All living organisms synthesize a group of proteins called heat shock proteins (HSPs) when exposed to elevated temperatures or other abiotic stresses such as cold, salinity, drought, oxidation, and heavy metals (Vierling, 1991). These proteins are classified into five conserved classes based on their molecular weights: HSP100, HSP90, HSP70, HSP60, and small (s) HSPs (12 to 42 kDa; Waters et al., 1996). The sHSPs are more abundant and diverse in plants compared with other organisms (Sun et al., 2002). Haslbeck et al. (2005) have reported that the number of genes encoding sHSPs is one or two in bacteria and archaea, four in Drosophila melanogaster, 16 in Caenorhabditis elegans, 10 in Homo sapiens, and 19 in Arabidopsis thaliana. They are divided into seven subclasses based on their cellular localization and sequence homology. Three classes (Class I, II, and III) are localized in the cytosol/nucleus, whereas the other four classes are present in the peroxisome (P), the endoplasmic reticulum (ER), the mitochondria (M), and the chloroplast (C). Such sHSPs are characterized by a conserved α-crystalline domain (90 a.a.), which is also present in the α-crystalline proteins of the vertebrate eye lens, located at their carboxyl-terminal. The α-crystalline domain is further divided into consensus I and II regions separated by hydrophobic regions of various lengths (Sun et al., 2002). In contrast, the amino-terminals of sHSPs vary greatly among the different classes. The α-crystalline domain is known to exert molecular chaperone activity, thereby preventing protein denaturation and/or correcting the folding of partially unfolded proteins under stress conditions.
Our recent studies have shown that sHSPs are expressed not only under heat stress, but also under various abiotic stresses, where DcHsp17.7 in carrot (Daucus carota L.) was expressed under conditions of heat (Kim and Ahn, 2009), cold (Song and Ahn, 2010), and salinity (Song and Ahn, 2011). Furthermore, the heterologous expression of DcHsp17.7 enhanced cell viability in transgenic E. coli under these environmental stress conditions, suggesting that DcHsp17.7 may confer multiple stress tolerances. If so, DcHsp17.7 would be a valuable candidate for the development of transgenic plants possessing multiple stress tolerance.
We examined here the expression and function of DcHsp17.7 under oxidative and osmotic stress conditions, which are abiotic stresses that frequently accompany heat stress (Vandenbroucke et al., 2008). In plants, oxidative stress results in the accumulation of reactive oxygen species (ROS; Desikan et al., 2001). Increased ROS such as H2O2 causes an imbalance in cellular redox state, thereby damaging the proteins, lipids, and nucleic acids of living cells (Vandenbroucke et al., 2008). Drought and desertification (i.e., osmotic stress) result from global warming and attendant climate changes (Ashraf, 1994). According to a report by Johnson et al. (2006), 44% of arable lands are affected by desertification.
To examine whether DcHsp17.7 is involved in conferring tolerance to oxidative and osmotic stresses, we studied the accumulation of DcHsp17.7 in stressed tissues using a polyclonal antibody raised against DcHsp17.7. Then, the DcHsp17.7 gene was introduced into E. coli to analyze its action under these abiotic stress conditions. In this case, the cell viability and protein solubility of transgenic E. coli expressing DcHsp17.7 were examined under stress conditions to determine whether the protein confers tolerance to oxidative and/or osmotic stresses.
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