Post-Transcriptional Regulation Prevents Accumulation of Glutathione Reductase Protein and Activity in the Bundle Sheath Cells of Maize^{<a href="#FN1" rid="FN1" class=" fn">1</a>}

Abstract

Glutathione (γ-glutamyl cysteinyl Gly) is a versatile regulator of cell metabolism and function (Rennenberg, 1982). Essential for plant growth and development, this antioxidant is a key cellular redox component that functions in the regulation of gene expression and the cell cycle (for review, see Noctor et al., 1998). The reduced glutathione (GSH)/oxidized glutathione (GSSG, or glutathione disulfide) redox couple is involved in the expression of defense genes (Dron et al., 1988; Wingate et al., 1988), in sulfur metabolism by regulation of sulfur uptake in the roots (Herschbach and Rennenberg, 1994; Lappartient and Touraine, 1996), in the detoxification of xenobiotics through GSH S-transferases (Lamoreaux and Rusness, 1993; Marrs, 1996), and in the redox control of cell division (Russo et al., 1995; Sanchez-Fernandez et al., 1997).

The enzyme glutathione reductase (GR; EC 1.6.4.2) is pivotal to the function of the glutathione system in eukaryotic cells (Noctor et al., 1998). This flavoprotein oxidoreductase catalyzes the reduction of GSSG to GSH in a NADPH-dependent reaction. GR has a central role in maintaining GSH within the cellular environment, particularly during stress. Most, if not all, stresses include an oxidative stress component (Wise and Naylor, 1987; Hodgson and Raison, 1991; McKersie, 1991; Prasad et al., 1994; Wise, 1995) that leads to tissue damage if antioxidative defenses are insufficient. Chilling sensitivity in maize (Zea mays) leaves has been linked to the antioxidant status of the cells (Doulis et al., 1997), while interspecific variations in cold tolerance have been correlated with antioxidant capacity (Jahnke et al., 1991; Kocsy et al., 1996; Prasad, 1996, 1997). In addition, chilling has been shown to cause H₂O₂ accumulation in the leaves of cereals including maize (Okuda et al., 1991; Kingston-Smith et al., 1998). GR is considered to be an important factor limiting the degree of photodamage experienced by maize leaves upon exposure to chilling temperatures (Jahnke et al., 1991; Massacci et al., 1995; Hodges et al., 1997; Leipner et al., 1997; Fryer et al., 1998).

Maize is one of the most important crops worldwide. Since it originated in tropical regions, it is not surprising that it is particularly sensitive to low-temperature stress. The optimal growth temperature for maize is between 20°C and 30°C. In northern Europe and other areas, suboptimal temperatures that cause chilling-induced damage are frequently encountered early in the mornings. The combination of high light intensities and low temperatures, such as those experienced on cold but sunny mornings in the spring, can cause dramatic damage to young maize seedlings (Fryer et al., 1998). Stress tolerance has therefore become a major selection criterion in maize breeding programs. The damage caused to mature and developing leaves by low-temperature stress occurs primarily in the chloroplasts, leading to inhibition of photosynthesis and premature senescence (Nie and Baker, 1991; Nie et al., 1992, 1995). Studies on the relationships between CO₂ assimilation, photosynthetic electron transport, and antioxidant enzyme activities in field-grown maize suggest that the donation of electrons to oxygen by the photosynthetic electron transport chain is increased by growth at low temperatures (Fryer et al., 1998).

The differential partitioning of antioxidants between photosynthetic cell types may be central to the inherent low-temperature sensitivity of maize (Doulis et al., 1997; Burgener et al., 1998) and to the sensitivity of proteins in the bundle sheath cells to oxidative damage (Kingston-Smith and Foyer, 2000). Maize has a specialized leaf anatomy that encompasses the C₄ photosynthetic cycle in addition to the C₃ pathway (Furbank and Foyer, 1988). The initial steps of CO₂ assimilation in the mesophyll cells are spatially separated from the enzymes of the Benson-Calvin cycle in the bundle sheath cells (Hatch and Osmond, 1976; Furbank and Foyer, 1988; Furbank and Taylor, 1995). Similarly, components of the antioxidant system are differentially distributed between the bundle sheath and the mesophyll cells in maize leaves (Doulis et al., 1997). GR activity has been found to be almost exclusively localized in the mesophyll cells, whereas ascorbate peroxidase and superoxide dismutase are largely absent from the mesophyll fraction. The enzymes of assimilatory sulfate reduction and GSH synthesis are also differentially compartmented between the bundle sheath and mesophyll cells (Burgener et al., 1998). ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase are localized in the bundle sheath cells, whereas GSH synthetase, Cys, γ-glutamyl-Cys, and GSH are found mainly in the mesophyll cells (Burgener et al., 1998).

The differential partitioning of antioxidants between bundle sheath and mesophyll cells has been explained in terms of the availability of reducing power and NADPH, because the bundle sheath cells are depleted in NADPH-producing capacity (Furbank and Foyer, 1988; Doulis et al., 1997). GR activity requires NADPH, so it is not surprising that it is localized in the mesophyll cells, where the availability of NADPH is sufficient for catalysis. The molecular mechanisms that determine this cell-specific partitioning of GR activity in maize leaves are largely unexplored, and the localization of the GR protein and GR mRNA in maize leaves is unknown. The aim of this work was to elucidate factors determining the intercellular distribution of GR between bundle sheath and mesophyll cells in maize leaves and to determine whether these phenomena are related to the low-temperature sensitivity of many maize genotypes.

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