Oil Body Formation Research Articles

Endosperm-specific expressed transcription factor protein WRINKLED1-mediated oil accumulative mechanism in woody oil peony Paeonia ostii var. lishizhenii

Paeonia ostii var. lishizhenii exhibits superiority of high α-linolenic acid in seed oils, yet, the low yield highlights the importance of enhancing oil accumulation in seeds for edible oil production. The transcription factor protein WRINKLED1 (WRI1) plays crucial roles in modulating oil content in higher plants; however, its functional characterization remains elusive in P. ostii var. lishizhenii. Herein, based on a correlation analysis of transcription factor transcript levels, FA accumulation rates, and interaction assay of FA biosynthesis associated proteins, a WRI1 homologous gene (PoWRI1) that potentially regulated oil content in P. ostii var. lishizhenii seeds was screened. The PoWRI1 exhibited an endosperm-specific and development-depended expression pattern, encoding a nuclear-localized protein with transcriptional activation capability. Notably, overexpressing PoWRI1 upregulated certain key genes relevant to glycolysis, FA biosynthesis and desaturation, and improved seed development, oil body formation and oil accumulation in Arabidopsis seeds, resulting an enhancement of total seed oil weight by 9.47–18.77 %. The defective impacts on seed phenotypes were rescued through ectopic induction of PoWRI1 in wri1 mutants. Our findings highlight the pivotal role of PoWRI1 in controlling oil accumulation in P. ostii var. lishizhenii, offering bioengineering strategies to increase seed oil accumulation and enhance its potential for edible oil production.

MpR2R3-MYB2 is a key regulator of oil body formation in Marchantia polymorpha.

MpMYB02, a regulator of marchantin accumulation, also acts as a key regulator of oil body formation. MpMYB02 induces the expression of MpSYP12B and promotes oil body formation, subsequently leading to marchantin accumulation. The oil body observed in Marchantia polymorpha is a cellular organelle surrounded by a unit membrane, accumulating various secondary metabolites such as marchantins and terpenes. We observed that oil body formation is regulated by MpMYB02, a key regulator of marchantin accumulation. In the Mpmyb02 mutant, no oil bodies were observed, although idioblast-like cells were present in the gemma. We introduced MpMYB02-glucocorticoid receptor (GR), a steroid-inducible transcriptional activator, into Mpmyb02 and assessed the effect of dexamethasone (DEX) on oil body formation. Following DEX treatment, transformed liverworts began forming oil bodies within 12h. During the initial stages of oil body development, we observed the aggregation of small globular structures. DEX treatment upregulated several genes implicated in oil body formation, including MpSYP12B. Our findings underscore that MpMYB02 plays a crucial role not only in marchantin accumulation but also in oil body formation.

Identification and Analysis of Expression Patterns of the Caleosin Genes in Hickory (Carya cathayensis Sarg.)

The deciduous tree hickory (Carya cathayensis) holds economic significance in China due to its high oil content, particularly in unsaturated fatty acids. Oil bodies are crucial for storing triacylglycerol (TAG), with caleosin serving as a predominant oil body protein that aids in oil body formation and stability maintenance. Our study utilized bioinformatics techniques to identify caleosin genes within Carya cathayensis, Carya illinoinensis, and Juglans regia. Three caleosin genes were discovered in the genomes of Carya cathayensis, Carya illi-noinensis, and Juglans regia. These genes encode hydrophilic proteins. Additionally, all caleosin proteins feature a single Ca2+-binding EF-hand, a conserved “proline knot” motif, and a C-terminal hydrophilic region with four potential phosphorylation sites. The caleosin proteins in Carya cathayensis consist of α-helix, β-corner, extended chain, and random curl structures. Cis-acting elements related to stress response and hormone signaling were identified in Carya cathayensis, Carya illinoinensis, and Juglans regia, with distinct cis-acting elements implicated in seed-specific regulation in Carya cathayensis. Additionally, subcellular localization analysis confirmed that CcaCLO1 and CcaCLO2 were localized within oil bodies. Transcriptome analysis and quantitative real-time polymerase chain reaction (qRT-PCR) data demonstrated a significant up-regulation of CcaCLO1 expression during the developmental stages of the Carya cathayensis embryo. Furthermore, qPCR findings indicated that caleosins from Carya cathayensis were responsive to salt stress, with a significant up-regulation of CcaCLO1 following exposure to salt stress treatment. Consequently, caleosin genes in Carya cathayensis, Carya illinoinensis, and Juglans regia share similar physicochemical characteristics and conserved motifs. Specifically, CcaCLO1 in Carya cathayensis primarily responds to embryo development and salt stress. These findings offer foundational insights for future investigations into the regulatory mechanisms of oil accumulation and response to salt stress in hickory.

MpTGA, together with MpNPR, regulates sexual reproduction and independently affects oil body formation in Marchantia polymorpha.

In angiosperms, basic leucine-zipper (bZIP) TGACG-motif-binding (TGA) transcription factors (TFs) regulate developmental and stress-related processes, the latter often involving NON EXPRESSOR OF PATHOGENESIS-RELATED GENES (NPR) coregulator interactions. To gain insight into their functions in an early diverging land-plant lineage, the single MpTGA and sole MpNPR genes were investigated in the liverwort Marchantia polymorpha. We generated Marchantia MpTGA and MpNPR knockout and overexpression mutants and conducted morphological, transcriptomic and expression studies. Furthermore, we investigated MpTGA interactions with wild-type and mutagenized MpNPR and expanded our analyses including TGA TFs from two streptophyte algae. Mptga mutants fail to induce the switch from vegetative to reproductive development and lack gametangiophore formation. MpTGA and MpNPR proteins interact and Mpnpr mutant analysis reveals a novel coregulatory NPR role in sexual reproduction. Additionally, MpTGA acts independently of MpNPR as a repressor of oil body (OB) formation and can thereby affect herbivory. The single MpTGA TF exerts a dual role in sexual reproduction and OB formation in Marchantia. Common activities of MpTGA/MpNPR in sexual development suggest that coregulatory interactions were established after emergence of land-plant-specific NPR genes and contributed to the diversification of TGA TF functions during land-plant evolution.

Hub Genes in Stable QTLs Orchestrate the Accumulation of Cottonseed Oil in Upland Cotton via Catalyzing Key Steps of Lipid-Related Pathways

Upland cotton is the fifth-largest oil crop in the world, with an average supply of nearly 20% of vegetable oil production. Cottonseed oil is also an ideal alternative raw material to be efficiently converted into biodiesel. However, the improvement in kernel oil content (KOC) of cottonseed has not received sufficient attention from researchers for a long time, due to the fact that the main product of cotton planting is fiber. Previous studies have tagged QTLs and identified individual candidate genes that regulate KOC of cottonseed. The regulatory mechanism of oil metabolism and accumulation of cottonseed are still elusive. In the current study, two high-density genetic maps (HDGMs), which were constructed based on a recombinant inbred line (RIL) population consisting of 231 individuals, were used to identify KOC QTLs. A total of forty-three stable QTLs were detected via these two HDGM strategies. Bioinformatic analysis of all the genes harbored in the marker intervals of the stable QTLs revealed that a total of fifty-one genes were involved in the pathways related to lipid biosynthesis. Functional analysis via coexpression network and RNA-seq revealed that the hub genes in the co-expression network that also catalyze the key steps of fatty acid synthesis, lipid metabolism and oil body formation pathways (ACX4, LACS4, KCR1, and SQD1) could jointly orchestrate oil accumulation in cottonseed. This study will strengthen our understanding of oil metabolism and accumulation in cottonseed and contribute to KOC improvement in cottonseed in the future, enhancing the security and stability of worldwide food supply.

Targeted mutagenesis of flavonoid biosynthesis pathway genes reveals functional divergence in seed coat colour, oil content and fatty acid composition in Brassica napus L.

Yellow-seed is widely accepted as a good-quality trait in Brassica crops. Previous studies have shown that the flavonoid biosynthesis pathway is essential for the development of seed colour, but its function in Brassica napus, an important oil crop, is poorly understood. To systematically explore the gene functions of the flavonoid biosynthesis pathway in rapeseed, several representative TRANSPARENT TESTA (TT) genes, including three structural genes (BnaTT7, BnaTT18, BnaTT10), two regulatory genes (BnaTT1, BnaTT2) and a transporter (BnaTT12), were selected for targeted mutation by CRISPR/Cas9 in the present study. Seed coat colour, lignin content, seed quality and yield-related traits were investigated in these Bnatt mutants together with Bnatt8 generated previously. These Bnatt mutants produced seeds with an elevated seed oil content and decreased pigment and lignin accumulation in the seed coat without any serious defects in the yield-related traits. In addition, the fatty acid (FA) composition was also altered to different degrees, i.e., decreased oleic acid and increased linoleic acid and α-linolenic acid, in all Bnatt mutants except Bnatt18. Furthermore, gene expression analysis revealed that most of BnaTT mutations resulted in the down-regulation of key genes related to flavonoid and lignin synthesis, and the up-regulation of key genes related to lipid synthesis and oil body formation, which may contribute to the phenotype. Collectively, our study generated valuable resources for breeding programs, and more importantly demonstrated the functional divergence and overlap of flavonoid biosynthesis pathway genes in seed coat colour, oil content and FA composition of rapeseed.

Normal oil body formation in Marchantia polymorpha requires functional coat protein complex I proteins.

Eukaryotic cells possess endomembrane organelles equipped with specific sets of proteins, lipids, and polysaccharides that are fundamental for realizing each organelle’s specific function and shape. A tightly regulated membrane trafficking system mediates the transportation and localization of these substances. Generally, the secretory/exocytic pathway is responsible for transporting cargo to the plasma membrane and/or the extracellular space. However, in the case of oil body cells in the liverwort Marchantia polymorpha, the oil body, a liverwort-unique organelle, is thought to be formed by secretory vesicle fusion through redirection of the secretory pathway inside the cell. Although their formation mechanism remains largely unclear, oil bodies exhibit a complex and bumpy surface structure. In this study, we isolated a mutant with spherical oil bodies through visual screening of mutants with abnormally shaped oil bodies. This mutant harbored a mutation in a coat protein complex I (COPI) subunit MpSEC28, and a similar effect on oil body morphology was also detected in knockdown mutants of other COPI subunits. Fluorescently tagged MpSEC28 was localized to the periphery of the Golgi apparatus together with other subunits, suggesting that it is involved in retrograde transport from and/or in the Golgi apparatus as a component of the COPI coat. The Mpsec28 mutants also exhibited weakened stiffness of the thalli, suggesting impaired cell–cell adhesion and cell wall integrity. These findings suggest that the mechanism of cell wall biosynthesis is also involved in shaping the oil body in M. polymorpha, supporting the redirection of the secretory pathway inward the cell during oil body formation.

Critical metabolic pathways and genes cooperate for epoxy fatty acid-enriched oil production in developing seeds of Vernonia galamensis, an industrial oleaginous plant

BackgroundVernonia galamensis native to Africa is an annual oleaginous plant of Asteraceae family. As a newly established industrial oil crop, this plant produces high level (> 70%) of vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid), which is an unusual epoxy fatty acid (EFA) with multiple industrial applications. Here, transcriptome analysis and fatty acid profiling from developing V. galamensis seeds were integrated to uncover the critical metabolic pathways responsible for high EFA accumulation, aiming to identify the target genes that could be used in the biotechnological production of high-value oils.ResultsBased on oil accumulation dynamics of V. galamensis seeds, we harvested seed samples from three stages (17, 38, and 45 days after pollination, DAP) representing the initial, fast and final EFA accumulation phases, and one mixed sample from different tissues for RNA-sequencing, with three biological replicates for each sample. Using Illumina platform, we have generated a total of 265 million raw cDNA reads. After filtering process, de novo assembly of clean reads yielded 67,114 unigenes with an N50 length of 1316 nt. Functional annotation resulted in the identification of almost all genes involved in diverse lipid-metabolic pathways, including the novel fatty acid desaturase/epoxygenase, diacylglycerol acyltransferases, and phospholipid:diacylglycerol acyltransferases. Expression profiling revealed that various genes associated with acyl editing, fatty acid β-oxidation, triacylglycerol assembly and oil-body formation had greater expression levels at middle developmental stage (38 DAP), which were consistent with the fast accumulation of EFA in V. galamensis developing seed, these genes were detected to play fundamental roles in EFA production. In addition, we isolated some transcription factors (such as WRI1, FUS3 and ABI4), which putatively regulated the production of V. galamensis seed oils. The transient expression of the selected genes resulted in a synergistic increase of EFA-enriched TAG accumulation in tobacco leaves. Transcriptome data were further confirmed by quantitative real-time PCR for twelve key genes in EFA biosynthesis. Finally, a comprehensive network for high EFA accumulation in V. galamensis seed was established.ConclusionsOur findings provide new insights into molecular mechanisms underlying the natural epoxy oil production in V. galamensis. A set of genes identified here could be used as the targets to develop other oilseeds highly accumulating valued epoxy oils for commercial production.

How plants solubilise seed fats: revisiting oleosin structure and function to inform commercial applications.

Plants store triacylglycerides in organelles called oil bodies, which are important fuel sources for germination. Oil bodies consist of a lipid core surrounded by an interfacial single layer membrane of phospholipids and proteins. Oleosins are highly conserved plant proteins that are important for oil body formation, solubilising the triacylglycerides, stabilising oil bodies, and playing a role in mobilising the fuel during the germination process. The domain structure of oleosins is well established, with N- and C-terminal domains that are hydrophilic flanking a long hydrophobic domain that is proposed to protrude into the triacylglyceride core of the oil body. However, beyond this general understanding, little molecular level detail on the structure is available and what is known is disputed. This lack of knowledge limits our understanding of oleosin function and concomitantly our ability to engineer them. Here, we review the state of play in the literature regarding oleosin structure and function, and provide some examples of how oleosins can be used in commercial settings.

Comprehensive mining of storage oil related genes in developing seed of Abelmoschus esculentus

Okra (Abelmoschus esculentus), famous for its desired nutritional and medicinal values, could accumulate approximately 20% (w/w) seed oil dominated with unsaturated fatty acids (UFAs). However, the mechanism of oil biosynthesis and metabolic regulation in its seeds remains poorly understood. Here, storage oil profiles and transcriptome of okra seeds at different developmental stages were analyzed. Seed oil accumulated at a high rate during 21 to 28 days after flowering (DAF). Total of 136.6 million clean reads were obtained and 109,171 unigenes were assembled from the seed transcriptome. Differentially expressed genes among three developing stages of seeds were functionally annotated into the storage lipid metabolism pathway. Comparative analysis was performed for structural genes and transcription factor genes that participate in fatty acid biosynthesis, triacylglycerol assembly, oil body formation, and triacylglycerol degradation. A complex regulating network was built accordingly. Additionally, expressions of 12 key functional genes responsible for okra oil accumulation were examined by quantitative real-time PCR (qRT-PCR), demonstrating that RNA-sequencing data were reliable. This study will facilitate further investigation of molecular mechanism responsible for unsaturated fatty acid biosynthesis and oil accumulation in plant seed, providing valued reference for creating new okra varieties with better lipid composition and high oil yield.

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.

The liverwort oil body is formed by redirection of the secretory pathway

Eukaryotic cells acquired novel organelles during evolution through mechanisms that remain largely obscure. The existence of the unique oil body compartment is a synapomorphy of liverworts that represents lineage-specific acquisition of this organelle during evolution, although its origin, biogenesis, and physiological function are yet unknown. We find that two paralogous syntaxin-1 homologs in the liverwort Marchantia polymorpha are distinctly targeted to forming cell plates and the oil body, suggesting that these structures share some developmental similarity. Oil body formation is regulated by an ERF/AP2-type transcription factor and loss of the oil body increases M. polymorpha herbivory. These findings highlight a common strategy for the acquisition of organelles with distinct functions in plants, via periodical redirection of the secretory pathway depending on cellular phase transition.

Oil Body Formation in Marchantia polymorpha Is Controlled by MpC1HDZ and Serves as a Defense against Arthropod Herbivores

The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their function and development are poorly understood. Oil bodies of Marchantia polymorpha develop within specialized cells as one single large organelle. Here, we show that a class I homeodomain leucine-zipper (C1HDZ) transcription factor controls the differentiation of oil body cells in two different ecotypes of the liverwort M.polymorpha, a model genetic system for early divergent land plants. In flowering plants, these transcription factors primarily modulate responses to abiotic stress, including drought. However, loss-of-function alleles of the single ortholog gene, MpC1HDZ, in M.polymorpha did not exhibit phenotypes associated with abiotic stress. Rather, Mpc1hdz mutant plants were more susceptible to herbivory, and total plant extracts of the mutant exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction in expression of genes related to secondary metabolism that was accompanied by a specific depletion of oil body terpenoid compounds. Through time-lapse imaging, we observed that MpC1HDZ expression maxima precede oil body formation, indicating that MpC1HDZ mediates differentiation of oil body cells. Our results indicate that M.polymorpha oil bodies, and MpC1HDZ, are critical for defense against herbivory, but not for abiotic stress tolerance. Thus, C1HDZ genes were co-opted to regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages.

Comparative transcriptome analysis of the genes involved in lipid biosynthesis pathway and regulation of oil body formation in Torreya grandis kernels

Torreya grandis (T. grandis) is an important economic tree species because of the high content of unsaturated fatty acids in its kernel oil. However, different T. grandis landraces vary significantly in their fatty acid and oil contents, and their molecular regulatory mechanisms remain unclear. To investigate the molecular basis of the fatty acid biosynthesis pathway and oil body formation in T. grandis kernels, transcriptome sequencing were performed based on the Illumina platform in ten different T. grandis landraces. In total, 112,699 unigenes were identified, among them, 175 unigenes related to lipid biosynthesis were found, including 126 unigenes for fatty acid biosynthesis, 37 unigenes for triacylglycerol assembly and 12 unigenes for oil body proteins. The correlation analysis between the oil content and the expression levels of unigenes suggested that biotin carboxylase, acyl-ACP thioesterase A and lysophosphatidic acid acyltransferase may play key roles in oil accumulation. Three candidate genes (TgOLEO1, TgCLO1 and TgSLO1) encoding oil body-associated proteins were further identified through phylogenetic analysis and amino acid sequence alignment. Further subcellular localization assay indicated that all of the proteins were localized in both of endoplasmic reticulum and oil body. Compared with wild type, oil body proteins transiently over-expressed tobacco leaves had a higher abundance of oil bodies. Furthermore, the candidate regulatory genes (including TgWRI1, TgFUS3, etc.) that may be involved in the regulation of lipid biosynthesis were also identified. This study will be critical for the molecular assisted screening and breeding of high content of oil and unsaturated fatty acids cultivars for T. grandis.

Maize Dek33 encodes a pyrimidine reductase in riboflavin biosynthesis that is essential for oil-body formation and ABA biosynthesis during seed development.

The maize (Zea mays) defective kernel 33 (dek33) mutant produces defective and occasionally viviparous kernel phenotypes. In this study, we cloned Dek33 by positional cloning and found that it encodes a pyrimidine reductase in riboflavin biosynthesis. In dek33, a single-base mutation (G to A) in the C-terminal COG3236 domain caused a premature stop codon (TGA), producing a weak mutant allele with only a truncated form of the DEK33 protein that occurred at much lower levels that the completed WT form, and with a reduced riboflavin content. The dek33 mutation significantly affected oil-body formation and suppressed endoreduplication. It also disrupted ABA biosynthesis, resulting in lower ABA content that might be responsible for the viviparous embryo. In addition, our results indicated that the COG3236 domain is important for the protein stability of DEK33. Yeast two-hybrid experiments identified several proteins that interacted with DEK33, including RGLG2 and SnRK1, suggesting possible post-translational regulation of DEK33 stability. The interaction between DEK33 and these proteins was further confirmed by luciferase complementation image assays. This study provides a weak mutant allele that can be utilized to explore cellular responses to impaired riboflavin biosynthesis during seed development. Our findings indicate that the COG3236 domain might be an essential regulatory structure for DEK33 stability in maize.

Identification of genes associated with ricinoleic acid accumulation in Hiptage benghalensis via transcriptome analysis

BackgroundRicinoleic acid is a high-value hydroxy fatty acid with broad industrial applications. Hiptage benghalensis seed oil contains a high amount of ricinoleic acid (~ 80%) and represents an emerging source of this unusual fatty acid. However, the mechanism of ricinoleic acid accumulation in H. benghalensis is yet to be explored at the molecular level, which hampers the exploration of its potential in ricinoleic acid production.ResultsTo explore the molecular mechanism of ricinoleic acid biosynthesis and regulation, H. benghalensis seeds were harvested at five developing stages (13, 16, 19, 22, and 25 days after pollination) for lipid analysis. The results revealed that the rapid accumulation of ricinoleic acid occurred at the early–mid-seed development stages (16–22 days after pollination). Subsequently, the gene transcription profiles of the developing seeds were characterized via a comprehensive transcriptome analysis with second-generation sequencing and single-molecule real-time sequencing. Differential expression patterns were identified in 12,555 transcripts, including 71 enzymes in lipid metabolic pathways, 246 putative transcription factors (TFs) and 124 long noncoding RNAs (lncRNAs). Twelve genes involved in diverse lipid metabolism pathways, including fatty acid biosynthesis and modification (hydroxylation), lipid traffic, triacylglycerol assembly, acyl editing and oil-body formation, displayed high expression levels and consistent expression patterns with ricinoleic acid accumulation in the developing seeds, suggesting their primary roles in ricinoleic acid production. Subsequent co-expression network analysis identified 57 TFs and 35 lncRNAs, which are putatively involved in the regulation of ricinoleic acid biosynthesis. The transcriptome data were further validated by analyzing the expression profiles of key enzyme-encoding genes, TFs and lncRNAs with quantitative real-time PCR. Finally, a network of genes associated with ricinoleic acid accumulation in H. benghalensis was established.ConclusionsThis study was the first step toward the understating of the molecular mechanisms of ricinoleic acid biosynthesis and oil accumulation in H. benghalensis seeds and identified a pool of novel genes regulating ricinoleic acid accumulation. The results set a foundation for developing H. benghalensis into a novel ricinoleic acid feedstock at the transcriptomic level and provided valuable candidate genes for improving ricinoleic acid production in other plants.

The wheat ABC transporter Lr34 modifies the lipid environment at the plasma membrane

Phospholipids (PLs) are emerging as important factors that initiate signal transduction cascades at the plasma membrane. Their distribution within biological membranes is tightly regulated, e.g. by ATP-binding cassette (ABC) transporters, which preferably translocate PLs from the cytoplasmic to the exoplasmic membrane leaflet and are therefore called PL-floppases. Here, we demonstrate that a plant ABC transporter, Lr34 from wheat (Triticum aestivum), is involved in plasma membrane remodeling characterized by an intracellular accumulation of phosphatidic acid and enhanced outward translocation of phosphatidylserine. In addition, the content of phosphatidylinositol 4,5-bisphosphate in the cytoplasmic leaflet of the plasma membrane was reduced in the presence of the ABC transporter. When heterologously expressed in Saccharomyces cerevisiae, Lr34 promoted oil body formation in a mutant defective in PL-transfer in the secretory pathway. Our results suggest that PL redistribution by Lr34 potentially affects the membrane-bound proteome and contributes to the previously reported stimuli-independent activation of biotic and abiotic stress responses and neutral lipid accumulation in transgenic Lr34-expressing barley plants.

Gene refashioning through innovative shifting of reading frames in mosses

Early-diverging land plants such as mosses are known for their outstanding abilities to grow in various terrestrial habitats, incorporating tremendous structural and physiological innovations, as well as many lineage-specific genes. How these genes and functional innovations evolved remains unclear. In this study, we show that a dual-coding gene YAN/AltYAN in the moss Physcomitrella patens evolved from a pre-existing hemerythrin gene. Experimental evidence indicates that YAN/AltYAN is involved in fatty acid and lipid metabolism, as well as oil body and wax formation. Strikingly, both the recently evolved dual-coding YAN/AltYAN and the pre-existing hemerythrin gene might have similar physiological effects on oil body biogenesis and dehydration resistance. These findings bear important implications in understanding the mechanisms of gene origination and the strategies of plants to fine-tune their adaptation to various habitats.

3D reconstruction of endoplasmic reticulum in a hydrocarbon-secreting green alga, Botryococcus braunii (Race B).

Based on 3D sections through cells of Botryococcus braunii, the structure of three domains of endoplasmic reticulum, and their spatial and functional relationships to other organelles are clarified. Oil production by photosynthetic microalgae has attracted attention since these oils can be converted into renewable, carbon-neutral fuels. The green alga B. braunii accumulates large amounts of hydrocarbons, 30-50% of cell dry weight, in extracellular spaces rather than its cytoplasm. To advance the knowledge of hydrocarbon biosynthesis and transport pathways in this alga, we utilized transmission EM combined with rapid freezing and image reconstruction. We constructed detailed 3D maps distinguishing three ER domains: rdER with ribosomes on both sides, rsER with ribosomes on one side, and sER without ribosomes. The rsER and sER domains were especially prominent during the oil body formation and oil secretion stages. The ER contacted the chloroplasts, oil bodies, or plasma membrane via the rsER domains, oriented with the ribosome-free surface facing the organelles. We discuss the following transport pathway for hydrocarbons and their precursors in the cytoplasm: chloroplast→endoplasmic reticulum (ER)→oil bodies→ER→plasma membrane→secretion. This study represents the first 3D study of the three-domain classification (rdER, rsER and sER) of the ER network among eukaryotic cells. Finally, we propose the novel features of the ERs in plant cells that are distinct from the latest proposed model for the ERs in mammalian cells.

MYB76 Inhibits Seed Fatty Acid Accumulation in Arabidopsis.

The MYB family of transcription factors is important in regulatory networks controlling development, metabolism and responses to biotic and abiotic stresses in Arabidopsis. However, their role in regulating fatty acid accumulation in seeds is still largely unclear. Here, we found that MYB76, localized in the nucleus, was predominantly expressed in developing seeds during maturation. The myb76 mutation caused a significant increase in the amounts of total fatty acids and several major fatty acid compositions in mature seeds, suggesting that MYB76 functioned as an important repressor during seed oil biosynthesis. RNA sequencing and quantitative real-time PCR analysis revealed remarkable alteration of numerous genes involved in photosynthesis, fatty acid biosynthesis, modification, and degradation, and oil body formation in myb76 seeds at 12 days after pollination. These results help us to understand the novel function of MYB76 and provide new insights into the regulatory network of MYB transcriptional factors controlling seed oil accumulation in Arabidopsis.