Induction of Freezing Tolerance in Spinach Is Associated with the Synthesis of Cold Acclimation Induced Proteins

Abstract

Spinach (Spinacia okracca L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold accfimation. Seedlings grown for 3 weeks postsowing on an inorganic-nutrientagar medium were able to increase their freezing tolerance when grown at 5°C. During cold acclimation at 5°C and deaccfimation at 25°C, the kinetics of freezing tolerance induction and loss were similar to that of soil-grown plants. Freezing tolerance increased after 1 day of cold acclimation and reached a maximum within 7 days. Upon deacclimation at 25°C, freezing tolerance declined within 1 day and was largely lost by the 7th day. Leaf proteins of intact plants grown at 5 and 25°C were in vivo radiolabeled, without wounding or injury, to high specific activities with 3ISimethionine. Leaf proteins were radiolabeled at 0, 1, 2, 3, 4, 7, and 14 days of cold acclimation and at 1, 3, and 7 days of deacclimation. Up to 500 labeled proteins were separated by two-dimensional gel electrophoresis and visnalized by fluorography. A rapid and stable change in the protein synthesis pattern was observed when seedlings were transferred to the low temperature environment. Cold-acclimated leaves contained 22 polypeptides not found in nonaccimated leaves. Exposure to 5°C induced the synthesis of three high molecular weight cold acclimation proteins (CAPs) (M, of about 160,000, 117,000, and 85,000) and greatly increased the synthesis of a fourth high molecular weight protein (M, 79,000). These proteins were synthesized during day 1 and throughout the 14 day exposure to 5°C. During deaccimation, the synthesis of CAPs 160, 117, and 85 was greatly reduced by the first day of exposure to 25°C. However, CAP 79 was synthesized throughout the 7 day deacclimation treatment. Thus, the induction at low temperature and termination at warm temperature of the synthesis of CAPs 160, 117, and 85 was highly correlated with the induction and loss of freezing tolerance. Cold acclimation did not result in a general posttranslational modification of leaf proteins. Most of the observed changes in the two-dimensional gel patterns could be attributed to the de novo synthesis of proteins induced by low temperature. In spinach leaf tissue, heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs). One polypeptide synthesized in cold-acclimated leaves had a molecular weight and net charge (M, 79,000, pl 4.8) similar to that of a HSP (M, 83,000, pl 4.8). However, heat shock did not increase the freezing tolerance, and cold acclimation did not increase heat tolerance over that of nonacclimated plants, but heat-shocked leaf tissue was more tolerant to high temperatures than nonacclimated or cold-acclimated leaf tissue. When protein extracts from heat-shocked and cold-acclimated leaves were mixed and separated in the same two-dimensional gel, the CAP and HSP were shown to be two separate polypeptides with slightly different isoelectric points and molecular weights.

Association of 70-Kilodalton Heat-Shock Cognate Proteins with Acclimation to Cold

“…After focusing, the fractions were collected and pH values determined. SDS-PAGE analyses revealed that free-solution-isoelectric focused fractions closely approximated analytical two-dimensional separations (12) for spinach cold acclimation proteins, which facilitated identification of fractions containing CAP79. 2 Fractions most enriched for CAP79 were selected for further purification.…”

“…Spinach seedlings (Spinacia oleracea L. cv Bloomsdale) were grown from seed in a controlled environment as previously described (12). Cold acclimation and deacclimation treatments were conducted with a 12-h photoperiod as previously described (12).…”

“…Cold acclimation and deacclimation treatments were conducted with a 12-h photoperiod as previously described (12).…”

“…For example, unlike the heat-shock response (3), plants exposed to low temperature continue synthesis of the vast majority of housekeeping proteins while simultaneously in-ducing or upregulating the synthesis of selected proteins (12). The purpose of this increased expression of certain proteins by plants in response to low temperature is not understood.…”

Cold Acclimation in Arabidopsis thaliana

“…In this regard, however, it is interesting to note the work of Guy et al (7,9). These investigators have shown that gene expression is altered during cold hardening of spinach and that one of the 'cold acclimation proteins' was a large acidic polypeptide with a mol wt of about 160 kD and pI about 4.5.…”