Isolation of a dehydrin cDNA from orange and grapefruit citrus fruit that is specifically induced by the combination of heat followed by chilling temperatures

Dehydrins (DHNs; late embryogenesis abundant D‐11) are a family of plant proteins induced in response to environmental stresses such as water stress, salinity and freezing or which occur during the late stages of embryogenesis. Previously, it was reported that citrus contains a small gene family encoding a unique class of dehydrins that differs from most other plant dehydrins in various respects, such as having an unusual K‐segment similar to that of gymnosperms. In the present study, we identified by cDNA differential display analysis a ‘Navel’ orange 202‐bp polymerase chain reaction (PCR) fragment, which encoded the typical plant angiosperm‐type K‐segment consensus sequence, and of which the expression was down‐regulated by exposure to low oxygen levels. The full‐length cDNA sequence of the orange DHN, designated csDHN (for Citrus sinensis DHN), was further isolated by 5′‐and 3′‐RACE; it had a total length of 933 bp and encoded a predicted polypeptide of 235 amino acids. In addition, the same 202‐bp ‘Navel’ dehydrin PCR fragment was used to screen a ‘Star Ruby’ grapefruit flavedo cDNA library, and its full‐length grapefruit homologue, designated cpDHN (for C. paradisi DHN) was isolated and found to have a total length of 1024 bp and to encode a predicted polypeptide of 234 amino acids. The defined orange and grapefruit DHN proteins were completely identical in the 196 amino acids of their N‐terminus but differed in their C‐terminus region. Overall, the csDHN and cpDHN proteins share 84% identity and contain the conserved dehydrin serine cluster (S‐segment) and a putative nuclear localization signal, but csDHN has one conserved dehydrin K‐segment consensus sequence, whereas cpDHN contains two dehydrin K‐segments. Both csDHN and cpDHN represent single copy genes, in ‘Navel’ orange and ‘Star Ruby’ grapefruit genomes, respectively. We found that the cpDHN gene was consistently expressed in the fruit peel tissue at harvest, but that its message levels dramatically decreased during storage at either ambient or low temperatures. However, a pre‐storage hot water treatment, given to enhance fruit‐chilling tolerance, increased cpDHN mRNA levels during the first 3 weeks of cold storage at 2°C, and enabled the message levels to be retained for up to a further 8 weeks of cold storage at 2°C. The hot water treatment by itself had no inductive effect on cpDHN gene expression when the fruits were held at non‐chilling temperatures. Other stresses applied to the fruit, such as wounding, UV irradiation, water stress, low oxygen and exposure to the stress hormone ethylene decreased DHN mRNA levels, whereas abscisic acid had no effect at all.