Molecular Genetic and Physiological Analysis of the Cold-Responsive Dehydrins of Blueberry

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Summary Blueberry plants, like many perennial plants, must undergo physiological changes in order to survive winter. Lack of winter hardiness and susceptibility to spring frosts have been identified as two of the most important genetic limitations of current blueberry cultivars. These traits are influenced by several factors such as the level of cold hardiness reached while plants are in the cold acclimated state and the chilling requirement or amount of low-temperature exposure required for breaking dormancy. How these factors are controlled genetically and how they interact with each other is not well understood. For these reasons, we have been using a combination of molecular, genetic, and physiological approaches to investigate genetic controls of chilling requirement, cold hardiness, and related factors in blueberry. Previously, we identified three dehydrins of 65, 60, and 14 kDa as the predominant proteins present in cold acclimated blueberry floral buds. Dehydrins are a group of heat-stable, glycine-rich plant proteins that are induced by environmental stimuli that have a dehydrative component, such as drought and low temperature. Levels of the blueberry dehydrins increase with cold acclimation and decrease with deacclimation and resumption of growth. Expression studies with whole plants indicate that blueberry dehydrins are induced by cold stress in all organs examined including floral buds, leaves, stems, and roots, and by drought stress in primarily stems. Although dehydrin accumulation correlates positively with cold hardiness levels, it does not correspond precisely to the degree of drought tolerance or drought avoidance. Our studies of dehydrin expression in cell suspension cultures indicate that cell cultures are not a good system for studying blueberry dehydrin expression. Peptide sequence information from the blueberry dehydrins has been used to prepare degenerate primers and amplify a portion of a gene encoding a dehydrin. This amplification product has been used to screen our cDNA library, prepared from RNA from cold acclimated blueberry floral buds, and has resulted in the isolation of a full-length cDNA clone thought to encode the 60 kDa dehydrin. The gene represented by this clone has been designated bbdhn1. This clone has now been used as a probe to further screen the cDNA library and has resulted, to date, in the isolation of four partial-length dehydrin cDNAs. All have been completely sequenced and the sequences compared to each other and to that of the bbdhn1 cDNA. The sequences are identical at the 3'end and diverge more and more as they approach the 5'end. Whether the cDNAs represent different genes or a combination of different alleles and different genes remains to be determined. Efforts are currently underway to complete cloning and sequencing the remaining unique dehydrin cDNAs, as well as isolate and characterize cDNA clones representing other cold-responsive messages from blueberry.