Expressed sequence tag analysis of the response of apple (Malus x domestica 'Royal Gala') to low temperature and water deficit

Leaf, bark, xylem and root tissues were used to make nine cDNA libraries from non-stressed (control) 'Royal Gala' apple trees, and from 'Royal Gala' trees exposed to either low temperature (5 degrees C for 24 h) or water deficit (45% of saturated pot mass for 2 weeks). Over 22 600 clones from the nine libraries were subjected to 5' single-pass sequencing, clustered and annotated using BLASTX. The number of clusters in the libraries ranged from 170 to 1430. Regarding annotation of the sequences, BLASTX analysis indicated that within the libraries 65-72% of the clones had a high similarity to known function genes, 6-15% had no functional assignment and 15-26% were completely novel. The expressed sequence tags were combined into three classes (control, low-temperature and water deficit) and the annotated genes in each class were placed into 1 of 10 different functional categories. The percentage of genes falling into each category was then calculated. This analysis indicated a distinct downregulation of genes involved in general metabolism and photosynthesis, while a significant increase in defense/stress-related genes, protein metabolism and energy was observed. In particular, there was a three-fold increase in the number of stress genes observed in the water deficit libraries indicating a major shift in gene expression in response to a chronic stress. The number of stress genes in response to low temperature, although elevated, was much less than the water deficit libraries perhaps reflecting the shorter (24 h) exposure to stress. Genes with greater than five clones in any specific library were identified and, based on the number of clones obtained, the fold increase or decrease in expression in the libraries was calculated and verified by semiquantitative polymerase chain reaction. Genes, of particular note, that code for the following proteins were overexpressed in the low-temperature libraries: dehydrin and metallothionein-like proteins, ubiquitin proteins, a dormancy-associated protein, a plasma membrane intrinsic protein and an RNA-binding protein. Genes that were upregulated in the water deficit libraries fell mainly into the functional categories of stress (heat shock proteins, dehydrins) and photosynthesis. With few exceptions, the overall differences in downregulated genes were nominal compared with differences in upregulated genes. The results of this apple study are similar to other global studies of plant response to stress but offer a more detailed analysis of specific tissue response (bark vs xylem vs leaf vs root) and a comparison between an acute stress (24-h exposure to low temperature) and a chronic stress (2 weeks of water deficit).