Integrative study on gene expression and metabolism in crop seed
The study of storage processes in seeds is complicated by the extensive non-homogeneity in gene expression, metabolic conversions and storage product accumulation. The development of tools to analyse tissue-specific gene expression and metabolism is therefore an important priority in the context of seed biotechnology. Main plant models in this studies are oilseed rape, oat and barley.
Efficiency of the most popular 35S-promoter in oilseed rape plants were visualised and quantified in more than 30 different tissues types within distinct vegetative and generative organs. Considerable differences in promoter efficiency within the tissues are related to the cell type and developmental state. To quantify the strengths of promoters at sub-tissue resolution of cell clusters a novel technique is introduced. The method combines laser assisted micro-dissection (LAM) sampling and improved fluorometric analysis of β-glucuronidase activity using CMUG (6-Chloro-4-methylumbelliferyl-ß-D-glucuronide) as a most efficient fluorescent substrate for kinetic studies in plants. Method can be applied on other crops and coupled with other compatible techniques (Jasik et al., 2011).
An original methodology has been proposed, which combines rapid analysis of metabolic intermediates, major storage products and transcripts in a tissue-specific manner (Schiebold et al. (2011). A guideline and detailed procedure description towards the quantitative analysis of laser micro-dissected (LM) tissues is provided. 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). Current developments are toward spatially resolved modelling of metabolism in rape seed and barley.
Funding: the German Federal Ministry of Education and Research, Deutsche Forschungsgemeinschaft and BayerCropScience.
Grain structure and lipid distribution in barley (A) and soy bean (B).
Cross-section at the median part of seed (left) and lipid content is color-coded and forms a gradient with a maximal level (red) and a minimum level (blue-green) within the tissues.
Jasik J, Schiebold S, Rolletschek H, Denolf P, Van Audenhove K, Altmann T, Borisjuk L. (2011). Sub-tissue specific evaluation of promoter efficiency by quantitative fluorometric assay in laser micro dissected tissues of rapeseed (Brassica napus). Plant Physiology 157: 563-573.
Schiebold S, Tschiersch H, Borisjuk L, Heinzel N, Radchuk R, Rolletschek H. (2011). A novel procedure for the quantitative analysis of metabolites, storage products and transcripts of laser microdissected seed tissues of Brassica napus. Plant Methods 7: 19.