Calcium-Dependent Regulation of Genes for Plant Nodulation in Rhizobium leguminosarum Detected by iTRAQ Quantitative Proteomic Analysis

Rhizobia, the nitrogen-fixing bacterial
symbionts of legumes, represent
an agricultural application of primary relevance and a model of plant–microbe
molecular dialogues. We recently described rhizobium proteome alterations
induced by plant flavonoids using iTRAQ. Herein, we further extend
that experimentation, proving that the transient elevation in cytosolic
calcium is a key signaling event necessary for the expression of the
nodulation (nod) genes. Ca2+ involvement in nodulation
is a novel issue that we recently flagged with genetic and physiological
approaches and that hereby we demonstrate also by proteomics. Exploiting
the multiple combinations of 4-plex iTRAQ, we analyzed Rhizobium leguminosarum cultures grown with or without
the nod gene-inducing plant flavonoid naringenin
and in the presence or absence of the extracellular Ca2+ chelator EGTA. We quantified over a thousand proteins, 189 of which
significantly altered upon naringenin and/or EGTA stimulation. The
expression of NodA, highly induced by naringenin, is strongly reduced
when calcium availability is limited by EGTA. This confirms, from
a proteomic perspective, that a Ca2+ influx is a necessary
early step in flavonoid-mediated legume nodulation by rhizobia. We
also observed other proteins affected by the different treatments,
whose identities and roles in nodulation and rhizobium physiology
are likewise discussed.

根瘤菌（Rhizobia）是与豆科植物共生的固氮细菌，既是一类具有核心农业应用价值的微生物，也是研究植物-微生物分子互作的经典模型体系。我们此前利用同位素相对和绝对定量技术（iTRAQ），解析了植物类黄酮诱导的根瘤菌蛋白质组变化。本研究进一步拓展了前述实验，证实胞质钙离子的瞬时升高是结瘤（nod）基因表达所必需的关键信号事件。钙离子参与结瘤过程是我们团队近期通过遗传与生理学方法提出的全新假说，本次研究亦通过蛋白质组学手段验证了这一结论。本研究利用4标iTRAQ（4-plex iTRAQ）的多重组合策略，对添加/未添加结瘤基因诱导型植物类黄酮柚皮素，以及存在/不存在细胞外钙离子螯合剂EGTA的培养条件下的豌豆根瘤菌（Rhizobium leguminosarum）进行了蛋白质组分析。我们共定量了超过1000种蛋白质，其中189种在柚皮素和/或EGTA处理下发生显著表达变化。柚皮素可强烈诱导结瘤蛋白A（NodA）的表达，而当EGTA限制钙离子可获得性时，该蛋白的表达水平会显著下调。这从蛋白质组学视角进一步证实，钙离子内流是类黄酮介导的根瘤菌诱导豆科植物结瘤过程中不可或缺的早期关键步骤。此外，本研究还发现了其他受不同处理影响的蛋白质，并对其身份以及在结瘤过程和根瘤菌生理中的作用进行了讨论。