Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance

Abstract

Cold acclimation in plants is associated with the expression of COR (cold-regulated) genes that encode polypeptides of unknown function. It has been widely speculated that products of these genes might have roles in freezing tolerance. Here we provide direct evidence in support of this hypothesis. We show that constitutive expression of COR15a, a cold-regulated gene of Arabidopsis thaliana that encodes a chloroplast-targeted polypeptide, enhances the in vivo freezing tolerance of chloroplasts in nonacclimated plants by almost 2 degrees C, nearly one-third of the increase that occurs upon cold acclimation of wild-type plants. Significantly, constitutive expression of COR15a also affects the in vitro freezing tolerance of protoplasts. At temperatures between -5 and -8 degrees C, the survival of protoplasts isolated from leaves of nonacclimated transgenic plants expressing COR15a was greater than that of protoplasts isolated from leaves of nonacclimated wild-type plants. At temperatures between -2 and -4 degrees C, constitutive expression of COR15a had a slight negative effect on survival. The implications of these data regarding possible modes of COR15a action are discussed.

Figure 2

Freezing tolerance of chloroplasts in A. thaliana RLD, T8, and T9 plants. Leaves from nonacclimated (NA) and cold-acclimated (CA) plants were subjected to a freeze–thaw cycle to various subzero temperatures and damage to chloroplasts was assessed by determining F_v/F_m. (A) Nonacclimated (n = 6) and cold-acclimated (n = 2) RLD and T8 plants. (B) Nonacclimated (n = 4) RLD and T9 plants and cold-acclimated (n = 2) RLD plants. Each experiment (n) included at least three replicate samples per point.

Figure 3

Freeze-induced damage to chloroplasts in F₃ lines of nonacclimated A. thaliana plants that either did (diagonal bars) or did not (solid bars) constitutively express COR15a. Leaves from nonacclimated plants were frozen to either −5°C or −6°C, thawed, and damage to chloroplasts was assessed by determining F_v/F_m. The percentage decrease in F_v/F_m was determined by dividing the mean F_v/F_m values for the frozen leaves (values presented in Table 1) by the mean F_v/F_m values for nonfrozen leaves (and multiplying by 100). The values for the nonfrozen leaf samples for COR15am positive and negative lines (six lines for each; three leaves for each line) were 0.808 ± 0.005 and 0.804 ± 0.004, respectively.

Figure 1

Presence of COR15am in the soluble protein fraction of chloroplasts isolated from A. thaliana RLD and T8 plants. Chloroplasts were purified from the leaves of nonacclimated (NA) and cold-acclimated (CA) T8 and RLD plants. Total soluble (25 μg) protein was fractionated by tricine SDS/PAGE. COR15am was detected by immunoblot analysis.

Figure 4

Effects of constitutive COR15a expression on the freezing tolerance of protoplasts isolated from leaves of nonacclimated A. thaliana plants. Isolated protoplasts were frozen to the indicated temperatures and thawed; survival was determined by staining with FDA (expressed as a percentage of the unfrozen control). Three hemocytometer samples were counted for each temperature in a given experiment. The results shown are the average and SD of individual experiments. (A) Transgenic T8 and wild-type RLD plants. (B) Plants that constitutively express COR15a (T8, T9, 1-5, and 2-11) and those that do not [RLD, GUS (35S–GUS), 1-11, and 2-5].