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Chilling and heat requirement of pomegranate (Punica granatum L.) trees grown under sustained deficit irrigation
Scientia Horticulturae 263 (2020) 109117
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Chilling and heat requirement of pomegranate (Punica granatum L.) trees grown under sustained deficit irrigation
T
Mohammadebrahim Nasrabadia, Asghar Ramezaniana,*, Saeid Eshghia, Ali Sarkhoshb a b
Department of Horticultural Science, School of Agriculture, Shiraz University, Shiraz, Iran Department of Horticultural Sciences, University of Florida, Gainesville, FL, 32611, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Stomatal density Leaf area Pomegranate cuttings Non-structural carbohydrate Drought stress
Soil moisture is one of the important factors affecting the chilling requirement of a plant, and many plants do not resume normal growth and set fruit without satisfying their required chilling. The effect of water deficit on chilling requirement, heat requirement, stomatal density, non-structural carbohydrate, and leaf area of two Iranian pomegranate cultivars, ‘Shishecap’ and ‘Malas-Yazdi’ was studied. The experiment was carried out during two successive years of 2015 and 2016. Two sustained deficit irrigation (SDI) regimes, 75% (moderate stress) and 50% (severe stress) of water requirement were applied, while 100% water supply served as the control treatment for each cultivar. The experiment was setup as a randomized complete block design with three replications and two trees were measured in each replicate. Results indicated the chilling requirement and stomatal density increased with greater water restriction in both cultivars and in both years. Non-structural carbohydrates and leaf area were reduced in water deficit treatments compared to the well irrigated trees in both cultivars. Based on our findings, water deficit treatments did not affect the heat requirement of either cultivar. Overall, these results strengthened the idea that leaf stomatal density, level of stem non-structural carbohydrate and leaf area at the end of the growing season are three key variables for pomegranate growth analysis, and correlate with chilling requirement under water stress conditions.
1. Introduction Pomegranate trees (Punica granatum L.) are cultivated in subtropical and tropical regions of the world including Iran, India, Turkey, Afghanistan, Spain, Egypt, and other parts of North Africa, China, Italy, France, and the United States (Holland et al., 2009) and are well adapted to poor soil conditions and wide ranging climates (Holland et al., 2009; Nasrabadi et al., 2019). It has been reported that quality and amount of irrigation are the two important limiting factors for agricultural expansion in the arid and semi-arid regions (Nasrabadi et al., 2019). It has also been noted that water scarcity has led to serious decreases in the growth and quality of many plants (Pérez-Pérez et al., 2008). Plants using specialized mechanisms such as reducing leaf production, increasing the rate of leaf senescence, and delaying leaf abscission can survive and stand against drought conditions (Hayatu et al., 2014). In addition to these mechanisms and according to previous reports, water deficiency in arid and semi-arid regions has led to the implementation of new methods for saving water, such as sustained deficit irrigation (SDI) (Peña‐Estévez et al., 2015). Dormancy is an important part of the development phase of
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temperate-zone deciduous fruit trees, which allows to them to tolerate and survive in adverse conditions throughout cold seasons (Campoy et al., 2011). On the other hand, cold temperatures in winter are vital for flowering and successful production of many fruit trees, and all temperate and subtropical trees have chilling requirements to promote their flowering, fruit set, and economically sufficient production (Luedeling et al., 2009). It has been reported that production and crop quality decrease severely when winter is not adequately cool, and failure to fulfil the chilling requirement can lead to irregular flowering, uneven fruit development, reduction in fruit size and maturity stage, subsequently reducing the quantity and quality of the product (Luedeling et al., 2009). In recent years as a result of global warming, the temperature of the world increased abnormally and in some parts of Iran (especially in Fars province in southern Iran). climate change has affected the rates of chilling and heat accumulation, which are vital for flowering and fruit set, and can lead to reduced production of fruit trees (Ministry of Jihad-e-Agriculture Agricultural Statistics, 2017). In many fruit trees such as pomegranate (Ghasemi Soloklui et al., 2017), apple (Jacobs et al., 2002) and almond (Djampor and Gregorian, 2000; Alonso et al., 2005) dormant cuttings have been used to determine the
Corresponding author. E-mail addresses: [email protected], [email protected] (A. Ramezanian).
https://doi.org/10.1016/j.scienta.2019.109117 Received 12 October 2019; Received in revised form 3 December 2019; Accepted 6 December 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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Vn = In × A × 1000
chilling requirement. In order to determine the plants chilling requirement many models have been used such as the Utah model, lowchilling model, Aron model, Dynamic model, North Carolina model, etc. (Ghasemi Soloklui et al., 2017). To the best of our knowledge, there is no data about the influence of water stress (diff ;erent levels of irrigation) on the chilling and heat requirement of pomegranate cultivars. The main objective of this study was to appraise the influence of two sustained deficit irrigation (SDI) levels, 75% (moderate stress) and 50% (severe stress) compared to the 100% (control) irrigation on some botanical traits of two commercial Iranian pomegranate ‘Shishecap’ and ‘Malas-Yazdi’ at the end of the growth season and on their chilling and heat requirements.
Where:
Vn: Netirrigation volume(Lper tree) and A: wetted area(m2per tree)
Vg =
Vn × T 100 × Ea
Where: Vg : Gross irrigation volume(Lper tree), Ea
: water application efficiency(%) and T: irrigation treatment(%). 2.3. Plant material One-year-old twigs (previous season growth) were obtained immediately after leaf abscission from each pomegranate tree cultivar in November (before chilling accumulation) of 2015 and 2016 when the mean temperature was ≈ 13 °C. The pomegranate twig samples were randomly collected by hand and were packed into plastic bags (wrapped in a damp cloth) in order to avoid de-hydration, then immediately transferred to the laboratory of Shiraz University for further evaluation.
2. Materials and methods 2.1. Experimental site characteristics This experiment was conducted during two successive years of 2015 and 2016 on 8 year old pomegranates (Punica granatum L. cvs. ‘Shishecap’ and ‘Malas-Yazdi’) in the main pomegranate repository of Iran, located at the pomegranate collection of Yazd Agricultural and Natural Resources Research Center (ANRRC). The ANRRC is in the central part of Iran (31°54′N and 54°24′E) with an annual average precipitation of less than 100 mm and an elevation of 1230 m above sea level. Trees prior to the study had been established in a 4 m × 3 m spacing. The growing season for pomegranate trees in the experiment site typically begins in late March and ends in late October. The study site irrigation water and soil saturation extract electrical conductivity and other physico-chemical characteristics of the soil and water are shown in Table 1. In the experiment location no precipitation was monitored throughout two growing seasons. The soil characteristics were determined from 6 locations of the experiment site (respectively in depths 0–30, 30–60 and 60–90 cm) and the irrigation water characteristics were measured in three stages during the growing seasons and then the average of the measured parameters are shown in Table 1.
2.4. Chilling treatments in the laboratory In the laboratory, pomegranate twigs were cut into ≈ 30 cm long segments. The cuttings were immersed in fungicide solution (4000 ppm of benomyl fungicide (Ariashimi Co., Zahedan, Iran)) for 5 min and then put into plastic bags in chilling conditions. Nine artificial chilling treatments including 100, 200, 300, 400, 500, 600, 700, 800, and 900 h at 4 ± 1 °C in a refrigerator (LG, model R-B601GM, R-B602GCWP, Korea) were used while the cuttings without any artificial chilling served as control for each cultivar. For each artificial chilling treatment, 30 cuttings of each irrigation level (in 3 replicates) were taken from each cultivar for determining the chilling requirement. The cuttings were wrapped in a humid cloth and then were transferred to plastic containers. 2.5. Evaluating chilling and heat requirements of cuttings
2.2. Irrigation treatments details After artificial chilling treatments, the cuttings were taken from the refrigerator and proximal ends of the cuttings were placed in 0.3 L pots that were previously filled with distilled water. Day and night temperatures of the laboratory were adjusted to 26 and 18 °C, respectively. The distilled water of each pot was replaced daily while proximal ends of each cutting were recut every other day. According to Richardson et al. (1974), budbreak was determined when the phenological stage was at first leaf sprouting. On the other hand, when 50% of buds of the cuttings were sprouted, it was considered as the release of bud dormancy. Budbreak data was monitored three times per week until end of the experiments. Finally, GDHs accumulated for each treatment in each cultivar were calculated by using the heat requirement of the cuttings
Two sustained deficit irrigation levels including 50% (severe stress) and 75% (moderate stress) of crop water requirement were used while 100% water supply served as control treatment for each cultivar. Net irrigation water depth calculated as follow (Allen et al., 1998):
In =
(θFc − θi ) × D 100
Where: In = Net irrigation 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)
Table 1 Physico-chemical characteristics of the soil and irrigation water used in the experiment. soil characteristics Soil depth (cm)
Texture
0–30 30–60 60–90
SL SL SL
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) 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 characteristics −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. 2
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immediately tubes were put into ice to cool. The samples absorption was recorded by spectrophotometer at 630 nm. Finally, sample starch concentration was calculated by the calibration curve drawn for glucose standard solutions and multiplied by 0.92 (McCready et al., 1950).
when the cuttings moved to the laboratory condition from the refrigerator. When 50% of buds reached the sprouting of first leaves it was considered the endpoint of the measurements. According to the previous reports, 10 °C is considered to be the phenological development threshold of the pomegranate (Melgarejo et al., 1997; Jackson, 1999; Ghasemi Soloklui et al., 2017). Richardson et al. (1974) reported that one GDH °C is equal to one hour above the plant base temperature, and GDH was computed when hourly temperature was between 10 and 25 °C.
2.8. Stomatal density For each treatment, nine fully expanded leaves were picked randomly from canopy mid-section of the current season and numbers of leaf stomata were determined by using the replica method (Soleimani et al., 2002). Using adhesive tape, the stellate hairs of the lower surface of the leaves were eliminated, and stomatal density was determined by a thin film of cellulose acetate that was directly applied to the middle part of each leaf (the lower epidermis of the leaf). The cellulose acetate film was allowed to dry at room temperature before removing the film. The film sheet was removed and was placed on coded slides and counting the stomatal of each leaf was done by binocular microscope at 40x magnification. Finally, stomatal density was reported in a field area of 1 mm2.
2.6. Leaf area For this case, 20 leaves of each treatment from different sections of the tree were collected randomly. Leaf area (LA) was measured with a leaf area meter (AAM-8, Hayashi Denko Co. Ltd., Japan). 2.7. Non-structural carbohydrates content The pomegranate stem fragments were used to measure the nonstructural carbohydrates (soluble carbohydrates i.e. sum of the glucose, fructose, sucrose and starch content). The non-structural carbohydrates were measured in the same samples that were used for chilling and heat requirement measurements. Finally, non-structural carbohydrates content of each sample was determined from the total amount of soluble carbohydrates and starch content of the same segment. The soluble carbohydrates content of each segment was measured by the Ranganna (1986) method with some modifications. Stem segments were ovendried at 70 °C for 24 h and then powdered, and 13 ml of 80% ethanol was added to 0.1 g of dry powder and then shaken. Afterward, samples were centrifuged at 5000 rpm for 10 min and the supernatant was separated. The supernatant was used for soluble sugar measurements. The phenol solution 5% (5 ml) and sulfuric acid (5 ml) were added to each test tube containing 1 ml of supernatant and the test tubes were vortexed (30 s). The tubes were then immediately placed into ice to reach room temperature. The absorption of each sample at 490 nm was recorded by spectrophotometer (BioTek, VT 05404-0998, USA). Finally, total sugars content was expressed as mg soluble carbohydrates g−1 dry weight after calculated by calibration curve drawn for glucose standard solutions. The segments starch content was determined through the following method. For this purpose, cold distilled water (5 ml) and perchloric acid 52% (6.5 ml) was added to each residual material that was used for sugar analysis. After that, the samples were mixed (15 min) and distilled water (20 ml) was added to each sample. The same method with residue was repeated 3 times and then all supernatants were mixed. The samples were centrifuged, and the supernatant separated and kept at 0 °C for 30 min. The supernatant was filtered and their volumes adjusted to 100 ml by using distilled water. Then, cold 2% anthron solution (10 ml) was added to supernatant (2.5 ml) in the test tubes. The samples were heated in a boiling water bath for 7.5 min at 100 °C and
2.9. Experimental design and statistical analysis This experiment was setup in a randomized complete block design with three replications and two trees per each replication. Bud chilling and heat requirement, non-structural carbohydrates of cuttings, LA, and stomatal density of leaf samples 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 bud chilling and heat requirement and non-structural carbohydrates content, LA and stomatal density was performed using Pearson’s correlation coefficient (PROC CORR). 3. Results and discussion 3.1. Effect of irrigation treatment on chilling and heat requirements Based on the results of the analysis of variance, chilling requirement was not significantly affected by the year, whereas it was significantly affected by cultivar, irrigation treatment and the interaction between cultivar × irrigation (Table 2). The results show that all restricted soil water conditions in both cultivars led to an increase in chilling requirement. However, there was no significant difference between the control irrigated trees and those exposed to moderate stress in ‘Shishecap’ cultivar. The results demonstrated that the chilling requirement was significantly different between deficit irrigation treatments (700 h and 694.44 h for the moderate and severe stress, respectively) and well irrigated treatment (605.56 h) in ‘Malas-Yazdi’ cultivar. However, there was no significant difference between the moderate and severe stressed trees in ‘Malas-Yazdi’ cultivar (Fig. 1). As shown in Table 4, according to results of the analysis of variance
Table 2 Results of the analysis of variance for the study year, cultivar and irrigation and their interaction effects on measured parameters (comparison of the means based on Duncan test, P ≤ 0.05). Source Year (CUL) (IRR) Year × CUL Year × IRR CUL × IRR Year × CUL × IRR Error C.V
DF 1 1 2 1 2 2 2 20
Chilling requirement (h) ns
277.83 19289.96 *** 25586.86 *** 1512.30 ns 92.54 ns 6512.38 *** 30.86 ns 524.66 3.55
Heat requirement GDH (°C) ns
Stomatal density (mm 2.) ns
3136.00 2637376.00 ns 529984.00 ns 254016.00 ns 3136.00 ns 1997632.00 * 65856.00 ns 533747.20 8.68
113.95 428.145 ** 771.22 *** 14.83 ns 20.47 ns 255.28 ** 30.92 ns 34.19 6.63
DF: degree of freedom CUL: Cultivar IRR: Irrigation. 3
Non-structural carbohydrate
Leaf area
150.70 * 369.51 ** 2217.26 *** 66.63 ns 76.27 ns 24.74 ns 31.50 ns 35.72 4.76
11.61 *** 0.98 ns 25.64 *** 0.06 ns 0.10 ns 0.90 ns 0.04 ns 0.61 9.50
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Fig. 1. Effect of different irrigation levels (a = control, b = moderate stress, and c = severe stress) on chilling requirement 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 (a = control, b = moderate stress, and c = severe stress) on stomatal density 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. 2. Effect of different irrigation levels (a = control, b = moderate stress, and c = severe stress) on heat requirement (GDH) 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. 4. Effect of different irrigation levels (a = control, b = moderate stress, and c = severe stress) on non-structural carbohydrate 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.
there was no significant difference between the two years (Table 2), therefore the average of the results of two years was presented (Fig. 2). As shown in Table 4, the interaction between cultivar × irrigation and cultivar on heat requirement was significant (P ≤ 0.05). According to our results, heat requirement was not significantly affected by the irrigation treatments. Also, the heat requirement of ‘Malas-Yazdi’ cultivar was higher than the ‘Shishecap’ cultivar by 11.06%.
between 46 and 108 stomata mm−2. Stomata are microscopic structures that are found on the aerial surfaces of the plant leaves and distribution of them is one of the important characteristics of plants in response to drought stress conditions by playing a basic role in adjusting the water status and obtaining carbon (Bertolino et al., 2019). Stomatal density is a quantitative characteristic and it has been reported that it is determined genetically (Gailing et al., 2008), but this feature can also be affected by environmental conditions (Bertolino et al., 2019). Our results were consistent with previous studies on the stomatal density, when olive trees were affected by water accessibility limitations (Bosabalidis and Kofidis, 2002). Also Bañon et al. (2004) reported that water stress conditions led to increase in the stomatal density on abaxial and adaxial surfaces of leaves compared to wellwatered plants in Lotus creticus. It has been reported that increasing the stomatal density in water stress conditions is due to the decrease in leaf area and it is not an adaptative mechanism (Ciha and Brun, 1975; Bañon et al., 2004). It has also been reported that decrease in transpiration rate is one of the main actions that occurs by closing the plant stomata in drought conditions (Bosabalidis and Kofidis, 2002). In fact, plants stomata will be closed under drought stress to prevent water loss, and higher leaf stomatal density of stressed pomegranate trees cannot increase the amount of carbohydrate storage in these plants, because when the plant stomata are closed the rate of photosynthesis decreases due to the reduction of the CO2 and O2 exchange between the inside and outside of the leaf. Correlation results showed that there was a positive correlation
3.2. Stomatal density Combined analysis of variance of two years showed that the effect of year was not significant for stomatal density (Table 2). Therefore, two year average results for the stomatal density was presented (Fig. 3). The interaction effect of cultivar × irrigation on stomatal density was significant (Table 2). In both cultivars, stomatal density increased by increasing water stress depending on cultivar (Fig. 4). However, the results showed that there was no significant difference between stomatal density of ‘Shishecap’ cultivar in well irrigated and moderate stressed trees, but the maximum and minimum of stomatal density were obtained in severe stressed and well irrigated trees, respectively (Fig. 4). The stomatal density of ‘Malas-Yazdi’ was not significantly different in moderate and severe stressed trees, but severe stressed and control treatment had the maximum and minimum of stomatal density, respectively (Fig. 4). Stomatal density ranged from 68 stomata mm−2 (well irrigated in ‘Shishecap’ cultivar) to 101 stomata mm−2 (severe stress in ‘Shishecap’ cultivar). This result is in agreement with previous report by Ghasemi Soloklui et al. (2017) in 20 Iranian pomegranate cultivars which ranged 4
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Table 3 Pearson correlation coefficients of the chilling requirement, heat requirement, stomatal density, non-structural carbohydrate and leaf area of two Iranian pomegranate cultivar. Variable
Chilling requirement
GDH
Stomatal density
Non-structural carbohydrate
Chilling requirement GDH Stomatal density Non-structural carbohydrate Leaf area
0.32 ns 0.75 *** −0.57 ** −0.81 ***
0.25 ns 0.19 ns −0.10 ns
−0.64 ** −0.78 ***
0.82 ***
Leaf area
GDH: growing degree hour.
(r = 0.75, P ≤ 0.001) between stomatal density and chilling requirement, whereas no strong correlation (r = 0.25) was observed between stomatal density and heat requirement (Table 3). Our results were consistent with previous reports on pomegranate (Ghasemi Soloklui et al., 2017). Stomata are flanked by a pair of specialized guard cells, that when the soil or air water is abundant, the turgor pressure increases, and increasing the turgor pressure leads to increases in the rate of the CO2 uptake for photosynthesis (Hetherington and Woodward, 2003). Conversely, water deficit signals decrease the hydraulic conductivity and increase the concentration of abscisic acid hormone, leading to decreases in the guard cell turgor pressure, resulting in the photosynthesis rate declining (Bertolino et al., 2019). The closure of stomata prevents the CO2 entry into leaf mesophyll, and leads to reducing the amount of photosynthesis and thus carbohydrates (Bosabalidis and Kofidis, 2002). It has been reported that decreases in photosynthesis rate can reduce the action of photosynthesis and decrease carbohydrate production (Anjum et al., 2011), and it has been found that decrease of carbohydrate production and its accumulation is an important parameter which can affect bud break and early season growth in spring (Wong et al., 2003). Based on our results, when pomegranate trees grow under water deficit conditions their leaf stomatal function may be affected, and as the stomata play a very important role in the production and accumulation of carbohydrates, production and accumulation of nonstructural carbohydrates is reduced, and leads to increasing the chilling requirement.
2009). It is known that the main cause of reducing the photosynthesis and its products in water deficit conditions is related to changes in the structure and performance of chloroplasts by demolition of chlorophyll structure (Anjum et al., 2011). Also it has been elucidated that formation of xylem embolisms increase in water deficit conditions, thus reduces the movement of water from the roots to the leaves, and the leaves respond by closing the stomata to prevent more water loss and subsequent embolism (Trifilò et al., 2017). It has been reported that closing the stomata leads to a progressive decrease of photosynthetic rates and potentially forcing plants to unload the non-structural carbohydrate reserves (Maguire and Kobe, 2015). According to the results, trees with highest chilling requirement had the lowest non-structural carbohydrates concentration at the end of the growth season in both cultivars. In this case correlation results showed that there was a negative correlation (r = −0.57, P ≤ 0.01) between non-structural carbohydrate concentration and chilling requirement and there was no significant correlation between non-structural carbohydrate concentration at the end of the growth season and heat requirement (r = 0.19) (Table 3). The reserved carbohydrates stored at the end of the growth season are the best indicator for forecasting the trees health, vitality and their productivity and reserved carbohydrates stored in early autumn have positive correlation with vitality of trees, and conversely, low levels of stored starch in early autumn is correlated with tree dieback and mortality (Wong et al., 2009). Also, it has been shown that 80% of the plants growth reserves consumed for re-growth are used from the reserved carbohydrates (Oosthuizen and Snyman, 2001). It has been well documented that summer drought conditions decrease the fixation of the photosynthetic carbon which reduces the carbohydrates and also the manufacture and accumulation of nonstructural carbohydrates which can affect deciduous trees cold-season physiology in leafless periods (Wong et al., 2009). It has been reported that the rate of stored starch is crucial for bud break and early season growth (Wong et al., 2003). According to results of this research, trees with higher levels of non-structural carbohydrates at the end of the growth season had lower chilling requirements because the level of reserved carbohydrates stored in their stems was more than the deficit treatments in two Iranian pomegranate cultivars.
3.3. Non-structural carbohydrate The results of the combined analysis of variance for two years showed that the effect of year was significant for non-structural carbohydrate (Table 2). Therefore, two year results were presented separately (Fig. 4). As shown in Table 2, the effect of irrigation (P ≤ 0.001) and cultivar (P ≤ 0.01) on non-structural carbohydrate was significant. In both cultivars, all water deficit levels led to a decrease in nonstructural carbohydrate compared to the well irrigated treatment in both years. According to results, maximum non-structural carbohydrate concentration was found in control trees of ‘Shishecap’ which was significantly different than moderate and severe stress treatments in the first and second year. In the second year, there was no significant diff ;erences between water deficit treatments in ‘Shishecap’ cultivar (Fig. 4). In ‘Malas-Yazdi’ cultivar, the maximum and the minimum of non-structural carbohydrate concentrations were obtained from the fully irrigated and severe stressed trees, respectively, in both years (Fig. 4). Based on our results, there was significant differences between control and moderate stress treatments and also between moderate and severe stress treatments in the ‘Malas-Yazdi’ cultivar in the first year, but there was no significant difference between moderate and severe stress treatments in ‘Malas-Yazdi’ cultivar in the second year (Fig. 4). Our results are in agreement with previous studies on the concentration of carbohydrate reserves of pomegranate, when trees were aff ;ected by drought stress (Ebtedaie and Shekafandeh, 2017). It has been found that reduction of reserve carbohydrates stored in water deficit conditions is due to the reduction of photosynthesis (Wong et al.,
3.4. Leaf area The combined analysis of variance of two years showed that there was a statistically significant difference between years for leaf area (Table 2), so the results of two years were presented separately (Fig. 5). According to results, the interaction effect of cultivar × irrigation and irrigation treatments were significant on the leaf area (Table 2). All water stress levels in both cultivars led to decreases in leaf area compared to the control in both cultivars, and in both years (Fig. 5). The maximum and the minimum leaf area were obtained from the severe stressed and fully irrigated, respectively, in ‘Shishecap’ cultivar but there was no significant difference between the moderate and fully irrigated trees in ‘Shishecap’ in both years (Fig. 5). In both years, the most leaf area was observed in fully irrigated treatment, while the lowest amount of leaf area was obtained in highest water deficit condition in ‘Malas-Yazdi’ cultivar (Fig. 5). According to results, all water 5
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4. Conclusion The influence of different irrigation treatments on chilling and heat requirements of two Iranian pomegranate cultivars after sustained deficit irrigation were detected by cutting stems in two successive years. Regarding the results, ‘Shishecap’ and ‘Malas-Yazdi’ cultivars showed a different tolerance to sustained deficit irrigation. Water accessibility limitation conditions caused negative influence on some parameters such as reduction of non-structural carbohydrates content and leaf area of these cultivars at end of the growth season. Based on the information provided, reducing the amount of soil water availability will increase the chilling requirement of pomegranate trees but does not influence on pomegranate trees heat requirement. Based on our findings, moderate deficit irrigation did not have significant effect on ‘Shishecap’ cultivar chilling requirement. Our findings showed that leaf area decreases with increasing drought stress, leading to increase of the stomatal density. Non-structural carbohydrates content can be used as the most important physiological marker to evaluate the chilling requirement under water deficit irrigation treatments. Overall, water limitation had different effects on chilling requirement in diff ;erent pomegranate cultivars, and that is very important in areas where the cold season is not adequately cool and can increase the flowering and successful production problems.
Fig. 5. Effect of different irrigation levels (a = control, b = moderate stress, and c = severe stress) on LA 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.
deficit levels showed significant decrease in leaf area compared to 100% water supply in both years in ‘Malas-Yazdi’ cultivar. Results from this study are similar to those founded by another study in which decrease in leaf area was reported under water stress (Bañon et al., 2004; Hayatu et al., 2014). Portes and Melo (2014) reported that changes in LAI parameter is related to some factors such as seasonal climate change, plant water availability, nutritional condition (specifically nitrogen) and atmospheric CO2 concentration. It has been reported that decreases in plant leaf area is an adaptation mechanism in order to avoid higher transpiration rate and also decreasing leaf surface exposed to solar radiation (Hayatu et al., 2014). Also, reduction in plant leaf area under water deficit is an avoidance mechanism that decreases the water loss from the leaf surfaces (Bañon et al., 2004). Also, Bañon et al. (2004) stated that irrigation can affect morphological and anatomical characteristics that associate with the mechanisms of avoidance, including changes in growth of plants aerial sections as well as size and shape of leaves. It has been found that increasing plant root depth for maximum water uptake, efficient root systems in order to maximize water absorption, decreasing water loss by reducing stomatal conductance, reducing radiation absorption by leaf rolling and also reducing evapo-transpiration are the efficient mechanisms for turgor enhancement in water stress conditions (Mitra, 2001). Also, it has been reported that plants use other mechanisms such as reducing leaf production, increasing leaf senescence and delaying leaf abscission against drought conditions (Hayatu et al., 2014). There was a strong negative correlation between leaf area and chilling requirement (r = -0.69, P ≤ 0.001). Also, according to correlation results, there was no significant correlation between leaf area of pomegranate trees at the end of the growth season and heat requirement (r = −0.05) (Table 3). The ability of CO2 fixation by plants in photosynthesis depends primarily on the LAI and also other leaf characteristics such as morphology, anatomy, and arrangement of leaves in the canopy (Portes and Melo, 2014). Previous researchers argued that there is a strong correlation between plant production and light, in which this relationship is mainly determined by the plant leaf area (Hirose, 2004; Lindquist et al., 2005; Portes and Melo, 2014). According to our findings, there was a strong significant correlation between leaf area and non-structural carbohydrates (r = 0.82, P ≤ 0.001) which confirms that when pomegranate trees grow under water deficit conditions, their leaf area decreases and therefore, the concentration of carbohydrate reserves decreases. Most likely, higher amounts of non-structural carbohydrates affected cold-season physiology of control pomegranate trees in leafless periods and led to their faster bud break and early season growth.
CRediT authorship contribution statement Mohammadebrahim Nasrabadi: Conceptualization, Formal analysis, Methodology, Writing - original draft. Asghar Ramezanian: Conceptualization, Project administration, Funding acquisition, Resources. Saeid Eshghi: Methodology, Software, Writing - review & editing. Ali Sarkhosh: Writing - review & editing. Declaration of Competing Interest The authors declare that there is no conflict of interest. Acknowledgements This project was made conceivable through a Ph.D. dissertation at School of Agriculture, Shiraz University (Mohammadebrahim Nasrabadi). The authors are truly thankful to Shiraz University for financial supports and Yazd Agricultural and Natural Resources Research Center, Yazd, Iran for providing the plant materials. References Ministry of Jihad-e-Agriculture Agricultural Statistics, 2017. Executive Committee on Management of Environmental Stresses for Horticultural Products. Allen, R.G., Pereira, L.S., Raes, D., Smith, M., 1998. Crop Evapotranspiration-Guidelines for Computing Crop Water Requirements-FAO Irrigation and Drainage Paper 56. FAO, Rome 300, D05109. Alonso, J.M., Ansón, J.M., Espiau, M.T., 2005. Determination of endodormancy break in almond flower buds by a correlation model using the average temperature of different day intervals and its application to the estimation of chill and heat requirements and blooming date. J. Am. Soc. Hortic. Sci. 130, 308–318. Anjum, S., Wang, L., Farooq, M., Khan, I., Xue, L., 2011. Methyl jasmonate‐induced alteration in lipid peroxidation, antioxidative defence system and yield in soybean under drought. J. Agron. Crop Sci. 197, 296–301. Bañon, S., Fernandez, J., Franco, J., Torrecillas, A., Alarcón, J., Sánchez-Blanco, M.J., 2004. Effects of water stress and night temperature preconditioning on water relations and morphological and anatomical changes of Lotus creticus plants. Sci. Hort. 101, 333–342. Bertolino, L.T., Caine, R.S., Gray, J.E., 2019. Impact of stomatal density and morphology on water-use efficiency in a changing world. Front. Plant Sci. 1–11. Bosabalidis, A.M., Kofidis, G., 2002. Comparative effects of drought stress on leaf anatomy of two olive cultivars. Plant Sci. 163, 375–379. Campoy, J., Ruiz, D., Egea, J., 2011. Dormancy in temperate fruit trees in a global warming context: a review. Sci. Hort. 130, 357–372. Ciha, A., Brun, W., 1975. Stomatal size and frequency in soybeans 1. Crop Sci. 15, 309–313. Djampor, J., Gregorian, V., 2000. Assessing the dormancy characteristics of some
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Phenological stages of the pomegranate tree (Punica granatum L.). Ann. Appl. Biol. 130, 135–140. Mitra, J., 2001. Genetics and genetic improvement of drought resistance in crop plants. Csci 758–763. Nasrabadi, M., Ramezanian, A., Eshghi, S., Kamgar-Haghighi, A.A., Vazifeshenas, M.R., Valero, D., 2019. Biochemical changes and winter hardiness in pomegranate (Punica granatum L.) trees grown under deficit irrigation. Sci. Hort. 251, 39–47. Oosthuizen, I., Snyman, H., 2001. The influence of water stress on non-structural carbohydrate concentration in Themeda triandra. S. Afr. J. Bot. 67, 53–57. Peña‐Estévez, M.E., Gómez, P.A., Artés, F., Aguayo, E., Martínez‐Hernández, G.B., Otón, M., Galindo, A., Artés‐Hernández, F., 2015. Quality changes of fresh‐cut pomegranate arils during shelf life as affected by deficit irrigation and postharvest vapour treatments. J. Sci. Food Agric. 95, 2325–2336. Pérez-Pérez, J., Romero, P., Navarro, J., Botía, P., 2008. Response of sweet orange cv ‘Lane late’to deficit irrigation in two rootstocks. I: water relations, leaf gas exchange and vegetative growth. Irrig. Sci. 26, 415–425. Portes, Td.A., Melo, H.Cd., 2014. Light interception, leaf area and biomass production as a function of the density of maize plants analyzed using mathematical models. Acta Sci. Agron. 36, 457–463. Ranganna, S., 1986. Handbook of Analysis and Quality Control for Fruit and Vegetable Products. Tata McGraw-Hill Education. Richardson, E.A., Seeley, S.D., Walker, D.R., 1974. A model for estimating the completion of rest for’Redhaven’and’Elberta’peach trees. HortScience 9, 331–332. Soleimani, A., Lessani, H., Talaie, A., 2002. Relationship between stomatal density and ionic leakage as indicators of cold hardiness in olive (Olea europaea L.). XXVI International Horticultural Congress: Environmental Stress and Horticulture Crops. pp. 521–525. Trifilò, P., Casolo, V., Raimondo, F., Petrussa, E., Boscutti, F., Gullo, M.A.L., Nardini, A., 2017. Effects of prolonged drought on stem non-structural carbohydrates content and post-drought hydraulic recovery in Laurus nobilis L.: the possible link between carbon starvation and hydraulic failure. Plant Physiol. Biochem. 120, 232–241. Wong, B., Baggett, K., Rye, A., 2003. Seasonal patterns of reserve and soluble carbohydrates in mature sugar maple (Acer saccharum). Can. J. Bot. 81, 780–788. Wong, B., Baggett, K., Rye, A., 2009. Cold-season patterns of reserve and soluble carbohydrates in sugar maple and ice-damaged trees of two age classes following drought. Botany. 87, 293–305.
commerical almond cultivars for different climates. Iran Agric. Res. 19, 181–192. Ebtedaie, M., Shekafandeh, A., 2017. Antioxidant and carbohydrate changes of two pomegranate cultivars under deficit irrigation stress. Span. J. Agric. Res. 14, 1–9. Gailing, O., LANGENFELD‐HEYSER, R., Polle, A., Finkeldey, R., 2008. Quantitative trait loci affecting stomatal density and growth in a Quercus robur progeny: implications for the adaptation to changing environments. Glob. Change Biol. Bioenergy 14, 1934–1946. Ghasemi Soloklui, A.A., Gharaghani, A., Oraguzie, N., Eshghi, S., Vazifeshenas, M., 2017. Chilling and heat requirements of 20 iranian pomegranate cultivars and their correlations with geographical and climatic parameters, as well as tree and fruit characteristics. HortScience 52, 560–565. Hayatu, M., Muhammad, S., Abdu, H.U., 2014. Effect of water stress on the leaf relative water content and yield of some cowpea (Vigna unguiculata (L) Walp.) genotype. IJSTR 3 (7), 148–152. Hetherington, A.M., Woodward, F.I., 2003. The role of stomata in sensing and driving environmental change. Nature. 424 (6951), 901–908. Hirose, T., 2004. Development of the Monsi–Saeki theory on canopy structure and function. Ann. Bot. 95, 483–494. Holland, D., Hatib, K., Bar-Ya’akov, I., 2009. Pomegranate: Botany, Horticulture, Breeding Horticultural Reviews Vol. 35 Edited by Jules Janick Copyright amp; John Wiley Sons. Inc. Jackson, D., 1999. Climate and fruit plants. Temparate and Subtropical Fruit Production. pp. 7–14. Jacobs, J.N., Jacobs, G., Cook, N.C., 2002. Chilling period influences the progression of bud dormancy more than does chilling temperature in apple and pear shoots. J. Hortic. Sci. Biotech. 77, 333–339. Lindquist, J.L., Arkebauer, T.J., Walters, D.T., Cassman, K.G., Dobermann, A., 2005. Maize radiation use efficiency under optimal growth conditions. Agron. J. 97, 72–78. Luedeling, E., Zhang, M., Girvetz, E.H., 2009. Climatic changes lead to declining winter chill for fruit and nut trees in California during 1950–2099. PLoS One 16 (7), e6166 4. Maguire, A.J., Kobe, R.K., 2015. Drought and shade deplete nonstructural carbohydrate reserves in seedlings of five temperate tree species. Ecol. Evol. 5, 5711–5721. McCready, R., Guggolz, J., Silviera, V., Owens, H., 1950. Determination of starch and amylose in vegetables. Anal. Chem. 22, 1156–1158. Melgarejo, P., Martinez‐valero, R., Guillamón, J.M., Miro, M., Amoros, A., 1997.
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Chilling and heat requirement of pomegranate (Punica granatum L.) trees grown under sustained deficit irrigation
T
Mohammadebrahim Nasrabadia, Asghar Ramezaniana,*, Saeid Eshghia, Ali Sarkhoshb a b
Department of Horticultural Science, School of Agriculture, Shiraz University, Shiraz, Iran Department of Horticultural Sciences, University of Florida, Gainesville, FL, 32611, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Stomatal density Leaf area Pomegranate cuttings Non-structural carbohydrate Drought stress
Soil moisture is one of the important factors affecting the chilling requirement of a plant, and many plants do not resume normal growth and set fruit without satisfying their required chilling. The effect of water deficit on chilling requirement, heat requirement, stomatal density, non-structural carbohydrate, and leaf area of two Iranian pomegranate cultivars, ‘Shishecap’ and ‘Malas-Yazdi’ was studied. The experiment was carried out during two successive years of 2015 and 2016. Two sustained deficit irrigation (SDI) regimes, 75% (moderate stress) and 50% (severe stress) of water requirement were applied, while 100% water supply served as the control treatment for each cultivar. The experiment was setup as a randomized complete block design with three replications and two trees were measured in each replicate. Results indicated the chilling requirement and stomatal density increased with greater water restriction in both cultivars and in both years. Non-structural carbohydrates and leaf area were reduced in water deficit treatments compared to the well irrigated trees in both cultivars. Based on our findings, water deficit treatments did not affect the heat requirement of either cultivar. Overall, these results strengthened the idea that leaf stomatal density, level of stem non-structural carbohydrate and leaf area at the end of the growing season are three key variables for pomegranate growth analysis, and correlate with chilling requirement under water stress conditions.
1. Introduction Pomegranate trees (Punica granatum L.) are cultivated in subtropical and tropical regions of the world including Iran, India, Turkey, Afghanistan, Spain, Egypt, and other parts of North Africa, China, Italy, France, and the United States (Holland et al., 2009) and are well adapted to poor soil conditions and wide ranging climates (Holland et al., 2009; Nasrabadi et al., 2019). It has been reported that quality and amount of irrigation are the two important limiting factors for agricultural expansion in the arid and semi-arid regions (Nasrabadi et al., 2019). It has also been noted that water scarcity has led to serious decreases in the growth and quality of many plants (Pérez-Pérez et al., 2008). Plants using specialized mechanisms such as reducing leaf production, increasing the rate of leaf senescence, and delaying leaf abscission can survive and stand against drought conditions (Hayatu et al., 2014). In addition to these mechanisms and according to previous reports, water deficiency in arid and semi-arid regions has led to the implementation of new methods for saving water, such as sustained deficit irrigation (SDI) (Peña‐Estévez et al., 2015). Dormancy is an important part of the development phase of
⁎
temperate-zone deciduous fruit trees, which allows to them to tolerate and survive in adverse conditions throughout cold seasons (Campoy et al., 2011). On the other hand, cold temperatures in winter are vital for flowering and successful production of many fruit trees, and all temperate and subtropical trees have chilling requirements to promote their flowering, fruit set, and economically sufficient production (Luedeling et al., 2009). It has been reported that production and crop quality decrease severely when winter is not adequately cool, and failure to fulfil the chilling requirement can lead to irregular flowering, uneven fruit development, reduction in fruit size and maturity stage, subsequently reducing the quantity and quality of the product (Luedeling et al., 2009). In recent years as a result of global warming, the temperature of the world increased abnormally and in some parts of Iran (especially in Fars province in southern Iran). climate change has affected the rates of chilling and heat accumulation, which are vital for flowering and fruit set, and can lead to reduced production of fruit trees (Ministry of Jihad-e-Agriculture Agricultural Statistics, 2017). In many fruit trees such as pomegranate (Ghasemi Soloklui et al., 2017), apple (Jacobs et al., 2002) and almond (Djampor and Gregorian, 2000; Alonso et al., 2005) dormant cuttings have been used to determine the
Corresponding author. E-mail addresses: [email protected], [email protected] (A. Ramezanian).
https://doi.org/10.1016/j.scienta.2019.109117 Received 12 October 2019; Received in revised form 3 December 2019; Accepted 6 December 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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Vn = In × A × 1000
chilling requirement. In order to determine the plants chilling requirement many models have been used such as the Utah model, lowchilling model, Aron model, Dynamic model, North Carolina model, etc. (Ghasemi Soloklui et al., 2017). To the best of our knowledge, there is no data about the influence of water stress (diff ;erent levels of irrigation) on the chilling and heat requirement of pomegranate cultivars. The main objective of this study was to appraise the influence of two sustained deficit irrigation (SDI) levels, 75% (moderate stress) and 50% (severe stress) compared to the 100% (control) irrigation on some botanical traits of two commercial Iranian pomegranate ‘Shishecap’ and ‘Malas-Yazdi’ at the end of the growth season and on their chilling and heat requirements.
Where:
Vn: Netirrigation volume(Lper tree) and A: wetted area(m2per tree)
Vg =
Vn × T 100 × Ea
Where: Vg : Gross irrigation volume(Lper tree), Ea
: water application efficiency(%) and T: irrigation treatment(%). 2.3. Plant material One-year-old twigs (previous season growth) were obtained immediately after leaf abscission from each pomegranate tree cultivar in November (before chilling accumulation) of 2015 and 2016 when the mean temperature was ≈ 13 °C. The pomegranate twig samples were randomly collected by hand and were packed into plastic bags (wrapped in a damp cloth) in order to avoid de-hydration, then immediately transferred to the laboratory of Shiraz University for further evaluation.
2. Materials and methods 2.1. Experimental site characteristics This experiment was conducted during two successive years of 2015 and 2016 on 8 year old pomegranates (Punica granatum L. cvs. ‘Shishecap’ and ‘Malas-Yazdi’) in the main pomegranate repository of Iran, located at the pomegranate collection of Yazd Agricultural and Natural Resources Research Center (ANRRC). The ANRRC is in the central part of Iran (31°54′N and 54°24′E) with an annual average precipitation of less than 100 mm and an elevation of 1230 m above sea level. Trees prior to the study had been established in a 4 m × 3 m spacing. The growing season for pomegranate trees in the experiment site typically begins in late March and ends in late October. The study site irrigation water and soil saturation extract electrical conductivity and other physico-chemical characteristics of the soil and water are shown in Table 1. In the experiment location no precipitation was monitored throughout two growing seasons. The soil characteristics were determined from 6 locations of the experiment site (respectively in depths 0–30, 30–60 and 60–90 cm) and the irrigation water characteristics were measured in three stages during the growing seasons and then the average of the measured parameters are shown in Table 1.
2.4. Chilling treatments in the laboratory In the laboratory, pomegranate twigs were cut into ≈ 30 cm long segments. The cuttings were immersed in fungicide solution (4000 ppm of benomyl fungicide (Ariashimi Co., Zahedan, Iran)) for 5 min and then put into plastic bags in chilling conditions. Nine artificial chilling treatments including 100, 200, 300, 400, 500, 600, 700, 800, and 900 h at 4 ± 1 °C in a refrigerator (LG, model R-B601GM, R-B602GCWP, Korea) were used while the cuttings without any artificial chilling served as control for each cultivar. For each artificial chilling treatment, 30 cuttings of each irrigation level (in 3 replicates) were taken from each cultivar for determining the chilling requirement. The cuttings were wrapped in a humid cloth and then were transferred to plastic containers. 2.5. Evaluating chilling and heat requirements of cuttings
2.2. Irrigation treatments details After artificial chilling treatments, the cuttings were taken from the refrigerator and proximal ends of the cuttings were placed in 0.3 L pots that were previously filled with distilled water. Day and night temperatures of the laboratory were adjusted to 26 and 18 °C, respectively. The distilled water of each pot was replaced daily while proximal ends of each cutting were recut every other day. According to Richardson et al. (1974), budbreak was determined when the phenological stage was at first leaf sprouting. On the other hand, when 50% of buds of the cuttings were sprouted, it was considered as the release of bud dormancy. Budbreak data was monitored three times per week until end of the experiments. Finally, GDHs accumulated for each treatment in each cultivar were calculated by using the heat requirement of the cuttings
Two sustained deficit irrigation levels including 50% (severe stress) and 75% (moderate stress) of crop water requirement were used while 100% water supply served as control treatment for each cultivar. Net irrigation water depth calculated as follow (Allen et al., 1998):
In =
(θFc − θi ) × D 100
Where: In = Net irrigation 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)
Table 1 Physico-chemical characteristics of the soil and irrigation water used in the experiment. soil characteristics Soil depth (cm)
Texture
0–30 30–60 60–90
SL SL SL
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) 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 characteristics −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. 2
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immediately tubes were put into ice to cool. The samples absorption was recorded by spectrophotometer at 630 nm. Finally, sample starch concentration was calculated by the calibration curve drawn for glucose standard solutions and multiplied by 0.92 (McCready et al., 1950).
when the cuttings moved to the laboratory condition from the refrigerator. When 50% of buds reached the sprouting of first leaves it was considered the endpoint of the measurements. According to the previous reports, 10 °C is considered to be the phenological development threshold of the pomegranate (Melgarejo et al., 1997; Jackson, 1999; Ghasemi Soloklui et al., 2017). Richardson et al. (1974) reported that one GDH °C is equal to one hour above the plant base temperature, and GDH was computed when hourly temperature was between 10 and 25 °C.
2.8. Stomatal density For each treatment, nine fully expanded leaves were picked randomly from canopy mid-section of the current season and numbers of leaf stomata were determined by using the replica method (Soleimani et al., 2002). Using adhesive tape, the stellate hairs of the lower surface of the leaves were eliminated, and stomatal density was determined by a thin film of cellulose acetate that was directly applied to the middle part of each leaf (the lower epidermis of the leaf). The cellulose acetate film was allowed to dry at room temperature before removing the film. The film sheet was removed and was placed on coded slides and counting the stomatal of each leaf was done by binocular microscope at 40x magnification. Finally, stomatal density was reported in a field area of 1 mm2.
2.6. Leaf area For this case, 20 leaves of each treatment from different sections of the tree were collected randomly. Leaf area (LA) was measured with a leaf area meter (AAM-8, Hayashi Denko Co. Ltd., Japan). 2.7. Non-structural carbohydrates content The pomegranate stem fragments were used to measure the nonstructural carbohydrates (soluble carbohydrates i.e. sum of the glucose, fructose, sucrose and starch content). The non-structural carbohydrates were measured in the same samples that were used for chilling and heat requirement measurements. Finally, non-structural carbohydrates content of each sample was determined from the total amount of soluble carbohydrates and starch content of the same segment. The soluble carbohydrates content of each segment was measured by the Ranganna (1986) method with some modifications. Stem segments were ovendried at 70 °C for 24 h and then powdered, and 13 ml of 80% ethanol was added to 0.1 g of dry powder and then shaken. Afterward, samples were centrifuged at 5000 rpm for 10 min and the supernatant was separated. The supernatant was used for soluble sugar measurements. The phenol solution 5% (5 ml) and sulfuric acid (5 ml) were added to each test tube containing 1 ml of supernatant and the test tubes were vortexed (30 s). The tubes were then immediately placed into ice to reach room temperature. The absorption of each sample at 490 nm was recorded by spectrophotometer (BioTek, VT 05404-0998, USA). Finally, total sugars content was expressed as mg soluble carbohydrates g−1 dry weight after calculated by calibration curve drawn for glucose standard solutions. The segments starch content was determined through the following method. For this purpose, cold distilled water (5 ml) and perchloric acid 52% (6.5 ml) was added to each residual material that was used for sugar analysis. After that, the samples were mixed (15 min) and distilled water (20 ml) was added to each sample. The same method with residue was repeated 3 times and then all supernatants were mixed. The samples were centrifuged, and the supernatant separated and kept at 0 °C for 30 min. The supernatant was filtered and their volumes adjusted to 100 ml by using distilled water. Then, cold 2% anthron solution (10 ml) was added to supernatant (2.5 ml) in the test tubes. The samples were heated in a boiling water bath for 7.5 min at 100 °C and
2.9. Experimental design and statistical analysis This experiment was setup in a randomized complete block design with three replications and two trees per each replication. Bud chilling and heat requirement, non-structural carbohydrates of cuttings, LA, and stomatal density of leaf samples 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 bud chilling and heat requirement and non-structural carbohydrates content, LA and stomatal density was performed using Pearson’s correlation coefficient (PROC CORR). 3. Results and discussion 3.1. Effect of irrigation treatment on chilling and heat requirements Based on the results of the analysis of variance, chilling requirement was not significantly affected by the year, whereas it was significantly affected by cultivar, irrigation treatment and the interaction between cultivar × irrigation (Table 2). The results show that all restricted soil water conditions in both cultivars led to an increase in chilling requirement. However, there was no significant difference between the control irrigated trees and those exposed to moderate stress in ‘Shishecap’ cultivar. The results demonstrated that the chilling requirement was significantly different between deficit irrigation treatments (700 h and 694.44 h for the moderate and severe stress, respectively) and well irrigated treatment (605.56 h) in ‘Malas-Yazdi’ cultivar. However, there was no significant difference between the moderate and severe stressed trees in ‘Malas-Yazdi’ cultivar (Fig. 1). As shown in Table 4, according to results of the analysis of variance
Table 2 Results of the analysis of variance for the study year, cultivar and irrigation and their interaction effects on measured parameters (comparison of the means based on Duncan test, P ≤ 0.05). Source Year (CUL) (IRR) Year × CUL Year × IRR CUL × IRR Year × CUL × IRR Error C.V
DF 1 1 2 1 2 2 2 20
Chilling requirement (h) ns
277.83 19289.96 *** 25586.86 *** 1512.30 ns 92.54 ns 6512.38 *** 30.86 ns 524.66 3.55
Heat requirement GDH (°C) ns
Stomatal density (mm 2.) ns
3136.00 2637376.00 ns 529984.00 ns 254016.00 ns 3136.00 ns 1997632.00 * 65856.00 ns 533747.20 8.68
113.95 428.145 ** 771.22 *** 14.83 ns 20.47 ns 255.28 ** 30.92 ns 34.19 6.63
DF: degree of freedom CUL: Cultivar IRR: Irrigation. 3
Non-structural carbohydrate
Leaf area
150.70 * 369.51 ** 2217.26 *** 66.63 ns 76.27 ns 24.74 ns 31.50 ns 35.72 4.76
11.61 *** 0.98 ns 25.64 *** 0.06 ns 0.10 ns 0.90 ns 0.04 ns 0.61 9.50
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Fig. 1. Effect of different irrigation levels (a = control, b = moderate stress, and c = severe stress) on chilling requirement 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 (a = control, b = moderate stress, and c = severe stress) on stomatal density 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. 2. Effect of different irrigation levels (a = control, b = moderate stress, and c = severe stress) on heat requirement (GDH) 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. 4. Effect of different irrigation levels (a = control, b = moderate stress, and c = severe stress) on non-structural carbohydrate 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.
there was no significant difference between the two years (Table 2), therefore the average of the results of two years was presented (Fig. 2). As shown in Table 4, the interaction between cultivar × irrigation and cultivar on heat requirement was significant (P ≤ 0.05). According to our results, heat requirement was not significantly affected by the irrigation treatments. Also, the heat requirement of ‘Malas-Yazdi’ cultivar was higher than the ‘Shishecap’ cultivar by 11.06%.
between 46 and 108 stomata mm−2. Stomata are microscopic structures that are found on the aerial surfaces of the plant leaves and distribution of them is one of the important characteristics of plants in response to drought stress conditions by playing a basic role in adjusting the water status and obtaining carbon (Bertolino et al., 2019). Stomatal density is a quantitative characteristic and it has been reported that it is determined genetically (Gailing et al., 2008), but this feature can also be affected by environmental conditions (Bertolino et al., 2019). Our results were consistent with previous studies on the stomatal density, when olive trees were affected by water accessibility limitations (Bosabalidis and Kofidis, 2002). Also Bañon et al. (2004) reported that water stress conditions led to increase in the stomatal density on abaxial and adaxial surfaces of leaves compared to wellwatered plants in Lotus creticus. It has been reported that increasing the stomatal density in water stress conditions is due to the decrease in leaf area and it is not an adaptative mechanism (Ciha and Brun, 1975; Bañon et al., 2004). It has also been reported that decrease in transpiration rate is one of the main actions that occurs by closing the plant stomata in drought conditions (Bosabalidis and Kofidis, 2002). In fact, plants stomata will be closed under drought stress to prevent water loss, and higher leaf stomatal density of stressed pomegranate trees cannot increase the amount of carbohydrate storage in these plants, because when the plant stomata are closed the rate of photosynthesis decreases due to the reduction of the CO2 and O2 exchange between the inside and outside of the leaf. Correlation results showed that there was a positive correlation
3.2. Stomatal density Combined analysis of variance of two years showed that the effect of year was not significant for stomatal density (Table 2). Therefore, two year average results for the stomatal density was presented (Fig. 3). The interaction effect of cultivar × irrigation on stomatal density was significant (Table 2). In both cultivars, stomatal density increased by increasing water stress depending on cultivar (Fig. 4). However, the results showed that there was no significant difference between stomatal density of ‘Shishecap’ cultivar in well irrigated and moderate stressed trees, but the maximum and minimum of stomatal density were obtained in severe stressed and well irrigated trees, respectively (Fig. 4). The stomatal density of ‘Malas-Yazdi’ was not significantly different in moderate and severe stressed trees, but severe stressed and control treatment had the maximum and minimum of stomatal density, respectively (Fig. 4). Stomatal density ranged from 68 stomata mm−2 (well irrigated in ‘Shishecap’ cultivar) to 101 stomata mm−2 (severe stress in ‘Shishecap’ cultivar). This result is in agreement with previous report by Ghasemi Soloklui et al. (2017) in 20 Iranian pomegranate cultivars which ranged 4
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Table 3 Pearson correlation coefficients of the chilling requirement, heat requirement, stomatal density, non-structural carbohydrate and leaf area of two Iranian pomegranate cultivar. Variable
Chilling requirement
GDH
Stomatal density
Non-structural carbohydrate
Chilling requirement GDH Stomatal density Non-structural carbohydrate Leaf area
0.32 ns 0.75 *** −0.57 ** −0.81 ***
0.25 ns 0.19 ns −0.10 ns
−0.64 ** −0.78 ***
0.82 ***
Leaf area
GDH: growing degree hour.
(r = 0.75, P ≤ 0.001) between stomatal density and chilling requirement, whereas no strong correlation (r = 0.25) was observed between stomatal density and heat requirement (Table 3). Our results were consistent with previous reports on pomegranate (Ghasemi Soloklui et al., 2017). Stomata are flanked by a pair of specialized guard cells, that when the soil or air water is abundant, the turgor pressure increases, and increasing the turgor pressure leads to increases in the rate of the CO2 uptake for photosynthesis (Hetherington and Woodward, 2003). Conversely, water deficit signals decrease the hydraulic conductivity and increase the concentration of abscisic acid hormone, leading to decreases in the guard cell turgor pressure, resulting in the photosynthesis rate declining (Bertolino et al., 2019). The closure of stomata prevents the CO2 entry into leaf mesophyll, and leads to reducing the amount of photosynthesis and thus carbohydrates (Bosabalidis and Kofidis, 2002). It has been reported that decreases in photosynthesis rate can reduce the action of photosynthesis and decrease carbohydrate production (Anjum et al., 2011), and it has been found that decrease of carbohydrate production and its accumulation is an important parameter which can affect bud break and early season growth in spring (Wong et al., 2003). Based on our results, when pomegranate trees grow under water deficit conditions their leaf stomatal function may be affected, and as the stomata play a very important role in the production and accumulation of carbohydrates, production and accumulation of nonstructural carbohydrates is reduced, and leads to increasing the chilling requirement.
2009). It is known that the main cause of reducing the photosynthesis and its products in water deficit conditions is related to changes in the structure and performance of chloroplasts by demolition of chlorophyll structure (Anjum et al., 2011). Also it has been elucidated that formation of xylem embolisms increase in water deficit conditions, thus reduces the movement of water from the roots to the leaves, and the leaves respond by closing the stomata to prevent more water loss and subsequent embolism (Trifilò et al., 2017). It has been reported that closing the stomata leads to a progressive decrease of photosynthetic rates and potentially forcing plants to unload the non-structural carbohydrate reserves (Maguire and Kobe, 2015). According to the results, trees with highest chilling requirement had the lowest non-structural carbohydrates concentration at the end of the growth season in both cultivars. In this case correlation results showed that there was a negative correlation (r = −0.57, P ≤ 0.01) between non-structural carbohydrate concentration and chilling requirement and there was no significant correlation between non-structural carbohydrate concentration at the end of the growth season and heat requirement (r = 0.19) (Table 3). The reserved carbohydrates stored at the end of the growth season are the best indicator for forecasting the trees health, vitality and their productivity and reserved carbohydrates stored in early autumn have positive correlation with vitality of trees, and conversely, low levels of stored starch in early autumn is correlated with tree dieback and mortality (Wong et al., 2009). Also, it has been shown that 80% of the plants growth reserves consumed for re-growth are used from the reserved carbohydrates (Oosthuizen and Snyman, 2001). It has been well documented that summer drought conditions decrease the fixation of the photosynthetic carbon which reduces the carbohydrates and also the manufacture and accumulation of nonstructural carbohydrates which can affect deciduous trees cold-season physiology in leafless periods (Wong et al., 2009). It has been reported that the rate of stored starch is crucial for bud break and early season growth (Wong et al., 2003). According to results of this research, trees with higher levels of non-structural carbohydrates at the end of the growth season had lower chilling requirements because the level of reserved carbohydrates stored in their stems was more than the deficit treatments in two Iranian pomegranate cultivars.
3.3. Non-structural carbohydrate The results of the combined analysis of variance for two years showed that the effect of year was significant for non-structural carbohydrate (Table 2). Therefore, two year results were presented separately (Fig. 4). As shown in Table 2, the effect of irrigation (P ≤ 0.001) and cultivar (P ≤ 0.01) on non-structural carbohydrate was significant. In both cultivars, all water deficit levels led to a decrease in nonstructural carbohydrate compared to the well irrigated treatment in both years. According to results, maximum non-structural carbohydrate concentration was found in control trees of ‘Shishecap’ which was significantly different than moderate and severe stress treatments in the first and second year. In the second year, there was no significant diff ;erences between water deficit treatments in ‘Shishecap’ cultivar (Fig. 4). In ‘Malas-Yazdi’ cultivar, the maximum and the minimum of non-structural carbohydrate concentrations were obtained from the fully irrigated and severe stressed trees, respectively, in both years (Fig. 4). Based on our results, there was significant differences between control and moderate stress treatments and also between moderate and severe stress treatments in the ‘Malas-Yazdi’ cultivar in the first year, but there was no significant difference between moderate and severe stress treatments in ‘Malas-Yazdi’ cultivar in the second year (Fig. 4). Our results are in agreement with previous studies on the concentration of carbohydrate reserves of pomegranate, when trees were aff ;ected by drought stress (Ebtedaie and Shekafandeh, 2017). It has been found that reduction of reserve carbohydrates stored in water deficit conditions is due to the reduction of photosynthesis (Wong et al.,
3.4. Leaf area The combined analysis of variance of two years showed that there was a statistically significant difference between years for leaf area (Table 2), so the results of two years were presented separately (Fig. 5). According to results, the interaction effect of cultivar × irrigation and irrigation treatments were significant on the leaf area (Table 2). All water stress levels in both cultivars led to decreases in leaf area compared to the control in both cultivars, and in both years (Fig. 5). The maximum and the minimum leaf area were obtained from the severe stressed and fully irrigated, respectively, in ‘Shishecap’ cultivar but there was no significant difference between the moderate and fully irrigated trees in ‘Shishecap’ in both years (Fig. 5). In both years, the most leaf area was observed in fully irrigated treatment, while the lowest amount of leaf area was obtained in highest water deficit condition in ‘Malas-Yazdi’ cultivar (Fig. 5). According to results, all water 5
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4. Conclusion The influence of different irrigation treatments on chilling and heat requirements of two Iranian pomegranate cultivars after sustained deficit irrigation were detected by cutting stems in two successive years. Regarding the results, ‘Shishecap’ and ‘Malas-Yazdi’ cultivars showed a different tolerance to sustained deficit irrigation. Water accessibility limitation conditions caused negative influence on some parameters such as reduction of non-structural carbohydrates content and leaf area of these cultivars at end of the growth season. Based on the information provided, reducing the amount of soil water availability will increase the chilling requirement of pomegranate trees but does not influence on pomegranate trees heat requirement. Based on our findings, moderate deficit irrigation did not have significant effect on ‘Shishecap’ cultivar chilling requirement. Our findings showed that leaf area decreases with increasing drought stress, leading to increase of the stomatal density. Non-structural carbohydrates content can be used as the most important physiological marker to evaluate the chilling requirement under water deficit irrigation treatments. Overall, water limitation had different effects on chilling requirement in diff ;erent pomegranate cultivars, and that is very important in areas where the cold season is not adequately cool and can increase the flowering and successful production problems.
Fig. 5. Effect of different irrigation levels (a = control, b = moderate stress, and c = severe stress) on LA 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.
deficit levels showed significant decrease in leaf area compared to 100% water supply in both years in ‘Malas-Yazdi’ cultivar. Results from this study are similar to those founded by another study in which decrease in leaf area was reported under water stress (Bañon et al., 2004; Hayatu et al., 2014). Portes and Melo (2014) reported that changes in LAI parameter is related to some factors such as seasonal climate change, plant water availability, nutritional condition (specifically nitrogen) and atmospheric CO2 concentration. It has been reported that decreases in plant leaf area is an adaptation mechanism in order to avoid higher transpiration rate and also decreasing leaf surface exposed to solar radiation (Hayatu et al., 2014). Also, reduction in plant leaf area under water deficit is an avoidance mechanism that decreases the water loss from the leaf surfaces (Bañon et al., 2004). Also, Bañon et al. (2004) stated that irrigation can affect morphological and anatomical characteristics that associate with the mechanisms of avoidance, including changes in growth of plants aerial sections as well as size and shape of leaves. It has been found that increasing plant root depth for maximum water uptake, efficient root systems in order to maximize water absorption, decreasing water loss by reducing stomatal conductance, reducing radiation absorption by leaf rolling and also reducing evapo-transpiration are the efficient mechanisms for turgor enhancement in water stress conditions (Mitra, 2001). Also, it has been reported that plants use other mechanisms such as reducing leaf production, increasing leaf senescence and delaying leaf abscission against drought conditions (Hayatu et al., 2014). There was a strong negative correlation between leaf area and chilling requirement (r = -0.69, P ≤ 0.001). Also, according to correlation results, there was no significant correlation between leaf area of pomegranate trees at the end of the growth season and heat requirement (r = −0.05) (Table 3). The ability of CO2 fixation by plants in photosynthesis depends primarily on the LAI and also other leaf characteristics such as morphology, anatomy, and arrangement of leaves in the canopy (Portes and Melo, 2014). Previous researchers argued that there is a strong correlation between plant production and light, in which this relationship is mainly determined by the plant leaf area (Hirose, 2004; Lindquist et al., 2005; Portes and Melo, 2014). According to our findings, there was a strong significant correlation between leaf area and non-structural carbohydrates (r = 0.82, P ≤ 0.001) which confirms that when pomegranate trees grow under water deficit conditions, their leaf area decreases and therefore, the concentration of carbohydrate reserves decreases. Most likely, higher amounts of non-structural carbohydrates affected cold-season physiology of control pomegranate trees in leafless periods and led to their faster bud break and early season growth.
CRediT authorship contribution statement Mohammadebrahim Nasrabadi: Conceptualization, Formal analysis, Methodology, Writing - original draft. Asghar Ramezanian: Conceptualization, Project administration, Funding acquisition, Resources. Saeid Eshghi: Methodology, Software, Writing - review & editing. Ali Sarkhosh: Writing - review & editing. Declaration of Competing Interest The authors declare that there is no conflict of interest. Acknowledgements This project was made conceivable through a Ph.D. dissertation at School of Agriculture, Shiraz University (Mohammadebrahim Nasrabadi). The authors are truly thankful to Shiraz University for financial supports and Yazd Agricultural and Natural Resources Research Center, Yazd, Iran for providing the plant materials. References Ministry of Jihad-e-Agriculture Agricultural Statistics, 2017. Executive Committee on Management of Environmental Stresses for Horticultural Products. Allen, R.G., Pereira, L.S., Raes, D., Smith, M., 1998. Crop Evapotranspiration-Guidelines for Computing Crop Water Requirements-FAO Irrigation and Drainage Paper 56. FAO, Rome 300, D05109. Alonso, J.M., Ansón, J.M., Espiau, M.T., 2005. Determination of endodormancy break in almond flower buds by a correlation model using the average temperature of different day intervals and its application to the estimation of chill and heat requirements and blooming date. J. Am. Soc. Hortic. Sci. 130, 308–318. Anjum, S., Wang, L., Farooq, M., Khan, I., Xue, L., 2011. Methyl jasmonate‐induced alteration in lipid peroxidation, antioxidative defence system and yield in soybean under drought. J. Agron. Crop Sci. 197, 296–301. Bañon, S., Fernandez, J., Franco, J., Torrecillas, A., Alarcón, J., Sánchez-Blanco, M.J., 2004. Effects of water stress and night temperature preconditioning on water relations and morphological and anatomical changes of Lotus creticus plants. Sci. Hort. 101, 333–342. Bertolino, L.T., Caine, R.S., Gray, J.E., 2019. Impact of stomatal density and morphology on water-use efficiency in a changing world. Front. Plant Sci. 1–11. Bosabalidis, A.M., Kofidis, G., 2002. Comparative effects of drought stress on leaf anatomy of two olive cultivars. Plant Sci. 163, 375–379. Campoy, J., Ruiz, D., Egea, J., 2011. Dormancy in temperate fruit trees in a global warming context: a review. Sci. Hort. 130, 357–372. Ciha, A., Brun, W., 1975. Stomatal size and frequency in soybeans 1. Crop Sci. 15, 309–313. Djampor, J., Gregorian, V., 2000. Assessing the dormancy characteristics of some
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