A survey of genes differentially expressed during long-term heat-induced chilling tolerance in citrus fruit

Long-term storage at low, non-freezing, temperature (1.5 degreesC) induces chilling injury in fruit of Fortune mandarin (Citrus clementina Hort. Ex Tanaka x Citrus reticulata, Blanco), manifested as pitting and brown depressed areas that may end up with local cell death. Pre-conditioning of fruit for 3 days at 37 degreesC prevented chilling injury. The use of suppression subtractive hybridization permitted the isolation of genes differentially expressed in heat-conditioned fruit exposed to chilling conditions, which may be candidates for heat-induced chilling tolerance. Northern blot analysis revealed that some genes were up-regulated by prolonged heat (3 days/37 degreesC) and their expression persisted in fruit cells upon subsequent chilling exposure. The expression of other genes was specifically induced by the combination of heat and cold. Among the putative tolerance-associated genes, we identified two transcription factors of the WRKY family and one TFIIB factor. Heat conditioning also altered the expression of genes encoding proteins involved in secondary metabolism, cell wall modification, oxidative damage and other stress-responsive proteins. These results illustrate the complexity of molecular mechanisms operating during heat-induced chilling tolerance in citrus fruit.