Isolation and characterization of the brassinosteroid receptor gene (GmBRI1) from Glycine max.
ABSTRACT: Brassinosteroids (BRs) constitute a group of steroidal phytohormones that contribute to a wide range of plant growth and development functions. The genetic modulation of BR receptor genes, which play major roles in the BR signaling pathway, can create semi-dwarf plants that have great advantages in crop production. In this study, a brassinosteroid insensitive gene homologous with AtBRI1 and other BRIs was isolated from Glycine max and designated as GmBRI1. A bioinformatic analysis revealed that GmBRI1 shares a conserved kinase domain and 25 tandem leucine-rich repeats (LRRs) that are characteristic of a BR receptor for BR reception and reaction and bear a striking similarity in protein tertiary structure to AtBRI1. GmBRI1 transcripts were more abundant in soybean hypocotyls and could be upregulated in response to exogenous BR treatment. The transformation of GmBRI1 into the Arabidopsis dwarf mutant bri1-5 restored the phenotype, especially regarding pod size and plant height. Additionally, this complementation is a consequence of a restored BR signaling pathway demonstrated in the light/dark analysis, root inhibition assay and BR-response gene expression. Therefore, GmBRI1 functions as a BR receptor to alter BR-mediated signaling and is valuable for improving plant architecture and enhancing the yield of soybean.
Project description:Brassinosteroids (BRs) play an essential role in plant growth, and BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) family transcription factors integrate a variety of plant signaling pathways. Despite the fact that BRs inhibit nodulation in leguminous plants, how BRs modulate rhizobia-host interactions and nodule morphogenesis is unknown. Here, we show that GmBEHL1, a soybean homolog of Arabidopsis BES1/BZR1 homolog 1 (BEH1), is an interacting partner of Nodule Number Control 1, a transcriptional repressor that mediates soybean nodulation. GmBEHL1 was highly expressed at the basal parts of emerging nodules, and its expression gradually expanded during nodule maturation. The overexpression and downregulation of GmBEHL1 inhibited and enhanced the number of nodules, respectively, in soybean. Intriguingly, alterations in GmBEHL1 expression repressed the expression of genes in the BR biosynthesis pathway, including homologs of Arabidopsis Constitutive Photomorphogenesis and Dwarf and Dwarf 4. We also detected an interaction between GmBEHL1 and GmBIN2, a putative BR-insensitive 2 (BIN2) homolog, in soybean. Moreover, BR treatment reduced the number, but increased the size, of soybean nodules. Our results reveal GmBEHL1 to be a potent gene that integrates BR signaling with nodulation signaling pathways to regulate symbiotic nodulation.
Project description:Brassinosteroids (BR) play important roles in plant growth and development. Although BR receptors have been intensively studied in Arabidopsis, the BR receptors in soybean remain largely unknown. Here, in addition to the known receptor gene Glyma06g15270 (GmBRI1a), we identified five putative BR receptor genes in the soybean genome: GmBRI1b, GmBRL1a, GmBRL1b, GmBRL2a, and GmBRL2b. Analysis of their expression patterns by quantitative real-time PCR showed that they are ubiquitously expressed in primary roots, lateral roots, stems, leaves, and hypocotyls. We used rapid amplification of cDNA ends (RACE) to clone GmBRI1b (Glyma04g39160), and found that the predicted amino acid sequence of GmBRI1b showed high similarity to those of AtBRI1 and pea PsBRI1. Structural modeling of the ectodomain also demonstrated similarities between the BR receptors of soybean and Arabidopsis. GFP-fusion experiments verified that GmBRI1b localizes to the cell membrane. We also explored GmBRI1b function in Arabidopsis through complementation experiments. Ectopic over-expression of GmBRI1b in Arabidopsis BR receptor loss-of-function mutant (bri1-5 bak1-1D) restored hypocotyl growth in etiolated seedlings; increased the growth of stems, leaves, and siliques in light; and rescued the developmental defects in leaves of the bri1-6 mutant, and complemented the responses of BR biosynthesis-related genes in the bri1-5 bak1-D mutant grown in light. Bioinformatics analysis demonstrated that the six BR receptor genes in soybean resulted from three gene duplication events during evolution. Phylogenetic analysis classified the BR receptors in dicots and monocots into three subclades. Estimation of the synonymous (Ks) and the nonsynonymous substitution rate (Ka) and selection pressure (Ka/Ks) revealed that the Ka/Ks of BR receptor genes from dicots and monocots were less than 1.0, indicating that BR receptor genes in plants experienced purifying selection during evolution.
Project description:Brassinosteroids (BRs) are important plant hormones that act synergistically with auxin to regulate a variety of plant developmental and physiological processes. In the past decade, genetic and biochemical studies have revealed a linear signaling pathway that relies on protein phosphorylation to transmit the BR signal into the nucleus, altering expression of hundreds of genes to promote plant growth. We conducted an activation-tagging based suppressor screen to look for Arabidopsis genes that, when overexpressed by inserted 35S enhancer elements, could suppress the dwarf phenotype of a weak BR receptor mutant bri1-301. This screen identified a total of six dominant activation-tagged bri1 suppressors (atbs-Ds). Using a plasmid rescue approach, we discovered that the bri1-301 suppression effect in four atbs-D mutants (atbs3-D to atbs6-D) was caused by overexpression of a YUCCA gene thought to be involved in tryptophan-dependent auxin biosynthesis. Interestingly, the three activation-tagged YUCCA genes belong to the YUCCA IIA subfamily that includes two other members out of 11 known Arabidopsis YUCCA genes. In addition, our molecular studies revealed a T-DNA insertion near a basic helix-loop-helix gene in atbs1-D and a T-DNA insertion in a region carrying a BR biosynthetic gene in atbs2-D. Further studies of these atbs-D mutants could lead to better understanding of the BR signaling process and the BR-auxin interaction.
Project description:Brassinosteroid hormones regulate many aspects of plant growth and development. The membrane receptor BRI1 is a central player in the brassinosteroid signaling cascade. Semi-dwarf 'uzu' barley carries a mutation in a conserved domain of the kinase tail of BRI1 and this mutant allele is recognised for its positive contribution to both yield and lodging resistance.Here we show that uzu barley exhibits enhanced resistance to a range of pathogens. It was due to a combination of preformed, inducible and constitutive defence responses, as determined by a combination of transcriptomic and biochemical studies. Gene expression studies were used to determine that the uzu derivatives are attenuated in downstream brassinosteroid signaling. The reduction of BRI1 RNA levels via virus-induced gene silencing compromised uzu disease resistance.The pathogen resistance of uzu derivatives may be due to pleiotropic effects of BRI1 or the cascade effects of their repressed BR signaling.
Project description: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.
Project description:Brassinosteroids (BRs) are plant hormones, fundamental for the growth and development of plants. A trans-membrane protein receptor kinase, Brassinosteroid-Insensitive 1 (BRI1), is known to interact with BRs and be directly involved in plant development. This study investigates the structural organization of BRI1 orthologs in several taxa, with a specific interest in Triticum aestivum. True orthologs of Arabidopsis thaliana BRI1 (AtBRI1) from seven-plant species showed sequence identity ranging from 54% to 95% at the protein level. All gene sequences lacked introns, leading to speculation that post-transcriptional processing in TaBRI1 is similar to AtBRI1. Based on in silico analysis, a single copy of BRI1 was present in each of the three wheat genomes on the long arm of chromosome 3. Domain structure of BRI1 orthologs among different taxa showed multiple leucine rich repeats (LRRs), an island domain (ID), a juxtamembrane/transmembrane domain (JTMD), a catalytic kinase domain (KD), C and N-Terminal domains. The KD showed the highest level of conservation while the LRRs and JTMD were most variable. Phosphorylation of residues in the juxtamembrane domain, known to be involved in the activation of the KD, is conserved in TaBRI1. While TaBRI1 has well-defined differences in the ID and LRR domains, many residues involved in ligand binding are conserved. The activation loop present in the KD showed 100% conservation in all taxa. Despite residue differences, hydrophobicity was conserved in the BR binding pocket across taxa, suggesting that function may not differ as drastically as residue identity may suggest. Predicted 3D structure of AtBRI1 and TaBRI1 showed a conserved super helical assembly, a feature essential in protein-protein interactions. An unrooted phylogram showed TaBRI1 in the monocot clade to be distinct from that of dicots. New insight in the structure and functions of BRI1 may help in targeting BR pathway for crop improvement.
Project description:Endoplasmic reticulum (ER)-associated degradation (ERAD) is an integral part of the ER quality-control system that removes toxic misfolded proteins via ubiquitin/proteasome-mediated degradation. Most of our knowledge on ERAD comes from biochemical and genetic studies in yeast and mammalian cells. Although ERAD is known to operate in plant cells, little is known about its molecular components and its biochemical mechanism. A genetic screen for suppressors of the Arabidopsis bri1-9, a weak dwarf mutant caused by ER retention of a structurally defective yet biochemically competent brassinosteroid (BR) receptor BRI1, resulted in identification of the EMS-mutagenized bri1 suppressor 5 (EBS5) gene that encodes an Arabidopsis homolog of the yeast Hrd3/mammalian Sel1L protein known to be involved in ERAD. Loss-of-function ebs5 mutations block the ERAD of bri1-9 and bri1-5, another ER-retained BR receptor. We showed that EBS5 complemented the ERAD defect of the yeast ?hrd3 mutant and interacted with the two mutated BR receptors in plant cells. Using a reverse genetic approach, we discovered that two Arabidopsis homologs of the yeast/mammalian Hrd1, an ER membrane-localized ubiquitin ligase, function redundantly in the ERAD of bri1-9. Together, our results revealed functional roles of two conserved ERAD components in degrading mutated/misfolded receptor-like kinases in Arabidopsis.
Project description:Brassinosteroids (BRs) are steroidal phytohormones that are involved in diverse physiological processes and affect many important traits, such as plant stature, stress tolerance, leaf angle, fertility, and grain filling. BR signaling and biosynthetic pathways have been studied in various plants, such as the model dicot Arabidopsis thaliana; however, relatively little is known about these pathways in monocots.To characterize BR-related processes in the model grass Brachypodium distachyon, we studied the response of these plants to the specific BR biosynthesis inhibitor, propiconazole (Pcz). We found that treatments with Pcz produced a dwarf phenotype in B. distachyon seedlings, similar to that observed in Pcz-treated Arabidopsis plants and in characterized BR-deficient mutants. Through bioinformatics analysis, we identified a list of putative homologs of genes known to be involved in BR biosynthesis and signaling in Arabidopsis, such as DWF4, BR6OX2, CPD, BRI1, and BIN2. Evaluating the response of these genes to Pcz treatments revealed that candidates for BdDWF4, BR6OX2 and, CPD were under feedback regulation. In addition, Arabidopsis plants heterologously expressing BdDWF4 displayed tall statures and elongated petioles, as would be expected in plants with elevated levels of BRs. Moreover, heterologous expression of BdBIN2 in Arabidopsis resulted in dwarfism, suggesting that BdBIN2 functions as a negative regulator of BR signaling. However, the dwarf phenotypes of Arabidopsis bri1-5, a weak BRI1 mutant allele, were not complemented by overexpression of BdBRI1, indicating that BdBRI1 and BRI1 are not functionally equivalent.We identified components of the BR biosynthetic and signaling pathways in Brachypodium, and provided examples of both similarities and differences in the BR biology of these two plants. Our results suggest a framework for understanding BR biology in monocot crop plants such as Zea mays (maize) and Oryza sativa (rice).
Project description:Maintaining active growth and effective immune responses is often costly for a living organism to survive. Fine-tuning the shared cross-regulators is crucial for metazoans and plants to make a trade-off between growth and immunity. The Arabidopsis regulatory receptor-like kinase BAK1 complexes with the receptor kinases FLS2 in bacterial flagellin-triggered immunity and BRI1 in brassinosteroid (BR)-mediated growth. BR homeostasis and signaling unidirectionally modulate FLS2-mediated immune responses at multiple levels. We have shown previously that BIK1, a receptor-like cytoplasmic kinase, is directly phosphorylated by BAK1 and associates with FLS2/BAK1 complex in transducing flagellin signaling. In contrast to its positive role in plant immunity, we report here that BIK1 acts as a negative regulator in BR signaling. The bik1 mutant displays various BR hypersensitive phenotypes accompanied with increased accumulation of de-phosphorylated BES1 proteins and transcriptional regulation of BZR1 and BES1 target genes. BIK1 associates with BRI1, and is released from BRI1 receptor upon BR treatment, which is reminiscent of FLS2-BIK1 complex dynamics in flagellin signaling. The ligand-induced release of BIK1 from receptor complexes is associated with BIK1 phosphorylation. However, in contrast to BAK1-dependent FLS2-BIK1 dissociation, BAK1 is dispensable for BRI1-BIK1 dissociation. Unlike FLS2 signaling which depends on BAK1 to phosphorylate BIK1, BRI1 directly phosphorylates BIK1 to transduce BR signaling. Thus, BIK1 relays the signaling in plant immunity and BR-mediated growth via distinct phosphorylation by BAK1 and BRI1, respectively. Our studies indicate that BIK1 mediates inverse functions in plant immunity and development via dynamic association with specific receptor complexes and differential phosphorylation events.
Project description:Cell elongation in plants is controlled by environmental cues such as light and internal growth regulators including plant steroid hormones, brassinosteroids (BRs). In this study, we found that 3 related receptor-like kinases (RLKs), HERCULES1, THESEUS1, and FERONIA, are transcriptionally induced by BRs and are down-regulated in the loss-of-function BR mutant bri1 and up-regulated in the constitutive BR-response mutant bes1-D. These RLKs belong to the CrRLK family that has 17 members in Arabidopsis. We hypothesize that these RLKs are involved in BR-regulated processes. Although 2 of the RLKs were recently found to mediate male-female interaction during pollen tube reception (FERONIA) and to sense cell wall integrity (THESEUS1), our genetic studies demonstrated that they are required for cell elongation during vegetative growth as herk1 the1 double and fer RNAi mutants displayed striking dwarf phenotypes. The herk1 the1 double mutant enhances the dwarf phenotype of bri1 and partially suppresses bes1-D phenotype, supporting a role of HERK1/THE1 in BR-mediated cell elongation. Microarray experiments demonstrated that these RLKs control the expression of a unique set of genes including those implicated in cell elongation and 16% of the genes affected in herk1 the1 are regulated by BRs. Our results, therefore, identify a previously unknown pathway that functions cooperatively with, but largely independent of the BR pathway to regulate cell elongation. The work establishes a platform to identify other signaling components in this important pathway for plant growth and provides a paradigm to study the coordination of independent pathways in the regulation of a common biological process.