Molecular cloning of a novel wound-induced gene from tomato:twi1

Damage to a leaf of a tomato plant is known to lead to changes in gene expression. A number of wound-responsive genes have already been identified, including those that encode prosystemin (McGurl et al., 1992), proteinase inhibitors (pins) (Graham et al., 1986), leucine aminopeptidase, polyphenol oxidase (Hildman et al., 1992) and enzymes involved in ethylene biosynthesis (Holdsworth et al., 1988; Olsen et al., 1991; van der Straeten et al., 1990). It is probable however, that the wound stimulus will produce a wide range of signals and that the total number of genes that are up-regulated by injury is likely to be considerable. As a way to identify novel wound-responsive genes we have used differential screening of a cDNA library constructed in Lambda Zap II from polyA+ mRNA extracted from leaf tissue of 21 day old tomato plants (Lycopersicon esculentum cv. Money Maker), two hours after wounding. This report describes a novel gene which is expressed rapidly and transiently in wounded tissue. Differential screening of the cDNA library with first strand cDNA prepared from mRNA from wounded leaves and unwounded leaves led to the identification of one differentially expressed cDNA clone. This was subsequently used as a probe to re-screen the wounded leaf cDNA library. Several positive clones were identified, partially sequenced, and found to be identical. The complete sequence of the largest of these cDNAs was then obtained, and the gene named twi1 (tomato wound- induced 1). Searching of the databases of known sequences with the nucleotide and predicted amino acid sequence of twi1 revealed that twi1 has sequence similarity with glucosyl transferase genes from a variety of different organisms. It has significant homology to M. esculenta Crantz cDNAs, mecgt1 and mecgt5, encoding UDP-glucose glucosyl transferases (54.3% and 52.2% amino acid similarity respectively) (Hughes and Hughes, 1994), and a Zea mays glucosyl transferase, identified as IAA-glu synthetase (52.8% amino acid similarity) (Szerszen et al., 1994). There is also some homology to a tomato ripening-related glucosyl transferase, ert1b (Picton et al., 1993). Upon comparing the sequnces it is interesting to note the higher regions of homology at the C-terminal end compared to the N- terminus. As all these proteins transfer glucose to their specific acceptors this region of higher homology probably represents those regions involved in UDP/UTP binding. The cDNA was used as a probe on northern analyses to investigate the changes in steady-state levels of twi1 transcripts on wounding. The twi1 gene was found to be rapidly induced and highly expressed in the tissue of the wounded leaf, reaching a maximum level approximately 1-2 hours after wounding. Preliminary data shows that twi1 is also developmentally-regulated, a transcript being expressed during fruit ripening, and to particularly high levels in ripe red fruit. Twi1 transcripts are also detectable in senescing leaves of a tomato plant, but no expression can be detected in root or stem tissue. A genomic Southern, under moderate stringency conditions, revealed that twi1 is likely to be a single copy gene in tomato. In potato, homologous bands were also detected. The rapid up-regulation of twi1 gene expression after tissue damage suggests the potential importance of the gene product in the mechanisms of plant defence. Further work is being carried out to investigate the role of twi1 in the wound response