磷脂酶D（phospholipase D, PLD）主要水解磷脂如磷脂酰胆碱（phosphocholine, PC）、磷脂酰乙醇胺（phosphoethanolamine, PE）等产生磷脂酸（phosphatidic acid, PA）和游离的头部基团。它们与磷脂酶C（phospholipase C, PLC）、磷脂酶A（phospholipase A, PLA）共同组成一个庞大的磷脂酶家族。模式植物拟南芥（Arabidopsis thaliana）基因组有12个PLD基因，分为PLDα(3)、PLDβ(2)、PLDγ(3)、PLDδ、PLDε和PLDζ(2)六类。AtPLDα1和AtPLDδ是含量最多的蛋白。PA是植物体内一种重要的信号分子，通过与靶蛋白的互作，广泛参与多种生理生化过程，如气孔运动、抗冷和脱水等。 微管（microtubule）与微丝（actin）共同组成植物细胞的骨架系统。微管骨架调控细胞形状的改变、细胞分裂、细胞内物质运输和信号转导等重要过程。在植物体内，由具有类似于动物的中心体功能的γ-tubulin驱动四种微管排列的形成，即间期排列（interphase cortical array）、早前期带（preprophase band，PPB）、有丝分裂纺锤体（mitotic spindle）和成膜体（phragmoplast）。在这些结构形成过程中，微管结合蛋白（microtubule-associated proteins，MAPs）起着重要的作用。 本实验室前期工作已经证明pldα1对盐胁迫敏感，且初步确定了PLDα1/PA是通过MAP65-1调控拟南芥盐响应过程的。本文进一步研究PLDα1/PA与MAP65-1结合及其互作机制。首先用10 μM oryzalin（微管解聚剂）处理WT（野生型）、pldα1、map65-1-1和map65-1-2，然后通过免疫荧光标记微管的方法观察到pldα1、map65-1-1和map65-1-2对oryzalin比WT敏感，微管解聚更严重。200 mM NaCl处理下， map65-1-1和map65-1-2存活率显著低于WT；pldα1和pldα1map65-1-1存活率也显著低于WT，微管解聚更严重。外源施加20 μM 16:0-18:2 PA（palmitoyl-linoleoyl PA）则能提高pldα1的耐盐能力，使其存活率上升，与WT无差异，微管数目也与WT相接近，而PA不能缓解map65-1-1和pldα1map65-1-1的盐敏感的表型，它们的微管数目显著低于PA和NaCl共处理下的WT和pldα1的微管数目。这些结果从遗传学角度证明在盐胁迫反应中，PLDα1/PA信号可能位于MAP65-1的上游。 我们进一步研究PA结合MAP65-1的位点。通过定点突变和脂-蛋白结合技术，证明MAP65-1的53-55位氨基酸残基KRK、61-63位的KSR和428-429位的SK是PA结合位点。随后通过脂微囊体免疫共沉淀、ELISA和原生质体过表达进一步证明这8个氨基酸是PA结合位点。然后将MAP65-6（与PA结合弱）与MAP65-1同源区域的氨基酸突变为KRK、KSR和SK，我们发现，突变的MAP65-6结合PA显著增强，进一步说明此8个氨基酸在PA 结合中的重要性。浊度法发现，虽然突变的MAP65-6结合PA增强了，但它对促进微管聚合没有影响，即使加入外源的PA，也不能促进微管聚合。这说明PA-MAP65-1互作在调控微管形态中的特异性。 我们接着研究PA与MAP65-1结合是否有利于耐盐性。25 mM NaCl处理原生质体后，过表达含有这8个氨基酸突变的MAP65-1（mutant）的原生质体细胞死亡率显著高于过表达MAP65-1（WT）的原生质体。与微管共沉淀、在原生质体过表达和对体外微管成束等实验结果表明，这8个氨基酸突变导致MAP65-1不能结合和成束微管。 最后，我们通过瞬时侵染拟南芥的方法观察MAP65-1与微管的共定位。对照下，无论在WT还是pldα1中，MAP65-1与微管共定位，微管数基本相同。50 mM NaCl处理24 h后，WT和pldα1的微管均发生解聚，但pldα1的微管解聚地更严重，几乎全部解聚，成点状结构，此时MAP65-1与解聚的微管共定位。外源施加20 μM 16:0-18:2 PA则使pldα1的微管恢复到WT的状态，MAP65-1重新与完整的微管结合。这些结果提示，盐诱导微管解聚以及MAP65-1与微管分离，作为耐盐机制的一部分，盐胁迫下，PLD1水解磷脂产生PA，后者结合MAP65-1并促进微管稳定，提高细胞耐盐能力。

Phospholipase Ds (PLDs) chiefly hydrolyze phospholipids, such as phosphocholine (PC) and phosphoethanolamine (PE) etc., into phosphatidic acid (PA) and free head group. The PLDs as well as their counterparts-phospholipase C (PLC) and phospholipase A (PLA) make up a huge phospholipase family. There are twelve PLDs in model plant-Arabidopsis thaliana genomes, namely PLDα(3), PLDβ(2), PLDγ(3), PLDδ, PLDε and PLDζ(2). AtPLDα1 and AtPLDδ are the most abundant proteins. PA, a signaling molecule of vita significance, is involved in various physiological and biochemical processes, e.g., stomatal movement, freezing tolerance and dehydration, by interacting with target proteins. The plant cells’ cytoskeleton system is composed of microtubules and actins. Microtubules regulate a broad range of processes such as dynamic change of cells, cell division, intracellular transportation and signal transduction etc. In planta, γ-tubulin, which possesses functionalities similar to animal centrosome, drives formation of four microtubule arrays, namely interphase cortical array, preprophase band (PPB), mitotic spindle and phragmoplast. During the formation of these arrays, microtubule-associated proteins (MAPs) play significant roles. Previous studies from our laboratory had demonstrated that pldα1 was more sensitive to salt stress compared to wild type (WT), and AtPLDα1/PA regulating responses to salt stress by interacting with MAP65-1, was preliminarily elucidated. This research focuses on further exploration of concrete PA binding sites of MAP65-1 and mechanisms of PA-MAP65-1 interaction. First of all, after exposure to 10 μM microtubule-disrupting drug oryzalin, microtubules of WT, pldα1, map65-1-1 and map65-1-2 were visualized by whole-mount immunofluorescence staining. As a consequence, pldα1, map65-1-1 and map65-1-2 were more sensitive to oryzalin than WT, followed by severe depolymerization of microtubules. Upon 200 mM NaCl treatment, the survival ratio of map65-1-1 and map65-1-2 was lower than that of WT. In addition to that, the survival ratio of pldα1 and pldα1map65-1-1 was lower than that of WT as well. Exogenous supplement with 20 μM 16:0-18:2 PA (palmitoyl-linoleoyl PA), salt tolerance of pldα1 was enhanced, accompanied by higher survival ratio and more microtubules which are similar to those of WT, respectively. However, the sensitive phenotype of pldα1 and pldα1map65-1-1 in response to salt stress was not alleviated by exogenous PA, with considerable decrease in the number of microtubules compared to that of WT and pldα1 which were co-treated with PA and NaCl. These results provided genetic evidence that there was a likelihood that PLDα1/PA signaling was upstream of MAP65-1. Next, PA binding sites of MAP65-1 were elaborate. Blending site-directed mutagenesis and fat-western, it is elucidated that 53-55 residues KRK, 61-63 residues KSR and 428-429 residues SK of MAP65-1 were PA binding sites. In the following experimentation using liposome coimmunoprecipitation, ELISA and protoplasts overexpression, these eight amino acids were deeply demonstrated as indeed PA binding sites. After that, the corresponding regions of MAP65-6 (having lowest affinity to PA than other members of MAP65 family), which were homologous to MAP65-1 with respect to PA binding regions, were mutated to KRK, KSR and SK, respectively. We found out capacity of MAP65-6 (mutant) binding to PA was improved, significantly surpassing that of MAP65-6 (WT) when using the same above approaches. Although MAP65-6 harbouring these eight amino acids, binding to PA was enhanced, it could not facilitate polymerization of microtubules when using turbidimetric analysis, even though with addition of exogenous PA. This result indicated PA-MAP65-1 interaction was of specificity in regulating organization of microtubules. Besides, whether PA-MAP65-1 binding and interaction were beneficial for strengthening salt tolerance of cells, was studied. Upon 25 mM NaCl treatment, the death ratio of protoplasts overexpressing MAP65-1 (mutant) harbouring mutation in these eight amino acids was significantly higher than that of protoplasts overexpressing MAP65-1 (WT). Surprisingly, in further studies, we found out MAP65-1 lost functionality of binding to and bundling microtubules when these eight amino acids were mutated by combination of microtubule cosedimention, protoplasts overexpression and microtubule polymerization. Finally, the colocalization of MAP65-1 with microtubules was investigated upon salt stress by transient transformation of Arabidopsis seedlings. MAP65-1 colocalized with microtubules well in WT and pldα1 seedlings and the number of microtubules in WT and pldα1 seedlings was similar. Nevertheless, upon 50 mM NaCl treatment for 24h, microtubules both became depolymerized in WT and pldα1 seedlings. However, in pldα1 seedlings, microtubules depolymerized more severely than those of WT, forming dot-like structures with few intact microtubules left. In the meanwhile, MAP65-1 colocalized with depolymerized microtubules. More importantly, exogenous 20 μM 16:0-18:2 PA could restore microtubule arrays of pldα1 seedlings to those of WT seedlings, and MAP65-1 recolocalized with intact microtubules. These results indicated that salt stress inducing depolymerization of microtubules and detachment of MAP65-1 from microtubules, were a part of mechanisms of salt tolerance. Salt stress stimulated PLD1’s activity, resulting in hydrolysis of phospholipids into PA. PA bound to MAP65-1, facilitating stability of microtubules and improving salt tolerance of cells.