Full-length transcriptome revealed the accumulation of polyunsaturated fatty acids in developing seeds of Plukenetia volubilis

Abstract

Background: Plukenetia volubilis is cultivated as a valuable oilseed crop, and its mature seeds are rich in polyunsaturated fatty acids (FAs), which are widely used in food and pharmaceutical industries. Recently, next-generation sequencing (NGS) transcriptome studies in P. volubilis indicated that some candidate genes were involved in oil biosynthesis. The NGS were inaccuracies in assembly of some candidate genes, leading to unknown errors in date analyses. However, single molecular real-time (SMRT) sequencing can overcome these assembled errors. Unfortunately, this technique has not been reported in P. volubilis.

Methods: The total oil content of P. volubilis seed (PVS) was determined using Soxhlet extraction system. The FA composition were analyzed by gas chromatography. Combining PacBio SMRT and Illumina technologies, the transcriptome analysis of developing PVS was performed. Functional annotation and differential expression were performed by BLAST software (version 2.2.26) and RSEM software (version 1.2.31), respectively. The lncRNA-targeted transcripts were predicted in developing PVS using LncTar tool.

Results: By Soxhlet extraction system, the oil content of superior plant-type (SPT) was 13.47% higher than that of inferior plant-type (IPT) at mature PVS. The most abundant FAs were C18:2 and C18:3, among which C18:3 content of SPT was 1.11-fold higher than that of IPT. Combined with PacBio and Illumina platform, 68,971 non-redundant genes were obtained, among which 7,823 long non-coding RNAs (lncRNAs) and 7,798 lncRNA-targeted genes were predicted. In developing seed, the expressions of 57 TFs showed a significantly positive correlation with oil contents, including WRI1-like1, LEC1-like1, and MYB44-like. Comparative analysis of expression profiles between SPT and IPT implied that orthologs of FAD3, PDCT, PDAT, and DAGT2 were possibly important for the accumulation of polyunsaturated FAs. Together, these results provide a reference for oil biosynthesis of P. volubilis and genetic improvement of oil plants.

Keywords: Full-length transcriptome; Oil accumulation; Plukenetia volubilis; Polyunsaturated fatty acids; Transcription factors.

Figure 1. Developmental changes in size, weight, and oil content of PVS.

(A) Intact SPT and IPT seeds from 10, 70, and 110 DAP. (B) Changes in fresh weight of the developing PVS. (C) Changes in size of the developing PVS. (D) Comparative analysis of oil content between SPT and IPT seeds. Values are mean ± standard deviation. Asterisks (**) indicate statistical significance oil contents (30DAF, P = 0.0137; 70 DAF, P = 0.0006; 110 DAF, P = 0.0008).

Figure 2. Analysis of differential gene expression in developing PVS.

(A) Pearson correlation coefficient of transcriptome samples. The greater the value, the greater the correlation. (B) The distribution of DEGs in different groups.

Figure 3. The expression heatmap of key TFs positively correlated with oil content in developing PVS.

Figure 4. RT-qPCR analysis of some key genes involved in oil accumulation.

The expression levels were calculated by 2^−ΔΔCt.

Figure 5. Transcript patterns for key enzymes involved in glycolysis.

In the line chart, the ordinate represents the TPM levels. Abbreviation: HK, hexokinase; PGI, Phosphoglucose isomerase; PFK, 6-phosphofructokinase; FBA, fructose-bisphosphate aldolase; GAPC, glyceraldehyde 3-phosphate dehydrogenase; TIM, triosephosphate isomerase; PGK, phosphoglycerate kinase; PGM, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase; ENO, enolase; PK, Pyruvate kinase; Glu, α-D-Glucose; G6P, α-D-Glucose-6-phosphate; F6P, β-D-Fructose-6-phosphate; F1,6P, β-D-Fructose-1,6-diphosphate; GAP, Glycerone-phosphate; DGAP, Glyceraldehyde-3-phosphate; 1,3PG, Glycerate-1,3-diphosphate; 3PGA, Glycerate-3 -phosphate; 2PGA, Glycerate-2-phosphate; PEP, Phosphoenol-pyruvate; PYR, Pyruvate.

Figure 6. Identification of genes related to FA desaturation and TAG assembly in developing PVS.

The heatmap from left to right represent the data from IPT-10 DAP, IPT-70 DAP, IPT-110 DAP, SPT-10 DAP, SPT-70 DAP, SPT-110 DAP, respectively. Abbreviations: LPCAT, Lysophospholipid acyltransferase; FAD, Fatty acid desaturase; GPAT, Glycerol-3-phosphate acyltransferase; LPAAT, 1-acylglycerol-3-phosphate O-acyltransferase; PP, Phosphatidate phosphatase; DAG-CPT, Diacylglycerol cholinephosphotransferase; PDCT, Phosphatidylcholine:diacylglycerol cholinephosphotransferase; PDAT, Phospholipid:diacylglycerol acyltransferase; DGAT, Diacylglycerol O-acyltransferase; PC, phosphatidylcholine; G3P, glycerol-3-phosphate; LPA, 1-acylglycerol-3-phosphate; PA, 1,2-diacylglycerol-3-phosphate; DAG, 1,2-diacylglycerol; TAG, triacylglycerol.