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...
The pipetting ofpear (Pyrus communis cv Bartlett) suspension cultures was followed by a substantial but transient decrease in heat sensitivity. During a culture cycle, pear cells were most sensitive to heat at day 3, which coincided with the period of most active cell division. To minimize serious artifacts, the influence of culture handling and age on parameters such as heat sensitivity must be standardized.In the study of complex phenomena such as temperature response, the convenience, simplicity, and uniformity of cell cultures make them valuable experimental systems. Although effects ofheat stress on photosynthesis are critical to whole plants, the absence of chloroplasts in simple cell cultures may further facilitate close examination of other metabolic responses. In considering tissue cultures as model systems, it is important to note that certain aspects of cultured cell response to heat are similar to those of intact plant tissues. Heat stress disrupts polysomes in cells of intact pear fruit (10) and in cultured pear fruit cells (Romani, personal communication). In both intact organs and tissue cultures, heat shock induces acclimation (8,12) and the synthesis of heat-shock proteins (1, 7). However, it is clear that the isolation and culture of plant cells alters physiological characteristics which may influence high temperature response. For example, batch-propagated suspension cultures are characterized by stages ofrelatively high mitotic activity (5). This is relevant to the study of heat stress because certain dividing cells are especially susceptible to heat injury (9, 1 1). Therefore, it is necessary to consider the relationship of culture age to heat sensitivity. Another inherent feature of tissue culture systems is the routine handling and manipulation involved in the transfer and treatment ofcells. Tissue handling has been shown to induce subtle but substantial metabolic changes (13,14), some ofwhich could influence heat tolerance.In this paper, we describe effects of suspension culture age and handling of the heat tolerance of pear cells. (13,14). Our procedure (15) involved pipetting aliquots to test tubes for imposition of heat stress. We found that handling (transfer by pipette) influenced heat sensitivity (Fig. 1)
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