All organisms studied to date respond to high temperatures by synthesizing a group of proteins known as heat shock proteins (HSPs). HSPs are divided into five classes based on their molecular weight: HSP100, HSP90, HSP70, HSP60, and small HSPs (sHSPs). sHSPs are ≈15 to 42 kDa in size and are characterized by a conserved α-crystallin domain of the vertebrate eye lens at their C-terminus (Haslbeck et al., 2005). In plants, sHSPs are the most abundant and diverse HSPs produced at high temperatures. The induction of sHSPs under heat stress and the correlation between sHSP accumulation and enhanced thermotolerance have been well established in a number of previous studies (Haslbeck et al., 2005; Sun et al., 2002). Furthermore, some sHSPs are also known to be induced by various abiotic stresses such as cold, salinity, drought, and chemical pollution (Vinocur and Altman, 2005).
Cold-induced accumulation of sHSPs and/or their transcripts has been reported in some plant species. An sHSP in sweet chestnut (Castanea sativa L.), CsHsp17.5, was constitutively expressed in stem and was upregulated by both high and low temperatures (Lopez-Matas et al., 2004; Soto et al., 1999). A more recent report showed that a rose sHSP (Rosa chinensis L.) was not expressed in the absence of stress but was induced under a cold stress condition (2 °C, 6 h; Jiang et al., 2009). These studies suggest that at least some plant sHSPs may confer cold tolerance.
A number of studies have shown that sHSPs function as molecular chaperones preventing heat-induced protein denaturation and promoting refolding of partially denatured proteins. Some plant sHSPs prevented thermal aggregation of model substrates such as malate dehydrogenase and citrate synthase (Lee et al., 1995, 1997). In vivo, Arabidopsis Hsp17.6 maintained firefly luciferase activity in an Arabidopsis cell suspension culture under heat stress (Forreiter et al., 1997). Under cold stress, which also causes protein denaturation in plant cells (Thomashow, 1999), it is possible that cold-induced sHSPs may function as molecular chaperones enhancing protein solubility. However, the precise functional mechanism of sHSPs under cold stress remains to be studied.
The carrot (Daucus carota L.) sHSP, DcHsp17.7, has been characterized in previous studies. Its constitutive expression enhanced cell viability and membrane stability in transgenic carrot cells and plants under heat stress (Malik et al., 1999). Furthermore, the expression of an antisense construct of the DcHsp17.7 gene resulted in reduced thermotolerance. This was the first demonstration that modification of the expression of a single sHSP can both increase and decrease thermotolerance in plants. When introduced into the cool-season crop potato (Solanum tuberosum L.), DcHsp17.7 enhanced cellular membrane stability and tuberization in vitro (Ahn and Zimmerman, 2006). More recently, heterologous expression of DcHsp17.7 in Escherichia coli showed that it functions as a molecular chaperone preventing aggregation of proteins and enhances cell viability under heat stress (Kim and Ahn, 2009).
In this study, we examined the expression and functional mechanism of DcHsp17.7 under the opposite temperature stress, the cold. DcHsp17.7 transcript and protein levels induced by low temperature were measured using semiquantitative reverse transcription polymerase chain reaction (PCR) and immunoblot analysis, respectively. Furthermore, to study the function of DcHsp17.7, the gene was introduced into E. coli. Under cold stress, cell viability and protein solubility of transformed E. coli expressing DcHsp17.7 were compared with those of controls. In addition, cold-induced dynamic association and dissociation of DcHsp17.7 complexes was also observed.
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Jiang, C. Xu, J. Zhang, H. Zhang, X. Shi, J. Li, M. Ming, F. 2009 A cytosolic class I small heat shock protein, RcHSP17.8, ofPlant Cell Environ. Rosa chinensisconfers resistance to a variety of stresses to Escherichia coli, yeast and Arabidopsis thaliana 32 1046 1059
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Lopez-Matas, M.-A. Nunez, P. Soto, A. Allona, I. Casado, R. Collada, C. Guevara, M.-A. Aragoncillo, C. Gomez, L. 2004 Protein cryoprotective activity of a cytosolic small heat shock protein that accumulates constitutively in chestnut stems and is up-regulated by low and high temperaturesPlant Physiol. 134 1708 1717
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Soto, A. Allona, I. Collada, C. Guevara, M.-A. Casado, R. Rodriguez-Cerezo, E. Aragoncillo, C. Gomez, L. 1999 Heterologous expression of a plant small heat-shock protein enhancesPlant Physiol. Escherichia coliviability under heat and cold stress 120 521 528
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