Analysis of oil synthesis pathway in Cyperus esculentus tubers and identification of oleosin and caleosin genes.

The tubers of the widely distributed Cyperus esculentus are rich in oil, and therefore, the plant is considered to have a high utilization value in the vegetable oil industry. Oleosins and caleosins are lipid-associated proteins found in oil bodies of seeds; however oleosins and caleosins genes have not been identified in C. esculentus. In this study, we performed transcriptome sequencing and lipid metabolome analysis of C. esculentus tubers at four developmental stages to obtain the information on their genetic profile, expression trends, and metabolites in oil accumulation pathways. Overall, 120,881 non-redundant unigenes and 255 lipids were detected; 18 genes belonged to the acetyl-CoA carboxylase (ACC), malonyl-CoA:ACP transacylase (MCAT), ß-ketoacyl-ACP synthase (KAS), and fatty acyl-ACP thioesterase (FAT) gene families involved in fatty acid biosynthesis, and 16 genes belonged to the glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol acyltransferase 3 (DGAT3), phospholipid:diacylglycerol acyltransferase (PDAT), FAD2, and lysophosphatidic acid acyltransferase (LPAAT) gene families playing important roles in triacylglycerol synthesis. We also identified 9 oleosin- and 21 caleosin-encoding genes in C. esculentus tubers. These results provide detailed information on the C. esculentus transcriptional and metabolic profiles, which can be used as reference for the development of strategies to increase oil content in C. esculentus tubers.

Exploring The Relative Astringency of Tea Catechins and Distinct Astringent Sensation of Catechins and Flavonol Glycosides via an In Vitro Assay Composed of Artificial Oil Bodies.

Artificial oil bodies covered by a recombinant surface protein, caleosin fused with histatin 3 (a major human salivary peptide), were employed to explore the relative astringency of eight tea catechins. The results showed that gallate-type catechins were more astringent than non-gallate-type catechins, with an astringency order of epicatechin gallate > epigallocatechin gallate > gallocatechin gallate > catechin gallate > epigallocatechin > epicatechin > gallocatechin > catechin. As expected, the extension of brewing time led to an increase in catechin content in the tea infusion, thus elevating tea astringency. Detailed analysis showed that the enhanced proportion of gallate-type catechins was significantly higher than that of non-gallate-type catechins, indicating that tea astringency was elevated exponentially, rather than proportionally, when brewing time was extended. Rough surfaces were observed on artificial oil bodies when they were complexed with epigallocatechin gallate (a catechin), while a smooth surface was observed on those complexed with rutin (a flavonol glycoside) under an atomic force microscope and a scanning electron microscope. The results indicate that catechins and flavonol glycosides induce the sensation of rough (puckering) and smooth (velvety) astringency in tea, respectively.

Multiple caleosins have overlapping functions in oil accumulation and embryo development.

Caleosins are lipid droplet- and endoplasmic reticulum-associated proteins. To investigate their functions in oil accumulation, expression levels of caleosins in developing seeds of Arabidopsis thaliana were examined and four seed-expressed caleosins (CLO1, CLO2, CLO4, and CLO6) were identified. The four single mutants showed similar minor changes of fatty acid composition in seeds. Two double mutants (clo1 clo2 and clo1×clo2) demonstrated distinct changes of fatty acid composition, a 16-23% decrease of oil content, and a 10-13% decrease of seed weight. Moreover, a 40% decrease of oil content, further fatty acid changes, and misshapen membranes of smaller lipid droplets were found in seeds of quadruple CLO RNAi lines. Notably, ~40% of quadruple CLO RNAi T1 seeds failed to germinate, and deformed embryos and seedlings were also observed. Complementation experiments showed that CLO1 rescued the phenotype of clo1 clo2. Overexpression of CLO1 in seedlings and BY2 cells increased triacylglycerol content up to 73.6%. Transcriptome analysis of clo1 clo2 developing seeds showed that expression levels of some genes related to lipid, embryo development, calcium signaling, and stress responses were affected. Together, these results suggest that the major seed-expressed caleosins have overlapping functions in oil accumulation and show pleiotropic effects on embryo development.

Identification and characterization of caleosin in Cycas revoluta pollen.

Oil bodies are essential energy storage organelles that are generally present in the seeds of plants. Caleosin protein has been identified in the seed oil bodies of Cycas revolutaseed. In this study, mature Cycas revoluta pollen grains were collected from cycad elliptical cones. However, the isolation and identification of oil bodies protein from mature Cycas revoluta grains have never been experimentally determined. Ultrastructural studies have shown that the oil bodies were present in pollen Cycas revoluta grains. Lipid analysis showed that oil bodies are predominantly composed of triacylglycerol. Complete cDNA fragments encoding the caleosin were obtained by PCR cloning. Phylogenetic tree analyzes showed that cycad pollen caleosin is closely related to the caleosin of Cycas revoluta seeds. Fresh Cycas revoluta pollen grains were allowed to germinate for 48 h in a germination medium containing 2% sucrose and 0.01% H3BO3. Pollen germination were recorded periodically from day one to day 10 and the results showed that the Cycas revoluta pollen elongate the tube and increasing of triacylglycerol(TAG) after 4 days.

Development of Indirect Competitive ELISA for Lithospermic Acid B of Salvia miltiorrhiza with Its Specific Antibodies Generated via Artificial Oil Bodies.

Lithospermic acid B (LSB), the major water-soluble ingredient of Salvia miltiorrhiza (Danshen), has been shown to be an active ingredient responsible for the therapeutic effects of this traditional Chinese herb used to treat cardiac disorders. This study aimed to develop an indirect competitive enzyme linked immunosorbent assay (ELISA) for the detection of LSB. Firstly, LSB was chemically conjugated to a modified oil-body protein, lysine-enriched caleosin, recombinantly expressed in Escherichia coli. Antibodies against LSB (Ab-LSB) were successfully generated by immunizing hens with artificial oil bodies constituted with the LSB-conjugated caleosin. Western blotting showed that Ab-LSB specifically recognized LSB, but not the carrier protein, lysine-enriched caleosin. To detect LSB via indirect competitive ELISA, LSB was conjugated with bovine serum albumin (LSB-BSA) and coated on a microplate. The binding between Ab-LSB and LSB-BSA on the microplate was competed dose-dependently in the presence of free LSB with a concentration ranging from 5 to 5 × 104 ng/mL. The IC50 value was approximately determined to be 120 ng/mL for LSB regardless of its complex with a metal ion of Na+, K+ or Mg2+.

In vitro assay to estimate tea astringency via observing flotation of artificial oil bodies sheltered by caleosin fused with histatin 3.

Astringency, a sensory characteristic of food and beverages rich in polyphenols, mainly results from the formation of complexes between polyphenols and salivary proteins, causing a reduction of the lubricating properties of saliva. To develop an in vitro assay to estimate the astringency of oolong tea infusion, artificial oil bodies were constituted with sesame oil sheltered by a modified caleosin fused with histatin 3, one of the human salivary small peptides. Aggregation of artificial oil bodies was induced when they were mixed with oolong tea infusion or its major polyphenolic compound, (-)-epigallocatechin gallate (EGCG) of 100µM as observed in light microscopy. The aggregated artificial oil bodies gradually floated on top of the solution and formed a visible milky layer whose thickness was in proportion to the concentrations of tea infusion. This assay system was applied to test four different oolong tea infusions with sensory astringency corresponding to their EGCG contents. The result showed that relative astringency of the four tea infusions was correlated to the thickness of floated artificial oil bodies, and could be estimated according to the standard curve generated by simultaneously observing a serial dilution of the tea infusion with the highest astringency.

Identification of caleosin and oleosin in oil bodies of pine pollen.

Unique proteins including steroleosin, caleosin, oleosin-L, and oleosin-G have been identified in seed oil bodies of pine (Pinus massoniana). In this study, mature pollen grains with wing-like bladders were collected from pine (Pinus elliottii). Ultrastructural studies showed that oil bodies were present in pollen grains, but not the attached bladders, and the presence of oil bodies was further confirmed by fluorescent staining with BODIPY 493/503. Stable oil bodies were successfully purified from pine pollen grains, and analyzed to be mainly composed of triacylglycerols. Putative oleosin and caleosin in pine pollen oil bodies were detected by immunoassaying with antibodies against sesame seed caleosin and lily pollen oleosin. Complete cDNA fragments encoding these two pollen oil-body proteins were obtained by PCR cloning. Sequence alignment showed that pine pollen caleosin (27 kDa) was highly homologous to pine seed caleosin (28 kDa) except for the lack of an appendix of eight residues at the C-terminus in accord with the 1 kDa difference in their molecular masses. Pine pollen oleosin (15 kDa) was highly homologous to pine seed oleosin-G (14 kDa) except for an insertion of eight residues at the N-terminus in accord with the 1 kDa difference in their molecular masses.

Identification of caleosin and two oleosin isoforms in oil bodies of pine megagametophytes.

Numerous oil bodies of 0.2-2 µm occupied approximately 80% of intracellular space in mature pine (Pinus massoniana) megagametophytes. They were stably isolated and found to comprise mostly triacylglycerols as examined by thin layer chromatography analysis and confirmed by both Nile red and BODIPY stainings. Fatty acids released from the triacylglycerols of pine oil bodies were mainly unsaturated, including linoleic acid (60%), adrenic acid (12.3%) and vaccenic acid (9.7%). Proteins extracted from pine oil bodies were subjected to immunological cross-recognition, and the results showed that three proteins of 28, 16 and 14 kDa were detected by antibodies against sesame seed caleosin, sesame oleosin-L and lily pollen oleosin-P, respectively. Complete cDNA fragments encoding these three pine oil-body proteins, tentatively named caleosin, oleosin-L and oleosin-G, were obtained by PCR cloning and further confirmed by mass spectrometric analysis. Consistently, phylogenetic tree analyses showed that pine caleosin was closely-related to the caleosin of cycad megagametophyte among known caleosin sequences. While pine oleosin-L was found clustered with seed oleosin isoforms of angiosperm species, oleosin-G was distinctively grouped with the oleosin-P of lily pollen. The oleosin-G identified in pine megagametophytes seems to represent a new class of seed oleosin isoform evolutionarily close to the pollen oleosin-P.

Genomic analysis and expression investigation of caleosin gene family in Arabidopsis.

Caleosin is a common lipid-droplet surface protein, which has the ability to bind calcium. Arabidopsis (Arabidopsis thaliana) is considered a model organism in plant researches. Although there are growing researches about caleosin in the past few years, a systemic analysis of caleosins in Arabidopsis is still scarce. In this study, a comprehensive investigation of caleosins in Arabidopsis was performed by bioinformatics methods. Firstly, eight caleosins in Arabidopsis are divided into two types, L-caleosin and H-caleosin, according to their molecular weights, and these two types of caleosin have many differences in characteristics. Secondly, phylogenetic tree result indicates that L-caleosin may evolve from H-caleosin. Thirdly, duplication pattern analysis shows that segmental and tandem duplication are main reasons for Arabidopsis caleosin expansion with the equal part. Fourthly, the expression profiles of caleosins are also investigated in silico in different organs and under various stresses and hormones. In addition, based on promoter analysis, caleosin may be involved in calcium signal transduction and lipid accumulation. Thus, the classification and expression analysis of caleosin genes in Arabidopsis provide facilities to the research of phylogeny and functions in this gene family.

Development of a protocol to solidify native and artificial oil bodies for long-term storage at room temperature.

BACKGROUND: Oil bodies isolated from sesame seeds coalesced to form large oil drops when they were solidified in a drying process commonly used for food products. The aim of this study was to develop a protocol to solidify oil bodies for long-term storage at room temperature. RESULTS: On the basis of testing several excipients, the coalescence of oil bodies could be effectively prevented when they were combined with mannitol. Sizes of oil bodies appeared similar under a light microscope before and after powderisation in combination with 70% or more mannitol. Artificial oil bodies were successfully generated with sesame oil, phospholipid and recombinant sesame caleosin. Following the developed protocol, native and artificial oil bodies were stably solidified in tablets. Both native and artificial oil bodies dissolved from the tablets remained stable after an accelerated stress test under a condition of 75% humidity at 40 °C for 4 months. CONCLUSION: A protocol was successfully developed for the solidification of native and artificial oil bodies in stable powder and tablet forms. This successful protocol is very likely to expedite the utilisation of artificial oil bodies in their potential applications.