Salicylic acid-activated BIN2 phosphorylation of TGA3 promotes Arabidopsis PR gene expression and disease resistance.

The plant defense hormone, salicylic acid (SA), plays essential roles in immunity and systemic acquired resistance. Salicylic acid induced by the pathogen is perceived by the receptor nonexpressor of pathogenesis-related genes 1 (NPR1), which is recruited by TGA transcription factors to induce the expression of pathogenesis-related (PR) genes. However, the mechanism by which post-translational modifications affect TGA's transcriptional activity by salicylic acid signaling/pathogen infection is not well-established. Here, we report that the loss-of-function mutant of brassinosteroid insensitive2 (BIN2) and its homologs, bin2-3 bil1 bil2, causes impaired pathogen resistance and insensitivity to SA-induced PR gene expression, whereas the gain-of-function mutant, bin2-1, exhibited enhanced SA signaling and immunity against the pathogen. Our results demonstrate that salicylic acid activates BIN2 kinase, which in turn phosphorylates TGA3 at Ser33 to enhance TGA3 DNA binding ability and NPR1-TGA3 complex formation, leading to the activation of PR gene expression. These findings implicate BIN2 as a new component of salicylic acid signaling, functioning as a key node in balancing brassinosteroid-mediated plant growth and SA-induced immunity.

Phosphorylation of the transcription factor SlBIML1 by SlBIN2 kinases delays flowering in tomato.

Brassinosteroids (BRs) are well known for their important role in the regulation of plant growth and development. Plants with deficiency in BR signaling show delayed plant development and exhibit late flowering phenotypes. However, the precise mechanisms involved in this process require investigation. In this study, we cloned homologs of BRASSINOSTEROID INSENSITIVE 2 (SlBIN2), the GSK3-like protein kinase in tomato (Solanum lycopersicum). We characterized growth-related processes and phenotypic changes in the transgenic lines and found that SlBIN2s transgenic lines have delayed development and slow growing phenotypes. SlBIN2s work redundantly to negatively regulate BR signaling in tomato. Furthermore, the transcription factor SlBIN2.1-INTERACTING MYB-LIKE 1 (SlBIML1) was identified as a downstream substrate of SlBIN2s that SlBIN2s interact with and phosphorylate to synergistically regulate tomato developmental processes. Specifically, SlBIN2s modulated protein stability of SlBIML1 by phosphorylating multiple amino acid residues, including the sites Thr266 and Thr280. This study reveals a branch of the BR signaling pathway that regulates the vegetative growth phase and delays floral transition in tomato without the feedback affecting BR signaling. This information enriches our understanding of the downstream transduction pathway of BR signaling and provides potential targets for adjusting tomato flowering time.

The F-box Protein KIB1 Mediates Brassinosteroid-Induced Inactivation and Degradation of GSK3-like Kinases in Arabidopsis.

The glycogen synthase kinase-3 (GSK3) family kinases are central cellular regulators highly conserved in all eukaryotes. In Arabidopsis, the GSK3-like kinase BIN2 phosphorylates a range of proteins to control broad developmental processes, and BIN2 is degraded through unknown mechanism upon receptor kinase-mediated brassinosteroid (BR) signaling. Here we identify KIB1 as an F-box E3 ubiquitin ligase that promotes the degradation of BIN2 while blocking its substrate access. Loss-of-function mutations of KIB1 and its homologs abolished BR-induced BIN2 degradation and caused severe BR-insensitive phenotypes. KIB1 directly interacted with BIN2 in a BR-dependent manner and promoted BIN2 ubiquitination in vitro. Expression of an F-box-truncated KIB1 caused BIN2 accumulation but dephosphorylation of its substrate BZR1 and activation of BR responses because KIB1 blocked BIN2 binding to BZR1. Our study demonstrates that KIB1 plays an essential role in BR signaling by inhibiting BIN2 through dual mechanisms of blocking substrate access and promoting degradation.

Recent advances in understanding the regulatory mechanism of plasma membrane H+-ATPase through the brassinosteroid signaling pathway.

The polyhydroxylated steroid phytohormone brassinosteroids (BRs) control many aspects of plant growth, development and responses to environmental changes. Plasma membrane (PM) H+-ATPase, the well-known PM proton pump, is a central regulator in plant physiology, which mediates not only plant growth and development, but also adaptation to stresses. Recent studies highlight that PM H+-ATPase is at least partly regulated via the BR signaling. Firstly, the BR cell surface receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and multiple key components of BR signaling directly or indirectly influence PM H+-ATPase activity. Secondly, the SMALL AUXIN UP RNA (SAUR) gene family physically interacts with BRI1 to enhance organ development of Arabidopsis by activating PM H+-ATPase. Thirdly, RNA-sequencing (RNA-seq) assays showed that the expression of some SAUR genes is upregulated under the light or sucrose conditions, which is related to the phosphorylation state of the penultimate residue of PM H+-ATPase in a time-course manner. In this review, we describe the structural and functional features of PM H+-ATPase, and summarize recent progress toward understanding the regulatory mechanism of PM H+-ATPase by BRs, and briefly introduce how PM H+-ATPase activity is modulated by its own biterminal regions and the post-translational modifications.

ATBS1-INTERACTING FACTOR 2 Positively Regulates Freezing Tolerance via INDUCER OF CBF EXPRESSION 1/C-REPEAT BINDING FACTOR-Induced Cold Acclimation Pathway.

The INDUCER OF CBF EXPRESSION 1/C-REPEAT BINDING FACTOR (ICE1/CBF) pathway plays a crucial role in plant responses to cold stress, impacting growth and development. Here, we demonstrated that ATBS1-INTERACTING FACTOR 2 (AIF2), a non-DNA-binding basic helix-loop-helix transcription factor, positively regulates freezing tolerance through the ICE1/CBF-induced cold tolerance pathway in Arabidopsis. Cold stress transcriptionally upregulated AIF2 expression and induced AIF2 phosphorylation, thereby stabilizing the AIF2 protein during early stages of cold acclimation. The AIF2 loss-of-function mutant, aif2-1, exhibited heightened sensitivity to freezing before and after cold acclimation. In contrast, ectopic expression of AIF2, but not the C-terminal-deleted AIF2 variant, restored freezing tolerance. AIF2 enhanced ICE1 stability during cold acclimation and promoted the transcriptional expression of CBFs and downstream cold-responsive genes, ultimately enhancing plant tolerance to freezing stress. MITOGEN-ACTIVATED PROTEIN KINASES 3 and 6 (MPK3/6), known negative regulators of freezing tolerance, interacted with and phosphorylated AIF2, subjecting it to protein degradation. Furthermore, transient co-expression of MPK3/6 with AIF2 and ICE1 downregulated AIF2/ICE1-induced transactivation of CBF2 expression. AIF2 interacted preferentially with BRASSINOSTEROID-INSENSITIVE 2 (BIN2) and MPK3/6 during the early and later stages of cold acclimation, respectively, thereby differentially regulating AIF2 activity in a cold acclimation time-dependent manner. Moreover, AIF2 acted additively in a gain-of-function mutant of BRASSINAZOLE-RESISTANT 1 (BZR1; bzr1-1D) and a triple knockout mutant of BIN2 and its homologs (bin2bil1bil2) to induce CBFs-mediated freezing tolerance. This suggests that cold-induced AIF2 coordinates freezing tolerance along with BZR1 and BIN2, key positive and negative components, respectively, of brassinosteroid signaling pathways.

BRASSINOSTEROID-INSENSITIVE 2 regulates salt stress tolerance in Arabidopsis by promoting AGL16 activity.

Salt stress is a negative environmental factors to affecting plants. Salinity inhibits seed germination and root growth, which reduces the biomass of agricultural plants. BRASSINOSTEROID-INSENSITIVE2 (BIN2) functions as a signalling hub to integrate the perception and transduction of plant growth and stress tolerance by the phosphorylation of target proteins. However, only a small number of target molecules have been discovered thus far. In this study, we present evidence that BIN2 controls the post-transcriptional activity of AGL16. BIN2 interacts and phosphorylates AGL16, which increases AGL16 stability and transcriptional activity. Genetic testing showed that the agl16 mutant can restore the reduction in the seed germination rate and primary root growth of the bin2-1 mutant, while the overexpression of AGL16 in the bin2-3bil1bil2 mutant reduced the salt tolerance compared with bin2-3bil1bil2 in response to salt stress. Taken together, our data identify a BIN2-AGL16 core protein module that is mediates the inhibition of seed germination and primary root growth under salt stress.

Brassinosteroid signaling positively regulates abscisic acid biosynthesis in response to chilling stress in tomato.

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.

Integration of multi-omics data reveals interplay between brassinosteroid and Target of Rapamycin Complex signaling in Arabidopsis.

Brassinosteroids (BRs) and Target of Rapamycin Complex (TORC) are two major actors coordinating plant growth and stress responses. Brassinosteroids function through a signaling pathway to extensively regulate gene expression and TORC is known to regulate translation and autophagy. Recent studies have revealed connections between these two pathways, but a system-wide view of their interplay is still missing. We quantified the level of 23 975 transcripts, 11 183 proteins, and 27 887 phosphorylation sites in wild-type Arabidopsis thaliana and in mutants with altered levels of either BRASSINOSTEROID INSENSITIVE 2 (BIN2) or REGULATORY ASSOCIATED PROTEIN OF TOR 1B (RAPTOR1B), two key players in BR and TORC signaling, respectively. We found that perturbation of BIN2 or RAPTOR1B levels affects a common set of gene-products involved in growth and stress responses. Furthermore, we used the multi-omic data to reconstruct an integrated signaling network. We screened 41 candidate genes identified from the reconstructed network and found that loss of function mutants of many of these proteins led to an altered BR response and/or modulated autophagy activity. Altogether, these results establish a predictive network that defines different layers of molecular interactions between BR- or TORC-regulated growth and autophagy.

HOP1 and HOP2 are involved in salt tolerance by facilitating the brassinosteroid-related nucleo-cytoplasmic partitioning of the HSP90-BIN2 complex.

The co-chaperone heat shock protein (HSP)70-HSP90 organizing protein (HOP) is involved in plant thermotolerance. However, its function in plant salinity tolerance was not yet studied. We found that Arabidopsis HOP1 and HOP2 play critical roles in salt tolerance by affecting the nucleo-cytoplasmic partitioning of HSP90 and brassinosteroid-insensitive 2 (BIN2). A hop1/2 double mutant was hypersensitive to salt-stress. Interestingly, this sensitivity was remedied by exogenous brassinolide application, while the application of brassinazole impeded growth of both wild-type (WT) and hop1/2 plants under normal and salt stress conditions. This suggested that the insufficient brassinosteroid (BR) content was responsible for the salt-sensitivity of hop1/2. After WT was transferred to salt stress conditions, HOP1/2, BIN2 and HSP90 accumulated in the nucleus, brassinazole-resistant 1 (BZR1) was phosphorylated and accumulated in the cytoplasm, and BR content significantly increased. This initial response resulted in dephosphorylation of BZR1 and BR response. This dynamic regulation of BR content was impeded in salt-stressed hop1/2. Thus, we propose that HOP1 and HOP2 are involved in salt tolerance by affecting BR signalling.

Brassinosteroid Signaling Downstream Suppressor BIN2 Interacts with SLFRIGIDA-LIKE to Induce Early Flowering in Tomato.

Brassinosteroid (BR) signaling is very important in plant developmental processes. Its various components interact to form a signaling cascade. These components are widely studied in Arabidopsis; however, very little information is available on tomatoes. Brassinosteroid Insensitive 2 (BIN2), the downstream suppressor of BR signaling, plays a critical role in BR signal pathway, while FRIGIDA as a key suppressor of Flowering Locus C with overexpression could cause early flowering; however, how the BR signaling regulates FRIGIDA homologous protein to adjust flowering time is still unknown. This study identified 12 FRIGIDA-LIKE proteins with a conserved FRIGIDA domain in tomatoes. Yeast two-hybrid and BiFC confirmed that SlBIN2 interacts with 4 SlFRLs, which are sub-cellularly localized in the nucleus. Tissue-specific expression of SlFRLs was observed highly in young roots and flowers. Biological results revealed that SlFRLs interact with SlBIN2 to regulate early flowering. Further, the mRNA level of SlBIN2 also increased in SlFRL-overexpressed lines. The relative expression of SlCPD increased upon SlFRL silencing, while SlDWF and SlBIN2 were decreased, both of which are important for BR signaling. Our research firstly provides molecular evidence that BRs regulate tomato flowering through the interaction between SlFRLs and SlBIN2. This study will promote the understanding of the specific pathway essential for floral regulation.

BIN2 negatively regulates plant defence against Verticillium dahliae in Arabidopsis and cotton.

Verticillium wilt is caused by the soil-borne vascular pathogen Verticillium dahliae, and affects a wide range of economically important crops, including upland cotton (Gossypium hirsutum). Previous studies showed that expression levels of BIN2 were significantly down-regulated during infestation with V. dahliae. However, the underlying molecular mechanism of BIN2 in plant regulation against V. dahliae remains enigmatic. Here, we characterized a protein kinase GhBIN2 from Gossypium hirsutum, and identified GhBIN2 as a negative regulator of resistance to V. dahliae. The Verticillium wilt resistance of Arabidopsis and cotton were significantly enhanced when BIN2 was knocked down. Constitutive expression of BIN2 attenuated plant resistance to V. dahliae. We found that BIN2 regulated plant endogenous JA content and influenced the expression of JA-responsive marker genes. Further analysis revealed that BIN2 interacted with and phosphorylated JAZ family proteins, key repressors of the JA signalling pathway in both Arabidopsis and cotton. Spectrometric analysis and site-directed mutagenesis showed that BIN2 phosphorylated AtJAZ1 at T196, resulting in the degradation of JAZ proteins. Collectively, these results show that BIN2 interacts with JAZ proteins and plays a negative role in plant resistance to V. dahliae. Thus, BIN2 may be a potential target gene for genetic engineering against Verticillium wilt in crops.

Exogenous Application of Low-Concentration Sugar Enhances Brassinosteroid Signaling for Skotomorphogenesis by Promoting BIN2 Degradation.

In plants, seedling growth is subtly controlled by multiple environmental factors and endogenous phytohormones. The cross-talk between sugars and brassinosteroid (BR) signaling is known to regulate plant growth; however, the molecular mechanisms that coordinate hormone-dependent growth responses with exogenous sucrose in plants are incompletely understood. Skotomorphogenesis is a plant growth stage with rapid elongation of the hypocotyls. In the present study, we found that low-concentration sugars could improve skotomorphogenesis in a manner dependent on BR biosynthesis and TOR activation. However, accumulation of BZR1 in bzr1-1D mutant plants partially rescued the defects of skotomorphogenesis induced by the TOR inhibitor AZD, and these etiolated seedlings displayed a normal phenotype like that of wild-type seedlings in response to both sucrose and non-sucrose treatments, thereby indicating that accumulated BZR1 sustained, at least partially, the sucrose-promoted growth of etiolated seedlings (skotomorphogenesis). Moreover, genetic evidence based on a phenotypic analysis of bin2-3bil1bil2 triple-mutant and gain-of-function bin2-1 mutant plant indicated that BIN2 inactivation was conducive to skotomorphogenesis in the dark. Subsequent biochemical and molecular analyses enabled us to confirm that sucrose reduced BIN2 levels via the TOR-S6K2 pathway in etiolated seedlings. Combined with a determination of the cellulose content, our results indicated that sucrose-induced BIN2 degradation led to the accumulation of BZR1 and the enhancement of cellulose synthesis, thereby promoting skotomorphogenesis, and that BIN2 is the converging node that integrates sugar and BR signaling.

A gain-of-function mutation in Brassinosteroid-insensitive 2 alters Arabidopsis floral organ development by altering auxin levels.

KEY MESSAGE: Auxin can alter the fertility of bin2-1 plants and depends on the expression of SHY2. Brassinosteroids (BRs) play important roles in plant growth and developmental processes. By systematically evaluating the phenotypes of BR biosynthesis and BR signaling mutants, researchers have reported that BRs positively regulate floral development. In this study, we found that brassinosteroid-insensitive 2 (bin2-1) and short-hypocotyl 2 (shy2-2) mutants exhibited significantly reduced fertility. These mutants had short inflorescences, decreased floral organ length (short petals, stamens, carpels, and stigmas), and short siliques. Exogenous auxin applications could partially rescue the shortened length of the floral organs and siliques of the bin2-1 mutants. Additional experiments revealed that a lack of SHY2 activity increased the fertility of the bin2-1 mutants. A search for downstream affected genes revealed that auxin influences the expression of ARFs and PINs in the bin2-1 mutants, suggesting that auxin plays a major role in the regulation of bin2-1 plant fertility. Thus, BIN2 plays a role in fertility by affecting auxin levels, mainly by altering the expression of SHY2.

Reactive oxygen species-mediated BIN2 activity revealed by single-molecule analysis.

Much evidence has shown that reactive oxygen species (ROS) regulate several plant hormone signaling cascades, but little is known about the real-time kinetics and the underlying molecular mechanisms of the target proteins in the brassinosteroid (BR) signaling pathway. In this study, we used single-molecule techniques to investigate the true signaling timescales of the major BR signaling components BRI1-EMS-SUPPRESSOR 1 (BES1) and BRASSINOSTEROID INSENSITIVE 2 (BIN2) of Arabidopsis thaliana. The rate constants of BIN2 associating with ATP and phosphorylating BES1 were determined to be 0.7 ± 0.4 mM-1  s-1 and 2.3 ± 1.4 s-1 , respectively. Interestingly, we found that the interaction of BIN2 and BES1 was oxygen-dependent, and oxygen can directly modify BIN2. The activity of BIN2 was switched on via modification of specific cysteine (Cys) residues, including C59, C95, C99 and C162. The mutation of these Cys residues inhibited the BR signaling outputs. These findings demonstrate the power of using single-molecule techniques to study the dynamic interactions of signaling components, which is difficult to be discovered by conventional physiological and biochemical methods.

Histone deacetylase HDA6 enhances brassinosteroid signaling by inhibiting the BIN2 kinase.

Glycogen synthase kinase 3 (GSK3)-like kinases play important roles in brassinosteroid (BR), abscisic acid, and auxin signaling to regulate many aspects of plant development and stress responses. The Arabidopsis thaliana GSK3-like kinase BR-INSENSITIVE 2 (BIN2) acts as a key negative regulator in the BR signaling pathway, but the mechanisms regulating BIN2 function remain unclear. Here we report that the histone deacetylase HDA6 can interact with and deacetylate BIN2 to repress its kinase activity. The hda6 mutant showed a BR-repressed phenotype in the dark and was less sensitive to BR biosynthesis inhibitors. Genetic analysis indicated that HDA6 regulates BR signaling through BIN2. Furthermore, we identified K189 of BIN2 as an acetylated site, which can be deacetylated by HDA6 to influence BIN2 activity. Glucose can affect the acetylation level of BIN2 in plants, indicating a connection to cellular energy status. These findings provide significant insights into the regulation of GSK3-like kinases in plant growth and development.

Brassinosteroid-Induced Transcriptional Repression and Dephosphorylation-Dependent Protein Degradation Negatively Regulate BIN2-Interacting AIF2 (a BR Signaling-Negative Regulator) bHLH Transcription Factor.

Brassinosteroids (BRs) are plant polyhydroxy-steroids that play important roles in plant growth and development via extensive signal integration through direct interactions between regulatory components of different signaling pathways. Recent studies have shown that diverse helix-loop-helix/basic helix-loop-helix (HLH/bHLH) family proteins are actively involved in control of BR signaling pathways and interact with other signaling pathways. In this study, we show that ATBS1-INTERACTING FACTOR 2 (AIF2), a nuclear-localized atypical bHLH transcription factor, specifically interacts with BRASSINOSTEROID-INSENSITIVE 2 (BIN2) among other BR signaling molecules. Overexpression of AIF2 down-regulated transcript expression of growth-promoting genes, thus resulting in retardation of growth. AIF2 renders plants hyposensitive to BR-induced root growth inhibition, but shows little effects on BR-promoted hypocotyl elongation. Notably, AIF2 was dephosphorylated by BR, and the dephosphorylated AIF2 was subject to proteasome-mediated degradation. AIF2 degradation was greatly induced by BR and ABA, but relatively slightly by other hormones such as auxin, gibberellin, cytokinin and ethylene. Moreover, AIF2 transcription was significantly suppressed by a BRI1/BZR1-mediated BR signaling pathway through a direct binding of BRASSINAZOLE RESISTANT 1 (BZR1) to the BR response element (BRRE) region of the AIF2 promoter. In conclusion, our study suggests that BIN2-driven AIF2 phosphorylation could augment the BIN2/AIF2-mediated negative circuit of BR signaling pathways, and the BR-induced transcriptional repression and protein degradation negatively regulate AIF2 transcription factor, reinforcing the BZR1/BES1-mediated positive BR signaling pathway.

Identification of brassinosteroid genes in Brachypodium distachyon.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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).

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

MAIN CONCLUSION: 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.

Brassinosteriod Insensitive 2 (BIN2) acts as a downstream effector of the Target of Rapamycin (TOR) signaling pathway to regulate photoautotrophic growth in Arabidopsis.

The components of the target of rapamycin (TOR) signaling pathway have been well characterized in heterotrophic organisms from yeast to humans. However, because of rapamycin insensitivity, embryonic lethality in tor null mutants and a lack of reliable ways of detecting TOR protein kinase in higher plants, the key players upstream and downstream of TOR remain largely unknown in plants. Using engineered rapamycin-sensitive Binding Protein 12-2 (BP12-2) plants, the present study showed that combined treatment with rapamycin and active-site TOR inhibitors (asTORis) results in synergistic inhibition of TOR activity and plant growth in Arabidopsis. Based on this system, we revealed that TOR signaling plays a crucial role in modulating the transition from heterotrophic to photoautotrophic growth in Arabidopsis. Ribosomal protein S6 kinase 2 (S6K2) was identified as a direct downstream target of TOR, and the growth of TOR-suppressed plants could be rescued by up-regulating S6K2. Systems, genetic, and biochemical analyses revealed that Brassinosteriod Insensitive 2 (BIN2) acts as a novel downstream effector of S6K2, and the phosphorylation of BIN2 depends on TOR-S6K2 signaling in Arabidopsis. By combining pharmacological with genetic and biochemical approaches, we determined that the TOR-S6K2-BIN2 signaling pathway plays important roles in regulating the photoautotrophic growth of Arabidopsis.

Transcription factor HAT1 is phosphorylated by BIN2 kinase and mediates brassinosteroid repressed gene expression in Arabidopsis.

Plant steroid hormones, brassinosteroids (BRs), play essential roles in modulating cell elongation, vascular differentiation, senescence and stress responses. BRs signal through plasma membrane-localized receptor and other components to modulate the BES1/BZR1 (BRI1-EMS SUPPRESSOR 1/BRASSINAZOLE RESISTANT 1) family of transcription factors that modulate thousands of target genes. Arabodopsis thaliana homeodomain-leucine zipper protein 1 (HAT1), which encodes a homeodomain-leucine zipper (HD-Zip) class II transcription factor, was identified through chromatin immunoprecipitation (ChIP) experiments as a direct target gene of BES1. Loss-of-function and gain-of-function mutants of HAT1 display altered BR responses. HAT1 and its close homolog HAT3 act redundantly, as the double mutant hat1 hat3 displayed a reduced BR response that is stronger than the single mutants alone. Moreover, hat1 hat3 enhanced the phenotype of a weak allele of the BR receptor mutant bri1 and suppressed the phenotype of constitutive BR response mutant bes1-D. These results suggest that HAT1 and HAT3 function to activate BR-mediated growth. Expression levels of several BR-repressed genes are increased in hat1 hat3 and reduced in HAT1OX, suggesting that HAT1 functions to repress the expression of a subset of BR target genes. HAT1 and BES1 bind to a conserved homeodomain binding (HB) site and BR response element (BRRE) respectively, in the promoters of some BR-repressed genes. BES1 and HAT1 interact with each other and cooperate to inhibit BR-repressed gene expression. Furthermore, HAT1 can be phosphorylated and stabilized by GSK3 (GLYCOGEN SYNTHASE KINASE 3)-like kinase BIN2 (BRASSINOSTEROID-INSENSITIVE 2), a well established negative regulator of the BR pathway. Our results thus revealed a previously unknown mechanism by which BR signaling modulates BR-repressed gene expression and coordinates plant growth.