Fatty Acid Biosynthesis In Seeds Research Articles

DELLA proteins BnaA6.RGA and BnaC7.RGA negatively regulate fatty acid biosynthesis by interacting with BnaLEC1s in Brassica napus.

SummarySeed oil content (SOC) and fatty acid (FA) composition determine the quality and economic value of rapeseed (Brassica napus). Little is known about the role of gibberellic acid (GA) in regulating FA biosynthesis in B. napus. Here, we discovered that four BnaRGAs (B. napus REPRESSOR OF GA), encoding negative regulators of GA signalling, were suppressed during seed development. Compared to the wild type, SOC was reduced in gain‐of‐function mutants bnaa6.rga‐D and ds‐3, which also showed reduced oleic acid and increased linoleic acid contents. By contrast, the loss‐of‐function quadruple mutant bnarga displayed higher SOC during early seed development than the wild type, with increased oleic acid and reduced linoleic acid contents. Notably, only BnaA6.RGA and BnaC7.RGA physically interacted with two BnaLEC1s, which function as essential transcription factors in FA biosynthesis. The FA composition did not significantly differ between bnarga bnalec1 sextuple mutants and bnalec1, suggesting that BnaLEC1s are epistatic to BnaRGAs in the regulation of FA composition. Furthermore, BnaLEC1‐induced activation of BnaABI3 expression was repressed by BnaA6.RGA, indicating that GA triggers the degradation of BnaRGAs to relieve their repression of BnaLEC1s, thus promoting the transcription of downstream genes to facilitate oil biosynthesis. Therefore, we uncovered a developmental stage‐specific role of GA in regulating oil biosynthesis via the GA‐BnaRGA‐BnaLEC1 signalling cascade, providing a novel mechanistic understanding of how phytohormones regulate FA biosynthesis in seeds. BnaRGAs represent promising targets for oil crop improvement.

English

WRINKLED1 belongs to AP2/EREB family of transcription factors whose role has been well established in seed oil biosynthesis. The objective of the study was to trace the role of fiber related Gbwri1 in seed development and fatty acid biosynthesis. In this study, we isolated a transcript from elite fiber producing cotton (Gossypium barbadense), which is over-expressed in G. barbadense fibers as compared to G. hirsutum and G. arboreum. The putative protein encoded by this transcript exhibited homology in specific domains and protein structure with WRINKLED1 of Arabidopsis thaliana and was thus designated as Gbwri1. In this study, we investigated the functional homology of fiber elongation related Gbwri1 with fatty acid biosynthesis regulator Atwri1. Ectopic expression of Gbwri1 in wri1-3 mutant of A. thaliana was analyzed. In the transgenic lines of A. thaliana, Gbwri1 resumed the seed weight, seed area, and surface morphology to the wild type. Gbwri1 transformation rescued the wrinkled phenotype of wri1-3 mutants by resuming the expression of fatty acid biosynthesis genes biotin carboxyl carrier protein isoform 2 (bccp2) and keto-ACP synthase 1 (kas1). Moreover, the seedling development of transgenic lines on non-sucrose medium demonstrated that the Gbwri1 was able to regulate the supply of sucrose for normal seedling establishment. Our results showed that the transformation of Gbwri1 in A. thaliana wri1-3 mutant was able to complement wri1-3 impaired phenotype. Thus, Gbwri1 is involved in cotton fiber development and fatty acid biosynthesis in seeds. © 2021 Friends Science Publishers

Regulation of FATTY ACID ELONGATION1 expression and production in Brassica oleracea and Capsella rubella.

The contribution of variations in coding regions or promoters to the changes in FAE1 expression levels have be quantified and compared in parallel by specifically designed swapping constructs. FATTY ACID ELONGATION1 (FAE1) is a key gene in control of erucic acid synthesis in plant seeds. The expression of FAE1 genes in Brassica oleracea and Capsella rubella, representatives of high and low erucic acid species, respectively, was characterized to provide insight into the regulation of very long-chain fatty-acid biosynthesis in seeds. Virtually, no methylation was detected either in B. oleracea or in C. rubella, suggesting that modification of promoter methylation might not be a predominant mechanism. Swapping constructs were specifically designed to quantify and compare the contribution of variations in coding regions or promoters to the changes in FAE1 expression levels in parallel. A significantly higher fold change in erucic acid content was observed when swapping coding regions rather than when swapping promoters, indicating that the coding region is a major determinant of the catalytic power of β-ketoacyl-CoA synthase proteins. Common motifs have been proposed as essential for the preservation of basic gene expression patterns, such as seed-specific expression. However, the occurrence of variation in common cis-elements or the presence of species-specific cis-elements might be plausible mechanisms for changes in the expression levels in different organisms. In addition, conflicting observations in previous reports associated with FAE1 expression are discussed, and we suggest that caution should be taken when selecting a plant transformation vector and in interpreting the results obtained from vectors carrying the CaMV 35S promoter.

Overexpression of the triose phosphate translocator (TPT) complements the abnormal metabolism and development of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase mutants.

The presence of two glycolytic pathways working in parallel in plastids and cytosol has complicated the understanding of this essential process in plant cells, especially the integration of the plastidial pathway into the metabolism of heterotrophic and autotrophic organs. It is assumed that this integration is achieved by transport systems, which exchange glycolytic intermediates across plastidial membranes. However, it is unknown whether plastidial and cytosolic pools of 3-phosphoglycerate (3-PGA) can equilibrate in non-photosynthetic tissues. To resolve this question, we employed Arabidopsis mutants of the plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) that express the triose phosphate translocator (TPT) under the control of the 35S (35S:TPT) or the native GAPCp1 (GAPCp1:TPT) promoters. TPT expression under the control of both promoters complemented the vegetative developmental defects and metabolic disorders of the GAPCp double mutants (gapcp1gapcp2). However, as the 35S is poorly expressed in the tapetum, full vegetative and reproductive complementation of gapcp1gapcp2 was achieved only by transforming this mutant with the GAPCp1:TPT construct. Our results indicate that the main function of GAPCp is to supply 3-PGA for anabolic pathways in plastids of heterotrophic cells and suggest that the plastidial glycolysis may contribute to fatty acid biosynthesis in seeds. They also suggest a 3-PGA deficiency in the plastids of gapcp1gapcp2, and that 3-PGA pools between cytosol and plastid do not equilibrate in heterotrophic cells.

MYB89 Transcription Factor Represses Seed Oil Accumulation.

In many higher plants, seed oil accumulation is precisely controlled by intricate multilevel regulatory networks, among which transcriptional regulation mainly influences oil biosynthesis. In Arabidopsis (Arabidopsis thaliana), the master positive transcription factors, WRINKLED1 (WRI1) and LEAFY COTYLEDON1-LIKE (L1L), are important for seed oil accumulation. We found that an R2R3-MYB transcription factor, MYB89, was expressed predominantly in developing seeds during maturation. Oil and major fatty acid biosynthesis in seeds was significantly promoted by myb89-1 mutation and MYB89 knockdown; thus, MYB89 was an important repressor during seed oil accumulation. RNA sequencing revealed remarkable up-regulation of numerous genes involved in seed oil accumulation in myb89 seeds at 12 d after pollination. Posttranslational activation of a MYB89-glucocorticoid receptor fusion protein and chromatin immunoprecipitation assays demonstrated that MYB89 inhibited seed oil accumulation by directly repressing WRI1 and five key genes and by indirectly suppressing L1L and 11 key genes involved in oil biosynthesis during seed maturation. These results help us to understand the novel function of MYB89 and provide new insights into the regulatory network of transcriptional factors controlling seed oil accumulation in Arabidopsis.

Overexpression of phosphoenolpyruvate carboxylase from Jatropha curcas increases fatty acid accumulation in Nicotiana tabacum

Jatropha curcas L. is an excellent biofuel crop, which displays a high efficiency of carbon absorption, and seed oil of Jatropha can be efficiently processed to produce high-quality biodiesel. Plant phosphoenolpyruvate carboxylases (PEPCs) play important roles not only in initial fixation of atmospheric CO2 in C4 and Crassulacean acid metabolism (CAM) plants, but also in fatty acid biosynthesis in seeds of oil plants by regulating carbon partitioning. Here, we identified JcPEPC1 from J. curcas L. by homology cloning, and alignment analysis of protein sequence revealed JcPEPC1 was a plant C3-type PEPC, and shared high similarity to PEPC of castor oil plant Ricinus communis. We implemented detailed functional characterization of JcPEPC1 by expression analysis and transgenic tobacco. JcPEPC1 gene expressed in the leaves and seeds of J. curcas L., and remarkable increase of expression level was also detected at seed oil-accumulating stages. We overexpressed JcPEPC1 in tobacco, and showed the enzymatic activity of PEPC in transgenic plants was notably higher than wild type. Gas chromatography (GC) analysis elucidated the composition and total content of fatty acids were also altered. This study indicated JcPEPC1 played a fundamental role in fatty acid biosynthesis in Jatropha seeds. Our results proposed enhanced PEPC activity of Jatropha could improve biosynthesis of fatty acid, which implied critical functions in primary metabolism of non-photosynthetic PEPC.

WRINKLED Transcription Factors Orchestrate Tissue-Specific Regulation of Fatty Acid Biosynthesis in Arabidopsis

Acyl lipids are essential constituents of all cells, but acyl chain requirements vary greatly and depend on the cell type considered. This implies a tight regulation of fatty acid production so that supply fits demand. Isolation of the Arabidopsis thaliana WRINKLED1 (WRI1) transcription factor established the importance of transcriptional regulation for modulating the rate of acyl chain production. Here, we report the isolation of two additional regulators of the fatty acid biosynthetic pathway, WRI3 and WRI4, which are closely related to WRI1 and belong to the APETALA2-ethylene-responsive element binding protein family of transcription factors. These three WRIs define a family of regulators capable of triggering sustained rates of acyl chain synthesis. However, expression patterns of the three WRIs differ markedly. Whereas only WRI1 activates fatty acid biosynthesis in seeds for triacylglycerol production, the three WRIs are required in floral tissues to provide acyl chains for cutin biosynthesis and prevent adherence of these developing organs and subsequent semisterility. The targets of these WRIs encode enzymes providing precursors (acyl chain and glycerol backbones) for various lipid biosynthetic pathways, but not the subsequent lipid-assembling enzymes. These results provide insights into the developmental regulation of fatty acid production in plants.

Enhancing the carotenoid content of Brassica napus seeds by downregulating lycopene epsilon cyclase

The accumulation of carotenoids in higher plants is regulated by the environment, tissue type and developmental stage. In Brassica napus leaves, beta-carotene and lutein were the main carotenoids present while petals primarily accumulated lutein and violaxanthin. Carotenoid accumulation in seeds was developmentally regulated with the highest levels detected at 35-40 days post anthesis. The carotenoid biosynthesis pathway branches after the formation of lycopene. One branch forms carotenoids with two beta rings such as beta-carotene, zeaxanthin and violaxanthin, while the other introduces both beta- and epsilon-rings in lycopene to form alpha-carotene and lutein. By reducing the expression of lycopene epsilon-cyclase (epsilon-CYC) using RNAi, we investigated altering carotenoid accumulation in seeds of B. napus. Transgenic seeds expressing this construct had increased levels of beta-carotene, zeaxanthin, violaxanthin and, unexpectedly, lutein. The higher total carotenoid content resulting from reduction of epsilon-CYC expression in seeds suggests that this gene is a rate-limiting step in the carotenoid biosynthesis pathway. epsilon-CYC activity and carotenoid production may also be related to fatty acid biosynthesis in seeds as transgenic seeds showed an overall decrease in total fatty acid content and minor changes in the proportions of various fatty acids.

Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase.

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. Previously, the cloning of a PDHK cDNA from Arabidopsis thaliana and the effects of constitutively down-regulating its expression on plant growth and development has been reported. The first detailed analyses of the biochemical and physiological effects of partial silencing of the mtPDHK in A. thaliana using antisense constructs driven by both constitutive and seed-specific promoters are reported here. The studies revealed an increased level of respiration in leaves of the constitutive antisense PDHK transgenics; an increase in respiration was also found in developing seeds of the seed-specific antisense transgenics. Both constitutive and seed-specific partial silencing of the mtPDHK resulted in increased seed oil content and seed weight at maturity. Feeding 3-(14)C pyruvate to bolted stems containing siliques (constitutive transgenics), or to isolated siliques or immature seeds (seed-specific transgenics) confirmed a higher rate of incorporation of radiolabel into all seed lipid species, particularly triacylglycerols. Neither constitutive nor seed-specific partial silencing of PDHK negatively affected overall silique and seed development. Instead, oil and seed yield, and overall plant productivity were improved. These findings suggest that a partial reduction of the repression of the mtPDC by antisense PDHK expression can alter carbon flux and, in particular, the contribution of carbon moieties from pyruvate to fatty acid biosynthesis and storage lipid accumulation in developing seeds, implicating a role for mtPDC in fatty acid biosynthesis in seeds.

Broad-Range and Binary-Range Acyl-Acyl-Carrier-Protein Thioesterases Suggest an Alternative Mechanism for Medium-Chain Production in Seeds

In the current model of medium-chain (C8-14) fatty acid biosynthesis in seeds, specialized FatB acyl-acyl-carrier-protein (ACP) thioesterases are responsible for the production of medium chains. We have isolated and characterized FatB cDNAs from the maturing seeds of elm (Ulmus americana) and nutmeg (Myristica fragrans), which accumulate predominantly caprate (10:0)- and myristate (14:0)-containing oils, respectively. In neither species were we able to find cDNAs encoding enzymes specialized for these chain lengths. Nutmeg FatB hydrolyses C14-18 substrates in vitro and expression in Brassica napus seeds leads to an oil enriched in C14-18 saturates. Elm FatB1 displays a binary specificity: one activity is centered on 10:0-ACP, and a second is centered on palmitate (16:0)-ACP. After expression in B. napus seeds the oil is enriched in C10-18 saturates, predominantly 16:0, 14:0, and 10:0. The composition of free fatty acids produced by elm FatB1 in Escherichia coli shifts from C14-16 to mostly C8-10 by increasing the rate of chain termination by this enzyme. These results suggest the existence of an alternative mechanism used in the evolution of medium-chain production, a model of which is presented.

Effect of low temperature on fatty acid biosynthesis in seeds

1. 1. By studying the effect of the incubation temperature on the formation of unsaturated fatty acids from [2- 14C]acetate in flax, sunflower and castor seeds, an increased formation of unsaturated fatty acids could be demonstrated as the temperature decreased. 2. 2. The magnitude of this effect increased in the order castor > sunflower > flax seeds. 3. 3. When the O 2 concentration was elevated at fixed temperatures, the formation of unsaturated fatty acids in castor and sunflower seeds increased with temperatures between 10° and 30°. This effect did not occur with flax. 4. 4. The failure of flax seeds to respond to changes in the concentration of O 2 was shown to be due to the presence of chloroplasts in the seed tissue. 5. 5. It is concluded that one of the major effects controlling an increase in the level of unsaturated fatty acids in seeds at low temperatures is the increase in available O 2 which is the rate-limiting factor for desaturation.