Proceedings of the XLVII Italian
Society of Agricultural Genetics - SIGA Annual Congress
Verona,
Italy - 24/27 September, 2003
ISBN 88-900622-4-X
Poster
Abstract - 1.17
CLONING
OF A WRKY TRANSCRIPTION ACTIVATOR INVOLVED IN COLD AND DROUGHT RESPONSE IN BARLEY
E. MAZZUCOTELLI, C. MARÈ, C. CROSATTI, G.
DALFINO, D. GUERRA, L. CATTIVELLI
Experimental
Institute for Cereal Research, Fiorenzuola d’Arda
barley, cold
acclimation, transcription factor
In
the past years a number of cold-responsive (cor) genes expressed during acclimation to
low temperature have been isolated and characterised in many crop species.
Nevertheless only the CBF/DREB1 gene family of Arabidopsis thaliana codes for transcription activators which
have been shown to control many cold and drought induced genes. CBF/DREB1 genes
are themselves cold-regulated and their transcripts begin to accumulate within
15 min of transfer to low temperature, suggesting that differential screening
techniques could be suitable for the isolation of new transcription factors.
By
differential display RT-PCR technique we have isolated, from barley leaves, a
cDNA induced after 3h of low temperature exposure. This transcript was
characterised by an early and rapid expression in comparison with other known
barley cor genes
usually detectable only after 24 h of cold treatment. By using the
differentially expressed cDNA the corresponding full length clone was recovered
from a cDNA library. Sequence analysis of isolated cDNA revealed that this gene
encodes for a 38kDa protein homologous to Avena fatua ABF2 a member of the large WRKY
(pronounced “worky”) family of plant transcription factors. The
barley gene, named corWRKY38,
is characterised by the WRKY domain together with a zinc-finger-like motif and
a leucine zipper sequence. The accumulation of corWRKY38 transcripts was investigated by northern
blot analysis. During hardening at +2°C the corWRKY38 expression is rapid and transient and
achieves its highest level after 12 hours in the leaves and 5 hours in the
roots. Since the corWRKY38
mRNAs disappear after 24h at +2°C, we have examined if a further
temperature decrease promotes a new induction of corWRKY38. Barley plants were hardened at +2°C
for one week, then submitted to –3°C or –8°C for 48 hours.
The temperature steps down induced a rapid and stable accumulation of corWRKY38 mRNA providing that frozen temperatures
still promote a molecular response. corWRKY38 mRNA accumulation is also promoted by
drought treatment, but not affected by ABA. Gel mobility shift assay experiments
demonstrated that an in vitro
synthesised WRKY38 protein binds oligonucleotides containing two W-box motifs
suggesting that corWRKY38
can potentially act as transcription factors also in vivo.
The WRKY proteins, defined by the conserved amino acid sequence WRKYGOK, are involved in the regulation of varied physiological processes: pathogen defence, senescence and trichome development. This work provides the first evidence of involvement of WRKY transcription factors in cold-stress response.