Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step of the acyl-CoA-dependent TAG biosynthesis and thus is a key target for manipulating oil production in microalgae. The microalga Chromochloris zofingiensis can accumulate substantial amounts of triacylglycerol (TAG) and represents a promising source of algal lipids. In this study, C. zofingiensis DGAT2s (CzDGAT2s) were characterized with in silico, in vivo (yeast), and in vitro assays. Putative CzDGAT2s were identified, and their functional motifs and evolutionary relationship with other DGAT2s were analyzed. When CzDGAT2s were individually expressed in a TAG-deficient Saccharomyces cerevisiae strain, only CzDGAT2C could restore the TAG biosynthesis. Further in vitro assays indicated that CzDGAT2C displayed typical DGAT activity, which was fitted to the Michaelis−Menten equation, and N-and C-terminals were important for the enzyme activity. In addition, membrane yeast twohybrid assay revealed a possible DGAT2 activity modulation via the formation of homodimer/heterodimer among different DGAT2 isoforms.

This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The P(II) proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of P(II) might play a role in the control of nitrogen metabolism in H. seropedicae.

SUMMARY Triacylglycerols have important physiological roles in photosynthetic organisms, and are widely used as food, feed and industrial materials in our daily life. Phospholipid:diacylglycerol acyltransferase (PDAT) is the pivotal enzyme catalyzing the acyl‐CoA‐independent biosynthesis of triacylglycerols, which is unique in plants, algae and fungi, but not in animals, and has essential functions in plant and algal growth, development and stress responses. Currently, this enzyme has yet to be examined in an evolutionary context at the level of the green lineage. Some fundamental questions remain unanswered, such as how PDATs evolved in photosynthetic organisms and whether the evolution of terrestrial plant PDATs from a lineage of charophyte green algae diverges in enzyme function. As such, we used molecular evolutionary analysis and biochemical assays to address these questions. Our results indicated that PDAT underwent divergent evolution in the green lineage: PDATs exist in a wide range of plants and algae, but not in cyanobacteria. Although PDATs exhibit the conservation of several features, phylogenetic and selection‐pressure analyses revealed that overall they evolved to be highly divergent, driven by different selection constraints. Positive selection, as one major driving force, may have resulted in enzymes with a higher functional importance in land plants than green algae. Further structural and mutagenesis analyses demonstrated that some amino acid sites under positive selection are critically important to PDAT structure and function, and may be central in lecithin:cholesterol acyltransferase family enzymes in general.