BRI1 Research Articles

A Temperature-Sensitive Misfolded bri1-301 Receptor Requires Its Kinase Activity to Promote Growth.

BRASSINOSTEROID-INSENSITIVE1 (BRI1) is a leucine-rich-repeat receptor-like kinase that functions as the cell surface receptor for brassinosteroids (BRs). Previous studies showed that BRI1 requires its kinase activity to transduce the extracellular BR signal into the nucleus. Among the many reported mutant bri1 alleles, bri1-301 is unique, as its glycine-989-to-isoleucine mutation completely inhibits its kinase activity in vitro but only gives rise to a weak dwarf phenotype compared with strong or null bri1 alleles, raising the question of whether kinase activity is essential for the biological function of BRI1. Here, we show that the Arabidopsis (Arabidopsis thaliana) bri1-301 mutant receptor exhibits weak BR-triggered phosphorylation in vivo and absolutely requires its kinase activity for the limited growth that occurs in the bri1-301 mutant. We also show that bri1-301 is a temperature-sensitive misfolded protein that is rapidly degraded in the endoplasmic reticulum and at the plasma membrane by yet unknown mechanisms. A temperature increase from 22°C to 29°C reduced the protein stability and biochemical activity of bri1-301, likely due to temperature-enhanced protein misfolding. The bri1-301 protein could be used as a model to study the degradation machinery for misfolded membrane proteins with cytosolic structural lesions and the plasma membrane-associated protein quality-control mechanism.

BES1 hinders ABSCISIC ACID INSENSITIVE5 and promotes seed germination in Arabidopsis.

Proper regulation of seed germination is essential for the successful propagation of a plant. The transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) of the abscisic acid (ABA) signaling pathway plays a central role in the inhibition of seed germination. ABI5 is precisely regulated by the core ABA signaling components and multiple other factors. However, the complex regulatory network of ABI5 remains largely unknown. In this study, we determined the biochemical interaction between ABI5 and the BRINSENSITIVE1 (BRI1)-EMS-SUPPRESSOR1 (BES1) transcription factor of the brassinosteroid (BR) signaling pathway, as well as the function of BES1 regulating ABI5 during seed germination in Arabidopsis. BES1 directly interacts with ABI5 both invitro and invivo. The bZIP domain of ABI5, which is responsible for DNA binding, is critical for ABI5 binding to BES1. The interaction of BES1 with ABI5 significantly suppressed the binding of ABI5 to the promoter regions of downstream genes, which resulted in their reduced expression and consequently facilitated seed germination. This study shed new light on the coordination of multiple signaling pathways during seed germination. In particular, BES1 directly binds to ABI5, which interferes with its transcriptional activity and suppresses ABA signaling output.

Phosphocode-dependent functional dichotomy of a common co-receptor in plant signalling.

Multicellular organisms employ cell-surface receptor kinases (RKs) to sense and process extracellular signals. Many plant RKs form ligand-induced complexes with shape-complementary co-receptors for their activation1. The best-characterized co-receptor is BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), which associates with numerous leucine-rich repeat (LRR)-RKs to control immunity, growth, and development2. Here, we report key regulatory events controlling the functionality of BAK1 and, more generally, LRR-RKs. Through a combination of phospho-proteomics and targeted mutagenesis, we identified conserved phosphosites that are required for BAK1 immune function in Arabidopsis thaliana (hereafter Arabidopsis). Strikingly, these phosphosites are not required for BAK1-dependent brassinosteroid (BR)-regulated growth. In addition to revealing a critical role for BAK1 C-terminal tail phosphorylation, we identified a conserved tyrosine phosphosite that may be required for functionality of the majority of Arabidopsis LRR-RKs, and separates them into two distinct functional classes. Our results suggest a phosphocode-based dichotomy of BAK1 functionality in plant signaling, and provide novel insights into receptor kinase activation, which have broad implications for our understanding of how plants respond to their changing environment.

BR-INSENSITIVE1 regulates hydrotropic response by interacting with plasma membrane H+-ATPases in Arabidopsis

ABSTRACTArabidopsis roots sense the moisture gradient in soils and grow toward an area with higher water potential – a process called hydrotropism. Our previous study has shown that the apoplastic proton extrusion in root tips is influenced by brassinosteroids (BRs) receptor BR-INSENSITIVE1 (BRI1) and is crucial for hydrotropic response in Arabidopsis. Here we show that BRI1 interacts directly not only with Arabidopsis plasma membrane H+-ATPase 2 (AHA2) but also with Arabidopsis plasma membrane H+-ATPase 7 (AHA7). Therefore, BRI1 may affect hydrotropic response via regulating the activities of AHA2 and AHA7.

Somatic embryogenesis-related gene expression and functional genomics in mangosteen

The atypical somatic embryogenesis of mangosteen leads us to enquire what is happening at each stage of somatic embryogenesis (SE) at the level of the molecular regulation controlling transition between embryogenic stages. The expression of selected genes somatic embryogenesis receptor-like kinase 1 (SERK1), brassinosteroid insensitive 1-associated kinase 1 (BAK1)/SERK3, brassinosteroid insensitive 1 (BRI1), Leafy cotyledon 1 (LEC1) and Wuschel (WUS) were examined during the stages of SE in mangosteen. Somatic embryos of mangosteen were induced on Murashige and Skoog medium with 3.08 μM 6-benzylaminopurine, 3.18 μM thidiazurone and 60 mg/L glucose. Selected genes were isolated from early, mid and late globular stages of somatic embryo development. Quantitative PCR analyses showed that the early globular stage is characterised by low level of expression of SERK1, BRI1 and WUS and no expression of LEC1. The mid globular stage is characterised by a higher level of expression of the SERK1 and BRI1 genes, while the late globular stage is characterised by a high level of expression of LEC1 and WUS genes with low expression of the BRI1 gene. This demonstrates a clear match between globular stages and the expression of key genes at each stage. Transcriptome data from embryogenic structures showed differentially expressed genes involved in hypothetical signal transduction (brassinosteroid, auxin, cytokinin, jasmonic acid, abscisic acid and glucose) and biosynthetic (glucose and polyamine) pathways. The five selected genes showed interconnection with differentially expressed unigenes (up-regulated) indicating molecular interaction involved in somatic embryogenesis of mangosteen.

Corrigendum: Arabidopsis BRASSINOSTEROID INACTIVATOR2 is a typical BAHD acyltransferase involved in brassinosteroid homeostasis

Brassinosteroids (BRs) are plant-specific steroidal hormones; BR homeostasis is crucial for various aspects of plant growth and development. However, to date, the BR inactivation process has not been thoroughly elucidated. In this study, we identified and characterized a novel BAHD family acyltransferase gene, BRASSINOSTEROID INACTIVATOR2 (BIA2), involved in BR inactivation. BIA2-overexpressing (OE-BIA2) plants displayed typical BR-deficient phenotypes, which were rescued by exogenous BR treatment. Real-time qRT-PCR and transcriptome analyses showed that expression levels of virtually all of the BR biosynthetic genes were increased, whereas the expression of many BR inactivation genes was reduced in OE-BIA2 plants. Root inhibition assays showed that the root growth of OE-BIA2 plants was inhibited. We obtained plants with an intermediate phenotype by crossing the OE-BIA2 plants with BRASSINOSTEROID-INSENSITIVE1 (BRI1)-overexpressing plants. The null BIA2 mutants had longer hypocotyls in the dark. BIA2 was predominantly expressed in roots, and its expression was induced by 24-epibrassinolide or dark treatment, but it exhibited a differential expression pattern compared with its homologue, BIA1. Furthermore, genetic transformation with point-mutant and deleted-BIA2 constructs confirmed that the HXXXD motif is essential for the function of BIA2. Taken together, these findings indicate that BIA2 is a typical BAHD acyltransferase that is involved in BR homeostasis and may inactivate bioactive BRs by esterification, particularly in roots and hypocotyls under dark conditions.

GmBEHL1, a BES1/BZR1 family protein, negatively regulates soybean nodulation

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.

The SERK3 elongated allele defines a role for BIR ectodomains in brassinosteroid signalling.

The leucine-rich repeat receptor kinase (LRR-RK) BRASSINOSTEROID INSENSITIVE 1 (BRI1) requires a shape-complementary SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) co-receptor for brassinosteroid sensing and receptor activation1. Interface mutations that weaken the interaction between receptor and co-receptor in vitro reduce brassinosteroid signalling responses2. The SERK3 elongated (elg) allele3-5 maps to the complex interface and shows enhanced brassinosteroid signalling, but surprisingly no tighter binding to the BRI1 ectodomain in vitro. Here, we report that rather than promoting the interaction with BRI1, the elg mutation disrupts the ability of the co-receptor to interact with the ectodomains of BRI1-ASSOCIATED-KINASE1 INTERACTING KINASE (BIR) receptor pseudokinases, negative regulators of LRR-RK signalling6. A conserved lateral surface patch in BIR LRR domains is required for targeting SERK co-receptors and the elg allele maps to the core of the complex interface in a 1.25 Å BIR3-SERK1 structure. Collectively, our structural, quantitative biochemical and genetic analyses suggest that brassinosteroid signalling complex formation is negatively regulated by BIR receptor ectodomains.

Brassinosteroids regulate vacuolar morphology in root meristem cells of Arabidopsis thaliana

ABSTRACTBrassinosteroids (BRs) are plant hormones that regulate plant development and environmental response. Brz-insensitive-long hypocotyl4 (BIL4) was identified as a positive regulator of BR signaling that interacts with the BR receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), and inhibits vacuolar degradation of BRI1 in Arabidopsis thaliana. Although BIL4 also localizes to the vacuolar membrane, the possible vacuolar function of BIL4 remains unknown. Here, we studied the effect of BIL4 and BR signaling on vacuole shape in root meristem cells using genetic and pharmacological approaches. In BIL4-deficient plants, vacuoles assumed a smaller luminal structure. Treatment with brassinolide (BL), the most active BR, resulted in visibly larger vacuoles, whereas treatment with the BR biosynthesis inhibitor Brz resulted in substantially smaller luminal vacuolar structures. In the bri1 mutant, vacuolar shapes exhibited small and fragmented structures. Our results suggest that BR signaling impacts vacuolar shape.

The systemin receptor SYR1 enhances resistance of tomato against herbivorous insects.

The discovery in tomato of systemin, the first plant peptide hormone1,2, was a fundamental change for the concept of plant hormones. Numerous other peptides have since been shown to play regulatory roles in many aspects of the plant life, including growth, development, fertilization and interactions with symbiotic organisms3-6. Systemin, an 18 amino acid peptide derived from a larger precursor protein 7 , was proposed to act as the spreading signal that triggers systemic defence responses observed in plants after wounding or attack by herbivores1,7,8. Further work culminated in the identification of a leucine-rich repeat receptor kinase (LRR-RK) as the systemin receptor 160 (SR160)9,10. SR160 is a tomato homologue of Brassinosteroid Insensitive 1 (BRI1), which mediates the regulation of growth and development in response to the steroid hormone brassinolide11-13. However, a role of SR160/BRI1 as systemin receptor could not be corroborated by others14-16. Here, we demonstrate that perception of systemin depends on a pair of distinct LRR-RKs termed SYR1 and SYR2. SYR1 acts as a genuine systemin receptor that binds systemin with high affinity and specificity. Further, we show that presence of SYR1, although not decisive for local and systemic wound responses, is important for defence against insect herbivory.

Arabidopsis BRASSINOSTEROID INACTIVATOR2 is a typical BAHD acyltransferase involved in brassinosteroid homeostasis.

Brassinosteroids (BRs) are plant-specific steroidal hormones; BR homeostasis is crucial for various aspects of plant growth and development. However, to date, the BR inactivation process has not been thoroughly elucidated. In this study, we identified and characterized a novel BAHD family acyltransferase gene, BRASSINOSTEROID INACTIVATOR2 (BIA2), involved in BR inactivation. BIA2-overexpressing (OE-BIA2) plants displayed typical BR-deficient phenotypes, which were rescued by exogenous BR treatment. Real-time qRT-PCR and transcriptome analyses showed that expression levels of virtually all of the BR biosynthetic genes were increased, whereas the expression of many BR inactivation genes was reduced in OE-BIA2 plants. Root inhibition assays showed that the root growth of OE-BIA2 plants was inhibited. We obtained plants with an intermediate phenotype by crossing the OE-BIA2 plants with BRASSINOSTEROID-INSENSITIVE1 (BRI1)-overexpressing plants. The null BIA2 mutants had longer hypocotyls in the dark. BIA2 was predominantly expressed in roots, and its expression was induced by 24-epibrassinolide or dark treatment, but it exhibited a differential expression pattern compared with its homologue, BIA1. Furthermore, genetic transformation with point-mutant and deleted-BIA2 constructs confirmed that the HXXXD motif is essential for the function of BIA2. Taken together, these findings indicate that BIA2 is a typical BAHD acyltransferase that is involved in BR homeostasis and may inactivate bioactive BRs by esterification, particularly in roots and hypocotyls under dark conditions.

Regulation of Arabidopsis brassinosteroid receptor BRI1 endocytosis and degradation by plant U-box PUB12/PUB13-mediated ubiquitination

Plants largely rely on plasma membrane (PM)-resident receptor-like kinases (RLKs) to sense extracellular and intracellular stimuli and coordinate cell differentiation, growth, and immunity. Several RLKs have been shown to undergo internalization through the endocytic pathway with a poorly understood mechanism. Here, we show that endocytosis and protein abundance of the Arabidopsis brassinosteroid (BR) receptor, BR INSENSITIVE1 (BRI1), are regulated by plant U-box (PUB) E3 ubiquitin ligase PUB12- and PUB13-mediated ubiquitination. BR perception promotes BRI1 ubiquitination and association with PUB12 and PUB13 through phosphorylation at serine 344 residue. Loss of PUB12 and PUB13 results in reduced BRI1 ubiquitination and internalization accompanied with a prolonged BRI1 PM-residence time, indicating that ubiquitination of BRI1 by PUB12 and PUB13 is a key step in BRI1 endocytosis. Our studies provide a molecular link between BRI1 ubiquitination and internalization and reveal a unique mechanism of E3 ligase-substrate association regulated by phosphorylation.

Investigating the Conformational Dynamics of Plant Protein Kinases

The receptor-like kinases Brassinosteroid Insensitive 1 (BRI1) and BRI1-Associated Kinase 1 (BAK1) are critical to plant growth and development signaling, serving as co-receptors for brassinosteroid hormones. Possibly because of their importance in these pathways, BRI1 and BAK1 are both highly regulated. Upon brassinosteroid binding, the BRI1 and BAK1 kinase domains (KDs) phosphorylate and activate one another, while among other negative regulatory mechanisms, BAK1 is deactivated in vitro by S-glutathionylation in oxidative conditions. However, the molecular mechanisms of BRI1 and BAK1 regulation and activation remain unclear. In order to investigate the dynamics of fully phosphorylated BRI1 and BAK1 KDs and to understand the mechanism of BAK1 deactivation by S-glutathionylation, we performed extensive all-atom molecular dynamics simulations on the BRI1 and BAK1 core KDs along with BAK1 in all viable singly glutathionylated forms. In non-glutathionylated BRI1 and BAK1, we found considerable disorder in the αC helix, a common regulatory domain in protein kinases (PKs) which must be folded in an active conformation. In order to validate our findings, we performed circular dichroism spectroscopy experiments on the BRI1 αC helix peptide, yielding results consistent with our simulations. Using disorder prediction software on all Arabidopsis thaliana PK sequences, we found that αC helix disorder may be a common feature in plant kinomes. From our simulations of glutathionylated BAK1, we found that S-glutathionylation of Cys408, neighboring the αC helix, destabilized active-like BAK1 conformations, while modification of other Cys residues had little effect. Using Kullback-Leibler divergence, we found that Cys408 S-glutationylation had long-range effects on individual residue conformations. Our results suggest that additional factors beyond phosphorylation are required for BRI1 and BAK1 activation, while supporting an allosteric mechanism of BAK1 deactivation by Cys408 S-glutathionylation. To our knowledge, our simulations represent the first insight into atomistic plant PK dynamics.

Brassinosteroids regulate pavement cell growth by mediating BIN2-induced microtubule stabilization.

Brassinosteroids (BRs), a group of plant steroid hormones, play important roles in regulating plant development. The cytoskeleton also affects key developmental processes and a deficiency in BR biosynthesis or signaling leads to abnormal phenotypes similar to those of microtubule-defective mutants. However, how BRs regulate microtubule and cell morphology remains unknown. Here, using liquid chromatography-tandem mass spectrometry, we identified tubulin proteins that interact with Arabidopsis BRASSINOSTEROID INSENSITIVE2 (BIN2), a negative regulator of BR responses in plants. In vitro and in vivo pull-down assays confirmed that BIN2 interacts with tubulin proteins. High-speed co-sedimentation assays demonstrated that BIN2 also binds microtubules. The Arabidopsis genome also encodes two BIN2 homologs, BIN2-LIKE 1 (BIL1) and BIL2, which function redundantly with BIN2. In the bin2-3 bil1 bil2 triple mutant, cortical microtubules were more sensitive to treatment with the microtubule-disrupting drug oryzalin than in wild-type, whereas in the BIN2 gain-of-function mutant bin2-1, cortical microtubules were insensitive to oryzalin treatment. These results provide important insight into how BR regulates plant pavement cell and leaf growth by mediating the stabilization of microtubules by BIN2.

Vacuole Integrity Maintained by DUF300 Proteins Is Required for Brassinosteroid Signaling Regulation

Brassinosteroid (BR) hormone signaling controls multiple processes during plant growth and development and is initiated at the plasma membrane through the receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) together with co-receptors such as BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). BRI1 abundance is regulated by endosomal recycling and vacuolar targeting, but the role of vacuole-related proteins in BR receptor dynamics and BR responses remains elusive. Here, we show that the absence of two DUF300 domain-containing tonoplast proteins, LAZARUS1 (LAZ1) and LAZ1 HOMOLOG1 (LAZ1H1), causes vacuole morphology defects, growth inhibition, and constitutive activation of BR signaling. Intriguingly, tonoplast accumulation of BAK1 was substantially increased and appeared causally linked to enhanced BRI1 trafficking and degradation in laz1 laz1h1 plants. Since unrelated vacuole mutants exhibited normal BR responses, our findings indicate that DUF300 proteins play distinct roles in the regulation of BR signaling by maintaining vacuole integrity required to balance subcellular BAK1 pools and BR receptor distribution.

Allosteric Control of a Plant Receptor Kinase through S-Glutathionylation

Growing evidence supports the importance of protein S-glutathionylation as a regulatory post-translational modification with functional consequences for proteins. Discoveries of redox-state-dependent protein kinase S-glutathionylation have fueled discussion of redox-sensitive signaling. Following previously published experimental evidence for S-glutathionylation induced deactivation of the Arabidopsis thaliana kinase BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR-LIKE KINASE 1 (BAK1), we investigated the consequences of S-glutathionylation on the equilibrium conformational ensemble of BAK1 using all-atom molecular dynamics simulations. We found that glutathionylation of C408 allosterically destabilizes the active-like state of BAK1 and stabilizes an inactive conformation known to recur in protein kinases. Glutathionylation of C408 also has structural consequences throughout the BAK1 kinase domain, whereas glutathionylation of C353 in the N-lobe and C374 near the ATP-binding site have few notable effects on BAK1 compared with the unmodified protein. Our results suggest an allosteric mechanism for inhibition of BAK1 by C408 S-glutathionylation, and more generally, support the notion of protein kinase S-glutathionylation as a means of redox signaling in plant cells.

Functional characterisation of brassinosteroid receptor MtBRI1 in Medicago truncatula

Brassinosteroids are phytohormones involved in plant development and physiological processes. Brassinosteroids Insensitive 1 (BRI1) is required for BR perception and initiation of subsequent signal transduction in Arabidopsis. In this study, the orthologue of BRI1 in the model legume species Medicago truncatula, MtBRI1, was identified and characterised. Three allelic Tnt1 insertion mutants, mtbri1-1, mtbri1-2, and mtbri1-3, were obtained from the M. truncatula Tnt1 insertion population. mtbri1 mutants displayed characteristic bri1 mutant phenotypes: extreme dwarfness, dark green curled leaves, short primary roots, less lateral roots, and insensitive to exogenous brassinolide (BL). Moreover, mtbri1 mutants show decreased total nodule number and defects in nitrogen fixation. MtBRI1 is able to complement an Arabidopsis BRI1 mutant, bri1-5. Similar to the interaction of BRI1 and BAK1 in Arabidopsis, MtBRI1 interacts with MtSERK1 in vivo. Global gene expression profiling revealed that the expression of BR biosynthesis genes and SAUR genes are significantly altered in mtbri1 mutants. MapMan analysis indicated that genes involved in signaling, hormone, cell wall, and biotic stress responses are over-represented in differentially expressed genes. Taken together, the results indicate that MtBRI1 is the BR receptor in M. truncatula and that BR signaling may play a conserved role in balancing plant growth and defenses.

Enhancing Brassinosteroid Signaling via Overexpression of Tomato (Solanum lycopersicum) SlBRI1 Improves Major Agronomic Traits.

Brassinosteroids (BRs) play important roles in plant growth, development, and stress responses through the receptor, Brassinosteroid-insensitive 1 (BRI1), which perceives BRs and initiates BR signaling. There is considerable potential agricultural value in regulating BR signaling in crops. In this study, we investigated the effects of overexpressing the tomato (Solanum lycopersicum) BRI1 gene, SlBRI1, on major agronomic traits, such as seed germination, vegetative growth, fruit ethylene production, carotenoid accumulation, yield, and quality attributes. SlBRI1 overexpression enhanced the endogenous BR signaling intensity thereby increasing the seed germination rate, lateral root number, hypocotyl length, CO2 assimilation, plant height, and flower size. The transgenic plants also showed an increase in fruit yield and fruit number per plant, although the mean weight of individual fruit was reduced, compared with wild type. SlBRI1 overexpression also promoted fruit ripening and ethylene production, and caused an increase in levels of carotenoids, ascorbic acid, soluble solids, and soluble sugars during fruit ripening. An increased BR signaling intensity mediated by SlBRI1 overexpression was therefore positively correlated with carotenoid accumulation and fruit nutritional quality. Our results indicate that enhancing BR signaling by overexpression of SlBRI1 in tomato has the potential to improve multiple major agronomic traits.

Tyrosine-610 in the Receptor Kinase BAK1 Does Not Play a Major Role in Brassinosteroid Signaling or Innate Immunity.

The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1) functions as a co-receptor with several receptor kinases including the brassinosteroid (BR) receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR), respectively, which are involved in immunity. BAK1 is a dual specificity protein kinase that can autophosphorylate on serine, threonine and tyrosine residues. It was previously reported that phosphorylation of Tyr-610 in the carboxy-terminal domain of BAK1 is required for its function in BR signaling and immunity. However, the functional role of Tyr-610 in vivo has recently come under scrutiny. Therefore, we have generated new BAK1 (Y610F) transgenic plants for functional studies. We first produced transgenic Arabidopsis lines expressing BAK1 (Y610F)-Flag in the homozygous bak1-4 bkk1-1 double null background. In a complementary approach, we expressed untagged BAK1 and BAK1 (Y610F) in the bak1-4 null mutant. Neither BAK1 (Y610F) transgenic line had any obvious growth phenotype when compared to wild-type BAK1 expressed in the same background. In addition, the BAK1 (Y610F)-Flag plants responded similarly to plants expressing BAK1-Flag in terms of brassinolide (BL) inhibition of root elongation, and there were only minor changes in gene expression between the two transgenic lines as monitored by microarray analysis and quantitative real-time PCR. In terms of plant immunity, there were no significant differences between plants expressing BAK1 (Y610F)-Flag and BAK1-Flag in the growth of the non-pathogenic hrpA- mutant of Pseudomonas syringae pv. tomato DC3000. Furthermore, untagged BAK1 (Y610F) transgenic plants were as responsive as plants expressing BAK1 (in the bak1-4 background) and wild-type Col-0 plants toward treatment with the EF-Tu- and flagellin-derived peptide epitopes elf18- and flg22, respectively, as measured by reactive oxygen species production, mitogen-activated protein kinase activation, and seedling growth inhibition. These new results do not support any involvement of Tyr-610 phosphorylation in either BR or immune signaling.

High-temperature promotion of callus formation requires the BIN2-ARF-LBD axis in Arabidopsis.

The auxin-brassinosteroid interaction involving the BIN2-ARF-LBD axis plays a key role in temperature-dependent callus formation in Arabidopsis. An extensive web of multiple hormone signaling pathways underlies callus formation. Here, we report that a brassinosteroid (BR) signaling component, BR-INSENSITIVE 2 (BIN2), positively regulates callus formation. The BIN2 kinase promotes transcriptional activities of AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 and subsequently activates expression of LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16) and LBD29 during callus formation. Consistently, the BIN2 activity is dependent on ARFs in the control of callus formation. Notably, this auxin-BR interaction is particularly relevant in temperature-dependent callus formation. Misexpression of BIN2 and ARFs resulted in the temperature insensitivity of callus formation. These results indicate that the BIN2-ARF-LBD axis plays a key role in temperature-dependent callus formation in Arabidopsis.