The roles of Phospholipase C in oil biosynthesis in oilseeds
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Research on the biosynthesis pathways of storage oil (triacylglycerol, or TAG) in plants has gained momentum for the last couple of decades. Despite significant achievements, a complete understanding of such pathways is still lacking. Also, the production of industrially important unusual oils such as hydroxy fatty acids (HFA) at the commercial level is limited in both native and transgenic plants. In this study, I have examined the roles of Phospholipase C in the TAG biosynthesis. To test roles of non-specific PLCs (NPCs) in TAG synthesis, FA composition was analyzed in mature seeds of T-DNA lines of 6 Arabidopsis npc T-DNA mutants. Among them, npc1, npc4 and npc6 showed increases in oleic acid (18:1) and decreases in lineoleic acid (18:2) and linolenic acid (18:3). The 18:1 was further increased in the npc1/npc6 double mutant compared to npc1 and npc6. These changes in FA profile suggest the role of NPCs in TAG biosynthesis. Given the possible roles of NPCs in TAG synthesis in the seeds of Arabidopsis, I searched for suitable PLC from Castor (RcPLC) for further investigation and to transform to the 7-1 line of Camelina sativa expressing a fatty acid hydroxylase (RcFAH12). From previously published Castor transcriptome, RcPLCL1 was the phospholipase C-like gene that was primarily expressed in the endosperm. Exploration of the RcPLCL1 protein using bioinformatics tools placed it to the PLC-like phosphodiesterase family which has not been characterized yet. Based on the sequence analyses, the protein contains the X domain for the PLC activity on phosphatidylinositols (PI), but it lacks the Y and C2 domains. Enzyme assays using heterologous expression in yeast showed that the protein had both PC- and PI-PLC activities. Based on the genomic sequence analysis, 8 genes were found in Arabidopsis that were predicted to encode PLC-like phosphodiesterases. The seeds of 5 T-DNA knockout lines were analyzed for fatty acid composition, which showed decreased linolenic acid (18:3) and increased oleic acid (18:1). The FA composition change suggested the involvement of these proteins in oil biosynthesis in Arabidopsis seeds. Upon transformation of RcPLCL1 into the 7-1 line, HFA content in the seeds was increased to as high as 24% from 15%. Concurrently the amount of HFA decreased in the membrane lipid phosphatidylcholine (PC). However, the Arabidopsis homologue, AtPLCL1, did not increase the HFA in seeds of 7-1. These results suggest that RcPLCL1 is involved in HFA accumulation. The HFA-producing 7-1 plants were tested in the field. Results from two-year experiments indicated that both wild type and 7-1 demonstrated better performance for traits like oil content and yield. However, the HFA level in 7-1 was significantly decreased in field-grown plants than greenhouse plants.