The Vacuolar Proton-Cation Exchanger EcNHX1 Generates pH Signals for the Expression of Secondary Metabolism in Eschscholzia californica1
The Vacuolar Proton-Cation Exchanger EcNHX1 Generates pH Signals for the Expression of Secondary Metabolism in Eschscholzia californica1
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DOI：
10.1104/pp.15.01570
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发表时间：
2015-11
期刊：
影响因子：
7.4
通讯作者：
Sophie Weigl;W. Brandt;R. Langhammer;W. Roos
中科院分区：
文献类型：
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作者：
Sophie Weigl;W. Brandt;R. Langhammer;W. Roos
由激发剂刺激磷脂酶A2产生的溶脂激活了一个独特的液泡Na+/H+反转运蛋白，由此产生的细胞质H+峰值诱导生物碱的生物合成。加州埃斯科尔齐亚的细胞培养反应真菌激发剂过量生产抗菌苯并苯胺生物碱。生物合成酶表达的信号级联包括(1)质膜磷脂酶A2的激活，导致溶血磷脂酰胆碱的峰值，以及(2)随后空泡质子的短暂外排，导致细胞质H+的峰值。这项研究表明Na + / H +的逆向转运代理在大肠californica介导细胞的液泡膜质子通量。从互补DNA （EcNHX1-EcNHX4）中分离并克隆了4个反转运蛋白编码基因。以RNA干扰为基础，同时沉默EcNHX1、EcNHX3和EcNHX4导致细胞系稳定，但(1)液泡质子的钠依赖性外排能力和(2)激发剂引发的生物碱过量生产能力大大降低。加州加利福尼亚州的四个EcNHX基因中的每一个都在酿酒酵母Δnhx零突变体中重建了Na+依赖的H+流出的缺乏。只有转化并表达EcNHX1基因的酵母菌株表现出受溶血磷脂酰胆碱刺激的Na+依赖质子通量，从而产生空泡H+的净流出。这一发现得到了三维蛋白同源模型的支持，该模型预测了仅在EcNHX1中溶血磷脂酰胆碱的合理识别位点。我们得出结论，EcNHX1反转运蛋白通过招募液泡质子池参与信号传导过程，在激发子启动的生物碱生物合成基因表达中发挥作用。
A lysolipid generated by an elicitor-stimulated phospholipase A2 activates a distinct vacuolar Na+/H+ antiporter and the resulting peak of cytosolic H+ induces alkaloid biosynthesis. Cell cultures of Eschscholzia californica react to a fungal elicitor by the overproduction of antimicrobial benzophenanthridine alkaloids. The signal cascade toward the expression of biosynthetic enzymes includes (1) the activation of phospholipase A2 at the plasma membrane, resulting in a peak of lysophosphatidylcholine, and (2) a subsequent, transient efflux of vacuolar protons, resulting in a peak of cytosolic H+. This study demonstrates that one of the Na+/H+ antiporters acting at the tonoplast of E. californica cells mediates this proton flux. Four antiporter-encoding genes were isolated and cloned from complementary DNA (EcNHX1–EcNHX4). RNA interference-based, simultaneous silencing of EcNHX1, EcNHX3, and EcNHX4 resulted in stable cell lines with largely diminished capacities of (1) sodium-dependent efflux of vacuolar protons and (2) elicitor-triggered overproduction of alkaloids. Each of the four EcNHX genes of E. californica reconstituted the lack of Na+-dependent H+ efflux in a Δnhx null mutant of Saccharomyces cerevisiae. Only the yeast strain transformed with and expressing the EcNHX1 gene displayed Na+-dependent proton fluxes that were stimulated by lysophosphatidylcholine, thus giving rise to a net efflux of vacuolar H+. This finding was supported by three-dimensional protein homology models that predict a plausible recognition site for lysophosphatidylcholine only in EcNHX1. We conclude that the EcNHX1 antiporter functions in the elicitor-initiated expression of alkaloid biosynthetic genes by recruiting the vacuolar proton pool for the signaling process.