Mechanism for Membrane Lipid Alteration upon Phosphate Starvation in Plants

1. 磷酸盐饥饿下的半乳糖脂合成植物在磷酸盐（Pi）饥饿期间，膜磷脂含量降低，同时非磷糖脂增加。虽然一些研究表明植物激素参与了Pi饥饿后的各种生理变化，但Pi饥饿诱导的膜脂改变的调控尚不清楚。此前，我们报道了B型单半乳糖二酰基甘油合成酶基因（atMGD2和atMGD3）对Pi饥饿的反应，并提出了这些基因在Pi饥饿期间半乳糖脂积累中的作用。在本项目中，我们研究了atMGD2/3对Pi饥饿的响应和膜脂组成变化的调控机制。外源生长素激活了Pi饥饿期间atMGD2/3的表达，而细胞分裂素在根中抑制了它们的表达。此外，生长素抑制剂和axr4 aux1双突变抑制了Pi饥饿时atMGD2/3表达的增加，表明atMGD2/3的激活需要生长素。在Pi饥饿期间，激素对膜脂组成的影响也被观察到，这表明生长素和细胞分裂素确实参与了Pi饥饿期间膜脂的动态变化。亚硝酸盐在植物体内不能代谢；然而，当我们向pi饥饿的植物提供亚磷酸盐时，在atMGD2/3的表达和膜脂的变化方面，pi饥饿反应消失了。这些结果表明，在Pi饥饿期间观察到的植物膜的整体变化不是由Pi饥饿引起的植物细胞损伤引起的，而是由Pi信号和生长素/细胞分裂素的交叉对话严格调节的。磷酸盐饥饿时磷脂分解代谢在磷酸盐饥饿期间，磷脂被降解，相反，一种非磷半乳糖双半乳糖二酰基甘油在植物根质膜中积累。在本项目中，我们发现了一种新的磷脂酶C，它可以水解磷脂酰胆碱，并在拟南芥中受到磷酸盐剥夺的强烈诱导。由于磷脂酶C对磷脂酰胆碱的水解活性在缺磷植物中被高度上调，部分磷脂酶C的基因表达可能在缺磷植物中被诱导。基于与细菌磷脂酰胆碱水解磷脂酶C的氨基酸序列相似性，在拟南芥基因组中鉴定出6个推定的磷脂酶C，其中一个NPC4在磷酸盐限制下表现出显著的转录激活。NPC4基因的分子克隆和功能表达证实，NPC4基因编码的功能性磷脂酰胆碱水解磷脂酶C不需要Ca^<2+>才能发挥活性。亚细胞定位分析表明，NPC4蛋白在质膜中高度富集。对T-DNA标记的npc4突变体的分析显示，在磷酸盐饥饿反应中，npc4的破坏严重降低了磷脂酰胆碱水解磷脂酶C的活性。这些结果表明，NPC4在无机磷酸盐和二酰基甘油的膜定位磷脂供应中起重要作用，这些磷脂在磷酸盐剥夺过程中用于补充磷酸盐和替代根质膜中的极性脂。少

1. Galactolipid synthesis under phosphate starvationDuring phosphate (Pi) starvation in plants, membrane phospholipid content decreases concomitantly with an increase in non-phosphorus glycolipids. Although several studies have indicated the involvement of phytohormones in various physiological changes upon Pi starvation, the regulation of Pi-starvation induced membrane lipid alteration remains unknown. Previously, we reported the response of type B monogalactosyl diacylglycerol synthase genes (atMGD2 and atMGD3) to Pi starvation, and suggested a role for these genes in galactolipid accumulation during Pi starvation. In this project, we performed an investigation of the regulatory mechanism for the response of atMGD2/3 and changes in membrane lipid composition to Pi starvation. Exogenous auxin activated atMGD2/3 expression during Pi starvation, whereas their expression was repressed by cytokinin treatment in the root. Moreover, auxin inhibitors and the axr4 aux1 double mutation in auxi … More n signaling impaired the increase of atMGD2/3 expression during Pi starvation, showing that auxin is required for atMGD2/3 activation. The fact that hormonal effects during Pi starvation were also observed with regard to changes in membrane lipid composition demonstrates that both auxin and cytokinin are indeed involved in the dynamic changes in membrane lipids during Pi starvation. Phosphite is not metabolically available in plants ; however, when we supplied phosphite to Pi-starved plants, the Pi-starvation response disappeared with respect to both atMGD2/3 expression and changes in membrane lipids. These results indicate that the observed global change in plant membranes during Pi starvation is not caused by Pi-starvation induced damage in plant cells but rather is strictly regulated by Pi signaling and auxin/cytokinin cross-talk.2. Phospholipid catabolism upon phosphate starvationDuring phosphate starvation, it is known that phospholipids are degraded, and conversely, a non-phosphorus galactolipid digalactosyldiacylglycerol accumulates in the root plasma membrane of plants. In this project, we found on a novel phospholipase C which hydrolyzes phosphatidylcholine and is greatly induced in response to phosphate deprivation in Arabidopsis. Since phosphatidylcholine-hydrolyzing activity by phospholipase C was highly up-regulated in phosphate-deprived plants, gene expression of some phospholipase C was expected to be induced during phosphate starvation. Based on amino acid sequence similarity to a bacterial phosphatidylcholine-hydrolyzing phospholipase C, six putative phospholipase Cs were identified in the Arabidopsis genome, one of which, NPC4, showed significant transcriptional activation upon phosphate limitation. Molecular cloning and functional expression of NPC4 confirmed that NPC4 gene encoded a functional phosphatidylcholine-hydrolyzing phospholipase C which did not require Ca^<2+> for its activity. Subcellular localization analysis showed that NPC4 protein was highly enriched in the plasma membrane. Analyses of T-DNA tagged npc4 mutants revealed that disruption of NPC4 severely reduces the phosphatidylcholine-hydrolyzing phospholipase C activity in response to phosphate starvation. These results suggest that NPC4 plays an important role in the supply of both inorganic phosphate and diacylglycerol from membrane-localized phospholipids that would be used for phosphate supplementation and the replacement of polar lipids in the root plasma membrane during phosphate deprivation. Less