| Ponkan (Citrus reticulata Blanco) is one of the most widely-cultivated loose skin mandarins in china. In production of Ponkan, postharvest diseases and quality degredation usually caused a serious economic loss. Therefore, fresh-keeping and anti-corrosion are the main tasks in citrus postharvest management. In practice, it is demonstrated that hot water treatment (HWT), a non-chemical treatment, is an effective method on improving storage characteristics of citrus, however the intrinsic mechanisms of HWT on anti-corrosion is still unclear. It was reported in plant, SNARE (N-ethylanaleimide-sensitive fusion protein attachment protein receptors) plays important regulatory roles in stress response. Vesicle trafficking mediated by SNAREs could recognize external stimuli, followed by activation of organism defense responses. This process was assumed to be closely related to the constructon of cell wall. Preliminary experiments revealed that the expression level of VAMP, a member of SNARE family, was significantly up-regulated in citrus fruit when exposed to HWT. However, the expression pattern and response of VAMP induced by HWT in citrus fruit was largely unknown. In present study, HWT at 52℃followed by storage experiments at ambient (10-20℃) and cold (4-6℃) conditions were conducted on mature Ponkan (Citrus reticulata Blanco) fruits. Cytological changes were observed under microscope via paraffin sectioning, meanwhile, alteration of the cell wall structure were also assyed by lignin contents analysis. To understand the specific expression patterns of VAMP7 in the treated fruit, semi-quantitative and quantitative RT-PCRs, as well as western blot were carried out. Our investigations were expected to provide molecular evidence on function of HWT to postharvest citrus. The main results were as follows:1. Two VAMP members were successfully indentified and characterized in sclio cloning and 3’-RACE from Ponkan fruit. The two members were named as CitVAMP71 and CitVAMP72, cDNA fragment in the size of 1028 bp and 1009 bp were obtained respectively. CitVAMP71 and CitVAMP72 are highly homologous to Arabidposis thaliana VAMP with an identity of 80% at the amino acdid level, which indicated that the CitVAMP7 proteins were members of VAMP family.2. Expression of CitVAMP71 and CitVAMP72 had been remarkable affected by HWT. Semi-quantitative RT-PCR and Real-time PCR shows that CitVAMP71 and CitVAMP72 was strongly but transiently induced by HWT, especially in flavedo. The expression of CitVAMP71 and CitVAMP72 in fruits stored in the subsequent cold condition is higher than that in ambient, and the expression level in peel and pulp showed contrary trends.3. Accumulation of CitVAMP71 protein was remarkably affected by HWT. Western blotting results showed that CitVAMP71 protein level in pulp is higher than in flavedo. CitVAMP71 protein accumulated at a relatively high level at 2 h and 12h after HWT in pulp. Compared to CitVAMP71, CitVAMP72 showed no significant difference. 4. HWT strengthens cell wall of the peel. According to the result of paraffin sectioning, HWT was demonstrated to significantly thickened the cell wall of the peel; lignin content as a main component of secondary cell wall, was also induced by HWT.In summary, the transcriptional level and translational level of CitVAMP71 were increased in citrus fruit when exposed to HWT, and the cell wall of fruit was significantly thickened, which reinforced the supporting structure to act as chemical defense. The expression alteration of CitVAMP71 may drive vesicle with material related to cell wall transporting to cell wall space, which constructed crude barrier to stress and pathogen. Results of this study provided therotic basis for improving storage characteristics of citrus by HWT. |
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