SUMMARYBrassinosteroids (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.

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 downregulated 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.brassinosteroid ͉ FERONIA ͉ HERCULES1 ͉ THESEUS1

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 group III WRKY transcription factors, WRKY46, WRKY54, and WRKY70, are involved in both BR-regulated plant growth and drought response as the 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.

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.

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 proteinpoor 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.QQS | NF-YC4 | carbon allocation | nitrogen allocation | orphan C arbon and nitrogen allocation to plant proteins, carbohydrates, and lipids is not controlled by a single gene but by many (1). Most of the enzymes promoting accumulation of these products have been identified; however, much less is understood about the mechanisms that regulate this complex metabolic network (2-8).Arabidopsis thaliana QQS (Qua-Quine Starch; At3g30720) lacks sequence similarity to any other protein-coding genes, and is considered an orphan gene that has arisen de novo from noncoding sequence since the divergence of A. thaliana from other species (9, 10). Although orphans typically comprise 2-8% of the genome of eukaryotic and prokaryotic species, their origin and biological function have not been well-explored (11)(12)(13)(14). Proteins encoded by some orphan genes provide a defensive capability by binding to a receptor of a predator organism (11). In contrast, QQS action is endogenous (3): Overexpression of QQS in Arabidopsis increases total protein content and decreases total starch content in leaf, whereas down-regulation of QQS has the converse effect. The increased starch content in QQS RNAi (RNA interference) mutants is due to increased starch accumu...