The Rice Basic Helix-Loop-Helix 79 (OsbHLH079) Determines Leaf Angle and Grain Shape.

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.

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

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.

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.

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.

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.

Background:Grain size is a direct determinant of grain weight and yield in rice; however, the genetic and molecular mechanisms determining grain size remain largely unknown.Findings:We identified a mutant, wide grain 3 (wg3), which exhibited significantly increased grain width and 1000-grain weight. Cytological analysis showed that WG3 regulates grain size by affecting cell proliferation. MutMap-based gene cloning and a transgenic experiment demonstrated that WG3 encodes a GRAS protein. Moreover, we found that WG3 directly interacts with DWARF AND LOW-TILLERING (DLT), a previously reported GRAS protein, and a genetic experiment demonstrated that WG3 and DLT function in a common pathway to regulate grain size. Additionally, a brassinosteroid (BR) sensitivity test suggested that WG3 has a positive role in BR signaling in rice. Collectively, our results reveal a new genetic and molecular mechanism for the regulation of grain size in rice by the WG3-DLT complex, and highlight the important functions of the GRAS protein complex in plants.Conclusion:WG3 functions directly in regulating grain size and BR signaling in rice.

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

Dof (DNA binding with one finger) proteins, a class of plant-specific transcription factors, are involved in plant growth and developmental processes and stress responses. However, their biological functions remain to be elucidated, especially in rice (Oryza sativa L.). Previously, we have reported that OsDof12 can promote rice flowering under long-day conditions. Here, we further investigated the other important agronomical traits of the transgenic plants overexpressing OsDof12 and found that overexpressing OsDof12 could lead to reduced plant height, erected leaf, shortened leaf blade, and smaller panicle resulted from decreased primary and secondary branches number. These results implied that OsDof12 is involved in rice plant architecture formation. Furthermore, we performed a series of Brassinosteroid (BR)-responsive tests and found that overexpression of OsDof12 could also result in BR hyposensitivity. Of note, in WT plants the expression of OsDof12 was found up-regulated by BR treatment while in OsDof12 overexpression plants two positive BR signaling regulators, OsBRI1 and OsBZR1, were significantly down-regulated, indicating that OsDof12 may act as a negative BR regulator in rice. Taken together, our results suggested that overexpression of OsDof12 could lead to altered plant architecture by suppressing BR signaling. Thus, OsDof12 might be used as a new potential genetic regulator for future rice molecular breeding.

Increasing grain yield is a major goal of breeders due to the rising global demand for food. We previously reported that the miR397-LACCASE (OsLAC) module regulates brassinosteroid (BR) signaling and grain yield in rice (Oryza sativa). However, the precise roles of laccase enzymes in the BR pathway remain unclear. Here, we report that OsLAC controls grain yield by preventing the turnover of TRANSTHYRETIN-LIKE (OsTTL), a negative regulator of BR signaling. Overexpressing OsTTL decreased BR sensitivity in rice, while loss-of-function of OsTTL led to enhanced BR signaling and increased grain yield. OsLAC directly binds to OsTTL and regulates its phosphorylation-mediated turnover. The phosphorylation site Ser226 of OsTTL is essential for its ubiquitination and degradation. Overexpressing the dephosphorylation-mimic form of OsTTL (OsTTLS226A) resulted in more severe defects than did overexpressing OsTTL. These findings provide insight into the role of an ancient laccase in BR signaling and suggest that the OsLAC-OsTTL module could serve as a target for improving grain yield.

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.

Brassinosteroids (BRs) are plant-specific steroid hormones which regulate plant growth, development, and adaptation. Transcriptional regulation plays key roles in plant hormone signaling. A mediator can serve as a bridge between gene-specific transcription factors and the RNA polymerase machinery, functioning as an essential component in regulating the transcriptional process. However, whether a mediator is involved in BR signaling is unknown. Here, we discovered that Oryza sativa mediator subunit 25 (OsMED25) is an important regulator of rice BR signaling. Phenotypic analyses showed that the OsMED25-RNAi and osmed25 mutant presented erect leaves, as observed in BR-deficient mutants. In addition, the OsMED25-RNAi and osmed25 mutant exhibited decreased BR sensitivity. Genetic analysis indicated that OsMED25-RNAi could suppress the enhanced BR signaling phenotype of Osbzr1-D. Further biochemical analysis showed that OsMED25 interacts with OsBZR1 in vivo, and OsMED25 is enriched on the promoter of OsBZR1 target genes. RNA sequencing analysis indicated that OsMED25 affects the expression of approximately 45% of OsBZR1-regulated genes and mainly functions as a corepressor of OsBZR1. Together, these findings revealed that OsMED25 regulates rice BR signaling by interacting with OsBZR1 and modulating the expression of OsBZR1 target genes, thus expanding our understanding of the roles of mediators in plant hormone signaling.

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

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.