| The surface of the land plants are covered with a layer of epicuticular wax(EW), acting as a protective barrier from excessive water loss. The EW plays an important role in increasing the drought tolerance of the plants by reducing the non-stomatal transpiration, which contributes to the evolution of aquatic plants to land plants. It’s reported that the water utility and the crop yield of plants which are covered with a layer of waxes are higher than the corresponding NILs without waxes under the same condition. Therefore, it’s esential to explore more wax-related genes and study their functions in crop breeding for drought tolerance. Here, we report the phenotypic characterization and gene cloning of maize mutant glossy6(gl6), and the primary functional analysis of gl6 gene. The main results are as below:The second leaves of gl6 mutant and the corresponding wild-type seedlings(three-leave stage) were collected separately for SEM analysis. Compared with the corresponding wild-type, the mutant of gl6 shows significant EW deficiency, and abnormal linear inclusions were observed in the leaf epidermal cells of mutant when further analyzed by TEM. The waxes component analysis of gl6 mutant suggests that the amount of total wax is reduced to 38.12% of wild-type, mainly because of the reduction of C29 components or longer ones, especially for the VLCFA C32:0 has reduced to 4.58%.The physiological studies show gl6 mutant has a lower capability of water retention and a faster chlorophyll leaching assays compared with the wild-type in normal watering condition. In addition, the gl6 mutant shows a lower relative leaf water content, a higher relative electrical conductivity and a lower survival ratio than the wild-type under water deficiency condition. Thermal imaging of seedlings shows that the leaf temperature of wild-type is higher than that of gl6 under normal or drought-stressed conditions. Further analysis shows that the stomata density, and stomatal index of gl6 mutant are unchanged, being similar to these of wild type. These results together suggest the dramatic decrease of gl6 mutant epicuticular wax leads to its weak drought tolerance.The D-L-A(Digestion-Ligation-Amplification) method is used for cloning the Mu-induced allele of gl6, combined with the gel selection of the amplicons that co-segregated with the glossy phenotypes, and followed by the Sanger sequencing of the positive PCR products. The sequenced non-redundant fragments are aligned to the B73 reference genome, and GRMZM2G139786 seems to be a gl6 candidate gene, which is further confirmed via PCR. It’s indicated that the gl6 expression of q RT-PCR analysis is consistent to the data from Maize GDB and the q Teller, which shows a relatively high expression of gl6 in the leaf, embryo, anthers, androecium and the male tassels. Subcellular analysis of the gl6 in maize protoplast shows that gl6 is mainly expressed in the cytoplasm and plasma membrane. The homolog analysis of the gl6 suggests that few homologs with annotation are found in the Arabidopsis or in the rice database. Overexpression of the gl6 in Arabidopsis doesn’t alter the drought resistance of transgenic lines.In conclusion, it’s speculated that gl6 may play an important role in elongation or transportation of waxes. For certain components, the retention in cytoplasm of mutant changes the normal accumulation of EW. Therefore, the gl6 seedlings show weak resistance toward drought stress. |
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