|Citrus unshiu (Satsuma mandarin) is considered one of the most cold hardy commercial Citrus species; whereas, Poncirus trifoliata (Trifoliate orange), a close relative of Citrus sp., is capable of surviving freezes to -26°C. Citrus develop plant water deficits during cold acclimation that may play a role in contrasting cold tolerance between these two species. The accumulation of 14-3-3 proteins has been associated with an increase in cold tolerance, and the genes expressed were thought to have an adaptive role in this process. 14-3-3 proteins have been identified and induced 2.3 fold when comparing cold-acclimated to non-acclimated Satsuma mandarin. This study was conducted to determine differences between these two species that may explain the differences in gene expression of 14-3-3.
C. unshiu and P. trifoliata trees were kept unacclimated by holding day/night air temperatures at 25°C/20°C, or acclimated by dropping day/night air temperatures weekly from 25°C/20°C to 20°C/15°C to 15°C/10°C, and then to 10°C/5°C for 9 weeks. During acclimation, stomata closed similarly for both species. Stomata never completely closed, which allowed low levels of transpiration and CO2 assimilation. Root resistance was higher for C. unshiu compared to P. trifoliata, and was higher for acclimated versus unacclimated plants by the end of the experiment. Plant water deficits, as measured by stem water potential, declined similarly for both species during cold acclimation, while remaining similar and constant when unacclimated. The similarity in plant water deficits between species during cold acclimation and the responses of stomates and root resistance cannot explain the contrasting cold hardiness of these two species. The difference of cold tolerance is likely due to a difference in gene expression during cold acclimation.
14-3-3 protein was observed using quantitative RT-PCR for both acclimated and unacclimated species. Both acclimated C. unshiu and P. trifoliata showed down-regulation of 14-3-3 protein when plants were fully acclimated to chambers at 5°C and held for 21d, which differs from a study that showed up-regulation of 14-3-3 protein in C. unshiu when acclimated to 7°C for 7d. A possible conclusion would be that 14-3-3 is an important factor during mid to late-acclimation, but the gene is no longer needed when plants are fully acclimated or cold-hardened. More detailed characterization of the signal transduction important 14-3-3 gene identified in C. unshiu and P. trifoliata will shed light on the understanding of the responsive mechanisms under cold acclimation.