Gene Function Analysis Of WRKY12and WRKY13in Arabidopsis Thaliana

The flowering time of plants is affected by both internal and external factors, with photoperiod being a very important factor. For model plant Arabidopsis thaliana, an earlier floral transition is observed under long-day than under short-day conditions, with flowering under the latter mainly regulated by the plant hormone gibberellin. We identified several wrky mutants with disruption in WRKY12or WKRY13gene. These mutants have phenotype of altered flowering time under short-day conditions. The wrky12mutants flowered later than the wild type, while the wrky13mutants flowered earlier than the wild type. We have the expression patterns of the two genes analyzed. The two genes expressed at the same locations, which were shoot apex and leaf veins. The expression time profiles were different. As plants grew, the expression level of WKRY12gene increased, while the transcript level of WRKY13gene decreased. This was consistent with the function of the two WRKY genes in regulating flowering time. WRKY13gene, as a flowering repressor, expressed in early stage, preventing plants from flowering during inappropriate development stage. WRKY12gene, as a flowering inducer, expressed in later stage, ensuring that plants flower at the appropriate development stage. In addition, we performed promoter-swap experiment. We fused the promoter region of WKRY12and the coding sequence of WRKY13into the same expression vector. Then the construct was transferred into wrkyl3mutant. Or the promoter region of WKRY13was fused with the coding sequence of WRKY12. Then the construct was transferred into wrky12mutant. The different restoration effects of different constructs on the flowering time confirmed that the staggered expression profiles of the two WRKY genes confered great roles in the regulation of flowering time.Key genes in flowering regulation pathway, such as FUL and SOC1, were downregulated in wrky12mutants while upregulated in wrky13mutants, comparing to that in wild type. The in vivo chromatin immunoprecipitation assays confirmed the direct binding of WKRY proteins to the promoter regions of FUL gene. Then the yeast-two-hybrid system screened out the interaction proteins of WKRY12and WRKY13, GAI and RGL1. Detailed analysis confirmed that GAI and RGL1bound to the DNA binding area of the two WRKY proteins, suggesting that GAI and RGL1can affect the transcription factor role of WRKY proteins to indirectly regulate the expression of downstream genes. GAI and RGL1belong to the DELLA family. The plant hormone gibberrellin promotes flowering under short time conditions through degradation of DELLA, releasing the DELLA interacting transcription factors and the inhibition of downstream genes by DELLA. We analysed the GA content in Arabidopsis thaliana at different time points and found that the GA content gradually rise as plants grow. Taken together, these evidences suggest a model regulating flowering time. When the plants develop into the appropriate stage, bioactive GA degrade repressor DELLA, releasing the interacting WRKY proteins. Then the WRKY equilibrium lean towards the flowering induction state, promoting plants to flower.In addition, we also found that wrkyl3mutants had lodging phenotype under long day conditions. The forces needed to pull stems apart were measured. We found that the breaking forces needed for wrky13mutant stems were less than that for wild type. This was consistent with the lodging phenotype of wrkyl3mutants. Gene expression analysis showed that WRKY13expressed strongly in stem internodes, where its transcript level increases with maturity. Thin cross-sections of stems were cut and subjected to toluidine blue O staining. We found that, compared with that of wild type, the diameters of wrky13mutant stems were smaller. The cross-sectional area containing sclerenchyma cells also decreased in wrky13mutant stems. Plant stem cells can be divided into two major categories, sclerenchyma cells and parenchyma cells. Unlike parenchyma cells, sclerenchyma cells have two kinds of walls, primary walls and secondarily thickened cell walls. Secondary walls, with a composition of cellulose, hemicellulose and lignin, are deposited inside the primary walls after the cells cease expansion. Several key factors in the process of the synthesis of secondary cell walls, such as PAL4,4CL1, NST2, CesA7and CesA8were downregulated in wrkyl3mutant. Further in vivo chromatin immunoprecipitation experiment confirmed that WRKY13protein could directly bind to the promoter regions of NST2gene. Taken together, these results proved that WRKY13gene can directly or indirectly regulate the sclerenchyma cell wall synthesis genes to affect the development of sclerenchyma cells in stems, which in addition affect the mechanical strength of stem and the growth of plants.