Differential Gene Expression in Chilling-Acclimated Maize Seedlings and Evidence for the Involvement of Abscisic Acid in Chilling Tolerance

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Abstract
An acclimation phenomenon was characterized in seedlings of chilling-sensitive maize (Zea mays L.) inbred G50 (Pioneer). Seedlings were germinated at 27[deg]C for 3 d and then exposed to chilling treatments of 4, 5, or 6[deg]C for 2, 4, 7, or 10 d in darkness. Damage symptoms in the more severe treatments included a waterlogged appearance and a discoloration of the tissue. The symptoms were most obvious in the mesocotyl. After a 10-d grow-out period in the greenhouse, moderately damaged seedlings exhibited chlorotic areas, an occasional disruption in leaf expansion, and a constriction of the mesocotyl. Growth and survival were improved by first exposing seedlings to a 14[deg]C acclimation treatment for 3 d before applying the chilling treatment. After chilling at 5[deg]C for 7 d, 79% of the acclimated seedlings survived, whereas only 22% of the nonacclimated seedlings survived. Differences in gene expression between acclimated and control seedlings were investigated using subtraction and differential screening techniques. Transcripts corresponding to three genes, car333, car30, and car757 (chilling acclimation responsive), were present in higher levels in seedlings after acclimation. Sequence analysis identified car333 as cat3, which encodes maize mitochondrial catalase isozyme 3. Characterization of these three clones revealed that all corresponding transcripts were elevated in acclimated seedlings in a manner that depended on the organ, i.e. coleoptile, mesocotyl, or root. Although transcripts were elevated in all three organs in response to acclimation, car30 was most abundant in the coleoptile and root, whereas cat3 and car757 were most abundant in the coleoptile and mesocotyl. Catalase activity followed the same general trend as cat3 transcript levels. Exogenous treatment with abscisic acid (ABA) resulted in an improvement in growth and survival of nonacclimated, chilled seedlings. Inhibition of ABA biosynthesis with fluridone abolished acclimation-induced chilling tolerance, and exogenous application of ABA to fluridone-treated seedlings restored chilling tolerance. Exogenous ABA treatment also resulted in increases in cat3, car30, and car757 transcript levels and catalase activity in the same organ-specific manner as in acclimated seedlings. These results indicate that ABA synthesis is essential for chilling tolerance. However, measurement of ABA levels in mesocotyls during acclimation and chilling revealed only a marginal increase during acclimation and a dramatic increase during chilling, regardless of whether or not seedlings were acclimated. Thus, although ABA may be required for chilling tolerance, we have no conclusive evidence that the acclimation process is mediated by ABA.