No abstract

The transcription factor LONG HYPOCOTYL5 (HY5) acts downstream of multiple families of the photoreceptors and promotes photomorphogenesis. Although it is well accepted that HY5 acts to regulate target gene expression, in vivo binding of HY5 to any of its target gene promoters has yet to be demonstrated. Here, we used a chromatin immunoprecipitation procedure to verify suspected in vivo HY5 binding sites. We demonstrated that in vivo association of HY5 with promoter targets is not altered under distinct light qualities or during light-to-dark transition. Coupled with DNA chip hybridization using a high-density 60-nucleotide oligomer microarray that contains one probe for every 500 nucleotides over the entire Arabidopsis thaliana genome, we mapped genome-wide in vivo HY5 binding sites. This analysis showed that HY5 binds preferentially to promoter regions in vivo and revealed >3000 chromosomal sites as putative HY5 binding targets. HY5 binding targets tend to be enriched in the early light-responsive genes and transcription factor genes. Our data thus support a model in which HY5 is a high hierarchical regulator of the transcriptional cascades for photomorphogenesis.

SUMMARY Brassinosteroids (BRs) regulate a wide range of developmental and physiological processes in plants through a receptor-kinase signaling pathway that controls the BZR transcription factors. Here we use transcript profiling and chromatin-immunoprecipitation microarray (ChIP-chip) experiments to identify 953 BR-regulated BZR1 target (BRBT) genes. Functional studies of selected BRBTs further demonstrate roles in BR-promotion of cell elongation. The BRBT genes reveal numerous molecular links between the BR signaling pathway and downstream components involved in developmental and physiological processes. Furthermore, the results reveal extensive crosstalk between BR and other hormonal and light signaling pathways at multiple levels. For example, BZR1 not only controls the expression of many signaling components of other hormonal and light pathways, but also co-regulates common target genes with light-signaling transcription factors. Our results provide a genomic map of steroid hormone actions in plants, which reveals a regulatory network that integrates hormonal and light signaling pathways for plant growth regulation.

Brassinosteroids (BRs) regulate plant development through a signal transduction pathway involving the BRI1 and BAK1 transmembrane receptor kinases. The detailed molecular mechanisms of phosphorylation, kinase activation, and oligomerization of the BRI1/BAK1 complex in response to BRs are uncertain. We demonstrate that BR-dependent activation of BRI1 precedes association with BAK1 in planta, and that BRI1 positively regulates BAK1 phosphorylation levels in vivo. BRI1 transphosphorylates BAK1 in vitro on specific kinase-domain residues critical for BAK1 function. BAK1 also transphosphorylates BRI1, thereby quantitatively increasing BRI1 kinase activity toward a specific substrate. We propose a sequential transphosphorylation model in which BRI1 controls signaling specificity by direct BR binding followed by substrate phosphorylation. The coreceptor BAK1 is then activated by BRI1-dependent transphosphorylation and subsequently enhances signaling output through reciprocal BRI1 transphosphorylation. This model suggests both conservation and distinct differences between the molecular mechanisms regulating phosphorylation-dependent kinase activation in plant and animal receptor kinases.

MYB proteins are a superfamily of transcription factors that play regulatory roles in developmental processes and defense responses in plants. We identified 198 genes in the MYB superfamily from an analysis of the complete Arabidopsis genome sequence, among them, 126 are R2R3-MYB, 5 are R1R2R3-MYB, 64 are MYB-related, and 3 atypical MYB genes. Here we report the expression profiles of 163 genes in the Arabidopsis MYB superfamily whose full-length open reading frames have been isolated. This analysis indicated that the expression for most of the Arabidopsis MYB genes were responsive to one or multiple types of hormone and stress treatments. A phylogenetic comparison of the members of this superfamily in Arabidopsis and rice suggested that the Arabidopsis MYB superfamily underwent a rapid expansion after its divergence from monocots but before its divergence from other dicots. It is likely that the MYB-related family was more ancient than the R2R3-MYB gene family, or had evolved more rapidly. Therefore, the MYB gene superfamily represents an excellent system for investigating the evolution of large and complex gene families in higher plants. Our comprehensive analysis of this largest transcription factor superfamily of Arabidopsis and rice may help elucidate the possible biological roles of the MYB genes in various aspects of flowering plants.

Brassinosteroids (BRs) are phytosteroid hormones controlling various physiological processes critical for normal growth and development. BRs are perceived by a protein complex containing two transmembrane receptor kinases, BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) [1-3]. BRI1 null mutants exhibit a dwarfed stature with epinastic leaves, delayed senescence, reduced male fertility, and altered light responses. BAK1 null mutants, however, only show a subtle phenotype, suggesting that functionally redundant proteins might be present in the Arabidopsis genome. Here we report that BAK1-LIKE 1 (BKK1) functions redundantly with BAK1 in regulating BR signaling. Surprisingly, rather than the expected bri1-like phenotype, bak1 bkk1 double mutants exhibit a seedling-lethality phenotype due to constitutive defense-gene expression, callose deposition, reactive oxygen species (ROS) accumulation, and spontaneous cell death even under sterile growing conditions. Our detailed analyses demonstrate that BAK1 and BKK1 have dual physiological roles: positively regulating a BR-dependent plant growth pathway, and negatively regulating a BR-independent cell-death pathway. Both BR signaling and developmentally controlled cell death are critical to optimal plant growth and development, but the mechanisms regulating early events in these pathways are poorly understood. This study provides novel insights into the initiation and crosstalk of the two signaling cascades.

The Arabidopsis thaliana Somatic Embryogenesis Receptor Kinases (SERKs) consist of five members, SERK1 to SERK5, of the leucine-rich repeat receptor-like kinase subfamily II (LRR-RLK II). SERK3 was named BRI1-Associated Receptor Kinase 1 (BAK1) due to its direct interaction with the brassinosteroid (BR) receptor BRI1 in vivo, while SERK4 has also been designated as BAK1-Like 1 (BKK1) for its functionally redundant role with BAK1. Here we provide genetic and biochemical evidence to demonstrate that SERKs are absolutely required for early steps in BR signaling. Overexpression of four of the five SERKs—SERK1, SERK2, SERK3/BAK1, and SERK4/BKK1—suppressed the phenotypes of an intermediate BRI1 mutant, bri1-5. Overexpression of the kinase-dead versions of these four genes in the bri1-5 background, on the other hand, resulted in typical dominant negative phenotypes, resembling those of null BRI1 mutants. We isolated and generated single, double, triple, and quadruple mutants and analyzed their phenotypes in detail. While the quadruple mutant is embryo-lethal, the serk1 bak1 bkk1 triple null mutant exhibits an extreme de-etiolated phenotype similar to a null bri1 mutant. While overexpression of BRI1 can drastically increase hypocotyl growth of wild-type plants, overexpression of BRI1 does not alter hypocotyl growth of the serk1 bak1 bkk1 triple mutant. Biochemical analysis indicated that the phosphorylation level of BRI1 in serk1 bak1 bkk1 is incapable of sensing exogenously applied BR. As a result, the unphosphorylated level of BES1 has lost its sensitivity to the BR treatment in the triple mutant, indicating that the BR signaling pathway has been completely abolished in the triple mutant. These data clearly demonstrate that SERKs are essential to the early events of BR signaling.

We present high-resolution maps of DNA methylation and H3K4 di-and trimethylation of two entire chromosomes and two fully sequenced centromeres in rice (Oryza sativa) shoots and cultured cells. This analysis reveals combinatorial interactions between these epigenetic modifications and chromatin structure and gene expression. Cytologically densely stained heterochromatin had less H3K4me2 and H3K4me3 and more methylated DNA than the less densely stained euchromatin, whereas centromeres had a unique epigenetic composition. Most transposable elements had highly methylated DNA but no H3K4 methylation, whereas more than half of protein-coding genes had both methylated DNA and di-and/or trimethylated H3K4. Methylation of DNA but not H3K4 was correlated with suppressed transcription. By contrast, when both DNA and H3K4 were methylated, transcription was only slightly reduced. Transcriptional activity was positively correlated with the ratio of H3K4me3/H3K4me2: genes with predominantly H3K4me3 were actively transcribed, whereas genes with predominantly H3K4me2 were transcribed at moderate levels. More protein-coding genes contained all three modifications, and more transposons contained DNA methylation in shoots than cultured cells. Differential epigenetic modifications correlated to tissue-specific expression between shoots and cultured cells. Collectively, this study provides insights into the rice epigenomes and their effect on gene expression and plant development.