Impaginato 319 Adv. Hort. Sci., 2017 31(4): 319-327 DOI: 10.13128/ahs-20545 Phenolic metabolism and antioxidant activity during endodormancy of Kiwifruit buds E. Abedi Gheshlaghi 1 (*), V. Rabiei 2, M. Ghasemi 3, J. Fattahi 3, F. Razavi 2 1 Horticulture Crops Research Department, Guilan Agricultural and Natural Resources Research and Education Center, AREEO, Rasht, Iran. 2 Department of Horticulture, Faculty of Agriculture, University of Zanjan, Iran. 3 Horticulture Research Institute, Citrus and Subtropical Research Center, Agricultural Research, Education and Extension Organization (AREEO), Ramsar, Iran. Key words: Actinidia deliciosa, biochemistry, chilling, enzyme. Abstract: Bud dormancy is an adaptability process in woody plants that enables them to survive in unfavorable conditions. In the present study, the phenols, antioxidant capacity, and activity of three enzymes were evaluated during endodormancy phases in two Hayward and Tomuri cultivars and two female and male Golden genotypes of kiwifruit buds. The buds were collected from ten-year-old own-rooted vines from the end of October 2015 until the end of January 2016 in the north of Iran. The results revealed that phenols, antioxi- dant capacity (RSA), phenylalanine ammonia-lyase (PAL), and polyphenol oxi- dase (PPO) activities of buds significantly increased at the beginning of endodormancy and subsequently decreased at the end of the endodormancy. The POD activity increased in Hayward and Tomuri from the onset of endodor- mancy and continued for two weeks after the endodormancy release. The total phenol had a positive and significant correlation with RSA and PAL enzyme activity. Furthermore, higher antioxidant capacity and phenols in both male and female Golden genotypes were attributed to the higher PAL enzyme activi- ty in both genotypes. This study proposes that the RSA%, PAL activity, and phe- nol concentration could be employed as a biomarker to indicate bud dormancy phases in kiwifruit. 1. Introduction the first axillary buds are initiated on the developing shoots of kiwifruit shortly after bud break in the first growing season (Walton et al., 1997). Similar to temperate fruits, the axillary buds of kiwifruit can be induced into endodormancy by short days and low temperatures at the end of the summer or the beginning of autumn (mcPherson et al., 1995). Bud endodormancy is an adaptability process in woody plants that (*) Corresponding author: eabedig@yahoo.com Citation: ABEDI GHESHLAGHI E., rABIEI V., GHASEmI m., fAttAHI j., rAzAVI f., 2017 - Phenolic metabo- lism and antioxidant activity during endodorman- cy of Kiwifruit buds. - Adv. Hort. Sci., 31(4): 319- 327 Copyright: © 2017 Abedi Gheshlaghi E., rabiei V., Ghasemi m., fattahi j., razavi f. this is an open access, peer reviewed article published by firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: the authors declare no competing interests. received for publication 16 April 2017 Accepted for publication 19 October 2017 AHS Advances in Horticultural Science Adv. Hort. Sci., 2017 31(4): 319-327 320 enables trees to survive in unfavorable conditions such as drought and extremely hot and cold weather (Arora et al., 2003); in addition, more importantly, it encourages the reproductive processes such as the formation of flowers and fruit set to be accomplished in favorable condition and guarantees the reproduc- tive growth and survival of the plants (Campoy et al., 2011). Endodormancy (winter dormancy) is a true dormancy in which the bud growth is prevented by an inhibitory system within the bud (Horvath et al., 2003). Overcoming endodormancy and maximum bud break and flowering in favorable environmental conditions are achieved by accumulating the mini- mum amount of chilling hours (mcPherson et al., 1997). After that endodormancy is released, the growth of buds is prevented directly by external envi- ronmental factors. this type of dormancy mainly occurs in late winter and is named ecodormancy (Horvath et al., 2003). Generally, horticultural specialists determine the t i m e o f b u d b r e a k b y c h i l l i n g a n d h e a t u n i t s . However, this method is based on ambient tempera- ture, varies according to the environmental condi- tions, and cannot express the internal situation of buds (Dennis, 2003). During bud endodormancy, there is no visible growth, but physiological changes in respiration, growth regulators, carbohydrate metabolism, the amount of water, and other com- pounds occur, influencing bud endodormancy control (Ben mohamed et al., 2010). A series of the changes occurring in the biochemistry of the buds appear to indicate the shift from the endodormancy stage to the ecodormancy stage (Pakish et al., 2009; Szecskó et al., 2002). richardson et al. (2010) stated that high and stable sucrose concentrations are likely to be a good indicator of the true dormancy of the buds in kiwifruit (Actinidia deliciosa). Investigating the relationship between biochemi- cal compounds and the beginning of the endodor- mancy in nine varieties of apricot revealed seasonal changes in phenolic compounds and peroxidase (Laslo and Vicas, 2012). these changes are caused by t h e a c c u m u l a t i o n o f c h i l l i n g u n i t s d u r i n g t h e endodormancy, that is necessary for the develop- ment of some phenological stages. Pakish et al. (2009) studied peroxidase and oxidase activities in the varieties of pistachio buds during the winter and observed seasonal variation in the November-march interval. there is a significant difference in terms of the phenols of buds in different dormancy stages within cultivars of the same species. the total phenol of the flowering buds in peach (Szalay et al., 2005) and apri- cot (Laslo and Vicas, 2012) increased at the beginning of endodormancy, showed a gradual increase in dor- mancy period, and disappeared during flowering. Large variations in the content of polyphenols can be observed in certain varieties of pistachio (Pistacia vera L.) in the November-march interval. the phenol of all cultivars was reduced in the swollen buds (Pakish et al., 2009). factors controlling the onset, maintenance, and termination of endodormancy are varied and have not been studied much (Luedeling et al., 2009). moreover, the starting point of endodormancy, the end of endodormancy, and the start of ecodormancy are not clearly defined in plants; therefore, the study of changes in biochemical compounds in this field could be useful (Pakish et al., 2009; Ben mohamed et al., 2010). A number of studies have been done on the changes in carbohydrates (richardson et al., 2007, 2010), nitrogen, and amino acid (Walton et al., 1991) of kiwifruit in the endodormancy period; how- ever, the activities of enzymatic and non-enzymatic antioxidants such as phenol and its metabolism have not been studied yet. thus, the aim of this study was to inspect the changes in antioxidant capacity, total p h e n o l , a n d a c t i v i t y o f t h r e e e n z y m e s d u r i n g endodormancy and the early ecodormancy in four cultivars and genotypes of kiwifruit. 2. Materials and Methods Plant material and sampling Bud samples of Hayward and tomuri kiwifruit cul- tivars (Actinidia deliciosa) and two male and female Golden genotypes (A. chinensis; mass selection of Golden kiwifruit cultivar seedlings) were collected from canes of ten-year-old own-rooted vines from the end of October 2015 until the end of january 2016 with a 7-day interval at 11 steps. from the first to the last sampling, the vines received 0, 222, 309, 443, 570, 740, 864, 1003, 1125, 1272, and 1450 chill- ing units, respectively (richardson et al., 1974). Kiwifruit vines were located at the National Citrus and Subtropical research Institute of Iran (latitude 75.36° North and longitude 33.51° East) and were trained on a t-bar training system with a planting dis- tance of 4×6 m. the samples were immediately frozen in liquid nitrogen and kept for subsequent analyses at -80°C. At every sampling date, 30 buds were collected from 1-year-old canes at nodes 6 to 20 starting from the basal end of canes. ten buds Abedi Gheshlaghi et al. - Phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds 321 were selected randomly in three replications for fur- ther analyses of biochemical compounds (richardson et al., 2010). the activities of phenylalanine ammo- nia-lyase (PAL), peroxides (POD), polyphenol oxidase (PPO), antioxidant capacity (rSA), and total phenol content were determined in buds during endodor- mancy. Estimation of endodormancy period Chilling requirements and bud endodormancy period of kiwifruit cultivars and genotypes were esti- mated via single-node cuttings test. Simultaneously, fifteen cuttings in three replications of each cultivar and genotype were collected and the buds were taken for biochemical measurements. Cuttings from each treatment were transferred into a forcing cham- ber at 25°C, with 16 h of light (Wall et al., 2008). the lowest mean time budburst (mtB) for half of the buds were considered as an endodormancy release (tisne-Agostini et al., 1992). Extraction and determination of total phenol and antioxidant capacity the total phenol of each extract was determined according to the folin-Ciocalteu procedure reported by meyers et al. (2003). moreover, the spectrophoto- metric method introduced by Wettasinghe and Shahidi (2000) was employed for the chemical deter- mination of antioxidant. Extraction and assay of PAL One hundred mg of kiwifruit buds powdered by liquid nitrogen were mixed with 2 ml of 1.0 mm borate buffer containing 1.0% of polyvinyl pyrroli- done. After homogenization by a homogenizer (model IKA-t8, Germany), the samples were cen- trifuged for 15 min at 4°C and 13,000 rpm. the super- natants were slowly transferred into the tubes by pipette. Extracts for the ensuing measurements were maintained at -80°C. PAL (EC 4.3.1.24) activity was determined according to the study carried out by Yu et al. (2012). Extraction and assay of PPO and POD activities the two hundred-mg samples of fresh buds, which were collected, were ground in liquid nitrogen and homogenated with 2 ml of potassium phosphate buffer (50 mm, pH= 7.0) containing 1% polyvinyl pyrrolidone (PVP) (W/V) and 0.05% EDtA at 4°C. the homogenate was centrifuged at 14000 rpm for 15 min at 4°C. the supernatant was used as a crude enzyme solution for assay and was maintained at -80°C for the following measurements. the activity of PPO enzyme (EC 1.14.18.1) was quantified by the method described by In et al. (2007). the activity of POD enzyme (E.C 1.11.1.7) was measured by the method employed by Srivastava et al. (1983). the activities of these enzymes were cal- culated, using the Beer-Lambert law on the basis of a single enzyme unit (μmol) per mg of fresh weight according to the following formula: U/g fWmin = absorption changes per minute × reaction mixtur/ supernatant volume × extinction coefficient Statistical analyses this study was conducted as a two-factor factorial in a completely randomized design. the first factor is 11 sampling dates, and the second factor is four culti- vars having three replications in the period of 2015- 2016. the ANOVAs and standard errors of the mean (SE) were generated, using SAS 9. All significant means were separated, using the Duncan (P≤0.01). the correlation between the total phenol and antiox- idant capacity, PAL, PPO, and POD activities was cal- culated via the software SPSS 22. 3. Results and Discussion the beginning date for the chilling accumulation was considered to be when a stable chilling accumu- lation occurred and the temperatures causing a nega- tive effect were infrequent (richardson et al., 1974; Guerriero et al., 1990). this date corresponded with 27th October 2015. the first samples were conduct- ed on this date. mean time budburst was more evi- dent on this date than on the date of the endodor- mancy release; thus, the kiwifruit axillary buds may inter endodormancy sooner than the end of the sum- mer or the beginning of autumn (mcPherson et al., 1995). the results revealed that the maximum depth of endodormancy in this study was in late November. the duration of bud endodormancy was different in the cultivars and genotypes. the end of endodor- mancy was 21st December 2015 for female Golden genotype (740-unit chilling), 28th December 2015 for male Golden genotype (864-unit chilling), and 4th january 2016 for Hayward and tomuri cultivars (1003-unit chilling) as shown in figure 1. the antioxidant capacity indicated a significant difference between the buds of genotypes and culti- vars sampled on different dates (P≤0.01). the highest value in the antioxidant capacity was observed in female Golden genotype on 28th December 2015 (fig. 2); however, there were no significant differ- ences in the antioxidant capacity of all buds samples Adv. Hort. Sci., 2017 31(4): 319-327 322 collected in December (i.e. 07th, 14th, 21st and 28th). the lowest value in the antioxidant capacity was observed in tomuri cultivar on 11th january after endodormancy release (fig. 2). the antioxidant capacity of buds changed considerably at the begin- ning and during the maintenance and release of endodormancy (P≤0.01) (fig. 2). the increase in antioxidant capacity from the end of October to the early November was simultaneous in four cultivars and genotypes; however, the peak period of the antioxidant capacity of buds was different between these cultivars and genotypes (fig. 2). the antioxi- dant capacity showed a significant reduction (P≤0.01) in the male and female genotypes at the end of December, being simultaneous with the end of bud endodormancy of both genotypes. the reduction in the antioxidant capacity in Hayward and tomuri culti- vars occurred about two weeks later, coinciding with the completion of their chilling requirements and the end of endodormancy in the buds (fig. 2). the activity of PAL enzyme has been shown in fig- ure 3 and demonstrated a significant difference in buds of all kiwifruit cultivars and genotypes (P≤ 0.01). the activity of this enzyme in the male and female G o l d e n g e n o t y p e s w a s m o r e t h a n t h a t o f t h e Hayward and tomuri cultivars (fig. 3). female Golden g e n o t y p e h a d t h e h i g h e s t P A L a c t i v i t y i n m i d - December. the lowest activity of this enzyme was in tomuri cultivar on 30th November 2015. PAL enzyme activity in all four kiwifruit cultivars and genotypes began an upward trend at the end of November (P≤0.01) (fig. 3). It remained relatively stable during endodormancy in cultivars and genotypes, but was associated with a fluctuation in male and female Golden genotypes during ecodormancy. However, the enzyme activity of all cultivar showed a signifi- cant decrease (P≤0.01) at the end of endodormancy compared to the stable periods of endodormancy. P l a n t s r e l e a s e h y d r o g e n p e r o x i d e ( H 2O 2) i n response to the environmental stress. Low tempera- ture stress has also been shown to induce H2O2 accu- mulation in cells (Okane et al., 1996). Hydrogen per- oxide, as the second messenger of the increase in the activity of PAL enzyme, can activate PAL enzyme activity, as a key enzyme in the phenylpropanoids pathway, ultimately leading to higher total phenol and accumulation of flavonoids (Wang et al., 2015). O x i d a t i v e s t r e s s c a u s e d b y c h i l l i n g d u r i n g t h e endodormancy of kiwifruit buds increased the activi- ty of this enzyme and the production of antioxidant compounds such as phenols, resulting in a higher antioxidant capacity in the buds during the endodor- mancy. It has been reported that cold acclimation of fig. 1 - Effect of sampling date of cuttings on average of 50% bud break and endodormancy end period in four kiwifruit cultivars and genotypes during dormant season in 2015-2016. the arrows show the endodormancy end date for each cultivar and genotype. fig. 2 - Effects of kiwifruit cultivars and different sampling dates on antioxidant capacity of axillary buds during dormant season in 2015-2016. Each data point represents the mean of three replicates, each containing ten buds. moreover, ± the standard error of mean is shown on the vertical bar. fig. 3 - Effects of kiwifruit cultivars and different sampling dates on phenylalanine ammonia-lyase activity of axillary buds during dormant season in 2015-2016. Each data point represents the mean of three replicates, each contain- ing ten buds moreover, ± the standard error of mean is shown on the vertical bar. Abedi Gheshlaghi et al. - Phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds 323 plants leads to a remarkable increase in PAL activity, depending upon the range of low temperature to which the plants are subjected (Stefanowska et al., 2002). the amount of total phenol have been shown in figure 4 and varied between cultivars and genotypes of kiwifruit, and there were significant differences in their buds (P≤0.01). male and female Golden geno- types had higher total phenol than Hayward and tomuri cultivars (fig. 4). total phenol content showed substantial changes at the beginning, during the maintenance, and at the end of the endodorman- cy of kiwifruit buds (P≤0.01) (fig. 4). the amount of total phenol showed an increasing trend from the late October. In the late November-early December period, coinciding with the onset of true endodor- mancy, the amount of total phenol reached its maxi- mum (fig. 4) and its value remained at a high level in the buds of cultivars and genotypes during this peri- od. By reaching the end of endodormancy, the amount of phenol had decreased significantly at the end of December and at the beginning of january in the buds of male and female Golden genotypes and Hayward and tomuri cultivars, respectively (P≤0.01). Phenolic compounds are a valuable piece of evi- dence used in determining the differences between diverse varieties of Myrtus communis and Pistacia lentiscus and have a key role in detecting the genetic differences in biochemical methods (tattini et al., 2006). thus, it appears that the significant differ- ences in terms of the total phenol content between Hayward and tomuri cultivars and male and female Golden genotypes (P≤0.01) (fig. 4) are the result of their genetic differences. Endodormancy is developed gradually after the cessation of growth; additionally, the severity of the endodormancy deepens in autumn and then gradual- ly disappears by removing the physiological barriers of growth through the chilling process (Dennis, 2003). total phenol concentration and antioxidant capacity enhance along with the development of endodormancy and reach their maximum value in the deepest stage of endodormancy (fig. 2, 4). the changes in these two variables are similar in the establishment, maintenance, and release of endodor- mancy. A high total antioxidant activity in male and female Golden genotypes may be attributed to the high amount of phenol. Phenolic compounds are syn- thesized in plant cells in favorable environmental conditions, but environmental stresses change their levels in cells (Kliebenstein, 2004). mid-autumn cold and the start of endodormancy period causes an increase in oxidative stress in plants. this stress results from reactive oxygen species that affect the growth of plants (Scalabrelli et al., 1991; mittler et al., 2004). Plants possess a protective system com- posed by the enzymatic antioxidant system such as peroxidase and catalase (Anderson et al., 1995) and the non-enzymatic systems (Agarwal and Pandey, 2004). Phenols are non-enzymatic antioxidants and their antioxidant activities are mainly due to their redox properties which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quencher (Huda-faujan et al., 2009). Phenols play an important role not only during c o l d r e s i s t a n c e , b u t a l s o d u r i n g b r e a k i n g t h e endodormancy of peach (Siller-Cepeda et al., 1992) and apricot (Viti and Bartolini, 1998) buds as an antioxidant. total phenol changes in kiwifruit buds are in agreement with the results of the studies on peach flower buds (Szalay et al., 2005), apricot vege- tative buds (Laslo and Vicas, 2012), and pistachio flower buds (Pakish et al., 2009). Correlation analyses showed a positive significant correlation between total phenol, antioxidant capaci- ty and PAL activity in kiwifruit cultivars and geno- types (table 1). reduction in antioxidant capacity (fig. 2) and total phenol (fig. 4) after receiving chill- ing and a stable period in winter could be considered as a biomarker of endodormancy release in the culti- vars which were studied. In cultivars and genotypes of kiwifruit buds, PPO activity changed substantially (P≤0.01) as shown in figure 5. this enzyme had the highest activity in the tomuri variety on 7th january 2016 and the lowest activity in male and female Golden genotypes at the fig. 4 - Effects of kiwifruit cultivars and different sampling dates on phenol content of axillary buds during dormant sea- son in 2015-2016. Each data point represents the mean of three replicates, each containing ten buds. moreover, ± the standard error of mean is shown on the vertical bar. Adv. Hort. Sci., 2017 31(4): 319-327 324 first sampling date (fig. 5). At the end of October 2015 and contemporaneous with the development of bud endodormancy, the activity of PPO enzyme, in all cultivars except Hayward, increased and reached its peak in mid-December and then decreased (P≤0.01; fig. 5). PPO activity in the Hayward variety peaked a week earlier than the other cultivars. In tomuri culti- var, PPO activity was stable for three weeks in the dormancy period and reduced at ecodormancy. PPO is a copper-containing enzyme which cat- alyzes the oxidation of phenolic compounds to quinone or quinine-like compounds in the presence of molecular oxygen. A high PPO enzyme activity after endodormancy is probably due to the removal of some growth-inhibiting phenols (Wang et al., 1991), and the phenolic substances such as inhibitors o r s t i m u l a n t s c h a n g e e n z y m e a c t i v i t y (thirugnanasambantham et al., 2013). the increased activity of the PPO enzyme at the early stage of endodormancy period and its declined activity at the end of the endodormancy of grape buds (Scalabrelli et al., 1991), plums (Szecskó et al., 2002), and pistachio flower buds (Pakish et al., 2009) were reported, corresponding with the results of this experiment. It appears that the effect of antioxidant compounds is to inhibit free radicals and reactive oxygen species in cultivars and genotypes during stress. P O D a c t i v i t y w a s n o t a b l y d i f f e r e n t i n b u d s (P≤0.01). this enzyme had the highest activity in the tomuri cultivar after the endodormancy release (fig. 6). the lowest activity was observed at all cultivars and genotypes at the first sampling date (the 27th October). the POD activity pattern was not constant during the endodormancy of buds in the cultivars and genotypes which were studied (fig. 6). POD activity increased in Golden genotypes later than that in the Hayward and tomuri cultivars. the activity of this enzyme increased significantly in early December and mid-December 2015 in male and female Golden genotype buds, respectively (P≤0.01). However, POD activity decreased significantly in both genotypes at t h e b u d e n d o d o r m a n c y r e l e a s e ( f i g . 6 ) . t h e increased activity of POD at the end of October, in ** Correlation is significant at the 0.01 level. table 1 - Correlation coefficient of kiwifruit cultivars and geno- types between phenol, antioxidant capacity (rSA %), phenylalanine ammonia-lyase (PAL), peroxidase (POD), and Polyphenoleoxidase (PPO) fig. 5 - Effects of kiwifruit cultivars and different sampling dates on polyphenol oxidase activity of axillary buds during dormant season in 2015-2016. Each data point repre- sents the mean of three replicates, each containing ten buds. moreover, ± the standard error of mean is shown on the vertical bar. fig. 6 - Effects of kiwifruit cultivars and different sampling dates on peroxidase activity of axillary buds during dormant season in 2015-2016. Each data point represents the mean of three replicates, each containing ten bud. moreover, ± the standard error of mean is shown on the vertical bar. Cultivars and genotypes Phenol PAL rSA POD PPO Hayward Phenol 1 0.25 NS 0.95 ** -0.11 NS -0.34 NS PAL 0.25 NS 1 0.17 NS 0.24 NS -0.18 NS rSA 0.95 ** 0.17 NS 1 -0.22 NS -0.27 NS POD -0.11 NS 0.24 * -0.22 NS 1 0.44 NS PPO -0.34 NS -0.18 NS -0.27 NS 0.44 NS 1 tomuri Phenol 1 0.38 NS 0.94 ** 0.38 NS 0.28 NS PAL 0.38 NS 1 0.42 NS 0.20 NS -0.22 NS rSA 0.94 ** 0.42 NS 1 0.27 NS 0.28 NS POD 0.38 NS 0.20 NS 0.27 NS 1 0.28 NS PPO 0.28 NS -0.22 NS 0.28 NS 0.28 NS 1 female Golden Phenol 1 0.76 ** 0.93 ** 0.20 NS 0.16 NS PAL 0.76 ** 1 0.67 * -0.19 NS -0.16 NS rSA 0.93 ** 0.67* 1 0.19 NS 0.14 NS POD 0.20 NS -0.19 NS 0.19 NS 1 0.88 ** PPO 0.16 NS -0.16 NS 0.14 NS 0.88 ** 1 male Golden Phenol 1 0.75 * 0.83 ** 0.36 NS 0.26 NS PAL 0.75 * 1 0.74 * 0.40 NS 0.37 NS rSA 0.83 ** 0.74 * 1 0.61 NS 0.49 NS POD 0.36 NS 0.40 NS 0.61 NS 1 0.95 ** PPO 0.26 NS 0.37 NS 0.49 NS 0.95 ** 1 Abedi Gheshlaghi et al. - Phenolic metabolism and antioxidant activity during endodormancy of kiwifruit buds 325 requires further investigation. this study indicated a significant and stable increase in the PAL and rSA activities. furthermore, we found that phenol con- centration could be associated with the transition to, and maintenance of, bud in true endodormancy. Due to the positive and significant correlation between total phenol, antioxidant capacity, and PAL activity, we concluded that antioxidant capacity, in both male and female Golden genotypes, is attributed to the high phenol which was a result of high PAL enzyme activity. Acknowledgements t h e p a p e r i s r e s u l t e d o f E b r a h i m A b e d i Gheshlaghi’s PhD thesis. this research was funded by the University of zanjan, Iran, which is gratefully acknowledged. References AGArWAL S., PANDEY V., 2004 - Antioxidant enzyme respones to NaCl stress in Cassia angustifolia. - Plant Biol., 48: 555-560. ANDErSON D., PrASAD K., StEWArt r., 1995 - Changes in isozyme profiles of catalase, peroxidase Glutathione reductase during acclimation to chilling in mesocotyls of maize seedlings. - Plant Physiol., 109: 1247-1257. ArOrA r., rOWLAND L.j., tANINO K., 2003 - Induction release of bud dormancy in woody perennials: A sci- ence comes of age. - HortScience, 38: 911-921. 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HAO S., fENG-WANG m., 2004 - Relationship between early endodormancy of buds, for Hayward and tomuri cultivars was in agreement with results of studies on grape buds (Scalabrelli et al., 1991), onion bulbs (Benkeblia and Shiomi, 2004), and floral buds of apricots (Laslo and Vicas, 2012). the increasing activity of this enzyme continued after an endodor- mancy breakdown in Hayward and tomuri cultivars; moreover, these results concurred with the antioxi- dant enzyme changes in pear flower buds, where POD activity increased both during and after the end of endodormancy (Hao and feng-Wang, 2004). there are reports on the POD activity associated with susceptibility to cold in pistachio (Pakish et al., 2009) and peach (Szalay et al., 2005), suggesting that the varieties resistant to cold have higher POD activi- ty than the susceptible cultivars. tomuri had the highest POD activity and its activity significantly increased at the ecodromancy period (P≤0.01) (fig. 6). therefore, tomuri cultivar could be the most tol- erant to cold, that needs to be investigated further. A significantly positive correlation was observed between phenols, PAL, and antioxidant capacity in Golden genotypes, and between phenols and antioxi- dant capacity in Hayward and tomuri cultivars (table 1). the lowest PPO and POD activities (P≤0.01) (figs. 5, 6) and the insignificant changes in these activities during early endodormancy could result from the higher non-enzymatic antioxidant capacity (for exam- ple, total phenol and PAL) in Golden genotypes (fig. 2 ) . t h e b u d s o f H a y w a r d a n d t o m u r i c u l t i v a r s showed lower PAL activity and total phenol and high- er POD and PPO activities than those in male and female Golden genotypes from the early stage of endodormancy to the ecodormancy stage (figs. 4-6). However, there was a significantly positive correla- tion between total phenol content and antioxidant capacity in two cultivars (table 1). It was concluded that the antioxidant capacity in Hayward and tomuri cultivars may be due to total phenol rather than POD and PPO activities. Gur et al. (1988) reported the dif- ference in PPO enzyme activity and phenol content in apple cultivars. 4. Conclusions Peroxidase activity increased with the onset of the buds endodormancy, but continued for two w e e k s a f t e r t h e e n d o f t h e e n d o d o r m a n c y i n Hayward and tomuri cultivars. this is probably due to the resistance of these cultivars to cold compared to the male and female genotypes and, therefore, Adv. Hort. Sci., 2017 31(4): 319-327 326 breaking of dormancy and reactive oxygen species metabolism in flower buds of pear. - Acta Photophysiol. Sin. (Abstr.). HOrVAtH D.P., ANDErSON j.V., CHAO W.S., fOLEY m.E., 2003 - Knowing when to grow: signals regulating bud dormancy. - trends Plant Sci., 8: 534-540. 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