During exposure to 2 OC, pea (Pisum sativum) seedlings cold acclimated to a killing temperature of -6 OC. Associated with this increase in freezing resistance was an increase in the weight of cell walls and changes in wall composition. Arabinosyl content increased by 100%, while other cell wall glycosyl residues and cellulose increased by about 20%. The cell wall hydroxyproline content increased by 80%. Arabinose and hydroxyproline are both major components of the structural cell wall glycoprotein, extensin. The increase in these components indicates that the level of extensin in the cell wall increases during cold acclimation. Northern blot analysis, using the pDC5A1 genomic clone as a probe, revealed a more than three-fold increase in total extensin mRNA during exposure to cold temperature. Specific extensin transcripts of 6.0, 4.5, 3.5, 2.6, 2.3, 1.8, and 1.5 kilobases were identified. Those at 6.0, 2.6, and 1.5 kilobases were especially promoted by low temperature treatment. The rise in extensin during cold acclimation may be regulated, at least in part, at the gene level. The possible structural role of this protein in freezing protection is discussed.During extracellular freezing, plant cells are exposed to stresses associated with dehydration and consequent volume reduction, direct effects of low temperature, and mechanical effects of extracellular ice. The plasmalemma response to protoplast freeze-thaw has been characterized by Steponkus (22) A cell wall change which could significantly alter relevant mechanical properties is the increased deposition of the glycoprotein extensin, a change known to be stress induced (27). A structural role for extensin was first proposed by Lamport and Northcote (13). The glycoprotein they discovered, later named extensin for its putative role in cessation of extension growth, was found to contain a great portion of the hydroxyproline in the cell. Extensin is viewed to contribute to the strength and rigidity of the cell wall by forming an interpeptide-linked network that is separate from, but complementary to the cellulose mesh (14).It was hypothesized that if structural changes in the cell wall increase its rigidity during acclimation, thereby possibly limiting freeze-induced cell water loss and resulting injury, then an increase in freezing resistance should occur. Data presented here quantify: cell wall weight, total glucan, noncellulosic glucan, cellulose, and glycosyl content during acclimation. A pronounced increase in arabinose content led us to suspect an increase in extensin. Cell wall hydroxyproline content and extensin mRNA were measured during acclimation to test this theory. MATERIALS AND METHODSUnless otherwise indicated, the conditions for growth, acclimation, and freezing tolerance measurement were as follows. Experiments were conducted with seedlings of Pisum sativum, cultivars 'Alaska' and 'Melrose.' In most experiments, seeds were germinated and seedlings grown in the dark at 22 C, using vermiculite moistened with deionized water. In certain ca...

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Antioxidant metabolism has been examined in red spruce growing at two sites with similar levels of ozone pollution but different winter temperatures. The northern site was Whiteface Mountain, New York with a latitude of 44° 3r (NY). The southern site was Whitetop Mountain, Virginia with a latitude of 36° 54' (VA). The northern site has lower winter temperatures. At the NY site two populations of trees were defined with respect to cold tolerance. An eflfort was made to establish a temporal relationship between the antioxidant capacity of conifer needles on one hand and the different stages of winter hardening/dehardening, and the induction of dormancy, on the other. The hypothesis tested was that winter injury of red spruce was due to an increased demand for glutathione at levels exceeding the inherent capacity of the needles and/or the altered activities of glutathione reductase. In the autumn, as temperatures began to decrease, photosynthetic potential decreased as spruce entered dormancy. At the same time, cold tolerance (measured as needle electrolyte leakage) gradually increased. Glutathione metabolism appeared to be coupled to these processes. At both sites, seasonal changes in glutathione levels were observed. These changes were found to coincide temporally with the onset and cessation of dormancy (as measured by decline in pbotosynthetic activity). The same pattern was observed at the northern and the southern site with the winter increases being greater at the former. Glutathione began to increase as soon as needles entered dormancy and began hardening. Glutathione reductase activities closely followed the dehardenmg process during spring, while glutathione concentration continued to stay relatively high with prehardening levels reached only by summer. At the northern site during the winter, cold-tolerant trees had significantly higher glutathione reductase activities compared with cold-sensitive ones. Based on these data, it is proposed that individuals that cannot respond to the increased demand for glutathione reductase activity during the hardening, winter and dehardening periods, become susceptible to winter injury.

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