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

Authors: Wisniewski, Michael; Bassett, Carole; Norelli, John; Macarisin, Dumitru; Artlip, Timothy - Tim; GASIC, KASENIJA - UNIV OF ILLINOIS; KORBAN, SCHULYER - UNIV OF ILLINOIS

Submitted to: Physiologia Plantarum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2007
Publication Date: 5/1/2007
Citation: Wisniewski, M.E., Bassett, C.L., Norelli, J.L., Macarisin, D., Artlip, T.S., Gasic, K., Korban, S. 2007. Expressed sequence tag analysis of the response of apple (Malus x domestica) to low temperature and water deficit. Physiologia Plantarum. 133:298-317.

Interpretive Summary: Freezing temperatures and drought are two abiotic stresses that can have a significant negative impact on the productivity and longevity of fruit trees. These losses can result in reduced revenues for fruit growers and higher prices for consumers. Cold and drought resistance are complex, multigenic traits. Therefore, there is a need to better understand what genes are involved in stress response and which of those sets of genes are responsible for greater resistance to environmental stress. Large scale (global) studies of gene expression in plants have provided invaluable information on many aspects of plant biology including resistance to diseases and stress, however, only recently have these technologies been applied to study the biology of fruit trees. In the current study, we obtained tissue samples from non-stressed (control), low-temperature stressed, and drought stressed apple trees and obtained a representative samples of the genes that were being expressed in these tissues by extracting RNA, making it into DNA, and cloning the resulting genes. Over 22,000 clones were obtained and sequenced. The sequences of these genes allowed us to determine which proteins these genes encoded and in many cases helped us to find the functional role of these proteins in plant metabolism. Over 65% of the cloned genes could be identified using this approach. The genes obtained from the different tissues were placed into one of ten different functional categories, and the changes in gene expression in response to low temperature and water deficit stress were then analyzed. Our results suggest that osmotic regulation, desiccation tolerance, and adjustments in photosynthesis are some of the key components of stress adaptation in apple. This information will be used to identify gene networks involved in stress response and help us to understand what genes are specific to low temperature stress vs. drought stress. The long range objective of this research is to identify candidate genes that can be used either in marker-assisted breeding programs or for genetic enhancement using biotechnology to improve stress resistance.

Technical Abstract: We constructed nine cDNA libraries from stressed and non-stressed tissues of apple. Specifically, leaf, bark, xylem, and root tissues were used to make the libraries from non-stressed (control) trees, and trees exposed to either low-temperature (5 deg C for 24 h) or water deficit (45% of saturated pot mass for two 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 170-1430. 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 ESTs were combined into three classes (control, low-temperature, water deficit), and the annotated genes in each class were placed into one of ten different functional categories. The percentage of genes falling into each category was then calculated. This analysis indicated a distinct down regulation 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 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 reflecting the shorter (24h) exposure to stress. Genes with greater than five clones in any specific library were identified and based on the number of clones obtained, the magnitude of increase or decrease in expression in the libraries was calculated. 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 up-regulated 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 down-regulated genes was nominal compared to differences in up-regulated 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).