DIFFERENTIAL DISPLAY-MEDIATED ISOLATION OF A GENOMIC SEQUENCE FOR A PUTATIVE MITOCHONDRIAL LMW HSP SPECIFICALLY EXPRESSED IN CONDITION OF INDUCED THERMOTOLERANCE IN ARABIDOPSIS-THALIANA (L) HEYNH

Plants of Arabinopsis thaliana pre-treated at 37 degrees C for 2 h can survive an otherwise lethal heat shock at 45 degrees C. Differential display reverse transcriptase-PCR (DDRT-PCR) was utilized to clone DNA fragments corresponding to mRNAs specifically expressed in conditions of induced thermotolerance or of expression of thermotolerance. One o f these DDRT-PCR fragments enabled the isolation of a genomic clone pA t1.3EX, containing the sequence Athsp23.5, the gene for a low-molecula r-weight (LMW) heat shock protein (HSP), AtHSP23.5. Athsp23.5 is low- or single-copy in the Arabidopsis genome and its open reading frame is interrupted by a 137 bp intron. Analysis of the sequence suggests AtH SP23.5 is targeted to the mitochondrion. The steady-state level of the AtHSP23.5 mRNA varied significantly according to the heat treatment, increasing on heat shock (transfer from 22 degrees C to 37 degrees C), with a further increase during expression of thermotolerance (transfe r from 22 degrees C to 37 degrees C and then to 45 degrees C). Express ion was low after an abrupt stress (from 22 degrees C to 45 degrees C) . This behaviour was different from that observed for other LMW HSP mR NAs that were present at high level at 37 degrees C, but did not incre ase significantly in condition of expression of thermotolerance, and r eached a considerable steady-state level also during the abrupt stress at 45 degrees C. The retrotranscription of AtHSP23.5 mRNA followed by amplification with two primers encompassing the intron allowed for th e isolation of an almost full-length cDNA sequence. The sequence analy sis of the two cDNAs obtained from condition 22 degrees C-->37 degrees C and condition 22 degrees C-->37 degrees C-->45 degrees C suggested that in both cases the intron had been correctly spliced. The importan ce of correct intron splicing in survival at high temperatures and the role of mitochondrial HSP in induction and expression of thermotolera nce are discussed.