Physiological, biochemical and gene-expressional responses to water deficit in apple subjected to partial root-zone drying (PRD)

Plant Physiology and Biochemistry 148 (2020) 333–346

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Research article

Physiological, biochemical and gene-expressional responses to water deficit in apple subjected to partial root-zone drying (PRD)

T

Hajar Ghafaria, Hamid Hassanpoura,∗, Morad Jafarib, Sina Besharatc a

Department of Horticultural Sciences, Faculty of Agricultural Sciences, Urmia University, Urmia, Iran Department of Plant Breeding and Biotechnology, Faculty of Agricultural Sciences, Urmia University, Urmia, Iran c Department of Water Engineering, Faculty of Agricultural Sciences, Urmia University, Urmia, Iran b

A R T I C LE I N FO

A B S T R A C T

Keywords: Apple Abscisic acid Antioxidant enzymes Abiotic stress-responsive transcription factors Drought stress

Water scarcity is one of the major factors limiting apple production. Partial root-zone drying (PRD) is a watersaving irrigation technique necessary to improve the efficiency of irrigation techniques to optimize the amount of fruit produced with the volume of water used. The apple trees cv. Red Delicious were exposed to four treatments, including (1) control with 100% of the crop evapotranspiration (ETc) needs; (2) alternate partial root-zone drying with 75% of the ETc needs (APRD75); (3) fixed partial root-zone drying with 75% of the ETc needs (FPRD75); (4) fixed partial root-zone irrigation with 50% of the ETc needs (FPRD50) in a semiarid region of Iran. Results showed that leaf water potential (Ψ leaf), and chlorophyll were significantly decreased in FPRD50 compared to control and other PRD treatments. APRD75 and FPRD75 treatments significantly enhanced (+) -catechin (+C), epicatechin (EC), chlorogenic acid (CGA), caffeic acid (CA) as well as increased water use efficiency (WUE) (by 30–40% compared to control) without significant reduction of yield. PRD reduced gibberellic acid (GA3) and kinetin, while, increased the abscisic acid (ABA) and salicylic acid (SA) levels. The abiotic stress-responsive transcription factors (TFs) MdoMYB121, MdoMYB155, MdbZIP2, and MdbZIP48 were highly expressed in all PRD treatments. Our results demonstrated that APRD75 and FPRD75 have the potential to stimulate antioxidant defense mechanisms, hormonal signaling pathways, and expression of drought-tolerance TFs to improve WUE while maintaining crop yield. Therefore, APRD75andFPRD75 with water savings as compared to full irrigation might be a suitable strategy for irrigation apple trees under water scarcity.

1. Introduction Water-saving in agriculture, due to global climate change and limited water resources, is considered as the most critical strategy in many countries (Marjanovic et al., 2012). To overcome the water scarcity in apple production and water saving in agriculture, the best irrigation practices must be implemented in order to reduce water losses. Deficit irrigation, defined as the application of water below maximum ETc, is an optimizing strategy under which plants are allowed to sustain some degree of drought and yield reduction (Ghrab et al., 2013). PRD is a deficit irrigation technique that allows half of the root system to be irrigated while keeping the other half dry in each irrigation before rewetting the root zone by shifting irrigation to the dry side (Qin et al., 2018). PRD, including APRD and FPRD, has been receiving considerable attention as a water-saving irrigation technique (Yang et al., 2012). APRD involves approximately half of the root system exposed to drying soil, while the remaining half is irrigated usually, and the wetting and

∗

drying sides of the root system are repeatedly alternated with a certain frequency. In FPRD, one side of the tree root is permanently irrigated, and the other side remains dry (Yang et al., 2012). PRD improves WUE, which usually decreases the potential yield, but with a significant reduction in the amount of water used, moreover, various fruit tree crops seem to be well adapted to these irrigation methods (Li and Zhang, 2017). During certain growth stages of apples, WUE is positively affected if the irrigation amount of the APRI method satisfies the water requirement (Du et al., 2016). Romero-Conde et al. (2014) reported that the citrus trees under PRD trigger a root-to-shoot signaling mechanism such as ABA that are transported to the leaves via transpiration and induced a partial stomatal closure. As a result, the leaf transpiration rate reduced without limiting CO2 assimilation and reducing the photosynthesis rate, and consequently, WUE was increased (Romero-Conde et al., 2014). PRD may moderate the efficiency of plants under water scarcity through the change of the gene expression, increasing the antioxidant enzyme activity, and decreasing the growth

Corresponding author. E-mail addresses: [email protected], [email protected] (H. Hassanpour).

https://doi.org/10.1016/j.plaphy.2020.01.034 Received 11 November 2019; Received in revised form 7 January 2020; Accepted 24 January 2020 Available online 25 January 2020 0981-9428/ © 2020 Elsevier Masson SAS. All rights reserved.

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to the results of previous studies, we studied the expression of MdoMYB121, MdoMYB155, MdbZIP2, and MdbZIP48 due to their stressinduced expression and the potential uses of these TFs in the improvement of the resistance of apple to water deficit. The yield of the crop is highly susceptible to climate change. Abiotic stress conditions like drought induce a wide array of physiological, morphological, and molecular changes that might provide cues towards drought-responsive/adaptive mechanisms (Sircar and Parekh, 2019). Overall, trees can activate biochemical and physiological mechanisms to regulate the water status under drought conditions (Selahvarzi et al., 2017). Nevertheless, extensive water resources restriction has severely reduced the yield and production of apple in most areas of Iran. Fewer studies about the effects of PRD on interactions between physiological, biochemical, and gene-expressional responses to water deficit were conducted for apple production in the semiarid regions. Hence, the objective of this research was to test the hypothesis that PRD treatments may affect yield, WUE, antioxidant enzyme activities, phenolic compounds content, endogenous hormones and gene-expressional responses to PRD in apple trees. The present study has high importance, since a better perception of phytohormones changes and gene expression response to drought can provide deeper insights into the adaptation of apple trees to water deficit and improved WUE and maintaining yield under water scarcity.

of shoots (Jovanovic and Stikic, 2018). The ability of plants to cope with water deficit relies mainly on its water status under changing climatic conditions. Incorporating drought stress tolerance in plants could be a promising approach to meet the globally increasing food demand (Joshi et al., 2016). For adaptation to drought, complex interactions between physiology, morphology, and biochemistry strategies are needed in plants that these strategies of drought adaptation and as well as interactions between them are under genetic control (Bassett et al., 2014). When the plants exposed to stress situations, first the signals are perceived by the plant cells receptors. Then, cells trigger a cascade of signaling and result in activation of the TFs, and expression of drought-related genes. Many of these signal transduction pathways mediated by the crosstalk of ABA, SA, JA with growth promoting hormones such as cytokinin (CK), GAs, and auxins (Tan et al., 2017). One of the essential signaling molecules which are produced and accumulated during water scarcity conditions is the ABA (Magalhaes et al., 2016). The transport of ABA in the xylem enhances with drying soil, which probably is associated with pH alteration. These signals stimulate several physiological adaptations within the leaves including, stomatal closure, lower respiration, and enhanced antioxidants to preserve water and protect the photosynthetic machinery (Dbara et al., 2016). The accumulation of the ABA leads to the activation of ABA-related TFs, such as HD-ZIP and MYB factors that play a pivotal role as droughtrelated signaling mediators (Ksouri et al., 2016). Stress-responsive genes in plants can be generally classified into two main groups based on their products, effector molecules, and regulator molecules. Regulator molecules such as antioxidant proteins and late embryogenesis abundant (LEA) proteins are known to prevent the oxidative damage. Among various regulatory molecules, TFs act as significant coordinators to transduce stress signals and to orchestrate the expression of functional genes that play a direct role in preventing plants from stressassociated damages (Li et al., 2017). When plants receive signaling events during water scarcity conditions, TFs play significant role in promoting multiple responses at the molecular level by modulating the expression of target genes and interactions with basal TFs at promoters of target gene and have an activation or repression effects on the function of target genes (Mittal et al., 2018). The MYB TFs, one of the largest gene families in plants, have been shown to regulate different biological processes like cell development and cell cycle, modulating plant primary and secondary metabolism, and different stress responses in plants and also participate in the synthesis of hormone and signal transduction (Li et al., 2016b). Hou et al. (2018) reported that MYB46 promotes the deposition of lignin and thickness of the secondary cell wall via increased gene expression of cell wall biosynthesis. PbrMYB21 and AtMYB96 are involved in drought tolerance by regulating polyamine levels and arginine decarboxylase in Pyrus betulaefolia and modulating of wax biosynthesis genes in Arabidopsis, which these mechanisms leading to ameliorate plant tolerance to abiotic stresses such as drought (Li et al., 2016a). Three MYB TF family cis-elements that may be contributing factors for the enhanced drought tolerance were detected (Zhang et al., 2018). The biosynthesis of lignin that is known as one of the first lines of defense against biotic and abiotic stresses, is regulated by MdoMYB121 and MdoMYB155 through transcriptional activation of the gene's promoter such as PAL in the phenylpropanoid pathway (Huang et al., 2012). Like the MYB transcription factor, the ZIP TFs also respond to various abiotic stresses such as drought, These TFs are induced by ABA and regulate the expression of stress-related genes in ABA-dependent manner by binding to specific ABA-responsive cis-acting elements (ABRE) in their promoter regions (Wang et al., 2016). Analysis of the expression of MdbZIPs under drought and salt stresses in apple leaves and roots indicated that the MdbZIP genes such as MdbZIP2 and MdbZIP48 showed a range of response patterns when exposed to abiotic stress, indicating that they may be involved in stress signaling (Li et al., 2016b; Zhao et al., 2016). Subsequently, according

2. Materials and methods 2.1. Study site, meteorological data and treatments The experiments were carried out during May to September 2016 and 2017 with 16-year-old apple trees cv. Red Delicious in an apple research orchard with drip irrigation system located in 15 km of Urmia (37○39′ N, 45○85′ E; 1377 m altitude), West Azerbaijan, Iran. The mean annual precipitation and temperature of 2016 and 2017 were 246 mm and 270 mm, and 13.5 °C and 11 °C, respectively. Trees were spaced at 3 × 4 m. Apple trees were chosen in three blocks. To reduce workloads, only 16 trees in each block were selected with similar trunk diameter, and located in the middle of the experimental site. The soil texture of orchard at depths of 0–50 and 50–100 cm is clay loam (24.28% sand, 36.55% silt, 39.16% clay) and silty clay loam (15.07% sand, 52.05% silt, 32.88% clay), respectively. Irrigation of the apple trees was conducted according to the equation of Penman–Monteith (ETC = KC × ET0) that ETc obtained from multiplying ET0 (reference evapotranspiration) by the crop coefficient (Kc) (Allen et al., 1998). The Kc values were calculated according to the methods of soil moisture balance and soil moisture lysimeter and micro-lysimeter (Besharat et al., 2010; Marsal et al., 2013). Meteorological data were prepared from a meteorological station near the garden. The total rainfall during the growing seasons in 2016 and 2017 was 104 and 112 mm, respectively. The apple trees were subjected to four irrigation treatments on the basis of potential ETc: control trees received full irrigation with ETc needs to both sides of the root system; the APRD75 irrigation supplied 75% of the volume of water required to meet ETc to one half of the root system, with the irrigated and drying halves of the root-zone alternated every seven days; the FPRD75 irrigation treatment provided 75% of the volume of water equivalent to ETc to only one side of the root-system permanently while the other side remains dry; the FPRD50 irrigation treatment provided 50% of the volume of water equivalent to ETc to only one side of the root-system permanently while the other side remains dry. A drip irrigation system was used to irrigate the apple trees. Each irrigated treatment was equipped with a time clock valve assembly to control water delivery. The emitters were installed that spaced at 1 m from the trunk, with a flow rate of 4 Lh-1 with a total of six emitters used for the control, four emitters per tree in the FPRD75 and APRD75 treatments and three emitters per tree in the FPRD50 treatment. Irrigation was scheduled at 2 days per week from mid-May to mid-September. 334

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(APX, EC 1.11.1.11) and peroxidase (POD, EC 1.11.1.7) activities, frozen powder leaves were homogenized with different buffers, including sodium phosphate buffer for SOD, potassium phosphate buffer (for APX) and sodium phosphate buffer (for POD). The homogenate was centrifuged at 15,000 rpm for 15 min at 4 °C and then kept at −20 °C until used for analysis. SOD and POD activities were assayed as described by Giannopolitis and Ries (1977), and Mac Adam et al. (1992), respectively. The absorbance was read at 560 and 470 nm for SOD and POD, respectively. The results for SOD and POD activities were defined as unit mg−1 protein and μM tetra guaiacol mg−1 protein min, respectively. APX activity was measured spectrophotometrically 290 nm as described by Nakano and Asada (1981). The results of APX activity were expressed nmol ascorbate mg−1 protein min. The H2O2 concentration assay was carried out with method of Junglee et al. (2014) at 390 nm. Briefly, frozen powder leaves were homogenized with trichloroacetic acid, 0.25 ml potassium phosphate buffer and 0.5 ml potassium iodide and then centrifuged at 12,000 rpm for 15 min. The H2O2 concentration was estimated using the calibration curve and the results were expressed as μmolg−1 FW.

2.2. Yield, crop water use or ET and WUE Yield per trees was calculated and weighted after harvesting of fruits on September 20, 2016 and September 24, 2017. Crop water use or ET was estimated by the soil water balance equation (an indirect method for estimating of ET):

ET = I+ P± ΔSW–Dp–Roff where ET is evapotranspiration (mm); I, the amount of irrigation water applied (mm); P, the precipitation (mm); ΔSW, the change in the soil water content (mm); Dp, the deep percolation (mm); and Roff is amount of run-off (mm). Since the amount of irrigation water was controlled, Dp and Roff were presumed to be negligible. The WUE based on tree evapotranspiration (WUEET) and irrigation amount (WUEI) was respectively calculated as (Bozkurt Çolak et al., 2017):

WUE =

1 Y × 10 ET

where Y and ET represent yield (kg ha−1) and tree evapotranspiration (mm), respectively.

2.6. Phenolic compounds

1 Y WUE = × 10 I

Some phenolic compounds of apple leaves, including + C, EC, CGA, and CA were evaluated with a high-performance liquid chromatography (HPLC) system (KNAUER, Germany) equipped with UV detector and a reversed phase C18 column (5 μm, 250 × 4.6 mm) was used for separation of phenolic compounds. The mobile phase gradient at the time 0–7 minutes was acetic acid 0.3% and methanol (55:45), at the time 12 to 17 was acetonitrile, acetic acid 0.3% and methanol (5:37:58), at the time 19 to 23 was acetonitrile, acetic acid 0.3% and methanol (8:22:70), and at the time 25 to 30 was acetic acid 0.3% and methanol (55:45). The flow rate and wavelength were 0.4 ml/min and 280 nm, respectively. Phenolic compounds of the samples were determined by the equation of calibration curves and were expressed as mg/g FW.

where Y and I represent yield (kg ha−1) and irrigation amount (mm), respectively. 2.3. Leaf area, shoot length and Ψleaf These parameters were measured in young fully expanded leaves. Leaf area was evaluated using a leaf area meter. To measure shoot length, fruit bearing shoots were tagged from each of four canopy sides. This parameter was evaluated on May 8 and 28, Jun 28, July 28 and Aug 28 in 2016 and 2017. Also, Ψ leaf was conducted monthly on mature leaves using the Scholander-type chamber (SKPM 1400, Skye instruments, UK). Measurements were taken from the selected leaves near the trunk. Three trees were used for each treatment. The Ψ leaf was measured at 9 fully expanded mature leaves per tree (i.e. 27 per treatment) between 8:00 a.m. to 9:00 a.m. on May 8 and 28, Jun 28, July 28 and Aug 28 (Rahmati et al., 2015).

2.7. Measurement of endogenous hormones Apple leaves were extracted with 80% cold methanol in a dark place for determining GA3, kinetin, ABA, SA, and indoleacetic acid (IAA). Endogenous hormones were measured with HPLC (KNAUER, Germany) equipped with UV detector at 288 nm. Separation of endogenous hormones was carried out using a reversed phase C18 column (5 μm, 250 × 4.6 mm). The mobile phase consisted of methanol, acetonitrile and acetic acid 0.3% (25:20:55). The results were expressed as mg/g FW.

2.4. Determination of pigment content These parameters were evaluated from mid-May to early September in 2016 and 2017. For the determination of chlorophyll and carotenoid pigments, the leaf samples were homogenized in methanol at 4 °C and then centrifuged for 10 min at 10,000 rpm. For determination of chlorophyll and carotenoid content, the absorbance was read at 662, 646 and 470 nm (Wellburn, 1994). Chlorophylls (a+b) and carotenoids were estimated by following formulas:

2.8. RNA isolation and quantitative real-time gene expression analysis Leaf samples were harvested randomly on May 8, 12 and 26, Jun 9 and frozen immediately in liquid nitrogen for gene expression analysis. Total RNA was isolated using the ethanol extraction protocol developed by Asif et al. (2006). RNA integrity and quantity were assessed using agarose gel electrophoresis and spectrophotometric measurements, respectively. Traces of genomic DNA from total RNA samples were removed by treatment with RNase-free DNase I (Thermo Scientific, USA). For quantitative real-time PCR, first-strand cDNAs were obtained from each total RNA sample (1 μg) using a Revert Aid First-Strand cDNA Synthesis kit (Thermo Scientific, USA) according to the manufacturer's instructions. The relative expression level of MdoMYB121, MdoMYB155, MdbZIP2, and MdbZIP48 genes were analyzed on RotorGene 6000 system (Corbett Life Science, QIAGEN, USA) using the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific, USA), following the manufacturer's instruction. The apple EF-1a gene was used as an internal control for expression normalization. The primer sequences corresponding to the genes are listed in Table 1. Relative

Chlorophyll a = 11.75 A662 - 2.350 A645 Chlorophyll b = 18.61 A645 - 3.960 A662 Carotenoids = 1000 A470 – (2.270 chlorophyll a - 81.4 chlorophyll b/227) 2.5. Antioxidant enzyme activities and H2O2 concentration After shoot length and Ψ leaf measurements, leaf samples were simultaneously harvested randomly on May 8 and 28, Jun 28, July 28 and Aug 28 and frozen immediately in liquid nitrogen for analysis of antioxidant enzyme activities, H2O2, phenolic compounds, and endogenous hormones. In order to extraction for the determination of antioxidant enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase 335

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Table 1 Gene-specific primers used for the real-time PCR analysis in this study. Genes

Sequences

Amplicon size (bp)

Accession no.

References

MdoMYB121

F-primer 5′-TCATCCCCCATCCTCACTACCA-3′ R-primer 5′-TCGTTTGCATCTTAGCACTTGC-3′ F-primer 5′-GGTGATCAGGAAGGATATGG -3′ R-primer 5′-ATCGACTTGCTGATCAGCGAC-3′ F-primer 5′-GGCTTCTTCAAGTGGGGTT-3′ R-primer 5′-CATTCTTTTGCGCTTTCTC-3′ F-primer 5′-CGAAAATGGCGGTGGTT-3′ R-primer 5′-TGCTTGTGGTGACGGTTGT-3′ F-primer 5′-ATTCAAGTATGCCTGGGTGC-3′ R-primer 5′-CAGT CAGCCTGTGATGTTCC-3′

112

KC834015

Cao et al. (2013)

120

XM_029108000

Cao et al. (2013)

105

HM122466

Li et al. (2016)

112

XM_008367053

Zhao et al. (2016)

174

DQ341381

Asif et al. (2006)

MdoMYB155 MdbZIP2 MdbZIP48 MdEF-1α

changes in gene expression levels were determined using the 2−ΔΔCT method (Livak and Schmittgen, 2001).

The experimental design was factorial in a randomized complete block design. Forty-eight trees were chosen and separated in three blocks as four replicates. Statistical analysis was performed using proc GLM in SAS (Version 9.2, SAS Institute, 2013) and the Tukey's honestly significant difference (HSD) test was applied to compare the means.

to control at the end of the season. According to obtained results, the leaf area in FPRD50 was lower than control (30–40%) at the end of the 2016 growing season. Also, the leaf area in FPRD75 was lower (7–11%) as compared to APRD75 at the end of the growing season. The results showed that the water status of apple trees was significantly affected by the irrigation regimes. As can be concluded from Fig. 2, all PRD treatments reached lower mean Ψ leaf values than control during the most measured times. Overall, FPRD50 treatment was the most stressed treatment. Similar results were observed in 2017 in leaf area, shoot growth, and Ψ leaf.

3. Results

3.3. Chlorophyll and carotenoid content

3.1. Yield and WUE The yield and WUE were affected by irrigation treatments. No significant difference in yield was found among APRD75, FPRD75, and control. Yield in FPRD50 was decreased by 54% as compared to control (Table 2). In the present study, the APRD75 and FPRD75 treatments compared to control increased WUEI and WUEET of apple trees by 20–35% in 2016. Similar results were observed in 2017 in yield and WUE. The results showed that WUE was improved by FPRD75 and APRD75 treatments compared to control and FPRD50.

As shown in Fig. 3, chlorophyll content was significantly affected by irrigation treatments. Also, the results showed that the carotenoid content of treated trees was similar to control. At the treatment onset, chlorophyll content was similar in all treatments, but after the early July, the chlorophyll content in FPRD75 and FPRD50 treatments was decreased. Compared to control, chlorophyll content in FPRD75 and FPRD50 treatments decreased by 55–85% at the end of the experiment. APRD75 presented similar chlorophyll content than control during the most irrigation period. Similar results were observed in 2017 in pigment content.

3.2. Leaf area, shoots length and Ψleaf

3.4. Antioxidant enzyme activities and H2O2content

The reduction of leaf area and shoot growth is a reaction to water scarcity conditions. Our results illustrated that the leaf area and shoot growth were markedly affected by deficit irrigation treatments in both years. As shown in Fig. 1, the leaf area and shoot growth had a temporal variation that showed an increasing trend throughout the entire irrigation season and generally has been decreased significantly under PRD compared to the control. During the irrigation season, both traits tended to decrease with the intensity of water deficit, as indicated by the significantly lower mean values of both traits in FPRD50 in 2016. The results showed that FPRD75, APRD75, and FPRD50 treatments reduced the length of shoots (25, 25, and 60%, respectively) compared

As shown in Fig. 4a, the SOD activity showed an increasing trend by about 40–60% during the growing season until late–Aug reaching a maximum value in 2016. The activities of SOD and APX enzymes were higher in all PRD treatments than control at the most measured times. FPRD50 had the highest effect on the activity of these enzymes. Compared to control trees, SOD activity in FPRD50, FPRD75, and APRD75 treatments was increased by 50, 30, and 14%, respectively at the end of the experiment. Also, at the end of the experiment, the APX activity in APRD75, FPRD75 and FPRD50 treatments was increased by 23, 30, and 50%, respectively, compared to control. As shown in Fig. 4b–d, the POD activity, and H2O2 content were similar in PRD treatments. Similar

2.9. Statistical analysis

Table 2 Total rainfall, evapotranspiration and total irrigation of the experimental orchard and also effect of irrigation on yield, WUEI and WUEET in 2016 and 2017. Irrigation treatment

control APRD75 FPRD75 FPRD50 ANOVA Irrigation treatment

Total Rainfall (mm)

ET + DP (mm)

Total Irrigation (mm)

Yield (t ha−1)

WUEI (kg m−3)

WUEET (kg m−3)

2016

2017

2016

2017

2016

2017

2016

2017

2016

2017

2016

2017

104 104 104 104

112 112 112 112

654 463 463 340

652 493 499 345

640 449 449 324

628 469 469 319

51.1 50.6 49.6 23.7

48.0a 47.5 a 47.1 a 22.7 b

7.9 c 11.2 a 11.0 b 7.3 d

7.6 c 10.1 a 10.0 b 7.1 d

7.8 c 10.9 a 10.7 b 6.9 d

7.3 9.6 9.4 6.5

**

**

*

**

**

**

a a a b

c a b d

Control with 100% of the ETc needs (control); alternate partial root-zone irrigation with 75% of the ETc needs (APRD75); fixed partial root-zone irrigation with 75% of the ETc needs (FPRD75); fixed partial root-zone irrigation with 50% of the ETc needs (FPRD50). 336

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Fig. 1. Seasonal pattern of shoots length and leaf area index (LAI) in 2016 (a, b) and 2017 (c, d). Vertical bars represent the Tukey's HSD values. NS: indicates no significant differences and asterisks indicate significant differences between the means, *: P ≤ 0.05, **: P ≤ 0.01.

results were observed in 2017 in antioxidant enzyme activities and H2O2 content (Fig. 5). 3.5. Phenolic compounds The phenolic compounds of the apple leaves were markedly affected by the irrigation regimes at the most measured times (Fig. 6). The phenolic compounds showed an increasing trend during the growing season. Based on HPLC assay, PRD increased significantly + C, EC, CGA, and CA compared to control reaching a maximum value of these compounds at the end of the experiment. Compared to control, +C content in APRD75 and FPRD75 treatments increased by 20–40% at the end of the experiment. Whereas CGA and CA contents in all PRD treatments were increased by 30–45% compared to control at the end of the experiment. PRD treatments reached the highest EC value throughout the entire growing season, the maximum differences in APRD75 (39%), FPRD75 (15%) and FPRD50 (20%) in compared to control were observed at the end of the experiment (Fig. 6). Similar results were observed in 2017 in phenolic compounds (Fig. 7). 3.6. Endogenous hormones PRD treatments reduced significantly leaf GA3 and kinetin concentrations compared to control. As shown in Fig. 8-a, All PRD treatments reached the lowest GA3 content than control throughout the entire growing season, the maximum differences in APRD75 (7%), FPRD75 (45%), and FPRD50 (60%) in compared to control were observed in the 28-Jun. Kinetin concentration tended to reduce with the intensity of water deficit, as indicated by the significantly lower values of kinetin concentration (25–75%) in FPRD50 as compared with control in the growing season. As can be concluded from Fig. 8-c, APRD75, FPRD75, and FPRD50 treatments significantly reduced the IAA level of the leaves compared to the control by about 20–38% until early June, then increased by about 45–75% until late–Aug. The beginning of the increase in SA levels under the PRD condition was observed several days after the treatment onset. The maximum differences in PRD

Fig. 2. Seasonal pattern of red delicious leaf water potential (Ψ leaf) for all irrigation treatments in 2016 (a) and 2017 (b). Data denote the average of three replicates per treatment. Vertical bars represent the Tukey's HSD values. NS: indicates no significant differences and asterisks indicate significant differences between the means, *: P ≤ 0.05, **: P ≤ 0.01.

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Fig. 3. Seasonal pattern of carotenoid and chlorophyll content in leaves of apple trees for all irrigation treatments in 2016 (a, b) and 2017 (c, d).). Vertical bars represent the Tukey's HSD values. NS: indicates no significant differences and asterisks indicate significant differences between the means, *: P ≤ 0.05, **: P ≤ 0.01.

treatments in compared to control were observed during late –June by about 42–97% compared to control (Fig. 8-d). Moreover, as shown in Fig. 8-e and 9-e, several days after the treatment onset, the ABA level increased sharply until late –May reaching a value of 0.07 mg g −1 FW in the leaves exposed to FPRD50

treatment. The ABA levels under PRD treatments were significantly higher than that of control during the most measured times. The maximum difference of ABA content in PRD treatments in compared to control was observed by about 60–80% during late –May. A higher ABA and SA content and lower GA3 and kinetin content were recorded in

Fig. 4. Seasonal changes of antioxidant enzyme activities and H2O2 content in the apple leaves for all irrigation treatments in 2016. Vertical bars represent the Tukey's HSD values. NS: indicates no significant differences and asterisks indicate significant differences between the means, *: P ≤ 0.05, **: P ≤ 0.01. 338

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Fig. 5. Seasonal changes of antioxidant enzyme activities and H2O2 content in the apple leaves for all irrigation treatments in 2017. Vertical bars represent the Tukey's HSD values. NS: indicates no significant differences and asterisks indicate significant differences between the means, *: P ≤ 0.05, **: P ≤ 0.01.

Fig. 6. Seasonal changes of catechin (+C), epicatechin (EC), caffeic acid (CA), and chlorogenic acid (CGA) in leaves of apple trees for all irrigation treatments in 2016. Vertical bars represent the Tukey's HSD values. NS: indicates no significant differences and asterisks indicate significant differences between the means, *: P ≤ 0.05, **: P ≤ 0.01.

PRD treatments than control. Similar results were observed in 2017 in endogenous hormone levels (Fig. 9).

3.7. Expression of drought-related TFs To gain the transcriptional response of apple cv. Red Delicious to drought stress, we examined the expression of four abiotic stress339

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Fig. 7. Seasonal changes of catechin (+C), epicatechin (EC), caffeic acid (CA), and chlorogenic acid (CGA) in leaves of apple trees for all irrigation treatments in 2017. Vertical bars represent the Tukey's HSD values. NS: indicates no significant differences and asterisks indicate significant differences between the means, *: P ≤ 0.05, **: P ≤ 0.01.

compared to control. In this study, FPRD50 treatment exhibited lower yield in compared to FPRD75, and APRD75 treatments, which was associated with stomatal closure due to the more increased ABA in FPRD50 plants. Indeed, the stomatal closure leads to reduce the photosynthesis rate and assimilates and consequently decreasing yield (Wu et al., 2018). The increasing of ABA in FPRD75 and APRD75 treatments was less than FPRD50, therefore it could cause partial closure of stomata. The partial opening of stomata under mild drought stress may result in the reduction of transpiration and maintenance of photosynthesis. Also, the low water use efficiency in FPRD50 compared to FPRD75 and APRD75 treatments was expected because of adverse effects associated with severe water deficit conditions on the photosynthesis and synthesis of assimilates as well as a reduction in the development of secondary roots and consequently cause impairment of water and nutrient absorption (Du et al., 2016; Li and Zhang, 2017). FPRD50 treatment also showed a more significant reduction in leaf area and chlorophyll content, which could result in a decrease in photosynthesis. The obtained results concurred with the findings of Egea et al. (2011), who reported PRD treated trees despite receiving less water and having less Ψleaf maintained the yield and fruit quality during the experiment because of limitation of net photosynthesis was lower in PRD trees. However, Zegbe and Serna-Pérez (2018) reported that yield was slightly reduced in PRD plants compared to full irrigated plants. The high-water use efficiency in FPRD75 and APRD75 treatments can be due to wetting one side of the root, reducing the evaporation, and slight reducing the relative conductivity of the stomatal and transpiration. These results are in agreement with Loveys et al. (1998), who reported that PRD saves irrigation water with a reduction in shoot growth without significant reduction of grapevine yield.

responsive TFs genes (MdoMYB121, MdoMYB155, MdbZIP2, and MdbZIP48). The results showed that all TFs expression were affected by irrigation regimes, and alteration of the expression level of the genes was dependent on deficit treatment as well as the period of stress (Fig. 10). The expression levels of the TFs was relatively stable throughout the entire irrigation season, whereas in the deficit treatments, MdbZIP2 and MdbZIP48 dropped during the irrigation season and one week after the treatment onset, then returned to control levels after three weeks (Fig. 10c and d). MdbZIP2 and MdbZIP48 showed a noticeable increase in expression during the first days of water scarcity. The expression of MdbZIP2 and MdbZIP48 was 37-45-fold higher in FPRD50 and 30-34-fold higher in FPRD75 and APRD75 compared to control in 2016 during the first days of water scarcity. In the deficit treatments, the MdoMYB121 expression presented an increasing trend from 3 days until one week after the treatment onset and reached to the peak, which was 23-fold higher in FPRD50 and10-12-fold higher in APRD75 and FPRD75 treatments compared to control in 2016, and then it dropped sharply during the rest of the season (Fig. 10 a). All PRD treatments reached the peak in MdoMYB155 expression in early Jun which was 60-fold higher in FPRD50 and 50-fold higher in FPRD75 and APRD75 treatments compared to control in 2016. Generally, all four TFs showed high levels of expression in all PRD treatments than control throughout the entire irrigation season. Similar results were also observed in the 2017 growing season in expression of TFs (Fig. 11). 4. Discussion 4.1. Yield and WUE

4.2. Leaf area, shoot length and Ψleaf

Based on the obtained results, WUE in FPRD75 and APRD75 treatments was increased by 20–35% compared to control, while yield was not affected by FPRD75 and APRD75. However, WUE and yield in FPRD50 treatment were reduced by 10 and 54%, respectively,

Water deficit significantly reduced the leaf area, and especially during the end of the irrigation season. Compared to control, leaf area 340

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Fig. 8. Seasonal changes of endogenous hormones in apple leaves for all irrigation treatments in 2016. Vertical bars represent the Tukey's HSD values. NS: indicates no significant differences and asterisks indicate significant differences between the means, *: P ≤ 0.05, **: P ≤ 0.01.

Also, plant hormones such as auxin and CK are necessary for cell cycle progression and cell division. The significant decrease in CK probably led to a decrease in growth. These results are in agreement with Prerostova et al. (2018), who reported that down-regulation of active CK due to overexpression of CK oxidase/dehydrogenase in response to drying soil resulted in the reduction of the growth rate and improved plant drought tolerance.

in FPRD50, FPRD75, and APRD75 treatments was reduced by 30–40, 13, and 4%, respectively. Moreover, the shoots grown under PRD conditions were shorter. Growth reduction appeared quickly after the treatment onset under PRD conditions and lasted until the end of the irrigation season by about 25–60%. Lower growth of the shoots and leaf area were associated with lower Ψ leaf. According to the results of this study, in all PRD treatments, a significant reduction in Ψ leaf was observed compared to the control during irrigation season. In a previous study, Zegbe and Serna-Pérez (2011) reported that the control trees had higher leaf xylem water potential (leaf) than PRD trees throughout the experiment. Our results, as reduction of shoot growth, are also consistent with the finding of Hipps et al. (1995), who showed that the shoot system architecture of young peach trees was significantly affected by water availability. This is directly attributed to the decrease in water potential. Water applications below the evapotranspiration requirements reduced water potential values. As a consequence of reducing the water potential, leaf turgor is lost in mature leaves, and expansive growth is impaired. This would significantly decrease vegetative growth and total leaf area in deficit PRD trees than control (Romero-Conde et al., 2014).

4.3. Chlorophyll and carotenoid content According to the results, chlorophyll content in APRD75 treatment was similar to control, while the chlorophyll content was sharply decreased in FPRD50 compared to other PRD treatments and control. Also, carotenoid content in treated trees was similar to control. The severe decrease of chlorophyll content in FPRD50 treatment (about 60–85%), especially at the end of the growing season, was probably due to the reduction of hormones such as GA3 and CK. Degradation of chlorophyll as an important biomolecule, which absorbs and transfers the light energy during photosynthesis, is prevented by auxins, GA3, and CK (Asghari and Zahedipour, 2016). Nevertheless, Sampathkumar 341

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Fig. 9. Seasonal changes of endogenous hormones in apple leaves for all irrigation treatments in 2017. Vertical bars represent the Tukey's HSD values. NS: indicates no significant differences and asterisks indicate significant differences between the means, *: P ≤ 0.05, **: P ≤ 0.01.

less reduction of chlorophyll content compared to the FPRD50.

et al. (2014) reported that the level of lower chlorophyll observed in water stress treatments could be due to degradation of chlorophyll by producing proteolytic enzymes such as chlorophyllase and cell membrane changes such as an increase in penetrability and leakage of cell solutes, which affect the stability of chlorophyll. Reduced photosynthetic pigments under water deficit may be due to two different mechanisms. One may be due to reduced synthesis of the main chlorophyll pigment complexes encoded by the cab gene family because of limitation of gas exchange and reduction of leaf area and consequently decreasing photosynthetic pigments (stomatal limitation). The second mechanism is linked to the destruction of the pigmentprotein complexes, which protect the photosynthetic apparatus or to oxidative damage of chloroplast lipids and proteins (not-stomatal limitations) (Mibei et al., 2017). The transfer of electrons from the electron transport chain to oxygen during photosynthesis resulted in producing reactive oxygen species (ROS) in chloroplast and carotenoids a vital part of the plant antioxidant defense system can play a protective role in the photosynthetic reaction center against photo-oxidation (Asghari and Zahedipour, 2016). The APRD75 and FPRD75 treatments probably prevent photosynthesis rate reduction via maintaining carotenoid and

4.4. Antioxidant enzyme activities and H2O2 concentration With increasing water scarcity, SOD and APX activities increased, while H2O2 content was similar to control. The maintaining of H2O2 content in PRD treatments at a constant level with control may be due to increased antioxidant enzymes. There is a parallel correlation between antioxidant enzymes and H2O2 in order to make a balance between the production of ROS and their elimination through antioxidant systems. These results are consistent with those of Haider et al. (2018), who illustrated that the water scarcity provokes the defense system of peach trees, including antioxidant enzymes such as SOD, POD, and APX against oxidative damage. Also, Egea et al. (2011) reported that the PRD can upregulate the antioxidant enzymes that may ameliorate the deleterious effects of water stress on the photosynthetic machinery. ROS act as inter- and intracellular messenger and modulate in a concentration-dependent manner, natural physiological processes in plants and stress signals generation resulting in acquiring resistance. However, these ROS became toxic when left unchecked, therefore plant 342

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Fig. 10. Relative expression of four abiotic stress-responsive TFs (MdoMYB121, MdoMYB155, MdbZIP2, and MdbZIP48) in apple cv. Red Delicious under drought stress in 2016. Vertical bars represent the Tukey's HSD values. NS: indicates no significant differences and asterisks indicate significant differences between the means, *: P ≤ 0.05, **: P ≤ 0.01.

Fig. 11. Relative expression of four abiotic stress-responsive TFs (MdoMYB121, MdoMYB155, MdbZIP2, and MdbZIP48) in apple cv. Red Delicious under drought stress in 2017. Vertical bars represent the Tukey's HSD values. NS: indicates no significant differences and asterisks indicate significant differences between the means, *: P ≤ 0.05, **: P ≤ 0.01. 343

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through the ABA-receptors such as PP2Cs, PYL and ABF. This ABA signal transduction pathway modulates the expression of various genes, including those encoding antioxidant enzymes and defense system proteins under water scarcity (Chen et al., 2019; Bankaji et al., 2019). ABA accumulation in soil drying under PRD irrigation may enhance the CK oxidase gene expression and subsequently, decrease the CK levels. The interaction of root-sourced ABA with CK signaling and ABA/ CK ratio leads to the regulation of stomatal aperture (Yan et al., 2019). It is known that ABA has interplay with other phytohormone signaling pathways such as ethylene, jasmonic acid, and SA. SA is recognized as a regulatory signal that produces and accumulates during stress conditions and induce the local and systemic acquired resistance (SAR), and production of pathogenesis-related proteins. On the other hand, SA plays an important role in modulating the redox balance that leads to protect the plant from oxidative stress against biotic and abiotic stresses (Shah et al., 2017). The IAA levels in PRD treatments compared to control were initially decreased by 20–38%, then this trend was changed, and an increasing trend of about 45–75% was observed in PRD treatments. In our study, the irregular trend of endogenous IAA levels may also be due to a negative regulatory role of ROS on active auxin levels. The auxin by inducing intracellular ROS production, probably through mediating of RBOH reaction with Rho-GTPases (RACs/ROPs), regulates the balance of free and conjugated auxin in a negative feedback loop under abiotic stress conditions (Novaković et al., 2018). The auxin: CK ratio has a decisive impact on the regulation of root growth as well as on the meristem size and the number of cells in the primary root during stress progression. Inhibition of root growth is the consequence of the antagonistic relationship between auxin and CK in the regulation of cell division and differentiation in the root apical meristem. CK inhibits root growth while auxin signaling antagonizes CK, increasing meristem size, and promoting growth under osmotic stress (Rowe et al., 2016). The secondary growth of roots can result in increased nutrient and water uptake and ultimately lead to maintain yield under mild stress conditions. Therefore, plants are very dynamic systems having a great ability to cope with drying conditions by triggering a network of interconnected signaling pathways (Batool et al., 2019).

cells to prevent or alleviate the toxicity of ROS, such as H2O2, and keep their intracellular concentration at a constant level by a series of mechanisms such as antioxidant system (Zahedipour et al., 2019). In order to detoxify stress-induced ROS, a set of ROS detoxification system, including several enzymes, maintaining the ROS homeostasis at a favorable situation, have been evolved in plants (Jin et al., 2017). Chloroplast as a primary cellular source of ROS should be rapidly scavenged to protect thylakoids and stroma targets; therefore, the ascorbate-glutathione cycle acts as one of the most essential parts of the chloroplast antioxidative system that includes several enzymes and non-enzyme molecules acting in an integrated manner. Moreover, since H2O2 capable of diffusing across membranes from their site of generation, also extra-chloroplastic detoxification systems considered as H2O2 scavenging pathways (Ramalho et al., 2018). Therefore, probably PRD through ABA could be activating the resistance mechanisms such as antioxidant enzymes to keep the ROS levels at a constant level and prevent ROS damage to the photosynthetic system. 4.5. Phenolic compounds The phenolic compounds of the apple leaves were affected by the irrigation treatments. In this study, the +C, EC, CGA, and CA contents were by about 15–45% higher in all PRD treatments than control at the end of the experiment. The increasing of phenolic compounds may be a consequence of the increased expression of phenolic biosynthetic genes, which their expression was significantly increased in this experiment. These results are in agreement with Castellarin et al. (2007), who reported that the expression of flavonoid and anthocyanin biosynthesis genes like CHS3, UFGT, F3H, and MYBA1 in response to water scarcity and increasing ABA production were upregulated. Following the upregulation of these genes, the content of phenolic compounds and flavonoids were promoted in grapevine. Besides, Akagi et al. (2012) suggested that some anthocyanin and flavonoid biosynthesis pathway genes are induced by an ABA-dependent manner, in a process mediated by the TFs of DkMyb2, DkMyb4 and DkbZIP5 in persimmon. In order to regulate the biosynthesis of anthocyanins through the phenylpropanoid pathway, the activity of the MYB–bHLH–WD40 (MBW) protein complex is required, In the apple, the MdoMYB121 region of the MBW complex interacts with ubiquitin ligase MdCOP1, and the MdbHLH3 region binds to the MdDFR and MdUFGT promoters (Wang et al., 2018). The MdoMYB121 gene can induce the biosynthesis of phenolic compounds such as + C, EC, and CA by ABA, CK, and ethylene mediated signaling. In addition, the biosynthesis of phenolic compounds at the transcriptional or the post-transcriptional level requires the reaction of these hormones with the MYB-bHLH-WD40 complexes (Wu et al., 2017).

4.7. Expression of drought-related TFs As an innovation aspect of the present study, we examined the expression of drought-related TFs. It is interesting that the expression levels of all four TFs were significantly increased (about 40–90% higher than control) during the experiment, and reached the highest level at certain times. For instance, in early Jun, the expression of MdoMYB155 was 50-60-fold higher in PRD treatments compared to control. The MdoMYB121 expression was 10-23-fold higher in PRD treatments compared to control from three days until one week after the treatment onset. The expression of MdbZIP2 and MdbZIP48 were 37-45-fold higher in FPRD50 and 30-34-fold higher in FPRD75 and APRD75 compared to control during the first days of water scarcity. The results are in good agreement with those of Cao et al. (2013), who reported that the MdoMYB121 transgenic tomato plants showed noticeably enhanced tolerance to water deficit by producing more proline, less MDA and less electrolyte leakage than the control plants. The upregulation of transcription factors can improve the plant's defense system and, through activating the antioxidant system, lead to ameliorating deleterious effect of water stress on photosynthetic machinery and maintaining carbon assimilation. For instance, it has been shown that the MdoMYB155 improved drought stress tolerance in plants by regulating the expression of proline biosynthesis and transport genes and activation of defense systems and antioxidant enzymes as well as regulating of plant senescence via the inhibition of CK-mediated branching at late stages of development. Besides, overexpression of MYB2 improved tolerance to drought by ABA-inducible gene expression and ABA-dependent responses,

4.6. Endogenous hormones Endogenous hormones were significantly affected by irrigation treatments. The ABA and SA increased significantly in PRD treatments compared to the control and reached 40–90% more than control during some times (May and June). The content of CK and GA were significantly decreased in PRD treatments compared to the control, so that in July and August, with the increase in severity of water scarcity, CK and GA contents were reduced by 10–60% compared to control, respectively. In the present study, the increasing of ABA in PRD treatments can reduce water loss through the stomata and ultimately lead to increased WUE in APRD75 and FPRD75 treatments, which undergo mild water stress. These results are consistent with the results of Du et al. (2016). The partial root-zone method in dry soil induces an increase in the ABA content and subsequently triggers the cascade events and initially induces stomatal closure to reduce the effect of water loss (Batool et al., 2019). ABA leads to plant tolerance, decreasing transpiration, and stomatal aperture. On the other hand, A series of signaling cascade by changing of ABA levels are triggered and modulated 344

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References

therefore confers advantages of ABA in drought tolerance (Baldoni et al., 2015). It was suggested that MdoMYB121 directly binds to ciselement in the promoters of genes that encode vacuolar proton pump subunits to regulate the anthocyanin/malate accumulation and cell pH, and thereby enhancing anthocyanins and polyphenolics in vacuoles (Hu et al., 2016). The activated expression of OsbZIP62 improved the drought tolerance through interacting with promoters of some genes such as OsGL1 that is involved in the wax accumulation of leaf cuticles, NAC transcription factor, and DSM2 that enhances the biosynthesis of lutein and ABA (Yang et al., 2019). Li et al. (2016b) and Zhao et al. (2016), through expression analyses of group A MdbZIP genes and changes in expression profiles in response to drought, identified MdbZIP genes that were responsive to drought. They found that expression of the majority of the MdbZIP genes studied, especially MdbZIP2 and MdbZIP48, were up-regulated in response to water deficit. The bZIP gene, ppa013046m, plays a role as transcriptional activators of the ProDH gene that can modulate the metabolism of proline to glutamate during drought. The P5CS gene, which induces the accumulation of proline, is stimulated by signals deriving from H2O2 and the ABA-dependent synthesis pathway during drought (Bielsa et al., 2016).

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5. Conclusion Alternating wet and dry parts of the root zone during PRD may create differences in root-to-shoot signaling that have not only effect on physiological characterization but also show a better adaptive response to drought. According to our results, PRD could activate the resistance mechanisms such as antioxidant enzymes like APX and SOD, and phenolic compounds. Phenolic compounds were significantly increased by 15–45% in PRD treatments, especially in APRD75 treatment, which can be due to increased expression of phenolic biosynthetic genes such as MdoMYB155 and MdoMYB121. Interestingly, all four TFs showed high levels of expression by about 40–90% in all PRD treatments than control throughout the entire irrigation season while Ψ leaf reached the minimum value, confirming a drought stress response. APRD75 and FPRD75 treatments were increased WUE by 20–35% compared to control without decreasing yield. Whereas FPRD50 was reduced WUE and Yield by 10% and 54%, respectively, compared to control. Probably, the drastic decrease in chlorophyll content as well as in leaf area in FPRD50 treatment leads to reduce photosynthesis and consequently yield. The decreasing of Ψ leaf, GA3, and kinetin resulted in reduce leaf area about 4–30%, and shoot growth was reduced by 25–60% in PRD treatments compared to control at the end of the growing seasons. The management of the efficiency of water use could be achieved through PRD techniques. FPRD75 and APRD75 strategies, in particular, might be a suitable water-saving strategy for irrigation under water scarcity without significant reduction of apple yield due to creating a series of defense mechanisms which, in turn, can ameliorate deleterious effects of water stress on the photosynthetic machinery.

Contributions Hamid Hassanpour conceived the original idea, Hajar Gafari and Hamid Hassanpour performed physiological and biochemical analyses and the majority of experiments, Morad Jafari and Hajar Gafari designed, performed the gene expression experiments and participated in data analyses, Sina Besharat designed PRD Treatments. All authors discussed the results and wrote the manuscript.

Declaration of competing interest The authors declare that they have no conflict of interests. 345

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