Molecular actions of two synthetic brassinosteroids, iso-carbaBL and 6-deoxoBL, which cause altered physiological activities between Arabidopsis and rice.
ABSTRACT: Brassinosteroid (BR) is an important plant hormone that is perceived by the BRASSINOSTEROID INSENSITIVE 1 (BRI1) receptor. BRI1 is conserved among dicot and monocot species; however, the molecular mechanism underlying BR perception in monocots is not fully understood. We synthesised two BRs, iso-carbabrassinolide (iso-carbaBL) and 6-deoxoBL, which have different BR activities in Arabidopsis thaliana (Arabidopsis) and rice. Our bioassay indicated that iso-carbaBL has relatively strong BR activity in Arabidopsis, but is inactive in rice and competitively inhibits BR activity. The bioactivity of 6-deoxoBL was similar to that of BL in Arabidopsis, but was much lower in rice. Binding experiments using recombinant Arabidopsis and rice BRI1 protein fragments suggested that iso-carbaBL and 6-deoxoBL bind to both receptors. These results showed that iso-carbaBL and 6-deoxoBL act as an antagonist and agonist, respectively, of BRs in rice. A docking simulation analysis suggested that iso-carbaBL fits deeper in the binding pocket to block the binding of active BR to rice BRI1. The simulated binding energy of 6-deoxoBL with rice BRI1 is much lower than that with Arabidopsis BRI1. The possible structural characteristics of rice BRI1 were determined based on the difference in the BR activities of iso-carbaBL and 6-deoxoBL in Arabidopsis and rice.
Project description:Internalization of cell surface receptors, followed by either recycling back to the plasma membrane or degradation, is crucial for receptor homeostasis and signaling. The plant brassinosteroid (BR) receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), undergoes constitutive cycling between the plasma membrane and the internal membranes. We show that protein phosphatase 2A (PP2A) dephosphorylated BRI1 and that Arabidopsis thaliana rcn1, a mutant for a PP2A subunit, caused an increase in BRI1 abundance and BR signaling. We report the identification, in A. thaliana, of a suppressor of bri1, sbi1, which caused selective accumulation of BR-activated BRI1, but not the BR co-receptor BAK1 (BRI1-ASSOCIATED KINASE 1), in the membranous compartment. SBI1 mRNA was induced by BRs, and SBI1 encodes a leucine carboxylmethyltransferase (LCMT) that methylated PP2A and controlled its membrane-associated subcellular localization. We propose that BRs increase production of SBI1, which methylates PP2A, thus facilitating its association with activated BRI1. This leads to receptor dephosphorylation and degradation, and thus to the termination of BR signaling.
Project description:To withstand ever-changing environmental stresses, plants are equipped with phytohormone-mediated stress resistance mechanisms. Salt stress triggers abscisic acid (ABA) signaling, which enhances stress tolerance at the expense of growth. ABA is thought to inhibit the action of growth-promoting hormones, including brassinosteroids (BRs). However, the regulatory mechanisms that coordinate ABA and BR activity remain to be discovered. We noticed that ABA-treated seedlings exhibited small, round leaves and short roots, a phenotype that is characteristic of the BR signaling mutant, brassinosteroid insensitive1-9 (bri1-9). To identify genes that are antagonistically regulated by ABA and BRs, we examined published Arabidopsis microarray data sets. Of the list of genes identified, those upregulated by ABA but downregulated by BRs were enriched with a BRRE motif in their promoter sequences. After validating the microarray data using quantitative RT-PCR, we focused on RD26, which is induced by salt stress. Histochemical analysis of transgenic Arabidopsis plants expressing RD26pro:GUS revealed that the induction of GUS expression after NaCl treatment was suppressed by co-treatment with BRs, but enhanced by co-treatment with propiconazole, a BR biosynthetic inhibitor. Similarly, treatment with bikinin, an inhibitor of BIN2 kinase, not only inhibited RD26 expression, but also reduced the survival rate of the plant following exposure to salt stress. Our results suggest that ABA and BRs act antagonistically on their target genes at or after the BIN2 step in BR signaling pathways, and suggest a mechanism by which plants fine-tune their growth, particularly when stress responses and growth compete for resources.
Project description:Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte Selaginella moellendorffii. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and epi-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based membrane receptor complexes evolved in these plants.
Project description:Plants and animals use innate immunity as a first defense against pathogens, a costly yet necessary tradeoff between growth and immunity. In Arabidopsis, the regulatory leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1 combines with the LRR-RLKs FLS2 and EFR in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and the LRR-RLK BRI1 in brassinosteroid (BR)-mediated growth. Therefore, a potential tradeoff between these pathways mediated by BAK1 is often postulated. Here, we show a unidirectional inhibition of FLS2-mediated immune signaling by BR perception. Unexpectedly, this effect occurred downstream or independently of complex formation with BAK1 and associated downstream phosphorylation. Thus, BAK1 is not rate-limiting in these pathways. BRs also inhibited signaling triggered by the BAK1-independent recognition of the fungal PAMP chitin. Our results suggest a general mechanism operative in plants in which BR-mediated growth directly antagonizes innate immune signaling.
Project description:Lamina inclination is a key agronomical character that determines plant architecture and is sensitive to auxin and brassinosteroids (BRs). Loose Plant Architecture1 (LPA1) in rice (Oryza sativa) and its Arabidopsis homologues (SGR5/AtIDD15) have been reported to control plant architecture and auxin homeostasis. This study explores the role of LPA1 in determining lamina inclination in rice. LPA1 acts as a positive regulator to suppress lamina bending. Genetic and biochemical data indicate that LPA1 suppresses the auxin signalling that interacts with C-22-hydroxylated and 6-deoxo BRs, which regulates lamina inclination independently of OsBRI1. Mutant lpa1 plants are hypersensitive to indole-3-acetic acid (IAA) during the lamina inclination response, which is suppressed by the brassinazole (Brz) inhibitor of C-22 hydroxylase involved in BR synthesis. A strong synergic effect is detected between lpa1 and d2 (the defective mutant for catalysis of C-23-hydroxylated BRs) during IAA-mediated lamina inclination. No significant interaction between LPA1 and OsBRI1 was identified. The lpa1 mutant is sensitive to C-22-hydroxylated and 6-deoxo BRs in the d61-1 (rice BRI1 mutant) background. We present evidence verifying that two independent pathways function via either BRs or BRI1 to determine IAA-mediated lamina inclination in rice. RNA sequencing analysis and qRT-PCR indicate that LPA1 influences the expression of three OsPIN genes (OsPIN1a, OsPIN1c and OsPIN3a), which suggests that auxin flux might be an important factor in LPA1-mediated lamina inclination in rice.
Project description:Metazoans and plants use pattern recognition receptors (PRRs) to sense conserved microbial-associated molecular patterns (MAMPs) in the extracellular environment. In plants, the bacterial MAMPs flagellin and elongation factor Tu (EF-Tu) activate distinct, phylogenetically related cell surface pattern recognition receptors of the leucine-rich repeat receptor kinase (LRR-RK) family called FLS2 and EF-Tu receptor, respectively. BAK1 is an LRR-RK coreceptor for both FLS2 and EF-Tu receptor. BAK1 is also a coreceptor for the plant brassinosteroid (BR) receptor, the LRR-RK BRI1. Binding of BR to BRI1 primarily promotes cell elongation. Here, we tune the BR pathway response to establish how plant cells can generate functionally different cellular outputs in response to MAMPs and pathogens. We demonstrate that BR can act antagonistically or synergistically with responses to MAMPs. We further show that the synergistic activities of BRs on MAMP responses require BAK1. Our results highlight the importance of plant steroid homeostasis as a critical step in the establishment of plant immunity. We propose that tradeoffs associated with plasticity in the face of infection are layered atop plant steroid developmental programs.
Project description:The phytohormones, brassinosteroids (BRs), play important roles in regulating cell elongation and cell size, and BR-related mutants in Arabidopsis display significant dwarf phenotypes. Cellulose is a biopolymer which has a major contribution to cell wall formation during cell expansion and elongation. However, whether BRs regulate cellulose synthesis, and if so, what the underlying mechanism of cell elongation induced by BRs is, is unknown. The content of cellulose and the expression levels of the cellulose synthase genes (CESAs) was measured in BR-related mutants and their wild-type counterpart. The chromatin immunoprecipitation (CHIP) experiments and genetic analysis were used to demonstrate that BRs regulate CESA genes. It was found here that the BR-deficient or BR-perceptional mutants contain less cellulose than the wild type. The expression of CESA genes, especially those related to primary cell wall synthesis, was reduced in det2-1 and bri1-301, and was only inducible by BRs in the BR-deficient mutant det2-1. CHIP experiments show that the BR-activated transcription factor BES1 can associate with upstream elements of most CESA genes particularly those related with the primary cell wall. Furthermore, over-expression of the BR receptor BRI1 in CESA1, 3, and 6 mutants can only partially rescue the dwarf phenotypes. Our findings provide potential insights into the mechanism that BRs regulate cellulose synthesis to accomplish the cell elongation process in plant development.
Project description:Brassinosteroids (BRs) are essential steroid hormones that have crucial roles in plant growth and development. BRs are perceived by the cell-surface receptor-like kinase brassinosteroid insensitive 1 (BRI1). In the absence of BRs, the cytosolic kinase domain (KD) of BRI1 is inhibited by its auto-inhibitory carboxyl terminus, as well as by interacting with an inhibitor protein, BRI1 kinase inhibitor 1 (BKI1). How BR binding to the extracellular domain of BRI1 leads to activation of the KD and dissociation of BKI1 into the cytosol remains unclear. Here we report the crystal structure of BRI1 KD in complex with the interacting peptide derived from BKI1. We also provide biochemical evidence that BRI1-associated kinase 1 (BAK1) plays an essential role in initiating BR signaling. Steroid-dependent heterodimerization of BRI1 and BAK1 ectodomains brings their cytoplasmic KDs in the right orientation for competing with BKI1 and transphosphorylation.
Project description:Brassinosteroids (BRs) regulate a variety of physiological processes in plants via extensive crosstalk with diverse biological signaling networks. Although BRs are known to reciprocally regulate circadian oscillation, the molecular mechanism underlying BR-mediated regulation of circadian clock remains unknown. Here, we demonstrate that the BR-activated transcription factor bri1-EMS-SUPPRESSOR 1 (BES1) integrates BR signaling into the circadian network in Arabidopsis. BES1 repressed expression of CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) at night by binding to their promoters, together with TOPLESS (TPL). The repression of CCA1 and LHY by BR treatment, which occurred during the night, was compromised in bes1-ko and tpl-8 mutants. Consistently, long-term treatment with BR shortened the circadian period, and BR-induced rhythmic shortening was impaired in bes1-ko and tpl-8 single mutants and in the cca1-1lhy-21 double mutant. Overall, BR signaling is conveyed to the circadian oscillator via the BES1/TPL-CCA1/LHY module, contributing to gating diurnal BR responses in plants.
Project description:Brassinosteroids (BRs), plant steroid hormones, play important roles in plant cell elongation and differentiation. To investigate the mechanisms of BR signaling, we previously used the BR biosynthesis inhibitor Brz as a chemical biology tool and identified the Brz-insensitive-long hypocotyl4 mutant (bil4). Although the BIL4 gene encodes a seven-transmembrane-domain protein that is evolutionarily conserved in plants and animals, the molecular function of BIL4 in BR signaling has not been elucidated. Here, we demonstrate that BIL4 is expressed in early elongating cells and regulates cell elongation in Arabidopsis. BIL4 also activates BR signaling and interacts with the BR receptor brassinosteroid insensitive 1 (BRI1) in endosomes. BIL4 deficiency increases the localization of BRI1 in the vacuoles. Our results demonstrate that BIL4 regulates cell elongation and BR signaling via the regulation of BRI1 localization.