UP-REGULATION OF 2 CDNA CLONES ENCODING METALLOTHIONEIN-LIKE PROTEINSIN APPLE FRUIT DURING COOL STORAGE

We are investigating the molecular basis of low temperature responses in apples, by identifying and characterising fruit genes which show al tered expression in response to cool-storage. Two independent cold-reg ulated clones (pAMT1 and pAMT2) were isolated from a cDNA library deri ved from cool-stored apple (Malus domestica Borkh. cv. Granny Smith) f ruit. These clones share only 27% amino acid identity with each other, but both show high similarity to plant metallothionein (MT)-like prot eins. The polypeptide encoded by pAMT1 shares similarity with type 2 M T-like sequences, while that encoded by pAMT2 is similar to others whi ch share a different distribution of cysteine residues. We suggest, th ese form a 'type 3' group of MT-like clones. Genomic Southern analysis confirmed that there is a family of MT-like genes in apple. There are differing patterns of pAMT1 and pAMT2 expression during apple fruit d evelopment, amt1 RNA was abundant in flowers and during the early stag es of development, and decreased as the fruit approached maturity, whi le amt2 RNA was barely detectable in flowers and young fruit and accum ulated with fruit development. In ripe fruit, amt1 expression was up-r egulated, while amt2 expression was down-regulated. In leaves, both ge nes showed increased expression with leaf age. In Granny Smith, Cox's Orange Pippin and Braeburn apple cultivars, both genes were up-regulat ed in cool-stored fruit. In Granny Smith cortical tissue, ami RNA leve ls were elevated within the first 45 min at both 0.5 degrees C and 4 d egrees C, but not al 12.5 degrees C. The different patterns of amt1 an d amt2 expression during fruit development and in different tissues su ggest that the respective genes have distinct controlling elements and may be functionally different. The in vivo roles of the encoded polyp eptides, particularly in relation to chilling tolerance or acclimation , are as yet unknown.