Oleosin Gene Research Articles

Molecular characterization of CeOLE6, a diverged SH oleosin gene, preferentially expressed in Cyperus esculentus tubers.

CeOLE6, a tuber-specific gene in tigernut, encodes a diverged SH oleosin that functions in oil accumulation via homo and heteromultimerization. Tigernut (Cyperus esculentus L.) is a rare example accumulating high levels of triacylglycerols (TAGs) in underground tubers; however, the mechanism underlying is poorly understood. Given essential roles of oleosins (OLEs) in oil accumulation, in this study, structural and functional analyses were conducted for CeOLE6, an oleosin gene preferentially expressed in tigernut tubers. Phylogenetic analysis revealed that CeOLE6 encodes a diverged oleosin in Clade SH, which also includes CeOLE4 and -5. Further synteny analysis and sequence comparison indicated that CeOLE6 is more likely to be a whole-genome duplication (WGD) repeat of CeOLE4, which underwent rapid evolution and deletion of the typical C-terminal insertion for SHs. Nevertheless, CeOLE6 retains the capacity of oligomerization and oil accumulation, because (i) CeOLE6 could not only interact with itself but also with CeOLE2 and -5, two tuber-dominant members belonging to Clades SL and SH, respectively, and (ii) overexpressing CeOLE6 in tobacco leaves could significantly enhance the TAG content. Though CeWRI1 exhibits a similar expression pattern as CeOLE6 during tuber development, both CeWRI1 and -3 could not activate the CeOLE6 promoter, implying that they are not transcription factors contributing tuber-specific activation of CeOLE6. These findings not only provide insights into CeOLE genes in tuber oil accumulation, but also lay a foundation for further genetic improvement in tigernut and other species.

Metabolic engineering-induced transcriptome reprogramming of lipid biosynthesis enhances oil composition in oat.

The endeavour to elevate the nutritional value of oat (Avena sativa) by altering the oil composition and content positions it as an optimal crop for fostering human health and animal feed. However, optimization of oil traits on oat through conventional breeding is challenging due to its quantitative nature and complexity of the oat genome. We introduced two constructs containing three key genes integral to lipid biosynthesis and/or regulatory pathways from Arabidopsis (AtWRI1 and AtDGAT1) and Sesame (SiOLEOSIN) into the oat cultivar 'Park' to modify the fatty acid composition. Four homozygous transgenic lines were generated with a transformation frequency of 7%. The expression of these introduced genes initiated a comprehensive transcriptional reprogramming in oat grains and leaves. Notably, endogenous DGAT, WRI1 and OLEOSIN genes experienced upregulation, while genes associated with fatty acid biosynthesis, such as KASII, SACPD and FAD2, displayed antagonistic expression patterns between oat grains and leaves. Transcriptomic analyses highlighted significant differential gene expression, particularly enriched in lipid metabolism. Comparing the transgenic oat plants with the wild type, we observed a remarkable increase of up to 34% in oleic acid content in oat grains. Furthermore, there were marked improvements in the total oil content in oat leaves, as well as primary metabolites changes in both oat grains and leaves, while maintaining homeostasis in the transgenic oat plants. These findings underscore the effectiveness of genetic engineering in manipulating oat oil composition and content, offering promising implications for human consumption and animal feeding through oat crop improvement programmes.

Exploring selection signatures in the divergence and evolution of lipid droplet (LD) associated genes in major oilseed crops

BackgroundOil bodies or lipid droplets (LDs) in the cytosol are the subcellular storage compartments of seeds and the sites of lipid metabolism providing energy to the germinating seeds. Major LD-associated proteins are lipoxygenases, phospholipaseD, oleosins, TAG-lipases, steroleosins, caleosins and SEIPINs; involved in facilitating germination and enhancing peroxidation resulting in off-flavours. However, how natural selection is balancing contradictory processes in lipid-rich seeds remains evasive. The present study was aimed at the prediction of selection signatures among orthologous clades in major oilseeds and the correlation of selection effect with gene expression.ResultsThe LD-associated genes from the major oil-bearing crops were analyzed to predict natural selection signatures in phylogenetically close-knit ortholog clusters to understand adaptive evolution. Positive selection was the major force driving the evolution and diversification of orthologs in a lineage-specific manner. Significant positive selection effects were found in 94 genes particularly in oleosin and TAG-lipases, purifying with excess of non-synonymous substitution in 44 genes while 35 genes were neutral to selection effects. No significant selection impact was noticed in Brassicaceae as against LOX genes of oil palm. A heavy load of deleterious mutations affecting selection signatures was detected in T-lineage oleosins and LOX genes of Arachis hypogaea. The T-lineage oleosin genes were involved in mainly anther, tapetum and anther wall morphogenesis. In Ricinus communis and Sesamum indicum > 85% of PLD genes were under selection whereas selection pressures were low in Brassica juncea and Helianthus annuus. Steroleosin, caleosin and SEIPINs with large roles in lipid droplet organization expressed mostly in seeds and were under considerable positive selection pressures. Expression divergence was evident among paralogs and homeologs with one gene attaining functional superiority compared to the other. The LOX gene Glyma.13g347500 associated with off-flavor was not expressed during germination, rather its paralog Glyma.13g347600 showed expression in Glycine max. PLD-α genes were expressed on all the tissues except the seed,δ genes in seed and meristem while β and γ genes expressed in the leaf.ConclusionsThe genes involved in seed germination and lipid metabolism were under strong positive selection, although species differences were discernable. The present study identifies suitable candidate genes enhancing seed oil content and germination wherein directional selection can become more fruitful.

Differential gene expression and potential regulatory network of fatty acid biosynthesis during fruit and leaf development in yellowhorn (Xanthoceras sorbifolium), an oil-producing tree with significant deployment values.

Xanthoceras sorbifolium (yellowhorn) is a woody oil plant with super stress resistance and excellent oil characteristics. The yellowhorn oil can be used as biofuel and edible oil with high nutritional and medicinal value. However, genetic studies on yellowhorn are just in the beginning, and fundamental biological questions regarding its very long-chain fatty acid (VLCFA) biosynthesis pathway remain largely unknown. In this study, we reconstructed the VLCFA biosynthesis pathway and annotated 137 genes encoding relevant enzymes. We identified four oleosin genes that package triacylglycerols (TAGs) and are specifically expressed in fruits, likely playing key roles in yellowhorn oil production. Especially, by examining time-ordered gene co-expression network (TO-GCN) constructed from fruit and leaf developments, we identified key enzymatic genes and potential regulatory transcription factors involved in VLCFA synthesis. In fruits, we further inferred a hierarchical regulatory network with MYB-related (XS03G0296800) and B3 (XS02G0057600) transcription factors as top-tier regulators, providing clues into factors controlling carbon flux into fatty acids. Our results offer new insights into key genes and transcriptional regulators governing fatty acid production in yellowhorn, laying the foundation for efforts to optimize oil content and fatty acid composition. Moreover, the gene expression patterns and putative regulatory relationships identified here will inform metabolic engineering and molecular breeding approaches tailored to meet biofuel and bioproduct demands.

Integrative Analysis of Oleosin Genes Provides Insights into Lineage-Specific Family Evolution in Brassicales.

Oleosins (OLEs) are a class of small but abundant structural proteins that play essential roles in the formation and stabilization of lipid droplets (LDs) in seeds of oil crops. Despite the proposal of five oleosin clades (i.e., U, SL, SH, T, and M) in angiosperms, their evolution in eudicots has not been well-established. In this study, we employed Brassicales, an economically important order of flowering plants possessing the lineage-specific T clade, as an example to address this issue. Three to 10 members were identified from 10 species representing eight plant families, which include Caricaceae, Moringaceae, Akaniaceae, Capparaceae, and Cleomaceae. Evolutionary and reciprocal best hit-based homologous analyses assigned 98 oleosin genes into six clades (i.e., U, SL, SH, M, N, and T) and nine orthogroups (i.e., U1, U2, SL, SH1, SH2, SH3, M, N, and T). The newly identified N clade represents an ancient group that has already appeared in the basal angiosperm Amborella trichopoda, which are constitutively expressed in the tree fruit crop Carica papaya, including pulp and seeds of the fruit. Moreover, similar to Clade N, the previously defined M clade is actually not Lauraceae-specific but an ancient and widely distributed group that diverged before the radiation of angiosperm. Compared with A. trichopoda, lineage-specific expansion of the family in Brassicales was largely contributed by recent whole-genome duplications (WGDs) as well as the ancient γ event shared by all core eudicots. In contrast to the flower-preferential expression of Clade T, transcript profiling revealed an apparent seed/embryo/endosperm-predominant expression pattern of most oleosin genes in Arabidopsis thaliana and C. papaya. Moreover, the structure and expression divergence of paralogous pairs was frequently observed, and a good example is the lineage-specific gain of an intron. These findings provide insights into lineage-specific family evolution in Brassicales, which facilitates further functional studies in nonmodel plants such as C. papaya.

Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density

BackgroundCommercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions.ResultsExpected relative increases in total fatty acids of 17–58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity.ConclusionsThese results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

Molecular characterization of oleosin genes in Cyperus esculentus, a Cyperaceae plant producing oil in underground tubers.

CeOLE genes exhibit a tuber-predominant expression pattern and their mRNA/protein abundances are positively correlated with oil accumulation during tuber development. Overexpression could significantly increase the oil content of tobacco leaves. Oleosins (OLEs) are abundant structural proteins of lipid droplets (LDs) that function in LD formation and stabilization in seeds of oil crops. However, little information is available on their roles in vegetative tissues. In this study, we present the first genome-wide characterization of the oleosin family in tigernut (Cyperus esculentus L., Cyperaceae), a rare example accumulating high amounts of oil in underground tubers. Six members identified represent three previously defined clades (i.e. U, SL and SH) or six out of seven orthogroups (i.e. U, SL1, SL2, and SH1-3)proposed in this study. Comparative genomics analysis reveals that lineage-specific expansion of Clades SL and SH was contributed by whole-genome duplication and dispersed duplication, respectively. Moreover, presence of SL2 and SH3 in Juncus effuses implies their appearance sometime before Cyperaceae-Juncaceae divergence, whereas SH2 appears to be Cyperaceae specific. Expression analysis showed that CeOLE genes exhibit a tuber-predominant expression pattern and transcript levels are considerably more abundant than homologs in the close relative Cyperus rotundus. Moreover, CeOLE mRNA and protein abundances were shown to positively correlate with oil accumulation during tuber development. Additionally, two dominant isoforms (i.e. CeOLE2 and -5) were shown to locate in LDs as well as the endoplasmic reticulum of tobacco (Nicotiana benthamiana)leaves, and are more likely to function in homo and heteromultimers. Furthermore, overexpression of CeOLE2 and -5 in tobacco leaves could significantly increase the oil content, supporting their roles in oil accumulation. These findings provide insights into lineage-specific family evolution and putative roles of CeOLE genes in oil accumulation of vegetative tissues, which facilitate further genetic improvement for tigernut.

The lineage-specific evolution of the oleosin family in Theaceae

Oleosins play essential roles in stabilization of lipid droplets (LDs) and seed oil production. However, evolution of this gene family has not been reported in Theaceae, a large plant family that contains many important tea and oil tea species. In this study, a total of 65 oleosin genes were identified in nine genome-sequenced Theaceae species. Among these genomes, the gene number of oleosin showed significant difference, with Camellia sinensis var. sinensis cv. Shuchazao and Camellia lanceoleosa displayed more oleosin numbers than other species. Phylogenetic analyses revealed that Theaceae oleosin genes were classified into three clades (U, SL, SH) respectively. Proteins within the same clade had similar gene structure and motif composition. Segmental duplication was the primary driving force for the evolution of oleosin genes in Shuchazao (SCZ), Huangdan (HD), C.lanceoleosa (Cla), and wild tea (DASZ). Synteny analysis showed that most oleosin genes displayed inter-species synteny among tea and oil tea species. Expression analysis demonstrated that oleosin genes were specifically expressed in seed and kernel of Huangdan (HD) and C.lanceoleosa. Moreover, expression divergence was observed in paralogous pairs and ∼1–2 oleosin genes in each clade have become activate. This study leads to a comprehensive understanding of evolution of oleosin family in Theaceae, and provides a rich resource to further address the functions of oleosin in tea and oil tea species.

Identification of Key Lipogenesis Stages and Proteins Involved in Walnut Kernel Development.

Walnuts are abundant in oil content, especially for polyunsaturated fatty acids, but the understanding of their formation is limited. We collected walnut (Juglans regia L.) kernels at 60, 74, 88, 102, 116, 130, and 144 days after pollination (designated S1-S7). The ultrastructure and accumulation of oil bodies (OBs) were observed using transmission electron microscopy (TEM), and the oil content, fatty acid composition, and proteomic changes in walnut kernels were determined. The oil content and OB accumulation increased during the development and rose sharply from S1 to S3 stages, which are considered the key lipogenesis stage. A total of 5442 proteins were identified and determined as differentially expressed proteins (DEPs) using label-free proteomic analysis. Fatty acid desaturases (FAD) 2, FAD3, oleosin, and caleosin were essential and upregulated from the S1 to S3 stages. Furthermore, the highly expressed oleosin gene JrOLE14.7 from walnuts was cloned and overexpressed in transgenic Brassica napus. The overexpression of JrOLE14.7 increased the oil content, diameter, hundred weight of seeds and changed the fatty acid composition and OB size of Brassica napus seeds. These findings provide insights into the molecular mechanism of oil biosynthesis and the basis for the genetic improvement of walnuts.

Transcriptome and Regional Association Analyses Reveal the Effects of Oleosin Genes on the Accumulation of Oil Content in Brassica napus.

Rapeseed stores lipids in the form of oil bodies. Oil bodies in the seeds of higher plants are surrounded by oleosins. Adjusting oleosin protein levels can prevent the fusion of oil bodies and maintain oil body size during seed development. However, oil contents are affected by many factors, and studies on the complex molecular regulatory mechanisms underlying the variations in seed oil contents of B. napus are limited. In this study, a total of 53 BnOLEO (B. napus oleosin) genes were identified in the genome of B. napus through a genome-wide analysis. The promoter sequences of oleosin genes consisted of various light-, hormone-, and stress-related cis-acting elements, along with transcription factor (TF) binding sites, for 25 TF families in 53 BnOLEO genes. The differentially expressed oleosin genes between two high- and two low-oil-content accessions were explored. BnOLEO3-C09, BnOLEO4-A02, BnOLEO4-A09, BnOLEO2-C04, BnOLEO1-C01, and BnOLEO7-A03 showed higher expressions in the high-oil-content accessions than in low-oil-content accessions, at 25, 35, and 45 days after pollination (DAP) in two different environments. A regional association analysis of 50 re-sequenced rapeseed accessions was used to further analyze these six BnOLEO genes, and it revealed that the nucleotide variations in the BnOLEO1-C01 and BnOLEO7-A03 gene regions were related to the phenotypic variations in seed oil content. Moreover, a co-expression network analysis revealed that the BnOLEO genes were directly linked to lipid/fatty acid metabolism, TF, lipid transport, and carbohydrate genes, thus forming a molecular network involved in seed oil accumulation. These favorable haplotypes can be utilized in molecular marker-assisted selection in order to further improve seed oil contents in rapeseed.

Oleosin Expression Patterns and Size of Oil Bodies as a Factor in Determining Oil Content in Safflower (Carthamus Tinctorius L.) Genotypes

The climate crisis and the Ukraine war have shown the vulnerability of various crop commodities. One of those badly affected is cooking oil, leading to a shortage in several countries. This coupled with the need for healthier cooking oil, increases proportionally with the world population and has resulted in escalated cooking oil prices. Thus, continued evaluation of alternative oil crops that can do well in marginal lands becomes a vital practice to undertake. Safflower is one of the marginalized oil crops with high-quality oil containing essential fatty acids beneficial to human health. Screening safflower genotypes for oil content is critical for its breeding and adoption in non-native areas. Therefore, this study delineates the relationship between oleosin genes and oil bodies in regulating the oil content of safflower seeds. Oleosin genes and oil bodies from the seeds of five safflower genotypes were isolated and quantified using qPCR and fluorescence microscope respectively, and evaluated against the seed oil content. The results showed an inverse relationship where smaller oil bodies were displayed by genotypes with high oil content. A high relative expression of oleosin genes was observed in genotypes with high oil content (Kenya-9819 and Gila). Of the eight Ctoleosin genes that were studied, it was observed that Ctoleosin genes (1, 4, 6, 7, and 8) were highly reliable in characterizing safflower genotypes based on the oil content. Kenya-9819 and Gila genotypes were found to have high oil potential and this was confirmed by a higher accumulation of the oleosin gene. A high correlation coefficient between oleosin, oil content, and oil body was also observed in this study. The findings suggest that selected oleosin genes and oil bodies are important traits to consider when characterizing oil seed crops for oil content.

RNAi and CRISPR-Cas silencing E3-RING ubiquitin ligase AIP2 enhances soybean seed protein content.

The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.

Genomic insights into lineage-specific evolution of the oleosin family in Euphorbiaceae

BackgroundLipid droplets (LDs) present in land plants serve as an essential energy and carbon reserve for seed germination and seedling development. Oleosins, the most abundant structural proteins of LDs, comprise a small family involved in LD formation, stabilization and degradation. Despite their importance, our knowledge on oleosins is still poor in Euphorbiaceae, a large plant family that contains several important oil-bearing species.ResultsTo uncover lineage-specific evolution of oleosin genes in Euphorbiaceae, in this study, we performed a genome-wide identification and comprehensive comparison of the oleosin family in Euphorbiaceae species with available genome sequences, i.e. castor bean (Ricinus communis), physic nut (Jatropha curcas), tung tree (Vernicia fordii), Mercurialis annua, cassava (Manihot esculenta) and rubber tree (Hevea brasiliensis), and a number of five, five, five, five, eight and eight members were found, respectively. Synteny analysis revealed one-to-one collinear relationship of oleosin genes between the former four (i.e. castor bean, physic nut, tung tree and M. annua) as well as latter two species (i.e. cassava and rubber tree), whereas one-to-one and one-to-two collinear relationships were observed between physic nut and cassava, reflecting the occurrence of one recent whole-genome duplication (WGD) in the last common ancestor of cassava and rubber tree. The presence of five ortholog groups representing three previously defined clades (i.e. U, SL and SH) dates back at least to the Malpighiales ancestor, because they are also conserved in poplar (Populus trichocarpa), a tree having experienced one Salicaceae-specific recent WGD. As observed in poplar, WGD was shown to be the main driver for the family expansion in both cassava and rubber tree. Nevertheless, same retention patterns of WGD-derived duplicates observed in cassava and rubber tree are somewhat different from that of poplar, though certain homologous fragments are still present in rubber tree. Further transcriptional profiling revealed an apparent seed-predominant expression pattern of oleosin genes in physic nut, castor bean and rubber tree. Moreover, structure and expression divergence of paralogous pairs were also observed in both cassava and rubber tree.ConclusionComparative genomics analysis of oleosin genes reported in this study improved our knowledge on lineage-specific family evolution in Euphorbiaceae, which also provides valuable information for further functional analysis and utilization of key members and their promoters.

Genome-wide identification and analysis of Oleosin gene family in four cotton species and its involvement in oil accumulation and germination

BackgroundCotton is not only a major textile fiber crop but also a vital oilseed, industrial, and forage crop. Oleosins are the structural proteins of oil bodies, influencing their size and the oil content in seeds. In addition, the degradation of oleosins is involved in the mobilization of lipid and oil bodies during seed germination. However, comprehensive identification and the systematic analysis of the Oleosin gene (OLEOs) family have not been conducted in cotton.ResultsAn in-depth analysis has enabled us to identify 25 and 24 OLEOs in tetraploid cotton species G. hirsutum and G. barbadense, respectively, while 12 and 13 OLEOs were identified in diploid species G. arboreum and G. raimondii, respectively. The 74 OLEOs were further clustered into three lineages according to the phylogenetic tree. Synteny analysis revealed that most of the OLEOs were conserved and that WGD or segmental duplications might drive their expansion. The transmembrane helices in GhOLEO proteins were predicted, and three transmembrane models were summarized, in which two were newly proposed. A total of 24 candidate miRNAs targeting GhOLEOs were predicted. Three highly expressed oil-related OLEOs, GH_A07G0501 (SL), GH_D10G0941 (SH), and GH_D01G1686 (U), were cloned, and their subcellular localization and function were analyzed. Their overexpression in Arabidopsis increased seed oil content and decreased seed germination rates.ConclusionWe identified OLEO gene family in four cotton species and performed comparative analyses of their relationships, conserved structure, synteny, and gene duplication. The subcellular localization and function of three highly expressed oil-related OLEOs were detected. These results lay the foundation for further functional characterization of OLEOs and improving seed oil content.

Abscisic acid activation of oleosin gene HvOle3 expression prevents the coalescence of protein storage vacuoles in barley aleurone cells.

Protein storage vacuoles (PSVs) in aleurone cells coalesce during germination, and this process is highly coupled with mobilization of PSV reserves, allowing de novo synthesis of various hydrolases in aleurone cells for endosperm degradation. Here we show that in barley (Hordeum vulgare L.) oleosins, the major integral proteins of oleosomes are encoded by four genes (HvOle1 to 4), and the expression of HvOle1 and HvOle3 is strongly up-regulated by abscisic acid (ABA), which shows antagonism to gibberellic acid. In aleurone cells, all HvOLEs were subcellularly targeted to the tonoplast of PSVs. Gain-of-function analyses revealed that HvOLE3 effectively delayed PSV coalescence, whereas HvOLE1 only had a moderate effect, with no notable effect of HvOLE2 and 4. With regard to longevity, HvOLE3 chiefly outperformed other HvOLEs, followed by HvOLE1. Experiments swapping the N- and C-terminal domain between HvOLE3 and other HvOLEs showed that the N-terminal region of HvOLE3 is mainly responsible, with some positive effect by the C-terminal region, for mediating the specific preventive effect of HvOLE3 on PSV coalescence. Three ACGT-core elements and the RY-motif were responsible for ABA induction of HvOle3 promoter activity. Transient expression assays using aleurone protoplasts demonstrated that transcriptional activation of the HvOle3 promoter was mediated by transcription factors HvABI3 and HvABI5, which acted downstream of protein kinase HvPKABA1.

Characterization of oleosin genes from forage sorghum in Arabidopsis and yeast reveals their role in storage lipid stability.

Overexpression of forage sorghum oleosin genes in Arabidopsis oleosin-deficient mutant and yeast showed increased germination rate, triacylglycerol content, and protection against lipase-mediated TAG degradation. Plant lipids are an important source of ration for cattle or other livestock animals to fulfil their energy needs. Poor energy containing green forages are still one of the major sources of food for livestock animals, leaving the animals undernourished. This lowers the milk and meat production efficiency, thereby affecting human consumption. Oleosin, an essential oil body surface protein, is capable of enhancing and stabilizing the lipid content in plants. We identified and functionally characterized three forage sorghum oleosin genes (SbOle1, SbOle2, and SbOle3) in Arabidopsis and yeast. Phylogenetic analysis of SbOle proteins showed a close relationship with rice and maize oleosins. Expression analysis of SbOle genes determined a higher expression pattern in embryo followed by endosperm, while its expression in the non-seed tissues remained negligible. Overexpression of SbOle genes in Arabidopsis ole1-deficient mutants showed restoration of normal germination whereas control mutant seeds showed lower germination rates. Heterologous overexpression of SbOle in yeast cells resulted in increased TAG accumulation. Additionally, the TAG turnover assay showed the effectiveness of SbOle genes in reducing the yeast endogenous and rumen bacterial lipase-mediated TAG degradation. Taken together, our findings not only provide insights into forage sorghum oleosin for increasing the energy content in non-seed organs but also opened up the direction towards implication of oleosin in rumen protection of fodders.

Lipid Body Dynamics in Shoot Meristems: Production, Enlargement, and Putative Organellar Interactions and Plasmodesmal Targeting.

Post-embryonic cells contain minute lipid bodies (LBs) that are transient, mobile, engage in organellar interactions, and target plasmodesmata (PD). While LBs can deliver γ-clade 1,3-β-glucanases to PD, the nature of other cargo is elusive. To gain insight into the poorly understood role of LBs in meristems, we investigated their dynamics by microscopy, gene expression analyzes, and proteomics. In developing buds, meristems accumulated LBs, upregulated several LB-specific OLEOSIN genes and produced OLEOSINs. During bud maturation, the major gene OLE6 was strongly downregulated, OLEOSINs disappeared from bud extracts, whereas lipid biosynthesis genes were upregulated, and LBs were enlarged. Proteomic analyses of the LB fraction of dormant buds confirmed that OLEOSINs were no longer present. Instead, we identified the LB-associated proteins CALEOSIN (CLO1), Oil Body Lipase 1 (OBL1), Lipid Droplet Interacting Protein (LDIP), Lipid Droplet Associated Protein1a/b (LDAP1a/b) and LDAP3a/b, and crucial components of the OLEOSIN-deubiquitinating and degradation machinery, such as PUX10 and CDC48A. All mRFP-tagged LDAPs localized to LBs when transiently expressed in Nicotiana benthamiana. Together with gene expression analyzes, this suggests that during bud maturation, OLEOSINs were replaced by LDIP/LDAPs at enlarging LBs. The LB fraction contained the meristem-related actin7 (ACT7), “myosin XI tail-binding” RAB GTPase C2A, an LB/PD-associated γ-clade 1,3-β-glucanase, and various organelle- and/or PD-localized proteins. The results are congruent with a model in which LBs, motorized by myosin XI-k/1/2, traffic on F-actin, transiently interact with other organelles, and deliver a diverse cargo to PD.

A transcriptional journey from sucrose to endosperm oil bodies in triple transgene oily wheat grain

Transgenic wheat expressing Wrinkled1 (Wri1), diacylglycerol acyltransferase 1 (DGAT1), and Oleosin genes with endosperm active promoters, in the companion paper were found to accumulate 5–10 times the quantity of triacylglyceride (TAG) in the endosperm compared to the control. In this study we track the pleiotropic effects on transcription at four grain development time points, of genes whose products are involved in the various pathways from sugar import to oil body formation, as well as other major metabolic pathways in the developing grain. The transcriptional impacts were substantial with early increases in genes for sucrose transport, hexose phosphate formation, cytoplasmic glycolysis, and ADP-glucose transport to the plastid. Corresponding to the late expression of the Wrinkled1 transgene, there was a late up-regulation of the entire suite of genes in the plastidial fatty acid synthesis pathway. Transport of fatty acid precursors to the ER was likely facilitated by early up-regulation of FAX1 (Fatty acid export) and LACS8 (long-chain acyl-CoA synthetase) genes. With the exception of the transgenic DGAT1, there were relatively few and moderate changes to transcription of other genes involved in TAG assembly in the ER, although the direct use of the acyl-PC pool appears to be down-regulated. A large up-regulation was observed at the three earliest time points of many genes associated with the formation of lipid droplets, including endogenous oleosins, caleosins, stereoleosins, seipins, and a variety of other lipid body associated proteins. Also unanticipated was the early down-regulation of a number of genes whose products are involved in lipid body turnover and peroxisomal β-oxidation of released fatty acids. Despite the substantial increase in TAG accumulation, the transgenic grains were not reduced in starch or protein and the transcriptional analysis demonstrated an early up-regulation of the entire suite of starch synthesis genes and protein storage genes.

Dynamic transcriptome analysis identifies genes related to fatty acid biosynthesis in the seeds of Prunus pedunculata Pall

BackgroundPrunus pedunculata Pall, the deciduous shrub of Amygdalus subgenus in Rosaceae, is a new kind of desert oil-bearing tree. It has a long story of being planted in the West and North of China for sand fixation and desert control. In addition, the seeds of P. pedunculata are rich of oil, especially the monounsaturated fatty acid and polyunsaturated fatty acid. However, little is known about the molecular mechanisms of oil accumulation during the seed development of P. pedunculata.ResultsThe seeds of P. pedunculata from three independent plants at 10, 18, 24, 31, 39, 45, 59 and 73 days after flowering (DAF) were obtained and the oil compositions were evaluated. It showed that oleic acid was the dominant type of oil content in the mature seeds (from 32.724% at 10DAF to 72.06% at 73DAF). Next, transcriptome sequencing for the developing seeds produced 988.795 million high quality reads and TRINITY assembled 326,271 genes for the first transcriptome for P. pedunculata. After the assembled transcriptome was evaluated by BUSCO with 85.9% completeness, we identified 195,342, 109,850 and 121,897 P. pedunculata genes aligned to NR, GO and KEGG pathway databases, respectively. Then, we predicted 23,229 likely proteins from the assembled transcriptome and identified 1917 signal peptides and 5512 transmembrane related proteins. In the developing seeds we detected 91,362 genes (average FPKM > 5) and correlation analysis indicated three possible development stages – early (10 ~ 24DAF), middle (31 ~ 45DAF) and late (59 ~ 73DAF). We next analyzed the differentially expressed genes (DEGs) in the developing seeds. Interestingly, compared to 10DAF the number of DEGs was increased from 4406 in 18DAF to 27,623 in 73DAF. Based on the gene annotation, we identified 753, 33, 8 and 645 DEGs related to the fatty acid biosynthesis, lipid biosynthesis, oil body and transcription factors. Notably, GPAT, DGD1, LACS2, UBC and RINO were highly expressed at the early development stage, ω6-FAD, SAD, ACP, ACCA and AHG1 were highly expressed at the middle development stage, and LACS6, DGD1, ACAT1, AGPAT, WSD1, EGY2 and oleosin genes were highly expressed at the late development stage.ConclusionsThis is the first time to study the developing seed transcriptome of P. pedunculata and our findings will provide a valuable resource for future studies. More importantly, it will improve our understanding of molecular mechanisms of oil accumulation in P. pedunculata.

The oleosin gene family in coffee: Analysis of oleosomes, oleosins and lipid-related gene expression during germination of Coffea arabica seeds

Little information is available about lipid metabolism in Coffea arabica L. seeds, which have an oil content between 11 and 16% w/w. This is surprising given the involvement of lipids in the physical, chemical, and physiological properties of the coffee beverage. To shed light on this subject, freshly harvested C. arabica seeds were sampled at different stages of germination. Oleosomes (the organelles responsible for lipid storage) were extracted and their structure was observed by differential interference-contrast microscopy. Triacylglycerol content of oleosomes was analyzed by thin-layer chromatography, and the protein fraction was evaluated by gel electrophoresis. A review of available genomic data on oleosin and lipase genes was performed, and expression of genes related to lipid metabolism was measured by Real-Time PCR.The results drew a coherent picture of coffee seed germination, revealing that oleosins have the highest concentration after 14 days of germination, followed by a marked decrease. This was backed up by molecular observations, showing how oleosin genes were downregulated after 6–9 days of germination, while genes related to lipid degradation were upregulated from day 9 onwards. Morphological evidence supported these findings, as after 14 days embryo growth became significant and cotyledons started to invade the endosperm and metabolize seed reserves. The present research will be an important first step in better understanding the biology of oleosomes in Coffea arabica L. and in elucidating the role of lipid metabolism in green coffee quality.