The present studies were performed to investigate genetic factors of more adaptable cultivars to changes of recent climatic condition during winter and/or early spring and to enhance our understanding of kinetics and mechanisms of cold acclimation, deacclimation, and reacclimation in the shoots of four peach cultivars (P. persica cvs. Daewol, Aikawanakajima, Fukuyokabijin, and Kiraranokiwami). Two experiments, (1) seasonal cold acclimation and deacclimation and (2) repeated deacclimation and reacclimation were carried out. The percent injury data based on electrolyte leakage analysis under various temperature conditions were transformed into LT50 values representing cold hardiness. The SDS-PAGE profiles of proteins from each cultivar were used to analyze changes of dehydrin proteins. Carbohydrates were analyzed using a HPLC. Transcript accumulation of different genes related to changes of dehydrins and carbohydrates were examined using a quantitative real-time RT-PCR.

Experiment 1. Changes of Dehydrins, Carbohydrates, and Gene Expressions during Cold Acclimation and Deacclimation in Adult Peach Trees
Cold hardiness of four peach cultivars increased during the autumn, reached a maximum in midwinter and then gradually decreased. Numerical changes of cold hardiness in ‘Daewol’, ‘Aikawanakajima’, ‘Fukuyokabijin’, and ‘Kiraranokiwami’ peach trees were modest during cold acclimation period from August 2011 to December 2011. However, numerical changes of cold hardiness in four cultivars during deacclimation period from January 2012 to April 2012 were significantly different; LT50 values of most freezing tolerant ‘Daewol’ showed differences in cold hardiness above -10oC compared to most freezing susceptible ‘Kiraranokiwami’.
Changes of a 60 kDa of dehydrin protein encoded by PpDhn1 gene were observed in four cultivars from August 2011 to April 2012. The SDS-PAGE profiles of proteins from four cultivars were very similar during investigated period. Data indicated that a 60 kDa protein of four cultivars accumulated to high level during fall and winter (from November 2011 to January 2012) followed by a complete disappearance in spring (from March to April 2012). A 30 kDa of polypeptide, assumed to be a dehydrin protein encoded by PpDhn2 gene, did not display discernible changes. A 16 kDa of polypeptide which was assumed as a “bark-storage protein” also exhibited a similar seasonal pattern in four cultivars.
PpDhn1 transcript accumulation occurred on seasonal basis, rising steadily during the autumn, reaching the maximum in midwinter and declining during the spring. On the other hand, PpDhn2 did not seem to be seasonally regulated. Expression patterns of PpDhn3 were similar to those of PpDhn1 in all cultivars (P ≤ 0.05). However, like changes of a 60 kDa of polypeptide, changes of dehydrin gene expressions among the cultivars at each sampling dates were not significantly different. Expression of PpNCED1 gene displayed apparent seasonal patterns in ‘Aikawanakajima’ and ‘Kiraranokiwami’, whereas did not in ‘Daewol’ and ‘Fukuyokabijin’. Expressions of PpDhn1 and PpDhn3 were highly correlated with cold hardiness of four cultivars, and the relation between PpDhn1 and cold hardiness (P ≤ 0.001) was stronger than the relation between PpDhn3 and cold hardiness (P ≤ 0.01). However, the relation between PpDhn2 and cold hardiness was statistically significant only in ‘Daewol’ and ‘Kiraranokiwami’ (P ≤ 0.01). Expression of PpNCED1 was highly correlated with cold hardiness in ‘Aikawanakajima’ and ‘Kiraranokiwami’ (P ≤ 0.001), and was positively correlated with expressions of PpDhn1 (P ≤ 0.001) and PpDhn3 (P ≤ 0.001) in ‘Aikawanakajima’ and ‘Kiraranokiwami’.
The seasonal changes of cold hardiness were closely correlated to the contents of sucrose and total soluble sugars in four cultivars (P ≤ 0.001). The content of sucrose and total soluble sugars in the shoots of four peach cultivars increased during cold acclimation and decreased during deacclimation. The contents of glucose and fructose in ‘Aikawanakajima’, ‘Fukuyokabijin’, and ‘Kiraranokiwami’ did not show any specific patterns, although slight changes were observed during cold acclimation and deacclimation. Especially, the contents of glucose and fructose in ‘Daewol’ significantly increased from December 2011 to February 2012 when average air temperatures were the lowest during investigated period. However, the contents of two sugars were much lower than those of sucrose and sorbitol.
Relative gene expression levels of β-amylase and (sucrose phosphate synthase) SPS in four peach cultivars significantly increased during cold acclimation, showed a transient decrease in midwinter, and decreased during deacclimation (P ≤ 0.01).

Experiment 2. Changes of Dehydrins, Carbohydrates, and Gene Expressions in Response to Deacclimation and Reacclimation in One-year-old Peach Trees
In our previous study (in Experiment 1), the difference in cold hardiness between ‘Daewol’ (relatively cold-tolerant) and ‘Kiraranokiwami’ (relatively cold-susceptible) was the most obvious throughout the investigated period. Patterns of cold hardiness in response to repeated deacclimation and reacclimation declined dramatically during the deacclimation and rose during the reacclimation in both cultivars. Particularly, ‘Kiraranokiwami’ which was more cold-susceptible than ‘Daewol’ in our previous study exhibited the numerical decrease of cold hardiness, evaulated by LT50 values, in response to repeated warm temperatures and their buds were burst earlier. Notably, despite of deacclimation and reacclimation treatments during the same period for four days in this experiment, loss of cold hardiness by deacclimation was higher than recovery of cold hardiness by reacclimation.
Accumulation patterns of a polypeptide with an estimated molecular mass of 60 kDa, known as a dehydrin protein coded by PpDhn1 gene, paralleled fluctuations of cold hardiness in two cultivars. The SDS-PAGE profiles of proteins between both cultivars were similar. Data indicated that a 60 kDa protein of two cultivars became faint during the deacclimation, but the band intensity increased during the reacclimation. However, changes of the 60 kDa of polypeptide among the deacclimation or reacclimation treatments could not be distinguished. A 30 kDa of polypeptide, assumed to be a dehydrin protein coded by PpDhn2 gene, did not show any visible changes. A 16 kDa of polypeptide exhibited a similar pattern to a 60 kDa of that in both cultivars.
Relative expression of PpDhn1 gene coincided with changes of cold hardiness, declining dramatically during the deacclimation, rising during the reacclimation. Expression patterns of PpDhn2 and PpDhn3 were similar to those of PpDhn1 in both cultivars. Expressions of PpDhn1 and PpDhn2 were highly correlated with cold hardiness of two cultivars (P ≤ 0.01). Although expression pattern of PpDhn3 was similar to those of PpDhn1 and PpDhn2 in both cultivars, the correlation between cold hardiness and expression pattern of PpDhn3 was not statistically significant.
All of the sugars including sucrose, sorbitol, glucose, and fructose were statistically correlated with cold hardiness in both cultivars (P ≤ 0.01). Starch was also correlated to cold hardiness in both cultivars (P ≤ 0.05). Interestingly, ‘Daewol’ which was relatively cold-tolerant in Experiment 1 showed more sensitive changes in the carbohydrates in response to warm and low temperatures compared to ‘Kiraranokiwami’ which was relatively cold-susceptible. ‘Daewol’ showed almost similar repeated down- and up- patterns in the contents of soluble sugars in response to repeated deacclimations and reacclimations, whereas showed repeated up- and down- patterns in the contents of starch. However, ‘Kiraranokiwami’ showed a consistent increase in the contents of soluble sugars and a consistent decrease in the contents of starch.
Relative expression of β-amylase gene in ‘Daewol’ decreased in approximately half during deacclimation treatments compared to before treatment (BT), whereas during the reacclimation treatments relative expression of β-amylase gene in ‘Daewol’ increased approximately 3- to 4-fold compared to BT. Relative expression of β-amylase gene in ‘Kiraranokiwami’ also showed pattern similar to that in ‘Daewol’. However, the levels of β-amylase gene expression in ‘Kiraranokiwami’ were much lower than in ‘Daewol’ in all the treatments.