Agronomy College, Sichuan Agricultural University, Chengdu 611130, China; Division of Applied Life Sciences (BK21+), Graduate School of Gyeongsang National University, Jinju 660-701, Republic of Korea.
Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843-2133, USA.
Plant Physiol Biochem. 2014 Jan;74:176-84. doi: 10.1016/j.plaphy.2013.10.022. Epub 2013 Nov 22.
The interaction of Obg (Spo0B-associated GTP-binding protein) GTPase and SpoT, which is a bifunctional ppGpp (guanosine 3',5'-bispyrophosphate) hydrolase/synthetase, is vital for the modulation of intracellular ppGpp levels during bacterial responses to environmental cues. It has been recently reported that the ppGpp level is also inducible by various stresses in the chloroplasts of plant cells. However, the function of the Obg-SpoT interaction in plants remains elusive. The results from the present and previous studies suggest that AtRSH1 is a putative bacterial SpoT homolog in Arabidopsis and that its transcription levels are responsive to wounding and salt stresses. In this study, we used a yeast two-hybrid analysis to map the regions required for the AtObgC-AtRSH1 interaction. Moreover, protein-protein docking simulations revealed reasonable geometric and electrostatic complementarity in the binding surfaces of the two proteins. The data support our experimental results, which suggest that the conserved domains in AtObgC and the N terminus of AtRSH1 containing the TGS domain contribute to their interaction. In addition, quantitative real-time PCR (qRT-PCR) analyses showed that the expression of AtObgC and AtRSH1 exhibit a similar inhibition pattern under wounding and salt-stress conditions, but the inhibition pattern was not greatly influenced by the presence or absence of light. Based on in vivo analyses, we further confirmed that the AtRSH1 and AtObgC proteins similarly localize in chloroplasts. Based on these results, we propose that the AtObgC-AtRSH1 interaction plays a vital role in ppGpp-mediated stress responses in chloroplasts.
Obg（Spo0B 相关 GTP 结合蛋白）GTP 酶与 SpoT 的相互作用对于细菌对外界环境刺激的反应过程中细胞内 ppGpp 水平的调节至关重要，SpoT 是一种具有双功能的 ppGpp（鸟苷 3'，5'-二磷酸）水解酶/合成酶。最近有报道称，植物细胞叶绿体中的各种应激也可以诱导 ppGpp 水平的变化。然而，Obg-SpoT 相互作用在植物中的功能仍然难以捉摸。本研究和之前的研究结果表明，AtRSH1 是拟南芥中一种假定的细菌 SpoT 同源物，其转录水平对创伤和盐胁迫有反应。在本研究中，我们使用酵母双杂交分析来绘制 AtObgC-AtRSH1 相互作用所需的区域。此外，蛋白质-蛋白质对接模拟显示，这两种蛋白质的结合表面具有合理的几何和静电互补性。数据支持我们的实验结果，表明 AtObgC 的保守结构域和含有 TGS 结构域的 AtRSH1 的 N 端有助于它们的相互作用。此外，实时定量 PCR（qRT-PCR）分析显示，AtObgC 和 AtRSH1 的表达在创伤和盐胁迫条件下表现出相似的抑制模式，但抑制模式不受光的存在与否的影响。基于体内分析，我们进一步证实了 AtRSH1 和 AtObgC 蛋白在叶绿体中具有相似的定位。基于这些结果，我们提出 AtObgC-AtRSH1 相互作用在叶绿体中 ppGpp 介导的应激反应中发挥着重要作用。
