Deposition Process Of Epicuticular Wax On Rice Leaves And Characterization Of Wax Associated Transcription Factor Gene OsWTF1 And OsWTF2

A mechanism that plants have adopted to survive in a terrestrial, aerial environment is the production of waxes deposited into and on the cuticular layer of their outmost surface. The epicuticular wax acts as the first protective barrier against UV radiation, bacterial and fungal attacks and regulates non-stomatal water loss. Studies have showed a positive relationship between the epicuticular wax content and plant stress resistance. Stress-related transcription factors play important roles in regulating the expression of special stress-related genes that allow plants to respond optimally to their changing environment. It has been suggested that manipulating of genes encoding transcription factors might be an effective method in plant anti-stress gene engineering.In this paper, epicuticular wax deposition during rice leaf development, among different cultivars, under drought and ultraviolet-B radiation stresses was analyzed respectively. Two putative wax related rice transcription factor genes, OsWTF1 and OsWTF2 were cloned. The expressions pattern of OsWTF1 and OsWTF2 were analyzed using their promoter driven GUS transgenic lines. The functions of OsWTF1 and OsWTF2 were characterized by overexpression and RNAi transgenic plants analyses. The following results were obtained:1) Deposition and crystalloid morphology of epicuticular wax were observed by scanning electronic microscopy during the development of rice leaf. Epicuticular wax was deposited well on the leaf before it was exposed to the aerial environment. The development process of epicuticular wax is from leaf apex to leaf base, from leaf adaxial to leaf abaxial. More wax crystalloids were deposited on adaxial than on abaxial. Wax crystalloid accumulation achieved to peak when leaves were fully developed. Beginning from the late mature stage, the epicuticular wax on leaves started to collapse from leaf margin to leaf center.2) The extraction method of rice epicuticular wax was optimized. High-temperature method with solution CHCl₃ was the most effective and simplest rice wax extraction method.3) Epicuticular wax crystalloids on leaves of three different rice (Oryza sativa L.) genotypes, including two wetland varieties (Japonica cultivar Nipponbare and Indica cultivar 9311) and one upland rice variety (drought resistance variety Han 209) were observed by scanning electronic microscope. The wax contents on leaves of three rice genotypes were compared at normal growth condition and under drought or ultraviolet-B radiation stress. The results indicated that upland rice cultivar had more epicuticular wax crystalloids and higher wax contents than wetland cultivars. Wax contents and crystalloid distribution were increased in all three cultivars under ultraviolet-B radiation and drought stresses. Uniform and dense epicuticular wax distribution on rice leaves were positively related with strong resistance to drought and ultraviolet-B radiation stresse.4) By BLAST Arabidopsis transcription factor gene WIN1(SHN1)involved in wax biosynthesis to the rice genome sequences, two homologous gene sequences were found in rice and were named OsWTF1 and OsWTF2 respectively. Bioinformatics analysis showed that both OsWTF1 and OsWTF2 contained a single conserved AP2 DNA-binding domain respectively, which belonging to ERF subfamily of AP2/ERF transcriptional factor super-family. Moreover, many cis-acting elements were found in controlling the spatial and temporal expression and the stress-responsive gene expression. These results suggested that OsWTF1 and OsWTF2 might participate in regulating the stress responses.5) The expression patterns of the OsWTF1 and OsWTF2 genes were examined by their putative promoter driven GUS expression in the transgenic rice plants. GUS expression was detected in most plant organs, including epidermis, pith of the root, vascular bundle or pith of the stem, stomata apparatu, trichomes, leaf sheath and stamen. OsWTF1 expressions were stronger in nutrient organs than that in reproductive organs, but reverse to OsWTF2.6) Full length cDNA sequence of OsWTF1 was cloned by RACE technology from rice mRNA and full length of OsWTF2 were bought from rice resource centre of Japan. OsWTF1 and OsWTF2 Overexpression vectors and OsWTF2 RNAi vector were constructed and separately transformed into rice by agrobacterium-mediated method. The transformations were confirmed by PCR and RT-PCR. OsWTF1 and OsWTF2 overexpression transgenic lines showed reduced plant heights and seed setting rates,,abnormal pollen morphology, decreased trichome numbers on leaf and chlorophyll leaching rates and increased leaf epicuticular wax These phenotypes suggested that OsWTF1 and OsWTF2 were involved in the wax metabolism. However, no epicuticular wax change was detected in silenced OsWTF2 RNAi transgenic lines.