Dale A Shelton

Analysis of grape ESTs: global gene expression patterns in leaf and berry.

Analysis of 2479 ESTs from Vitis vinifera berry tissue and 2438 from leaf revealed that 1% of the ESTs match to known Vitis proteins, 72% to plant proteins, 11% to non-plant, and 16% had no match (P[N]>0.5). The levels of redundancy were similar in the leaf and berry libraries. Only 12% of the genes matched by the ESTs were common to both libraries indicating marked differences in the genes expressed in the two tissues. The abundance of transcripts with predicted cellular roles in leaf and berry were estimated by classifying the primary BLAST matches to known proteins (score >80) into functional categories. Thirty-six percent of the leaf transcripts were involved in photosynthesis, compared to 3% in the berry. This is a much higher proportion of transcripts involved with a function limited to specialized cells, than was found when transcripts of 33 human tissues were compared using a similar approach, suggesting plant cells may involve their cellular machinery to a greater extent in specialized activities than animal cells. Relatively enhanced expression of specific transcription factors, and genes involved in defense, detoxification, stress response, proteolysis, trafficing, and signal transduction, suggests berry tissue is actively engaged in responding to environmental stimuli.

Isolation of genes involved in secondary metabolism from Melaleuca alternifolia (Cheel) using expressed sequence tags (ESTs)

Melaleuca alternifolia (Cheel) is a commercially important, cultivated Australian native tree, harvested for its monoterpene-rich essential oil. To isolate genes involved in terpene biosynthesis from this tree, 454 randomly selected clones from a cDNA library derived from flush growth were sequenced and analysed. The five most abundant sequences included cDNAs that encoded Rubisco and photosystem II, as expected. The remaining three abundant sequences included a GATA-binding transcription factor homolog, a sequence with low similarity to a hypothetical protein from a plastid genome of evening primrose (Oenothera elata) and a short sequence with no similarity. These latter three pose interesting targets for functional genomic studies, as their function is unknown and their relative high abundance infer a pivotal role in young tissue of M. alternifolia. Only three ESTs involved in terpene biosynthesis were isolated, a cycloartenol synthase-like protein, a limonene synthase-like protein and farnesyl pyrophosphate synthase, of which only the latter two are involved in the production of the valuable essential oils. However, five ESTs were isolated for phenylpropanoid biosynthesis, excluding those that are committed to lignin biosynthesis. Other secondary metabolite-related ESTs included genes for carotenoid and plant growth regulator biosynthesis, as well as various cytochrome p450s of unknown function. A total of 5% of ESTs isolated in this study are involved directly with secondary metabolite production. This study illustrates the usefulness of ESTs as a method for bio-prospecting for secondary metabolites in simple plant tissues.

Genetic control of monoterpene composition in the essential oil of Melaleuca alternifolia (Cheel)

Abstract.Previous studies into the population structure of Melaleuca alternifolia by both isozyme and microsatellite analysis revealed little evidence for genetic structuring within genetic provenances. In contrast, analysis of the oil composition within these same regions showed distinct clustering of chemotypes within the provenances suggesting either that chemotype was not under genetic control, or that there is strong environmental selection for plant chemotypes. To investigate the level of genetic control of monoterpene composition in the essential oil of M. alternifolia, individuals representing the three extreme chemotypes of high terpinen-4-ol, high 1,8-cineole and high terpinolene were crossed with an individual with the commercially desirable high terpinen-4-ol oil profile. The progeny resulting from these crosses displayed oil profiles that were intermediate to that of the parent. Further analysis of the survey of oil chemotypes within the natural population also suggests that these intermediate chemotypes may arise naturally between regions containing high proportions of the extreme chemotypes. These results imply that there is a level of genetic structure for chemotype determination within the genetic provenance that is undetected by isozyme and microsatellite analysis. This information could play a vital role in the selection of appropriate genetic material to be used in future essential oil selection and breeding programs.

Isopentenyl pyrophosphate isomerases from Melaleuca alternifolia (Cheel) and their role in isoprenoid biosynthesis

Summary Two cDNA clones, idil and idi2, representing different isoforms of isopentenyl pyrophosphate isomerase (IPP isomerase) were isolated from Melaleuca alternifolia (Cheel) by functional complementation of carotenoid biosynthesis in E. coli. Excluding the putative transit peptide region, the isoforms share 89.5% predicted protein sequence identity. The high level of conservation between the isoforms indicates that these genes may share a common ancestral origin and supports the proposition that cytosolic and plastid targeted IPP isomerase may be differentially translated from a single gene. This study supports recent evidence suggesting that isopentenyl pyrophosphate and dimethylallylic pyrophosphate are both immediate products of the deoxyxylulose pathway and that IPP isomerase may have a more central role in the biosynthesis of carotenoids than in the biosynthesis of monoterpenes.