Biochemical changes and winter hardiness in pomegranate (Punica granatum L.) trees grown under deficit irrigation

Scientia Horticulturae 251 (2019) 39–47

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Biochemical changes and winter hardiness in pomegranate (Punica granatum L.) trees grown under deficit irrigation

T

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Mohammadebrahim Nasrabadia, Asghar Ramezaniana, , Saeid Eshghia, Ali Akbar Kamgar-Haghighib, Mohammad Reza Vazifeshenasc, Daniel Valerod a

Department of Horticultural Science, School of Agriculture, Shiraz University, Shiraz, Iran Water Engineering Department, School of Agriculture, Shiraz University, Shiraz, Iran c Improvement Plant and Seed Department, Yazd Agricultural and Natural Resources Research and Education Center Research, AREEO, Yazd, Iran d Department of Food Technology, University Miguel Hernández, Orihuela, Alicante, Spain b

A R T I C LE I N FO

A B S T R A C T

Keywords: Antioxidant activity Lethal temperature Phenolic content Soluble carbohydrate Starch content

Drought and cold temperature as the most important abiotic factors reduce the agricultural productivity in the world. In this research, the influence of irrigation levels on biochemical changes and cold hardiness of two commercial Iranian pomegranate cultivars (‘Shishecap’ and ‘Malas-Yazdi’) investigated during 2016 and 2017. A factorial experiment based on randomized complete block design was used with three irrigation treatments including full irrigation (control), 75% of crop water requirement (moderate stress) and 50% of crop water requirement (severe stress). At the end of the growing season, some biochemical traits such as proline, soluble carbohydrate, starch, and total phenolic content as well as antioxidant activity and 50% lethal temperature (LT50) were measured in pomegranate stems. Proline, total phenolics, soluble carbohydrate content and antioxidant activity increased and starch content decreased with reducing the irrigation level. The most autumn cold hardiness in November for ‘Shishecap’ (−16.26 °C and −15.56 °C, respectively for the first and second year) and ‘Malas-Yazdi’ (−13.50 °C and −12.33 °C, respectively for the first and second year) was found in control trees, and the lowest cold hardiness for ‘Shishecap’ (−12.63 °C and −11.65 °C, respectively for the first and second year) and ‘Malas-Yazdi’ (−12.85 °C and −11.50 °C, respectively for the first and second year) were recorded in severe stressed plants. Winter cold hardiness in ‘Shishecap’ was affected by irrigation levels during both years; however in ‘Malas-Yazdi’, it was affected by irrigation levels only in the first year of study. Based on the Pearson correlation coefficients, starch content had a positive correlation with cold hardiness at the end of the growing season. Also, correlation coefficients between soluble carbohydrate and LT50 in November (r = 0.82, P ≤ 0.001) were negative. Therefore, it can be concluded that irrigation levels affect the pomegranate trees cold hardiness. Full irrigation resulted in an increase in the cold hardiness in the pomegranate trees, depending on cultivar.

1. Introduction

et al., 2000) in places that winter temperature is not lower than −15 °C, but central Asian cultivars can even survive at temperatures of −25 °C or −30 °C (Melgarejo et al., 1997). Cold temperature, as the most important abiotic factor limit the distribution of plant species and reduces the agricultural productivity on earth (Rodrigo, 2000; Cansev et al., 2012). Sorely cold winter temperatures or untimely frost in cold season can significantly reduce productivity in many tropical and subtropical crops (Cansev et al., 2012) and also lead to reduction of biosynthetic activity and inhibit physiological processes and may cause constant injuries and finally death (Bañuelos et al., 2008). In this case, severe cold season frost in 2007 and 2016 destroyed 36,931 ha and 35,000 ha of pomegranate orchards in Iran, respectively (Ministry of Jihad-e-

The pomegranate tree (Punica granatum L.) is a favourite fruit of tropical and subtropical climates (Pourghayoumi et al., 2017). Pomegranates are native to the area between Iran and Himalayas in northern India (Ergun and Ergun, 2009; Viuda‐Martos et al., 2010) and due to these features commercial orchards of pomegranate trees are now widely cultivated in Iran, India, Mediterranean countries, the drier regions of Southeast Asia, Malaysia, the East Indies, tropical Africa, United States, China, Japan, and Russia (Fadavi et al., 2005). It is well adapted to bad conditions and it can survive and grow in nutritionally poor soils conditions and undesirable climatic conditions (Sepulveda

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Corresponding author. E-mail address: [email protected] (A. Ramezanian).

https://doi.org/10.1016/j.scienta.2019.03.005 Received 6 January 2019; Received in revised form 1 March 2019; Accepted 4 March 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

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blocks design was used. The average of growing seasons, and physicochemical properties of the soil and irrigation water are presented in Table 2. In order to determine the soil parameters, sampling was carried out from 6 places of the experiment site at the mentioned depths (0–30, 30–60 and 60–90 cm). Also irrigation water parameters was measured in three steps at the growing seasons.

Agriculture, 2017). So, tolerance to chilling stress is crucial for preservation of agricultural crops. For pomegranate and other plants, selecting cold tolerant genotypes and agricultural practices such as irrigation levels and understanding the mechanism by which cold hardiness can improve cold resistance is necessary. There are many biochemical tests for evaluating resistance to cold hardiness in plants such as measurement of the phenolic compounds (Pennycooke et al., 2005; Bafeel and Ibrahim, 2008; Cansev et al., 2012), electrolyte leakage (La Porta et al., 1994; Soleimani et al., 2002; Moshtaghi et al., 2009; Aslamarz et al., 2011), stomatal size (Roselli and Venora, 1989), stomatal density (Roselli et al., 1989; Soleimani et al., 2002; Aslamarz and Vahdati, 2009) and photosynthetic activity (Antognozzi et al., 1990). Production of reactive oxygen species (ROS) in freezing damage, like other stresses in plant cells, causes oxidative stress (Mittler, 2002). In higher plants, the ability to scavenge the ROS is essential for resistance to environmental stresses (Guo et al., 2006). It has been reported a correlation between the increased capacity to scavenge ROS and tolerance to adverse environmental conditions (Cansev et al., 2012). Some reports showed that changes in the biochemical compounds such as increasing total carbohydrates (Morin et al., 2007), proline (Lalk and Dörffling, 1985), proteins (Guy, 1990), soluble carbohydrate (Pennycooke et al., 2003), phenolic compound (Pennycooke et al., 2005; Bafeel and Ibrahim, 2008; Cansev et al., 2012), decreasing of starch content (Yano et al., 2005) and increasing and then decreasing of soluble carbohydrate concentrations (Ghasemi Soloklui et al., 2012) occurs in plants during cold acclimation. Accumulation of proline and carbohydrates in plants during low temperature stress, can induce tolerance to dehydration through induction of osmotic adjustment and water potential decline (Ghasemi Soloklui et al., 2012). In addition to low temperature, another major limiting factor for the agricultural extension in the arid and semi- arid areas of the world is the amount and quality of available irrigation water. In some reports, it has been noted that pomegranate trees are tolerant to soil water deficit (Holland et al., 2009; Intrigliolo et al., 2011; Khattab et al., 2012). In arid and semi-arid regions of the world, water scarcity has led to the expansion of new strategies for the water-saving such as the sustained deficit irrigation (SDI) (Peña‐Estévez et al., 2015). High relative apoplastic water content and the ability to deal with drought stress by developing complementary stress avoidance and stress tolerance mechanisms has led to the drought resistance characteristics in pomegranates (Galindo et al., 2014). ‘Shishecap’, and ‘Malas-Yazdi’ are two important commercial cultivars of pomegranate in Iran (Pourghayoumi et al., 2017). To the best of our knowledge, there is no data about the effect of drought stress (different levels of irrigation) on the cold hardiness of pomegranate cultivars. The present study aimed to appraise the influence of the different levels of irrigation, well irrigated (control), 75% of crop water requirement (moderate stress) and 50% of crop water requirement (severe stress), on biochemical changes at the end of the growing season and cold hardiness of two commercial Iranian pomegranate ‘Shishecap’ and ‘Malas-Yazdi’.

2.2. Pomegranate orchard and irrigation treatments details The experiment was carried out on 8 years old pomegranates (Punica granatum L. cv. ‘Shishecap’ and ‘Malas-Yazdi’). Trees were planted in a 4 m × 3 m pattern. Growing season of pomegranate in the research site usually begins in late March and ends in late October. The irrigation treatments were including full irrigation (control), 75% of crop water requirement (moderate stress) and 50% of crop water requirement (severe stress). Net irrigation water depth was calculated as follow (Allen et al., 1998):

In =

(θFc − θi ) × D 100

Where: In : Net irrigation water depth (m), θFc : Soil water content at field capacity (volumetric percentage), θi : soil water content before irrigation (volumetric percentage) and D: effective root depth (m)

Vn = In × A × 1000 Where: Vn : Net irrigation volume (litter/tree) and A: wetted area (m2/tree)

Vg =

Vn × T 100 × Ea

Where: Vg : Gross irrigation volume (litter/tree), Ea: water application efficiency (%) and T: irrigation treatment (%). 2.3. Plant material One-year-old shoots (previous season growth) from 8 year old pomegranate trees were collected during autumn (in November) and winter (in late January) in 2016 and 2017. The samples were randomly collected from three pomegranate trees, then packed on ice, and brought to the laboratory of Shiraz University. 2.4. Low temperature treatments About 10–15 cm long segments of the one year old shoots were cut and covered by aluminum foil with humid paper in order to protect against desiccation, then they were exposed to low temperatures (Ghasemi Soloklui et al., 2012). Briefly, after transferring pomegranate branches to the laboratory, they washed with deionized water, cut into 1 cm long pieces, and finally six pieces put into 50 ml plastic tubes per replicate. For each sample, in order to immediate ice formation, 1 ml of deionized water was added. Tubes were exposed to low temperatures, in freezing chamber (Kimia Rahavard, Iran). The start temperature was 5 °C and the rate of cooling was 2 °C h–1. Temperatures of two acclimation stages (in 2016 & 2017) were as follow: First acclimation stage (25 Nov 2016 & 27 Nov 2017) was done with temperatures of –6, – 9, –12, –15, and −18 °C. Second acclimation stage (28 Jan 2016 & 2017) was done with temperatures of –12, –15, –18, –21, and −24 °C. At the end of the acclimation stage (at final temperature) samples were kept for 1 h and then exited from the freezing chamber.

2. Materials and methods 2.1. Experimental site The experiment was carried out during two successive seasons of 2015 and 2016 on 8 years old mature pomegranate trees in pomegranate collection of Yazd agricultural and natural resources research center, Yazd province in central part of Iran (31°54′N and 54°24′E) with an altitude of 1230 m above sea level. Trees were grown in a sandy loam soil. Soil saturation extract and irrigation water electrical conductivity were 4.97 and 3.99 dS m−1, respectively (Table 1). In the research site, no rainfall was recorded during two growing seasons. Each treatment of experiment was replicated three times and for each replicate, two trees were considered and the randomized complete

2.5. Electrolyte leakage (EL) Forty ml of deionized water was added to each tube and then they were shaken for 1 h (250 rpm) and tubes were kept for 24 h at room 40

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Table 1 Physico-chemical properties of the soil and irrigation water used in the experiment. soil properties Soil depth (cm)

Texture

Ece (dS m−1)

CaCO3 %

pH

OC%

P

K

Cu

Mn

Fe

Zn

110 150 165

0.34 0.86 0.87

1.8 3.4 3.8

4.2 5.8 5.8

0.64 0.70 0.76

(mg kg−1 soil) 0-30 30-60 60-90

SL SL SL

3.85 4.90 6.18

7.9 7.8 7.8

23.2 22.6 21.7

0.19 0.09 0.17

9.8 11.3 11.7

Irrigation water properties -1 HCO− 3 (meq L )

pH

EC (dS m−1)

Cl (meq L−1)

SO4−2 (meq L-1)

Ca2+ (meq L−1)

Mg2+ (meq L−1)

Na+(meq L−1)

2.7

7.35

3.99

24.5

13.9

13.3

10.3

17.5

OC = Organic Carbon, SL = Sandy loam, ECe = Saturated soil paste electrical conductivity, EC = Electrical conductivity. Table 2 Some meteorological data of the experimental site (Pomegranate collection of Yazd province). Month

Temperature (°C) Minimum

April May June July August September October November

Maximum

2015

2016

2015

2016

14.1 19.2 24.7 26.4 23.3 18.7 17.0 10.3

12.5 19.5 19.0 25.9 23.8 22.2 16.7 10.7

26.4 31.9 38.5 40.0 37.8 33.5 30.6 24.8

24.9 32.8 35.4 39.6 38.3 37.0 31.0 25.3

Evaporation (mm) Average 2015

2015

2016

2015

2016

8.96 10.3 14 16.2 14.4 13.4 8.5 5.4

7.6 10.5 12 15.9 14.8 11.4 8.6 5.6

28.6 20.5 10 11 11 23 17.2 21.5

28 21 15.1 11.7 10.5 13 17 22

2016

20.25 25.55 31.6 33.2 30.55 26.1 23.8 17.55

18.7 26.15 27.2 32.75 31.05 29.6 23.85 18

temperature (In dark place) before measurement of the first electrical conductivity (EC1). In order to obtain maximum ion leakage, tubes were autoclaved at 120 °C for 20 min and then tubes cooled at room temperature for 2 h. Then electrical conductivity (EC2) was measured and finally the relative electrolyte leakage (REL) was calculated by the following formula: REL= (EC1/EC2) × 100. Lethal temperature where 50% of the total ion leakage happened (LT50) used for calculating the cold hardiness by the following logistic sigmoid function (Ghasemi Soloklui et al., 2012).

R=

Average of relative humidity

Y = 0.000966X + 0.0204(R2 = (0.989) Where: Y: Absorbance of medium, X: Concentration of glucose (mg/L) 2.7. Starch content Respectively, 5 ml of cold distilled water and 6.5 ml of perchloric acid (52%) was added to each residual material that, used for sugar analysis and then was mixed for 15 min, and 20 ml distilled water was added to each sample. The same procedure was repeated 3 times with residue and obtained supernatants were combined. The samples were centrifuged and supernatant were separated and left for 30 min at 0 °C. Afterward, supernatants were filtered and their volumes adjusted to 100 ml with distilled water. Ten ml of cold 2% anthron solution was added to 2.5 ml of supernatant in the test tubes and then heated at 100 °C in a boiling water bath for 7.5 min. Tubes were cooled on ice bath rapidly. Absorption of the samples were recorded at 630 nm with spectrophotometer. Finally, to calculate the starch concentration, the calibration curve drawn for glucose standard solutions and multiplied them with 0.92 (McCready et al., 1950).

a +d 1 + e b (x − c )

Where, R was REL based on LT50 estimation method used; x was treatment temperature; b was slope of the function at the inflection point c, a and d determine the upper and lower asymptotes of the function, respectively. 2.6. Soluble carbohydrate content Soluble sugar was measured as described by Ranganna (1986), with some modifications. Stem samples were dried and 13 ml of 80% ethanol was added to 0.1 g of dry powder and then was shaken. Samples were centrifuged at 5000 rpm for10 min, then the supernatant was separated. Afterward, 5 ml of phenol solution (5%) and 5 ml of sulfuric acid was added to about 1 ml of supernatant in test tube and vortexed for 30 s. Then tubes were transferred into ice immediately to reach room temperature. The samples absorption was recorded by spectrophotometer (Bio Tek VT 05404-0998, USA) at 490 nm. Finally, total sugars concentration expressed as mg soluble carbohydrates g−1 dry weight after calculated by calibration curve drawn for glucose standard solutions. The equation of the calibration curve for soluble carbohydrate content was calculated as follow:

2.8. Proline content Stem samples were powdered by using liquid nitrogen. 0.5 g of powder was homogenized in 10 ml of 3% (w/v) aqueous sulfosalicylic acid and this homogenate solution was filtered by Whatman No. 1 filter paper. 2 ml ninhydrin and 2 ml glacial acetic acid were added to two ml of filtered extract in glass tubes. The samples were heated at 100 °C in a boiling water bath for 60 min and immediately were transferred to in an ice bath and toluene (4 ml) was added to the mixture and then was mixed vigorously for 15–20 s. Finally, absorption of the mixture was recorded at 520 nm using visible spectrophotometer (Toluene was used 41

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Table 3 The analysis of variance for the study year, cultivar (CUL), irrigation level (IRL) and their interaction effects on measured biochemical parameters and LT50 (comparison of the means based on Duncan’s multiple range test, P ≤ 0.05). Source

Year (CUL) (IRR) Year × CUL Year × IRL CUL × IRL Year × CUL × IRL

DF

Proline

1 1 2 1 2 2 2

Source

Year (CUL) (IRL) Year × CUL Year × IRL CUL × IRL Year × CUL × IRL

DF

1 1 2 1 2 2 2

Starch

Soluble carbohydrate

Antioxidant activity

F value

P value

F value

P value

F value

P value

F value

P value

0.14 213.85 228.54 20.20 0.26 31.58 0.96

> < < < > < >

1.85 0.34 125.43 0.75 1.18 8.70 3.14

> > < > > < >

1.73 25.46 21.77 6.04 1.80 1.43 0.80

> < < < > > >

9.73 93.49 12.01 7.42 0.23 3.58 0.04

< < < < > < >

0.05 0.05 0.05 0.05 0.05 0.05 0.05

Phenolic content

0.05 0.05 0.05 0.05 0.05 0.05 0.05

November LT50 (°C)

0.05 0.05 0.05 0.05 0.05 0.05 0.05

0.05 0.05 0.05 0.05 0.05 0.05 0.05

January LT50 (°C)

F value

P value

F value

P value

F value

P value

156.43 22.26 17.47 23.47 1.21 2.86 0.92

< < < < > > >

3.04 17.56 8.51 3.37 2.92 18.83 1.37

> < < > > < >

6.79 24.75 21.72 68.74 2.09 35.40 0.77

< < < < > < >

0.05 0.05 0.05 0.05 0.05 0.05 0.05

0.05 0.05 0.05 0.05 0.05 0.05 0.05

0.05 0.05 0.05 0.05 0.05 0.05 0.05

DF: Degree of freedom CUL: Cultivar IRL: Irrigation level.

methanol was used as blank to determine the concentration of remaining DPPH. Antioxidant activity was calculated from a standard curve of Trolox and results were expressed as mmol Trolox equivalent (TE) per g FW (Cansev et al., 2012). The percentage inhibition of the DPPH radical was calculated with the following equation:

for blank). Proline concentration was calculated by a calibration curve and results were expressed as micromole proline per gram of fresh weight as follow: (Bates et al., 1973). [(μg proline/ml × ml toluene) / 115.5 μg/μmole]/[(g sample)/5] = μ moles proline/g of fresh weight material.

DPPH Scavenging Effect % = [(A control – A sample) /A control] × 100

2.9. Extraction method for determination of total phenolic compounds and antioxidant activity Two g of stem samples was homogenized by blender and was extracted in 25 ml of 80% methanol by using shaker (at 250 rpm) at lab temperature for 24 h. Afterwards, these extracts were centrifuged at 4000 rpm for 15 min. The same procedure was repeated 3 time on the remaining part of the pomegranate stems and all extracts were combined and the volumes of extracts were adjusted to 100 ml using the same solvent (Cansev et al., 2012). These obtained extracts were used for determination of total phenolic compounds and antioxidant activity in pomegranate stems.

2.12. . Statistical analyses Proline, starch, soluble carbohydrates and phenolic content, antioxidant activity and LT50 values of stem samples measured at each acclimation stage (November and January) were analysed using a three-way analysis of variance (PROC GLM, SAS Institute, Cary, NC). Means were separated using Duncan’s multiple range tests (P ≤ 0.05). Correlation analysis between proline concentration, soluble carbohydrates content, phenolic compounds concentration, antioxidant activity and LT50 values in November and January was performed using Pearson’s correlation coefficient (PROC CORR).

2.10. Total phenolics Five ml of H2O and 0.5 ml of Folin-Ciocalteu reagent were added to 1 ml of extracts into the test tubes and the tubes were mixed and 1 ml of 7.5% Na2CO3 was added and test tube were shaken for 60 min in dark at room temperature. Absorbance of the samples were recorded at 750 nm with spectrophotometer. Finally, total phenolic content was calculated using the standard curve of gallic acid and results were expressed as mg of gallic acid equivalents (GAE) per g of fresh weight (FW) (Cansev et al., 2012). The equation of the calibration curve for total phenolics is present thus:

3. Result and discussion 3.1. Effect of irrigation treatment on cold hardiness The results showed that November LT50 was not significantly affected by the influence of year, whereas it was significantly affected by cultivar and irrigation treatments (Table 3). During these both years, all the water stress levels in both cultivars led to a decrease in November LT50. However, there was no significant difference for November cold hardiness between the irrigation treatments in ‘Malas-Yazdi’ cultivar (Table 4). The results of November LT50 in the first year of study showed that, the cold hardiness was significantly different between control (-16.26 °C) and deficit irrigation treatments (-12.61 °C and12.63 °C for the moderate and severe stress, respectively) in ‘Shishecap’. However, in the second year, there was no significant difference between the moderate stressed and well irrigated trees (Table 4). As shown in Table 4, water stress decreased January cold hardiness in ‘Shishecap’ pomegranate trees. However there was no significant difference for January cold hardiness between the deficit irrigation treatments during both years in ‘Shishecap’. The most winter cold

Y = 0.01X − 0.0101(R2 = (0.9934) Where: Y: Absorbance of medium, X: Concentration of gallic acid (mg/ L) 2.11. Antioxidant activity Briefly, 3.9 ml of 6 × 10−5 M methanolic solution of DPPH radicals were added to 0.1 ml of extracts into the test tubes. The tubes were placed in dark for 60 min. The DPPH free radical scavenging activity of each sample was recorded at 515 nm using spectrophotometer, whereas 42

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Table 4 Effect of different irrigation levels on November LT50 (◦C) and January LT50 (◦C) of two Iranian cultivars (‘Shishecap’ and ‘Malas-Yazdi’). Means in columns with the same letter are not significantly different at P ≤ 0.05. November LT50 (°C)

January LT50 (°C)

Year 1

Year 1

Table 5 Pearson correlation coefficients of the proline, starch, soluble carbohydrate concentration, antioxidant activity, phenolic content with November LT50 values, January LT50 values and average of these two stages in the two Iranian pomegranate. Variables

‘Shishecap’

‘Malas-Yazdi’

100 % 75 % 50% 100 % 75 % 50%

−16.26 −12.61 −12.63 −13.50 −13.01 −12.85

Year 2 a b b b b b

−15.56 −14.38 −11.65 −12.33 −12.26 −11.50

a a b b b b

−22.83 −19.73 −19.53 −20.45 −19.36 −19.71

November LT50

January LT50

All two stage

0.7 *** −0.74 *** 0.29 ns 0.02 ns 0.32 ns

0.64 ** −0.85 *** 0.63 ** 0.39 ns 0.56 **

Year 2 a bc bc b c bc

−21.16 −16.05 −16.45 −21.75 −21.13 −21.05

Proline Starch Soluble carbohydrate Antioxidant activity Phenolic content

a b b a a a

ns

0. 36 −0.69 *** 0.82 *** 0.69 *** 0.66 **

Number of observations in each level was 18, ns, *, **, *** Non-significant or significant at P ≤ 0.05, 0.01 or 0.001, respectively.

hardiness in ‘Malas-Yazdi’ cultivar was detected in control trees during both years (-20.45 and -21.75 °C, respectively). Also, there was no significant difference between the moderate stress and severe stress in the first year and among irrigation levels in the second year in ‘MalasYazdi’ winter cold hardiness (Table 4).

proline content and LT50 values in January was obtained (Table 5). However there was no significant correlation between proline concentration and November LT50 values at the end of growth season. Deficit irrigation treatments led to a significant increase in the proline content of the ‘Shishecap’ pomegranate stems. During both years, deficit irrigation treatment showed the lowest November and January cold hardiness in ‘Shishecap’. November cold hardiness was not significantly different in all irrigation treatments in ‘Malas-Yazdi’ cultivar during both years. Also, in the first year of experiment, the January cold hardiness was not significantly different between the moderate and severe stress trees and also, between control and severe stress treatment (Table 4). Pomegranate cold hardiness was not affected by stem proline content. According to our results, stem proline content increased by increasing the water restriction level. Whereas pomegranate cold hardiness was decreased in the treatments with the highest proline content in both cultivars. Little is known about the effect of proline content on cold hardiness, especially about the effect of the proline content on cold hardiness after the water stress season. However, several reports have shown the proline role in cold hardiness such as Arabidopsis (Xin, 1998), winter wheat (Petcu et al., 2000) and Persian walnut (Aslamarz et al., 2011). On the other hand, another study showed that there was not strong relationship between proline concentration of stems and cold hardiness in the pomegranate cultivars (Ghasemi Soloklui et al., 2012). Furthermore, it has been shown that proline induces chilling tolerance in chilling-sensitive plants (Xin and Li, 1993) and it is less efficient under severe cold stress (Ghasemi Soluklui et al., 2014). In addition, another research showed that when the cell membranes is intact, proline accumulation in the cytoplasm is useful in cold hardiness (Chen and Li, 2002). In our study, may be cell membranes were affected by water stress condition after the stress season in the deficit water treatment.

3.2. Proline content As show in Table 3, the interaction effect of year and cultivar on proline content was significant (P ≤ 0.05). All water stress levels in both cultivars led to an increase in proline content compared to the control. The maximum and the minimum proline contents were obtained from the severe stressed and fully irrigated trees, respectively, in both cultivars and both years (Fig. 1). During both years, the highest proline content observed in severe stress treatment in ‘Shishecap’ (1.71 and 1.58 μM g–1 FW, first and second year respectively), while the least amount (0.38 and 0.49 μM g–1 FW, first and second year respectively) was found in full irrigation treatment in ‘Malas-Yazdi’ cultivar. Results demonstrated that, proline content showed a narrow range of variation in ‘Malas-Yazdi’ compared to ‘Shishecap’ during both years. ‘MalasYazdi’ did not show the significant change between the moderate stress and control. Our findings were consistent with previous reports in pea (Alexieva et al., 2001), ckickpea (Mafakheri et al., 2010) and pomegranate (Catola et al., 2016). Osmolytes with low-molecular weight, including proline, glycinebetaine, organic acids and polyols are vital to maintain functions of cells under drought stress condition. Also under osmotic stress, osmotic adjustment has a key role for maintaining water status by involvement in the accumulation of other osmotically active molecules/ions such as proline, soluble sugars, sugar alcohols, glycinebetaine, organic acids, calcium, potassium, chloride ions, etc. (Farooq et al., 2009; Zhang et al., 2010). According to results, proline content at the end of growth season did not increase the pomegranate November cold hardiness (Table 5). A strong correlation between

3.3. Starch content The interaction effect of irrigation levels and cultivar on starch content was significant (P ≤ 0.05). By increasing water stress in pomegranate trees, starch content decreased in both cultivars during two successive years (Fig. 2). The highest and the lowest starch content were observed in control and severe stress trees, respectively, in both cultivars. In the first year, maximum starch concentration was found in control tress of ‘Shishecap’ (47.83 mg g–1 FW) which was not significantly different than ‘Malas-Yazdi’ (43.50 mg g–1 FW) control trees. Besides, in the second year, the maximum starch concentration was obtained in control trees in both cultivars. Our finding is in accordance with previous study on the starch content ratio, when pomegranate trees were affected by water accessibility limitations (Ebtedaie and Shekafandeh, 2017). Previous studies shown that the decrease in the carbohydrate reserve was related to the reduction of photosynthesis in drought stressed trees (Epron and Dreyer, 1993; Wong et al., 2009). However, it has been elucidated that functional and structural changes

Fig. 1. Effect of different irrigation levels (C = control, M = moderate stress and S = severe stress) on proline content of two Iranian pomegranate cultivars (SH= ‘Shishecap’ and M= ‘Malas-Yazdi’). Similar letters above the columns indicate non-significant difference among the irrigation levels at P ≤ 0.05. 43

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Fig. 2. Effect of different irrigation levels (C = control, M = moderate stress and S = severe stress) on starch concentration of two Iranian pomegranate cultivars (SH= ‘Shishecap’ and M= ‘Malas-Yazdi’). Similar letters above the columns indicate non-significant differences among the irrigation levels at P ≤ 0.05.

Fig. 3. Effect of different irrigation levels (C = control, M = moderate stress and S = severe stress) on soluble carbohydrate content of two Iranian pomegranate cultivars (SH= ‘Shishecap’ and M= ‘Malas-Yazdi’). Similar letters above the columns indicate non-significant differences among the irrigation levels at P ≤ 0.05.

in chloroplast leading to destruction of chlorophyll structure is the main reason of photosynthesis inactivation and reducing its products under drought stress condition (Anjum et al., 2011). According to results, decreasing the starch content led to a decrease in the November and January cold hardiness. The most autumn cold hardiness was obtained in control trees containing the highest starch content (Fig. 2). Also, strong negative correlation was observed between the starch content and November LT50, January LT50 and LT50 values when two stages (November LT50 and January LT50) were merged (Table 5). Some of researchers proposed that starch is determining factor in freezing tolerance due to its act as an energy reserve during winter (Rogers et al., 1975; Cai et al., 2004). Furthermore, it is proposed that, starch degradation as a source of energy during cold acclimation lead to enhancing freezing tolerance (Yano et al., 2005). In another research, it has been noted that the conserved carbohydrates in autumn after defoliation is the main source of carbon that may be used during the cold acclimation, development and preservation of cold resistance and cellular respiration in the cold season (Wong et al., 2009). It has been reported that low levels of storage starch implicate in dieback and mortality of the tree in the autumn (Wong et al., 2009). Our results showed that, the irrigation treatments, resulted in the highest levels of starch at the end of the growing season in control trees in both cultivars, increased cold hardiness. Sufficient storage of the main source of carbon is important for cold hardiness during the cold-season period (Wong et al., 2009).

the amount of soluble carbohydrates in pomegranate (Ebtedaie and Shekafandeh, 2017). According to results, most tolerant trees to autumn and winter cold hardiness had lowest soluble carbohydrate concentration at the end of the growth season during both years in ‘Shishecap’. But November cold hardiness in ‘Malas-Yazdi’ cultivar was not affected by irrigation treatments during both years and also, January cold hardiness in the second year. It has been reported that, conservation of soluble carbohydrate is an important prerequisite for increasing cell membranes cryostability that is one of the necessary prerequisites of cold hardiness (Ghasemi Soluklui et al., 2014). Furthermore, soluble carbohydrates have an important role in osmotic adjustment and support the cell against intercellular freezing (Ghasemi Soloklui et al., 2012). On the contrary, some studies showed that soluble carbohydrates have no significant relationship with freezing tolerance in woody plants (Cox and Stushnoff, 2001; Pagter et al., 2008; Ghasemi Soloklui et al., 2012). Generally, increasing in the soluble sugars concentration and decreasing in the starch concentration at the beginning of cold acclimation can be useful in a number of woody plants such as grapes (Hamman et al., 1996). The results of this research demonstrated that trees were more freeze tolerant when concentration of soluble carbohydrate was low or starch concentration was high in control trees at the end of the growth season. This case suggests a probable interaction between starch and soluble carbohydrate with other biochemicals such as proline, phenolic compounds or antioxidant activity that occur during cold acclimation in pomegranate. The mentioned events may be influenced by the different irrigation levels in growth season. Furthermore, it has been reported that, total concentration of soluble sugars increases significantly during acclimation which can provide the cryoprotectants (Pennycooke et al., 2003). According to results, the summation of total non-structural carbohydrates at the end of the growth season was more important than the effects of soluble carbohydrate and starch content individually on the cold hardiness of pomegranate trees, when the pomegranate trees were exposed to water scarcity (Table 5). Correlation results show that, there was a high correlation between soluble carbohydrates and LT50 values in November and there was no significant correlation between soluble carbohydrates concentration at the end of the growth season and LT50 values in the January (r = 0.29). These relationships indicate that, trees with less soluble carbohydrate but higher starch content at the end of the growth season in control trees may produce more soluble carbohydrate through the conversion of starch to carbohydrate during cold acclimation than trees with high soluble carbohydrate content and less starch content at the end of the growth season in water stress treatments during cold acclimation period.

3.4. Soluble carbohydrate concentration As shown in Table 3, the interaction effect of year × cultivar on soluble carbohydrate was significant (P ≤ 0.05). Also, the effect of irrigation levels on soluble carbohydrate was significant (P ≤ 0.05). The results showed that, the concentration of soluble carbohydrate increased in ‘Shishecap’ cultivar under water stress condition during both years (Fig. 3). But there was no significant difference between control and moderate stress treatments in the first year and between moderate and severe stress treatments in the second year. Soluble carbohydrate had a narrow range of variation among the irrigation treatments in ‘Malas-Yazdi’, but an increase in the soluble carbohydrate content was observed by increasing the water stress levels during both years (Fig. 3). However, different irrigation levels did not influence on soluble carbohydrate in ‘Malas-Yazdi’ cultivar in first year. Plants increase the concentration of low molecular-mass organic solutes such as soluble sugars, proline or other amino acids in order to regulate the osmotic potential of cells. This mechanism helps them to improve the water absorption under the drought stress (Zhang et al., 2010). These results are in accordance with previous study on the effects of drought stress on 44

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Fig. 4. Effect of different irrigation levels (C = control, M = moderate stress and S = severe stress) on antioxidant activity of two Iranian pomegranate cultivars (SH= ‘Shishecap’ and M= ‘Malas-Yazdi’). Similar letters above the columns indicate non-significant differences among the irrigation levels at P ≤ 0.05.

Fig. 5. Effect of different irrigation levels (C = control, M = moderate stress and S = severe stress) on phenolic content of two Iranian pomegranate cultivars (SH= ‘Shishecap’ and M= ‘Malas-Yazdi’). Similar letters above the columns indicate non-significant differences among the irrigation levels at P ≤ 0.05.

content was significant (Table 3). In both cultivars and years, the phenolic content increased by reducing the plant water supply (Fig. 5). However, irrigation levels did not affect phenolic content in both cultivars, in first year. However in the second year, phenolic content was significantly different between the moderate stress (468.70 mg g−1 FW) and severe stress (481.99 mg g−1 FW) in ‘Malas-Yazdi’ cultivar. In ‘Shishecap’ cultivar, a significant difference between different irrigation treatments was found in the second year. An increase in phenolics content was reported under abiotic stress conditions in other plants (Christie et al., 1994; Tahi et al., 2008; Oh et al., 2010; Gharibi et al., 2016). The phenolics content of ‘Malas-Yazdi’ was not significantly changed in control and moderate stressed trees in two years. Also, the amount of phenolics in ‘Malas-Yazdi’ was not significantly different between different irrigation levels in the first year. Genetic and environmental conditions such as high temperature, pathogens, herbicides and light (including ultraviolet light) can affect the biosynthesis of secondary metabolites such as phenolics (Cansev et al., 2012). Drought stress leads to the oxidation of membrane lipids, the increasing the activity of ROS and damaging to the photosynthetic system (Sofo et al., 2008). The ROS damage is determined by the balance between the production of ROS and their neutralization by plant antioxidant system (Gharibi et al., 2016). High correlations was revealed between phenolic content and November LT50 values (Table 5). Relationship between the phenolic compounds and low temperature tolerance reported in petunia (Pennycooke et al., 2005), alfalfa (Bafeel and Ibrahim, 2008) and olive (Cansev et al., 2012). Cansev et al. (2012), reported that cold-acclimation induced accumulation of phenolic compounds in leaves of the olive trees in which was positively correlated with antioxidant capacity. Also, Pennycooke et al. (2005) reported that, cold acclimation occurs due to the increasing of the total phenolic compounds. Several secondary metabolites such as phenolic compounds accumulate to regulate the cold stress in plants (Christie et al., 1994).

3.5. Antioxidant activity The interaction effect of year × cultivar on antioxidant activity was significant (Table 3). Generally, antioxidant activity increased by increasing water stress depending on cultivar (Fig. 4). The results indicated that different irrigation levels did not influence on antioxidant activity, in ‘Malas-Yazdi’ cultivar. Minimum and maximum antioxidant activities were obtained in control and severe stressed trees, respectively (Fig. 4). Probably, it depends on cultivar origin. This cultivar is originated from Yazd province (desert zone). Also, the experiment was carried out in this zone and this advantage might be due to its original condition. In the first year, there was a significant difference between ‘Shishecap’ severe stressed (81.21%) and control (68.48%) trees. However, there was no significant difference in antioxidant activity between ‘Shishecap’ control and moderate stressed trees (Fig. 4) and between ‘Shishecap’ moderate and severe stressed trees in the first year in. In the second year, changes in antioxidant activity was significant between the moderate stressed (64.42%) and severe stressed (73.54%) trees. The previous studies indicated that reactive oxygen species (ROS) increased under drought stress condition (Sofo et al., 2008) and accordingly high antioxidant components are necessary to compensate the drought stress damage and to improve the stress tolerance (Gharibi et al., 2016). Meantime, cell membrane damage under drought stress condition can lead to a decrease in free radical scavenging in plants (Lin et al., 2006). Indeed, drought stress imposes injury to the membrane during the growing season which can increase the sensitivity of membrane during reaction with cold weather. There was no significant correlation between January LT50 and antioxidant activity, neither between antioxidant activity and LT50 value when the two stages were pooled (Table 5). Higher correlations were shown between antioxidant activity and LT50 value in November stage (r = 0.69, P ≤ 0.001). One of the main factors for tolerance to stress condition in higher plants is their ability to scavenge the ROS (Guo et al., 2006). These data suggest that pomegranate tissues are damaged irreversibly during drought stress, especially in ‘Shishecap’ cultivar which results in decreased cold resistance during the cold season. Freezing damage like drought stress by producing ROS can cause cell injury in plant tissues (Mittler, 2002) and in this condition the rate of produce of ROS can suppress the plant’s antioxidant capacity (Becana et al., 1998; Cansev et al., 2012). Pennycooke et al. (2005), expressed that the effect of plant antioxidant capacity function is medium in the chilling tolerance.

4. Conclusion The effects of different irrigation levels on cold hardiness of two Iranian pomegranate cultivars after sustained deficit irrigation were detected by measurement of EL in two years. According to the results, deficit irrigation increased proline, total phenolics, soluble carbohydrates content as well as antioxidant activity in stems at the end of the growth season. But there were differences between the mentioned parameters on cold hardiness of two cultivars. The results showed that, ‘Malas-Yazdi’ cultivar was more resistant to drought stress. Our finding showed that moderate deficit irrigation didn’t has significant effect on the measured parameters, except ‘Malas-Yazdi’ cultivar starch content.

3.6. Phenolic content According to results, the interaction of year × cultivar on phenolic content was significant (P ≤ 0.05). The effect of year on phenolic 45

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According to the results, November LT50 in both years and January LT50 in second year of experiment were not affected by irrigation treatments in ‘Malas-Yazdi’ cultivar. On the contrary, November and January LT50 were affected by deficit irrigation in the ‘Shishecap’ cultivar in both years. Our findings showed that, more starch content in fully irrigated trees of both cultivars was involved in more cold hardiness. Starch content at the end of the growth season seems to be positively related with pomegranate cold hardiness. Overall, water limitation had different effects on November and January cold hardiness in different pomegranate cultivars which is very important in areas with critical low temperature during winter.

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