Study On The Mechanisms Of Ethylene In Regulating Brassica Napus Leaf Cuticular Waxes

Plant leaf epicuticular waxes are the boundaries between plants and environments, and they are believed to be the initial defenses to biological and abiotic stresses. Phytohormones play important regulatory roles in plant growth and development and answering defense reaction to stress. In the defense mechanism of Brassica nupas rape, we are not clear whether or not the epicuticular waxes was taking part in the ethylene induction defense process. Therefore, in the current experiment, two B. napus cultivars, resistant cultivar Zhongshuang 9 and susceptible cultivar Yuyou 19, were selected to analyze the effects of ethylene on epicuticular wax components and gas exchange parameters in B. napus. We also analyzed the effects of 1-Aminocylopropane-l-carboxylic acid (ACC) on the metabolism of epicuticular wax and the regulation related gene expression. The influence of the epicuticular wax induced by inoculating Sclerotinia scleotiorum on plant resistance was also analyzed. The main results were shown as follows:1. Results from scanning electron microscope showed that spraying ethephon had significant effects on epicuticular wax morphological structure. Fifteen days after ethephon treatment, the wax crystals melted on leaves of Zhongshuang 9 and Yuyou 19, and the number of short tubular structure increased. Twenty five days after ethephon treatment, the melting wax crystal enlarged, parts of the stomata was hidden, and the number of short tubular structure increased further.2. Compared with the spraying water, the content of total epicuticular waxes in Zhongshuang 9 was significantly reduced by ethylene treatment. The contents of alkanes, secondary alcohols and acids were also significantly reduced, whereas other components changed insignificantly. The total epicuticular waxes and alkanes contents in yuyou 19 were also significantly reduced after treated with ethylene, with others insignificantly changed.3. Compared with the spraying water, photo synthetic rate and stomatal conductance in Zhongshuang9 and photo synthetic rate in Yuyou 19 significant increased after treated with ethylene for 15 days. Twenty five days after treatment, photo synthetic rate, stomatal conductance, transpiration rate, and intercellular CO2 concentration in zhongshuang9 were significantly reduced, however the photo synthetic rate, stomatal conductance and transpiration rate in yuyoul9 significant increased.4. Compared with the spraying water, the size of lesion on plant leaf was less in treatment with ethylene after inoculating Sclerotinia sclerotiorum for 48h and 72h. There was no obvious difference in the resistance induced by ethylene between zhongshuang9 and yuyoul9.5. The results of the gene expression analysis showed that, the expression of ethylene response factor ERF, wax-related regulatory factor gene CER7, WIN1 and Myb96, and wax-related metabolic gene KCS1, in the two tested cultivars, were significantly up-regulated after 0.5h induced by ACC (WIN1 in zhongshuang9 insignificantly changed). The expression ofMAHl which is responsible for secondary alcohols synthesis was significantly down-regulated. The expression of the other wax-related genes including KCR, ECR, CER4, CER1 and CER3 were significantly up-regulated in yuyoul9 (ECR expression level was unchanged), while down-regulated in zhongshuang9 with significance in ECR and CER1. Two hours after ACC induction, the gene expression of ERF, CER7, KCR, ECR, CER4, CER1 and CER3 in yuyoul9, and the gene expression of WIN], Myb96, ERF, KCS1, ECR, CER4 and CER3 in zhongshuang9 kept up-regulated. The gene expression of MAH1 kept down-regulated. Six hours after ACC induction, though ERF expression levels kept up-regulated, the rising extent reduced when compared with treatment for 2h. The expression of regulatory factor changed insignificantly when compared to control. The other wax-related genes significantly down-regulated, with CER3 in both cultivars and KCS1 in Yuyoul9 insignificantly changed.Based on the main results stated above, ethephon could induce the melting of epicuticular wax structures and reduce the contents of alkanes and total epicuticular wax in B.napus. The variations of the wax component contents differed between two tested cultivars. The changes of epicuticular wax characteristic induced by ethephon might affect gas exchange parameters and plant disease resistance. The ethylene signaling pathways might be involved in regulating the expression of epicuticular wax gene, and thus the epicuticular wax deposition in B.napus.