Plants, being sessile, are more vulnerable to daily and/or seasonal temperature elevation in their natural environment. Moreover, average global surface temperature is on the rise as a result of global warming. The Intergovernmental Panel on Climate Change projected that annual global temperature will likely increase by 1.1 to 6.4 °C during the 21st century (Keller, 2007). In plants, elevated temperatures can disrupt essential metabolic processes such as photosynthesis, respiration, and carbon fixation (Pollock et al., 1993). Accordingly, heat stress negatively impacts agricultural crop production and product quality. Lobell and Field (2007) reported that over the last two decades, warming temperatures have caused annual losses of ≈40 million metric tons, worth $5 billion, for the three major agricultural crops, wheat, maize, and barley. Accordingly, it is important to understand plant heat stress physiology and develop crop plants that show enhanced thermotolerance and production under heat stress.
All living organisms, including plants, respond to heat stress (10 to 15 °C above their optimal growth temperatures) by producing a set of proteins called heat shock proteins (HSPs; Wahid et al., 2007). In eukaryotic organisms, HSPs are classified into five different classes, HSP100, HSP90, HSP70, HSP60, and small (sm) HSPs (15 to 42 kDa), based on their molecular masses. In plants, smHSPs are the most dominant and abundant HSPs produced on heat stress (Waters et al., 1996). It was reported that there are 19 open reading frames, which code for smHSP-related proteins in the Arabidopsis genome (Scharf et al., 2001). Under normal growth temperatures, most smHSPs are not detected in vegetative tissues. However, on heat stress, smHSPs can be synthesized within minutes and accumulate up to 1% of total cellular proteins (Hsieh et al., 1992). Even after heat stress has been eased, some smHSPs are very stable with half-lives of 30 to 50 h (DeRocher et al., 1991). These results suggest that smHSPs play an important role in enhancing thermotolerance in plants during heat stress and/or subsequent recovery.
Although the correlation between the synthesis of smHSPs and thermotolerance has been reported, their cellular and molecular mechanism is not yet fully understood. Some smHSPs are reported to function as molecular chaperones mainly in vitro when analyzed using model substrates (Basha et al., 2006; Giese and Vierling, 2002). Under heat stress, molecular chaperones bind to partially unfolded/denatured proteins to prevent further aggregation and/or promote renaturation of the proteins. Accordingly, the role of molecular chaperones under heat stress is critical for cell viability and survival of organisms.
To understand the function of smHSPs, it is important to examine their structure during heat stress. One of the notable characteristics of smHSPs is their organization into large oligomeric structures (Haslbeck et al., 2005). In a cellular environment, smHSPs are found in large complexes with molecular masses of 200 to 500 kDa (Kirschner et al., 2000; Young et al., 1999). These complexes are known to be homo-oligomeric (Suzuki et al., 1998). Moreover, on prolonged heat stress, some plant smHSPs are found in much larger insoluble structures (greater than 1 MDa, 40 nm in diameter) named heat shock granules (HSGs; Sun et al., 2002). They are believed to be reservoirs for unfolded proteins bound to smHSP oligomers when the refolding capacity of smHSPs is exceeded. HSGs found in tomato (Solanum lycopersicum) consist of cytosolic smHSPs, HSP70, and heat stress transcription factors (Scharf et al., 1998). It is likely that the formation of complexes and conformational changes during heat stress are closely related to the function of smHSPs.
We have examined DcHSP17.7, a smHSP from carrot (Daucus carota L.). DcHSP17.7 has been characterized in a number of studies. Its constitutive expression enhanced cell viability and membrane stability in transgenic carrot cells and plants under heat stress (Malik et al., 1999). Furthermore, expression of an antisense construct of DcHSP17.7 gene resulted in reduced thermotolerance. It was the first demonstration that modification of the expression of a single smHSP can both increase and decrease thermotolerance in plants. When introduced into potato (Solanum tuberosum L.), a cool-season crop, across species lines, DcHSP17.7 enhanced cellular membrane stability and tuberization in vitro (Ahn and Zimmerman, 2006). These results suggested that DcHSP17.7 is a promising candidate to be used for the genetic engineering of heat-resistant crops. To understand the possible function and structural dynamics of DcHSP17.7, in this study, we introduced a coding sequence of DcHSP17.7 into Escherichia coli and studied its function in the transformed E. coli cells under heat stress.
Ahn, Y.-J. & Zimmerman, J.L. 2006 Introduction of the carrot HSP17.7 into potato (Solanum tuberosum L.) enhances cellular membrane stability and tuberization in vitro Plant Cell Environ. 29 95 104
Basha, E., Friedrich, K.L. & Vierling, E. 2006 The N-terminal arm of small heat shock proteins is important for both chaperone activity and substrate specificity J. Biol. Chem. 281 39943 39952
Bradford, M.M. 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal. Biochem. 72 248 254
Cho, E.K. & Bae, S.-J. 2007 ATP-independent thermoprotective activity of Nicotiana tabacum heat shock protein 70 in Escherichia coli J. Biochem. Mol. Biol. 40 107 112
DeRocher, A.E., Helm, K.W., Lauzon, L.M. & Vierling, E. 1991 Expression of a conserved family of cytoplasmic low molecular weight heat-shock proteins during heat stress and recovery Plant Physiol. 96 1038 1047
Giese, K.C. & Vierling, E. 2002 Changes in oligomerization are essential for the chaperone activity of a small heat shock protein in vivo and in vitro J. Biol. Chem. 277 46310 46318
Haslbeck, M., Franzmann, T., Weinfurtner, D. & Buchner, J. 2005 Some like it hot: The structure and function of small heat-shock proteins Nat. Struct. Mol. Biol. 12 842 846
Hsieh, M., Chen, J., Jinn, T., Chen, Y. & Lin, C. 1992 A class of soybean low molecular weight heat-shock proteins Plant Physiol. 99 1279 1284
Keller, C.F. 2007 Global warming 2007. An update to global warming: The balance of evidence and its policy implications Scientific World Journal 9 381 399
Kirschner, M., Winkelhaus, S., Thierfelder, J.M. & Nover, L. 2000 Transient expression and heat-stress-induced co-aggregation of endogenous and heterologous small heat-stress proteins in tobacco protoplasts Plant J. 24 397 411
Lee, G.J., Roseman, A.M., Saibil, H.R. & Vierling, E. 1997 A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state EMBO J. 16 659 671
Lindner, R.A., Carver, J.A., Ehrnsperger, M., Buchner, J., Esposito, G., Behlke, J., Lutsch, G., Kotlyarov, A. & Gaestel, M. 2000 Mouse Hsp25, a small shock protein. The role of its C-terminal extension in oligomerization and chaperone action Eur. J. Biochem. 267 1923 1932
Lobell, D.B. & Field, C.B. 2007 Global scale climate-crop yield relationships and the impacts of recent warming Environ. Res. Lett. 2 014002
Malik, M.K., Slovin, J.P., Hwang, C.H. & Zimmerman, J.L. 1999 Modified expression of a carrot small heat shock protein gene, hsp17.7, results in increased or decreased thermotolerancedouble Plant J. 20 89 99
Pollock, C.J., Eagles, C.F., Howarth, C.J., Schunmann, P.H.D. & Stoddart, L.L. 1993 Temperature stress 109 132 Fowden L., Mansfield T. & Stoddart J. Plant adaptation to environmental stress Chapman & Hall London, UK
Scharf, K.D., Heider, H., Höhfeld, I., Lyck, R., Schmidt, E. & Nover, L. 1998 The tomato Hsf system: HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules Mol. Cell. Biol. 18 2240 2251
Scharf, K.D., Siddique, M. & Vierling, E. 2001 The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing alpha-crystallin domains (Acd proteins) Cell Stress Chaperones 6 225 237
Shashidharamurthy, R., Koteiche, H.A., Dong, J. & McHaourab, H.S. 2004 Mechanism of chaperone function in small heat shock proteins: Dissociation of the HSP27 oligomer is required for recognition and binding of destabilized T4 lysozyme J. Biol. Chem. 280 5281 5289
Sun, W., Van Montagu, M. & Verbruggen, N. 2002 Small heat shock proteins and stress tolerance in plants Biochim. Biophys. Acta 1577 1 9
Suzuki, T.C., Krawitz, D.C. & Vierling, E. 1998 The chloroplast small heat-shock protein oligomer is not phosphorylated and does not dissociate during heat stress in vivo Plant Physiol. 116 1151 1161
Waters, E.R., Lee, G.J. & Vierling, E. 1996 Evolution, structure and function of the small heat shock proteins in plants J. Expt. Bot. 47 325 338
Yang, H., Huang, S., Dai, H., Gong, Y., Zheng, C. & Chang, Z. 1999 The Mycobacterium tuberculosis small heat shock protein Hsp16.3 exposes hydrophobic surfaces at mild conditions: Conformational flexibility and molecular chaperone activity Protein Sci. 8 174 179
Yeh, C.H., Chang, P.F., Yeh, K.W., Lin, W.C., Chen, Y.M. & Lin, C.Y. 1997 Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshsp16.9, in Escherichia coli enhances thermotolerance Proc. Natl. Acad. Sci. USA 94 10967 10972
Young, L.S., Yeh, C.H., Chen, Y.M. & Lin, C.Y. 1999 Molecular characterization of Oryza sativa 16.9 kDa heat shock protein Biochem. J. 344 31 38