Hebei Key Laboratory of Molecular and Cellular Biology, College of Life Science, Hebei Normal University, Shijiazhuang 050016, China.
Plant J. 2012 Feb;69(4):689-700. doi: 10.1111/j.1365-313X.2011.04823.x. Epub 2011 Dec 2.
Intracellular calcium (Ca(2+)) increases rapidly after heat shock (HS) in the Ca(2+)/calmodulin (Ca(2+)/CaM) HS signal transduction pathway: a hypothesis proposed based on our previous findings. However, evidence for the increase in Ca(2+) after HS was obtained only through physiological and pharmacological experiments; thus, direct molecular genetic evidence is needed. The role of phosphoinositide-specific phospholipase C (PI-PLC) is poorly understood in the plant response to HS. In this work, atplc9 mutant plants displayed a serious thermosensitive phenotype compared with wild-type (WT) plants after HS. Complementation of atplc9 with AtPLC9 rescued both the basal and acquired thermotolerance phenotype of the WT plants. In addition, thermotolerance was even improved in overexpressed lines. The GUS staining of AtPLC9 promoter:GUS transgenic seedlings showed that AtPLC9 expression was ubiquitous. The fluorescence distribution of the fusion protein AtPLC9 promoter:AtPLC9:GFP revealed that the subcellular localization of AtPLC9 was restricted to the plasma membrane. The results of a PLC activity assay showed a reduction in the accumulation of inositol-1,4,5-trisphosphate (IP(3)) in atplc9 during HS and improved IP(3) generation in the overexpressed lines. Furthermore, the heat-induced increase in intracellular Ca(2+) was decreased in atplc9. Accumulation of the small HS proteins HSP18.2 and HSP25.3 was downregulated in atplc9 and upregulated in the overexpressed lines after HS. Together, these results provide molecular genetic evidence showing that AtPLC9 plays a role in thermotolerance in Arabidopsis.
细胞内钙（Ca（2+））在热激（HS）后迅速增加，这是 Ca（2+）/钙调蛋白（Ca（2+）/CaM）HS 信号转导途径中的一个假说，这是基于我们以前的发现提出的。然而，HS 后 Ca（2+）增加的证据仅通过生理和药理学实验获得；因此，需要直接的分子遗传学证据。磷脂酶 C（PI-PLC）在植物对 HS 的反应中的作用知之甚少。在这项工作中，与野生型（WT）植物相比，atplc9 突变体植物在 HS 后表现出严重的热敏表型。用 AtPLC9 互补 atplc9 挽救了 WT 植物的基础和获得的耐热性表型。此外，过表达系的耐热性甚至得到了改善。AtPLC9 启动子：GUS 转基因幼苗的 GUS 染色表明 AtPLC9 表达普遍存在。融合蛋白 AtPLC9 启动子：AtPLC9：GFP 的荧光分布表明 AtPLC9 的亚细胞定位局限于质膜。PLC 活性测定的结果表明，HS 期间 atplc9 中肌醇-1,4,5-三磷酸（IP（3））的积累减少，而过表达系中 IP（3）的产生得到改善。此外，atplc9 中的热诱导细胞内 Ca（2+）增加减少。HS 后，atplc9 中小热休克蛋白 HSP18.2 和 HSP25.3 的积累下调，而过表达系中上调。综上所述，这些结果提供了分子遗传学证据，表明 AtPLC9 在拟南芥耐热性中起作用。
