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Laser microdissection of conifer stem tissues: isolation and analysis of high quality RNA, terpene synthase enzyme activity and terpenoid metabolites from resin ducts and cambial zone tissue of white spruce (Picea glauca).

ABSTRACT: Laser microdissection (LMD) has been established for isolation of individual tissue types from herbaceous plants. However, there are few reports of cell- and tissue-specific analysis in woody perennials. While microdissected tissues are commonly analyzed for gene expression, reports of protein, enzyme activity and metabolite analysis are limited due in part to an inability to amplify these molecules. Conifer stem tissues are organized in regular patterns with xylem, phloem and cortex development controlled by the activity of the cambial zone (CZ). Defense responses of conifer stems against insects and pathogens involve increased accumulation of terpenoids in cortical resin ducts (CRDs) and de novo formation of traumatic resin ducts from CZ initials. These tissues are difficult to isolate for tissue-specific molecular and biochemical characterization and are thus good targets for application of LMD.We describe robust methods for isolation of individual tissue-types from white spruce (Picea glauca) stems for analysis of RNA, enzyme activity and metabolites. A tangential cryosectioning approach was important for obtaining large quantities of CRD and CZ tissues using LMD. We report differential expression of genes involved in terpenoid metabolism between CRD and CZ tissues and in response to methyl jasmonate (MeJA). Transcript levels of beta-pinene synthase and levopimaradiene/abietadiene synthase were constitutively higher in CRDs, but induction was stronger in CZ in response to MeJA. 3-Carene synthase was more strongly induced in CRDs compared to CZ. A differential induction pattern was observed for 1-deoxyxyulose-5-phosphate synthase, which was up-regulated in CRDs and down-regulated in CZ. We identified terpene synthase enzyme activity in CZ protein extracts and terpenoid metabolites in both CRD and CZ tissues.Methods are described that allow for analysis of RNA, enzyme activity and terpenoid metabolites in individual tissues isolated by LMD from woody conifer stems. Patterns of gene expression are demonstrated in specific tissues that may be masked in analysis of heterogeneous samples. Combined analysis of transcripts, proteins and metabolites of individual tissues will facilitate future characterization of complex processes of woody plant development, including periodic stem growth and dormancy, cell specialization, and defense and may be applied widely to other plant species.

SUBMITTER: Abbott E

PROVIDER: S-EPMC3095273 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Laser microdissection of conifer stem tissues: isolation and analysis of high quality RNA, terpene synthase enzyme activity and terpenoid metabolites from resin ducts and cambial zone tissue of white spruce (Picea glauca).

Abbott Eric E Hall Dawn D Hamberger Björn B Bohlmann Jörg J

BMC plant biology 20100612

<h4>Background</h4>Laser microdissection (LMD) has been established for isolation of individual tissue types from herbaceous plants. However, there are few reports of cell- and tissue-specific analysis in woody perennials. While microdissected tissues are commonly analyzed for gene expression, reports of protein, enzyme activity and metabolite analysis are limited due in part to an inability to amplify these molecules. Conifer stem tissues are organized in regular patterns with xylem, phloem and ...[more]

PMID: 20540781

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Project description:BACKGROUND: Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. RESULTS: We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. CONCLUSION: We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes.

Project description:BackgroundMembers of the pine family (Pinaceae), especially species of spruce (Picea spp.) and pine (Pinus spp.), dominate many of the world's temperate and boreal forests. These conifer forests are of critical importance for global ecosystem stability and biodiversity. They also provide the majority of the world's wood and fiber supply and serve as a renewable resource for other industrial biomaterials. In contrast to angiosperms, functional and comparative genomics research on conifers, or other gymnosperms, is limited by the lack of a relevant reference genome sequence. Sequence-finished full-length (FL)cDNAs and large collections of expressed sequence tags (ESTs) are essential for gene discovery, functional genomics, and for future efforts of conifer genome annotation.ResultsAs part of a conifer genomics program to characterize defense against insects and adaptation to local environments, and to discover genes for the production of biomaterials, we developed 20 standard, normalized or full-length enriched cDNA libraries from Sitka spruce (P. sitchensis), white spruce (P. glauca), and interior spruce (P. glauca-engelmannii complex). We sequenced and analyzed 206,875 3'- or 5'-end ESTs from these libraries, and developed a resource of 6,464 high-quality sequence-finished FLcDNAs from Sitka spruce. Clustering and assembly of 147,146 3'-end ESTs resulted in 19,941 contigs and 26,804 singletons, representing 46,745 putative unique transcripts (PUTs). The 6,464 FLcDNAs were all obtained from a single Sitka spruce genotype and represent 5,718 PUTs.ConclusionThis paper provides detailed annotation and quality assessment of a large EST and FLcDNA resource for spruce. The 6,464 Sitka spruce FLcDNAs represent the third largest sequence-verified FLcDNA resource for any plant species, behind only rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the only substantial FLcDNA resource for a gymnosperm. Our emphasis on capturing FLcDNAs and ESTs from cDNA libraries representing herbivore-, wound- or elicitor-treated induced spruce tissues, along with incorporating normalization to capture rare transcripts, resulted in a rich resource for functional genomics and proteomics studies. Sequence comparisons against five plant genomes and the non-redundant GenBank protein database revealed that a substantial number of spruce transcripts have no obvious similarity to known angiosperm gene sequences. Opportunities for future applications of the sequence and clone resources for comparative and functional genomics are discussed.

Dataset Information

Laser microdissection of conifer stem tissues: isolation and analysis of high quality RNA, terpene synthase enzyme activity and terpenoid metabolites from resin ducts and cambial zone tissue of white spruce (Picea glauca).

Publications

Laser microdissection of conifer stem tissues: isolation and analysis of high quality RNA, terpene synthase enzyme activity and terpenoid metabolites from resin ducts and cambial zone tissue of white spruce (Picea glauca).

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