Functional analysis of phospholipase Dδ family in tobacco pollen tubes

Phosphatidic acid (PA), an important signalling and metabolic phospholipid, is predominantly localized in the subapical plasma membrane (PM) of growing pollen tubes. PA can be produced from structural phospholipids by phospholipase D (PLD), but the isoforms responsible for production of PM PA were not identified yet and their functional roles remain unknown. Following genome‐wide bioinformatic analysis of the PLD family in tobacco, we focused on the pollen‐overrepresented PLDδ class. Combining live‐cell imaging, gene overexpression, lipid‐binding and structural bioinformatics, we characterized five NtPLDδ isoforms. Distinct PLDδ isoforms preferentially localize to the cytoplasm or subapical PM. Using fluorescence recovery after photobleaching, domain deletion and swapping analyses we show that membrane‐bound PLDδs are tightly bound to PM, primarily via the central catalytic domain. Overexpression analyses suggested isoform PLDδ3 as the most important member of the PLDδ subfamily active in pollen tubes. Moreover, only PLDδ3 shows significant constitutive PLD activity in vivo and, in turn, PA promotes binding of PLDδ3 to the PM. This forms a positive feedback loop leading to PA accumulation and the formation of massive PM invaginations. Tightly controlled production of PA generated by PLDδ3 at the PM is important for maintaining the balance between various membrane trafficking processes that are crucial for plant cell tip growth.     

Effect of transgenic alfalfa plants with introduced gene for Alfalfa Mosaic Virus coat protein on rhizosphere microbial community composition and physiological profile

The aim of this study was to evaluate the effect of transgenic alfalfa (Medicago sativa L.) plants, in comparison to their non-transgenic counterpart, on the density and physiological profiles of aerobic bacteria in the rhizosphere. Plants of transgenic alfalfa expressing the AMVcp-s gene coding for Alfalfa Mosaic Virus coat protein were cultivated in a climatic chamber. Two methods were used to determine the microbial diversity in rhizospheres of transgenic plants. First, the cultivation-dependent plating method, based on the determination of the density of colony-forming bacteria, and second, a biochemical method using the Biolog™ system, based on the utilization of different carbon sources by soil microorganisms. Statistically significant differences in densities of rhizospheric bacteria between transgenic and non-transgenic alfalfa clones were observed in ammonifying bacteria (GTL4/404-1), cellulolytic bacteria (GTL4/404-1, GTL4/402-2, A5-3-3), rhizobial bacteria (GTL4/402-2), denitrifying bacteria (A5-3-3) and Azotobacter spp. (GTL4/402-2). The highest values of substrate utilization by microbial communities and average respiration of C-sources were determined in non-transgenic alfalfa plants of the isogenic line SE/22-GT2. Carbohydrates, carboxylic acids and amino-acids were the most utilized carbon substrates by both Gram-negative and Gram-positive bacteria. Both, the community metabolic diversity and the utilization of C-sources increased in all alfalfa lines with culture time and regardless of transgenic or non-transgenic nature of lines.