Background: Nitrogen limitation can induce neutral lipid accumulation in microalgae, as well as inhibiting their growth. Therefore, to obtain cultures with both high biomass and high lipid contents, and explore the lipid accumulation mechanisms, we implemented nitrogen deprivation in a model diatom Phaeodactylum tricornutum at late exponential phase. Results: Neutral lipid contents per cell subsequently increased 2.4-fold, both the number and total volume of oil bodies increased markedly, and cell density rose slightly. Transcriptional profile analyzed by RNA-Seq showed that expression levels of 1213 genes (including key carbon fixation, TCA cycle, glycerolipid metabolism and nitrogen assimilation genes) increased, with a false discovery rate cut-off of 0.001, under N deprivation. However, most light harvesting complex genes were down-regulated, extensive degradation of chloroplast membranes was observed under an electron microscope, and photosynthetic efficiency declined. Further identification of lipid classes showed that levels of MGDG and DGDG, the main lipid components of chloroplast membranes, dramatically decreased and triacylglycerol (TAG) levels significantly rose, indicating that intracellular membrane remodeling substantially contributed to the neutral lipid accumulation. Conclusions: Our findings shed light on the molecular mechanisms of neutral lipid accumulation and the key genes involved in lipid metabolism in diatoms. They also provide indications of possible strategies for improving microalgal biodiesel production.

To obtain fast growing oil-rich microalgal strains has been urgently demanded for microalgal biofuel. Malic enzyme (ME), which is involved in pyruvate metabolism and carbon fixation, was first characterized in microalgae here. Overexpression of Phaeodactylum tricornutum ME (PtME) significantly enhanced the expression of PtME and its enzymatic activity in transgenic P. tricornutum. The total lipid content in transgenic cells markedly increased by 2.5-fold and reached a record 57.8% of dry cell weight with a similar growth rate to wild type, thus keeping a high biomass. The neutral lipid content was further increased by 31% under nitrogen-deprivation treatment, still 66% higher than that of wild type. Transgenic microalgae cells exhibited obvious morphological changes, as the cells were shorter and thicker and contained larger oil bodies. Immuno-electron microscopy targeted PtME to the mitochondrion. This study markedly increased the oil content in microalgae, suggesting a new route for developing ideal microalgal strains for industrial biodiesel production.

Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the last step of triacylglyceride (TAG) biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.

Background: Plants can recruit beneficial microbes to enhance their ability to defend against pathogens. However, in contrast to the intensively studied roles of the rhizosphere microbiome in suppressing plant pathogens, the collective community-level change and effect of the phyllosphere microbiome in response to pathogen invasion remains largely elusive. Results: Here, we integrated 16S metabarcoding, shotgun metagenomics and culture-dependent methods to systematically investigate the changes in phyllosphere microbiome between infected and uninfected citrus leaves by Diaporthe citri, a fungal pathogen causing melanose disease worldwide. Multiple microbiome features suggested a shift in phyllosphere microbiome upon D. citri infection, highlighted by the marked reduction of community evenness, the emergence of large numbers of new microbes, and the intense microbial network. We also identified the microbiome features from functional perspectives in infected leaves, such as enriched microbial functions for iron competition and potential antifungal traits, and enriched microbes with beneficial genomic characteristics. Glasshouse experiments demonstrated that several bacteria associated with the microbiome shift could positively affect plant performance under D. citri challenge, with reductions in disease index ranging from 65.7 to 88.4%. Among them, Pantoea asv90 and Methylobacterium asv41 identified as “recruited new microbes” in the infected leaves, exhibited antagonistic activities to D. citri both in vitro and in vivo, including inhibition of spore germination and/or mycelium growth. Sphingomonas spp. presented beneficial genomic characteristics and were found to be the main contributor for the functional enrichment of iron complex outer membrane receptor protein in the infected leaves. Moreover, Sphingomonas asv20 showed a stronger suppression ability against D. citri in iron-deficient conditions than iron-sufficient conditions, suggesting a role of iron competition during their antagonistic action. Conclusions: Overall, our study revealed how phyllosphere microbiomes differed between infected and uninfected citrus leaves by melanose pathogen, and identified potential mechanisms for how the observed microbiome shift might have helped plants cope with pathogen pressure. Our findings provide novel insights into understanding the roles of phyllosphere microbiome responses during pathogen challenge.