Data_Sheet_3_The Role of Heterotrimeric G-Protein Beta Subunits During Nodulation in Medicago truncatula Gaertn and Pisum sativum L..PDF

Heterotrimeric G-proteins regulate plant growth and development as master regulators of signaling pathways. In legumes with indeterminate nodules (e.g., Medicago truncatula and Pisum sativum), the role of heterotrimeric G-proteins in symbiosis development has not been investigated extensively. Here, the involvement of heterotrimeric G-proteins in M. truncatula and P. sativum nodulation was evaluated. A genome-based search for G-protein subunit-coding genes revealed that M. truncatula and P. sativum harbored only one gene each for encoding the canonical heterotrimeric G-protein beta subunits, MtG beta 1 and PsG beta 1, respectively. RNAi-based suppression of MtGbeta1 and PsGbeta1 significantly decreased the number of nodules formed, suggesting the involvement of G-protein beta subunits in symbiosis in both legumes. Analysis of composite M. truncatula plants carrying the pMtGbeta1:GUS construct showed β-glucuronidase (GUS) staining in developing nodule primordia and young nodules, consistent with data on the role of G-proteins in controlling organ development and cell proliferation. In mature nodules, GUS staining was the most intense in the meristem and invasion zone (II), while it was less prominent in the apical part of the nitrogen-fixing zone (III). Thus, MtG beta 1 may be involved in the maintenance of meristem development and regulation of the infection process during symbiosis. Protein–protein interaction studies using co-immunoprecipitation revealed the possible composition of G-protein complexes and interaction of G-protein subunits with phospholipase C (PLC), suggesting a cross-talk between G-protein- and PLC-mediated signaling pathways in these legumes. Our findings provide direct evidence regarding the role of MtG beta 1 and PsG beta 1 in symbiosis development regulation.

异三聚体G蛋白（Heterotrimeric G-proteins）作为信号通路的核心调控因子，调控植物的生长与发育。对于具有不定型根瘤（indeterminate nodules）的豆科植物（如蒺藜苜蓿（Medicago truncatula）和豌豆（Pisum sativum）），异三聚体G蛋白在共生发育中的作用尚未得到广泛研究。本研究针对异三聚体G蛋白在蒺藜苜蓿与豌豆根瘤形成中的参与情况进行了评估。基于基因组的G蛋白亚基编码基因检索结果显示，蒺藜苜蓿与豌豆各自仅携带1个编码经典异三聚体G蛋白β亚基的基因，分别为MtGβ1与PsGβ1。通过RNA干扰（RNAi）抑制MtGβ1与PsGβ1的表达，可显著降低形成的根瘤数量，这表明G蛋白β亚基在两种豆科植物的共生过程中均发挥作用。对携带pMtGβ1:GUS载体的复合蒺藜苜蓿植株进行分析，结果显示在发育中的根瘤原基与幼嫩根瘤中出现β-葡萄糖苷酸酶（β-glucuronidase，GUS）染色信号，这与G蛋白调控器官发育与细胞增殖的研究结论相符。在成熟根瘤中，GUS染色在分生组织与侵染区（II）中最为强烈，而在固氮区（III）的顶端区域染色较弱。据此推测，MtGβ1可能参与维持共生过程中的分生组织发育，并调控侵染进程。通过免疫共沉淀（co-immunoprecipitation）开展的蛋白质相互作用研究，揭示了G蛋白复合物的潜在组成，以及G蛋白亚基与磷脂酶C（phospholipase C，PLC）的相互作用，这表明在这些豆科植物中，G蛋白介导的信号通路与PLC介导的信号通路之间存在交叉对话。本研究的发现为MtGβ1与PsGβ1在共生发育调控中的作用提供了直接证据。