Characterisation of Mal d 1-related genes in <Emphasis Type="Italic">Malus</Emphasis>

It has been suggested that there are at least 15 Mal d 1-related (PR10) genes in one genotype of apple (Malus×domestica Borkh.). We sequenced cDNA libraries of cultivar Royal Gala and identified 12 members of the Mal d 1 family, including the previously reported Mal d 1b and Mal d 1d, an allelic variant of the previously reported Mal d 1a. Eight Mal d 1 gene products were expressed in tree-ripened fruit, in either the cortex or the skin, and most of these were also expressed in leaves in response to challenge with Venturia inaequalis—a fungal disease of apple. Mal d 1 gene products were identified from a large number of different tissues. Degree of ripeness as measured by standard parameters was shown not to predict either the amount of protein able to bind to a specific monoclonal antibody 5H8, previously shown to bind to an allergenic epitope in Mal d 1b and a/d, or the amount of Mal d 1 mRNA present. Mal d 1d and Mal d 1b were the most highly expressed isoforms in Royal Gala, particularly in the skin of fruit, and these isoforms were also predominant in other cultivars and species of apple. Genotypes, however, differed in relative predominance of Mal d 1b and Mal d 1d. The predominantly expressed Mal d 1 genes in ripe apple fruit were translated in vivo into proteins and proteins binding to the antibody were found in all cultivars and species examined. New Mal d 1 proteins were identified that bound to the 5H8 antibody. At least two new subfamilies have been identified, and while some structural differences are predicted between groups of isoforms, the P-loop motif is identical in all except two isoforms. A role in intracellular signalling in plants is suggested and in vitro expression of the isoforms should help in assessing their relative roles in disease, allergic responses, senescence and nucleotide-, cytokinin- and brassinosteroid-binding.