Analysis of Thermoprotective Activity Induced by Heat Shock Protein 70 in Plants Using Two-Dimensional Difference Gel Electrophoresis

The effect of heat shock protein 70 (HSP70) on soluble proteins extracted from seedlings exposed to high temperature was examined by two-dimensional gel electrophoresis (2-DE). NtHSP70-1 (AY372069), which is an HSP70 that is induced by heat stress in Nicotiana tabacum, was studied to analyze the protective role of plant HSP70. First, transgenic tobacco plants that constitutively expressed elevated levels of the tobacco HSP70, NtHSP70-1, and transgenic plants containing either the vector alone or the NtHSP70-1 gene in the antisense orientation were constructed. Plants with altered NtHSP70-1 levels were subjected to immunoblotting, and seedlings with enhanced levels of NtHSP70-1 in their transgenic sense lines were observed to show tolerance to heat stress. In the analysis using 2-DE, 28 putative proteins were differentially expressed in the comparison of three kinds of transgenic seedlings after high temperature/recovery treatment (45℃, 2 hr; 26℃C, 5 day) with the seedlings under normal condition (26°C). In transgenic sense seedlings, the proteins were maintained or increased, whereas proteins in the transgenic seedlings, containing either the vector alone or the NtHSP70-1 in the antisense orientation, were present in decreased amounts or had disappeared. These results suggested that NtHSP70-1 as molecular chaperone protecting proteins from aggregation induced by heat stress may play an important role in acquisition of thermotolerance.