Transcriptional mechanisms of brassinosteroid regulated plant growth and stress responses

Plant Steroid hormones, Brassinosteroids (BRs), play important roles in plant growth, development and responses to various stresses. BR signal through receptor BRI1 and BAK1 and a series signaling intermediates to control the activities of BES1/BZR1 family transcription factors that control the expression of thousands of genes, half of which are induced and the other half repressed by BR. While BES1 is known to activate BR-induced genes by itself or cooperating with co-activators, such as transcription factors, histone modification enzymes and transcription elongation factors, how BES1 mediates the BR-repressed gene expression is not known. In chapter Ⅱ, MYBL2, a small MYB family transcription repressor, was found to interact with BES1 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, suggesting that suppression of BR-repressed gene expression is required for optimal BR response. Moreover, MYBL2 is a substrate of GSK3-like kinase BIN2, a negative regulator functioning in inhibiting the activities of BES1/BZR1 through its phosphorylation in BR pathway. Unlike BIN2 phosphorylation of BES1/BZR1 leading to protein degradation, BIN2 phosphorylation stabilizes MYBL2, which demonstrated a dual role of BIN2 phosphorylation in BR pathway, similar to the function of GSK3 in WNT signaling pathway. Our results thus establish the mechanisms for BR-repressed gene expression and the integration of BR signaling and BR transcriptional network. In addition to promote the growth, BRs are known to be involved in drought response, but the mechanism of interactions between these two pathways remains to be established. In chapter Ⅲ, the NAC family transcription factor RD26 and its close homologs mediate crosstalk between drought and BR signaling pathway. RD26 is a direct target of BES1 and functions to inhibit BR-regulated growth as overexpression of RD26 leads to decreased plant growth and knockout of RD26 and its close homologs results in increased BR response. Global gene expression analysis revealed that RD26 modulates BR-regulated gene expression in a complex way. RD26 represses many BR-induced genes including BR-activated cell elongation genes and activates many BR-repressed genes, thereby inhibiting BR functions. On the other hand, BR signaling also inhibits drought responses through repressing the expression of RD26, its homologs and RD26-mediated drought-induced genes. The reciprocal inhibitory effects of BES1 and RD26 are mediated by their interactions on different promoter elements. This mechanism ensures that BR-induced plant growth is inhibited under drought condition that induced RD26 expression, while this mechanism also prevents unnecessary activation of drought response when plants undergo BR-induced growth, during which BES1 accumulates. Our results thus revealed a previously unknown mechanism coordinating plant growth and drought tolerance

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 zmbril complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbriI-RNAi transgenic lines have compromised BR signaling. zmbriI-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbriI-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 zmbriI-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 zmbriI-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

Citation: Wang, X., Chen, J., Xie, Z., Liu, S., Nolan, T., Ye, H., et al. (2014). 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 4,000-5,000 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 tri-methylation (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 SDG8 plays a major role in BR-regulated gene expression as 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

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

Transcriptional mechanisms of brassinosteroid regulated plant growth and stress responses

Plant Steroid hormones, Brassinosteroids (BRs), play important roles in plant growth, development and responses to various stresses. BR signal through receptor BRI1 and BAK1 and a series signaling intermediates to control the activities of BES1/BZR1 family transcription factors that control the expression of thousands of genes, half of which are induced and the other half repressed by BR. While BES1 is known to activate BR-induced genes by itself or cooperating with co-activators, such as transcription factors, histone modification enzymes and transcription elongation factors, how BES1 mediates the BR-repressed gene expression is not known. In chapter Ⅱ, MYBL2, a small MYB family transcription repressor, was found to interact with BES1 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, suggesting that suppression of BR-repressed gene expression is required for optimal BR response. Moreover, MYBL2 is a substrate of GSK3-like kinase BIN2, a negative regulator functioning in inhibiting the activities of BES1/BZR1 through its phosphorylation in BR pathway. Unlike BIN2 phosphorylation of BES1/BZR1 leading to protein degradation, BIN2 phosphorylation stabilizes MYBL2, which demonstrated a dual role of BIN2 phosphorylation in BR pathway, similar to the function of GSK3 in WNT signaling pathway. Our results thus establish the mechanisms for BR-repressed gene expression and the integration of BR signaling and BR transcriptional network. In addition to promote the growth, BRs are known to be involved in drought response, but the mechanism of interactions between these two pathways remains to be established. In chapter Ⅲ, the NAC family transcription factor RD26 and its close homologs mediate crosstalk between drought and BR signaling pathway. RD26 is a direct target of BES1 and functions to inhibit BR-regulated growth as overexpression of RD26 leads to decreased plant growth and knockout of RD26 and its close homologs results in increased BR response. Global gene expression analysis revealed that RD26 modulates BR-regulated gene expression in a complex way. RD26 represses many BR-induced genes including BR-activated cell elongation genes and activates many BR-repressed genes, thereby inhibiting BR functions. On the other hand, BR signaling also inhibits drought responses through repressing the expression of RD26, its homologs and RD26-mediated drought-induced genes. The reciprocal inhibitory effects of BES1 and RD26 are mediated by their interactions on different promoter elements. This mechanism ensures that BR-induced plant growth is inhibited under drought condition that induced RD26 expression, while this mechanism also prevents unnecessary activation of drought response when plants undergo BR-induced growth, during which BES1 accumulates. Our results thus revealed a previously unknown mechanism coordinating plant growth and drought tolerance.</p

Water-gas ratio characteristics and development concepts for water-producing gas reservoirs

Water production from gas wells is a key factor affecting the effectiveness of gas-reservoir development, and it poses serious challenges in terms of increasing the degree of recovery during the waterless production stage and reducing the impact of water production on gas-reservoir development in the middle and later periods. Thus, gas reservoirs must be efficiently exploited on the basis of identifying gas-water layers accurately, defining gas-water relationships, and understanding gas-water production performance. Accordingly, this study analyzes the production characteristics in gas reservoirs with different gas-water relationships, and it summarizes the rules that determine water-gas ratios. The results reveal that the water-gas ratio increases rapidly in the early stage of water production, but after a period of time, it enters a relatively stable state in which it is almost a fixed value. According to the material balance equation, the theoretically calculated water-gas ratio is fully consistent with the production rules for an entire confined gas reservoir. This shows that the reality of gas-well-water production must be faced, and that the development of water-bearing gas reservoirs must accommodate gas and water co-production. The gas-water relationship, water body scale, and reservoir heterogeneity determine the time of water breakthrough and the water-gas ratio. Therefore, we should change the traditional “water fear” concept in gas-field development, aim for an overall improvement in recovery, face up to the fact that gas wells produce water, and coordinate the development of multi-wells for entire gas reservoirs, all of which will achieve the ultimate goal of improved gas recovery

A coupling model for gas diffusion and seepage in SRV section of shale gas reservoirs

A prerequisite to effective shale gas development is a complicated fracture network generated by extensive and massive fracturing, which is called SRV (stimulated reservoir volume) section. Accurate description of gas flow behaviors in such section is fundamental for productivity evaluation and production performance prediction of shale gas wells. The SRV section is composed of bedrocks with varying sizes and fracture networks, which exhibit different flow behaviors – gas diffusion in bedrocks and gas seepage in fractures. According to the porosity and permeability and the adsorption, diffusion and seepage features of bedrocks and fractures in a shale gas reservoir, the material balance equations were built for bedrocks and fractures respectively and the continuity equations of gas diffusion and seepage in the SRV section were derived. For easy calculation, the post-frac bedrock cube was simplified to be a sphere in line with the principle of volume consistency. Under the assumption of quasi-steady flow behavior at the cross section of the sphere, the gas channeling equation was derived based on the Fick's laws of diffusion and the density function of gas in bedrocks and fractures. The continuity equation was coupled with the channeling equation to effectively characterize the complicated gas flow behavior in the SRV section. The study results show that the gas diffusivity in bedrocks and the volume of bedrocks formed by volume fracturing (or the scale of fracturing) jointly determines the productivity and stable production period of a shale gas well. As per the actual calculation for the well field A in the Changning–Weiyuan Block in the Sichuan Basin, the matrix has low gas diffusivity – about 10−5 cm2/s and a large volume with an equivalent sphere radius of 6.2 m, hindering the gas channeling from bedrocks to fractures and thereby reducing the productivity of the shale gas well. It is concluded that larger scale of volume fracturing and higher fracture density in the SRV section are important guarantees for efficient development of shale gas reservoirs