CORDIS Archive : Agriculture and Fisheries / Contract Nr : FAIRCT965055

THE EFFECTS OF DIFFERENTIAL COMPARTMENTATION OF THE ANTIOXIDANT DEFENCE SYSTEM ON THE SENSITIVITY OF THE BUNDLE SHEATH AND MESOPHYLL CELLS OF MAIZE TO LOW TEMPERATURE (ASSOCIATED WITH AIR1-CT920205, PROJECT ESTIM)

Host Laboratory	Scientific Supervisor
Institute of Grassland and Environmental Research Plas Gogerddan SY23 3EB Aberystwyth - Dyfed United Kingdom	Dr. Christine Foyer Tel : +44 1970828255 / Fax : +44 1970828357 Email : foyer@bbsrc.ac.uk
	Grant Holder
	Dr. Gabriela Maria Pastori (Italian) Tel : / Fax :
Abstract Acclimation to prevailing environmental conditions is an essential facet of metabolic regulation that facilitates survival in sub-optimal climates. While acclimation is a multifactorial process, the induction of improved antioxidant defences to overcome the increased production of active oxygen species is a vital feature of the acclimatory strategy. Antioxidants such as ascorbate and glutathione remove H202 and maintain the correct redox balance of the cell. They are also important factors in other fundamental processes related to adaptation to low temperatures. Ascorbic acid for example, has been implicated in plant growth and development. It is considered to be a necessary factor in cell proliferation that enables already competent cells to progress through the cell cycle phases. Both ascorbate and glutathione may be involved in gene expression. Our initial results suggest that the antioxidant defence system in maize may not be equally partitioned between the mesophyll and bundle sheath cells. Until recently, experiments on the photosynthetic processes and associated metabolism in the mesophyll and bundle sheath cells from maize were largely performed on isolated cell fractions purified by lengthy digestion and separation procedures. Such techniques modify the protein composition and antioxidant capacity of the isolated tissues. To our knowledge, no-one has undertaken a comprehensive study of the susceptibility of the photosynthetic processes or the antioxidant system to low temperature in rapidly purified (that is extracted within seconds) mesophyll and bundle sheath extracts. This project will address this question and involve precise measurements of the photosynthetic electron transport capacity, the activities of Benson-Calvin cycle enzymes and the components of the antioxidant defence system in optimal conditions, following short-term exposure to low temperature (8 C) and during long-term acclimation to growth at sub-optimal temperatures (10-14 C). This will involve chlorophyll a fluorescence analysis (for PSII), measurement of the 820 nm absorption changes (for PSI), oxygen electrode measurements (electron transport, photosynthesis and respiration), infra-red gas analysis (CO2 assimilation) and biochemical assays for enzymes and metabolites. In order to remove reactive oxygen species, plant cells are equipped with protective systems composed of low molecular weight antioxidants such as ascorbate, glutathione and a-tocopherol, and enzymes such as superoxide dismutases (SOD), catalases and peroxidases. Superoxide dismutases efficiently dismutate 02'- to H202 and molecular oxygen. Different isoforms of SOD, distinguished by different prostatic metals in the reaction centre, are localized in mitochondria (Mn-SOD), cytosol (CuZnSOD), peroxisomes (Mn-SOD or Fe-SOD) and chloroplasts (CuZn-SOD or Fe-SOD). In this project the total activities of these enzymes will be studied in maize leaves from plants exposed to optimal and suboptimal temperatures. Since discrete changes in the composition of isoenzyme forms with different properties may be more important than overall changes in activity, the isoenzyme profiles of the different cell types from stressed and unstressed leaves will be compared using isoelectrofocusing gels followed by activity staining, transfer to nitrocellulose and western blotting with specific antibodies. Since SODs merely transform one active oxygen species ( 02'- ) to another (H202) the effective destruction of H202 is a prerequisite for efficient protection to adverse environmental conditions. There are two related systems found in plants that destroy H202. H202 produced in peroxisomes is scavenged by catalases. In the cytosol and in chloroplasts, ascorbate-specific peroxidases fulfil this vital function. The activities and isoenzyme composition of foliar catalases and ascorbate peroxidases will be examined in maize leaves following exposure to different temperatures in order to explore the acclimatory responses of these systems to low temperatures. The amounts of the antioxidants, ascorbic acid and glutathione in the different cell types following such treatments will be measured. These measurements will allow us to determine (a) whether the photosynthetic components in the mesophyll and bundle sheath cells differ in their sensitivity to low temperatures and (b) the response of the antioxidant system to the imposition of limiting temperatures in these cell types. As transgenic plants modified in the enzymes of the antioxidant system are produced by the partners in the ESTIM project, the above techniques will be employed to characterize the effects of the transformation on the responses of the bundle sheath and mesophyll cells. The expression of the enzymes of the antioxidative defence system in the individual cell types following exposures to different temperatures in untransformed and transgenic maize will be measured. This will provide information on the molecular basis for the differential responses of the bundle sheath and mesophyll cells and allow the determination of the effects of overexpression of single enzymes of the antioxidant defences on the native forms of those enzymes in each compartment as well as pleiotropic effects on other enzymes of the pathway. It will provide essential information leading to the selection of transgenic lines to be used for field trials. 2 Contract number : FAIRCT965055 Duration : 24 Months from 02-02-97 -- Category 30 (B30)

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