Transcriptome Analysis Reveals Key Genes Involved in Fatty Acid and Triacylglycerol Accumulation in Developing Sunflower Seeds

Abstract

Background/objectives: Sunflower (Helianthus annuus L.) is one of the four major global oilseed crops. Understanding the molecular mechanisms regulating fatty acid synthesis and triacylglycerol (TAG) accumulation is crucial for improving oil yield and quality. In this study, the oilseed sunflower cultivar 'T302', which was wild-cultivated in the northwestern region of China, was analyzed for fatty acid content by targeted lipidomic analysis. RNA sequencing (RNA-seq) was performed on 15 cDNA libraries from sunflower embryos at five developmental stages (10, 17, 24, 31, and 38 days after flowering) to investigate gene expression patterns during oil accumulation. Differentially expressed genes (DEGs) related to fatty acid and triacylglycerol accumulation in developing sunflower seeds were identified. WGCNA was used to gain deeper insights into the mechanisms underlying lipid metabolism.

Results: The oil composition of 'T302' consisted of 86.61% unsaturated fatty acids (UFA), mainly linoleic acid (48.47%) and oleic acid (37.25%). Saturated fatty acids (SFAs) accounted for 13.39%, with palmitic acid (7.46%) and stearic acid (5.04%) being the most abundant. A total of 81,676 unigenes were generated from RNA-seq data, and 91 DEGs associated with lipid metabolism were identified, including key enzymes such as FAD2-1, SAD, FATA, LACS, PDAT2, and DGAT2. In addition, we identified several novel candidate transcription factor genes, including WRI1, LEC1, FUS3, and ABI3, which were found to regulate TAG synthesis during seed maturation and are worthy of further investigation. This study provides valuable insights into the molecular mechanisms of seed oil biosynthesis in oilseed sunflower. The identified key genes and transcription factors provide potential targets for molecular breeding strategies to increase oil content and modify fatty acid compositions in sunflower and other oilseed crops.

Keywords: developing seeds; lipid biosynthesis; sunflower (Helianthus annuus L.); transcriptome; unsaturated fatty acids.

Figure 1

KEGG pathway enrichment analysis of the unigenes. The X-axis represents the names of the KEGG metabolic pathways, while the Y-axis denotes the count of genes or transcripts annotated to each respective pathway.

Figure 2

PCA analysis of sunflower transcriptome samples and cluster analysis of DEGs. (A) Principal component analysis (PCA) of all 15 samples was performed based on RNA-seq FPKM. (B) The number of genes in sunflower seeds at different developmental stages: 10, 17, 24, 31, and 38 days after flowering (DAF). (C) Pairwise comparisons of the number of DEGs between samples at each time point during sunflower seed development were performed. A p-value < 0.05 and a fold change ≥ 1 were used as criteria for DEG identification.

Figure 3

WGCNA analysis of RNA-seq and physiological trait data. (A) WGCNA was calculated from 15 samples, and the 23,396 DEGs were partitioned into six modules. Columns represent the module eigengenes of the means. For each module, the number of DEGs and the number of KEGG pathways were listed. (B) The expression profiles of the modules were related to the date of sampling (DAF) and the physiological traits in Figure 1. The data in the boxes represent the number of differentially expressed genes.

Figure 4

Fatty acid and TAG biosynthetic pathways in sunflower seeds. The expression levels of potential candidate genes (represented by Log10 FPKM) in sunflower seeds at various developmental stages (10, 17, 24, 31, and 38 DAF) are highlighted through color coding, ranging from blue (low expression) to red (high expression). An asterisk (*) denotes the differentially expressed genes (DEGs) associated with FA synthesis, as identified through WGCNA analysis. Key genes involved in lipid metabolism include ACCase (acetyl-CoA carboxylase), BC (biotin carboxylase), BCCP (biotin carboxyl carrier protein), α-CT (carboxyl transferase subunit α), MACT (malonyl-CoA-acyl carrier protein transacylase), ACP (acid phosphatase), KAS III (3-oxoacyl-ACP synthase III), KAR (3-oxoacyl-ACP reductase), HAD (3-hydroxyacyl-ACP dehydratase), ENR (Enoyl-CoA reductase), KAS II (3-oxoacyl-ACP synthase II), FATB (fatty acyl-ACP thioesterase B), FATA (fatty acyl-ACP thioesterase A), SAD (stearoyl-ACP desaturase), FAD (fatty acid desaturase), LACS (long-chain acyl-CoA synthetase), LPCAT (lysophosphatidylcholine acyltransferase), PLA2 (phospholipase A2), GPAT (glycerol-3-phosphate acyltransferase), LPAAT (1-acyl-sn-glycerol-3-phosphate acyltransferase), PLD (phospholipase D), PAP (phosphatide phosphatase), CPT (CDP-choline: diacylglycerol cholinephosphotransferase), PDCT (phosphatidylcholine: diacylglycerol cholinephosphotransferase), PLC (phospholipase C), DGAT (diacylglycerol acyltransferase), PDAT (phospholipid: diacylglycerol acyltransferase), OLE (oleosin), CLO (caleosin), WRI1 (wrinkled 1), and FUS3 (FUSCA3).

Figure 5

DEGs related to FA and TAG biosynthetic pathways identified in developing sunflower seeds. The Y-axis represents reads per kilobase per million mapped reads (FPKM) values and the X-axis represents five developmental stages of sunflower seeds: 10, 17, 24, 31, and 38 days after anthesis (DAF). The error bar line represents the standard deviation (SD) calculated from three biological replicates. (A) Acetyl-CoA carboxylase subunits (BC, BCCP, α-CT); (B) β-Ketoacyl-ACP synthase isoforms (KASI, KASII, KASIII); (C) Acyl-ACP thioesterases (FATA, FATB); (D) Long-chain acyl-CoA synthetases (LACS4, LACS7, LACS9); (E) Triacylglycerol assembly enzymes (GPAT, DGAT2, DGAT3, PDAT); (F) Oil body-associated proteins (OLE, CLO); (G) Fatty acid desaturases (SAD, FAD2, FAD3); (H) Seed development regulators (LEC1, LEC2, ABI3, FUS3, WRI1).

Figure 6

Fatty acid desaturase genes identified in developing sunflower seeds. (A) Heat map showing the expression levels of desaturase genes. (B) Phylogenetic analysis of desaturase genes in sunflower and Arabidopsis, performed using ClustalX2 for nucleotide sequence alignment and MEGA 7.0 Neighbor Joining (NJ) method for tree construction. (C) Pathway map illustrating the desaturase gene network. The asterisk (*) represents the major DEGs associated with FA synthesis.

Figure 7

Transcription factors (TFs) involved in lipid synthesis in sunflower. (A) TFs identified in developing sunflower seeds. A total of 44 families were predicted, and the red columns indicate TFs that may be involved in FA and TAG biosynthesis. (B) Regulatory model of seed oil accumulation by TFs. (C) Heat map of differential expression of TF genes with FA and TAG biosynthesis. The asterisk (*) represents the major DEGs associated with TAG synthesis.

Figure 8

Expression validation of genes between RT-qPCR and RNA-seq at three stages of seed development in sunflower. Red line and blue volume represent RNA-seq and data, respectively. Sunflower 18S rRNA gene (AF1057577) was used as an internal control. Means with different letters indicate a significant difference (p < 0.05); error bars lines indicate SD (n = 3).