| The breeding period for apple is comparatively long mainly because of its long juvenile phase. The process of individual development in higher plants was necessarily accompanied by a series of gene differential expression and protein qualitative or quantitative changes, but the physiological and molecualr mechanisms during the developmental process of individuals in perennial woody angiosperm plants are still unclear. Previous studies showed that post-translational modifications happened for some proteases in individual apple seedling plant during its developmental processes. Protein phosphorylation is an important post-translational modification affecting protein function and metabolism; therefore it is posssible to uncover the molecular mechanism of phase change at protein post-translational level by studying the various phosphorylations in apple seedling plant during different developmental stages, which is important to further understand the individual development in woody perennial angiosperm plants.In this study, to confirm the accordance of protein phosphorylation among different apple seedlings derived from different hybrid crosses, proteins from three different developmental stages’ leaves of three apple seedlings derived from two hybrid crosses (’Jonathan’ x ’Golden Delicious’ and ’Jonathan’ x’Zisai Pearl’ x ’Red Fuji’)were extracted, then optimize the Pro-Q Diamond staining and protein dephosphorylation methods. Combine2-DE and western blotting to preliminary identified the phosphorylation of S6PDH and then confirmed the testing system. Proteins were extracted and subjected to alkaline phosphatase pre-treatment, following separated by two-dimensional gel electrophoresis, meanwhile used Pro-Q Diamond to stain the protein that have not dephosphorylation, then find out the differentially expressed phosphoproteins and identified by MALDI-TOF-TOF mass spectrometry. The results were shown as follows:1. The modified dephosphorylation method combined with the TCA/acetone protocol can easyily get clear background and phosphoprotein spots could be visualized on2-DE map. Combined2-DE with western blotting to identified phosphorylation of S6PDH during ontogeny in the Malus seedlings, we found it is feasible to identified the phosphorylation of protein by using alkaline phosphatase to dephosphorylation of proteins2. Five groups of seven groups’ total of107phosphorylated protein spots in three apple seedlings were identified by MALDI-TOF-TOF:Group A6, A33, A48contains55spots and they were identified as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments, they are varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases:the global abundance of A6in the reproductive phase of the three seedlings was significantly lower than in juvenile and adult vegetative phases; A33-1expression was relatively weak during the juvenile phase of the three seedlings, the expression abundance of A33-2was higher during the adult vegetative phase than in juvenile and adult reproductive phases, the protein in spot A33-3was expressed more abundantly during the adult vegetative phase than in juvenile and adult reproductive phases and the expression abundance of A33-3 was greater than that of A33-1and A33-2; The expression of protein in A48-1was relatively lower in the reproductive phase, A48-2was expressed more abundantly in the adult vegetative phase than in juvenile and adult reproductive phases. The expression abundance of A48-3was likewise lower in the adult vegetative phase than in juvenile and reproductive phases.Group B38contains27spots and they were identified as Rubisco activase, the abundance of Rubisco activase was characterized by a declining gradient from juvenile to the reproductive phases; The expression of spot B38-2was more abundant than that of B38-1and B38-3during all ontogenetic phases of the three seedlings.Group C24contains25spots and they were identified as Tubulin beta chain. More extensively phosphorylated tubulin beta chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases. Spot C24-3, with a higher isoelectric point, was expressed more abundantly than spots with lower isoelectric points during all three phases in the three seedlings.From the results we can see that protein phosphorylation varied significantly during vegetative phase change in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations.3. For the three apple seedlings, net photosynthesis rate of node40,80,140(07-07-115), node30,90,120(07-07-133) and node30,90,140(07-09-141) were peak. With the increase of leaf s node, net photosynthetic rate also changes, but the net photosynthesis rate did not gradient increasing with the rise of the node. Thus we thought the phosphorylation events on Rubisco large subunit and Rubisco activase were affected by the different ontogenetic stages but not the leaf’s level. |
