Research On Mechanisms Of Phosphalipaseα1/Phosphatidic Acid And Microtubule-Associated Protein Map65-1Mediating Microtubules In Response To Salt Stress In Arabidopsis Thaliana

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 PLDa(3), PLDβ(2), PLDy(3), PLDδ, PLDε and PLDζ(2). AtPLDal 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, y-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 pldal was more sensitive to salt stress compared to wild type (WT), and AtPLDal/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 MAP65-1and mechanisms of PA-MAP65-1interaction. First of all, after exposure to10μM microtubule-disrupting drug oryzalin, microtubules of WT, pldal, map65-1-1and map65-1-2were visualized by whole-mount immunofluorescence staining. As a consequence, pldal, map65-1-1and map65-1-2were more sensitive to oryzalin than WT, followed by severe depolymerization of microtubules. Upon200mM NaCl treatment, the survival ratio of map65-1-1and map65-1-2was lower than that of WT. In addition to that, the survival ratio of pldal and pldalmap65-1-1was lower than that of WT as well. Exogenous supplement with20μM16:0-18:2PA (palmitoyl-linoleoyl PA), salt tolerance of pldal was enhanced, accompanied by higher survival ratio and more microtubules which are similar to those of WT, respectively. However, the sensitive phenotype of pldal and pldalmap65-1-1in response to salt stress was not alleviated by exogenous PA, with considerable decrease in the number of microtubules compared to that of WT and pldal which were co-treated with PA and NaCl. These results provided genetic evidence that there was a likelihood that PLDal/PA signaling was upstream of MAP65-1.Next, PA binding sites of MAP65-1were elaborate. Blending site-directed mutagenesis and fat-western, it is elucidated that53-55residues KRK,61-63residues KSR and428-429residues SK of MAP65-1were 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 MAP65family), which were homologous to MAP65-1with 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 MAP65-6harbouring 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-MAP65-1interaction was of specificity in regulating organization of microtubules.Besides, whether PA-MAP65-1binding and interaction were beneficial for strengthening salt tolerance of cells, was studied. Upon25mM 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 MAP65-1lost 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 MAP65-1with microtubules was investigated upon salt stress by transient transformation of Arabidopsis seedlings. MAP65-1colocalized with microtubules well in WT and pldal seedlings and the number of microtubules in WT and pldal seedlings was similar. Nevertheless, upon50mM NaCl treatment for24h, microtubules both became depolymerized in WT and pldal seedlings. However, in plda1seedlings, microtubules depolymerized more severely than those of WT, forming dot-like structures with few intact microtubules left. In the meanwhile, MAP65-1colocalized with depolymerized microtubules. More importantly, exogenous20μM16:0-18:2PA could restore microtubule arrays of plda1seedlings to those of WT seedlings, and MAP65-1recolocalized with intact microtubules. These results indicated that salt stress inducing depolymerization of microtubules and detachment of MAP65-1from microtubules, were a part of mechanisms of salt tolerance. Salt stress stimulated PLDal’s activity, resulting in hydrolysis of phospholipids into PA. PA bound to MAP65-1, facilitating stability of microtubules and improving salt tolerance of cells.