Seasonal changes in leaf and twig antioxidant systems in Damask rose ( Rosa damascena Mill . )

Department of Horticulture, Islamic Azad UniversityScience and Research Branch, P. O. Box 14515775, Tehran, Iran. Biotechnology Research Department, Research Institute of Forests and Rangelands, P. O. Box 13185-116, Tehran, Iran. Department of Forest, Research Institute of Forests and Rangelands, P. O. Box 13185-116, Tehran, Iran. Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box 141556343, Tehran, Iran. Department of Gene Bank, Research Institute of Forests and Rangelands, P. O. Box 13185-116, Tehran, Iran.

antimicrobial activities of essential oil R. damascena have been demonstrated (Achuthan et al., 2003;Ozkan et al., 2004).Antioxidants are compounds that can delay/inhibit the oxidation of lipids or other molecules by inhibiting the initiation or propagation of oxidative chain reactions (Velioglu et al., 1998) or alternatively scavenging the free radicals generated during oxidative destruction.Antioxidant defense system in plants contains enzymatic and non-enzymatic antioxidants.The enzymatic system consists of enzymes such as POD, CAT, SOD, ARx, MDAR, DHAR and GR (Keles and Oncel, 2002).Antioxidant activity is mainly related to production of chemical agents that are used for the protection of the plant.Observation of the free radical scavenging action of the plant may serve as a monitor for stressor or conditions (e.g.seasonal and climatic changes) that lead to generation of these antioxidant compounds.In fact, changes of antioxidant compounds and enzyme activities reflect the impact of environmental stresses on plant metabolism and affected the antioxidant defense capacity of cells.As indicated above, antioxidant activity exhibits variations depending upon the physiological and biochemical effects (Herbinger et al., 2002;Syvacy and Sokmen, 2004;Beltagi, 2008).Meanwhile, enzyme activities aimed as important index of plant response and behavior to seasonal alterations.Variable in both time and location makes the seasonal environment difficult to predict responses of plants to the changing conditions of the environment (Sen and Mukherji, 2009).
POD is an important multifunctional enzyme and constitutes about 15% of all cellular proteins (Chibbar et al., 1984).It is involved in a number of important physiological processes and ubiquitous in plants (Sergeeva et al., 1987).PODs regulate H 2 O 2 levels and ROS production within cells (Passardi et al., 2005) and catalyse the reduction of H 2 O 2 by employing various substrates (De Gara, 2004;Hiraga et al., 2001;Passardi et al., 2005).CATs are consisting of monofunctional proteins that are specifically localized in peroxisomes (Engel et al., 2006;Feierabend, 2005;Willekens et al., 1997).Activities of CAT and non-specific POD is directly countered to production of H 2 O 2 (Foyer and Halliwell, 1976) and decompose H 2 O 2 to H 2 O at different cellular locations (Asada, 1999;Mittler, 2002).The balance between ROS production and activities of antioxidative enzyme determines whether oxidative signaling and/or damage will occur (Moller et al., 2007).
Consequently, the present study aimed to investigate the antioxidant activity of the leaves and twigs (various tissue types) in R. damascena and to determine the seasonal variations.Damask rose accessions were collected from 10 provinces of Iran and cultivated in Damavand region.Previous studies showed that they may include multiple genotypes (Tabaei-Aghdaei et al., 2006;2007).This research was directed towards the study of seasonal and annual changes in 12 accessions of Damask rose in relation to enzyme activities.Since enzymatic activities and the responses of the accessions were cleared difference and emphasis on the interaction among them.Also, it detected that one of them are sensitive and need more changes in enzymatic activities for protecting themselves in new condition.On the other hand, different tissues have various behaviors to the environmental changes.Therefore, this effort was performed for the reaction of each tissue to these alterations among the accessions.

Plant materials
Twelve Damask Rose accessions originating from ten provinces of Iran were cultivated under randomized complete block design with three replications at Rangelands Research Station of Hamand Absard, Damavand., Iran.The state is located at 65 km from Tehran (co-ordinates, 35°44´N, 52°05´E and elevation 1960 m).
The minimum and maximum temperature was -24°C in January-February and 37°C in July-August.Average rainfall of a year was 333 mm and of first 3 months of year (development and flowering stage of plants) were 13 and 130 mm in 2008 and 2009, respectively.In this research, samples were harvested at critical phenological stages of plant growth.Enzymatic activities in leaf and twig were evaluated during 2008 and 2009.The 3 phenological stages for leaf sampling included 50% flowering in spring, warmest time in summer and before rains in autumn.Twigs were taken at 4 stages, including above stages and in winter.Fresh leaves and twigs were sampled and the measurements were carried out.

Enzyme assays
For enzyme extracts and assays, about 1 g of leaves was rubbed and plugged in extraction buffer (pH 7.5) for 24 to 72 h (Ebermann and Stich, 1982).The homogenate was centrifuged at 3000 rpm for 15 min, and supernatant was collected and used for enzyme assays.POD activity assay was based on the method of Chance and Maehly (1955) with phosphate buffer (pH 6.0) which was determined by measuring the oxidation of guaiacol as an electron donor to tetraguaiacol in the presence of H2O2 at 420 nm.CAT activity was determined according to the method described by Eising and Gerhardt (1989) containing phosphate buffer (pH 7.0) with modifications which measure the decline of a decrease in absorbance at 240 nm for 1 min following the decomposition of H2O2.

Statistical analyses
Data presented are mean values ± S.E.M. for three replicates.The means of the treatments were separated using Duncan's new multiple ranges test (DMRT) at a 0.05 significant level.GLM procedure analysis was used in Statistical Analysis System (SAS) (SAS Institute, Inc., Cary, NC, USA).Cluster analysis was performed based on all characteristics, using Ward's method (Ward, 1963).

RESULTS
Significant differences were observed for specific activities of enzymes among R. damascena accessions, various harvesting time, and two years (Table 1).There were significant interactions among years, accessions and sampling stages.POD activities of leaves varied annually in the same condition and were higher in 2008 compared to 2009, except for Tehran1 that showed the highest activity (4.65 activity/g FW) in the second year.Strongest POD activities were showed in Fars1 and Tehran1 (3.54 and 3.39 activity/g FW, respectively) in the first year and the lowest ones was in Kermanshah1 (1.81 activity/g FW) in the second year (Figure 1).Results were expressed various effects of CAT activity among acessions in two years.So that Ardebil1 and Fars1 had maximum and Tehran1 had minimum CAT activities in two years (Figure 2).These results elucidated an inverse trend of POD and CAT activities among accessions, especially in Tehran1.The content of enhanced POD and CAT in leaves has been shown to undergo seasonal changes, with relatively low levels in young leaves in spring and strong activities in mature leaves in autumn  (Figure 3).Comparing the accessions, Tehran1 showed the highest POD activities in 3 stages (3.26,3.49 and 5.31 activity/g FW) and Fars1 and Ardebil1 (1.26 and 1.75 activity/g FW, respectively) had the lowest activities in spring.Ardebil1 showed exceptional activities, whereas it had strong activities (3.05 activity/g FW) in warmest time in summer (Figure 4).Meanwhile, Ardebil1 and Fars1 (2.17 and 1.81 activity per min) had highest CAT activity and Tehran1 indicated lowest activities (0.67 activity per min) in autumn, respectively (Figure 5).6).The difference was indicated that the season was expansion from spring to winter, POD activities were decreased, but CAT activities were enhanced to raining in autumn then it decreased (Figure 7).On the other hand, highest twig CAT activity was observed in Arak1, before rains in autumn and the lowest activity occurred in Tehran1 when leaves were fallen in winter, in contrast of POD activity (Figure 8).It is interesting that, POD activity exhibited higher and CAT activity detected lower by the R. damascena twig extract than the leaf extract (Figures 9 and 10).

DISCUSSION
Seasonal alteration can be illustrated with changes in plant metabolism and affect on biosynthetic pathways (Hatano et al., 1986;Salminen et al., 2001).The results presented in this research were obtained from plants undergoing 2-year study of seasonal changes effects on the enzymatic activities in 12 Damask rose accessions.Antioxidants protect cells from biochemical damage caused by ROS, whose production increases during imbalances within and between bioenergetics' pathways (Wise, 1995).Swanberg and Verhoeven (2002) reported the seasonal changes at antioxidant activity in the leaves of Taxus cuspidate.The existence of statistically significant differences in the specific activities of enzymes among R. damascena accessions could be attributed to the alteration in the amount of these enzymes in leaves on various harvesting time in two years.Also, main effect of seasonal acclimatization on the difference in POD and CAT enzymatic activities, suggesting a general capability of R. damascena plants of flexible adjustment in environmental conditions.The result was reinforced increase of leaf POD and CAT activities from spring to autumn, so that plants can be acclimated for high temperature in summer and prepared for winter conditions.So, decrease of raining in the first year was the reason of enhanced POD activity compare to the second year.On the other hand, these parallel changes of enzymes activities, indicating the synergistic function in protecting leaf cells against oxidative injury.Increases in the activity of leaf antioxidant systems have been suggested to be important in the acclimation of plants to winter conditions (Anderson et al., 1992;Logan et al., 1998).Vuleta et al. (2010) showed greater POD activity in leaves harvested in summer relative to their spring or autumn counterparts.Many studies have indicated the importance of guaiacol PODs in plant protection against various abiotic stresses.Mehlhorn et al. (1996) would render the guaiacol PODs in other less acidic-cellular sites effective as ascorbatedependent H 2 O 2 -detoxifying enzymes.Esfandiari et al. (2009) detected increased activities of CAT, APX and GR bring ROS production and scavenging systems activity into a balance, hence preventing oxidative stress in the cells.Finally, damage to membranes is decreased and the cell is rendered into more desirable conditions.
A comparison of various responses in leaf and twig during the season indicated the expected differences in antioxidant activity.Perhaps different behaviors of tissue type could affect and caused these changes.However, in contrast to the CAT enzyme that removes the bulk of H 2 O 2 in peroxisomes, PODs, like other peroxidases, scavenge the H 2 O 2 that is produced in the cell wall, vacuoles and the apoplast (De Gara, 2004;Willekens et al., 1997).Results showing POD and CAT activities in twigs were counterpart of the leaves.It may be due to the relative presence or distribution of active components in the extracts.Another hypothesis might be explained by the fact that during development of twig cells (high rate of assimilation) a higher level of peroxidase will be required.Therefore, it showed higher POD activity in twig compare to leaf and CAT activity was inversed.Different responses of accessions to seasonal changes were related to their originated and adaptation region.Antioxidants have to play a direct role in protecting plants in an unsuitable environment (Lohrmann et al., 2004).Plants are less sensitive to climate changes, possibly because of their natural property and very high adaptability to external conditions (Sen and Mukherji, 2009;Khabarova et al., 2010).However, sensitive accessions increased their Kashefi et al. 2581 enzyme activities to protecting the vital cycle and continue their life.POD is an important enzyme for defensive purposes (Passardi et al., 2005;Herbinger et al., 2002).The observations emphasized that Tehran1 had the greatest activity of POD and where found in the leaf among all accessions examined, as well as in the twig during annual and seasonal patterns.It is able to explain this result, since it may be that this location has few distances to the Damavand region.Therefore, they are more similar condition than the other regions.So, we can introduce Tehran1 as the most compatible Damask rose accessions to this condition.

Conclusion
Subsuming these results suggested that in face of seasonal changes and acclimatization to environment, different responses of tissue type, leaf and twig, in R. damascena accessions are activated as consequences of antioxidative enzymes activities.Also, this research allows documenting responses of Damask Rose acessions to genotypic differences by enzymatic activities.However, further separation and identification should be carried out for a better understanding.Other test for antioxidant activity will be carried out in the future to discover the full potential of these accessions.Further important traits are recommended to be evaluated and needs to be continued to understand complex characteristic responses of the plant and indicate genes involved in plant adaptation and tolerance to environmental conditions.

Figure 1 .
Figure 1.Changes of leaf peroxidase activity (activity/g FW) in 12 Damask Rose accessions in 2 years.Vertical bars represent the ±SEM.Different letters in each bar differ significantly according to DMRT test (0.05 level).

Figure 2 .Figure 3 .
Figure 2. Changes of leaf catalase activity (activity per min) in 12 Damask Rose accessions in 2 years.Vertical bars represent the ±SEM.Different letters in each bar differ significantly according to DMRT test (0.05 level).

Figure 5 .
Figure 5. Seasonal changes of leaf catalase activity (activity per min) at 3 stages in 12 Damask rose accessions in 2 years.Vertical bars represent the ±SEM.Different letters in each bar differ significantly according to DMRT test (0.05 level).

Figure 7 .Figure 8 .
Figure 7. Seasonal changes of twig peroxidase (activity/g FW) and catalase activities (activity per min) in 12 Damask rose accessions.Vertical bars represent the ±SEM.Different letters in each bar differ significantly according to DMRT test (0.05 level).

Figure 9 .Figure 10 .
Figure 9. Changes of leaf and twig peroxidase activity (activity/g FW) in 12 Damask Rose accessions.Vertical bars represent the ± SEM.Different letters in each bar differ significantly according to DMRT test (0.05 level).
(Table2).POD activities in twigs were highest for Tehran1 at 50% flowering in spring (12.05 activity/g FW) and lowest ones were obtained for Ardebil1 when leaves fallen in winter (2.59 activity/g FW), except for Fars1 that showed the lowest activity (1.21 activity/g FW) before rains in autumn.Altogether, Tehran1 had maximum(5.15,12.05,11.3 and 6.78 activity/g FW) and Fars1 showed the minimum (2.72, 3.19, 3.42 and 1.21 activity/g FW) twig POD activities among harvesting time in winter, spring, summer and autumn, respectively (Figure