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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.

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Abstract

Plants of Arabidopsis thaliana pre-treated at 37 °C for 2 h can survive an otherwise lethal heat shock at 45 °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 of these DDRT-PCR fragments enabled the isolation of a genomic clone pAt1.3EX, containing the sequence Athsp23.5, the gene for a low-molecular-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 AtHSP23.5 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 °C to 37 °C), with a further increase during expression of thermotolerance (transfer from 22 °C to 37 °C and then to 45 °C). Expression was low after an abrupt stress (from 22 °C to 45 °C). This behaviour was different from that observed for other LMW HSP mRNAs that were present at high level at 37 °C, but did not increase significantly in condition of expression of thermotolerance, and reached a considerable steady-state level also during the abrupt stress at 45 °C. The retrotranscription of AtHSP23.5 mRNA followed by amplification with two primers encompassing the intron allowed for the isolation of an almost full-length cDNA sequence. The sequence analysis of the two cDNAs obtained from condition 22 °C→37 °C and condition 22 °C→37 °C→45 °C suggested that in both cases the intron had been correctly spliced. The importance of correct intron splicing in survival at high temperatures and the role of mitochondrial HSP in induction and expression of thermotolerance are discussed.

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  1. Laboratory of Environmental Biotechnologies, Department of Environmental Sciences, University of Parma, Viale delle Scienze, 43100, Parma, Italy

    Giovanna Visioli, Elena Maestri & Nelson Marmiroli

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  1. Giovanna Visioli
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Visioli, G., Maestri, E. & Marmiroli, N. 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.. Plant Mol Biol 34, 517–527 (1997). https://doi.org/10.1023/A:1005824314022

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