Molecular characterization of GhPLDα1 and its relationship with secondary cell wall thickening in cotton fibers.

Phospholipase D (PLD) hydrolyzes phospholipids to generate a free polar head group (e.g., choline) and a second messenger phosphatidic acid and plays diverse roles in plant growth and development, including seed germination, leaf senescence, root hair growth, and hypocotyl elongation. However, the function of PLD in cotton remains largely unexplored. Here, the comprehensive molecular characterization of GhPLD alpha 1 was explored with its role in upland cotton (Gossypium hirsutum) fiber development. The GhPLD alpha 1 gene was cloned successfully, and a sequence alignment showed that GhPLD alpha 1 contains one C2 domain and two HKD (HxKxxxxD) domains. Quantitative reverse transcriptase-polymerase chain reaction measured the expression of GhPLD alpha 1 in various cotton tissues with the highest level in fibers at 20 days post anthesis (d.p.a.). Fluorescent microscopy and immunoblotting in tobacco epidermis showed the GhPLD alpha 1 distribution in both cell membranes and the cytoplasm. An activity assay indicated changes in PLD alpha enzyme activity in developing fiber cells with a peak level at 20 d.p.a., coinciding with the onset of cellulose accumulation and the increased H2O2 content during fiber development. Furthermore, the inhibition of PLD alpha activity obviously decreased the cellulose and H2O2 contents of in vitro-cultured cotton fibers. These results provide important evidence explaining the relationship of GhPLD alpha 1 with secondary cell wall thickening in cotton fibers in that GhPLD alpha 1 may correlate with the increased H2O2 content at the onset of secondary cell wall thickening, ultimately promoting cellulose biosynthesis.