No 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.

Summary Nitrogen‐fixing rhizobia and legumes have developed complex mutualistic mechanism that allows to convert atmospheric nitrogen into ammonia. Signalling by mitogen‐activated protein kinases (MAPKs) seems to be involved in this symbiotic interaction. Previously, we reported that stress‐induced MAPK (SIMK) shows predominantly nuclear localization in alfalfa root epidermal cells. Nevertheless, SIMK is activated and relocalized to the tips of growing root hairs during their development. SIMK kinase (SIMKK) is a well‐known upstream activator of SIMK. Here, we characterized production parameters of transgenic alfalfa plants with genetically manipulated SIMK after infection with Sinorhizobium meliloti. SIMKK RNAi lines, causing strong downregulation of both SIMKK and SIMK, showed reduced root hair growth and lower capacity to form infection threads and nodules. In contrast, constitutive overexpression of GFP‐tagged SIMK promoted root hair growth as well as infection thread and nodule clustering. Moreover, SIMKK and SIMK downregulation led to decrease, while overexpression of GFP‐tagged SIMK led to increase of biomass in above‐ground part of plants. These data suggest that genetic manipulations causing downregulation or overexpression of SIMK affect root hair, nodule and shoot formation patterns in alfalfa, and point to the new biotechnological potential of this MAPK.