Institute for Horticultural Plants, China Agricultural University, Beijing 100193, China.
Changli Institute of Pomology, Hebei Academy of Agricultural and Forestry Sciences, Changli 066600, China.
Proteome Sci. 2014 May 25;12:31. doi: 10.1186/1477-5956-12-31. eCollection 2014.
Although protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms.
Phosphorylated proteins from leaves of three apple seedlings at juvenile, adult vegetative and reproductive stages were extracted and subjected to alkaline phosphatase pre-treatment. After separating the proteins by two-dimensional gel electrophoresis and phosphoprotein-specific Pro-Q Diamond staining, differentially expressed phosphoproteins were identified by MALDI-TOF-TOF mass spectrometry.
A total of 107 phosphorylated protein spots on nine gels (three ontogenetic phases × three seedlings) were identified by MALDI-TOF-TOF mass spectrometry. The 55 spots of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases. Abundances of the 27 spots corresponding to Rubisco activase declined between juvenile and reproductive phases. More extensively, phosphorylated β-tubulin chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases.
Protein phosphorylation varied significantly during vegetative phase change and floral transition in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations.
尽管蛋白质磷酸化是影响蛋白质功能和代谢的一种重要的翻译后修饰，但木质被子植物在个体发生过程中这一过程的动态变化仍未得到探索。
从幼年、成年营养和生殖阶段的三个苹果幼苗叶片中提取磷酸化蛋白，并进行碱性磷酸酶预处理。通过二维凝胶电泳和磷酸蛋白特异性 Pro-Q Diamond 染色分离蛋白质后，用 MALDI-TOF-TOF 质谱鉴定差异表达的磷酸化蛋白。
通过 MALDI-TOF-TOF 质谱鉴定，在 9 张凝胶（三个个体发生阶段×三个幼苗）上共鉴定到 107 个磷酸化蛋白点。107 个磷酸化蛋白点中有 55 个为核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）大亚基片段，在个体发生阶段其蛋白丰度和磷酸化程度差异显著。与幼年和生殖阶段相比，对应 Rubisco 激活酶的 27 个斑点的丰度下降。更广泛地说，在幼年和成年营养阶段，具有较低等电点的磷酸化β-微管蛋白链斑点最为丰富。
在苹果幼苗的营养阶段变化和花转变过程中，蛋白质磷酸化发生了显著变化。大多数观察到的变化在幼苗之间以及杂种群体之间是一致的。
