Identification and characterization of genes involved in metabolism of n5 monoene precursors to n5 anacardic acids in the trichomes of Pelargonium x hortorum.

Abstract

Unusual monoenoic fatty acids (UMFA’s) and specialized metabolites called anacardic acids (AnAc) are produced in glandular trichomes of Pelargonium ´ hortorum (geranium). The UMFA’s, 16:1∆11 and 18:1∆13 are precursors for the synthesis of unsaturated AnAc 22:1n5and 24:1n5 that contribute to pest resistance in geraniums. UMFAs and their derived AnAc metabolites not only provide a useful biological marker that differentiates the biosynthetic pathway for unusual mononenes from the common fatty acids (i.e. stearic, palmitic, oleic, linoleic and linolenic) but also have industrial, medical and agricultural applications. Fatty acid biosynthesis enzymes like acyl carrier proteins (ACPs); thioesterases (TEs) and β-ketoacyl-ACP synthases (KASs) are required for common fatty acid as well as the UMFA biosynthesis. Based on this, it is hypothesized that the specific isoforms of the fatty acid biosynthesis enzymes are highly expressed in trichomes and are involved specifically in metabolic channeling of UMFAs to anacardic acid synthesis within trichomes of geranium. This hypothesis is based on the knowledge that there is a novel Δ9 myristoyl-ACP desaturase (MAD) that directs acyl-ACP into UMFA biosynthesis and the products of MAD are correlated with the dominant congeners of AnAc (22:1n5 and 24:1n5). Transcription of MAD as well as production of 16:1∆11 and 18:1∆13 and AnAc 22:1n5 and AnAc 24:1n5 has been found to be highly trichome specific. This dissertation reports the identification of the complete nucleotide and protein sequences of genes for 2 ACPs, 3 FAT-As, 3 FAT-Bs, 4 KAS Is, 1 KAS II and 1 KAS III from a geranium EST database. Quantitative real-time PCR (qRT-PCR) was used to analyze tissue-specific expression patterns of the target genes, which indicated that ACP 1, ACP 2, KAS I-a/b, KAS Ic, FAT-A1, and FAT-A2 are highly expressed in trichomes. To further this research, a de novo RNA and micro-RNA transcriptome was generated from trichomes and bald pedicle of geranium, which helped in identification of several genetic components involved in UMFA synthesis. Bioinformatics analysis of RNA-transcriptome along with qRT-PCR and biochemical assays (HPLC and GC) were used to correlate the effect of temperature (18°C, 23°C and 28°C) on gene expression (ACPs, KASs, FAT-As) and changes in production of 16:1Δ11 and 18:1Δ13 UMFAs and 22:1n5and 24:1n5 AnAc. Results of this work show that expression of ACP 1, ACP 2, KAS I-c, KAS I-a/b were correlated with changes in UMFAs and AnAc production with temperature, thus indicating their potential role in UMFA metabolism. We also determined that 23°C is an optimal temperature for production of UMFAs and AnAcs as compared to 18°C and 28°C. To determine and verify the function of ACP 1 and ACP 2, we co-expressed these genes in conjunction with a Δ9 myristoyl-ACP (MAD) desaturase in both E. coli and tobacco. E. coli assay results show that expression of ACP 2 with MAD increased the production of UMFAs significantly, thus validating the novel role of ACP 2 in UMFA production. This work, in addition to the generation of a de novo transcriptome, provides a platform for further defining UMFA metabolism within trichomes of geranium