling pathway is complex and includes many unknown members. Here, we propose that Oryza sativa BRASSINOSTEROID-RESPONSIVE LEAF ANGLE REGULATOR 1 (OsBLR1) encodes a bHLH transcription factor, and positively regulates BR signalling to increase the leaf angle and grain length in rice (Oryza sativa L.). Lines overexpressing OsBLR1 (blr1-D and BLR1-OE-1/2/3) had similar traits, with increased leaf angle and grain length. Conversely, OsBLR1-knockout mutants (blr1-1/2/3) had erect leaves and shorter grains. Lamina joint inclination, coleoptile elongation, and root elongation assay results indicated that these overexpression lines were more sensitive to BR, while the knockout mutants were less sensitive. There was no significant difference in the endogenous BR contents of blr1-1/2 and wild-type plants. These results suggest that OsBLR1 is involved in BR signal transduction. The blr1-D mutant, with increased cell growth in the lamina joint and smaller leaf midrib, showed significant changes in gene expression related to the cell wall and leaf development compared with wild-type plants; furthermore, the cellulose and protopectin contents in blr1-D were reduced, which resulted in the increased leaf angle and bent leaves. As the potential downstream target gene of OsBLR1, the REGULATOR OF LEAF INCLINATION1 (OsRLI1) gene expression was up-regulated in OsBLR1-overexpression lines and down-regulated in OsBLR1-knockout mutants. Moreover, we screened OsRACK1A as an interaction protein of OsBLR1 using a yeast two-hybrid assay and glutathione-S-transferase pull-down.

Brassinosteroids (BRs) and abscisic acid (ABA) are essential regulators of plant growth and stress tolerance. Although the antagonistic interaction of BRs and ABA is proposed to ensure the balance between growth and defense in model plants, the crosstalk between BRs and ABA in response to chilling in tomato (Solanum lycopersicum), a warm-climate horticultural crop, is unclear. Here, we determined that overexpression of the BR biosynthesis gene DWARF (DWF) or the key BR signaling gene BRASSINAZOLE-RESISTANT1 (BZR1) increases ABA levels in response to chilling stress via positively regulating the expression of the ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE1 (NCED1). BR-induced chilling tolerance was mostly dependent on ABA biosynthesis. Chilling stress or high BR levels decreased the abundance of BRASSINOSTEROID-INSENSITIVE2 (BIN2), a negative regulator of BR signaling. Moreover, we observed that chilling stress increases BR levels and results in the accumulation of BZR1. BIN2 negatively regulated both the accumulation of BZR1 protein and chilling tolerance by suppressing ABA biosynthesis. Our results demonstrate that BR signaling positively regulates chilling tolerance via ABA biosynthesis in tomato. The study has implications in production of warm-climate crops in horticulture.

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

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.

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.

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.

CNGC20 plays dual roles in regulating plant growth and immunity in Brassica napus

Modifications of plant architecture can increase planting density, regulate photosynthesis, and improve crop yields. Many basic helix-loop-helix (bHLH) transcription factors participate in the brassinosteroid (BR) signaling pathway and are critical for plant architecture morphogenesis in rice. However, the number of identified bHLH genes suitable for improving production value is still limited. In this study, we cloned Lam1, encoding the typical bHLH transcription factor OsbHLH92. OsbHLH92 knockout (KO) lines exhibit erect leaves. Decreases in the number and size of parenchyma cell layers on the adaxial side of the lamina joint in KO lines were the main reason for the decreased leaf angle. Genetic experiments verify that OsBU1 and its homologs are downstream of OsbHLH92, which is involved in the noncanonical RGA1-mediated BR signaling pathway. OsbHLH91, an OsbHLH92 homolog, plays both conserved and differentiated roles relative to OsbHLH92. Notably, OsbHLH92-KO lines show erect leaves without the acquisition of adverse agronomic traits. Moreover, by driving a specific panicle promoter, OsbHLH92 can greatly increase productivity by at least 10%. This study identifies new components of the BR signaling pathway, demonstrates the importance of OsbHLH92 in improving planting density and crop productivity, and broadens our knowledge of typical and atypical bHLH family members in rice.