Proteomic analysis of grafted and non-grafted melon at different growth stages

Abstract:【Objective】Melon (Cucumis melo L.) is popular among peoples for fresh and sweet fruits, aromaticflavor and abundant nutrition. With the progress producing conditions and scientific research,melon production has been rapidly developed in China. In practice, grafting has been widely used inmelon production to deal with soil-borne diseases and to improve plant vigor, and the absorption of waterand fertilizer. In order to reveal the molecular mechanism of the the effects of melon grafting on scionvariety, we analyzed the protein abundance changes in leaves of grafted and non-grafted melon atdifferent growth stages and to screen out the key metabolic pathways and important difference proteinsusing the approach of proteomics. 【Methods】The thin melon variety‘Guangmi No.1’and the pumpkinvariety‘Xiangzhen No.1’were respectively chosen as the scion and rootstock for grafting in the exper- iment, and the non- grafted melon was used as a control group. Their stem diameter and leaf area inboth groups were respectively measured on the 10th, 15th, 20th, 40th, 50th and 70th d after planting.The yield of melon was measured on the 30th d after florescence. The physiological traits, including solubleprotein content, lignin content, chlorophyll content, peroxidase (POD) activity, superoxide dismutase(SOD) activity, proline(Pro) content and malondialdehyde(MAD) content, were observed byplant biochemistry methods at the seedling stage(10 d after planting), flowering stage (40 d after planting)and fruit ripening stage(70 d after planting). The differential proteome expression was analyzed byTMT-labeled LC-MS/MS (TMT-LC-MS/MS) quantitative proteomics techniques and six important proteinswere verified by parallel reaction monitoring technique (PRM) at the three growth stages. 【Results】The stem diameter of grafted melon increased significantly by 26.88%, 18.29%, 27.78% and 28.66%,and the leaf area increased significantly by 15.9 cm2, 20.94 cm2, 42.67 cm2 and 30.15 cm2, respectively,on the 20th, 40th, 50th and 70th d after planting. But no significant change was found on the 10th d andthe 15th d after planting. The yield increased significantly by 10.52%. At the seedling stage, floweringstage and fruit ripening stage, the soluble protein content of grafted melon increased very significantlyby 39.22%, 57.83% and 51.73%; the Lignin content increased very significantly by 40.80%, 38.51%and 37.08%; the chlorophyll a content increased significantly by 19.91% and 19.00% at seedling stageand flowering stage, but there was no significant change at the fruit ripening stage; the chlorophyll bcontent increased significantly by 40.51%, 52.22% and 19.19%; the chlorophyll a+b content increasedsignificantly by 24.85%, 26.68% and 17.83%; the POD increased significantly by 102.74%, 40.15%and 15.57%; the SOD activity increased significantly by 22.97% and 11.80% at seedling stage and floweringstage, but there was no significant change at the fruit ripening stage; the Pro content increased significantlyby 40.42%, 14.19% and 18.88%; the MAD content decreased significantly by 18.09%,23.64% and 26.27%. The proteomics analysis indicated that 6 023 proteins were identified at the seedlingstage, the flowering stage and the fruit ripening stage. 5 150 of them had quantitative information,accounting for 85.5% of total proteins. The differentially expressed proteins standard was the protein expressiondifference change more than 1.3 times (up-regulation > 1.3) or less than 0.77 times (down-regulation＜ 0.77) with p ＜ 0.05. Statistical analysis revealed a total of 2 187 differential proteins including1 142 up-regulated and 1 045 down-regulated proteins, and the number of differential proteins at theseedling stage, the flowering stage and the fruit ripening stage were 1 141 (657 up-regulated and 484down-regulated proteins), 716 (334 up-regulated and 382 down-regulated proteins) and 330(151 up-regulatedand 179 down-regulated proteins), respectively. The differential proteins at three growth stagesrespectively accounted for 52.2%, 32.7% and 15.1% of total differential proteins. The GO and theKEGG analyses revealed that the differential proteins were mainly related to the molecular functions ofthe catalytic activity and the binding, metabolic and the cellular processes, and the single-organism process.The proteins from phenylpropanoid biosynthesis, ribosome and porphyrin and chlorophyll metabolismwere up-regulated, while the ones in the photosynthesis pathway were down-regulated. Interestingly,the expression of some proteins involved in the phenylpropanoid biosynthesis showed significant differencesat the three growth stages. It was found that the change trend of 6 selected differential proteinsusing PRM target verification was consistent with that of TMT quantitative analysis.【Conclusions】Thepumpkin rootstock might increase the osmotic adjustment and the protective enzyme system, and reducethemembrane lipid peroxidation and up-regulate the expression of the key proteins involved in thephenylpropanoid biosynthesis pathway, the ribosome pathway and the porphyrin and the chlorophyllmetabolism pathway to improve the growth and resistance of the scion variety of the melon.