Intercellular Distribution of Glutathione Synthesis in Maize Leaves and Its Response to Short-Term Chilling<sup><a href="#fn1" rid="fn1" class=" fn">1</a></sup>
Abstract
To investigate the intercellular control of glutathione synthesis and its influence on leaf redox state in response to short-term chilling, genes encoding γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GSH-S) were cloned from maize (Zea mays) and specific antibodies produced. These tools were used to provide the first information on the intercellular distribution of γ-ECS and GSH-S transcript and protein in maize leaves, in both optimal conditions and chilling stress. A 2-d exposure to low growth temperatures (chill) had no effect on leaf phenotype, whereas return to optimal temperatures (recovery) caused extensive leaf bleaching. The chill did not affect total leaf GSH-S transcripts but strongly induced γ-ECS mRNA, an effect reversed during recovery. The chilling-induced increase in γ-ECS transcripts was not accompanied by enhanced total leaf γ-ECS protein or extractable activity. In situ hybridization and immunolocalization of leaf sections showed that γ-ECS and GSH-S transcripts and proteins were found in both the bundle sheath (BS) and the mesophyll cells under optimal conditions. Chilling increased γ-ECS transcript and protein in the BS but not in the mesophyll cells. Increased BS γ-ECS was correlated with a 2-fold increase in both leaf Cys and γ-glutamylcysteine, but leaf total glutathione significantly increased only in the recovery period, when the reduced glutathione to glutathione disulfide ratio decreased 3-fold. Thus, while there was a specific increase in the potential contribution of the BS cells to glutathione synthesis during chilling, it did not result in enhanced leaf glutathione accumulation at low temperatures. Return to optimal temperatures allowed glutathione to increase, particularly glutathione disulfide, and this was associated with leaf chlorosis.
Stress survival strategies are essential for sedentary organisms, and evolution has conferred on plants a high degree of plasticity that underpins survival in a constantly changing environment (Pastori and Foyer, 2002). One of the most important and fluctuating environmental factors limiting the geographic distribution of plant species is temperature, and the rapidity with which plants can respond to temperature changes is key to growth and reproductive success (Levitt, 1962). Plants vary greatly in their ability to tolerate low growth temperatures, and they have different capacities for regulating metabolism and maintaining redox homeostasis at low temperatures. Species can be classified according to the threshold below which chilling injury is observed. Chilling-sensitive plants usually show decreased photosynthesis and growth between 10°C and 15°C (Levitt, 1962). Many cereals are able to withstand long periods of subzero temperatures by virtue of the process called cold acclimation (Hughes and Dunn, 1990). However, maize (Zea mays), a C4 species of subtropical origin, is chilling sensitive and shows little or no cold acclimation traits.
The chilling sensitivity of maize leaves is likely related to their high degree of cellular specialization. Maize leaves have Kranz anatomy and show extreme cellular differentiation between bundle sheath (BS) and mesophyll (M) cells, which have specialized metabolic roles. The Benson-Calvin cycle is restricted to the BS cells, minimizing photorespiration and allowing increased carbon, nitrogen, and water use efficiency. However, BS cells produce relatively little reductant, and, thus, antioxidant enzymes that require NADH or NADPH are restricted to the maize M cells, rendering BS proteins more susceptible to oxidative damage (Kingston-Smith and Foyer, 2000). The extreme structural and functional specialization of the BS and M cells requires rapid intercellular exchange of metabolites (Leegood, 1985), including antioxidants (Doulis et al., 1997), and this poses important problems for the operation and regulation of photosynthesis and antioxidant metabolism at low temperatures (Kingston-Smith et al., 1999). We have extensively investigated the mechanism of the sensitivity of photosynthesis to chilling in the H99 variety (Doulis et al., 1997; Kingston-Smith et al., 1999; Kingston-Smith and Foyer, 2000; Pastori et al., 2000a, 2000b), which we have chosen to study because it is a chilling-sensitive, dent-type maize that is relatively easy to transform (Van Breusegem, 1998).
Chilling resistance requires effective up-regulation of the antioxidant system because limitations on leaf metabolism at low temperature promote increased electron transport to oxygen and hence increased production of active oxygen species, including superoxide, H2O2, and hydroxyl radicals (Foyer and Harbinson, 1994; Fryer et al., 1998). Avoidance of active oxygen species-induced cell death and senescence requires efficient antioxidant protection (Foyer and Noctor, 2000). Among the battery of antioxidants found in leaves, the tripeptide thiol glutathione (GSH; γ-Glu-Cys-Gly) is strongly implicated in chilling tolerance, particularly in maize (Kocsy et al., 1996, 2000a, 2000b, 2001a, 2001b). Glutathione is the major thiol redox buffer in the soluble phase of most aerobic cells and in plants is involved in redox signaling, modulation of enzyme activity, control of root development, and in processes that modify and transport hormones and other endogenous compounds and xenobiotics, via formation of glutathione S-conjugates (May et al., 1998a; Noctor and Foyer, 1998a). Especially at low temperatures, the cyclic interconversion of dithiols and disulfides is key to cell defense processes (Levitt, 1962; Kunert and Foyer, 1993).
In all organisms studied so far, biosynthesis of GSH occurs from constituent amino acids via γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GSH-S; Hell and Bergmann, 1988, 1990; Meister, 1988; Rennenberg, 1997). In recent years, studies in model systems have increased our understanding of how glutathione synthesis is controlled. Genes encoding the above enzymes have been identified from a number of C3 dicotyledonous plants such as Arabidopsis, tomato (Lycopersicon esculentum), Brassica juncea, and Medicago trunculata. In Arabidopsis γ-ECS appears to be encoded by a single gene, gsh1, while the gene that encodes GSH-S produces both cytosolic and plastidic isoforms by alternative mRNA splicing. All the plant enzymes display high sequence homology. Plant transformation and expression studies in C3 plants have identified two main factors likely to control the accumulation of GSH: γ-ECS abundance and Cys availability (Noctor et al., 1996, 1998a, 1998b, 2002; Xiang and Oliver, 1998; Xiang and Bertrand, 2000; Xiang et al., 2001). Despite the importance of this thiol in chilling stress, there is limited knowledge of the factors that determine or limit glutathione accumulation and redox potential in these conditions. In maize, our knowledge of the regulation of glutathione biosynthesis during the chilling response is limited by the lack of gene sequences for any C4 or monocotyledonous species and by the absence of expression data for γ-ECS and GSH-S during chilling. The regulation of glutathione synthesis in maize is further complicated by data that suggest that, whereas Cys synthesis occurs exclusively in the BS, GSH-S activity is located primarily in the M cells (Burgener et al., 1998). No data has yet appeared on γ-ECS localization in maize leaves.
Leaf glutathione contents correlate with chilling resistance in maize (Kocsy et al., 1996, 2000a, 2000b, 2001a, 2001b). Furthermore, our previous data suggest that one factor in the sensitivity of maize to both short-term and long-term chilling may be restrictions over glutathione reduction and cycling between cell types (Doulis et al., 1997; Pastori et al., 2000a). The aims of this study were (1) to investigate the intercellular distribution of the expression of γ-ECS and GSH-S in maize leaves and (2) to explore how these enzymes and glutathione content and redox state respond to short-term chilling stress.
Extractable leaf activities are shown for plants maintained at 25°C (day)/19°C (night; control) or transferred to 10°C/8°C for 2 d (chill). All activities are expressed as nmol mgprotein min and are means ± se of three independent extracts from different plants.
Notes
This work was supported by the National Research Council, Argentina (Consejo Nacional de Investigaciones Científicas y Técnicas; fellowship to L.D.G.) and the Biotechnology and Biological Sciences Research Council, UK.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.033027.