Intron-mediated enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 expression in energycane promotes a step change for lipid accumulation in vegetative tissues.

BACKGROUND: Metabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG. RESULTS: In this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 (WRI1) and the TAG protect factor oleosin (OLE1). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic "push pull and protect" factors correlated with biomass reduction. CONCLUSIONS: Intron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels.

Seed-specific expression of the class 2 Phytoglobin (Pgb2) increases seed oil in Brassica napus.

To examine the function of phytoglobin 2 (Pgb2) on seed oil level in the oil-producing crop Brassica napus L., we generated transgenic plants in which BnPgb2 was over-expressed in the seeds using the cruciferin1 promoter. Over-expression of BnPgb2 elevated the amount of oil, which showed a positive relationship with the level of BnPgb2, without altering the oil nutritional value, as evidenced by the lack of major changes in composition of fatty acids (FA), and key agronomic traits. Two key transcription factors, LEAFY COTYLEDON1 (LEC1) and WRINKLED1 (WRI1), known to promote the synthesis of fatty acids (FA) and potentiate oil accumulation, were induced in BnPgb2 over-expressing seeds. The concomitant induction of several enzymes of sucrose metabolism, SUCROSE SYNTHASE1 (SUS) 1 and 3, FRUCTOSE BISPHOSPHATE ALDOLASE (FPA), and PHOSPHOGLYCERATE KINASE (PGK), and starch synthesis, ADP-GLUCOSE PHOSPHORYLASE (AGPase) suggests that BnPgb2 favors sugar mobilization for FA production. The two plastid FA biosynthetic enzymes SUBUNIT A OF ACETYL-CoA CARBOXYLASE (ACCA2), and MALONYL-CoA:ACP TRANSACYLASE (MCAT) were also up-regulated by the over-expression of BnPgb2. The requirement of BnPgb2 for oil deposition was further evidenced in natural germplasm by the higher levels of BnPgb2 in seeds of high-oil genotypes relative to their low-oil counterparts.

Improved bioenergy value of residual rice straw by increased lipid levels from upregulation of fatty acid biosynthesis.

BACKGROUND: Rice (Oryza sativa) straw is a common waste product that represents a considerable amount of bound energy. This energy can be used for biogas production, but the rate and level of methane produced from rice straw is still low. To investigate the potential for an increased biogas production from rice straw, we have here utilized WRINKLED1 (WRI1), a plant AP2/ERF transcription factor, to increase triacylglycerol (TAG) biosynthesis in rice plants. Two forms of Arabidopsis thaliana WRI1 were evaluated by transient expression and stable transformation of rice plants, and transgenic plants were analyzed both for TAG levels and biogas production from straw. RESULTS: Both full-length AtWRI1, and a truncated form lacking the initial 141 amino acids (including the N-terminal AP2 domain), increased fatty acid and TAG levels in vegetative and reproductive tissues of Indica rice. The stimulatory effect of the truncated AtWRI1 was significantly lower than that of the full-length protein, suggesting a role for the deleted AP2 domain in WRI1 activity. Full-length AtWRI1 increased TAG levels also in Japonica rice, indicating a conserved effect of WRI1 in rice lipid biosynthesis. The bio-methane production from rice straw was 20% higher in transformants than in the wild type. Moreover, a higher producing rate and final yield of methane was obtained for rice straw compared with rice husks, suggesting positive links between methane production and a high amount of fatty acids. CONCLUSIONS: Our results suggest that heterologous WRI1 expression in transgenic plants can be used to improve the metabolic potential for bioenergy purposes, in particular methane production.

Ectopic Expression of Perilla frutescens WRI1 Enhanced Storage Oil Accumulation in Nicotiana benthamiana Leaves.

Vegetable oils are indispensable in human and animal diets and have been widely used for the production of detergents, lubricants, cosmetics, and biofuels. The seeds of an allotetraploid Perilla frutescens contain approximately 35 to 40% oils with high levels of polyunsaturated fatty acids (PUFAs). WRINKELD1 (WRI1) encoding an AP2/ERF-type transcription factor is known to upregulate the expression of genes involved in glycolysis and fatty acid biosynthesis and TAG assembly. In this study, two WRI1 isoforms, PfWRI1A, and PfWRI1B were isolated from Perilla and predominantly expressed in developing Perilla seeds. The fluorescent signals from PfWRI1A:eYFP and PfWRI1B:eYFP driven by the CaMV 35S promoter were detected in the nucleus of the Nicotiana benthamiana leaf epidermis. Ectopic expression of each of PfWRI1A and PfWRI1B increased the levels of TAG by approximately 2.9- and 2.7-fold in N. benthamiana leaves and particularly, the enhanced levels (mol%) of C18:2, and C18:3 in the TAGs were prominent with the concomitant reduction in the amounts of saturated fatty acids. The expression levels of NbPl-PKß1, NbKAS1, and NbFATA, which were known to be target genes of WRI1, significantly increased in tobacco leaves overexpressing PfWRI1A or PfWRI1B. Therefore, newly characterized PfWRI1A and PfWRI1B can be potentially useful for the enhanced accumulation of storage oils with increased PUFAs in oilseed crops.

Overexpression of WRINKLED1 improves the weight and oil content in seeds of flax (Linum usitatissimum L.).

Seeds of flax (Linum usitatissimum L.) are highly rich in both oil and linolenic acid (LIN). It is crucial for flax agricultural production to identify positive regulators of fatty acid biosynthesis. In this study, we find that WRINKLED1 transcription factors play important positive roles during flax seed oil accumulation. Two WRINKLED1 genes, LuWRI1a and LuWRI1b, were cloned from flax, and LuWRI1a was found be expressed predominantly in developing seeds during maturation. Overexpression of LuWRI1a increased seed size, weight, and oil content in Arabidopsis and increased seed storage oil content in transgenic flax without affecting seed production or seed oil quality. The rise in oil content in transgenic flax seeds was primarily attributable to the increase in seed weight, according to a correlational analysis. Furthermore, overexpression or interference of LuWRI1a upregulated the expression of genes in the fatty acid biosynthesis pathway and LAFL genes, and the expression level of WRI1 was highly significantly positively associated between L1L, LEC1, and BCCP2. Our findings give a theoretical scientific foundation for the future application of genetic engineering to enhance the oil content of plant seeds.

Characterisation of Grains and Flour Fractions from Field Grown Transgenic Oil-Accumulating Wheat Expressing Oat WRI1.

Wheat (Triticum aestivum L.) is one of the major staple crops in the world and is used to prepare a range of foods. The development of new varieties with wider variation in grain composition could broaden their use. We characterized grains and flours from oil-accumulating transgenic wheat expressing the oat (Avena sativa L.) endosperm WRINKLED1 (AsWRI1) grown under field conditions. Lipid and starch accumulation was determined in developing caryopses of AsWRI1-wheat and X-ray microtomography was used to study grain morphology. The developing caryopses of AsWRI1-wheat grains had increased triacylglycerol content and decreased starch content compared to the control. Mature AsWRI1-wheat grains also had reduced weight, were wrinkled and had a shrunken endosperm and X-ray tomography revealed that the proportion of endosperm was decreased while that of the aleurone was increased. Grains were milled to produce two white flours and one bran fraction. Mineral and lipid analyses showed that the flour fractions from the AsWRI1-wheat were contaminated with bran, due to the effects of the changed morphology on milling. This study gives a detailed analysis of grains from field grown transgenic wheat that expresses a gene that plays a central regulatory role in carbon allocation and significantly affects grain composition.

The Sunflower WRINKLED1 Transcription Factor Regulates Fatty Acid Biosynthesis Genes through an AW Box Binding Sequence with a Particular Base Bias.

Sunflower is an important oilseed crop in which the biochemical pathways leading to seed oil synthesis and accumulation have been widely studied. However, how these pathways are regulated is less well understood. The WRINKLED1 (WRI1) transcription factor is considered a key regulator in the control of triacylglycerol biosynthesis, acting through the AW box binding element (CNTNG(N)7CG). Here, we identified the sunflower WRI1 gene and characterized its activity in electrophoretic mobility shift assays. We studied its role as a co-regulator of sunflower genes involved in plastidial fatty acid synthesis. Sunflower WRI1-targets included genes encoding the pyruvate dehydrogenase complex, the α-CT and BCCP genes, genes encoding ACPs and the fatty acid synthase complex, together with the FATA1 gene. As such, sunflower WRI1 regulates genes involved in seed plastidial fatty acid biosynthesis in a coordinated manner, establishing a WRI1 push and pull strategy that drives oleic acid synthesis for its export into the cytosol. We also determined the base bias at the N positions in the active sunflower AW box motif. The sunflower AW box is sequence-sensitive at the non-conserved positions, enabling WRI1-binding. Moreover, sunflower WRI1 could bind to a non-canonical AW-box motif, opening the possibility of searching for new target genes.

ABA-INSENSITIVE 3 with or without FUSCA3 highly up-regulates lipid droplet proteins and activates oil accumulation.

ABA-INSENSITIVE 3 (ABI3) has long been known for activation of storage protein accumulation. A role of ABI3 on oil accumulation was previously suggested based on a decrease of oil content in seeds of abi3 mutant. However, this conclusion could not exclude possibilities of indirect or pleiotropic effects, such as through mutual regulatory interactions with FUSCA3 (FUS3), an activator of oil accumulation. To identify that ABI3 functions independent of the effects of related seed transcription factors, we expressed ABI3 under the control of an inducible promoter in tobacco BY2 cells and Arabidopsis rosette leaves. Inducible expression of ABI3 activated oil accumulation in these non-seed cells, demonstrating a general role of ABI3 in regulation of oil biosynthesis. Further expressing ABI3 in rosette leaves of fus3 knockout mutant still caused up to 3-fold greater triacylglycerol accumulation, indicating ABI3 can activate lipid accumulation independently of FUS3. Transcriptome analysis revealed that LIPID DROPLET PROTEIN (LDP) genes, including OLEOSINs and CALEOSINs, were up-regulated up to 1000-fold by ABI3 in the absence of FUS3, while the expression of WRINKLED1 was doubled. Taken together, our results provide genetic evidence that ABI3 activates oil accumulation with or without FUS3, most likely through up-regulating LDPs and WRINKLED1.

Towards oilcane: preliminary field evaluation of metabolically engineered sugarcane with hyper-accumulation of triacylglycerol in vegetative tissues.

We recently generated oilcane, a metabolically engineered sugarcane with hyper-accumulation of energy dense triacylglycerol in vegetative tissues. Refinement of this strategy in high biomass crops like sugarcane may result in elevated lipid yields that exceed traditional oilseed crops for biodiesel production. This is the first report of agronomic performance, stable co-expression of lipogenic factors, and TAG accumulation in transgenic sugarcane under field conditions. Co-expression of WRI1; DGAT1, OLE1, and RNAi suppression of PXA1 was stable during the 2-year field evaluation and resulted in TAG accumulation up to 4.4% of leaf DW. This TAG accumulation was 70-fold higher than in non-transgenic sugarcane and more than 2-fold higher than previously reported for the same line under greenhouse conditions. TAG accumulation correlated highest with the expression of WRI1. However, constitutive expression of WRI1 was negatively correlated with biomass accumulation. Transgenic lines without WRI1 expression accumulated TAG up to 1.6% of leaf DW and displayed no biomass yield penalty in the plant cane. These findings confirm sugarcane as a promising platform for the production of vegetative lipids and will be used to inform strategies to maximize future biomass and lipid yields. The main conclusion is that constitutive expression of WRI1 in combination with additional lipogenic factors (DGAT1-2, OLE1, PXA1) in sugarcane under field conditions leads to hyper-accumulation of TAG and reduces biomass yield. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01333-5.

Analysis and Functional Verification of PoWRI1 Gene Associated with Oil Accumulation Process in Paeonia ostii.

The plant transcription factor WRINKLED1 (WRI1), a member of AP2/EREBP, is involved in the regulation of glycolysis and the expression of genes related to the de novo synthesis of fatty acids in plastids. In this study, the key regulator of seed oil synthesis and accumulation transcription factor gene PoWRI1 was identified and cloned, having a complete open reading frame of 1269 bp and encoding 422 amino acids. Subcellular localization analysis showed that PoWRI1 is located at the nucleus. After the expression vector of PoWRI1 was constructed and transformed into wild-type Arabidopsis thaliana, it was found that the overexpression of PoWRI1 increased the expression level of downstream target genes such as BCCP2, KAS1, and PKP-ß1. As a result, the seeds of transgenic plants became larger, the oil content increased significantly, and the unsaturated fatty acid content increased, which provide a scientific theoretical basis for the subsequent use of genetic engineering methods to improve the fatty acid composition and content of plant seeds.

Sunflower (Helianthus annuus) fatty acid synthase complex: ß-Ketoacyl-[acyl carrier protein] reductase genes.

Fatty acids play many roles in plants, but the function of some key genes involved in fatty acid biosynthesis in plant development are not yet properly understood. Here, we clone two ß-ketoacyl-[ACP] reductase (KAR) genes from sunflower, HaKAR1 and HaKAR2, and characterize their functional roles. The enzymes cloned were the only two copies present in the sunflower genome. Both displayed a high degree of similarity, but their promoters infer different regulation. The two sunflower KAR genes were constitutively expressed in all tissues examined, being maximum in developing cotyledons at the start of oil synthesis. Over-expression of HaKAR1 in E. coli changed the fatty acid composition by promoting the elongation of C16:0 to C18:0 fatty acids. The enzymatic characterization of HaKAR1 revealed similar kinetic parameters to homologues from other oil accumulating species. The results point to a partially functional redundancy between HaKAR1 and HaKAR2. This study clearly revealed that these genes play a prominent role in de novo fatty acids synthesis in sunflower seeds.

Functional and Predictive Structural Characterization of WRINKLED2, A Unique Oil Biosynthesis Regulator in Avocado.

WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors regulates fatty acid biosynthesis and triacylglycerol (TAG) accumulation in plants. Among the four known Arabidopsis WRI1 paralogs, only WRI2 was unable to complement and restore fatty acid content in wri1-1 mutant seeds. Avocado (Persea americana) mesocarp, which accumulates 60-70% dry weight oil content, showed high expression levels for orthologs of WRI2, along with WRI1 and WRI3, during fruit development. While the role of WRI1 as a master regulator of oil biosynthesis is well-established, the function of WRI1 paralogs is poorly understood. Comprehensive and comparative in silico analyses of WRI1 paralogs from avocado (a basal angiosperm) with higher angiosperms Arabidopsis (dicot), maize (monocot) revealed distinct features. Predictive structural analyses of the WRI orthologs from these three species revealed the presence of AP2 domains and other highly conserved features, such as intrinsically disordered regions associated with predicted PEST motifs and phosphorylation sites. Additionally, avocado WRI proteins also contained distinct features that were absent in the nonfunctional Arabidopsis ortholog AtWRI2. Through transient expression assays, we demonstrated that both avocado WRI1 and WRI2 are functional and drive TAG accumulation in Nicotiana benthamiana leaves. We predict that the unique features and activities of ancestral PaWRI2 were likely lost in orthologous genes such as AtWRI2 during evolution and speciation, leading to at least partial loss of function in some higher eudicots. This study provides us with new targets to enhance oil biosynthesis in plants.

The Role of Sugar Signaling in Regulating Plant Fatty Acid Synthesis.

Photosynthates such as glucose, sucrose, and some of their derivatives play dual roles as metabolic intermediates and signaling molecules that influence plant cell metabolism. Such sugars provide substrates for de novo fatty acid (FA) biosynthesis. However, compared with the well-defined examples of sugar signaling in starch and anthocyanin synthesis, until recently relatively little was known about the role of signaling in regulating FA and lipid biosynthesis. Recent research progress shows that trehalose 6-phosphate and 2-oxoglutarate (2-OG) play direct signaling roles in the regulation of FA biosynthesis by modulating transcription factor stability and enzymatic activities involved in FA biosynthesis. Specifically, mechanistic links between sucrose non-fermenting-1-related protein kinase 1 (SnRK1)-mediated trehalose 6-phosphate (T6P) sensing and its regulation by phosphorylation of WRI1 stability, diacylglycerol acyltransferase 1 (DGAT1) enzyme activity, and of 2-OG-mediated relief of inhibition of acetyl-CoA carboxylase (ACCase) activity by protein PII are exemplified in detail in this review.

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.

WRINKLED1 homologs highly and functionally express in oil-rich endosperms of oat and castor.

Oat (Avena sativa) and castor (Ricinus communis) accumulate a large amount of lipids in their endosperms, however the molecular mechanism remains unknown. In this study, differences in oil regulators between oat and wheat (Triticum aestivum) as well as common features between oat and castor were tested by analyzing their transcriptomes with further q-PCR analysis. Results indicated that WRINKLED1 (WRI1) homologs and their target genes highly expressed in the endosperms of oat and castor, but not in the starchy endosperms of wheat. Expression pattern of WRI1s was in agreement with that of oil accumulation. Three AsWRI1s (AsWRI1a, AsWRI1b and AsWRI1c) and one RcWRI1 were identified in the endosperms of oat and castor, respectively. AsWRI1c lacks VYL motif, which is different from the other three WRI1s. Expressions of these four WRI1s all complemented the phenotypes of Arabidopsis wri1-1 mutant. Overexpression of these WRI1s in Arabidopsis and tobacco BY2 cells increased oil contents of seeds and total fatty acids of the cells, respectively. Moreover, this overexpression also resulted in up-regulations of WRI1 target genes, such as PKp-ß1. Taken together, our results suggest that high and functional expression of WRI1 play a key role in the oil-rich endosperms and the VYL motif is dispensable for WRI1 function.

Atypical Splicing Accompanied by Skipping Conserved Micro-exons Produces Unique WRINKLED1, An AP2 Domain Transcription Factor in Rice Plants.

WRINKLED1 (WRI1), an AP2 domain transcription factor, is a master regulator of oil synthesis in plant seeds. Its closely related proteins (WRIs) are also involved in regulating the synthesis of fatty acids, which play a role in producing oils, membranes, and other important components in plants. We found two WRI1 genes, OsWRI1-1 and OsWRI1-2, and two additional WRI1 homologs, OsWRI3 and OsWRI4, in the rice genome. OsWRI1 was ubiquitously expressed in rice plants, including developing seeds. However, OsWRI3 was only significantly expressed in the leaf blade and OsWRI4 was not expressed at all. OsWRI1-1 contains amino acid sequence GCL instead of VYL, which is encoded by an independent 9-bp micro-exon that is conserved in many plant species. We found that the GCL sequence was produced by an atypical splicing accompanied by skipping of the micro-exon. Furthermore, OsWRI1-1 highly activates the transcription of the promoter for the biotin carboxyl transferase 2 gene in Arabidopsis, but its activity was reduced by amino acid replacement or deletion of the GCL sequence in a transient assay using Arabidopsis cells. Our results indicated that atypical splicing produced unique WRI1 in rice plants.

Genome-wide analysis reveals the evolution and structural features of WRINKLED1 in plants.

WRINKLED1 (WRI1), an AP2/ERE transcription factor, is one of the most important regulators of oil accumulation. It has been extensively studied in angiosperms, but its evolution and overview features in plants remain unknown. In this study, WRI1s, as well as WRI1-likes in non-WRI1 species, were investigated in 64 genome-sequenced plants. Their origin, distribution, duplication, evolution, functional domains, motifs, properties, and cis-elements were analyzed. Results suggest that WRI1 and WRI1-like may originate from Chlorophyta, and WRI1-likes in angiosperms resemble phylogenetically and structurally WRI1s from Chlorophyta and non-vascular plants. WRI1 or WRI1-like may be essential to vascular plants but not to non-vascular plants. Two YRG elements and two RAYD elements, as well as their phosphorylation sites and the 14-3-3 binding motif, are relatively conserved from Chlorophyta to angiosperm. The predicted DNA-binding domains are slightly shorter than the combination of one YRG element and one RAYD element. WRI1 gradually evolves from alkalinity to acidity. More motifs were developed in N-terminuses and C-terminuses in vascular plants. A short acidic amino-acid-enriched domain in the C-terminal region is predicted to be the putative transactivation domain. The VYL exon appears randomly in different WRI1 transcripts and it is not important for the function of WRI1. In addition, more cis-elements developed during WRI1 evolution may suggest its more complicated regulation and physiological functions. These results will assist future function studies of WRI1 and evolution studies of fatty acid biosynthesis regulation in plants.

Design of a Seed-Specific Chimeric Promoter with a Modified Expression Profile to Improve Seed Oil Content.

Increasing the yield of plant oil is an important objective to meet the demand for sustainable resources and energy. Some attempts to enhance the expression of genes involved in oil synthesis in seeds have succeeded in increasing oil content. In many cases, the promoters of seed-storage protein genes have been used as seed-specific promoters. However, conventional promoters are developmentally regulated and their expression periods are limited. We constructed a chimeric promoter that starts to express in the early stage of seed development, and high-level expression is retained until the later stage by connecting the promoters of the biotin carboxyl carrier protein 2 (BCCP2) gene encoding the BCCP2 subunit of acetyl-CoA carboxylase and the fatty acid elongase 1 (FAE1) gene from Arabidopsis. The constructed promoter was ligated upstream of the TAG1 gene encoding diacylglycerol acyltransferase 1 and introduced into Arabidopsis. Seeds from transgenic plants carrying AtTAG1 under the control of the chimeric promoter showed increased oil content (up by 18⁻73%) compared with wild-type seeds. The novel expression profile of the chimeric promoter showed that this could be a promising strategy to manipulate the content of seed-storage oils and other compounds.

Ectopic expression of EuWRI1, encoding a transcription factor in E. ulmoides, changes the seeds oil content in transgenic tobacco.

The WRINKLED1 (WRI1) gene is a well-established key transcriptional regulator involved in the regulation of fatty acid biosynthesis in developing seeds. In this study, a new WRI1 gene was isolated from seeds of Eucommia ulmoides and named EuWRI1. A close link between gibberellins signaling and EuWRI1 gene expression was suggested in this study. Functional characterization of EuWRI1 was elucidated through seed-specific expression in tobacco. In transgenic tobacco, the expression of EuWRI1 in eight independent transgenic lines was detected by semiquantitative RT-PCR. The relative mRNA accumulation of genes encoding enzymes involved in fatty acid biosynthesis (biotin carboxyl carrier protein and keto-ACP synthase 1) was also assayed in tobacco seeds. Analysis of the seeds oil content and starch content indicated that the transgenic lines showed a significant increase in seeds oil content, whereas starch content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (16:0), linoleic acid (18:2) and linolenic acid (18:3) increased significantly in seeds of transgenic tobacco lines, but stearic acid (18:0) levels significantly declined. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:337-346, 2018.

WRINKLED1 and ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE1 regulate tocochromanol metabolism in Arabidopsis.

Photosynthetic organisms such as plants, algae and some cyanobacteria synthesize tocochromanols, a group of compounds that encompasses tocopherols and tocotrienols and that exhibits vitamin E activity in animals. While most vitamin E biosynthetic genes have been identified in plant genomes, regulatory genes controlling tocopherol accumulation are currently unknown. We isolated by forward genetics Arabidopsis enhanced vitamin E (eve) mutants that overaccumulate the classic tocopherols and plastochromanol-8, and a tocochromanol unknown in this species. We mapped eve1 and eve4, and identified the unknown Arabidopsis tocochromanol by using a combination of analytical tools. In addition, we determined its biosynthetic pathway with a series of tocochromanol biosynthetic mutants and transgenic lines. eve1 and eve4 are two seed lipid mutants affecting the WRINKLED1 (WRI1) and ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) genes, respectively. The unknown tocochromanol is 11'-12' γ-tocomonoenol, whose biosynthesis is VITAMIN E 1 (VTE1) - and VTE2-dependent and is initiated by the condensation of homogentisate (HGA) and tetrahydrogeranylgeranyl pyrophosphate. This study identifies the first two regulatory genes, WRI1 and DGAT1, that control the synthesis of all tocochromanol forms in seeds, and shows the existence of a metabolic trade-off between lipid and tocochromanol metabolisms. Moreover, it shows that Arabidopsis possesses a tocomonoenol biosynthetic pathway that competes with tocopherol synthesis.