The complex hexaploid oil-Camellia genome traces back its phylogenomic history and multi-omics analysis of Camellia oil biosynthesis.

Oil-Camellia (Camellia oleifera), belonging to the Theaceae family Camellia, is an important woody edible oil tree species. The Camellia oil in its mature seed kernels, mainly consists of more than 90% unsaturated fatty acids, tea polyphenols, flavonoids, squalene and other active substances, which is one of the best quality edible vegetable oils in the world. However, genetic research and molecular breeding on oil-Camellia are challenging due to its complex genetic background. Here, we successfully report a chromosome-scale genome assembly for a hexaploid oil-Camellia cultivar Changlin40. This assembly contains 8.80 Gb genomic sequences with scaffold N50 of 180.0 Mb and 45 pseudochromosomes comprising 15 homologous groups with three members each, which contain 135 868 genes with an average length of 3936 bp. Referring to the diploid genome, intragenomic and intergenomic comparisons of synteny indicate homologous chromosomal similarity and changes. Moreover, comparative and evolutionary analyses reveal three rounds of whole-genome duplication (WGD) events, as well as the possible diversification of hexaploid Changlin40 with diploid occurred approximately 9.06 million years ago (MYA). Furthermore, through the combination of genomics, transcriptomics and metabolomics approaches, a complex regulatory network was constructed and allows to identify potential key structural genes (SAD, FAD2 and FAD3) and transcription factors (AP2 and C2H2) that regulate the metabolism of Camellia oil, especially for unsaturated fatty acids biosynthesis. Overall, the genomic resource generated from this study has great potential to accelerate the research for the molecular biology and genetic improvement of hexaploid oil-Camellia, as well as to understand polyploid genome evolution.

Interaction between phenylpropane metabolism and oil accumulation in the developing seed of Brassica napus revealed by high temporal-resolution transcriptomes.

BACKGROUND: Brassica napus is an important oilseed crop providing high-quality vegetable oils for human consumption and non-food applications. However, the regulation between embryo and seed coat for the synthesis of oil and phenylpropanoid compounds remains largely unclear. RESULTS: Here, we analyzed the transcriptomes in developing seeds at 2-day intervals from 14 days after flowering (DAF) to 64 DAF. The 26 high-resolution time-course transcriptomes are clearly clustered into five distinct groups from stage I to stage V. A total of 2217 genes including 136 transcription factors, are specifically expressed in the seed and show high temporal specificity by being expressed only at certain stages of seed development. Furthermore, we analyzed the co-expression networks during seed development, which mainly included master regulatory transcription factors, lipid, and phenylpropane metabolism genes. The results show that the phenylpropane pathway is prominent during seed development, and the key enzymes in the phenylpropane metabolic pathway, including TT5, BAN, and the transporter TT19, were directly or indirectly related to many key enzymes and transcription factors involved in oil accumulation. We identified candidate genes that may regulate seed oil content based on the co-expression network analysis combined with correlation analysis of the gene expression with seed oil content and seed coat content. CONCLUSIONS: Overall, these results reveal the transcriptional regulation between lipid and phenylpropane accumulation during B. napus seed development. The established co-expression networks and predicted key factors provide important resources for future studies to reveal the genetic control of oil accumulation in B. napus seeds.

PfbZIP85 Transcription Factor Mediates ω-3 Fatty Acid-Enriched Oil Biosynthesis by Down-Regulating PfLPAT1B Gene Expression in Plant Tissues.

The basic leucine zipper (bZIP) transcription factor (TF) family is one of the biggest TF families identified so far in the plant kingdom, functioning in diverse biological processes including plant growth and development, signal transduction, and stress responses. For Perilla frutescens, a novel oilseed crop abundant in polyunsaturated fatty acids (PUFAs) (especially α-linolenic acid, ALA), the identification and biological functions of bZIP members remain limited. In this study, 101 PfbZIPs were identified in the perilla genome and classified into eleven distinct groups (Groups A, B, C, D, E, F, G, H, I, S, and UC) based on their phylogenetic relationships and gene structures. These PfbZIP genes were distributed unevenly across 18 chromosomes, with 83 pairs of them being segmental duplication genes. Moreover, 78 and 148 pairs of orthologous bZIP genes were detected between perilla and Arabidopsis or sesame, respectively. PfbZIP members belonging to the same subgroup exhibited highly conserved gene structures and functional domains, although significant differences were detected between groups. RNA-seq and RT-qPCR analysis revealed differential expressions of 101 PfbZIP genes during perilla seed development, with several PfbZIPs exhibiting significant correlations with the key oil-related genes. Y1H and GUS activity assays evidenced that PfbZIP85 downregulated the expression of the PfLPAT1B gene by physical interaction with the promoter. PfLPAT1B encodes a lysophosphatidate acyltransferase (LPAT), one of the key enzymes for triacylglycerol (TAG) assembly. Heterogeneous expression of PfbZIP85 significantly reduced the levels of TAG and UFAs (mainly C18:1 and C18:2) but enhanced C18:3 accumulation in both seeds and non-seed tissues in the transgenic tobacco lines. Furthermore, these transgenic tobacco plants showed no significantly adverse phenotype for other agronomic traits such as plant growth, thousand seed weight, and seed germination rate. Collectively, these findings offer valuable perspectives for understanding the functions of PfbZIPs in perilla, particularly in lipid metabolism, showing PfbZIP85 as a suitable target in plant genetic improvement for high-value vegetable oil production.

Variety of Plant Oils: Species-Specific Lipid Biosynthesis.

Plant oils represent a large group of neutral lipids with important applications in food, feed and oleochemical industries. Most plants accumulate oils in the form of triacylglycerol within seeds and their surrounding tissues, which is comprised of three fatty acids attached to a glycerol backbone. Different plant species accumulate unique fatty acids in their oils, serving a range of applications in pharmaceuticals and oleochemicals. To enable the production of these distinctive oils, select plant species have evolved specialized oil metabolism pathways, involving differential gene co-expression networks and structurally divergent enzymes/proteins. Here, we summarize some of the recent advances in our understanding of oil biosynthesis in plants. We compare expression patterns of oil metabolism genes from representative species, including Arabidopsis thaliana, Ricinus communis (castor bean), Linum usitatissimum L. (flax), and Elaeis guineensis (oil palm) to showcase the co-expression networks of relevant genes for acyl metabolism. We also review several divergent enzymes/proteins associated with key catalytic steps of unique oil accumulation, including fatty acid desaturases, diacylglycerol acyltransferases, and oleosins, highlighting their structural features and preference towards unique lipid substrates. Lastly, we briefly discuss protein interactomes and substrate channeling for oil biosynthesis and the complex regulation of these processes.

Protein interactomes for plant lipid biosynthesis and their biotechnological applications.

Plant lipids have essential biological roles in plant development and stress responses through their functions in cell membrane formation, energy storage and signalling. Vegetable oil, which is composed mainly of the storage lipid triacylglycerol, also has important applications in food, biofuel and oleochemical industries. Lipid biosynthesis occurs in multiple subcellular compartments and involves the coordinated action of various pathways. Although biochemical and molecular biology research over the last few decades has identified many proteins associated with lipid metabolism, our current understanding of the dynamic protein interactomes involved in lipid biosynthesis, modification and channelling is limited. This review examines advances in the identification and characterization of protein interactomes involved in plant lipid biosynthesis, with a focus on protein complexes consisting of different subunits for sequential reactions such as those in fatty acid biosynthesis and modification, as well as transient or dynamic interactomes formed from enzymes in cooperative pathways such as assemblies of membrane-bound enzymes for triacylglycerol biosynthesis. We also showcase a selection of representative protein interactome structures predicted using AlphaFold2, and discuss current and prospective strategies involving the use of interactome knowledge in plant lipid biotechnology. Finally, unresolved questions in this research area and possible approaches to address them are also discussed.

Genome-wide exploration of oil biosynthesis genes in cultivated olive tree varieties (Olea europaea): insights into regulation of oil biosynthesis.

Genome-wide oil biosynthesis was explored by de novo sequencing two cultivated olive tree (Olea europaea) varieties (cv. Ayvalik and Picual). This is the first report of the former variety sequencing. As outgroups, raw reads of cv. Leccino and scaffold-level assembly of cv. Farga were also retrieved. Each of these four cultivars was chromosome-scale assembled into 23 pseudochromosomes, with 1.31 Gbp (Farga), 0.93 Gbp (Ayvalik), 0.7 Gbp (Picual), and 0.54 Gbp (Leccino) in size. Ab initio gene finding was performed on these assemblies, using wild olive tree (oleaster)-trained parameters. High numbers of gene models were predicted and anchored to the pseudochromosomes: 69,028 (Ayvalik), 55,073 (Picual), 63,785 (Farga), and 40,449 (Leccino). Using previously reported oil biosynthesis genes from wild olive tree genome project, the following homologous sequences were identified: 1,355 (Ayvalik), 1,269 (Farga), 812 (Leccino), and 774 (Picual). Of these, 358 sequences were commonly shared by all cultivars. Besides, some sequences were cultivar unique: Ayvalik (126), Farga (118), Leccino (46), and Picual (52). These putative sequences were assigned to various GO terms, ranging from lipid metabolism to stress tolerance, from signal transactions to development, and to many others, implicating that oil biosynthesis is synergistically regulated with involvement of various other pathways.

An integrated omics analysis reveals the gene expression profiles of maize, castor bean, and rapeseed for seed oil biosynthesis.

BACKGROUND: Seed storage lipids are valuable for human diet and for the sustainable development of mankind. In recent decades, many lipid metabolism genes and pathways have been identified, but the molecular mechanisms that underlie differences in seed oil biosynthesis in species with developed embryo and endosperm are not fully understood. RESULTS: We performed comparative genome and transcriptome analyses of castor bean and rapeseed, which have high seed oil contents, and maize, which has a low seed oil content. These results revealed the molecular underpinnings of the low seed oil content in maize. First of all, transcriptome analyses showed that more than 61% of the lipid- and carbohydrate-related genes were regulated in castor bean and rapeseed, but only 20.1% of the lipid-related genes and 22.5% of the carbohydrate-related genes were regulated in maize. Then, compared to castor bean and rapeseed, fewer lipid biosynthesis genes but more lipid metabolism genes were regulated in the maize embryo. More importantly, most maize genes encoding lipid-related transcription factors, triacylglycerol (TAG) biosynthetic enzymes, pentose phosphate pathway (PPP) and Calvin Cycle proteins were not regulated during seed oil synthesis, despite the presence of many homologs in the maize genome. Additionally, we observed differential regulation of vital oil biosynthetic enzymes and extremely high expression levels of oil biosynthetic genes in castor bean, which were consistent with the rapid accumulation of oil in castor bean developing seeds. CONCLUSIONS: Compared to high-oil seeds (castor bean and rapeseed), less oil biosynthetic genes were regulated during the seed development in low-oil seed (maize). These results shed light on molecular mechanisms of lipid biosynthesis in maize, castor bean, and rapeseed. They can provide information on key target genes that may be useful for future experimental manipulation of oil production in oil plants.

Oil plant genomes: current state of the science.

Vegetable oils are an indispensable nutritional component of the human diet as well as important raw materials for a variety of industrial applications such as pharmaceuticals, cosmetics, oleochemicals, and biofuels. Oil plant genomes are highly diverse, and their genetic variation leads to a diversity in oil biosynthesis and accumulation along with agronomic traits. This review discusses plant oil biosynthetic pathways, current state of genome assembly, polyploidy and asymmetric evolution of genomes of oil plants and their wild relatives, and research progress of pan-genomics in oil plants. The availability of complete high-resolution genomes and pan-genomes has enabled the identification of structural variations in the genomes that are associated with the diversity of agronomic and environment fitness traits. These and future genomes also provide powerful tools to understand crop evolution and to harvest the rich natural variations to improve oil crops for enhanced productivity, oil quality, and adaptability to changing environments.

Sunflower WRINKLED1 Plays a Key Role in Transcriptional Regulation of Oil Biosynthesis.

Sunflower (Helianthus annuus) is one of the most important oilseed crops worldwide. However, the transcriptional regulation underlying oil accumulation in sunflower is not fully understood. WRINKLED1 (WRI1) is an essential transcription factor governing oil accumulation in plant cells. Here, we identify and characterize a sunflower ortholog of WRI1 (HaWRI1), which is highly expressed in developing seeds. Transient production of HaWRI1 stimulated substantial oil accumulation in Nicotiana benthamiana leaves. Dual-luciferase reporter assay, electrophoretic mobility shift assay, fatty acid quantification, and gene expression analysis demonstrate that HaWRI1 acts as a pivotal transcription factor controlling the expression of genes involved in late glycolysis and fatty acid biosynthesis. HaWRI1 directly binds to the cis-element, AW-box, in the promoter of biotin carboxyl carrier protein isoform 2 (BCCP2). In addition, we characterize an 80 amino-acid C-terminal domain of HaWRI1 that is crucial for transactivation. Moreover, seed-specific overexpression of HaWRI1 in Arabidopsis plants leads to enhanced seed oil content as well as upregulation of the genes involved in fatty acid biosynthesis. Taken together, our work demonstrates that HaWRI1 plays a pivotal role in the transcriptional control of seed oil accumulation, providing a potential target for bioengineering sunflower oil yield improvement.

Extended mining of the oil biosynthesis pathway in biofuel plant Jatropha curcas by combined analysis of transcriptome and gene interactome data.

BACKGROUND: Jatropha curcas L. is an important non-edible oilseed crop with a promising future in biodiesel production. However, little is known about the molecular biology of oil biosynthesis in this plant when compared with other established oilseed crops, resulting in the absence of agronomically improved varieties of Jatropha. To extensively discover the potentially novel genes and pathways associated with the oil biosynthesis in J. curcas, new strategy other than homology alignment is on the demand. RESULTS: In this study, we proposed a multi-step computational framework that integrates transcriptome and gene interactome data to predict functional pathways in non-model organisms in an extended process, and applied it to study oil biosynthesis pathway in J. curcas. Using homologous mapping against Arabidopsis and transcriptome profile analysis, we first constructed protein-protein interaction (PPI) and co-expression networks in J. curcas. Then, using the homologs of Arabidopsis oil-biosynthesis-related genes as seeds, we respectively applied two algorithm models, random walk with restart (RWR) in PPI network and negative binomial distribution (NBD) in co-expression network, to further extend oil-biosynthesis-related pathways and genes in J. curcas. At last, using k-nearest neighbors (KNN) algorithm, the predicted genes were further classified into different sub-pathways according to their possible functional roles. CONCLUSIONS: Our method exhibited a highly efficient way of mining the extended oil biosynthesis pathway of J. curcas. Overall, 27 novel oil-biosynthesis-related gene candidates were predicted and further assigned to 5 sub-pathways. These findings can help better understanding of the oil biosynthesis pathway of J. curcas, as well as paving the way for the following J. curcas breeding application.

A GPAT1 Mutation in Arabidopsis Enhances Plant Height but Impairs Seed Oil Biosynthesis.

Glycerol-3-phosphate acyltransferases (GPATs) play an important role in glycerolipid biosynthesis, and are mainly involved in oil production, flower development, and stress response. However, their roles in regulating plant height remain unreported. Here, we report that Arabidopsis GPAT1 is involved in the regulation of plant height. GUS assay and qRT-PCR analysis in Arabidopsis showed that GPAT1 is highly expressed in flowers, siliques, and seeds. A loss of function mutation in GPAT1 was shown to decrease seed yield but increase plant height through enhanced cell length. Transcriptomic and qRT-PCR data revealed that the expression levels of genes related to gibberellin (GA) biosynthesis and signaling, as well as those of cell wall organization and biogenesis, were significantly upregulated. These led to cell length elongation, and thus, an increase in plant height. Together, our data suggest that knockout of GPAT1 impairs glycerolipid metabolism in Arabidopsis, leading to reduced seed yield, but promotes the biosynthesis of GA, which ultimately enhances plant height. This study provides new evidence on the interplay between lipid and hormone metabolism in the regulation of plant height.

TILLING-by-Sequencing+ to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis.

Reverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their DNA sequence. Chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. The current study uses an optimized high-throughput TILLING by target capture sequencing technology, or TILLING-by-Sequencing+ (TbyS+), coupled with universal bioinformatic tools to identify population-wide mutations in soybeans. Four ethyl methanesulfonate mutagenized populations (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. The mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. To test the efficiency of TbyS+ in complex genomes, we used soybeans as a model with a focus on three desaturase gene families, GmSACPD, GmFAD2, and GmFAD3, that are involved in the soybean fatty acid biosynthesis pathway. We successfully isolated mutants from all the six gene family members. Unsurprisingly, most of the characterized mutants showed significant changes either in their stearic, oleic, or linolenic acids. By using TbyS+, we discovered novel sources of soybean oil traits, including high saturated and monosaturated fatty acids in addition to low polyunsaturated fatty acid contents. This technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. The obtained soybean mutants from this study can be used in subsequent soybean breeding programs for improved oil composition traits.

Transitions in wheat endosperm metabolism upon transcriptional induction of oil accumulation by oat endosperm WRINKLED1.

BACKGROUND: Cereal grains, including wheat (Triticum aestivum L.), are major sources of food and feed, with wheat being dominant in temperate zones. These end uses exploit the storage reserves in the starchy endosperm of the grain, with starch being the major storage component in most cereal species. However, oats (Avena sativa L.) differs in that the starchy endosperm stores significant amounts of oil. Understanding the control of carbon allocation between groups of storage compounds, such as starch and oil, is therefore important for understanding the composition and hence end use quality of cereals. WRINKLED1 is a transcription factor known to induce triacylglycerol (TAG; oil) accumulation in several plant storage tissues. RESULTS: An oat endosperm homolog of WRI1 (AsWRI1) expressed from the endosperm-specific HMW1Dx5 promoter resulted in drastic changes in carbon allocation in wheat grains, with reduced seed weight and a wrinkled seed phenotype. The starch content of mature grain endosperms of AsWRI1-wheat was reduced compared to controls (from 62 to 22% by dry weight (dw)), TAG was increased by up to nine-fold (from 0.7 to 6.4% oil by dw) and sucrose from 1.5 to 10% by dw. Expression of AsWRI1 in wheat grains also resulted in multiple layers of elongated peripheral aleurone cells. RNA-sequencing, lipid analyses, and pulse-chase experiments using 14C-sucrose indicated that futile cycling of fatty acids could be a limitation for oil accumulation. CONCLUSIONS: Our data show that expression of oat endosperm WRI1 in the wheat endosperm results in changes in metabolism which could underpin the application of biotechnology to manipulate grain composition. In particular, the striking effect on starch synthesis in the wheat endosperm indicates that an important indirect role of WRI1 is to divert carbon allocation away from starch biosynthesis in plant storage tissues that accumulate oil.

Arachis hypogaea gene expression atlas for fastigiata subspecies of cultivated groundnut to accelerate functional and translational genomics applications.

Spatio-temporal and developmental stage-specific transcriptome analysis plays a crucial role in systems biology-based improvement of any species. In this context, we report here the Arachis hypogaea gene expression atlas (AhGEA) for the world's widest cultivated subsp. fastigiata based on RNA-seq data using 20 diverse tissues across five key developmental stages. Approximately 480 million paired-end filtered reads were generated followed by identification of 81 901 transcripts from an early-maturing, high-yielding, drought-tolerant groundnut variety, ICGV 91114. Further, 57 344 genome-wide transcripts were identified with ≥1 FPKM across different tissues and stages. Our in-depth analysis of the global transcriptome sheds light into complex regulatory networks namely gravitropism and photomorphogenesis, seed development, allergens and oil biosynthesis in groundnut. Importantly, interesting insights into molecular basis of seed development and nodulation have immense potential for translational genomics research. We have also identified a set of stable expressing transcripts across the selected tissues, which could be utilized as internal controls in groundnut functional genomics studies. The AhGEA revealed potential transcripts associated with allergens, which upon appropriate validation could be deployed in the coming years to develop consumer-friendly groundnut varieties. Taken together, the AhGEA touches upon various important and key features of cultivated groundnut and provides a reference for further functional, comparative and translational genomics research for various economically important traits.

Substrate preferences of long-chain acyl-CoA synthetase and diacylglycerol acyltransferase contribute to enrichment of flax seed oil with α-linolenic acid.

Seed oil from flax (Linum usitatissimum) is enriched in α-linolenic acid (ALA; 18:3Δ9cis,12cis,15cis ), but the biochemical processes underlying the enrichment of flax seed oil with this polyunsaturated fatty acid are not fully elucidated. Here, a potential process involving the catalytic actions of long-chain acyl-CoA synthetase (LACS) and diacylglycerol acyltransferase (DGAT) is proposed for ALA enrichment in triacylglycerol (TAG). LACS catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which in turn may serve as an acyl-donor in the DGAT-catalyzed reaction leading to TAG. To test this hypothesis, flax LACS and DGAT cDNAs were functionally expressed in Saccharomyces cerevisiae strains to probe their possible involvement in the enrichment of TAG with ALA. Among the identified flax LACSs, LuLACS8A exhibited significantly enhanced specificity for ALA over oleic acid (18:1Δ9cis ) or linoleic acid (18:2Δ9cis,12cis ). Enhanced α-linolenoyl-CoA specificity was also observed in the enzymatic assay of flax DGAT2 (LuDGAT2-3), which displayed ∼20 times increased preference toward α-linolenoyl-CoA over oleoyl-CoA. Moreover, when LuLACS8A and LuDGAT2-3 were co-expressed in yeast, both in vitro and in vivo experiments indicated that the ALA-containing TAG enrichment process was operative between LuLACS8A- and LuDGAT2-3-catalyzed reactions. Overall, the results support the hypothesis that the cooperation between the reactions catalyzed by LACS8 and DGAT2 may represent a route to enrich ALA production in the flax seed oil.

WRI1-1, ABI5, NF-YA3 and NF-YC2 increase oil biosynthesis in coordination with hormonal signaling during fruit development in oil palm.

The oil biosynthesis pathway must be tightly controlled to maximize oil yield. Oil palm accumulates exceptionally high oil content in its mesocarp, suggesting the existence of a unique fruit-specific fatty acid metabolism transcriptional network. We report the complex fruit-specific network of transcription factors responsible for modulation of oil biosynthesis genes in oil palm mesocarp. Transcriptional activation of EgWRI1-1 encoding a key master regulator that activates expression of oil biosynthesis genes, is activated by three ABA-responsive transcription factors, EgNF-YA3, EgNF-YC2 and EgABI5. Overexpression of EgWRI1-1 and its activators in Arabidopsis accelerated flowering, increased seed size and oil content, and altered expression levels of oil biosynthesis genes. Protein-protein interaction experiments demonstrated that EgNF-YA3 interacts directly with EgWRI1-1, forming a transcription complex with EgNF-YC2 and EgABI5 to modulate transcription of oil biosynthesis pathway genes. Furthermore, EgABI5 acts downstream of EgWRKY40, a repressor that interacts with EgWRKY2 to inhibit the transcription of oil biosynthesis genes. We showed that expression of these activators and repressors in oil biosynthesis can be induced by phytohormones coordinating fruit development in oil palm. We propose a model highlighting a hormone signaling network coordinating fruit development and fatty acid biosynthesis.

Genome-wide identification of oil biosynthesis-related long non-coding RNAs in allopolyploid Brassica napus.

BACKGROUND: Long noncoding RNAs (lncRNAs) are transcripts longer than 200 bp that do not encode proteins but nonetheless have been shown to play important roles in various biological processes in plants. Brassica napus is an important seed oil crop worldwide and the target of many genetic improvement activities. To understand better the function of lncRNAs in regulating plant metabolic activities, we carried out a genome-wide lncRNA identification of lncRNAs in Brassica napus with a focus on lncRNAs involved in lipid metabolism. Twenty ribosomal RNA depleted strand specific RNA-seq (ssRNA-seq) datasets were generatred using RNAs isolated from B. napus seeds at four developmental stages. For comparison we also included 30 publically available RNA-seq datasets generated from poly(A) enriched mRNAs isolated from from various Brassica napus tissues in our analysis. RESULTS: A total of 8905 lncRNA loci were identified, including 7100 long intergenic noncoding RNA (lincRNA) loci and 1805 loci generating long noncoding natural antisense transcript (lncNAT). Many lncRNAs were identified only in the ssRNA-seq and poly(A) RNA-seq dataset, suggesting that B. napus has a large lncRNA repertoire and it is necessary to use libraries prepared from different tissues and developmental stages as well as different library preparation approaches to capture the whole spectrum of lncRNAs. Analysis of coexpression networks revealed that among the regulatory modules are networks containing lncRNAs and protein-coding genes related to oil biosynthesis indicating a possible role of lncRNAs in the control of lipid metabolism. One such example is that several lncRNAs are potential regulators of BnaC08g11970D that encodes oleosin1, a protein found in oil bodies and involved in seed lipid accumulation. We also observed that the expression levels of B. napus lncRNAs is positively correlated with their conservation levels. CONCLUSIONS: We demonstrated that the B. napus genome has a large number of lncRNA and that these lncRNAs are expressed broadly across many developmental times and in different tissue types. We also provide evidence indicating that specific lncRNAs appear to be important regulators of lipid biosynthesis forming regulatory networks with transcripts involved in lipid biosynthesis. We also provide evidence that these lncRNAs are conserved in other species of the Brassicaceae family.

Transcriptome analysis reveals crucial genes involved in the biosynthesis of nervonic acid in woody Malania oleifera oilseeds.

BACKGROUND: Malania oleifera Chun et Lee (Olacaceae), an evergreen broad-leaved woody tree native to southwest China, is an important oilseed tree. Its seed oil has a high level of nervonic acid (cis-tetracos-15-enoic acid, over 60%), which is essential for human health. M. oleifera seed oil is a promising source of nervonic acid, but little is known about the physiological and molecular mechanisms underlying its biosynthesis. RESULTS: In this study, we recorded oil accumulation at four stages of seed development. Using a high-throughput RNA-sequencing technique, we obtained 55,843 unigenes, of which 29,176 unigenes were functionally annotated. By comparison, 22,833 unigenes had a two-fold or greater expression at the fast oil accumulation stage than at the initial stage. Of these, 198 unigenes were identified as being functionally involved in diverse lipid metabolism processes (including de novo fatty acid synthesis, carbon chain elongation and modification, and triacylglycerol assembly). Key genes (encoding KCS, KCR, HCD and ECR), putatively responsible for nervonic acid biosynthesis, were isolated and their expression profiles during seed development were confirmed by quantitative real-time PCR analysis. Also, we isolated regulatory factors (such as WRI1, ABI3 and FUS3) that are putatively involved in the regulation of oil biosynthesis and seed development. CONCLUSION: Our results provide novel data on the physiological and molecular mechanisms of nervonic acid biosynthesis and oil accumulation in M. oleifera seeds, and will also serve as a starting point for biotechnological genetic engineering for the production of nervonic acid resources.

Splice Variants of the Castor WRI1 Gene Upregulate Fatty Acid and Oil Biosynthesis When Expressed in Tobacco Leaves.

The plant-specific WRINKLED1 (WRI1) is a member of the AP2/EREBP class of transcription factors that positively regulate oil biosynthesis in plant tissues. Limited information is available for the role of WRI1 in oil biosynthesis in castor bean (Ricinus connunis L.), an important industrial oil crop. Here, we report the identification of two alternatively spliced transcripts of RcWRI1, designated as RcWRI1-A and RcWRI1-B. The open reading frames of RcWRI1-A (1341 bp) and RcWRI1-B (1332 bp) differ by a stretch of 9 bp, such that the predicted RcWRI1-B lacks the three amino acid residues "VYL" that are present in RcWRI1-A. The RcWRI1-A transcript is present in flowers, leaves, pericarps and developing seeds, while the RcWRI1-B mRNA is only detectable in developing seeds. When the two isoforms were individually introduced into an Arabidopsiswri1-1 loss-of-function mutant, total fatty acid content was almost restored to the wild-type level, and the percentage of the wrinkled seeds was largely reduced in the transgenic lines relative to the wri1-1 mutant line. Transient expression of each RcWRI1 splice isoform in N. benthamiana leaves upregulated the expression of the WRI1 target genes, and consequently increased the oil content by 4.3-4.9 fold when compared with the controls, and RcWRI1-B appeared to be more active than RcWRI1-A. Both RcWRI1-A and RcWRI1-B can be used as a key transcriptional regulator to enhance fatty acid and oil biosynthesis in leafy biomass.

14-3-3 protein mediates plant seed oil biosynthesis through interaction with AtWRI1.

Plant 14-3-3 proteins are phosphopeptide-binding proteins, belonging to a large family of proteins involved in numerous physiological processes including primary metabolism, although knowledge about the function of 14-3-3s in plant lipid metabolism is sparse. WRINKLED1 (WRI1) is a key transcription factor that governs plant oil biosynthesis. At present, AtWRI1-interacting partners remain largely unknown. Here, we show that 14-3-3 proteins are able to interact with AtWRI1, both in yeast and plant cells. Transient co-expression of 14-3-3- and AtWRI1-encoding cDNAs led to increased oil biosynthesis in Nicotiana benthamiana leaves. Stable transgenic plants overproducing a 14-3-3 protein also displayed increased seed oil content. Co-production of a 14-3-3 protein with AtWRI1 enhanced the transcriptional activity of AtWRI1. The 14-3-3 protein was found to increase the stability of AtWRI1. A possible 14-3-3 binding motif was identified in one of the two AP2 domains of AtWRI1, which was also found to be critical for the interaction of AtWRI1 with an E3 ligase linker protein. Thus, we hypothesize a regulatory mechanism by which the binding of 14-3-3 to AtWRI1 interferes with the interaction of AtWRI1 and the E3 ligase, thereby protecting AtWRI1 from degradation. Taken together, our studies identified AtWRI1 as a client of 14-3-3 proteins and provide insights into a role of 14-3-3 in mediating plant oil biosynthesis.