Molecular Plant Biophysics and Biochemistry, Department of Molecular Biology, University of Salzburg, Billrothstr. 11, A-5020 Salzburg, Austria.
Biol Chem. 2011 Mar;392(3):249-62. doi: 10.1515/BC.2011.026.
14-3-3 proteins are major regulators in plant development and physiology including primary metabolism and signal transduction pathways, typically via a phosphorylation-dependent interaction with a target protein. Four full-length 14-3-3 isoforms were identified in pollen grains of Lilium longiflorum by screening of a cDNA library and RACE (rapid amplification of cDNA ends)-PCR. Mass spectrometry analysis of partially purified 14-3-3s confirmed the presence of the four isoforms but also indicated the presence of additional, less abundant 14-3-3 isoforms in lily pollen. Separation of partially purified 14-3-3 proteins by two-dimensional gel electrophoresis resulted in nine spots that mainly contained the four major 14-3-3 isoforms. In a first step to examine putative physiological roles of specific 14-3-3 isoforms, their subcellular expression profile during pollen germination and tube growth was monitored using a characterized set of antibodies against 14-3-3 proteins with distinct crossreactivity. The abundance profile of 14-3-3 proteins associated with the cytosol, endomembranes (tonoplast, endoplasmic reticulum, Golgi, mitochondria) and plasma membrane showed high spatial-temporal dynamics. This indicates different targets of 14-3-3 proteins at different organelles and time points during pollen germination and growth.
14-3-3 蛋白是植物发育和生理的主要调节剂，包括初级代谢和信号转导途径，通常通过与靶蛋白的磷酸化依赖性相互作用来实现。通过筛选 cDNA 文库和 RACE（快速扩增 cDNA 末端）-PCR，在百合花粉粒中鉴定出四个全长 14-3-3 同工型。部分纯化的 14-3-3 的质谱分析证实了四种同工型的存在，但也表明百合花粉中存在其他丰度较低的 14-3-3 同工型。通过二维凝胶电泳分离部分纯化的 14-3-3 蛋白，得到了九个斑点，主要包含四种主要的 14-3-3 同工型。为了初步研究特定 14-3-3 同工型的潜在生理作用，使用针对具有不同交叉反应性的 14-3-3 蛋白的一组经过表征的抗体，监测其在花粉萌发和管生长过程中的亚细胞表达谱。与细胞质、内质网（液泡膜、内质网、高尔基体、线粒体）和质膜相关的 14-3-3 蛋白的丰度谱显示出高时空动态性。这表明在花粉萌发和生长过程中不同细胞器和时间点有不同的 14-3-3 蛋白靶标。
