Tissue-Specific Whole Transcriptome Sequencing in Castor, Directed at Understanding Triacylglycerol Lipid Biosynthetic Pathways

Abstract

BackgroundStorage triacylglycerols in castor bean seeds are enriched in the hydroxylated fatty acid ricinoleate. Extensive tissue-specific RNA-Seq transcriptome and lipid analysis will help identify components important for its biosynthesis.Methodology/FindingsStorage triacylglycerols (TAGs) in the endosperm of developing castor (Ricinus communis) seeds are highly enriched in ricinoleic acid (18:1-OH). We have analysed neutral lipid fractions from other castor tissues using TLC, GLC and mass spectrometry. Cotyledons, like the endosperm, contain high levels of 18:1-OH in TAG. Pollen and male developing flowers accumulate TAG but do not contain 18:1-OH and leaves do not contain TAG or 18:1-OH. Analysis of acyl-CoAs in developing endosperm shows that ricinoleoyl-CoA is not the dominant acyl-CoA, indicating that either metabolic channelling or enzyme substrate selectivity are important in the synthesis of tri-ricinolein in this tissue. RNA-Seq transcriptomic analysis, using Illumina sequencing by synthesis technology, has been performed on mRNA isolated from two stages of developing seeds, germinating seeds, leaf and pollen-producing male flowers in order to identify differences in lipid-metabolic pathways and enzyme isoforms which could be important in the biosynthesis of TAG enriched in 18:1-OH. This study gives comprehensive coverage of gene expression in a variety of different castor tissues. The potential role of differentially expressed genes is discussed against a background of proteins identified in the endoplasmic reticulum, which is the site of TAG biosynthesis, and transgenic studies aimed at increasing the ricinoleic acid content of TAG.Conclusions/SignificanceSeveral of the genes identified in this tissue-specific whole transcriptome study have been used in transgenic plant research aimed at increasing the level of ricinoleic acid in TAG. New candidate genes have been identified which might further improve the level of ricinoleic acid in transgenic crops.

Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly

“…The proportion of oleoyl-CoA was similar in all lines (Fig. 3A), and HFA-CoA accumulated to less than 10% in CL37, similar to levels of HFA in developing castor seeds (39). Additionally, the acyl-ACP compositions of all four lines were very similar (Fig.…”

The Phosphatidylcholine Diacylglycerol Cholinephosphotransferase Is Required for Efficient Hydroxy Fatty Acid Accumulation in Transgenic Arabidopsis

“…18:2/3-PC, 18:1OH-PC over 18:1-PC; 18:1-DAG over 18:2/3-DAG, 18:1OH-DAG), since the desaturases and the hydroxylase will enrich the PC pool with the PUFAs and hydroxylated FAs. On the other hand, although the RcROD1 protein has been detected in castor endosperm (Brown et al, 2012), previous results suggested that the castor PDCT unlikely preferred HFA-containing substrates, since microsomal preparations from castor endosperm rapidly turned the glycerol-labeled diricinoleoyl-DAG to TAG, whereas dioleoyl-DAG was preferentially used in PC synthesis (Bafor et al, 1991). Moreover, Dahlqvist et al (2000) showed that castor bean microsomes preferentially metabolized ricinoleoyl-PC to TAG (probably via PDAT), whereas oleoyl-PC was preferentially metabolized to DAG (probably via PDCT).…”

Biochemical and Molecular Characterization of RcSUS1, a Cytosolic Sucrose Synthase Phosphorylated in Vivo at Serine 11 in Developing Castor Oil Seeds

“…2, A-C) (40,41). RcSUS1 belongs to the dicot SUS1 group and has high sequence identity with soybean root nodule (GmSUS1) and Arabidopsis anoxiainducible (AtSUS1 and AtSUS4) orthologs (Table 2) (5, 21).…”

“…C, cotyledon; dE and dC, endosperm and cotyledon, respectively, from stage V developing COS that had been depodded for 48 h; D, dry (mature) COS; G, germinating COS endosperm harvested at 5 days postimbibition. pair of transcriptome sequencing studies (40,41). Both reports clearly established RcSUS1 as the dominant SUS isozyme expressed in the endosperm of developing COS and that its expression is maximal during the early stages of COS development (Fig.…”