Barjaktarović Zarko, Schütz Wolfgang, Madlung Johannes, Fladerer Claudia, Nordheim Alfred, Hampp Rüdiger
University of Tübingen, Botany Institute, Physiological Ecology of Plants, Auf der Morgenstelle 1, D-72076 Tübingen, Germany.
J Exp Bot. 2009;60(3):779-89. doi: 10.1093/jxb/ern324. Epub 2009 Jan 6.
In a recent study it was shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by changes in protein expression. Using ESI-MS/MS for proteins with differential abundance after separation by 2D-PAGE, 28 spots which changed reproducibly and significantly in amount (P <0.05) after 2 h of hypergravity (18 up-regulated, 10 down-regulated) could be identified. The corresponding proteins were largely involved in stress responses, including the detoxification of reactive oxygen species (ROS). In the present study, these investigations are extended to phosphorylated proteins. For this purpose, callus cell cultures of Arabidopsis thaliana were exposed to hypergravity (8 g) and simulated weightlessness (random positioning; RP) for up to 30 min, a period of time which yielded the most reliable data. The first changes, however, were visible as early as 10 min after the start of treatment. In comparison to 1 g controls, exposure to hypergravity resulted in 18 protein spots, and random positioning in 25, respectively, with increased/decreased signal intensity by at least 2-fold (P <0.05). Only one spot (alanine aminotransferase) responded the same way under both treatments. After 30 min of RP, four spots appeared, which could not be detected in control samples. Among the protein spots altered in phosphorylation, it was possible to identify 24 from those responding to random positioning and 12 which responded to 8 g. These 12 proteins (8 g) are partly (5 out of 12) the same as those changed in expression after exposure to 2 h of hypergravity. The respective proteins are involved in scavenging and detoxification of ROS (32%), primary metabolism (20.5%), general signalling (14.7%), protein translation and proteolysis (14.7%), and ion homeostasis (8.8%). Together with our recent data on protein expression, it is assumed that changes in gravitational fields induce the production of ROS. Our data further indicate that responses toward RP are more by post-translational protein modulation (most changes in the degree of phosphorylation occur under RP-treatment) than by protein expression (hypergravity).
最近的一项研究表明，拟南芥愈伤组织细胞培养物会通过蛋白质表达的变化来响应重力场强度的改变。利用电喷雾串联质谱（ESI-MS/MS）分析经二维聚丙烯酰胺凝胶电泳（2D-PAGE）分离后丰度有差异的蛋白质，在超重力处理2小时后，可鉴定出28个斑点，其数量发生了可重复且显著的变化（P<0.05）（18个上调，10个下调）。相应的蛋白质主要参与应激反应，包括活性氧（ROS）的解毒。在本研究中，这些研究扩展到了磷酸化蛋白质。为此，将拟南芥愈伤组织细胞培养物暴露于超重力（8g）和模拟失重（随机定位；RP）环境中长达30分钟，这段时间能产生最可靠的数据。然而，最早在处理开始后10分钟就可见到最初的变化。与1g重力对照组相比，暴露于超重力环境导致18个蛋白质斑点出现变化，随机定位导致25个蛋白质斑点出现变化，信号强度增加/降低至少2倍（P<0.05）。只有一个斑点（丙氨酸转氨酶）在两种处理下反应相同。在随机定位处理30分钟后，出现了4个在对照样品中未检测到的斑点。在磷酸化改变的蛋白质斑点中，可从对随机定位有反应的斑点中鉴定出24个，从对8g重力有反应的斑点中鉴定出12个。这12种蛋白质（8g重力处理）部分（12种中的5种）与暴露于2小时超重力后表达发生变化的蛋白质相同。相应的蛋白质参与ROS的清除和解毒（32%）、初级代谢（20.5%）、一般信号传导（14.7%）、蛋白质翻译和蛋白水解（14.7%）以及离子稳态（8.8%）。结合我们最近关于蛋白质表达的数据，推测重力场的变化会诱导ROS的产生。我们的数据进一步表明，对随机定位的反应更多是通过蛋白质翻译后修饰（大多数磷酸化程度的变化发生在随机定位处理下）而非蛋白质表达（超重力处理）。
