Tissue-specific whole transcriptome sequencing in castor, directed at understanding triacylglycerol lipid biosynthetic pathways.

Abstract

Storage 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.Storage 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.Several 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.