Lipid Droplets Mediate Salt Stress Tolerance in Parachlorella kessleri.

Microalgae are known to respond to salinity stress via mechanisms that include accumulation of compatible solutes and synthesis of antioxidants. Here, we describe a salinity-tolerance mechanism mediated by lipid droplets (LDs). In the alga Parachlorella kessleri grown under salt-stress conditions, we observed significant increases in cell size and LD content. LDs that were closely grouped along the plasma membrane shrank as the plasma membrane expanded, and some LDs were engulfed by vacuoles. Transcriptome analysis showed that genes encoding lysophospholipid acyltransferases (LPLATs) and phospholipase A2 were significantly up-regulated following salt stress. Diacylglycerol kinase and LPLAT were identified in the proteome of salt-induced LDs, alongside vesicle trafficking and plastidial proteins and histone H2B. Analysis of fatty acid composition revealed an enrichment of C18:1 and C18:2 at the expense of C18:3 in response to salt stress. Pulse-chase experiments further suggested that variations of fatty acid composition were associated with LDs. Acetate stimulation research further confirmed a positive role of LDs in cell growth under salt stress. These results suggest that LDs play important roles in salt-stress tolerance, through harboring proteins, participating in cytoplasmic component recycling, and providing materials and enzymes for membrane modification and expansion.

Increasing DNA content for cost-effective oil production in Parachlorella kessleri.

The aim of this work was to study salt stress effects on DNA content and oil production processes integrating harvesting, lipid accumulation and oil extraction. Salt-induced enlargement of Parachlorella kessleri cells, with increasing content of DNA and neutral lipid were found. The 34.77% neutral lipid content and biomass concentration of 0.83 g L-1 were obtained after 7 days of salt treatment, compared with that of 13.57% and 0.89 g L-1 cultivated under normal condition. Sedimentation efficiency increased markedly from 15% to 90% due to the cell enlargement. Disruption fraction and the recovery rate of total lipids of wet cells under salt stress were significantly higher than that of normal conditions (100% and 82.4% for salt stress vs.76.8% and 51.1% for normal conditions). This work demonstrated that salt-induced increase in cell size and DNA content was an effective strategy for the enhancement of oil production, microalgae harvesting and oil extraction.

Variation of fatty acid desaturation in response to different nitrate levels in Auxenochlorella pyrenoidosa.

Microalgae are promising feedstocks for biodiesel, where the high proportion of monounsaturated fatty acid such as oleic acid (C18:1) is preferred. To regulate fatty acid desaturation in microalgae, the relationship among nitrate concentration, fatty acid composition and the expression levels of desaturase genes was explored. Dynamic variations of fatty acid profiles suggested nitrate could induce desaturation of C18 fatty acids. The content of C18:1 in Auxenochlorella pyrenoidosa was 30.88% at 0 g l-1 nitrate concentration compared with 0.48% at 1.5 g l-1. The expressions of relative delta-9, 12 and 15 fatty acid desaturase genes (Δ9, Δ12 and Δ15FADs) were further investigated. The 330% upregulated expression of Δ9FAD in logarithmic phase at 0 g l-1 resulted in C18:1 accumulation. Moreover, nitrate replenishment caused a sharp reduction of C18:1 from 34.79% to 0.22% and downregulation of Δ9FAD expression to 1% of the nitrate absence level, indicating the pivotal role of Δ9FAD in C18:1 accumulation. Finally, overexpression of Δ9FAD in Escherichia coli and Saccharomyces cerevisiae resulted in an increase of C18:1, confirming its ability of desaturating C18:0. The results could provide a new approach and scientific guidance for the improvement of biodiesel quality and industrialization of high-valued chemicals by means of metabolic engineering.

Spatiotemporal expression profile of the Pumilio gene in the embryonic development of silkworm.

We previously identified a pumilio gene in silkworm (Bombyx mori L.), designated BmPUM, which was specifically expressed in the ovary and testis. To further characterize this gene's involvement in silkworm development, we have determined the spatiotemporal expression pattern of BmPUM during all embryonic stages. Real-time polymerase chain reaction (RT-PCR) analysis revealed that BmPUM was expressed in all stages of silkworm embryos and that its transcript levels displayed two distinct peaks. The first was observed at the germ-band formation stage (1 d after oviposition) and dropped to a low level at the gonad formation stage (5 d after oviposition). The second was detected at the stage of bristle follicle occurrence (6 d after oviposition), which was confirmed by Western blot analysis and immunohistochemistry. Nanos (Nos), functioning together with Pum in abdomen formation of Drosophila embryos, was also highly expressed at the beginning (0 h to 1 d after oviposition) of embryogenesis, but its transcript levels were very low after the stage of germ-band formation. These results suggest that BmPUM functions with Bombyx mori nanos (Bm-nanos) at the early stages of silkworm embryonic development, and may then play a role in gonad formation and the occurrence of bristle follicles. Our data thus provide a foundation to uncover the role of BmPUM during silkworm development.

A novel method of silkworm embryo preparation for immunohistochemistry.

It is difficult to obtain intact embryos, especially intact early embryos, from insect eggs because of their small sizes. Based on the means traditionally used to get silkworm embryos and the previous approaches used for getting Drosophila embryos, we established a novel method of silkworm embryo preparation. The new method is straightforward and easy to operate. Silkworm embryos could be prepared without severe damage in large quantities by this new protocol. In addition, the novel method of silkworm embryo preparation is quite suitable for immunohistochemistry.