Abstract
Phospholipase D alpha 1 (PLDα1, At3g15730) and its product phosphatidic acid (PA) are involved in a variety of cellular and physiological processes, such as cytoskeletal remodeling, regulation of stomatal closure and opening, as well as biotic and abiotic stress signaling. Here we aimed to study developmental expression patterns and subcellular localization of PLDα1 in Arabidopsis using advanced microscopy methods such as light-sheet fluorescence microscopy (LSFM) and structured illumination microscopy (SIM). We complemented two knockout pldα1 mutants with a YFP-tagged PLDα1 expressed under the PLDα1 native promoter in order to study developmental expression pattern and subcellular localization of PLDα1 in Arabidopsis thaliana under natural conditions. Imaging of tissue-specific and developmentally-regulated localization of YFP-tagged PLDα1 by LSFM in roots of growing seedlings showed accumulation of PLDα1-YFP in the root cap and the rhizodermis. Expression of PLDα1-YFP in the rhizodermis was considerably higher in trichoblasts before and during root hair formation and growth. Thus, PLDα1-YFP accumulated in emerging root hairs and in the tips of growing root hairs. PLDα1-YFP showed cytoplasmic subcellular localization in root cap cells and in cells of the root transition zone. In aerial parts of plants PLDα1-YFP was also localized in the cytoplasm showing enhanced accumulation in the cortical cytoplasmic layer of epidermal non-dividing cells of hypocotyls, leaves, and leaf petioles. However, in dividing cells of root apical meristem and leaf petiole epidermis PLDα1-YFP was enriched in mitotic spindles and phragmoplasts, as revealed by co-visualization with microtubules. Finally, super-resolution SIM imaging revealed association of PLDα1-YFP with both microtubules and clathrin-coated vesicles (CCVs) and pits (CCPs). In conclusion, this study shows the developmentally-controlled expression and subcellular localization of PLDα1 in dividing and non-dividing Arabidopsis cells.
Highlights
The major function of the phospholipase D (PLD) enzymes is to hydrolyse phospholipids such as phosphatidylcholine, resulting in the production of phosphatidic acid (PA) by transphosphatidylation of water and a free soluble head group, e.g., choline (Munnik and Musgrave, 2001)
In order to characterize the roles of PLDα1 in plant development, we performed in vivo cell- and tissue-specific expression analysis of the PLDα1-YFP driven by native PLDα1 promoter in two stably transformed pldα1 mutants of A. thaliana
To prove the expression of PLDα1YFP fusion protein in experimental plants, we performed SDS-PAGE with immunoblot analysis using anti-phospholipase D alpha 1/2 and anti-GFP antibodies
Summary
The major function of the phospholipase D (PLD) enzymes is to hydrolyse phospholipids such as phosphatidylcholine, resulting in the production of phosphatidic acid (PA) by transphosphatidylation of water and a free soluble head group, e.g., choline (Munnik and Musgrave, 2001). Later studies on transgenic Arabidopsis cell lines using pull-down assay with GFP tagged PLDδ identified PLDδ as a cortical microtubule-binding protein (Andreeva et al, 2009; Ho et al, 2009; Hong et al, 2016). Such regulatory interactions among microtubules and PLD isoforms ( the Arabidopsis PLDα1 isoform) proved to have functional consequences during plant responses to salt and hyperosmotic stress (Zhang et al, 2012), ABA-induced stomatal closure (Jiang et al, 2014), and drug-induced microtubule reorganization (Zhang et al, 2017a). Overall regulation of developmental expression pattern in cell- and tissue-specific context and subcellular localization of PLDα1 in dividing cells is not known
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