Jia-Ying Zhu

Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses

Plant growth is coordinately regulated by environmental and hormonal signals. Brassinosteroid (BR) plays essential roles in growth regulation by light and temperature, but the interactions between BR and these environmental signals remain poorly understood at the molecular level. Here, we show that direct interaction between the dark- and heat-activated transcription factor phytochrome-interacting factor 4 (PIF4) and the BR-activated transcription factor BZR1 integrates the hormonal and environmental signals. BZR1 and PIF4 interact with each other in vitro and in vivo, bind to nearly 2,000 common target genes, and synergistically regulate many of these target genes, including the PRE family helix–loop–helix factors required for promoting cell elongation. Genetic analysis indicates that BZR1 and PIFs are interdependent in promoting cell elongation in response to BR, darkness or heat. These results show that the BZR1–PIF4 interaction controls a core transcription network, enabling plant growth co-regulation by the steroid and environmental signals.

Antagonistic HLH/bHLH Transcription Factors Mediate Brassinosteroid Regulation of Cell Elongation and Plant Development in Rice and Arabidopsis

In rice (Oryza sativa), brassinosteroids (BRs) induce cell elongation at the adaxial side of the lamina joint to promote leaf bending. We identified a rice mutant (ili1-D) showing an increased lamina inclination phenotype similar to that caused by BR treatment. The ili1-D mutant overexpresses an HLH protein homologous to Arabidopsis thaliana Paclobutrazol Resistance1 (PRE1) and the human Inhibitor of DNA binding proteins. Overexpression and RNA interference suppression of ILI1 increase and reduce, respectively, rice laminar inclination, confirming a positive role of ILI1 in leaf bending. ILI1 and PRE1 interact with basic helix-loop-helix (bHLH) protein IBH1 (ILI1 binding bHLH), whose overexpression causes erect leaf in rice and dwarfism in Arabidopsis. Overexpression of ILI1 or PRE1 increases cell elongation and suppresses dwarf phenotypes caused by overexpression of IBH1 in Arabidopsis. Thus, ILI1 and PRE1 may inactivate inhibitory bHLH transcription factors through heterodimerization. BR increases the RNA levels of ILI1 and PRE1 but represses IBH1 through the transcription factor BZR1. The spatial and temporal expression patterns support roles of ILI1 in laminar joint bending and PRE1/At IBH1 in the transition from growth of young organs to growth arrest. These results demonstrate a conserved mechanism of BR regulation of plant development through a pair of antagonizing HLH/bHLH transcription factors that act downstream of BZR1 in Arabidopsis and rice.

Brassinosteroid signaling network and regulation of photomorphogenesis.

In plants, the steroidal hormone brassinosteroid (BR) regulates numerous developmental processes, including photomorphogenesis. Genetic, proteomic, and genomic studies in Arabidopsis have illustrated a fully connected BR signal transduction pathway from the cell surface receptor kinase BRI1 to the BZR1 family of transcription factors. Genome-wide analyses of protein-DNA interactions have identified thousands of BZR1 target genes that link BR signaling to various cellular, metabolic, and developmental processes, as well as other signaling pathways. In controlling photomorphogenesis, BR signaling is highly integrated with the light, gibberellin, and auxin pathways through both direct interactions between signaling proteins and transcriptional regulation of key components of these pathways. BR signaling also cross talks with other receptor kinase pathways to modulate stomata development and innate immunity. The molecular connections in the BR signaling network demonstrate a robust steroid signaling system that has evolved in plants to orchestrate signal transduction, genome expression, metabolism, defense, and development.

Cell elongation is regulated through a central circuit of interacting transcription factors in the Arabidopsis hypocotyl

As the major mechanism of plant growth and morphogenesis, cell elongation is controlled by many hormonal and environmental signals. How these signals are coordinated at the molecular level to ensure coherent cellular responses remains unclear. In this study, we illustrate a molecular circuit that integrates all major growth-regulating signals, including auxin, brassinosteroid, gibberellin, light, and temperature. Analyses of genome-wide targets, genetic and biochemical interactions demonstrate that the auxin-response factor ARF6, the light/temperature-regulated transcription factor PIF4, and the brassinosteroid-signaling transcription factor BZR1, interact with each other and cooperatively regulate large numbers of common target genes, but their DNA-binding activities are blocked by the gibberellin-inactivated repressor RGA. In addition, a tripartite HLH/bHLH module feedback regulates PIFs and additional bHLH factors that interact with ARF6, and thereby modulates auxin sensitivity according to developmental and environmental cues. Our results demonstrate a central growth-regulation circuit that integrates hormonal, environmental, and developmental controls of cell elongation in Arabidopsis hypocotyl. DOI: http://dx.doi.org/10.7554/eLife.03031.001

Integration of Light- and Brassinosteroid-Signaling Pathways by a GATA Transcription Factor in Arabidopsis

Light and brassinosteroid (BR) antagonistically regulate the developmental switch from etiolation in the dark to photomorphogenesis in the light in plants. Here, we identify GATA2 as a key transcriptional regulator that mediates the crosstalk between BR- and light-signaling pathways. Overexpression of GATA2 causes constitutive photomorphogenesis in the dark, whereas suppression of GATA2 reduces photomorphogenesis caused by light, BR deficiency, or the constitutive photomorphogenesis mutant cop1. Genome profiling and chromatin immunoprecipitation experiments show that GATA2 directly regulates genes that respond to both light and BR. BR represses GATA2 transcription through the BR-activated transcription factor BZR1, whereas light causes accumulation of GATA2 protein and feedback inhibition of GATA2 transcription. Dark-induced proteasomal degradation of GATA2 is dependent on the COP1 E3 ubiquitin ligase, and COP1 can ubiquitinate GATA2 in vitro. This study illustrates a molecular framework for antagonistic regulation of gene expression and seedling photomorphogenesis by BR and light.

Dynamics of brassinosteroid response modulated by negative regulator LIC in rice.

Brassinosteroids (BRs) regulate rice plant architecture, including leaf bending, which affects grain yield. Although BR signaling has been investigated in Arabidopsis thaliana, the components negatively regulating this pathway are less well understood. Here, we demonstrate that Oryza sativa LEAF and TILLER ANGLE INCREASED CONTROLLER (LIC) acts as an antagonistic transcription factor of BRASSINAZOLE-RESISTANT 1 (BZR1) to attenuate the BR signaling pathway. The gain-of-function mutant lic-1 and LIC–overexpressing lines showed erect leaves, similar to BZR1–depleted lines, which indicates the opposite roles of LIC and BZR1 in regulating leaf bending. Quantitative PCR revealed LIC transcription rapidly induced by BR treatment. Image analysis and immunoblotting showed that upon BR treatment LIC proteins translocate from the cytoplasm to the nucleus in a phosphorylation-dependent fashion. Phosphorylation assay in vitro revealed LIC phosphorylated by GSK3–like kinases. For negative feedback, LIC bound to the core element CTCGC in the BZR1 promoter on gel-shift and chromatin immunoprecipitation assay and repressed its transcription on transient transformation assay. LIC directly regulated target genes such as INCREASED LEAF INCLINATION 1 (ILI1) to oppose the action of BZR1. Repression of LIC in ILI1 transcription in protoplasts was partially rescued by BZR1. Phenotypic analysis of the crossed lines depleted in both LIC and BZR1 suggested that BZR1 functionally depends on LIC. Molecular and physiology assays revealed that LIC plays a dominant role at high BR levels, whereas BZR1 is dominant at low levels. Thus, LIC regulates rice leaf bending as an antagonistic transcription factor of BZR1. The phenotypes of lic-1 and LIC–overexpressing lines in erect leaves contribute to ideal plant architecture. Improving this phenotype may be a potential approach to molecular breeding for high yield in rice.

Information Integration and Communication in Plant Growth Regulation

Plants are equipped with the capacity to respond to a large number of diverse signals, both internal ones and those emanating from the environment, that are critical to their survival and adaption as sessile organisms. These signals need to be integrated through highly structured intracellular networks to ensure coherent cellular responses, and in addition, spatiotemporal actions of hormones and peptides both orchestrate local cell differentiation and coordinate growth and physiology over long distances. Further, signal interactions and signaling outputs vary significantly with developmental context. This review discusses our current understanding of the integrated intracellular and intercellular signaling networks that control plant growth.

TOPLESS mediates brassinosteroid-induced transcriptional repression through interaction with BZR1

Brassinosteroid (BR) regulates plant development by activating the transcription factor BRASSINAZOLE RESISTANT1 (BZR1), which activates and represses different target genes to switch cellular programs. The mechanisms that determine BZR1’s transcriptional activities remain largely unknown. Here we show that BZR1 represses target genes by recruiting the Groucho/TUP1-like transcriptional corepressor TOPLESS (TPL). Specific deletion or mutation of an evolutionarily conserved ERF-ASSOCIATED AMPHIPHILIC REPRESSION (EAR) motif at the C-terminus abolishes BZR1’s abilities to regulate gene expression and cell elongation, but these defects are rescued by TPL fusion to the EAR motif-mutated BZR1. The EAR motif in BZR1 mediates recruitment of TPL to BZR1-repressed promoters. A triple tpl mutant (tpl;tpr1;tpr4) shows reduced BR sensitivity and suppresses the gain-of-function bzr1-1D mutant phenotype. BR repression of gene expression also requires histone deacetylases that interact with TPL. Our study demonstrates key roles of the EAR motif and TPL in BR regulation of gene expression and plant growth.

BR Signal Influences Arabidopsis Ovule and Seed Number through Regulating Related Genes Expression by BZR1

Ovule and seed developments are crucial processes during plant growth, which are affected by different signaling pathways. In this paper, we demonstrate that the brassinosteroid (BR) signal is involved in ovule initiation and development. Ovule and seed numbers are significantly different when comparing BR-related mutants to wild-type controls. Detailed observation indicates that BR regulates the expression level of genes related to ovule development, including HLL, ANT, and AP2, either directly by targeting the promoter sequences or indirectly via regulation by BR-induced transcription factor BZR1. Also, Western blot demonstrates that the dephosphorylation level of BZR1 is consistent with ovule and seed number. The intragenic bzr1-1D suppressors bzs247 and bzs248 have much fewer ovules and seeds than bzr1-1D, which are similar to wild-type, suggesting that the phenotype can be rescued. The molecular and genetic experiments confirm that BZR1 and AP2 probably affect Arabidopsis ovule number determination antagonistically.

Genome-Wide Identification of Transcription Factor-Binding Sites in Plants Using Chromatin Immunoprecipitation Followed by Microarray (ChIP-chip) or Sequencing (ChIP-seq)

Nearly all signal transduction pathways lead to regulation of gene expression by controlling specific transcription factors (TFs). Chromatin immunoprecipitation (ChIP) is a powerful method for studying TF-DNA interactions in vivo. To identify all binding sites of a TF in the genome, the DNA obtained in ChIP experiments needs to be analyzed by hybridization to genome-tiling microarrays (ChIP-chip) or by next-generation sequencing (ChIP-seq). Here, we provide detailed protocols of ChIP for two model plant species Arabidopsis and rice, procedures of DNA sample preparation for ChIP-chip or ChIP-seq, and a general guide for computational data analysis. We have used these protocols to successfully identify direct target genes of the BZR1 TF of the brassinosteroid signaling pathway in both Arabidopsis and rice.