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A novel procedure for the quantitative analysis of metabolites, storage products and transcripts of laser microdissected seed tissues of Brassica napus.

Schiebold S, Tschiersch H, Borisjuk L, Heinzel N, Radchuk R, Rolletschek H - Plant Methods (2011)

Bottom Line: The described methodology allows for the rapid, combined analysis of metabolic intermediates, major storage products and transcripts in a tissue-specific manner.The protocols are robust for oilseed rape, and should be readily adjustable for other crop species.The suite of methods applied to LM tissues represents an important step in the context of both the systems biology and the biotechnology of oilseeds.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr, 3, 06466 Gatersleben, Germany. rollet@ipk-gatersleben.de.

ABSTRACT

Background: The biology of the seed is complicated by the extensive non-homogeneity (spatial gradients) in gene expression, metabolic conversions and storage product accumulation. The detailed understanding of the mechanisms underlying seed growth and storage therefore requires the development of means to obtain tissue-specific analyses. This approach also represents an important priority in the context of seed biotechnology.

Results: We provide a guideline and detailed procedures towards the quantitative analysis of laser micro-dissected (LM) tissues in oilseed rape (Brassica napus). This includes protocols for laser microdissection of the seed, and the subsequent extraction and quantitative analysis of lipids, starch and metabolites (sugars, sugar phosphates, nucleotides, amino acids, intermediates of glycolysis and citric acid cycle). We have also developed a protocol allowing the parallel analysis of the transcriptome using Brassica-specific microarrays. Some data are presented regarding the compartmentation of metabolites within the oilseed rape embryo.

Conclusion: The described methodology allows for the rapid, combined analysis of metabolic intermediates, major storage products and transcripts in a tissue-specific manner. The protocols are robust for oilseed rape, and should be readily adjustable for other crop species. The suite of methods applied to LM tissues represents an important step in the context of both the systems biology and the biotechnology of oilseeds.

No MeSH data available.


Experimental workflow. Schematic overview of the laser-microdissection based analysis of metabolites, storage products and transcripts in seeds of Brassica napus: (I) Seeds are removed out of the silique and immediately snap-frozen in liquid nitrogen. Two seeds mounted for cryosectioning are cut in 20 μm thick cross sections. (II) Sections are transferred to PET membrane slides and freeze-dried for one week. (III) Tissues of interest are laser microdissected from the cross sections. Light microscopy pictures show a cryosection of a 28 day old rape seed (bar = 1 mm) (IV) Isolated tissues are processed in parallel for subsequent biochemical and transcript analyses. Abbreviations: hy-hypocotyl; ic-inner cotyledon; oc-outer cotyledon.
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Figure 1: Experimental workflow. Schematic overview of the laser-microdissection based analysis of metabolites, storage products and transcripts in seeds of Brassica napus: (I) Seeds are removed out of the silique and immediately snap-frozen in liquid nitrogen. Two seeds mounted for cryosectioning are cut in 20 μm thick cross sections. (II) Sections are transferred to PET membrane slides and freeze-dried for one week. (III) Tissues of interest are laser microdissected from the cross sections. Light microscopy pictures show a cryosection of a 28 day old rape seed (bar = 1 mm) (IV) Isolated tissues are processed in parallel for subsequent biochemical and transcript analyses. Abbreviations: hy-hypocotyl; ic-inner cotyledon; oc-outer cotyledon.

Mentions: The first requirement was to optimize the preparation of tissue from the oilseed rape seed, appropriate for both transcriptome and biochemical analysis. Standard fixation and embedding procedures have been designed to preserve the morphological integrity of the sample, as well as to guarantee a sufficient quality of the RNA needed for gene expression analyses. However, these procedures entail the loss of many metabolites from the sample [20,25]. A cryogenic approach was therefore adopted in which the whole seed was first snap-frozen in liquid nitrogen, before sectioning without any additional chemical treatment. Sections of thickness 15, 20 or 30 μm were mounted either on standard microscope glass slides, or on various types of membrane-covered slides. The sections were dried at -20°C and stored at -80°C until required. The resulting morphology was adequate to distinguish between the various seed organs and even, to some extent at least, to enable the recognition of cell types such as the vascular tissue inside the hypocotyl, or the various cell layers of the seed coat. The dried sections were then subjected to LM (Figure 1), in which the target was first identified microscopically and then excised from the section with a focused UV laser. Sections of thickness 15 or 20 μm proved to be the most appropriate for the LM procedure.


A novel procedure for the quantitative analysis of metabolites, storage products and transcripts of laser microdissected seed tissues of Brassica napus.

Schiebold S, Tschiersch H, Borisjuk L, Heinzel N, Radchuk R, Rolletschek H - Plant Methods (2011)

Experimental workflow. Schematic overview of the laser-microdissection based analysis of metabolites, storage products and transcripts in seeds of Brassica napus: (I) Seeds are removed out of the silique and immediately snap-frozen in liquid nitrogen. Two seeds mounted for cryosectioning are cut in 20 μm thick cross sections. (II) Sections are transferred to PET membrane slides and freeze-dried for one week. (III) Tissues of interest are laser microdissected from the cross sections. Light microscopy pictures show a cryosection of a 28 day old rape seed (bar = 1 mm) (IV) Isolated tissues are processed in parallel for subsequent biochemical and transcript analyses. Abbreviations: hy-hypocotyl; ic-inner cotyledon; oc-outer cotyledon.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141804&req=5

Figure 1: Experimental workflow. Schematic overview of the laser-microdissection based analysis of metabolites, storage products and transcripts in seeds of Brassica napus: (I) Seeds are removed out of the silique and immediately snap-frozen in liquid nitrogen. Two seeds mounted for cryosectioning are cut in 20 μm thick cross sections. (II) Sections are transferred to PET membrane slides and freeze-dried for one week. (III) Tissues of interest are laser microdissected from the cross sections. Light microscopy pictures show a cryosection of a 28 day old rape seed (bar = 1 mm) (IV) Isolated tissues are processed in parallel for subsequent biochemical and transcript analyses. Abbreviations: hy-hypocotyl; ic-inner cotyledon; oc-outer cotyledon.
Mentions: The first requirement was to optimize the preparation of tissue from the oilseed rape seed, appropriate for both transcriptome and biochemical analysis. Standard fixation and embedding procedures have been designed to preserve the morphological integrity of the sample, as well as to guarantee a sufficient quality of the RNA needed for gene expression analyses. However, these procedures entail the loss of many metabolites from the sample [20,25]. A cryogenic approach was therefore adopted in which the whole seed was first snap-frozen in liquid nitrogen, before sectioning without any additional chemical treatment. Sections of thickness 15, 20 or 30 μm were mounted either on standard microscope glass slides, or on various types of membrane-covered slides. The sections were dried at -20°C and stored at -80°C until required. The resulting morphology was adequate to distinguish between the various seed organs and even, to some extent at least, to enable the recognition of cell types such as the vascular tissue inside the hypocotyl, or the various cell layers of the seed coat. The dried sections were then subjected to LM (Figure 1), in which the target was first identified microscopically and then excised from the section with a focused UV laser. Sections of thickness 15 or 20 μm proved to be the most appropriate for the LM procedure.

Bottom Line: The described methodology allows for the rapid, combined analysis of metabolic intermediates, major storage products and transcripts in a tissue-specific manner.The protocols are robust for oilseed rape, and should be readily adjustable for other crop species.The suite of methods applied to LM tissues represents an important step in the context of both the systems biology and the biotechnology of oilseeds.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr, 3, 06466 Gatersleben, Germany. rollet@ipk-gatersleben.de.

ABSTRACT

Background: The biology of the seed is complicated by the extensive non-homogeneity (spatial gradients) in gene expression, metabolic conversions and storage product accumulation. The detailed understanding of the mechanisms underlying seed growth and storage therefore requires the development of means to obtain tissue-specific analyses. This approach also represents an important priority in the context of seed biotechnology.

Results: We provide a guideline and detailed procedures towards the quantitative analysis of laser micro-dissected (LM) tissues in oilseed rape (Brassica napus). This includes protocols for laser microdissection of the seed, and the subsequent extraction and quantitative analysis of lipids, starch and metabolites (sugars, sugar phosphates, nucleotides, amino acids, intermediates of glycolysis and citric acid cycle). We have also developed a protocol allowing the parallel analysis of the transcriptome using Brassica-specific microarrays. Some data are presented regarding the compartmentation of metabolites within the oilseed rape embryo.

Conclusion: The described methodology allows for the rapid, combined analysis of metabolic intermediates, major storage products and transcripts in a tissue-specific manner. The protocols are robust for oilseed rape, and should be readily adjustable for other crop species. The suite of methods applied to LM tissues represents an important step in the context of both the systems biology and the biotechnology of oilseeds.

No MeSH data available.