Plant Molecular Biology 34: 517–527, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
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.
Giovanna Visioli, Elena Maestri and Nelson Marmiroli
Laboratory of Environmental Biotechnologies, Department of Environmental Sciences, University of Parma, Viale
delle Scienze, 43100 Parma, Italy (
author for correspondence)
Received 7 August 1996; accepted in revised form 9 April 1997
Key words: Arabidopsis thaliana, DDRT-PCR, induced thermotolerance, expression of thermotolerance, intron
splicing, mitochondrial LMW HSP
Plants of Arabidopsis thaliana pre-treated at 37
C for 2 h can survive an otherwise lethal heat shock at 45
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 variedsigniﬁcantly accordingtotheheat treatment, increasing on heat shock (transferfrom 22
with a further increase during expression of thermotolerance (transfer from 22
C and then to 45
Expression was low after an abrupt stress (from 22
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
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 and condition 22
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.
The heat shock (HS) response is one of the best char-
acterized model systems, at the molecular level, of
a genotype-environment interaction. Three aspects of
the HS response are of particular interest: (1) tran-
scriptional activation of a small set of genes, referred
toasheatshockgenes (hsps): (2) translation ofnew HS
tion of thermotolerance, which allows the organism to
GenBank and DDBJ Nucleotide Sequence Databases under the
accession numbers X98375, Y11864, Y11865 and Y11866.
withstand an otherwise lethal heat stress [28, 29, 32,
HSPs are evolutionary conserved in all organisms
so far studied, and they are found in all cellular com-
partments.Abiochemicalrole duringHShas beenpro-
posed for certain of the HSPs: (1) as molecular chaper-
ones, by assisting the folding of nascent polypeptides
and thus preventing incorrect association; (2) assisting
in proteolytic degradation; (3) protection of organelle
structure and function (in particular chloroplasts and
mitochondria); and (4) protection of RNA processing
and splicing [11, 12].
GR: 201001948, Pips nr. 138660 BIO2KAP
plan3634.tex; 5/06/1997; 8:24; v.7; p.1