Molecular and biochemical investigation of the mechanisms of acclimation to chilling stress in maize seedlings

Seedlings of chilling-sensitive maize inbred G50 (Pioneer) survived poorly when exposed to a chilling stress. Acclimation or ABA treatment caused a dramatic improvement of chilling tolerance. Differences in gene expression between acclimated and unacclimated seedlings were investigated and three cDNA clones (designated car333, car30, and car757) were isolated, representing transcripts that were up-regulated during acclimation. Car30 and car757 were not homologous to any known sequence but car333 was 97.8% homologous to cat3, maize mitochondrial catalase. The up-regulation of CAT3 during acclimation suggested that chilling stress caused the generation of H[subscript]2O[subscript]2 in the mitochondria and increased catalase was needed to prevent oxidative damage. Measurement of H[subscript]2O[subscript]2 revealed a 4-fold increase in the coleoptile+leaf and mesocotyl of chilled seedlings but H[subscript]2O[subscript]2 was maintained near unchilled levels in seedlings that were acclimated before chilling. In effort to explain the acclimation-induced protection from oxidative stress, isozyme profiles of catalase, guaiacol peroxidase, superoxide dismutase, ascorbate peroxidase, and glutathione reductase were examined. None of the enzymes responded to acclimation in the coleoptile+leaf. However, examination of ascorbate and glutathione pools revealed an increased synthesis of glutathione, which might be responsible for the protection of the coleoptile+leaf from oxidative stress. In the mesocotyl, superoxide dismutase and ascorbate peroxidase were unaffected but catalase, peroxidase, and glutathione reductase were altered by acclimation. It was proposed that induction of CAT3 may serve as the first line of defense against mitochondria-generated H[subscript]2O[subscript]2. Induction of cell wall peroxidases may be responsible for the observed increase in lignin content, which would serve to improve the mechanical strength of mesocotyls and allow them to remain intact when exposed to a chilling stress. Cytosolic glutathione reductase may have been shifted to forms that were more active at low temperature. All of these changes illustrated the numerous ways that chilling tolerance was enhanced in the mesocotyl. In contrast to acclimation, the response of antioxidant enzymes to ABA was dramatically different. Despite the fact that both ABA and acclimation induced chilling tolerance, the mechanisms of action were clearly different, at least in terms of antioxidant defenses