GSK3-like kinase BIN2 phosphorylates RD26 to potentiate drought signaling in Arabidopsis.

Plant steroid hormones brassinosteroids (BRs) regulate plant growth and development at many different levels. Recent research has revealed that stress-responsive NAC (petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) transcription factor RD26 is regulated by BR signaling and antagonizes BES1 in the interaction between growth and drought stress signaling. However, the upstream signaling transduction components that activate RD26 during drought are still unknown. Here, we demonstrate that the function of RD26 is modulated by GSK3-like kinase BIN2 and protein phosphatase 2C ABI1. We show that ABI1, a negative regulator in abscisic acid (ABA) signaling, dephosphorylates and destabilizes BIN2 to inhibit BIN2 kinase activity. RD26 protein is stabilized by ABA and dehydration in a BIN2-dependent manner. BIN2 directly interacts and phosphorylates RD26 in vitro and in vivo. BIN2 phosphorylation of RD26 is required for RD26 transcriptional activation on drought-responsive genes. RD26 overexpression suppressed the brassinazole (BRZ)  insensitivity of BIN2 triple mutant bin2 bil1 bil2, and BIN2 function is required for the drought tolerance of RD26 overexpression plants. Taken together, our data suggest a drought signaling mechanism in which drought stress relieves ABI1 inhibition of BIN2, allowing BIN2 activation. Sequentially, BIN2 phosphorylates and stabilizes RD26 to promote drought stress response.

Arabidopsis WRKY46, WRKY54, and WRKY70 Transcription Factors Are Involved in Brassinosteroid-Regulated Plant Growth and Drought Responses.

Plant steroid hormones, brassinosteroids (BRs), play important roles in growth and development. BR signaling controls the activities of BRASSINOSTERIOD INSENSITIVE1-EMS-SUPPRESSOR1/BRASSINAZOLE-RESISTANT1 (BES1/BZR1) family transcription factors. Besides the role in promoting growth, BRs are also implicated in plant responses to drought stress. However, the molecular mechanisms by which BRs regulate drought response have just begun to be revealed. The functions of WRKY transcription factors in BR-regulated plant growth have not been established, although their roles in stress responses are well documented. Here, we found that three Arabidopsis thaliana group III WRKY transcription factors, WRKY46, WRKY54, and WRKY70, are involved in both BR-regulated plant growth and drought response as the wrky46 wrky54 wrky70 triple mutant has defects in BR-regulated growth and is more tolerant to drought stress. RNA-sequencing analysis revealed global roles of WRKY46, WRKY54, and WRKY70 in promoting BR-mediated gene expression and inhibiting drought responsive genes. WRKY54 directly interacts with BES1 to cooperatively regulate the expression of target genes. In addition, WRKY54 is phosphorylated and destabilized by GSK3-like kinase BR-INSENSITIVE2, a negative regulator in the BR pathway. Our results therefore establish WRKY46/54/70 as important signaling components that are positively involved in BR-regulated growth and negatively involved in drought responses.

RD26 mediates crosstalk between drought and brassinosteroid signalling pathways.

Brassinosteroids (BRs) regulate plant growth and stress responses via the BES1/BZR1 family of transcription factors, which regulate the expression of thousands of downstream genes. BRs are involved in the response to drought, however the mechanistic understanding of interactions between BR signalling and drought response remains to be established. Here we show that transcription factor RD26 mediates crosstalk between drought and BR signalling. When overexpressed, BES1 target gene RD26 can inhibit BR-regulated growth. Global gene expression studies suggest that RD26 can act antagonistically to BR to regulate the expression of a subset of BES1-regulated genes, thereby inhibiting BR function. We show that RD26 can interact with BES1 protein and antagonize BES1 transcriptional activity on BR-regulated genes and that BR signalling can also repress expression of RD26 and its homologues and inhibit drought responses. Our results thus reveal a mechanism coordinating plant growth and drought tolerance.

Light intensity affects chlorophyll synthesis during greening process by metabolite signal from mitochondrial alternative oxidase in Arabidopsis.

Although mitochondrial alternative oxidase (AOX) has been proposed to play essential roles in high light stress tolerance, the effects of AOX on chlorophyll synthesis are unclear. Previous studies indicated that during greening, chlorophyll accumulation was largely delayed in plants whose mitochondrial cyanide-resistant respiration was inhibited by knocking out nuclear encoded AOX gene. Here, we showed that this delay of chlorophyll accumulation was more significant under high light condition. Inhibition of cyanide-resistant respiration was also accompanied by the increase of plastid NADPH/NADP(+) ratio, especially under high light treatment which subsequently blocked the import of multiple plastidial proteins, such as some components of the photosynthetic electron transport chain, the Calvin-Benson cycle enzymes and malate/oxaloacetate shuttle components. Overexpression of AOX1a rescued the aox1a mutant phenotype, including the chlorophyll accumulation during greening and plastidial protein import. It thus suggests that light intensity affects chlorophyll synthesis during greening process by a metabolic signal, the AOX-derived plastidial NADPH/NADP(+) ratio change. Further, our results thus revealed a molecular mechanism of chloroplast-mitochondria interactions.

QQS orphan gene regulates carbon and nitrogen partitioning across species via NF-YC interactions.

The allocation of carbon and nitrogen resources to the synthesis of plant proteins, carbohydrates, and lipids is complex and under the control of many genes; much remains to be understood about this process. QQS (Qua-Quine Starch; At3g30720), an orphan gene unique to Arabidopsis thaliana, regulates metabolic processes affecting carbon and nitrogen partitioning among proteins and carbohydrates, modulating leaf and seed composition in Arabidopsis and soybean. Here the universality of QQS function in modulating carbon and nitrogen allocation is exemplified by a series of transgenic experiments. We show that ectopic expression of QQS increases soybean protein independent of the genetic background and original protein content of the cultivar. Furthermore, transgenic QQS expression increases the protein content of maize, a C4 species (a species that uses 4-carbon photosynthesis), and rice, a protein-poor agronomic crop, both highly divergent from Arabidopsis. We determine that QQS protein binds to the transcriptional regulator AtNF-YC4 (Arabidopsis nuclear factor Y, subunit C4). Overexpression of AtNF-YC4 in Arabidopsis mimics the QQS-overexpression phenotype, increasing protein and decreasing starch levels. NF-YC, a component of the NF-Y complex, is conserved across eukaryotes. The NF-YC4 homologs of soybean, rice, and maize also bind to QQS, which provides an explanation of how QQS can act in species where it does not occur endogenously. These findings are, to our knowledge, the first insight into the mechanism of action of QQS in modulating carbon and nitrogen allocation across species. They have major implications for the emergence and function of orphan genes, and identify a nontransgenic strategy for modulating protein levels in crop species, a trait of great agronomic significance.

RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture.

Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling.

Histone lysine methyltransferase SDG8 is involved in brassinosteroid-regulated gene expression in Arabidopsis thaliana.

The plant steroid hormones, brassinosteroids (BRs), play important roles in plant growth, development, and responses to environmental stresses. BRs signal through receptors localized to the plasma membrane and other signaling components to regulate the BES1/BZR1 family of transcription factors, which modulates the expression of thousands of genes. How BES1/BZR1 and their interacting proteins function to regulate the large number of genes are not completely understood. Here we report that histone lysine methyltransferase SDG8, implicated in histone 3 lysine 36 di- and trimethylation (H3K36me2 and me3), is involved in BR-regulated gene expression. BES1 interacts with SDG8, directly or indirectly through IWS1, a transcription elongation factor involved in BR-regulated gene expression. The knockout mutant sdg8 displays a reduced growth phenotype with compromised BR responses. Global gene expression studies demonstrated that, while BR regulates about 5000 genes in wild-type plants, the hormone regulates fewer than 700 genes in sdg8 mutant. In addition, more than half of BR-regulated genes are differentially affected in sdg8 mutant. A Chromatin Immunoprecipitation (ChIP) experiment showed that H3K36me3 is reduced in BR-regulated genes in the sdg8 mutant. Based on these results, we propose that SDG8 plays an essential role in mediating BR-regulated gene expression. Our results thus reveal a major mechanism by which histone modifications dictate hormonal regulation of gene expression.

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.

MYBL2 is a substrate of GSK3-like kinase BIN2 and acts as a corepressor of BES1 in brassinosteroid signaling pathway in Arabidopsis.

Plant steroid hormones, brassinosteroids (BRs), play important roles in plants. BRs regulate the expression of several thousand genes, half of which are induced and the other half repressed by the hormone. BRs signal through plasma membrane-localized receptor kinase brassinosteroid-insensitive 1 (BRI1), BRI1-associated receptor kinase (BAK1), and several intermediates to regulate the protein levels, cellular localizations, and/or DNA binding of BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) family transcription factors. Although BES1 is known to interact with other transcription factors, histone-modifying enzymes, and transcription elongation factors to activate BR-induced genes, how BES1 mediates the BR-repressed gene expression is not known. Here, we show that BES1 interacts with myeloblastosis family transcription factor-like 2 (MYBL2), a transcription repressor, to down-regulate BR-repressed gene expression. The loss-of-function mybl2 mutant enhances the phenotype of a weak allele of bri1 and suppresses the constitutive BR-response phenotype of bes1-D. The results suggest that suppression of BR-repressed gene expression is required for optimal BR response. Moreover, MYBL2 is a substrate of glycogen synthase kinase 3 (GSK3)-like kinase brassinosteroid-insensitive 2 (BIN2), which has been well established as a negative regulator in the BR pathway by phosphorylating and inhibiting the functions of BES1/BZR1. Unlike BIN2 phosphorylation of BES1/BZR1 leading to protein degradation, BIN2 phosphorylation stabilizes MYBL2. Such dual role of phosphorylation has also been reported in WNT signaling pathway in which GSK3 phosphorylation destabilizes ß-catenin and stabilizes Axin, a scaffolding protein facilitating the phosphorylation of ß-catenin by GSK3. Our results thus establish the mechanisms for BR-repressed gene expression and the integration of BR signaling and BR transcriptional network.

Recent advances in the regulation of brassinosteroid signaling and biosynthesis pathways.

Brassinosteroids (BRs) play important roles in plant growth, development and responses to environmental cues. BRs signal through plasma membrane receptor BRI1 and co-receptor BAK1, and several positive (BSK1, BSU1, PP2A) and negative (BKI1, BIN2 and 14-3-3) regulators to control the activities of BES1 and BZR1 family transcription factors, which regulate the expression of hundreds to thousands of genes for various BR responses. Recent studies identified novel signaling components in the BR pathways and started to establish the detailed mechanisms on the regulation of BR signaling. In addition, the molecular mechanism and transcriptional network through which BES1 and BZR1 control gene expression and various BR responses are beginning to be revealed. BES1 recruits histone demethylases ELF6 and REF6 as well as a transcription elongation factor IWS1 to regulate target gene expression. Identification of BES1 and BZR1 target genes established a transcriptional network for BR response and crosstalk with other signaling pathways. Recent studies also revealed regulatory mechanisms of BRs in many developmental processes and regulation of BR biosynthesis. Here we provide an overview and discuss some of the most recent progress in the regulation of BR signaling and biosynthesis pathways.

A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana.

Brassinosteroids (BRs) are important regulators for plant growth and development. BRs signal to control the activities of the BES1 and BZR1 family transcription factors. The transcriptional network through which BES1 and BZR regulate large number of target genes is mostly unknown. By combining chromatin immunoprecipitation coupled with Arabidopsis tiling arrays (ChIP-chip) and gene expression studies, we have identified 1609 putative BES1 target genes, 404 of which are regulated by BRs and/or in gain-of-function bes1-D mutant. BES1 targets contribute to BR responses and interactions with other hormonal or light signaling pathways. Computational modeling of gene expression data using Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) reveals that BES1-targeted transcriptional factors form a gene regulatory network (GRN). Mutants of many genes in the network displayed defects in BR responses. Moreover, we found that BES1 functions to inhibit chloroplast development by repressing the expression of GLK1 and GLK2 transcription factors, confirming a hypothesis generated from the GRN. Our results thus provide a global view of BR regulated gene expression and a GRN that guides future studies in understanding BR-regulated plant growth.

Arabidopsis IWS1 interacts with transcription factor BES1 and is involved in plant steroid hormone brassinosteroid regulated gene expression.

Plant steroid hormones, brassinosteroids (BRs), regulate essential growth and developmental processes. BRs signal through membrane-localized receptor BRI1 and several other signaling components to regulate the BES1 and BZR1 family transcription factors, which in turn control the expression of hundreds of target genes. However, knowledge about the transcriptional mechanisms by which BES1/BZR1 regulate gene expression is limited. By a forward genetic approach, we have discovered that Arabidopsis thaliana Interact-With-Spt6 (AtIWS1), an evolutionarily conserved protein implicated in RNA polymerase II (RNAPII) postrecruitment and transcriptional elongation processes, is required for BR-induced gene expression. Loss-of-function mutations in AtIWS1 lead to overall dwarfism in Arabidopsis, reduced BR response, genome-wide decrease in BR-induced gene expression, and hypersensitivity to a transcription elongation inhibitor. Moreover, AtIWS1 interacts with BES1 both in vitro and in vivo. Chromatin immunoprecipitation experiments demonstrated that the presence of AtIWS1 is enriched in transcribed as well as promoter regions of the target genes under BR-induced conditions. Our results suggest that AtIWS1 is recruited to target genes by BES1 to promote gene expression during transcription elongation process. Recent genomic studies have indicated that a large number of genes could be regulated at steps after RNAPII recruitment; however, the mechanisms for such regulation have not been well established. The study therefore not only establishes an important role for AtIWS1 in plant steroid-induced gene expression but also suggests an exciting possibility that IWS1 protein can function as a target for pathway-specific activators, thereby providing a unique mechanism for the control of gene expression.

A family of receptor-like kinases are regulated by BES1 and involved in plant growth in Arabidopsis thaliana.

Plant growth is dictated by both developmental and environmental cues, many of which are perceived by receptor-like kinases (RLKs). In Arabidopsis, there are more than 600 RLKs; but the functions of most of them are unknown. We recently found that several members of CrRLK1L family RLKs are regulated by plant steroid hormone Brassinosteroids (BRs). Two of the RLKs, FERONIA (FER) and THESEUS1 (THE1) have been previously found to inhibit cell elongation during pollen tube/synergid cell recognition and in sensing cell wall integrity after damage, respectively. However, we found that HERCULES1 (HERK1), another member in the family, as well as THE1 and FER, are regulated by BRs and required for cell elongation during vegetative growth. Here we provide additional evidence for the regulation of the family members by BR effector protein BES1. We also show that another member in the family, designated as HERCULES2 (HERK2), functions redundantly with HERK1 and THE1 to promote stem elongation. Our results, together with those from others, provide compelling evidence that the CrRLK1L family members play important role in plant growth.

Three related receptor-like kinases are required for optimal cell elongation in Arabidopsis thaliana.

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.

Cloning and characterization of a novel Delta12-fatty acid desaturase gene from the tree Sapium sebiferum.

A new full-length cDNA (Ssd12) encoding a Delta12-fatty acid desaturase (Delta12-FAD) was cloned from Sapium sebiferum using RT-PCR and RACE methods. Ssd12 contained a 1146 bp open reading frame encoding a protein of 381 amino acids. The amino acid sequence showed a much higher match with microsomal Delta12-FAD amino acid sequences than chloroplast Delta12-FAD amino acid sequences. Genomic Southern blot analysis suggested that Ssd12 had at least two copies. Ssd12 transcripts were detected in roots, leaves, stems, and seeds by real time PCR.