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© PhD Students Symposium 2005. All rights reserved.
Prof. Dr. Mark Tester
Australian Centre for Plant Functional Genomics, University of Adelaide, Australia
It is intuitively obvious that specific cell types have different structures and functions, often determined by their position within the organism, yet most genetic engineering of plants employs the use of constitutive (or nearly constitutive) promoters. Although more subtle (sensible?!) approaches are often limited by the paucity of cell type-specific promoters, genetic engineering seems to focus more on the genes per se than on the spatial and temporal control of their expression. In this talk, a case study will be presented where it is argued that such control is central to the successful manipulation of many processes in a plant. Tools for such manipulation will then be described, and results to date from the research laboratory presented.
The specific case being examined involves the control of the delivery of a solute to the shoot. To control this, opposite transport processes need to be maximized in the inner and outer halves of the root. The endodermis, therefore, acts as the root equivalent of an epithelium. Thus, it is proposed that constitutive activation of a gene involved in controlling plasma membrane transport of a solute will be less likely to alter accumulation of that solute than cell type-specific gene activation. For example, consider a case where Na+ efflux is maximized in all cells. Although less Na+ would reach the cells adjacent to the xylem (as it would be pumped out of cells in the outer half of the root), the Na+ present would then be loaded at a higher rate (as cells around the dead xylem would be extruding Na+ into the xylem). The net effect of these two opposing processes is hard to predict, but it is clear that net exclusion from the shoot is not necessarily the outcome of engineering net efflux from all cells. Thus, to most effectively alter accumulation of a solute, activation of genes controlling plasma membrane transport of that solute should be cell-specific. The aim of our research program is to test this theoretical proposal and exploit outcomes to enable targeted manipulation of genes to alter shoot nutrient accumulation.
Because our knowledge of the genes catalyzing and controlling the transport of most solutes in plants is still rudimentary, the approach we are taking is to generate lines with activation of random genes in specific cell types, and to measure the shoot solute accumulation of resultant lines. Target cell types are the cells found within mature tissues of the epidermis, cortex, endodermis and pericycle. This is being done by exploiting a system developed by Jim Haseloff (Plant Sciences, Cambridge) and already used for specific gene activation in specific cell types (Kiegle et al., 2000: Plant J. 23, 267-78; Johnson et al., 2005: Plant J. 41, 779-89). Libraries of transgenic lines of Arabidopsis and rice have been made, in which plants are transformed with a gene encoding the yeast transcription factor, GAL4:VP16 and a second gene which encodes the green fluorescent protein, GFP. Expression of the GFP is driven by expression of the GAL4:VP16. Lines with the expression pattern of interest are then transformed a second time with the upstream activation sequence to which GAL4 binds, activating random genes (depending on the site of insertion of this second construct) in specific cell types (in which the GAL4 and GFP are expressed). The shoots of these lines are then analyzed for altered accumulation of 23 elements using ICP-MS and outliers identified for further study.