Intercellular Distribution of Glutathione Synthesis in Maize Leaves and Its Response to Short-Term Chilling

To investigate the intercellular control of glutathione synthesis and its influence on leaf redox state in response to short-term chilling, genes encoding (gamma)-glutamylcysteine synthetase ((gamma)-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 (gamma)-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 (gamma)-ECS mRNA, an effect reversed during recovery. The chilling-induced increase in (gamma)-ECS transcripts was not accompanied by enhanced total leaf (gamma)-ECS protein or extractable activity. In situ hybridization and immunolocalization of leaf sections showed that (gamma)ECS and GSH-S transcripts and proteins were found in both the bundle sheath (BS) and the mesophyll cells under optimal conditions. Chilling increased (gamma)-ECS transcript and protein in the BS but not in the mesophyll cells. Increased BS (gamma)-ECS was correlated with a 2-fold increase in both leaf Cys and (gamma)-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.