The demand for vegetable oils for food, fuel (bio-diesel) and bio-product applications is increasing rapidly [20]. As one of the main sources of edible oil, oil crops are vital to human health. Oil crops are the major source of edible oil [21]. Plant oils have great significance in agricultural production, and most are mainly used as edible oil in food processing and preparation [22].Although the synthesis and composition of lipids are similar in some oil crops, there are significant differences in their oil content and fatty acid composition.
Most plants contain accumulated oil (mainly TAG) in their seeds to provide energy for seed germination. Our results found general agreement with previous studies [6, 23] that the FAs in the seeds of Hibiseu manihot are dominated by UFAs, and the FA components remained basically unchanged during the development of Hibiseu manihot seeds, including MA, PA, SA, OA and LA, but the contents of each component changed. As the two main unsaturated fatty acids, OA and LA, account for more than 63% of the total FAs. OA began to accumulate from the early stage of development and accounted for the largest proportion in the middle stage of development. LA accounts for the largest proportion in the early stage of development, and is next to OA and PA in the late stage of development. Interestingly, the content of UFAs in different developmental stages is different, which may be related to the activities of enzymes involved in fatty acid biosynthesis and metabolism. The OA content in Hibiseu manihot seeds was the lowest at 5 DAF, and then gradually increased. Oil and lipids in plants are collectively referred to as lipids, and TAG is the main form of plant seed lipids the main form of plant seed lipids [24], and most plant seeds are dominated by 18C UFAs, with oil seed rape (Brassica napu L) [25], C.oleifera [19] (Lin et al., 2018), siberian apricot (Prunus sibirica L.) [26], and peanut dominated by C18:1, while soybean [7], cotton [27], walnut (Juglans regia L.) [28], and Artemisia sphaerocephala [17] are dominated by C18: 2 dominated. Their regulation differs by species, for example, among species with OA as the main FA, the main regulatory mechanism in siberian apricot relies on the up-regulation of genes encoding KASII and SAD6 [26], while both Carya cathayensis [29] and C.oleifera [19] have increased OA accumulation due to high levels of SAD in concert with low level of FAD2 expression. In the species with C18:2 as the main fatty acid, high expression of FAD2 and very low expression of FAD3 are the main reasons for the formation of LA in cotton seeds [27], while in walnut FAD3 expression levels are lower than FAD2.In this study, the FA components of seeds from different developmental stages of Hibiseu manihot seeds were analyzed. A total of six FAs were detected, which differed greatly in content, with chain lengths of 16 and 18 carbons, respectively. This result was also present in woody oil crops such as Jatropha curcas [30] and yellow horn (Xanthoceras sorbifolium) [31].However, the genetic changes during seed development of Malvaceae and their molecular mechanisms with FA synthesis have not been investigated.
TAG, the main storage form of lipid, is acylated by DAG and then stored in oil body. The changes of lipid involved in TAG synthesis during the development of siberian apricot kernel showed that PDAT may play an important role in TAG synthesis [26]. DGAT and PDAT are key enzymes for TAG synthesis in the acyl-CoA-dependent and acyl-CoA-independent pathways, respectively. In walnut, cotton, and Siberian apricot, PDAT can direct unusual FA into the TAG synthesis machinery compared to DGAT and was used to enhance the accumulation of oxygen-containing FA in transgenic seeds, whose high expression correlates with seed oil content and plays a more important role in TAG biosynthesis[26–28]. On the contrary, in oil seed rape, DGAT is more important for TAG synthesis than PDAT [25]. In the seeds of Hibiseu manihot, two major DGAT were up-regulated at 5v30 and one major PDAT was up-regulated at 5v15, which indicated that these two pathways cooperated to regulate the biosynthesis of TAG in the seeds of Hibiseu manihot. This is consistent with the research results of Carya cathayensis and perilla (Perilla frutescens L.) [29, 32]. In addition, DEGs encoding PDCT was found, which indicated that this pathway also affected the synthesis of TAG during the development of Hibiseu manihot seeds, in agreement with the results of perilla studies[32]. The number and cross-sectional area of oil bodies are positively correlated with the oil content of seeds [33]. Meanwhile, this study found that the oleosins in the late development stage of Hibiseu manihot seeds has a high expression level, which is consistent with the increase of oil content in seeds.
WRI1 is a key transcriptional regulator of fatty acid biosynthesis genes in a variety of oil-bearing tissues and is a member of the APETALA2 (AP2)/EREBP (ethylene responsive element binding protein) family of transcription factors [34]. In wri1, seed oil accumulation is reduced, while gene expression of FA biosynthetic enzymes is reduced. WRI1 is a key candidate for enhancing vegetable oil production is highly expressed during mid and late development of goldenrod, consistent with the process of oil content accumulation. We suggest that WRI1 may be involved and play an important role in the process of oil accumulation in goldenrod. The LEC2 gene plays an important regulatory role in the early and late stages of embryonic development and is involved in storage product accumulation, was ectopically expressed during senescence in the fatty acid breakdown mutant COMATOSE (cts2). This resulted in accumulation of seed oil type species of TAG in senescing tissue[35]. The AtLEC2 gene induced TAGs accumulation and changed the FA composition in vegetative tissues of Arabidopsis by up-regulating LEC1, ABI3, FUS3, and WRI1 gene expression [36]. The expression levels of LEC2 in Hibiseu manihot seeds were low in the first and middle stages and significantly increased in the later stages of development, corresponding to the expression levels of ABI3 and WRI1, and LEC2 positively regulated WRI1. The identification and study of these transcription factors will deepen our understanding of the regulatory mechanisms of oil biosynthesis in goldenrod.
The transcriptome has been used to understand the biosynthesis genes in oil seed, gene regulatory networks, regulatory factors and interactions [37], etc. By sequencing transcriptome, genes involved in seed development and their relationship with oil content can be screened and identified at molecular level. In order to understand the changes of FA composition during the development of Hibiseu manihot, this study conducted transcriptome sequencing and bioinformatics analysis based on Illumina Hiseq high-throughput sequencing technology and Hibiseu manihot seeds at different developmental stages. A total of 92,848 unigeness were obtained, 28,811 unigeness were classified into 52 subclasses by GO database and 17,285 unigeness could be assigned to 6 categories and 128 metabolic pathways by KEGG database. Meanwhile, the screening and expression pattern analysis of key genes involved in fatty acid biosynthesis and lipid accumulation pathways will help to lay the foundation for further research on the metabolic pathways and genetic mechanisms of of Hibiseu manihot. Here we screen the relevant DEGs for the enzymes involved in the synthetic pathway starting from the synthesis of malonyl-CoA by acetyl-CoA until the production of C16 or C18 FAs, which include ACCase, MCMT, KAS, KAR, HAD, FATA, FATB, FAD2 and other related enzymes involved. Genetic engineering to over-express/repress specific genes encoding enzymes and other proteins involved in the flow of carbon into seed oil has led to the development of transgenic lines with significant increases in seed oil content [20].Fatty acid desaturase is widely found in organ tissues and the main function of FAD is to remove hydrogen from carbon chains in the biosynthesis of UFAS, and producing C = C bonds. In plants, FADs can be divided into two categories based on their solubility: soluble desaturases and membrane integrins [38]. The higher plant-specific stearoyl ACP desaturase (SAD) is the only known FAD in the plastid matrix, the most studied FAD in plants, and the only soluble enzyme subfamily in the FAD family; the other types of fatty acid desaturase are membrane integrin [39], which catalyze the desaturation of stearic acid to produce OA-ACP, which in turn can produce free oleic acid through FATA to generate free OA, while FATB can remove from PA-ACP and SA-ACP to SFAs. FATA and FATB have different substrate specificity can hydrolyze the acyl groups of acyl ACP and release free FAs. The specificity of lipids largely determines the length and degree of unsaturation of plant fat chains in glycerolipid and TAG [40]. We showed that the expression of FATA was up-regulated in the whole seed development period, while FATB was up-regulated in the late seed development period. The expression of KASII was proportional to the 18C FA content, and KASI was only up-regulated in the late development stage, which promoted C18:0 to be used as substrate to catalyze the formation of free OA through SAD and FATA. Up-regulated expression of FATA synergizes with high expression of KASII and SAD to generate high level of oleic acid, which was further used for the synthesis of 18C UFAs. Another key enzyme, FAD2 plays a role in the desaturation process and is functionally responsible for the conversion of oleic acid to linoleic acid in oil crops plants [41]. Linoleic acid, the second most abundant unsaturated fatty acid after oleic acid in Hibiseu manihot seeds, varies in content at different stages of seed development. The high expression of FAD2 synergized with the low expression of FAD3 to promote the accumulation of LA, which is consistent with the continuous accumulation of LA content, while the low expression of FAD2 and FAD3 synergized with the high expression of SAD to promote the accumulation of OA in the late stage of seed development. Our results are in good agreement with the discovery in hickory, which suggests that perhaps a similar regulatory mechanism is involved [29]. PDAT can catalyze phospholipids to produce TAG, and PDAT is up-regulated in the pre- and mid-developmental stages. The expression of KASII, SAD, FATA, PDAT and FAD2 may be one of the reasons for the high accumulation of OA and LA.