Pei-Luen Jiang

A unique caleosin serving as the major integral protein in oil bodies isolated from Chlorella sp. cells cultured with limited nitrogen

Accumulation of oil bodies was successfully induced in a microalga, Chlorella sp., cultured in a nitrogen-limited medium. The oil bodies were initially assembled as many small entities (mostly 0.1-1 μm), and lately found as a major irregular compartment (>3 μm) occupying more than half of the cell space. Approximately, two thirds of oil bodies isolated from Chlorella cells were broken and formed a transparent oil layer on top of the milky compact layer of the remaining stable oil bodies after being washed with 0.1% triton X-100. The stable oil bodies mainly comprised triacylglycerols as examined by thin layer chromatography analysis and confirmed by both Nile red and BODIPY stainings. Integrity of these stable oil bodies was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Immunological cross-recognition revealed that a major protein of 29 kDa, tentatively identified as caleosin, was exclusively present in Chlorella oil bodies. Mass spectrometric analysis showed that the putative caleosin possessed a trypic fragment of 13 residues matching to that of a hypothetical caleosin in Picea sitchensis. With the aid of a degenerate primer designed according to the tryptic peptide, a complete cDNA fragment encoding this putative caleosin was obtained by PCR. Phylogenetic tree analysis supports that Chlorella caleosin is the most primitive caleosin found in oil bodies to date.

Stable oil bodies sheltered by a unique caleosin in cycad megagametophytes.

Stable oil bodies of smaller sizes and higher thermostability were isolated from mature cycad (Cycas revoluta) megagametophytes compared with those isolated from sesame seeds. Immunological cross-recognition revealed that cycad oil bodies contained a major protein of 27 kDa, tentatively identified as caleosin, while oleosin, the well-known structural protein, was apparently absent. Mass spectrometric analysis showed that the putative cycad caleosin possessed a tryptic fragment of 15 residues matching to that of a theoretical moss caleosin. A complete cDNA fragment encoding this putative caleosin was obtained by PCR cloning using a primer designed according to the tryptic peptide and another one designed according to a highly conservative region among diverse caleosins. The identification of this clone was subsequently confirmed by immunodetection and MALDI-MS analyses of its recombinant fusion protein over-expressed in Escherichia coli and the native form from cycad oil bodies. Stable artificial oil bodies were successfully constituted with triacylglycerol, phospholipid and the recombinant fusion protein containing the cycad caleosin. These results suggest that stable oil bodies in cycad megagametophytes are mainly sheltered by a unique structural protein caleosin.

Nitrogen-Deprivation Elevates Lipid Levels in Symbiodinium spp. by Lipid Droplet Accumulation: Morphological and Compositional Analyses

Stable cnidarian-dinoflagellate (genus Symbiodinium) endosymbioses depend on the regulation of nutrient transport between Symbiodinium populations and their hosts. It has been previously shown that the host cytosol is a nitrogen-deficient environment for the intracellular Symbiodinium and may act to limit growth rates of symbionts during the symbiotic association. This study aimed to investigate the cell proliferation, as well as ultrastructural and lipid compositional changes, in free-living Symbiodinium spp. (clade B) upon nitrogen (N)-deprivation. The cell proliferation of the N-deprived cells decreased significantly. Furthermore, staining with a fluorescent probe, boron dipyrromethane 493/503 (BODIPY 493/503), indicated that lipid contents progressively accumulated in the N-deprived cells. Lipid analyses further showed that both triacylglycerol (TAG) and cholesterol ester (CE) were drastically enriched, with polyunsaturated fatty acids (PUFA; i.e., docosahexaenoic acid, heneicosapentaenoic acid, and oleic acid) became more abundant. Ultrastructural examinations showed that the increase in concentration of these lipid species was due to the accumulation of lipid droplets (LDs), a cellular feature that have previously shown to be pivotal in the maintenance of intact endosymbioses. Integrity of these stable LDs was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Proteomic analyses of these LDs identified proteins putatively involved in lipid metabolism, signaling, stress response and energy metabolism. These results suggest that LDs production may be an adaptive response that enables Symbiodinium to maintain sufficient cellular energy stores for survival under the N-deprived conditions in the host cytoplasm.

Diel rhythmicity of lipid-body formation in a coral-Symbiodinium endosymbiosis

The biogenesis of intracellular lipid bodies (LBs) is dependent upon the symbiotic status between host corals and their intracellular dinoflagellates (genus Symbiodinium), though aside from this observation, little is known about LB behavior and function in this globally important endosymbiosis. The present research aimed to understand how LB formation and density are regulated in the gastrodermal tissue layer of the reef-building coral Euphyllia glabrescens. After tissue fixation and labeling with osmium tetroxide, LB distribution and density were quantified by imaging analysis of serial cryo-sections, and a diel rhythmicity was observed; the onset of solar irradiation at sunrise initiated an increase in LB density and size, which peaked at sunset. Both LB density and size then decreased to basal levels at night. On a seasonal timescale, LB density was found to be significantly positively correlated with seasonal irradiation, with highest densities found in the summer and lowest in the fall. In terms of LB lipid composition, only the concentration of wax esters, and not triglycerides or sterols, exhibited diel variability. This suggests that the metabolism and accumulation of lipids in LBs is at least partially light dependent. Ultrastructural examinations revealed that the LB wax ester concentration correlated with the number of electron-transparent inclusion bodies. Finally, there was a directional redistribution of the LB population across the gastroderm over the diel cycle. Collectively, these data reveal that coral gastrodermal LBs vary in composition and intracellular location over diel cycles, features which may shed light on their function within this coral–dinoflagellate mutualism.

Caleosin serves as the major structural protein as efficient as oleosin on the surface of seed oil bodies

Oleosin, the most abundant protein in oil bodies of all examined angiosperm seeds, has been demonstrated to serve as a structural protein to maintain the integrity of these lipid storage organelles. Caleosin, a minor protein in oil bodies of angiosperm seeds, is assumed to anchor to the organelles in a manner similar to oleosin, i.e., via its central hydrophobic domain with a unique proline-knot motif presumably responsible for the targeting to nascent oil bodies. Recently, we found that stable oil bodies in cycad seeds were mainly sheltered by caleosin in the absence of oleosin. This finding suggests that caleosin is competent as the major structural protein, just like oleosin, on the surface of seed oil bodies. A search of literature shows that genes putatively encoding for caleosin-, but not oleosin-like proteins, are present in more primitive species, such as algae and fungi. We hypothesize that oleosin may have been derived from caleosin after a long term of divergent evolution.

A Unique Caleosin in Oil Bodies of Lily Pollen

In view of the recent isolation of stable oil bodies as well as a unique oleosin from lily pollen, this study examined whether other minor proteins were present in this lipid-storage organelle. Immunological cross-recognition using antibodies against three minor oil-body proteins from sesame suggested that a putative caleosin was specifically detected in the oil-body fraction of pollen extract. A cDNA fragment encoding this putative pollen caleosin, obtained by PCR cloning, was confirmed by immunodetection and MALDI-MS analyses of the recombinant protein over-expressed in Escherichia coli and the native form. Caleosin in lily pollen oil bodies seemed to be a unique isoform distinct from that in lily seed oil bodies.

Nitrogen Deprivation Induces Lipid Droplet Accumulation and Alters Fatty Acid Metabolism in Symbiotic Dinoflagellates Isolated from Aiptasia pulchella

The stability of cnidarian-dinoflagellate (genus Symbiodinium spp.) endosymbioses depends on the regulation of nutrient transport between Symbiodinium populations and their hosts. Previously, we successfully induced the production of lipid droplets in the free-living cultured Symbiodinium (clade B) under the nitrogen-deprivation condition for 5 days. Therefore, the present study aimed at understanding the disruption of the endosymbiotic relationship between the cnidarians and dinoflagellates by nitrogen deprivation using Aiptasia pulchella as an example. Transmission electron micrographs revealed the formation of lipid droplets induced by nitrogen deprivation, and the lipid analyses further showed that polyunsaturated fatty acids were drastically enriched in Symbiodinium after 30 days of nitrogen deprivation, although these were unaffected after 5 days of nitrogen starvation. The present study also suggested that the host provided nitrogen to the symbiotic cells during short-term environmental stress. However, the relationship started to deteriorate after 30 days. These findings provide a more detailed understanding of the mechanisms of the symbiotic relationship between the symbiotic dinoflagellates in terms of the nitrogen source, which might provide more information for the explanation of the regulatory mechanism underlying endosymbiotic associations.

Assessment of metabolic modulation in free-living versus endosymbiotic Symbiodinium using synchrotron radiation-based infrared microspectroscopy

The endosymbiotic relationship between coral hosts and dinoflagellates of the genus Symbiodinium is critical for the growth and productivity of coral reef ecosystems. Here, synchrotron radiation-based infrared microspectroscopy was applied to examine metabolite concentration differences between endosymbiotic (within the anemone Aiptasia pulchella) and free-living Symbiodinium over the light–dark cycle. Significant differences in levels of lipids, nitrogenous compounds, polysaccharides and putative cell wall components were documented. Compared with free-living Symbiodinium, total lipids, unsaturated lipids and polysaccharides were relatively enriched in endosymbiotic Symbiodinium during both light and dark photoperiods. Concentrations of cell wall-related metabolites did not vary temporally in endosymbiotic samples; in contrast, the concentrations of these metabolites increased dramatically during the dark photoperiod in free-living samples, possibly reflecting rhythmic cell-wall synthesis related to light-driven cell proliferation. The level of nitrogenous compounds in endosymbiotic cells did not vary greatly across the light–dark cycle and in general was significantly lower than that observed in free-living samples collected during the light. Collectively, these data suggest that nitrogen limitation is a factor that the host cell exploits to induce the biosynthesis of lipids and polysaccharides in endosymbiotic Symbiodinium.

Characterization of Oil Bodies in Adlay (Coix lachryma-jobi L)

Oil bodies were observed in cells of both embryo and aleurone layers of mature adlay grains (Coix lachryma-jobi L. var. ma-yuen Stapf). Stable oil bodies were successfully isolated from the adlay grains. Thin-layer chromatography revealed that the contents stored in the adlay oil bodies were mainly neutral lipids (>90% triacylglycerols and about 5% diacylglycerols). The integrity of the isolated oil bodies was presumably maintained via electronegative repulsion and steric hindrance provided by their surface proteins. Immunological cross-recognition using antibodies against sesame oil-body proteins indicated that two oleosin isoforms (termed oleosin-H and oleosin-L) and one caleosin were present in the adlay oil bodies. Full-length cDNA fragments encoding these three unique oil-body proteins were obtained by PCR cloning. MALDI-MS analyses confirmed that the three full-length cDNA fragments encoded the two oleosin isoforms and one caleosin observed in the oil bodies isolated from the adlay grains.

Characterization of oil bodies in jelly fig achenes.

Thin-layer chromatography analysis revealed that the contents stored in oil bodies isolated from jelly fig (Ficus awkeotsang Makino) achenes were mainly neutral lipids (>90% triacylglycerols and approximately 5% diacylglycerols). Fatty acids released from the neutral lipids of achene oil bodies were highly unsaturated (62.65% alpha-linolenic acid, 18.24% linoleic acid, and 10.62% oleic acid). The integrity of isolated oil bodies was presumably maintained via electronegative repulsion and steric hindrance provided by their surface proteins. Immunological cross-recognition using antibodies against sesame oil-body proteins indicated that two oleosin isoforms and one caleosin were present in these oil bodies. MALDI-MS analyses confirmed that the three full-length cDNA fragments obtained by PCR cloning from maturing achenes encoded the two jelly fig oleosin isoforms and one caleosin identified by immunological screening.