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)

被引:70
作者
Abbott, Eric [1 ,2 ]
Hall, Dawn [1 ]
Hamberger, Bjoern [1 ]
Bohlmann, Joerg [1 ,2 ]
机构
[1] Univ British Columbia, Michael Smith Labs, Vancouver, BC V6T 1Z4, Canada
[2] Univ British Columbia, Dept Bot, Vancouver, BC V6T 1Z4, Canada
来源
BMC PLANT BIOLOGY | 2010年 / 10卷
基金
加拿大自然科学与工程研究理事会;
关键词
WEEVILS PISSODES-STROBI; NORWAY SPRUCE; METHYL JASMONATE; SITKA SPRUCE; CAPTURE MICRODISSECTION; TRANSCRIPTOME ANALYSIS; CATHARANTHUS-ROSEUS; GLANDULAR TRICHOMES; DEFENSE RESPONSES; MASS-SPECTROMETRY;
D O I
10.1186/1471-2229-10-106
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
Background: 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. Results: 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 b-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. Conclusions: 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 heterogenous 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.
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页数:16
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