The involvement of chloroplast HSP100/ClpB in the acquired thermotolerance in tomato.
The chloroplast HSP100/ClpB is a newly documented member of the ClpB family, but little was known about its role in imparting thermotolerance to cells. A cDNA coding for a HSP100/ClpB homolog has been cloned from Lycopersicon esculentum and termed as Lehsp100/ClpB (the cDNA sequence of Lehsp100/ClpB has been submitted to the GenBank database under accession number: AB219939). The protein encoded by the cDNA was most similar to the putative chloroplast HSP100/ClpBs in higher plants and the ClpB from Cyanobacterium Synechococcus sp. A 97 kDa protein, which matched the predicted size of mature LeHSP100/ClpB, was immunologically detected in chloroplast isolated from heat-treated tomato plants. In addition, the fusion protein, combining the transit sequence of LeHSP100/ClpB and GFP, was found to be located in chloroplast based on the observations of fluorescent microscope images. These results indicated the chloroplast-localization of LeHSP100/ClpB. Both the transcript and the protein of Lehsp100/ClpB were not detected under normal growth conditions, but they were induced by increasingly higher temperatures. An antisense Lehsp100/ClpB cDNA fragment was introduced into the tomato by Agrobacterium-mediated transformation. Antisense lines exhibited an extreme repression of heat-induced expression of Lehsp100/ClpB. The levels of chloroplast HSP60 and small HSP in antisense lines were identical to those of the control plants. After plants preconditioned at 38 degrees C for 2 h were exposed to a lethal heat shock at 46 degrees C for 2 h, the antisense lines were greatly impaired and withered in 21 days of the recovery phase, whereas the untransformed control plants and the vector-transformed plants survived. Furthermore, chlorophyll fluorescence measurements showed that PS II in antisense lines were more susceptible to the thermal irreversible inactivation than the untransformed and vector-transformed control plants. This work provides the first example that induction of chloroplast LeHSP100/ClpB contributes to the acquisition of thermotolerance in higher plants.
Yang
Jin-ying
JY
College of Life Science, Shandong Normal University, Jinan, Shandong, 250014, PR China.
Sun
Ying
Y
Sun
Ai-qing
AQ
Yi
Shu-ying
SY
Qin
Jia
J
Li
Ming-hui
MH
Liu
Jian
J
D000222:Adaptation, Physiological / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D002736:Chloroplasts / Q000378:metabolism; D017931:DNA Primers; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006358:Hot Temperature; D018551:Solanum lycopersicum / Q000235:genetics / Q000502:physiology*; D010802:Phylogeny; D030821:Plants, Genetically Modified
D017931:DNA Primers; D006360:Heat-Shock Proteins