We have taken advantage of an acclimation phenomenon in a chilling-sensitive maize inbred to investigate the molecular, biochemical, and physiological responses to chilling in preemergent maize seedlings. Three-day-old seedlings were exposed to 4[deg]C for 7 days and did not survive chilling stress unless they were preexposed to 14[deg]C for 3 days. cDNAs representing three chilling acclimation-responsive (CAR) genes were isolated by subtraction hybridization and differential screening and found to be differentially expressed during acclimation. Identification of one of these CAR genes as cat3, which encodes the mitochondrial catalase3 isozyme, led us to hypothesize that chilling imposes oxidative stress in the seedlings. Hydrogen peroxide levels were elevated during both acclimation and chilling of nonacclimated seedlings. Further molecular and biochemical analyses indicated that whereas superoxide dismutase activity was not affected, the levels of cat3 transcripts and the activities of catalase3 and guaiacol peroxidase were elevated in mesocotyls during acclimation. Accumulation of H2O2 following a short treatment with aminotriazole, a catalase inhibitor, indicated that catalase3 seems to be an important H2O2-scavenging enzyme in the seedlings. Control 3-day-old seedlings pretreated with H2O2 or menadione, a superoxide-generating compound, at 27[deg]C induced chilling tolerance. Both of these chemical treatments also increased cat3 transcripts and catalase3 and guaiacol peroxidase activities. We suggest that peroxide has dual effects at low temperatures. During acclimation, its early accumulation signals the production of antioxidant enzymes such as catalase3 and guaiacol peroxidase. At 4[deg]C, in nonacclimated seedlings, it accumulates to damaging levels in the tissues due to low levels of these, and perhaps other, antioxidant enzymes.

Pioneer Hi-Bred International, Johnston, lowa 50131We have taken advantage of an acclimation phenomenon in a chilling-sensitive maize inbred to investigate the molecular, blochemical, and physiological responses to chilling in preemergent maize seedlings. Three-day-old seedlings were exposed to 4OC for 7 days and did not survlve chilling stress unless they were preexposed to 14OC for 3 days. cDNAs representing three chllling acclimation-responsive (CAR) genes were isolated by subtraction hybridization and differentlal screening and found to be differentially expressed during acclimation. ldentification of one of these CAR genes as cata, whlch encodes the mitochondrial catalase3 isozyme, led us to hypothesize that chilling imposes oxidative stress ln the seedlings. Hydrogen peroxide levels were elevated during both acclimation and chilling of nonacclimated seedlings. Further molecular and biochemical analyses indicated that whereas superoxide dismutase activity was not affected, the levels of cata transcripts and the activities of catalase3 and guaiacol peroxidase were elevated in mesocotyls during acclimation. Accumulation of H202 followlng a short treatment with aminotriazole, a catalase inhibitor, indicated that catalase3 seems to be an important H202-scavenging enzyme in the seedlings. Control3-day-old seedlings pretreated with H202 or menadione, a superoxide-generating compound, at 27OC induced chilling tolerance. 90th of these chemical treatments also increased cat3 transcripts and catalase3 and guaiacol peroxidase activities. We suggest that peroxide has dual effects at low temperatures. During acclimation, its early accumulation signals the production of antioxidant enzymes such as catalase3 and guaiacol peroxidase. At 4OC, in nonacclimated seedlings, it accumulates to damaging levels in the tissues due to low levels of these, and perhaps other, antioxidant enzymes.

l h e response of antioxidants to acclimation and chilling in various tissues of dark-grown maize (Zea mays 1.) seedlings was examined in relation t o chilling tolerance and protection from chillinginduced oxidative stress. Chilling caused an accumulation of H,O, in both the coleoptile + leaf and the mesocotyl (but not roots), and acclimation prevented this accumulation. None of the antioxidant enzymes were significantly affected by acclimation or chilling i n the coleoptile + leaf or root. However, elevated levels of glutathione i n acclimated seedlings may contribute to an enhanced ability to scavenge H 2 0 2 i n the coleoptile + leaf. I n the mesocotyl (visibly most susceptible to chilling), catalase3 was elevated in acclimated seedlings and may represent the first line of defense from mitochondria-generated H,O,. Nine of the most prominent peroxidase isozymes were induced by acclimation, two of which were located in the cell wall, suggesting a role in lignification. Lignin content was elevated i n mesocotyls of acclimated seedlings, likely improving the mechanical strength of the mesocotyl. One cytosolic glutathione reductase isozyme was greatly decreased in acclimated seedlings, whereas two others were elevated, possibly resulting i n improved effectiveness of the enzyme at low temperature. When taken together, these responses to acclimation illustrate the potential ways in which chilling tolerance may be improved in preemergent maize seedlings.Stand establishment in maize (Zea mays L.) is greatly affected by exposure of seedlings to low temperature during germination and early seedling growth. Pioneer inbred G50 is particularly sensitive. By utilizing dark-grown seedlings to simulate a preemergent condition, Anderson et al. (1994) demonstrated that exposure of seedlings to a noninjuriou low temperature induced a degree of chilling tolerance, allowing them to survive subsequent exposure to more severe low temperatures. This acclimation phenomenon provided an opportunity to examine the molecular basis for the improvement in chilling tolerance. The discovery that cat3 (mitochondrial catalase) transcripts were up-regulated in response to acclimation led to inves-

Summary The mechanisms involved in chilling injury or tolerance in developing maize seedlings subjected to low temperature stress have been investigated. Although acclimation‐induced chilling tolerance was developmentally regulated, no tolerance was observed in non‐acclimated developing seedlings subjected to 7 days of 4°C stress. Consistent with previous results, chilling induced oxidative stress in the seedlings. Whereas acclimated seedlings had elevated levels (35–120%) of antioxidant enzymes, such as catalase, glutathione reductase and guaiacol peroxidase, non‐acclimated seedlings had lower levels of these enzyme activities during chilling conditions. The increase in the activity of mitochondrial catalase3 isozyme, a major anti‐oxidant enzyme in maize, was due to an increase in cat3 transcripts and CAT3 protein, as indicated by Northern and Western blot analyses. Besides, non‐acclimated seedlings had increased levels of oxidized proteins (two to threefold) and lipids (35–65%) during chilling and recovery compared with control or acclimated seedlings. Low‐temperature stress also inhibited two proteases, endopeptidase and aminopeptidase, by 35% in non‐acclimated seedlings. Since seedlings treated with prooxidants also promoted the oxidation of proteins and lipids and the inhibition of proteases, it was concluded that in vivo generated oxidative stress was likely responsible for affecting these processes. These results provide a correlative evidence to suggest that: (i) in non‐acclimated seedlings, chilling injury was partly due to the build up of reactive oxygen species (ROS) that promoted the oxidation of proteins and lipids and inhibited protease activity; (ii) in acclimated seedlings, chilling tolerance was partly due to an enhanced anti‐oxidant system that prevented the accumulation of ROS and therefore, prevented the oxidation of proteins and lipids and the inhibition of protease activities during 4°C stress and recovery.

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