CBF gene expression in peach leaf and bark tissues is gated by a circadian clock

Authors: Artlip, Timothy - Tim; Wisniewski, Michael; Bassett, Carole; Norelli, John

Submitted to: Tree Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/4/2013
Publication Date: 8/15/2013
Publication URL: http://handle.nal.usda.gov/10113/57993
Citation: Artlip, T.S., Wisniewski, M.E., Bassett, C.L., Norelli, J.L. 2013. CBF gene expression in peach leaf and bark tissues is gated by a circadian clock. Tree Physiology. 33:866-877.

Interpretive Summary: It is increasingly acknowledged climate change is occurring, and there are potentially serious consequences for many agricultural interests. Erratic conditions such as early spring warming followed by frosts or freezing events are particularly devastating to fruit and vegetable growers. If chilling requirements have been met in a fruit tree, warm spells permit the tree to deacclimate from a cold tolerant condition and foster bud break. Blossoms and nascent fruit are particularly susceptible to freeze damage. In an effort to better understand the mechanisms behind cold acclimation and deacclimation, we have investigated several genes in apple and peach that respond to low temperature. The c-repeat binding factor (CBF) family encodes transcription factors that act as higher-level “on switches” for many genes known for their importance in cold acclimation and tolerance. We have isolated the peach CBF1 gene and constitutively over-expressed it in apple, with the result that the apple trees have delayed vegetative bud break in the spring, typically avoiding some frost events. This result was unexpected and had not been observed in other plant systems. While the exact mechanism is unknown, the peach CBF1 gene appears to provide a novel sensitivity to daylength that apple is not known to have. We investigated several of the CBF genes in peach seedlings in order to understand whether the peach CBF1 gene and its relatives are normally responsive to the circadian rhythm that is set by sunrise. Sunrise and daylength play a crucial role in dormancy and cold acclimation in peaches. We determined that the peach CBF genes are cold responsive and their increase in expression in response to low temperature is regulated by the circadian clock. We also investigated dehydrin genes that are known to be cold responsive and possibly regulated by CBFs. Increases in dehydrin expression occurred hours after the expression peaks of CBFs. Together with other previous data, this timing in expression indicates that one or more of the dehydrins are regulated by CBFs. Additional studies on the genes may provide additional understanding as to how the peach CBF1 gene imparts the novel daylength sensitivity in apple. This improved knowledge may provide a means by which damage from frosts during bloom or early fruit set can be mitigated.

Technical Abstract: CBF transcription factors are part of the AP2/ERF domain family of DNA-binding proteins that recognize a C-repeat response cis-acting element that regulates a number of cold-responsive genes (CBF-regulon). In peach (Prunus persica), five CBF genes are situated in tandem on scaffold (Linkage Group) five of the peach genome (GDR transcripts ppa014628m, ppa010909m, ppa010800m, ppa017761m, and ppa021197m). The induction of CBF gene expression by low temperature has also been shown to be gated by a circadian clock in Arabidopsis. Since CBF gene regulation has been shown to be more complex in woody plants than in herbaceous plants, the present study was conducted to determine if CBF gene expression in peach leaf and bark tissues was also influenced by a circadian clock. One-year-old ‘Loring’ peach trees grafted on ‘Bailey’ rootstocks were entrained to a 12h day/12h night photoperiod at 25 degrees C. After two weeks, trees were exposed to four degrees C under continuous light for up to 48 h. Low temperature exposure was initiated at either subjective dawn plus four h (ZT4) or ZT plus 16 h (ZT16), and leaf and bark tissues were harvested at various time points. RNA was extracted from leaf and tissue samples and expression levels of the five peach CBF genes on LG5 and a DREB2 gene on LG2 (GDR transcript ppa007606m) were determined using RT-qPCR. Results indicated a distinct gating of CBF gene expression by a circadian clock for four CBF genes in both leaf and bark tissues. CBF genes were highly induced in ZT plus four samples after being placed at four degrees C, with expression peaking in leaf samples at six to to 24 h in a CBF gene-specific manner, while CBF expression peaked at various times in bark tissues. In contrast, CBF gene expression in ZT plus 16 samples was highly attenuated in leaves and less so in bark. These results are in agreement with similar studies in Arabidopsis. Further experiments were conducted to verify environmental influence on the induction of CBF and DREB2 genes. In contrast to DREB2 genes from other dicots, the peach DREB2 gene homologue was induced by both low temperature and dehydration. CBF genes that were low temperature inducible were generally not inducible by dehydration. Induction of the peach CBF and DREB2 genes appeared to correspond to the promoter elements that bioinformatic analyses indicate exist for four of the CBF genes and the DREB2 gene. Additional low temperature and dehydration induction data for three peach dehydrin genes indicates that regulation of these genes is complex, with individual dehydrin gene expression correlated to the expression of one or more peach CBFs.