Molecular Mechanism Involved In Gene Regulation Of Leaf Curvature

The identity of leaf polarity determines leaf flatness development directly, and abnormal polarity usually results in the abnormal leaf flatness, subsequently affect on many physiological functions, such as photosynthesis, transpiration and anti-stress. Furthermore, leaf curvature is an important and usable agricultural character for some crops. Leafy head, which characters the leaf curvature upward and inward, is the edible organ of Chinese cabbage, cabbage and brussels sprouts. Hence, understanding the genetic mechanism of leaf polarity or leaf curvature has the theoretic significance for controlling leaf form and shape, and for the leaf development theory. It also has applied significance for improving the quantity and quality of crops by improving theirs genetic characters such as leaf flatten or leaf curvature. Early in 1996, our lab has isolated a double-stranded RNA binding protein gene BcpLH, which is related to the curvature of leaf, by the method of different expression hybridization (Yu et al., 2000). In this research, to understand the molecular mechanism involved in the gene regulation of leaf curvature, we used the comparative biological method to analyze the BcpLH gene in Chinese cabbage and HYL1 gene in Arabidopsis basing on the recent studied of the leaf polarity and curvature. Firstly, we chose hyl1 mutant of Arabidopsis (Lu and Fedoroff, 2000) as a model for dissection of leaf venation pattern and adaxial/abaxial polarity. In leaves of hyl1 mutants that were hyponastic and curved upward, the complexity of the secondary veins was reduced, and the discontinuity of veins increased. In the lateral areas of the leaves where transverse curvature arises, dorsoventral polarity was lost due to the unclear spongy cells, and the epidermal cells became smaller on the adaxial surface than those of the abaxial surface, whereas the number of epidermal cells on the two surfaces were almost the same. In this case, it could be proposed that the less complexity of venation, decreased cell growth on the adaxial surface was attributed to leaf curvature. To depict the role of HYL1 in leaf venation and polarity, we isolated the HYL1 promoter and constructed pHYL1:: GUS to drive the uidA(beta-glucuronidase) gene, and observed the GUS signal in Arabidopsis. GUS signals appear primarily in the petioles and mid-veins of rosette leaves, and are restricted to vascular tissues, demonstrating that HYL1 promoter directs the process of leaf venation by the uneven expression of the HYL1gene in leaves. It is consist with the In situ hybridization results. Furthermore, the GUS expression pattern in cauline leaves is similar with the rosette leaves. In the sepal and stamen as well as the embryo also have the GUS signals. Semi-quantity RT-PCR results suggests that the expression level of adaxial identity gene REV is increased and that of the abaxial identity gene FIL is decreased in curved leaves of hyl1 mutants comparing to the wild type plants. In situ hybridization results indicated the expression position of REV restricted mainly in vasculature and on both sides of hyl1 growing leaves close to the margins while expression of the miR165 gene was remarkably reduced by Northern blotting analysis, suggesting that HYL1 maintains polarity and flatness of growing leaves by controlling the biogenesis and accumulation of microRNA that direct the cleavage of REV transcripts. The protein encoded by HYL1gene, which is the homolog of the double-stranded binding protein RDE-4 in Caenorhabditis elegans and R2D2 in Drosophila, contains two A type double-stranded RNA binding motifs (DSRM). In addition, a bipartite nuclear localization sequence and a six repeat sequence are detected in the C-terminus of HYL1. In order to study the different biological function in the different domains of HYL1, we construct fragments of HYL1 gene with different length and transform them back into the hyl1 mutants respectively. The assays of phenotype of transgenic plants, the response to the exogenous ABA and IAA, as well as the expression of miR165 and miR160 in the seedling or mature leaves of transgenic plants were performed. These results suggest that the both two DSRMs neither the DSRM1 nor the C-terminal fragnent are essential for HYL1 biology functions, including the leaf flatten, miRNA accumulation and hormone response. Moreover, HYL1 gene function...