Roles Of Two Phospholipases, GhPLA2α And GhPLA2β,in Cotton Fiber Development

Cotton is an important natural fiber crop in the world. Cotton fibers, derived from ovule epidermal cells, are unicellular and highly elongated. In some species, length of cotton fibers can reach over6cm, which is1000to3000times than their width. Besides, mature fibers are primarily composed of cellulose which accounts for more than95%of their dry weight. Therefore, cotton fiber is an ideal model for studying cell differentiation, elongation and cellulose biosynthesis.Development of cotton fiber consist of4sequential yet overlapping stages: initiation, elongation, secondary wall deposition and maturation. With the development, especially that of elongation stages, membrane of fiber cell exaggerates and reconstructs. There is a large demand for materials of cell membrane, such as phospholipid and protein.Phospholipase A2(PLA2) which catalyzes the hydrolysis of phospholipids at their sn-2positions participates in several biological process of cell membrane morphologic change. Some studies indicate that sPLA2β,a submember of PLA2, plays an important role in cell elongation. Furthermore, recent work shows that lysophospholipids and free fatty acid, two PLA2hydrolysis products, acts as signaling molecules involving in regulation of cell development. However, roles of PLA2in cell development remains elusive. So, we aimed to further reveal their roles using cotton fiber cells. At present, gene expression of a sPLA2a homologs was detected up-regulated in fiber initials compared with non fiber cells in epidermal layers of ovules, indicating potential roles of PLA2in cotton fiber development. Here, we cloned two genes of sPLA2isoforms GhPLA2a and GhPLA2β, and studied their PLA2activity, expression pattern and cotton fiber development upon up-or down-regulation of their expression in ovule epidermis.The main results are as follows: 1. Inhibitors of Phospholipase A impeded fiber elongation in vitroPLA2inhibitors (ONO-RS-082, HELSS and ETYA) were used to treat-1-dpa:otton ovule in vitro. Observation of fiber development indicated that PLA2inhibitors lave no effect on cotton fiber initiation under the treatment, even in a high dosage. In contrast, fiber elongation was considerably impaired with increasing addition of the nhibitors. This suggested roles of PLA2in regulating cotton fiber elongation.2. Cloning and expression pattern of GhPLA2a and GhPLA2βTwo cDNA of sPLA2homologs were isolated from cotton, denoted as GhPLA2a ind GhPLA2β. Real-time quantitative PCR analysis indicated that GhPLA2a was expressed preferentially in8to15dpa fibers. This suggested GhPLA2a might play a role in cotton fiber elongation. In contrast, GhPLA2β was predominantly expressed in leaves and fibers of20and23dpa although there was relative high expression at8-to15-dpa fiber, suggesting GhPLA2β might be involved in cellulose synthesis. Furthermore, promoter of GhPLA2a was cloned, fused with GUS reporter gene, and introduced into tomato. Histochemical analysis of GUS activity indicated that the promoter direct gene expressing in out integument of ovules specifically.3. GhPLA2a and GhPLA2β could hydrolyze phosphatidyl choline in vitroGhPLA2a and GhPLA2β fused with GST were expressed in prokaryotic expression system. Then, they were used to hydrolyze phosphatidyl choline in vitro. Products of the reaction was determined by GC-MS. The result showed that GhPLA2a and GhPLA2β fusion protein could hydrolyze phosphatidyl choline at sn-2specifically.4. Up-and down-regulating expression of GhPLA2a and GhPLA2β in the ovule epidermisIn order to investigate roles of GhPLA2a and GhPLA2β in plant, these two gene were up-and down-regulated in cotton under control of ovule-specific promoter BAN Observation by scanning electron microscopy showed there was no change was found in fiber initiation of the transgenic cotton compared with wild type. However,2-dpa fiber length of transgenic cotton interfering expression of GhPLA2a and GhPLA2β was shorter than that of wild type, indicating fiber elongation was inhibited. This results was confirmed by length of cotton mature fibers.In addition, compared to wild type micronaire value of BAN::GhPLA2β-RNAi transgenic cotton fibers was reduced. Since GhPLA2β was highly expressed in20and23-dpa fibers, it is possible that GhPLA2β plays another role in cellulose synthesis.All these results above proved that GhPLA2a and GhPLA2β encoded functional Phospholipase A2and important for cotton cell elongation. Besides, GhPLA2β might have another role in regulating cellulose synthesis in cotton fibers.