Identification of Genes Involved in Lipid Biosynthesis through de novo Transcriptome Assembly from Cocos nucifera Developing Endosperm

Abstract

Cocos nucifera (coconut), a member of the Arecaceae family, is an economically important woody palm that is widely grown in tropical and subtropical regions. The coconut palm is well known for its ability to accumulate large amounts of oil, approximately 63% of the seed weight. Coconut oil varies significantly from other vegetable oils as it contains a high proportion of medium-chain fatty acids (MCFA; 85%). The unique composition of coconut oil raises interest in understanding how the coconut palm produces oil of a high saturated MCFA content, and if such an oil profile could be replicated via biotechnology interventions. Although some gene discovery work has been performed there is still a significant gap in the knowledge associated with coconut's oil production pathways. In this study, a de novo transcriptome was assembled for developing coconut endosperm to identify genes involved in the synthesis of lipids, particularly triacylglycerol. Of particular interest were thioesterases, acyltransferases and oleosins because of their involvement in the processes of releasing fatty acids for assembly, esterification of fatty acids into glycerolipids and protecting oils from degradation, respectively. It is hypothesized that some of these genes may exhibit a strong substrate preference for MCFA and hence may assist the future development of vegetable oils with an enriched MCFA composition. In this study, we identified and confirmed functionality of five candidate genes from the gene families of interest. This study will benefit future work in areas of increasing vegetable oil production and the tailoring of oil fatty acid compositions.

Keywords: De novo assembly; Nicotiana; Coconut; DGAT; Endosperm; FATA; Fatty acid; GPAT9; Medium chain; Oleosin; PDAT; Thioesterase; Transcriptome; Triacylglycerol.

Fig. 1

Graphical representation of the primary pathways involved in triacylglycerol (TAG) production, highlighting the lipid synthesis genes of interest (grey bubbles). The asterisk represents those genes that have been investigated in this study following the construction of the Cocos nucifera (coconut palm) endosperm transcriptome. FAS, fatty acid synthase; FAT, fatty acid thioesterase, class A (FATA) and class B (FATB); GPAT, glycerol phosphate acyltransferase; LPAAT, lysophosphatidic acid acyltransferase; DGAT, diacylglycerol acyltransferase; PDAT, phospholipid:diacylglycerol acyltransferase; PC,phosphatidylcholine; DAG, diacylglycerol.

Fig. 2

Analysis of coconut oil composition. Extracted coconut oil from developing Cocos nucifera endosperm was analyzed by thin-layer chromatography (TLC) (A). The fatty acid composition of triacylglycerol (TAG) was compared between commercial coconut oil (Murray River Organics, Australia) and oil extracted from freshly harvested developing coconut endosperm (B). LPA, lysophosphatidic acid; PC, phosphatidylcholine; PA, phosphatidic acid; MAG, monoacylglycerol; DAG, diacylglycerol; FFA, free fatty acid; TAG, triacylglycerol.

Fig. 3

(A) Examining Cocos nucifera DGAT1 candidates for functionality through the quantification of changes in the accumulation of triacylglycerols (TAG), using the Nicotiana benthamiana transient expression assay. Error bars are representative of standard deviation (n = 4). (B) Analysis of major fatty acids in the total fatty acid profile from the N.benthamiana transient expression assay, associated with the testing of different diacylglycerol acyltransferase (DGAT1) candidates. Error bars are representative of standard deviation (n = 4). (C) Yeast (Saccharomyces cerevisiae) complementation assay of quadruple mutant strain H1246, investigating the functionality of DGAT1 through restoration of function. Transformants were fed C18:1Δ9 free fatty acids. Yeast transformants were resuspended in sterile water and diluted to OD600 = 1. Samples were further diluted in four consecutive dilutions, each at 1:10. From each dilution 5 μL was spotted on to YPD+FFA plates. The plates were incubated at 28�C for 3 d. Negative control, H1246::pYES2; Positive control, H1246::AtDGAT1; pKR.B1, H1246::CnDGAT1_V1. At, Arabidopsis thaliana; Cn, Cocos nucifera.

Fig. 4

Investigating the substrate specificity of Cocos nucifera DGAT1 candidates for functionality through analyzing the changes in both the total triacylglycerol (TAG; block dots) content and the TAG fatty acid profile, using the Nicotiana benthamiana transient expression assay (n = 4). The results were grouped by the corresponding class B acyl-ACP thioesterase (FATB), where control refers to the expression of p19 only. On the x-axis, the treatments were grouped into respective diacylglycerol acyltransferase (DGAT) expression. Control refers to samples that were infiltrated with the corresponding FATB only (Control = p19 only). The annotation of CnDGAT represents those treatments that exhibited CnDGAT1+AtWRI1 co-expression. At, Arabidopsis thaliana; Cc, Cinnamomum camphora; Cn, Cocos nucifera; Uc, Umbellularia californica.

Fig. 5

(A) Investigating the ability of phosphatidylcholine-diacylglycerol acyltransferase (PDAT) to increase production of triacylglycerol (TAG) in the leaves of Nicotiana benthamiana, following 5 d of expression. Error bars are representative of standard deviation (n = 3). Treatments were grouped by their respective PDAT expression including no PDAT, PDAT from Arabidopsis thaliana (AtPDAT) and Cocos nucifera (CnPDAT). (B) Investigating the changes in fatty acid composition of different lipid groups associated with the overexpression of PDAT, using the N.benthamiana transient expression assay. The lipid pools analyzed included were TAG, phospholipids and total fatty acids (TFA). Treatments were grouped by their respective PDAT expression including no PDAT, PDAT from Arabidopsis thaliana (AtPDAT) and from C.nucifera (CnPDAT) (n = 3).

Fig. 6

Confocal microscopy showing the localization of CnOLEOSIN-mCherry in epidermal cells of Nicotiana benthamiana leaves, 3 d post-infiltration (DPI). Lipid droplets were stained with BODIPY-505/515 and are shown in green, chloroplast autofluorescence in blue and CnOLEOSIN-mCherry in magenta. In all panels, white arrowheads point at lipid droplets, while empty arrowheads highlight CnOLEOSIN signal in the endoplasmic reticulum (ER), and stars highlight the nucleus of the cell (M–P). In leaves infiltrated with p19 only (A–D), the lipid droplets were very small. In leaves infiltrated with p19+CnOLEOSIN-mCherry (E–H), CnOLEOSIN localized to the ER and lipid droplets, and the size of the lipid droplets was not increased compared to p19 alone. In leaves infiltrated with p19+AtWRI1+AtDGAT1 (I–L), the lipid droplets increased in both size and abundance. Finally, in leaves infiltrated with p19+AtWRI1+AtDGAT1+CnOLEOSIN-mCherry (M–T), CnOLEOSIN was still detected in the ER (M–P) and was also found to coat the lipid droplets (Q–T). TL is transmitted light. Scale bars represent 20 μm except the lower row where they represent 10 μm. At, Arabidopsis thaliana; Cn, Cocos nucifera.

Fig. 7

(A) Investigating the ability of CnOLEOSIN to enable greater accumulation of triacylglycerol (TAG) in the leaves of Nicotiana benthamiana, following 5 d of expression. The WRI1 and DGAT1 genes were both from Arabidopsis thaliana. Error bars are representative of standard deviation (n = 6). (B) Investigating the ability of CnOLEOSIN in protecting lipid droplets by minimizing the turnover of TAG, by measuring the incorporation of [14C] labelled acetate into TAG. Error bars are representative of standard error (n = 3). At, Arabidopsis thaliana; Cn, Cocos nucifera.

Fig. 8

(A) Examining Cocos nucifera class A fatty acid thioesterase (FATA) candidates for functionality through the quantification of changes in the accumulation of triacylglycerols (TAG) content, using the Nicotiana benthamiana transient expression assay. The Arabidopsis thaliana homolog (AtFATA1) was used as a positive control. The sample ‘p19 Only’ refers to the background infiltration of AtWRI1 and AtDGAT1. Error bars are representative of standard deviation (n = 4). (B) Analysis of the major fatty acids in the TAG fatty acid composition, associated with the testing different FATA candidates, using the N.benthamiana transient expression assay. All treatments were performed in the background of AtWRI1 and AtDGAT1 co-expression. The A.thaliana homolog (AtFATA1) was used as a positive control. Error bars are representative of standard deviation (n = 4). At, Arabidopsis thaliana; Cn, Cocos nucifera.