Transcriptomic analysis of Perilla frutescens seed to insight into the biosynthesis and metabolic of unsaturated fatty acids

Abstract

Background: Perilla frutescens is well known for its high α-linolenic acid (ALA) accumulation in seeds and medicinal values as well as a source of edible and general-purpose oils. However, the regulatory mechanisms of the biosynthesis of fatty acid in its seeds remain poorly understood due to the lacking of sequenced genome. For better understanding the regulation of lipid metabolism and further increase its oil content or modify oil composition, time-course transcriptome and lipid composition analyses were performed.

Results: Analysis of fatty acid content and composition showed that the α-linolenic acid and oleic acid accumulated rapidly from 5 DAF to 15 DAF and then kept relatively stable. However, the amount of palmitic acid and linoleic acid decreased quickly from 5 DAF to 15DAF. No significant variation of stearic acid content was observed from 5 DAF to 25DAF. Our transcriptome data analyses revealed that 110,176 unigenes were generated from six seed libraries at 5, 10, 20 DAF. Of these, 53 (31 up, 22 down) and 653 (259 up, 394 down) genes showed temporal and differentially expression during the seed development in 5 DAF vs 10 DAF, 20 vs 10 DAF, respectively. The differentially expressed genes were annotated and found to be involved in distinct functional categories and metabolic pathways. Deep mining of transcriptome data led to the identification of key genes involved in fatty acid and triacylglycerol biosynthesis and metabolism. Thirty seven members of transcription factor family AP2, B3 and NFYB putatively involved in oil synthesis and deposition were differentially expressed during seed development. The results of qRT-PCR for selected genes showed a strong positive correlation with the expression abundance measured in RNA-seq analysis.

Conclusions: The present study provides valuable genomic resources for characterizing Perilla seed gene expression at the transcriptional level and will extend our understanding of the complex molecular and cellular events of oil biosynthesis and accumulation in oilseed crops.

Keywords: Fatty acid biosynthesis; Gene expression profiling; Perilla frutescens; RNA-seq; Seed development.

Fig. 1

Seed development stages, lipid content and composition, and differentially expressed genes analysis during seed development. a: The three developing stage after flowing used for lipid content and composition analysis; b: lipid content and composition; c: Differentially expressed genes in three paired comparisons (10 DAF vs 5 DAF, 20 DAF vs 10 DAF, and 20 DAF vs 5 DAF); d-f: Distribution of differentially expressed genes based on log2 FC values in three paired comparisons

Fig. 2

Gene ontology categories of all assembled unigenes. Unigenes were assigned into three main categories: biological processes, cellular components or molecular functions. The y-axis indicates the number of unigenes in a given category

Fig. 3

KOG functional classification of all unigenes. The unigenes were classified into different functional clusters based on KOG annotations

Fig. 4

Histogram of cluster of KEGG pathways of assembled unigenes in P. frutescens seed. The horizontal axis is the gene number; and vertical axis is the name of cluster of KEGG. A: Cellular processes; B: Environmental information processing; C: Genetic information processing; D: Metabolism; and E: Organismal systems

Fig. 5

Scatterplot of KEGG pathway enrichment analysis of differential expressed genes in paired comparisons of 10 DAF vs 5 DAF and 20 DAF vs 10 DAF. a Gene numbers enriched in the pathways are less than 2.0. b Gene numbers enriched in the pathways are more than 2.5

Fig. 6

qRT-PCR validation of selected genes. The relative expression levels of unigenes were normalized with internal reference gene actin and 18sRNA. Values are means±SE with three replicated for each samples in qRT-PCR

Fig. 7

The reconstructed pathways of fatty acids biosynthesis in plastid and triacylglycerol biosynthesis in ER based on the de novo assembly and annotation of P. frutescens transcriptome. The icons besides the key enzymes represent the relative expression levels of their transcripts in seeds between 10DAF and 5DAF, 20DAF and 10DAF. The identified enzymes involved in fatty acid biosynthesis include α-CT, carboxyl transferase α-subunit; β-CT, carboxyl transferase β-subunit; BC, biotin carboxylase; BCCP, biotin carboxyl carrier protein; MCMT, malonyl-CoA ACP transacylase; KAS, ketoacyl-ACP synthase; KAR, ketoacyl-ACP reductase; HAD, hydroxyacyl-ACP dehydrase; EAR, enoyl-ACP reductase; SAD, stearoyl-ACP desaturase; FATA/B, acyl-ACP thioesterase A/B; FAD6, oleate desaturase (chloroplast-type); FAD7/8, linoleate desaturase (chloroplast-type). Enzymes involved in triacylglycerol synthesis are LPCAT: lysophosphatidylcholine acyltransferase; FAD2, oleate desaturase; FAD3, linoleate desaturase; GPAT, glycerol-3-phosphate acyltransferase (EC2.3.1.15);PAP, PA phosphatase (EC: 3.1.3.4); DGAT, acyl-CoA: diacylglycerol acyltransferase; PDAT, phospholipid:diacylglycerol acyltransferase (EC: 2.3.1.20); PDCT, phosphatidylcholine: diacylglycerol cholinephosphotransferase (EC:2.7.8.*)

Fig. 8

An integrated view of α-linolenic acid metabolism and in P. frutescens. HPOT, hydroperoxyoctadeca-9,11,15-trienoate; EOTE, 12,13-epoxyoctadeca- 9,11,15-trienoic acid; 12-OPDA, 12-oxophyto-10,15-dienoate; OPC-8, 8-[(1R,2R)-3-Oxo-2- {(Z)-pent-2-enyl} cyclopentyl]octanoate; 3-O-OPC-CoA, 3-Oxo-OPC8-CoA; t-E-OPC-CoA, trans-2-enoyl- OPC-8- CoA; JA-CoA, 7-isojasmonic acid CoA; Me-JA, methyl jasmonate; LOX, lipoxygenase; AOS, hydroperoxide dehydratase; AOC, allene oxide cyclase; OPR, 12-oxophytodienoic acid reductase; OPCL1, OPC-8:0 CoA ligase 1; ACX, acyl-CoA oxidase; MFP2, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; ACAA, acetyl-CoA acyltransferase; JOM, jasmonate o-methyltransferase

Fig. 9

Expression profiles of differentially expressed members of transcription factor (TFs) family AP2, B3 and NFY putatively involved in oil biosynthesis and accumulation during seed development. a: Hierarchical cluster of expression levels of 37 TFs. Value of the color key refers to the log base 2 of gene expression level (RPKM).5 DAF (days after flowering), 10 DAF and 20 DAF were three stages of seed development. b-e: qRT-PCR validation of the expression of FUS3, LEC1, ABI3 and WRI1. The relative expression levels were normalized with internal reference gene Actin and 18 s RNA. Values are means ± SE with three replicated for each samples