Revealing Drought Tolerance Strategies in Pistachio Clonal Hybrids: Role of Osmotic Adjustment

Revealing Drought Tolerance Strategies in Pistachio Clonal Hybrids: Role of Osmotic Adjustment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Revealing Drought Tolerance Strategies in Pistachio Clonal Hybrids: Role of Osmotic Adjustment Mozhdeh Osku, Mahmoud Reza Roozban, Saadat Sarikhani, Mohammad Mehdi Arab, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5905176/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 May, 2025 Read the published version in BMC Plant Biology → Version 1 posted 8 You are reading this latest preprint version Abstract Background Pistachio ( Pistacia vera L.) growth, yield and quality are affected by abiotic stress especially drought. Understanding the strategies that improve dehydration tolerance is essential for developing resistant pistachio rootstocks. In the experiment, nine-month-old saplings of seven clonal interspecies hybrids of Pistacia atlantica × P. integerrima (C1, C2, C16-1, C8-3, C4-2, C9-4 and UCB1) were assessed for growth and physiological responses to water withholding and recovery. Result Water deficit negatively impacted growth parameters, including shoot dry weight, root dry weight and leaf area, in all hybrids; however, the C1 demonstrated relatively minor reductions compared to the other hybrids. Glycine betaine content in leaves increased by 49.4% in C9-4 and 47% in C1, while only 7% and 11% increases were found in the most sensitive clones, C8-3 and C4-2. Notably, C9-4, identified as the most tolerant clone, displayed the highest proline levels, with increases of 29.5% in leaves and 41.5% in roots, in contrast to C8-3, which showed minimal increases of 6% and 11% in leaves and roots, respectively. Clones with higher compatible solutes maintained higher relative water content (RWC), lower osmotic potential and smaller reductions in leaf water potential. RWC declined by just 6% in C9-4, whereas it dropped by 88% in C8-3. Osmotic potentials in C9-4 were − 1.61 MPa in leaves and − 0.271 MPa in roots, while in C8-3, they were − 0.93 MPa and − 0.11 MPa in leaves and roots, respectively. Following recovery, evaluations of growth, physiological traits and visual observations indicated that C8-3 had poor recovery ability. Heatmap and PCA analyses categorized the clones into three groups: "tolerant" (C9-4, C1 and C2), "moderately tolerant" (UCB1) and "sensitive" (C8-3, C4-2 and C16-1). Conclusion The results of this study underscore the significance of osmotic adjustment as a more critical trait compared to growth and stomatal parameters in effectively differentiating tolerant clones from sensitive ones. Osmolyte accumulation stomatal parameters water withholding rootstock UCB1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 1. Introduction Pistachio ( Pistacia vera L.) is a wind-pollinated, deciduous nut tree belonging to the Anacardiaceae family, primarily cultivated in Mediterranean regions but likely originating from central and southwestern Asia [ 1 ]. A significant abiotic stress affecting plant physiology and development is water scarcity. Pistachio trees are known for their ability to endure prolonged periods without water [ 2 ], yet their performance can decline under dry conditions [ 3 ]. In Iran, where pistachio cultivation is prevalent, these trees are often grown in arid and semi-arid regions. Rootstocks play a key role in water absorption and stress resistance. Drought-resistant rootstocks improve scion hydraulic function by enhancing xylem structure and reducing cavitation [ 4 ]. These rootstocks boost scion drought resistance through increasing non-structural carbohydrate (NSC) storage, altering photosynthesis, and adjusting stomatal conductance via hydraulic and hormonal changes [ 5 ]. The ability of rootstocks to tolerate drought stress depends on various factors, including their specific traits and adaptability to local climatic conditions, alongside regional characteristics such as soil composition and water availability [ 6 ]. For instance, rootstocks derived from Pistacia atlantica are well-known for their deep root systems, enabling access to water in lower soil layers during periods of drought. Moreover, these rootstocks play a vital role in regulating non-structural carbohydrate (NSC) accumulation, supporting plants in maintaining energy reserves and structural stability under water stress conditions [ 7 ]. Acclimation to water stress in plants involves the production of compatible solutes which stabilize membranes, enzymes and cellular osmotic balance [ 8 ]. These solutes play a critical role in dehydration tolerance by enabling plants to sustain cellular functions during water scarcity and maintaining turgor pressure to prevent dehydration [ 9 ]. Key biomarkers for drought resistance, including relative water content, osmotic potential and leaf water potential, help assess a plant’s ability to retain water and adjust osmotic pressure under stress [ 10 , 11 ]. These traits highlight important mechanisms such as turgor maintenance and, which contribute to identifying drought-tolerant clones and enhancing breeding programs [ 12 , 13 ]. Stomatal responses are among the first reactions to abiotic stress, occurring within seconds to minutes [ 14 ]. Stomata are essential for controlling water loss during drought stress, with abscisic acid (ABA) playing a key role in inducing stomatal closure to reduce transpiration. Additionally, leaf temperature serves as a reliable indicator of drought stress, as plants under such conditions close their stomata, resulting in elevated leaf temperatures compared to non-stressed plants. This approach is widely used in fruit trees to evaluate drought stress [ 15 , 16 ]. Drought-resistant rootstocks are essential for sustainable pistachio production in water-scarce areas. While the Badami-Zarand rootstock is widely used in Iran, it has drawbacks like slow growth and disease susceptibility. On the other hand, the hybrid rootstock UCB1, developed at UC Berkeley [ 17 ], offers advantages in drought tolerance and disease resistance. In recent decades, Pistacia atlantica and Pistacia integerrima have been widely utilized as parental species in hybridization programs, owing to their superior drought and salinity tolerance, rapid growth and increased yield potential. A new hybrid rootstock, Arota, developed from a cross between P. atlantica and P. integerrima , shows great promise [ 18 ]. It is essential to evaluate the drought resistance of these rootstocks before their widespread adoption in orchards. This study aims to understand the drought tolerance strategies of seven clonal interspecific hybrids of Pistacia atlantica Dscf. × Pistacia integerrima Stewart. Given the key role of rootstocks in supporting pistachio production under increasing water scarcity, we assessed the pistachio hybrids responses to water withholding, focusing on osmotic adjustment, growth characteristics, water relations and stomatal behavior. By combining the unique traits of each hybrid with consideration of regional environmental factors, such as soil type and local climate, this study offers valuable insights into the role of these rootstocks in promoting sustainable pistachio production in drought-prone regions. 2. Materials and methods 2.1. Plant materials and experimental conditions This research was carried out in the research greenhouse at the Faculty of Agricultural Technology, University of Tehran, from June to september 2023. The plant materials used in this study originated from a rootstock breeding program aimed at promoting high growth, carried out by the Pistat Research Center in partnership with ITA Sadra® Company at the Green Tat field in Boein-Zahra, Ghazvin, Iran. In this project, a total of 222 controlled hybridizations were executed, involving 37 female Pistacia atlantica Dscf. and six male Pistacia integerrima Stewart trees. The most vigorous and uniform hybrids resulting from these crosses were assessed over a four-year trial based on their relative growth metrics, as opposed to UCB1 standards [18]. From the top 15 hybrids identified during this evaluation, six high-performing hybrids labeled as C1, C2, C16-1, C8-3, C4-2 and C9-4 were selected and clonally micropropagated at ITA Sadra ® Biotechnology Co. serving as the plant material for this experiment beside UCB1. Nine-month-old saplings of the hybrids were transferred to 10 L pots filled with a mix of 50 % soil and 50 % perlite and sand (1:1 V/V). The pots were arranged in a randomized complete block design (RCBD) with three replications for each treatment. The plants were grown in the greenhouse for four weeks to facilitate the development of their canopy and root systems. All plants were fed weekly with a full-strength Hoagland’s nutrient solution [19] to provide essential nutrients and promote plant establishment before applying treatments. The volumetric water content at field capacity was 15% V/V and at wilting point 5% V/V that they were measured following the protocol outlined by Benedicto Ottoni [20]. Plants of the each clonal hybrid were divided into two groups: one for control and the other for withholding irrigation. The first group (control) received manual irrigation every three days until reaching soil to field capacity. The second group (no irrigation) experienced a period of water stress by withholding irrigation [21]. To monitor the soil moisture during the stress period, pot weights were recorded every three days. Water withholiding was imposed for 30 days, during which sensitive plants exhibited pronounced water stress symptoms, including severe turgor loss, wilting and extensive leaf discoloration. Following this period, the stressed plants were rehydrated to field capacity and allowed a 30-day recovery phase, which provided sufficient time to clearly distinguish different recovery responses among the clones (Fig. 1). The greenhouse temperature was kept at 28 °C during the day and 20 °C at night, with RH ranging from 40% to 60% and a light/dark photoperiod of 16/8 hours, providing a flux density of 500–650 µmol m −2 s −1 . Sampling and data collection were conducted in three phases: 15 days after onset of water withholding (mild stress), when the first visible signs of dehydration appeared in the sensitive plants; 30 days after withholding irrigation (severe stress); and at the end of the recovery period (Fig. 1). Water potential measurements were conducted prior to harvesting the plants. A part of the harvested plant material was stored at −80 °C, while the remainder was stored in dried form (72 h at 70 °C). 2.2. Experimental design The experiment was structured as factorial within a randomized complete block design (RCBD) framework, featuring two factors: irrigation (control and no irrigation) and clonal hybrids, with three biological replicates. This resulted in a design comprising 7 clonal hybrids × 3 replicates × 2 irrigation treatments. Analysis of variance (ANOVA) was conducted with R software (R 4.3.2), and means were compared using Duncan’s Multiple Range Test ( P<0.05 ). The normality of each trait was tested using the Shapiro-Wilk approach. Correlation network plot based on Pearson coefficients was constructed using ggraph package under R program. Principal component analysis (PCA) was conducted via factoextra package in R software. Heatmap cluster analysis (HCA) was depicted using the pheatmap package in the same software [22]. 2.3. Measurements 2.3.1. Grwoth parameters a. Shoot and root dry weight , root to shoot ratio At the end of the 30-day water withholding and subsequent rehydration, the plants were removed from the soil and separated into shoots and roots. They were then oven-dried at 70 °C for 72 h to determine their dry weight. Dry weights were measured using a digital scale with a precision of ± 0.1 mg. Root to shoot ratio was calculated by measuring the dry root and shoot weight [2]. b. Growth and recovery rate The growth rate (GR) of the aerial biomass is determined by comparing the dry weight of the plant's aerial parts at two intervals: before the onset of drought stress (day 0) and at the end of the drought stress period (day 30). The recovery rate (RR) is determined by comparing the aer s dry weight on the 30th day of the recovery period with the dry weight measured on the first day of the recovery period, following the drought stress phase [23]. c. Leaf area After water withholding and rehydration periods, all the leaves of each plant were arranged on a sheet of white paper alongside a scale. Photographs were taken using a digital camera. Subsequently, the images were analyzed using with Digimizer v.6.4.3 ® software to measure the leaf area [24]. 2.3.2. Water relations a. Relative water content (RWC) RWC was determined from gravimetric measurements using the formula (FW−DW) / (TW− DW) × 100, where FW represents the fresh weight of the leaf, DW is the dry weight obtained by oven-drying the leaves at 80 °C for 24 hours, and TW is the turgid weight measured after re-hydrating the leaves at 4 °C [25]. RWC was assessed during three time periods: mild drought, severe drought and recovery. b. Leaf water potential (Ψ w ) Water relations were evaluated by measuring leaf water potential (LWP) using a Pressure Chamber (Santa Barbara, Ca., USA, Made in Italy). Measurements were taken between 9:00-11:00 A.M. on three mature leaves from the mid-shoot of each plant. [26]. Assessments were conducted at three stages: 15 days after water withholding (mild stress, slight leaf wilting in sensitive plants), 30 days after water withholding (severe stress), and after a recovery period . c. Leaf and root osmotic potential (Ψ S ) Leaf and root samples were collected at the end of drought and rehydration periods, cut into small pieces, and frozen in liquid nitrogen. Samples were thawed for 30 minutes, centrifuged at 15,000g (4°C) for 15 minutes, and the tissue sap was analyzed for osmotic potential (Ψ S ). Osmolarity (C) was measured using an osmometer and converted from milliosmoles per kilogram (m osmoles/kg) to megapascals (MPa) using the appropriate formula [25]: Ψ S (MPa)= -C (m osmoles kg -1 )×2.58×10 -3 2.3.3. Osmoregulators asseys a. Glycine betaine concentration Dried leaf and root samples (0.5 g) were shaken with 25 mL distilled water for 48 hours at 25 °C. Afterward, 1 mL of extract was mixed with 2 N sulfuric acid, and 0.5 mL of the mixture was cooled in a water bath for 1 hour. Potassium iodide solution (0.2 mL) was added, and the mixture was stored at 4 °C for 14 hours. Samples were centrifuged at 10,000 rpm for 15 min, and granules were dissolved in 9 mL dichloromethane, shaken for 2 hours, and analyzed via UV-Visible spectrophotometry (HITACHI U-1900, Japan) at 365 nm. The concentration of glycine betaine was determined using a standard curve and reported as µmol g −1 dry weight [27]. b. Proline concentration Roots and leaves from control, drought-stressed and recovered pistachio saplings were collected. Acid-ninhydrin reagent was prepared by dissolving 1.25 g ninhydrin in a mix of 30 mL glacial acetic acid and 20 mL 6 M phosphoric acid, stored at 4 °C for up to 24 hours. Plant material (0.5 g) was homogenized in 10 mL 3% sulfosalicylic acid, filtered, and 2 mL of the filtrate was mixed with 2 mL acid-ninhydrin solution and 2 mL glacial acetic acid. The mixture was heated at 100 °C for 1 hour, cooled in an ice bath, and extracted with 4 mL toluene. The toluene phase containing the chromophore was separated and left to reach room temperature. Absorbance at 520 nm was measured using toluene as a blank. Proline concentration was determined from a standard curve and calculated based on the fresh weight of the sample [28]. c. Total soluble carbohydrate content (TSC) TSC assay was conducted using 95% ethanol extracts from root and leaf tissues. Fresh samples (0.5 g) were ground in 5 mL of 95% ethanol, washed twice with 5 mL of 70% ethanol, and centrifuged at 3,500 g for 10 minutes. Supernatants were stored at 4 °C. For TSC measurement, 0.1 mL of extract was mixed with 3 mL anthrone solution (150 mg anthrone in 100 mL 72% H₂SO₄), heated in a boiling water bath for 10 minutes, cooled, and absorbance was measured at 625 nm [29]. 2.3.4. Stomatal asseys a. Morphological characteristics of stomata Stomata morphological parameters (stomatal density, stomatal length, stomatal width, pore aperture, pore length) was assessed on the 15th day after the applying drought stress (mild stress), 30 days after the application of drought stress (severe stress) and after the recovery phase in clonal hybrids. This was done using the nail polish impression method on the lower epidermal layer of the second leaf closest to the bud apex and observed using a Nicon digital camera (DXM-1200) attached to a microscope. Per treatment, in total 90 stomata were used for studying stomata morphological parameters. Images were analyzed by using the public domain image processing program ImageJ (ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/) [30]. 2.3.5. Leaf temperature Leaf temperature was measured using an infrared thermometer (Benetech GM550E). Three separate readings were taken per plant to ensure accuracy, and arithmetic means of these measurements used for all subsequent analyses. Measurements were conducted during the late morning to early afternoon (10 AM to 12 PM), a period when gas exchange is in stable form [31, 32]. Efforts were directed towards maintaining stable environmental conditions during the measurement. 3. Results 3.1. Growth responses to withholding irrigation a. Shoot dry weight (SDW) and Root dry weight (RDW) Growth parameters (SDW and RDW) were assessed during water withholding and recovery. Significant differences in the interaction effect of irrigation treatments and clonal hybrid were observed ( P≤0.01 ) during water withholding. Under control conditions, C1 and C4-2 had the highest SDW. Water stress reduced SDW in all clones, with the lowest values in C4-2 and C8-3. C1 showed minimal reduction (Fig. 2A). Drought stress responses in RDW varied. UCB1 and C2 increased RDW by 96.3% and 50%, respectively. Other clones showed a decrease in RDW, with C16-1 having the highest RDW under control and C1 the lowest during drought (Fig. 2B). 30 days after recovery, SDW and RDW were significantly influenced by treatment interactions ( P≤0.01 ). C4-2 and C1 exhibited strong regrowth, while C8-3 showed the lowest SDW, indicating limited recovery (Fig. 2C). RDW showed no significant differences between rehydration and control treatments for most clones, except UCB1, C9-4 and C16-1, which had higher RDW in control (Fig. 2D). b. Root to shoot ratio, growth and recovery rate The root-to-shoot ratio, growth rate, and recovery rate were significantly influenced by the interaction between irrigation and clonal hybrids ( P≤0.01 ). Under stress conditions, the root-to-shoot ratio increased in the C2, C8-3, UCB1 and C4-2 clones compared to the control group. However, for the other clones, no significant differences were observed between the stress treatment and the control. Following the recovery period, the root-to-shoot ratio showed no significant differences between the stressed clones and their respective controls across all clones (Fig. 3A,B). Our findings revealed that the C1 and C4-2 clones exhibited the highest growth rates under control conditions. However, under water stress, growth was nearly completely suppressed across all clones, with no significant differences observed among them in this condition (Fig. 3C). The C4-2, identified as sensitive cline in this study, exhibited the highest recovery rate, surpassing even the resistant clones, C9-4 and C1. Although both C4-2 and C8-3 were categorized as sensitive, C4-2 demonstrated rapid recovery, while C8-3 had the lowest recovery rate. The UCB1, C2 and C9-4 showed no significant differences in recovery rate, with their performance being statistically similar (Fig. 3D). c. Leaf area During the water withholding period, leaf area decreased significantly ( P <0.01 ) compared to controls. In the irrigated group, the C1 and C2 displayed larger leaf areas compared to the other clones. Under water stress, C1 and C2 experienced only slight reductions in leaf area, whereas C8-3 and C4-2 were the most severely impacted (Fig. 4A). At the end of the rehydration period, significant differences in leaf area responses were observed ( P≤0.01 ). C1 and C9-4 had larger leaf areas in the rehydration treatment compared to controls, while C8-3 and C4-2 had the smallest leaf areas under recovery (Fig. 4B). 3.2. Water relations a. Relative water content (RWC) RWC was significantly influenced by the interaction effect between irrigation and clonal hybrids ( P≤0.01 ) across all stages: mild stress, severe stress, and recovery. Under mild stress, no significant differences were found in RWC for C9-4, C16-1, and C1 between drought and well-watered treatments, but RWC in C2, C4-2, C8-3 and UCB1 significantly decreased. RWC was 79.5% for UCB1 and 70% for C8-3, with C9-4 showing the highest RWC and C8-3 the lowest (Fig. 5A). After 30 days of water withholding (severe stress), RWC decreased significantly in all clones compared to the controls. C8-3 showed the greatest decrease (88%), followed by C4-2 (76.5%) and C16-1 (59.3%). C9-4 and C1 had minimal changes (6% and 13.48%) (Fig. 5B). These results indicate that C8-3 was most affected by drought, while C9-4 and C1 were more resilient. In the recovery stage, RWC did not differ significantly between control and recovery conditions for C1, C16-1, C2, and C9-4, suggesting their strong ability to recover. C8-3 had the lowest recovery RWC (50.5%), and UCB1 showed a 20% reduction in RWC compared to its control, indicating a significant decrease in recovery relative to other samples (Fig. 5C). b. Leaf water potential Significant differences in leaf water potential (LWP) were observed between hybrids and irrigation treatments ( P≤0.01 ) across all stages: mild stress, severe stress and recovery. Fifteen days after mild drought stress, LWP decreased, with most clones showing no significant changes, except for C8-3, which had a notably lower LWP. Under drought, LWP ranged from -0.43 MPa (C2) to -1.73 MPa (C8-3). (Fig. 6A). End of the drought stress period, LWP significantly decreased in C8-3, C4-2, C16-1, and UCB1. The lowest LWP was recorded in C8-3 (-4.43 MPa), followed by C4-2 (-3.19 MPa), C16-1 (-2.95 MPa) and UCB1 (-1.5 MPa) (Fig. 6B). At the end of the rehydration period, C4-2, C9-4 and UCB1 showed no significant differences with controls, indicating a quick return to stable conditions. The highest LWP was observed in C1 (-0.15 MPa), while the lowest was in C8-3 (-1.26 MPa) (Fig. 6C). c. Leaf and root osmotic potential Leaf and root samples were collected at the end of drought and rehydration periods. Significant differences in leaf osmotic potential (LOP) and root osmotic potential (ROP) were observed due to the interaction of clonal hybrids and irrigation ( P≤0.01 ). After 30-day drought stress, LOP significantly decreased, with the most notable decline in clone C9-4 (-1.61 MPa), indicating higher osmolyte accumulation under stress (Fig. 7A). In roots, ROP significantly decreased in C1 under drought stress, with the lowest ROP recorded in C1 (-0.61 MPa). No significant effect on ROP was found in other clones. Overall, osmolyte accumulation in C1 roots increased significantly under drought stress (Fig. 7B). There were no significant differences in LOP between recovery and control treatments for any clone. The lowest LOP was observed in UCB1 (-1.12 MPa), which did not differ significantly from C16-1 and C2 under stress (Fig. 7C). For ROP, only UCB1, with the lowest root osmotic potential (-0.5 MPa), showed a significant difference compared to C16-1 under recovery. No significant differences were found in other treatments (Fig. 7D). 3.3. Osmoregulators a. Glycine betaine concentration leaf and root glycine betaine content was significantly influenced by the interaction effect of irrigation and clonal hybrids ( P≤0.01 ) under the drought stress and recovery. Under stress, glycine betaine levels increased in all clones compared to the control, but the increase was significant only in C1, C9-4 and C2. In the commercial UCB1 clone, there was no increase in glycine betaine levels. Glycine betaine increased by 49.4% in C9-4, 47% in C1 and 22.5% in C2 (Fig. 8A). A similar trend was found in the roots, with higher accumulation in the leaves than in the roots. The highest glycine betaine content was 236 µmol·g⁻¹ DW in the leaves and 188 µmol·g⁻¹ DW in the roots of C9-4 under drought stress (Fig. 8B). During recovery, glycine betaine levels decreased in both leaves and roots compared to the stress phase. C9-4, C1 and C2 clones maintained the highest glycine betaine levels in both organs (Fig. 8C,D). b. Proline concentration Proline concentration in both leaves and roots increased in all clones under water withholding, except for the UCB1 clone (Fig. 9A,B). The highest proline content was found in C9-4 and C1, which were the most drought-tolerant rootstocks in this study. The highest proline concentrations were 83 µmol·g⁻¹ FW in leaves and 60 µmol·g⁻¹ FW in roots. In C9-4 clones, proline increased by 29.5% in leaves and 41.5% in roots compared to their controls under drought stress. At the end of rehydration period, significant differences in leaf and root proline, were observed among the hybrids ( P≤0.01 ), but no significant for irrigation treatments and their interaction. The highest proline levels were found in UCB1 clones, with 73 µmol·g⁻¹ FW in leaves and 66 µmol·g⁻¹ FW in roots, significantly different from the other clones. No significant differences in proline levels were observed in the leaves and roots of the other clones (Fig. 10A,B). c. Total soluble carbohydrate content (TSC) Under drought stress, all clones showed an increase in TSC in both leaf and root tissues. The increase was significant in C1, C2 and C9-4 compared to controls (Fig. 11A,B). C9-4 and C1 had the highest TSC levels, with increases of 97.3% in leaves and 129.3% in roots. After recovery, TSC levels decreased in both leaves and roots, but C9-4, C2 and C1 still had higher TSC than their controls (Fig. 11C,D). 3.4. Stomatal traits and leaf temperature Stomata morphological parameters (stomatal density, stomatal length, stomatal width, pore aperture, pore length) was assessed in three stage (mild stress, severe stress and revovery stage) (Fig. 12). Effect of clonal hybrid, irrigation and their interaction for stomata parameters and leaf temperature was not significant at any of the stages (Supplementary table 1, 2 and 3). The results of this study indicated that stomatal regulation does not play a role in the development of drought stress resistance in our clones; it is likely that other mechanisms are involved in conferring resistance in the resistant clones. 3.5. Heatmap and cluster analyses The study used cluster heat map analysis to classify pistachio clonal hybrids based on their response to water stress, evaluating traits like water status, stomatal characteristics, osmotic adjustment and growth. Seven hybrids were categorized into three groups: tolerant (I), moderately tolerant (II) and sensitive (III). The tolerant group (C9-4, C1 and C2) showed high levels of RWC, LGb, rGb, proline, and other stress-related traits, alongside low LOP and ROP indicating increased solutes for stress tolerance. In contrast, the sensitive group exhibited reduced growth traits such as leaf area, SDW, RDW and LWP, with minimal reduction in the tolerant group. Stomatal parameters and leaf temperature were largely unaffected across groups (Fig. 13). Under re-watering condition, pistachio hybrid were classified into the “strong (I)”, “weak (II)” and “moderate (III)” recovery groups including (C1), (C8-3), (UCB1, C2, C16-1, C9-4 and C4-2) respectively. Groups I and III showed rapid recovery in LWP and ROP, with quick growth recovery in C1 and C4-2. Group II showed minimal recovery, with most hybrids recovering well except those in the weak group (Fig. 14,15). 3.6. Correlation and principal component analyses Significant correlations were found between the drought stress index (DSI) and various physiological and growth traits under water stress (Fig. 16). LWP showed a negative correlation with DSI of ROP (r = -0.47 * ), and positive correlations with DSI of SDW, RWC, LTSC, rTSC, rGb, and LGb (r = 0.45 * , r = 0.906 *** , r = 0.75 *** , r = 0.64 ** , r = 0.5 ** , r = 0.64 * , respectively). RWC was positively correlated with SDW, LTSC, rTSC, rGb, LGb, Lproline and rproline (r = 0.56 ** , r = 0.86 ** , r = 0.76 *** , r = 0.69 *** , r = 0.79 *** , r = 0.57 ** , r = 0.6 ** , respectively), and negatively correlated with the DSI of ROP (r = -0.55 ** ) and LOP (r = -0.57 ** ). A strong positive correlation was found between LArea and SDW. LTSC, rTSC, LGb, rGb, rproline, and Lproline all showed significant positive correlations with each other, while negatively correlating with LOP, ROP, and RDW (Fig. 16). Principal Component Analysis (PCA) was applied to all physiological and growth traits of the seven pistachio clonal hybrids under water stress to identify key parameters and relationships among traits. PCA using DSI (value of trait under stressed conditions / value under well-watered conditions × 100) identified 18 significant principal components (PCs), with the first six components explaining over 88% of the total variation under severe drought stress. The first principal component (PC1), explaining more than 45% of the variations, was negatively correlated with LGb (96%), rGb (91%), LTSC (94%), rTSC (91%), rproline (89%), Lproline (85%), RWC (86%), and LWP (71%), making these traits the most sensitive indicators of drought effects on pistachio hybrids. The second principal component (PC2), explaining over 16% of the variation, was negatively correlated with LArea (74%), RDW (66%) and SDW (63%) (Fig 17). 4. Discussion Drought stress significantly affects pistachio ( Pistacia vera L.), reducing its growth, yield, and quality [33]. Plants respond to drought through various morphological, physiological and molecular adaptations, which vary among species and genotypes [34]. Understanding drought resistance mechanisms is crucial for improving crop resilience, ensuring food security, and promoting sustainable agriculture under water scarcity conditions [35]. In this study, we used nine-month saplings of seven clonal interspecific hybrids of Pistacia atlantica Dscf. × Pistacia integerrima Stewart labeled C1, C2, C16-1, C8-3, C4-2, C9-4 and UCB1. These clones were examined to assess their responses to drought stress, focusing on osmotic regulation in their leaves and roots, as well as stomatal indices. The decrease in shoot dry weight (SDW) under drought stress is mainly due to limited water, impaired photosynthesis, stomatal closure and survival-oriented physiological responses, leading to lower shoot biomass. [36–38]. Our results showed that under drought stress conditions, SDW decreased in all clones, but this decrease was minimal in C1 and maximal in C8-3. Additionally, the growth rate analysis indicated that under control conditions, C1 and C4-2 had the highest growth rates, which is consistent with the findings of Akbari [18] who reported the superiority of C1 over the commercial clone UCB1 in terms of growth. According to Blum [39], drought stress reduces root dry weight (RDW) by limiting water availability and reallocating resources to survival, impairing root growth, and reducing nutrient absorption. In our experiment, different clones showed varying responses to water withholding, with UCB1 and C2 showing increases in RDW by 96.3% and 50%, respectively, compared to controls. In contrast, the other clones experienced a decrease in RDW. The increased root biomass observed in UCB1 under drought conditions reflects a strategy to enhance water access. However, this adaptation likely comes with elevated metabolic costs, potentially delaying shoot recovery [40]. However, as noted by Sánchez-Blanco [41], root systems do not consistently respond to drought stress by undergoing changes. For instance, a study on the drought-resistant pistachio variety 'Sarakhs' reported no increase in root biomass under water stress conditions [42]. Similarly, our findings align with these observations, as the most resistant clones (C9-4 and C1) also showed no increase in root biomass. Recent studies suggest that plants need extended periods of stress exposure to adjust their root systems. For example, Vives-Peris reported that changes in root morphology, such as increased depth and biomass, require prolonged stress exposure [43]. Bhargava and Sawant, [44] noted that during short-term drought, plants prioritize physiological changes over morphological ones, using stored resources to maintain metabolic functions. In our experiment, the 30-day water withholding period might not have allowed sufficient time for morphological changes in roots, so saplings responded mainly with physiological adaptations. Following the recovery period, SDW increased across all clones, with the sensitive clone C4-2 exhibiting the most vigorous growth and achieving the highest recovery rate. Rapid recovery is typically associated with efficient stress management mechanisms, such as osmotic adjustment, photosynthesis, and stomatal control, while weaker genotypes struggle due to limitations in these traits [45]. Certain drought-sensitive genotypes may exhibit notable growth improvements following rehydration, utilizing recovery mechanisms that enable them to capitalize on the renewed availability of water [46]. However, our results showed that C9-4 (the most resistant clone) had higher RDW values in the control state, while the other clones exhibited no significant difference in RDW between the rehydration treatment and their respective controls. According to Flexas and Medrano [38], some resistant genotypes may fail to significantly increase root dry weight after recovery due to physiological and biochemical limitations. Drought stress may cause lasting effects on root architecture and function, preventing full recovery even in typically resistant genotypes. The increase in the root-to-shoot ratio under stress likely indicates a plant's effort to optimize resource allocation [47], particularly in sensitive clones such as C8-3 and C4-2. However, this adjustment did not align with enhanced resistance, suggesting that other mechanisms, such as osmotic regulation or antioxidant responses, may contribute to stress tolerance. In contrast, resistant clones exhibited no significant changes in this ratio, indicating their reliance on alternative strategies, such as metabolic stability, for enduring stress. Following the recovery phase, no notable differences were observed between recovery and control, likely because vegetative growth resumed quickly, emphasizing the temporary nature of stress-induced changes and the critical role of recovery mechanisms in restoring normal conditions [41]. Our results also revealed a negative relationship between drought stress and leaf area, although responses varied among clones. C1 and C2 showed slight reductions in leaf area, while C8-3 and C4-2 exhibited the most significant reductions. These findings are consistent with reports by Khoyerdi [2] and Kasmani [48], on pistachio. Reducing leaf area is a typical response to water scarcity, primarily due to reduced cell division [49]. As water potential decreases, cell expansion becomes the most sensitive process affected by water deficits [50]. During drought stress, plants limit leaf expansion to reduce water loss through transpiration, conserving water and supporting survival. This reduction in leaf area helps balance metabolic needs with available water, enabling more efficient resource allocation [51–53]. After the recovery period, only the C1 and C9-4 clones exhibited a greater leaf area during the rehydration period compared to their controls. Some clones may increase leaf area after recovery due to better growth and photosynthesis, while others maintain reduced leaf area due to impaired recovery mechanisms following drought stress [54, 55]. Rapid regrowth during recovery highlights an adaptive trade-off, where the short-term cost of osmotic adjustment is offset by long-term benefits in growth and yield. This underscores the importance of selecting efficient osmotic adjustment mechanisms in breeding programs to enhance drought resilience [39, 56, 57]. To evaluate plant water status, assessing leaf water potential and relative water content (RWC) is essential for understanding physiological responses to drought stress [2]. Literature suggests that RWC is higher in drought-resistant plants, indicating a strong correlation with the amount of water absorbed or retained by the leaves [58]. In our study, under mild drought stress, no significant differences in RWC were observed between drought-stressed and well-watered treatments for clones C9-4, C16-1 and C1. However, for clones C2, C4-2, C8-3 and UCB1, RWC significantly declined compared to their controls. After 30 days of withheld irrigation (severe stress), RWC significantly decreased for all clones compared to controls. This reduction was less severe in resistant clones, with C9-4 and C1 showing decreases of 6% and 13%, respectively. In contrast, the sensitive clones C8-3 and C4-2 experienced much larger reductions of 88% and 76%, respectively. Previous studies have shown that, similar to drought stress, salinity stress also reduces RWC in pistachio rootstocks, with a more significant decrease observed in sensitive varieties [59]. Our findings align with studies by Liu [60] and Fathi [61], which reported that while mild stress only slightly affects RWC, severe drought stress leads to a significant reduction in RWC across all genotypes. Water potential is widely recognized as a key indicator of water status and essential for irrigation scheduling in fruit and nut trees [62]. A decline in leaf water potential (LWP) typically signals increased water stress, which can negatively impact photosynthesis, growth, and overall plant health. Moderate and severe water stress have distinct effects on LWP and plant physiology [63]. Early signs of moderate stress can be detected through changes in root osmotic potential, increased root turgor pressure, or a difference between root and leaf turgor pressure [64]. In our experiment, LWP decreased under drought stress, with the reduction being more pronounced under severe stress. The C8-3 and C4-2 clones were more affected, showing a significant decline in LWP. In contrast, resistant clones exhibited a smaller decline in water potential overall. At the end of the rehydration period, LWP increased in all clones, approaching control levels. However, C8-3 still had the lowest value, indicating its limited ability to resume growth after recovery. According to Pita [65] ensitive clones typically experience a greater decline in LWP than resistant ones under drought stress. This is due to their inability to maintain turgor pressure and regulate water loss effectively, resulting in more severe physiological stress that impacts growth and development [66, 67]. Osmotic adjustment is a net increase in solute content per cell that is independent of the volume changes that result from loss of water. It’s a crucial mechanism that helps plants adapt to drought stress by reducing their osmotic potential [68]. This process primarily involves the accumulation of compatible solutes. The increased accumulation of these solutes lowers osmotic potential, improving the plant's tolerance to drought stress [69]. In our experiments, under drought stress conditions, the C9-4 and C2 clones accumulated more osmolytes in their leaves compared to other clones. Additionally, osmolyte accumulation in the roots of the C1 clone significantly increased with water withholding. However, in other clones, drought stress did not significantly affect root osmotic potential (ROP). After rehydration, there were no significant differences in leaf osmotic potential (LOP) or ROP between the recovery and control plants for any of the clones. Some studies have also found no significant differences in leaf and root osmotic potential between recovered and control plants [70]. These findings suggest that, while osmotic adjustment is an important drought response mechanism, its effects may not persist after rehydration in certain cases. Proline and glycine betaine are key organic osmolytes that help plants tolerate drought by maintaining water potential and protecting cellular structures [71]. Total soluble carbohydrates (TSC) also play a vital role in drought tolerance, with drought-tolerant genotypes accumulating higher levels of TSC than sensitive ones [72, 73]. Our research demonstrated that, under stress, levels of glycine betaine, proline, and TSC increased in all clones, with resistant clones showing a significantly greater increase. Glycine betaine accumulation was three times higher than proline, and this trend was similar in the roots, though with higher accumulation in the leaves. After recovery, levels of compatible solutes (glycine betaine, proline, TSC) decreased in both leaves and roots compared to the drought stress stage. However, C9-4, C2, and C1 still had higher levels of these solutes compared to their control plants. Naser reported that proline, glycine betaine, and TSC accumulation is particularly notable in response to drought [74]. Our findings align with those of Behzadi Rad, who showed that high levels of proline and TSC in Pistacia vera L. ‘Ghazvini’ rootstock may explain its higher tolerance to salinity compared to other rootstocks [59]. Our results are consistent with previous research on pistachio [75, 76]. Drought stress significantly impacts stomatal morphology in many crops. Studies show that water deficit increases stomatal density while reducing their size (length and width) [77, 78]. These changes negatively affect photosynthesis and transpiration but improve water use efficiency [77]. Under water stress, stomatal apertures tend to close. Interestingly, stomatal density and size vary across growth stages, with later stages showing higher density [78]. Drought-tolerant varieties typically exhibit lower stomatal density and smaller stomata compared to susceptible ones [77]. Our findings indicate that stomatal parameters (density, length, width, pore aperture and pore length) and leaf temperature were not significantly affected by drought stress or recovery treatments. This suggests that stomatal regulation may not play a key role in dehydration tolerance of pistachio in our clones, and other mechanisms could be at play. Previous research on isohydric and anisohydric plants indicates that pistachios are likely categorized as anisohydric. These plants do not close their stomata during water stress; instead, they utilize strategies like enhanced photosynthesis and osmotic adjustment to improve their stress tolerance [79]. Studies have demonstrated that pistachios and almonds maintain high photosynthetic rates under drought conditions [80], supporting the notion that enhanced photosynthesis and osmotic adjustments are critical for drought tolerance in these species [80–82]. Similarly, our findings suggest that pistachio's drought tolerance might rely on mechanisms beyond stomatal regulation, potentially involving osmotic adjustments. This indicates that the importance of stomatal regulation in drought resistance may vary between species and could be less significant in species like pistachio [82]. Studies also suggest that while stomatal regulation helps control water loss and gas exchange, it may not be the sole factor in drought resistance. Plants often use a combination of strategies, like osmotic adjustment and root architecture changes, to cope with water stress. [83]. Since leaf temperature in our study remained unaffected, it further confirms that stomatal regulation is not crucial in drought resistance for our clones. The cluster heat map, PCA and correlation analyses in this study provide valuable insights into the phenotypic responses of pistachio clones under drought stress. These analyses grouped the hybrids into three categories based on drought resistance, highlighting important traits like leaf glycine betaine, RWC, and leaf water potential. These traits are strongly linked to drought sensitivity, suggesting that they should be prioritized in breeding programs. It is also noteworthy that plants typically adopt various strategies to manage drought stress, including escape, avoidance, and tolerance [34]. In this study, the clonal hybrids showed an increase in osmolytes, such as glycine betaine, which clearly indicates a dehydration tolerance strategy [84]. This finding suggests that the hybrids rely on preserving cellular functions and maintaining osmotic balance under water-deficit conditions, rather than employing escape or avoidance mechanisms. The accumulation of osmolytes underscores the tolerance strategy and highlights the critical role of osmotic adjustment in improving drought resistance. 5. Conclusion Our study highlights the complexities of drought stress responses in pistachio clonal hybrids, revealing significant differences in mechanisms underlying drought resistance. While drought stress adversely affected shoot and root dry weights across all clones, the C1 demonstrated superior tolerance with minimal reductions. Notably, the resistant clones, such as C9-4 and C1, exhibited less pronounced declines in relative water content and osmotic potential, suggesting effective osmotic regulation and other adaptive strategies. The accumulation of compatible solutes, particularly glycine betaine and proline, was significantly higher in resistant clones, reinforcing their ability to withstand drought conditions. Furthermore, the lack of significant changes in stomatal parameters indicates that stomatal regulation may not be a primary mechanism for drought tolerance in the pistachio hybrids. Instead, the traits such as osmotic adjustment, likely play a more critical role. These findings emphasize the importance of identifying and utilizing resilient genotypes in agricultural industry to enhance pistachio productivity in the face of increasing water scarcity. These findings also offer valuable insights for breeding drought-resistant cultivars and selecting appropriate rootstocks for drought-prone regions. With the growing adoption of clonal rootstocks due to their benefits in disease resistance, stress tolerance and overall performance, utilizing resistant clonal rootstocks could be an effective strategy for areas facing water scarcity. 5. Conclusion Our study highlights the complexities of drought stress responses in pistachio clonal hybrids, revealing significant differences in mechanisms underlying drought resistance. While drought stress adversely affected shoot and root dry weights across all clones, the C1 demonstrated superior tolerance with minimal reductions. Notably, the resistant clones, such as C9-4 and C1, exhibited less pronounced declines in relative water content and osmotic potential, suggesting effective osmotic regulation and other adaptive strategies. The accumulation of compatible solutes, particularly glycine betaine and proline, was significantly higher in resistant clones, reinforcing their ability to withstand drought conditions. Furthermore, the lack of significant changes in stomatal parameters indicates that stomatal regulation may not be a primary mechanism for drought tolerance in the pistachio hybrids. Instead, the traits such as osmotic adjustment, likely play a more critical role. These findings emphasize the importance of identifying and utilizing resilient genotypes in agricultural industry to enhance pistachio productivity in the face of increasing water scarcity. These findings also offer valuable insights for breeding drought-resistant cultivars and selecting appropriate rootstocks for drought-prone regions. With the growing adoption of clonal rootstocks due to their benefits in disease resistance, stress tolerance and overall performance, utilizing resistant clonal rootstocks could be an effective strategy for areas facing water scarcity. Declarations Acknowledgements The authors would like to express their gratitude to the University of Tehran and the Iran National Science Foundation (Project No: 4014915) for their valuable support in the completion of this study. We would also like to extend our sincere appreciation to the research Center of Royeshe Sabze Farda (Pistat) which provided us plant materials. Data Availability Statement The data presented in this study are available on request from the corresponding author. Author Contributions Conceptualization: M.R.R and S.S; methodology: M.O and M.M.A; software: M.O; validation: M.R.R; K.V; M.M.A; investigation: M.M.R; M.M.A; resources: M.R.R; M.M.A; data curation: M.O; writing original draft preparation: M.O; writing review and editing: M.O; M.R.R; M.M.A; K.V; visualization: M.O; supervision: M.R.R; S.S.; project administration: M.R.R; funding acquisition: M.R.R. and S.S. All authors have read and agreed to the published version of the manuscript. Funding: The financial support provided by the Iran National Science Foundation (Project No: 4014915) and the grant of the University of Tehran. Compliance with ethical standards. Conflicts of Interest: The authors declare no conflict of interest. Ethical approval: Not applicable. Consent to participate: Not Applicable. Consent for publication: Not Applicable. Availability of data and material: All data generated or analyzed during this study are included this published paper. References 1. Colaço AF, Molin JP, Rosell-Polo JR, Escolà A. Application of light detection and ranging and ultrasonic sensors to high-throughput phenotyping and precision horticulture: Current status and challenges. Hortic Res. 2018;5. https://doi.org/10.1038/s41438-018-0043-0. 2. Khoyerdi FF, Shamshiri MH, Estaji A. Changes in some physiological and osmotic parameters of several pistachio genotypes under drought stress. Sci Hortic (Amsterdam). 2016;198:44–51. https://doi.org/10.1016/j.scienta.2015.11.028. 3. Spiegel-Roy P, Mazigh D, Evenari M. Response of Pistachio to Low Soil Moisture Conditions1. J Am Soc Hortic Sci. 2022;102:470–3. https://doi.org/10.21273/jashs.102.4.470. 4. Miranda MT, Pires GS, Pereira L, de Lima RF, da Silva SF, Mayer JLS, et al. Rootstocks affect the vulnerability to embolism and pit membrane thickness in Citrus scions. Plant Cell Environ. 2024;47:3063–75. https://doi.org/10.1111/pce.14924. 5. Han Q, Guo Q, Korpelainen H, Niinemets Ü, Li C. Rootstock determines the drought resistance of poplar grafting combinations. Tree Physiol. 2019;39:1855–66. https://doi.org/10.1093/treephys/tpz102. 6. Chen Y, Fei Y, Howell K, Chen D, Clingeleffer P, Zhang P. Rootstocks for Grapevines Now and into the Future: Selection of Rootstocks Based on Drought Tolerance, Soil Nutrient Availability, and Soil pH. Aust J Grape Wine Res. 2024;2024. https://doi.org/10.1155/2024/6704238. 7. Liu Q, Wu C, Gao L, Ying L. Coordination between non-structure carbohydrates and hydraulic function under different drought duration, intensity, and post-drought recovery. Environ Exp Bot. 2023;215:105485. https://doi.org/10.1016/j.envexpbot.2023.105485. 8. Cui M, Lin Y, Zu Y, Efferth T, Li D, Tang Z. Ethylene increases accumulation of compatible solutes and decreases oxidative stress to improve plant tolerance to water stress in Arabidopsis. J Plant Biol. 2015;58:193–201. https://doi.org/10.1007/s12374-014-0302-z. 9. Blum A. Osmotic adjustment is a prime drought stress adaptive engine in support of plant production. Plant Cell Environ. 2017;40:4–10. https://doi.org/10.1111/pce.12800. 10. Guizani A, Askri H, Amenta ML, Defez R, Babay E, Bianco C, et al. Drought responsiveness in six wheat genotypes: identification of stress resistance indicators. Front Plant Sci. 2023;14 September:1–17. https://doi.org/10.3389/fpls.2023.1232583. 11. Condorell GE, Newcomb M, Groli EL, Maccaferri M, Forestan C, Babaeian E, et al. Genome Wide Association Study Uncovers the QTLome for Osmotic Adjustment and Related Drought Adaptive Traits in Durum Wheat. Genes (Basel). 2022;13:1–19. ttps://doi.org/10.3390/genes13020293. 12. Ratzmann G, Zakharova L, Tietjen B. Optimal leaf water status regulation of plants in drylands. Sci Rep. 2019;9:1–9. https://doi.org/10.1038/s41598-019-40448-2. 13. Merga D, Beksisa L. Mechanisms of Drought Tolerance in Coffee (Coffea arabica L.): Implication for Genetic Improvement Program: Review. Am J Biosci. 2023;11:63–70. https://doi.org/10.11648/j.ajbio.20231103.12. 14. Matkowski H, Daszkowska-Golec A. Update on stomata development and action under abiotic stress. Front Plant Sci. 2023;14 October:1–14. https://doi.org/10.3389/fpls.2023.1270180. 15. Marchin RM, Ossola A, Leishman MR, Ellsworth DS. A Simple Method for Simulating Drought Effects on Plants. Front Plant Sci. 2020;10 January:1–14. https://doi.org/10.3389/fpls.2019.01715. 16. Daszkowska-Golec A, Szarejko I. Open or close the gate - Stomata action under the control of phytohormones in drought stress conditions. Front Plant Sci. 2013;4 MAY:1–16. https://doi.org/10.3389/fpls.2013.00138. 17. Ferguson L, Sanden B, Grattan S, Epstein L, Krueger B. Pistachio Rootstocks. Pist Prod Man. 2005;:67–73. https://doi.org/10.21273/hortsci.30.4.855g. 18. Akbari M, Hokmabadi H, Heydari M, Ghorbani A. ‘Arota’: A New Interspecific Hybrid Pistachio Rootstock. HortScience. 2020;55:965–6. https://doi.org/10.21273/HORTSCI14845-20. 19. Hoagland DR, Arnon DI. The water culture method for growing plants without soil. Miscellaneous Publications n o 354. Circular of California Agricultural Experimental Station. 1941;347:461. https://doi.org/10.5962/bhl.title.60957. 20. Benedicto Ottoni Filho T, Vasconcelos Ottoni M, Batista de Oliveira M, Ronaldo de Macedo J, Reichardt K, Redentor Av Zulamith F. Theophilo Benedicto Ottoni Filho et al. REVISITING FIELD CAPACITY (FC): VARIATION OF DEFINITION OF FC AND ITS ESTIMATION FROM PEDOTRANSFER FUNCTIONS (1). 2014;38:1750–64. https://doi.org/10.1590/S0100-06832014000600010. 21. Dghim F, Abdellaoui R, Boukhris M, Neffati M, Chaieb M. Physiological and biochemical changes in Periploca angustifolia plants under withholding irrigation and rewatering conditions. South African J Bot. 2018;114:241–9. https://doi.org/10.1016/j.sajb.2017.11.007. 22. Arab MM, Askari H, Aliniaeifard S, Mokhtassi-Bidgoli A, Estaji A, Sadat-Hosseini M, et al. Natural variation in photosynthesis and water use efficiency of locally adapted Persian walnut populations under drought stress and recovery. Plant Physiol Biochem. 2023;201 January:107859. https://doi.org/10.1016/j.plaphy.2023.107859. 23. Abid M, Tian Z, Ata-Ul-Karim ST, Wang F, Liu Y, Zahoor R, et al. Adaptation to and recovery from drought stress at vegetative stages in wheat (Triticum aestivum) cultivars. Funct Plant Biol. 2016;43:1159–69. https://doi.org/10.1071/FP16150. 24. Zhang W. Digital image processing method for estimating leaf length and width tested using kiwifruit leaves (Actinidia chinensis Planch). PLoS One. 2020;15:1–14. https://doi.org/10.1371/journal.pone.0235499. 25. Martìnez JP, Lutts S, Schanck A, Bajji M, Kinet JM. Is osmotic adjustment required for water stress resistance in the Mediterranean shrub Atriplex halimus L? J Plant Physiol. 2004;161:1041–51. https://doi.org/10.1016/j.jplph.2003.12.009. 26. da Silva AA, Silva CO, do Rosario Rosa V, Santos MFS, Kuki KN, Dal-Bianco M, et al. Metabolic adjustment and regulation of gene expression are essential for increased resistance to severe water deficit and resilience post-stress in soybean. PeerJ. 2022. https://doi.org/10.7717/peerj.13118. https://doi.org/10.7717/peerj.13118. 27. Sithtisarn S, Harinasut P, Pornbunlualap S, Cha-Um S, Carillo P, Gibon Y. PROTOCOL : Extraction and determination of glycine betaine Initiating Author Name : Kasetsart J - Nat Sci. 2009;43 5 SUPPL.:146–52. https://doi.org/10.1002/aris.2009.1440430113. 28. Claussen W. Proline as a measure of stress in tomato plants. Plant Sci. 2005;168:241–8. https://doi.org/10.1016/j.plantsci.2004.07.039. 29. Irigoyen JJ, Einerich DW, Sánchez‐Díaz M. Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativd) plants. Physiol Plant. 1992;84:55–60. https://doi.org/10.1111/j.1399-3054.1992.tb08764.x. 30. Aliniaeifard S, Van Meeteren U. Stomatal characteristics and desiccation response of leaves of cut chrysanthemum (Chrysanthemum morifolium) flowers grown at high air humidity. Sci Hortic (Amsterdam). 2016;205:84–9. https://doi.org/10.1016/j.scienta.2016.04.025. 31. Kathpalia R, Bhatla SC. Plant Water Relations. 2018. https://doi.org/10.1007/978-981-13-2023-1_1. 32. Radiation I. 2.1 Introduction. 2017. https://doi.org/10.1016/j.semradonc.2017.05.001. 33. Raoufi A, Salehi H, Rahemi M, Shekafandeh A, Khalili S. In vitro screening: The best method for salt tolerance selection among pistachio rootstocks. J Saudi Soc Agric Sci. 2021;20:146–54. https://doi.org/10.1016/j.jssas.2020.12.010. 34. Fang Y, Xiong L. General mechanisms of drought response and their application in drought resistance improvement in plants. Cell Mol Life Sci. 2015;72:673–89. https://doi.org/10.1007/s00018-014-1767-0. 35. Venkateswarlu B, Shanker AK, Shanker C, Maheswari M. Crop stress and its management: Perspectives and strategies. 2012. https://doi.org/10.1007/978-94-007-2220-0. 36. Aroca R. Front Matter: Plant responses to drought stress. Plant Responses to Drought Stress. 2012. https://doi.org/10.1007/978-3-642-32653-0. 37. Chaves MM, Pereira JS, Maroco J, Rodrigues ML, Ricardo CPP, Osório ML, et al. How plants cope with water stress in the field. Photosynthesis and growth. Ann Bot. 2002;89 SPEC. ISS.:907–16. https://doi.org/10.1093/aob/mcf105. 38. Flexas J, Medrano H. Drought-inhibition of photosynthesis in C3 plants: Stomatal and non-stomatal limitations revisited. Ann Bot. 2002;89:183–9. https://doi.org/10.1093/aob/mcf027. https://doi.org/10.1093/aob/mcf027. 39. Blum A. Drought resistance, water-use efficiency, and yield potential - Are they compatible, dissonant, or mutually exclusive? Aust J Agric Res. 2005;56:1159–68. https://doi.org/10.1071/AR05069. 40. Sutka MR, Manzur ME, Vitali VA, Micheletto S, Amodeo G. Evidence for the involvement of hydraulic root or shoot adjustments as mechanisms underlying water deficit tolerance in two Sorghum bicolor genotypes. J Plant Physiol. 2016;192:13–20. https://doi.org/10.1016/j.jplph.2016.01.002. 41. Sánchez-Blanco MJ, Álvarez S, Ortuño MF, Ruiz-Sánchez MC. Root System Response to Drought and Salinity: Root Distribution and Water Transport. 2014;:325–52. https://doi.org/10.1007/978-3-642-54276-3_15. 42. Saadatmand AR, Banihashemi Z, Maftoun M, Sepaskhah AR. Interactive effect of soil salinity and water stress on growth and chemical compositions of pistachio nut tree. J Plant Nutr. 2007;30:2037–50. https://doi.org/10.1080/01904160701700483. 43. Vives-Peris V, López-Climent MF, Pérez-Clemente RM, Gómez-Cadenas A. Root involvement in plant responses to adverse environmental conditions. Agronomy. 2020;10. https://doi.org/10.3390/agronomy10070942. 44. Bhargava S, Sawant K. Chapter Plant Responses to Drought Stress: Morphological, Physiological, Molecular Approaches, and Drought Resistance and regulation of gene expression. Plant Breed. 2013;132:21–32. https://doi.org/10.1111/pbr.12004. 45. Liu Y, He Z, Xie Y, Su L, Zhang R, Wang H, et al. Drought resistance mechanisms of Phedimus aizoon L. Sci Rep. 2021;11:13600. https://doi.org/10.1038/s41598-021-93118-7. 46. Deka D, Singh AK, Singh AK. Effect of Drought Stress on Crop Plants with Special Reference to Drought Avoidance and Tolerance Mechanisms: A Review. Int J Curr Microbiol Appl Sci. 2018;7:2703–21. https://doi.org/10.20546/ijcmas.2018.709.336. 47. Nwaogu, EN. Soil fertility changes and their effects on ginger (Zingiber officinale Rosc.) yield response in an ultisol under different pigeon pea hedgerow alley management in South Eastern Nigeria. African J Agric Res. 2014;9:2158–66. https://doi.org/10.5897/ajar2013.7291. 48. Kasmani RM, Andrews GE, Phylaktou HN. Experimental study on vented gas explosion in a cylindrical vessel with a vent duct. Process Saf Environ Prot. 2013;91:245–52. https://doi.org/10.1016/j.psep.2012.05.006. 49. Koch G, Rolland G, Dauzat M, Bédiée A, Baldazzi V, Bertin N, et al. Leaf production and expansion: A generalized response to drought stresses from cells to whole leaf biomass—a case study in the tomato compound leaf. Plants. 2019;8:1–17. https://doi.org/10.3390/plants8100409. 50. Biglouei MH, Assimi MH, Akbarzadeh A. Effect of water stress at different growth stages on quantity and quality traits of Virginia (flue-cured) tobacco type. Plant, Soil Environ. 2010;56:67–75. https://doi.org/10.17221/163/2009-pse. 51. Farquharson KL. Fine-tuning plant growth in the face of drought. Plant Cell. 2017;29:4. https://doi.org/10.1105/tpc.17.00038. 52. Claeys H, Inzé D. of choice: How plants balance growth and survival under water-limiting conditions. Plant Physiol. 2013;162:1768–79. https://doi.org/10.1104/pp.113.220921. 53. Zhao Y, Gao J, Kim JI, Chen K, Bressan RA, Zhu JK. Control of plant water use by ABA induction of senescence and dormancy: An overlooked lesson from evolution. Plant Cell Physiol. 2017;58:1319–27. https://doi.org/10.1093/pcp/pcx086. 54. Zhang YJ, Xie ZK, Wang YJ, Su PX, An LP, Gao H. Effect of water stress on leaf photosynthesis, chlorophyll content, and growth of oriental lily. Russ J Plant Physiol. 2011;58:844–50. https://doi.org/10.1134/s1021443711050268. 55. Marron N, Dreyer E, Boudouresque E, Delay D, Petit JM, Delmotte FM, et al. Impact of successive drought and re-watering cycles on growth and specific leaf area of two Populus x canadensis (Moench) clones, “Dorskamp” and “Luisa_Avanzo.” Tree Physiol. 2003;23:1225–35. https://doi.org/10.1093/treephys/23.18.1225. 56. Chaves MM, Oliveira MM. Mechanisms underlying plant resilience to water deficits: Prospects for water-saving agriculture. J Exp Bot. 2004;55:2365–84. https://doi.org/10.1093/jxb/erh269. 57. Farooq M, A.Wahid, Kobayashi N, Fujita D, Basra SMA. Review article Plant drought stress : e ff ects , mechanisms and management. Agron Sustain Dev. 2009;29:185–212. https://doi.org/10.1051/agro:2008021. 58. Afrousheh M, Javanshah A. The effect of humic acid on the growth and physiological indices of pistachio seedling (Pistacia vera) under drought stress. J Nuts. 2020;11:1–12. https://doi.org/10.22034/jon.2020.1879490.1069 59. Behzadi Rad P, Roozban MR, Karimi S, Ghahremani R, Vahdati K. Osmolyte accumulation and sodium compartmentation has a key role in salinity tolerance of pistachios rootstocks. Agric. 2021;11. https://doi.org/10.3390/agriculture11080708. 60. Wan T, Feng Y, Liang C, Pan L, He L, Cai Y. Metabolomics and transcriptomics analyses of two contrasting cherry rootstocks in response to drought stress. Biology (Basel). 2021;10:1–20. https://doi.org/10.3390/biology10030201. 61. Fathi H, Esmailamiri M, Imani A, Hajilou J, Nikbakht J. PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES IN SOME ALMOND (PRUNUS AMYGDALUS BATSCH.) GENOTYPES (GRAFTED ON/GN15) SUBMITTED TO DROUGHT STRESS. 2018. 62. Memmi H, Couceiro JF, Gijón C, Pérez-López D. Impacts of water stress, environment and rootstock on the diurnal behaviour of stem water potential and leaf conductance in pistachio (Pistacia vera L.). Spanish J Agric Res. 2016;14. https://doi.org/10.5424/sjar/2016142-8207. 63. Sharkey TD, Seemann JR. Mild Water Stress Effects on Carbon-Reduction-Cycle Intermediates, Ribulose Bisphosphate Carboxylase Activity, and Spatial Homogeneity of Photosynthesis in Intact Leaves. Plant Physiol. 1989;89:1060–5. https://doi.org/10.1104/pp.89.4.1060. 64. Setum P, Des T, Mcpherson BHG. Sensitivity of root and leaf water status in maize ( Zea mays ) subjected to mild soil dryness. 2017; January 1998. https://doi.org/10.1071/PP97047. https://doi.org/10.1071/pp97047. 65. Pita P, Gascó A, Pardos JA. Xylem cavitation, leaf growth and leaf water potential in Eucalyptus globulus clones under well-watered and drought conditions. Funct Plant Biol. 2003;30:891–9. https://doi.org/10.1071/fp03055. 66. Costa E Silva F, Shvaleva A, Maroco JP, Almeida MH, Chaves MM, Pereira JS. Responses to water stress in two Eucalyptus globulus clones differing in drought tolerance. Tree Physiol. 2004;24:1165–72. https://doi.org/10.1093/treephys/24.10.1165. 67. Luiz Ferraresso Conti Junior J, José de Araujo M, Cesar de Paula R, Barroso Queiroz T, Eiji Hakamada R, Hubbard RM. Quantifying turgor loss point and leaf water potential across contrasting Eucalyptus clones and sites within the TECHS research platform. For Ecol Manage. 2020;475 June:118454. https://doi.org/10.1016/j.foreco.2020.118454. 68. Shabala S, Shabala L. Ion transport and osmotic adjustment in plants and bacteria. Biomol Concepts. 2011;2:407–19. https://doi.org/10.1515/bmc.2011.032. 69. Hsiao TC, Otoole JC, Yambao EB, Turner NC. Influence of Osmotic Adjustment. 1984;:338–41. https://doi.org/10.1104/pp.75.2.338. 70. Mokotedi MEO. Water relations of Eucalyptus nitens × Eucalyptus grandis: Is there interclonal variation in response to experimentally imposed water stress? South For. 2013;75:213–20. https://doi.org/10.2989/20702620.2013.858212. 71. Rana V, Ram S, Nehra K. Review Proline Biosynthesis and its Role in Abiotic Stress. Int J Agric Innov Res. 2017;6:2319–1473. https://doi.org/10.1556/0806.44.2016.009. 72. Kerepesi I, Galiba G, Bányai É. Osmotic and Salt Stresses Induced Differential Alteration in Water-Soluble Carbohydrate Content in Wheat Seedlings. J Agric Food Chem. 1998;46:5347–54. https://doi.org/10.1021/jf980455w. 73. Kerepesi,I. and Galiba G. Osmotic and Salt Stress-Induced Alteration in Soluble Carbohydrate Content in Wheat Seedlings ´. Crop Sci J. 2014;40:482–7. https://doi.org/10.2135/cropsci2000.402482x. 74. Escalante-Magaña C, Aguilar-Caamal LF, Echevarría-Machado I, Medina-Lara F, Cach LS, Martínez-Estévez M. Contribution of glycine betaine and proline to water deficit tolerance in pepper plants. HortScience. 2019;54:1044–54. https://doi.org/10.21273/hortsci13955-19. 75. Reyhani Haghighi S, Hosseininaveh V, Maali-Amiri R, Talebi K, Irani S. Improving the Drought Tolerance in Pistachio (Pistacia vera) Seedlings by Foliar Application of Salicylic Acid. Gesunde Pflanz. 2021;73:495–507. https://doi.org/10.1007/s10343-021-00569-z. 76. Jamshidi Goharrizi K, Amirmahani F, Salehi F. Assessment of changes in physiological and biochemical traits in four pistachio rootstocks under drought, salinity and drought + salinity stresses. Physiol Plant. 2020;168:973–89. https://doi.org/10.1111/ppl.13042. 77. Zhao W, Sun Y, Kjelgren R, Liu X. Response of stomatal density and bound gas exchange in leaves of maize to soil water deficit. Acta Physiol Plant. 2015;37. https://doi.org/10.1007/s11738-014-1704-8. 78. Mehri N, Fotovat R, Saba J, Jabbari F. Variation of stomata dimensions and densities in tolerant and susceptible wheat cultivars under drought stress. J Food, Agric Environ. 2009;7:167–70. http://www.world-food.net/. 79. Sade N, Gebremedhin A, Moshelion M. Risk-taking plants. Plant Signal Behav. 2012;7:767–70. https://doi.org/10.4161/psb.20505. 80. Sedaghati N, Hokmabadi H. Optimizing pistachio irrigation management using the relationship between Echo-Physiological characteristics and water stress. J Agric Sci Technol. 2015;17:189–200. http://dorl.net/dor/20.1001.1.16807073.2015.17.1.8.9. 81. Durán-Zuazo VH, Rodriguez BC, Gutiérrez-Gordillo S, Bilbao M, Sacristán PC, Pérez Parra J, et al. Rethinking irrigated almond and pistachio intensification: a shift towards a more sustainable water management paradigm. Rev Ciências Agrárias. 2020;43 Especial 2:24–49. https://doi.org/10.19084/rca.19651 82. Bellvert J, Adeline K, Baram S, Pierce L, Sanden BL, Smart DR. Monitoring Crop Evapotranspiration and Crop Coefficients Over an Almond and Pistachio Orchard Throughout Remote Sensing. Remote Sens. 2018;10. https://doi.org/10.3390/rs10122001. 83. Neumann PM. Coping mechanisms for crop plants in drought-prone environments. Ann Bot. 2008;101:901–7. https://doi.org/10.1093/aob/mcn018. 84. Li N, Gao Y, pu K, Zhang M, Wang T, Li J, et al. Glycine betaine enhances tolerance of low temperature combined with low light in pepper (Capsicum annuum L.) by improving the antioxidant capacity and regulating GB metabolism. Plant Physiol Biochem. 2025;222:109705. https://doi.org/10.1016/j.plaphy.2025.109705. Additional Declarations No competing interests reported. Supplementary Files Supplementary.xlsx Cite Share Download PDF Status: Published Journal Publication published 02 May, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 15 Apr, 2025 Reviews received at journal 12 Apr, 2025 Reviews received at journal 08 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers agreed at journal 30 Mar, 2025 Reviewers invited by journal 28 Mar, 2025 Submission checks completed at journal 27 Mar, 2025 First submitted to journal 26 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5905176","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436013088,"identity":"4ac87d02-a18d-484b-97e2-db78be09c239","order_by":0,"name":"Mozhdeh Osku","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Mozhdeh","middleName":"","lastName":"Osku","suffix":""},{"id":436013089,"identity":"54acd7bb-e9e6-461b-8361-9d620f1f77a7","order_by":1,"name":"Mahmoud Reza Roozban","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYLACxgYGBn4om4d4LZINzKRqMTjATKSbdGcfPvjw5w67POMb+QcYftQwyJg3ENBidi4t2UDyTHKx2Y1kBsaeYww8MgcIaTnDYyZh2MacuA2ohYG3gYFHgpDDwFoS2+oTN88A2vKXaC0H2w4nbpBIZmAm0ha2ZMPGM8cTZ5x5bHBY5pgEMVqYQSFWndjfnvjw4ZsaG3uCWlDAAQYG0jSMglEwCkbBKMABAJ9HOYTMr7gRAAAAAElFTkSuQmCC","orcid":"","institution":"University of Tehran","correspondingAuthor":true,"prefix":"","firstName":"Mahmoud","middleName":"Reza","lastName":"Roozban","suffix":""},{"id":436013090,"identity":"d1d24773-3ea2-4d99-a809-cf65bf6a0a60","order_by":2,"name":"Saadat Sarikhani","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Saadat","middleName":"","lastName":"Sarikhani","suffix":""},{"id":436013091,"identity":"ce6e427a-87a8-4644-8fb4-c19a2a3bf851","order_by":3,"name":"Mohammad Mehdi Arab","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Mehdi","lastName":"Arab","suffix":""},{"id":436013092,"identity":"5139bb12-b1d7-4870-810d-298adb0008c0","order_by":4,"name":"Mohammad Akbari","email":"","orcid":"","institution":"Nazari Business Group","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Akbari","suffix":""},{"id":436013093,"identity":"6d7ed902-261a-4dd4-b5c8-3afe577b41c1","order_by":5,"name":"Kourosh Vahdati","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Kourosh","middleName":"","lastName":"Vahdati","suffix":""}],"badges":[],"createdAt":"2025-01-26 08:08:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5905176/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5905176/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-06583-x","type":"published","date":"2025-05-02T15:57:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79565997,"identity":"59a0767b-2e44-4d2d-8a36-ad7c5d73f660","added_by":"auto","created_at":"2025-03-31 09:35:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":164911,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the experimental design. Sampling Time 1: data collection 15 days after the onset of the water withholding (mild stress). Sampling Time 2: data collection 30 days after applying withholding irrigation (severe stress) and Sampling Time 3 is data collection at the end of recovery period.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/f31c0ef0fe17af240a0d4ece.png"},{"id":79565998,"identity":"71b67d35-c29b-4154-8f14-d2d1cf4d73dc","added_by":"auto","created_at":"2025-03-31 09:35:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7289374,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of water withholding and recovery on shoot dry weight (SDW) and root dry weight (RDW) of pistachio clonal hybrids (A,B: SDW and RDW under water withholding ; C,D: SDW and RDW under recovery respectively). Error bars represent standard error.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/7c179027532274abd9f0a520.png"},{"id":79565999,"identity":"8f1897f2-ab9a-4151-9b08-2b30ff549c62","added_by":"auto","created_at":"2025-03-31 09:35:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1760268,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of water withholding and recovery on the root to shoot ratio (A,B), growth rate (C) and recovery rate (D) of pistachio clonal hybrids. Error bars represent standard error.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/740efc5b4d957345ead891b6.png"},{"id":79565644,"identity":"0fff64b8-9fea-4b60-8103-ef8e9d8db618","added_by":"auto","created_at":"2025-03-31 09:27:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4564980,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of water withholding (A) and recovery (B) on leaf area in pistachio clonal hybrids.\u003c/p\u003e\n\u003cp\u003eError bars represent standard error.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/2ad963d731537979dd456f1c.png"},{"id":79565654,"identity":"7e043106-6c4d-4340-a597-80981006cfaa","added_by":"auto","created_at":"2025-03-31 09:27:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6307573,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of water stress (mild (A), severe (B) stress) and recovery (C) on relative water content (RWC) of pistachio clonal hybrids. Error bars represent standard error.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/8970bed33849dda753b81a89.png"},{"id":79565652,"identity":"c986f6ab-17e1-4022-91e6-3096a9f5c0dc","added_by":"auto","created_at":"2025-03-31 09:27:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6128052,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of water stress (mild (A), severe (B) stress) and recovery (C) on leaf water potential of pistachio clonal hybrids. Error bars represent standard error.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/b3716feec96a2f324410753f.png"},{"id":79565664,"identity":"c7f73f8f-0cbe-4117-aaf8-da648a95d44f","added_by":"auto","created_at":"2025-03-31 09:27:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9339731,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of water withholding and recovery on leaf and root osmotic potential (LOP and ROP) of pistachio clonal hybrids (A,B: LOP and ROP under water withholding; C,D: LOP and ROP under recovery respectively). Error bars represent standard error.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/08afd545b6f878c7606a7725.png"},{"id":79565656,"identity":"8f7ab30d-9372-4df8-b06d-56a2575bfae6","added_by":"auto","created_at":"2025-03-31 09:27:42","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":9305224,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of water withholding and recovery on leaf and root glycine betaine content of pistachio clonal hybrids (A,B: leaf and root glycine betaine under water withholding; C,D: leaf and root glycine betaine under recovery respectively). Error bars represent standard error.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/468eaf1e26c2f4e457caa5ed.png"},{"id":79566008,"identity":"22247d92-38bc-4a43-9a53-1c79e07774a3","added_by":"auto","created_at":"2025-03-31 09:35:42","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4591472,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of water withholding on leaf (A) and root (B) proline content of pistachio clonal hybrids.\u003c/p\u003e\n\u003cp\u003eError bars represent standard error.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/d2a713cd3bd4b3f31c7a5336.png"},{"id":79565662,"identity":"f835120b-664f-471e-8417-a2c6252c40e3","added_by":"auto","created_at":"2025-03-31 09:27:42","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":5501726,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of clonal hybrids on leaf (A) and root (B) proline. Error bars represent standard error.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/eeee43a1c1837404bee7d66a.png"},{"id":79567130,"identity":"6110cedc-f3fb-4bde-bb2a-fd0ac060fac0","added_by":"auto","created_at":"2025-03-31 09:43:42","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1216394,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of water withholding and recovery on leaf and root total soluble carbohydrate (TSC) content of pistachio clonal hybrids (A,B: leaf and root TSC under water withholding; C,D: leaf and root TSC under recovery respectively). Error bars represent standard error.\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/9452cd04c52c987882b651f3.png"},{"id":79565678,"identity":"da2c1608-5d1b-494d-b124-b92e71537bb3","added_by":"auto","created_at":"2025-03-31 09:27:43","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":609639,"visible":true,"origin":"","legend":"\u003cp\u003eStomatal characteristics of the most resistant (C9-4) and sensitive (C8-3) clones under control and drought stress conditions.\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/0079728f7acded8b886c294a.png"},{"id":79565663,"identity":"40789c19-9765-447e-a8a1-5f185a805ea0","added_by":"auto","created_at":"2025-03-31 09:27:42","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":974965,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap and hierarchical clustering of phenotypic plasticity (DSI) for all physiological and growth traits studied in this research in the seven pistachio clonal hybrids under water stress conditions(after 30 days of water-withholding). Clustering analysis of pistachio hybrid (left) showed three main groups where the groups I, II and III represent resistant, moderately resistant and sensitive under the water stress treatment respectively. The clustering analysis of studied parameters (top) showed five major groups. Group a (LWP: leaf water potential, SDW:shoot dry weight, RWC: relative water content, LArea: leaf area), group b (rGb: root glycine betaine, rTSC: root total soluble carbohydrate, RDW: root dry weight), group c (PA: pore aperture, SL: stomata, PL: pore length, LTemp:leaf tempreture, SW: stomata width), group d (LTSC: leaf total soluble carbohydrate, Lproline: leaf proline, rproline: root proline, LGb: leaf glycine betaine), group e (LOP: leaf osmotic potential, ROP: root osmotic potential).\u003c/p\u003e","description":"","filename":"image13.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/439fb6d427cc4eafa63a7ed9.png"},{"id":79565687,"identity":"135af6b5-0f05-4ac2-b0f4-51fd17e205f3","added_by":"auto","created_at":"2025-03-31 09:27:43","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":1125509,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap and hierarchical clustering of phenotypic plasticity (DRI) for all physiological and growth traits studied in this research in the seven pistachio clonal hybrids under recovery conditions (after 30 days of re-watering). Clustering analysis of pistachio hybrids (left) showed three main groups where the groups I, II and III represent with strong, weak and moderate recovery under the re-watering treatment respectively. The clustering analysis of studied parameters (top) showed three major groups: group a (LWP: leaf water potential, LArea: leaf area, rGb: root glycine betaine, LGb: leaf glycine betaine), group b (PA: pore aperture, PL: pore length, SW: stomata width, rproline: root proline, L proline: leaf proline, LTemp: leaf tempreture, SL: stomata length), group c (RWC: relative water content, LOP: leaf osmotic potential, SDW: shoot dry weight, RDW: root dry weight, ROP: root osmotic potential, rTSC: root total soluble carbohydrate, LTSC: leaf total soluble carbohydrate).\u003c/p\u003e","description":"","filename":"image14.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/d4295cdb60776c473d3db6bc.png"},{"id":79567132,"identity":"c65ee61c-a5c2-4ad3-81b0-e8ea1c5825aa","added_by":"auto","created_at":"2025-03-31 09:43:43","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":563240,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of moderate and severe drought stress and recovery on the most resistant (C9-4) and sensitive (C8-3) and UCB1 clones. [C: Control, D: Drought, R:Recovery]\u003c/p\u003e","description":"","filename":"image16.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/db96c02c4d02baae7f11e604.png"},{"id":79567134,"identity":"4e9b6bbc-54dc-4b0e-8b4b-be9c3da19d9c","added_by":"auto","created_at":"2025-03-31 09:43:43","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":1357648,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation coefficient of plasticity in trait value (DSI), between all physiological and growth traits studied in this research in the seven pistachio clonal hybrids grown in a greenhouse under water stress condition. The color spectrum, bright blue to bright red represents highly positive to highly negative correlations. Stars in circle indicate the significance of correlations (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001). Ltempreture: leaf tempreture, SDW: shoot dry weight, LArea: leaf area, LWP: leaf water potemtial, RWC: relative water content, LTSC: leaf total soluble carbohydrate, rTSC: root total soluble carbohydrate, LGb: leaf glycine betaine, rGb: root glycine betaine, Lproline: leaf proline, rproline: root proline, PL: pore length, ROP: root osmotic potential, LOP: leaf osmotic potential, SW: stomata width, RDW: root dry weight, SL: stomata length, PA: pore aperture.\u003c/p\u003e","description":"","filename":"image17.png","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/d70170c6f5e3dfacfc23eee9.png"},{"id":81987747,"identity":"9f449ca8-c431-4055-ac9b-7f3ccd8fc760","added_by":"auto","created_at":"2025-05-05 16:05:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":61910994,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/4e59a31a-ef6a-4232-82ed-3ad85f9364fd.pdf"},{"id":79565647,"identity":"1a52f5bf-66f5-424d-9822-dd18ab76b497","added_by":"auto","created_at":"2025-03-31 09:27:42","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14108,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5905176/v1/f3eb8fb8cbff8946e53c7bc4.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Revealing Drought Tolerance Strategies in Pistachio Clonal Hybrids: Role of Osmotic Adjustment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePistachio (\u003cem\u003ePistacia vera\u003c/em\u003e L.) is a wind-pollinated, deciduous nut tree belonging to the Anacardiaceae family, primarily cultivated in Mediterranean regions but likely originating from central and southwestern Asia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. A significant abiotic stress affecting plant physiology and development is water scarcity. Pistachio trees are known for their ability to endure prolonged periods without water [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], yet their performance can decline under dry conditions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In Iran, where pistachio cultivation is prevalent, these trees are often grown in arid and semi-arid regions.\u003c/p\u003e \u003cp\u003eRootstocks play a key role in water absorption and stress resistance. Drought-resistant rootstocks improve scion hydraulic function by enhancing xylem structure and reducing cavitation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These rootstocks boost scion drought resistance through increasing non-structural carbohydrate (NSC) storage, altering photosynthesis, and adjusting stomatal conductance via hydraulic and hormonal changes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The ability of rootstocks to tolerate drought stress depends on various factors, including their specific traits and adaptability to local climatic conditions, alongside regional characteristics such as soil composition and water availability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For instance, rootstocks derived from \u003cem\u003ePistacia atlantica\u003c/em\u003e are well-known for their deep root systems, enabling access to water in lower soil layers during periods of drought. Moreover, these rootstocks play a vital role in regulating non-structural carbohydrate (NSC) accumulation, supporting plants in maintaining energy reserves and structural stability under water stress conditions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAcclimation to water stress in plants involves the production of compatible solutes which stabilize membranes, enzymes and cellular osmotic balance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These solutes play a critical role in dehydration tolerance by enabling plants to sustain cellular functions during water scarcity and maintaining turgor pressure to prevent dehydration [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Key biomarkers for drought resistance, including relative water content, osmotic potential and leaf water potential, help assess a plant\u0026rsquo;s ability to retain water and adjust osmotic pressure under stress [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These traits highlight important mechanisms such as turgor maintenance and, which contribute to identifying drought-tolerant clones and enhancing breeding programs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStomatal responses are among the first reactions to abiotic stress, occurring within seconds to minutes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Stomata are essential for controlling water loss during drought stress, with abscisic acid (ABA) playing a key role in inducing stomatal closure to reduce transpiration. Additionally, leaf temperature serves as a reliable indicator of drought stress, as plants under such conditions close their stomata, resulting in elevated leaf temperatures compared to non-stressed plants. This approach is widely used in fruit trees to evaluate drought stress [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDrought-resistant rootstocks are essential for sustainable pistachio production in water-scarce areas. While the Badami-Zarand rootstock is widely used in Iran, it has drawbacks like slow growth and disease susceptibility. On the other hand, the hybrid rootstock UCB1, developed at UC Berkeley [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], offers advantages in drought tolerance and disease resistance. In recent decades, \u003cem\u003ePistacia atlantica\u003c/em\u003e and \u003cem\u003ePistacia integerrima\u003c/em\u003e have been widely utilized as parental species in hybridization programs, owing to their superior drought and salinity tolerance, rapid growth and increased yield potential. A new hybrid rootstock, Arota, developed from a cross between \u003cem\u003eP. atlantica\u003c/em\u003e and \u003cem\u003eP. integerrima\u003c/em\u003e, shows great promise [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It is essential to evaluate the drought resistance of these rootstocks before their widespread adoption in orchards.\u003c/p\u003e \u003cp\u003eThis study aims to understand the drought tolerance strategies of seven clonal interspecific hybrids of \u003cem\u003ePistacia atlantica\u003c/em\u003e Dscf. \u0026times; \u003cem\u003ePistacia integerrima\u003c/em\u003e Stewart. Given the key role of rootstocks in supporting pistachio production under increasing water scarcity, we assessed the pistachio hybrids responses to water withholding, focusing on osmotic adjustment, growth characteristics, water relations and stomatal behavior. By combining the unique traits of each hybrid with consideration of regional environmental factors, such as soil type and local climate, this study offers valuable insights into the role of these rootstocks in promoting sustainable pistachio production in drought-prone regions.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Plant materials and experimental conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was carried out in the research greenhouse at the Faculty of Agricultural Technology, University of Tehran, from June to september 2023. The plant materials used in this study originated from a rootstock breeding program aimed at promoting high growth, carried out by the Pistat Research Center in partnership with ITA Sadra\u0026reg; Company at the Green Tat field in Boein-Zahra, Ghazvin, Iran. In this project, a total of 222 controlled hybridizations were executed, involving 37 female \u003cem\u003ePistacia atlantica\u003c/em\u003e Dscf. and six male \u003cem\u003ePistacia integerrima\u003c/em\u003e Stewart trees. The most vigorous and uniform hybrids resulting from these crosses were assessed over a four-year trial based on their relative growth metrics, as opposed to UCB1 standards [18]. From the top 15 hybrids identified during this evaluation, six high-performing hybrids labeled as C1, C2, C16-1, C8-3, C4-2 and C9-4 were selected and clonally micropropagated at ITA Sadra\u003csup\u003e\u0026reg;\u003c/sup\u003e Biotechnology Co. serving as the plant material for this experiment beside UCB1.\u003c/p\u003e\n\u003cp\u003eNine-month-old saplings of the hybrids were transferred to 10 L pots filled with a mix of 50 % soil and 50 % perlite and sand (1:1 V/V).\u0026nbsp;The pots were arranged in a randomized complete block design (RCBD) with three replications for each treatment.\u0026nbsp;The plants were grown in the greenhouse for four weeks to facilitate the development of their canopy and root systems.\u0026nbsp;All plants were fed weekly with a full-strength Hoagland\u0026rsquo;s nutrient solution [19] to provide essential nutrients and promote plant establishment before applying treatments. The volumetric water content at field capacity was 15% V/V and at wilting point 5% V/V that they were measured following the protocol outlined by Benedicto Ottoni [20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlants of the each clonal hybrid were divided into two groups: one for control and the other for withholding irrigation. The first group (control) received manual irrigation every three days until reaching soil to field capacity. The second group (no irrigation) experienced a period of water stress by withholding irrigation [21]. To monitor the soil moisture during the stress period, pot weights were recorded every three days.\u003c/p\u003e\n\u003cp\u003eWater withholiding was imposed for 30 days, during which sensitive plants exhibited pronounced water stress symptoms, including severe turgor loss, wilting and extensive leaf discoloration. Following this period, the stressed plants were rehydrated to field capacity and allowed a 30-day recovery phase, which provided sufficient time to clearly distinguish different recovery responses among the clones (Fig. 1). The greenhouse temperature was kept at 28 \u0026deg;C during the day and 20 \u0026deg;C at night, with RH ranging from 40% to 60% and a light/dark photoperiod of 16/8 hours, providing a flux density of 500\u0026ndash;650 \u0026micro;mol m\u003csup\u003e\u0026minus;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;1\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSampling and data collection were conducted in three phases: 15 days after onset of water withholding (mild stress), when the first visible signs of dehydration appeared in the sensitive plants; 30 days after withholding irrigation (severe stress); and at the end of the recovery period (Fig. 1). Water potential measurements were conducted prior to harvesting the plants. A part of the harvested plant material was stored at \u0026minus;80 \u0026deg;C, while the remainder was stored in dried form (72 h at 70 \u0026deg;C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment was structured as factorial within a randomized complete block design (RCBD) framework, featuring two factors: irrigation (control and no irrigation) and clonal hybrids, with three biological replicates. This resulted in a design comprising 7 clonal hybrids \u0026times; 3 replicates \u0026times; 2 irrigation treatments.\u003c/p\u003e\n\u003cp\u003eAnalysis of variance (ANOVA) was conducted with R software (R 4.3.2), and means were compared using Duncan\u0026rsquo;s Multiple Range Test (\u003cem\u003eP\u0026lt;0.05\u003c/em\u003e). The normality of each trait was tested using the Shapiro-Wilk approach. Correlation network plot based on Pearson coefficients was constructed using ggraph package under R program. Principal component analysis (PCA) was conducted via factoextra package in R software. Heatmap cluster analysis (HCA) was depicted using the pheatmap package in the same software [22].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eGrwoth\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eparameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. Shoot and root dry weight\u003c/strong\u003e\u003cstrong\u003e, root to shoot ratio\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the 30-day water withholding and subsequent rehydration, the plants were removed from the soil and separated into shoots and roots. They were then oven-dried at 70 \u0026deg;C for 72 h to determine their dry weight. Dry weights were measured using a digital scale with a precision of \u0026plusmn; 0.1 mg. Root to shoot ratio was calculated by measuring the dry root and shoot weight [2].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. Growth and recovery rate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth rate (GR) of the aerial biomass is determined by comparing the dry weight of the plant\u0026apos;s aerial parts at two intervals: before the onset of drought stress (day 0) and at the end of the drought stress period (day 30). The recovery rate (RR) is determined by comparing the aer s dry weight on the 30th day of the recovery period with the dry weight measured on the first day of the recovery period, following the drought stress phase [23].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAYoAAAA5CAYAAAAoV2x/AAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAAFiUAABYlAUlSJPAAAArYSURBVHhe7d0xa9vOGwfwr//vIGQrpRTLS4dSKCFDSTtk6IXOgaQZiofSoLyApoSOpSC/gCaEDp6iFLwG6gwuxCJDmyHZLRNCyWToS7jf8Ncdp/NJdiIncezvBwTxSWcrsp57JN1ZKkkpJYiIiDL8zy4gIiIyMVEQEVEuJgoiIsrFREFERLmYKIiIKBcTBRER5WKiICKiXEwURESUi4mCiIhyMVEQEVGuqUoUpVLJLpo63AZ0X1UqFXS7XbuYrqjb7aJSqdjFuaYiUURRhFKphDiOsb+/j1KppCe1wcyyUqmE/f19Xd9VXqvVUuUu5mdtbGzYs/ssLS2l3rNWq/Wtr/lZ9jyl2+3qMnuHiOMYpVIJURSlyonGldqf6/U6Li8vnfFQqVRSZbVaTdc356lyO3ZcCUi1G6VSCUtLS/bsGzOqdiBr/cvlMur1eub/7SSnAAAZx3GqzPM8KYRIlfm+71xWSimFELLdbksppQyCQAZBoOe53isMQ2luXiGE9H0/tYxLu92WAGQYhqlyAH2fIaWUcRxLz/NSrwGk1tWcby7j+j+Jxo3neXp/VoQQfft1EASpfd/k+76OqTAMU7Hoei8VhypGfN93xt9NKdoODLP+aplhDLfUPeb7fqpRV+yGXBoNqL18u91ObWS7gQ3DsG9H8zwv9SXbX1weV1JRQWDXNwNAvbbruna4IAj6dhyicRMEQd/+LHPiCUDf8nYjatdxNZhCiFQ7YB+A3YYi7cCw6+9qL1wmOlHkHTmreXYDCqCv0Q+CoG85kz0/aye2k0cWtTOYVGKzk5i9rq7/ybUz5G0bonHhatyUrHhwxY69nMmen9Wo2o3vTbtuO3CV9XclSZeJ7qP4/fs3kFyTs5XLZQghUK/XdVkURfA8D3Ecp67h7+7uYnV1Vb82qWue5vy/f/8CGZ97dHRkF/VZXl4GkmuPytHRETzPw+7uri6LogivX7/Wr9X1xocPH+oy5fDwMPW6XC7D8zw0Go1UOdG4UDH44MEDexYAwPf9VDyY19vN2KnX6zqmbPv7+zg6OsLHjx912eXlJZDxua1Wyy66MddtB66y/gsLC4D1GS4TnSjURs1SrVbRbDb1Dra3t6cTx/HxMZDsfOaXoKgOts3NTWxubg7VWT0sVxLrdrv48OED4jjW63t8fIy1tTWj5tVUKhWcn5/bxURjQcWg64ALANbW1lIHdY1GQ8eMfUDmeo9SqYS3b99ie3v7Vjurh3Vb7YDnebi4uLCLUyY6UQwyPz8PJDuYsrCwACGEztiNRsP5JZTLZSSX7gAA29vbIx1JtLi4qJNYFEWoVqv6CEOtb6vV0kcERNNG7ft7e3sAgPPzcywsLMD3fWxvbwPJkXK1Wk3VU1T8ep6HZrM58Kj6LoxLOzDViUJl7N3dXURRhMePHwPJl6My9jBfQhzHqdfq0o899CyOY7x69QoYMBwXxmlno9HA3t4e5ufn9eWiVquFKIqwuLiYqqOOmtSlLyXrrIjovlNJwYw1FWNRFKFer2deNlY6nU7qtbpkoy7hKJ1OR8dcrVbD0tKSHsoaRZG+ymAPU4U15DVr6L09D9dsB4ZZ/yuzOy0miWtkk00tI4TQHbuqM8jVCZzF7hi2O66zOrjzqGF75jqoDi7f9/s6q2RGxzUcHdwyWUe7c4toXAwTM2oZYQxfl8bop2FH9tlDcO2OX7ODWLUZdoex2aFsxqFvjLwMHMPVB7lOO5C3/ras9sGU24qGYShXVlb0SAIkI4J835cHBwey1+ulVkZ9aXmT+gd6vV7qs25C3sZR1DL2lyeEGFhXcTXOdpKyv7hhqJ3B9YXb66vY/3OQMQyWo54mT9H4K1r/JmCYRixZN5Of/CZqUF2ZESN2krJj3K7j2nYqRs33uY7rtAOD1t9ebhDnEnEc64bSzlhxHMswDOXMzIwEILe2tlJ1pfGPwWpoDw4OdNKZm5u7lZ3NzOZZXI14kJP5zcSJnJ3R3A72+w8jqzEfdCZg7rR2ACj2jk6To2j8Fa0/SkHG7yhMrkbQPlAzqbZtUGyaZw72+9vxEzp+SyWNGC7iuu1A3vorrm3n0vcfnJ6e6iSQdzTd6/Xk3NxcZmPj2tGk8U/nrfyouTbytOM2mWxF469o/VHyHL/Mvmt2osg7Mndtx3FwlSTW15n9/v17/Pv3Dzs7O7mduLOzs/j+/btdPFC5XMbKygqQjBS6DXEcw/O8kY5Kuq9Uh1u73XYOGaTJVjT+ita/jk6ng5cvX/Z19I4T1Vaaw+TV30IIfPnyRZfXarW+gS63zfzN2DBSiaJWq+Hk5ASe52F9fd2c5fTs2bNr9aI/f/7cLrpRaihr1jC5aeJ5HqSUuQcBNNmKxl/R+tchpUS9Xr/zBhbJkNvNzU00m83U7y/iOMb29rYewaRGX/38+ROdTkeXn5+f3+lBWrfbRbVahZRy+PUwTy/U9UdXv8NVZZ26Sinl3NycBCBnZmbsWUQ0AkXjr2h9miz6jOLs7Eyfhjx9+tTMJSMVRRFOTk4AAJ8+feqbZ48rHjTxchLR8PLibxh59Rm/E0xljKyxwTZzRIQ52Z3a9nudnp6m6t5WRxjRNCoaf0Xr02TRiSJrSJxLr9eTKysrevmDgwN7ET3PnuzhtjfJ/mxO2RNNFvv7VdOw8WfXu2r9UbA/m9PNTnn0pacXL17oswx1M64ss7OzePfunX795s2b1HxTu92GlBJCCADAnz9/8OTJE3sxgKeuRCN3lfhzuUp9xu8EUxmj1+vpzLKyspJOJw5q3HBWJlLz1NFHr9fTv8/gaSvRzSoaf0Xr02TRZxSzs7N6fPSPHz9GPgxtdnYWv379ApLx1+YzbYnoZhWNv6L16X5L/Y7i69evmJmZAQBsbW2Zs0bi2bNn2NnZAQBsbm6O9Q9oiCZN0fgrWp/ur1SiKJfL+PbtG5CcVYzyYTzK+vq6PnPZ2NjA2dmZvQgR3ZCi8Ve0Pt1T9rUomfQ/qB/fCSFkGIb6BmC9Xk8eHBzoG2u5boRljqBy/Xiv1+ulbqwXBIE8PT21FyOiaygaf0Xr0+RxJgrFdZtxJHeO9JNbjZvMDm57su9yaN58MGsZIhpe0fjzk1tzu6Zh6tvL0OQoyf+PcCAiInLqu3ssXY1rXPjGxkaqzLxxmDnPLK/VarqcI0qIxgdjHO4+CroadU3XfL6DugzgOh0PwzA1Fj2wHpI06IEkRHS7pj3GmShGBI4n3YnkWbc2z/NSOxys26bkPZ2LiO7GNMc4Lz2NiBAC9Xo9VVYulxHHceo2Bd1uF5VKRd8HXo1FN58Psbq6CiS3RCCi8TDNMc5EMSLVahXNZjP1i3b1997eni5rNBqpByhdXFzA8zz92jTonltEdHumOcaZKEZEHSE0Gg0gOVJYXFyEEAKHh4d6uVarpZclovtjmmOciWKEhBDY3d0FkiOF5eVlVKtVfWra7XaHf/QgEY2daY1xJooRMneYVquFcrmM+fl5INmpGo0G1tbWUnUePXrU94BzdTpr3vqdiO7e1Ma43btNxSC57Yk59E2NjLCfAqjc9xERRNNkGmOcZxQjJoRAs9lMHSmoo5CsU9IgCFKdX58/f0YYhqlliGg8TGWM25mDignDsG9cdRzHfUcUNvM+O/ZYbSIaH9MY47zXExER5eKlJyIiysVEQUREuZgoiIgoFxMFERHlYqIgIqJcTBRERJSLiYKIiHL9B4oOtUpga3MZAAAAAElFTkSuQmCC\" width=\"394\" height=\"57\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. Leaf area\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter water withholding and rehydration periods, all the leaves of each plant were arranged on a sheet of white paper alongside a scale. Photographs were taken using a digital camera. Subsequently, the images were analyzed using with Digimizer v.6.4.3\u003csup\u003e\u0026reg;\u003c/sup\u003e software to measure the leaf area [24].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2. Water relations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. Relative water content (RWC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRWC was determined from gravimetric measurements using the formula (FW\u0026minus;DW) / (TW\u0026minus; DW) \u0026times; 100, where FW represents the fresh weight of the leaf, DW is the dry weight obtained by oven-drying the leaves at 80 \u0026deg;C for 24 hours, and TW is the turgid weight measured after re-hydrating the leaves at 4 \u0026deg;C [25].\u0026nbsp;RWC was assessed during three time periods: mild drought, severe drought and recovery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. Leaf water potential (\u0026Psi;\u003csub\u003ew\u003c/sub\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWater relations were evaluated by measuring leaf water potential (LWP) using a Pressure Chamber (Santa Barbara, Ca., USA, Made in Italy). Measurements were taken between 9:00-11:00 A.M. on three mature leaves from the mid-shoot of each plant. [26]. Assessments were conducted at three stages: 15 days after water withholding (mild stress, slight leaf wilting in sensitive plants), 30 days after water withholding (severe stress), and after a recovery period\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. Leaf and root osmotic potential (\u0026Psi;\u003csub\u003eS\u003c/sub\u003e)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeaf and root samples were collected at the end of drought and rehydration periods, cut into small pieces, and frozen in liquid nitrogen. Samples were thawed for 30 minutes, centrifuged at 15,000g (4\u0026deg;C) for 15 minutes, and the tissue sap was analyzed for osmotic potential (\u0026Psi;\u003csub\u003eS\u003c/sub\u003e). Osmolarity (C) was measured using an osmometer and converted from milliosmoles per kilogram (m osmoles/kg) to megapascals (MPa) using the appropriate formula [25]:\u003c/p\u003e\n\u003cp\u003e\u0026Psi;\u003csub\u003eS\u003c/sub\u003e (MPa)= -C (m osmoles kg\u003csup\u003e-1\u003c/sup\u003e)\u0026times;2.58\u0026times;10\u003csup\u003e-3\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.3.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eOsmoregulators asseys\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. Glycine betaine concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDried leaf and root samples (0.5 g) were shaken with 25 mL distilled water for 48 hours at 25 \u0026deg;C. Afterward, 1 mL of extract was mixed with 2 N sulfuric acid, and 0.5 mL of the mixture was cooled in a water bath for 1 hour. Potassium iodide solution (0.2 mL) was added, and the mixture was stored at 4 \u0026deg;C for 14 hours. Samples were centrifuged at 10,000 rpm for 15 min, and granules were dissolved in 9 mL dichloromethane, shaken for 2 hours, and analyzed via UV-Visible spectrophotometry (HITACHI U-1900, Japan) at 365 nm. The concentration of glycine betaine was determined using a standard curve and reported as \u0026micro;mol g\u003csup\u003e\u0026minus;1\u003c/sup\u003e dry weight [27].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. Proline concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRoots and leaves from control, drought-stressed and recovered pistachio saplings were collected. Acid-ninhydrin reagent was prepared by dissolving 1.25 g ninhydrin in a mix of 30 mL glacial acetic acid and 20 mL 6 M phosphoric acid, stored at 4 \u0026deg;C for up to 24 hours. Plant material (0.5 g) was homogenized in 10 mL 3% sulfosalicylic acid, filtered, and 2 mL of the filtrate was mixed with 2 mL acid-ninhydrin solution and 2 mL glacial acetic acid. The mixture was heated at 100 \u0026deg;C for 1 hour, cooled in an ice bath, and extracted with 4 mL toluene. The toluene phase containing the chromophore was separated and left to reach room temperature.\u0026nbsp;Absorbance at 520 nm was measured using toluene as a blank. Proline concentration was determined from a standard curve and calculated based on the fresh weight of the sample [28].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. Total soluble carbohydrate content (TSC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTSC assay was conducted using 95% ethanol extracts from root and leaf tissues. Fresh samples (0.5 g) were ground in 5 mL of 95% ethanol, washed twice with 5 mL of 70% ethanol, and centrifuged at 3,500 g for 10 minutes. Supernatants were stored at 4 \u0026deg;C. For TSC measurement, 0.1 mL of extract was mixed with 3 mL anthrone solution (150 mg anthrone in 100 mL 72% H₂SO₄), heated in a boiling water bath for 10 minutes, cooled, and absorbance was measured at 625 nm [29].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eStomatal\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003easseys\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. Morphological characteristics of stomata\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStomata morphological parameters (stomatal density, stomatal length, stomatal width, pore aperture, pore length)\u0026nbsp;was assessed on the 15th day after the applying drought stress (mild stress), 30 days after the application of drought stress (severe stress) and after the recovery phase in clonal hybrids. This was done using the nail polish impression method on the lower epidermal layer of the second leaf closest to the bud apex and observed using a Nicon digital camera (DXM-1200) attached to a microscope. Per treatment, in total 90 stomata were used for studying stomata morphological parameters. Images were analyzed by using the public domain image processing program ImageJ (ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,\u0026nbsp;http://imagej.nih.gov/ij/)\u0026nbsp;[30].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.5. Leaf temperature\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeaf temperature was measured using an infrared thermometer (Benetech GM550E). Three separate readings were taken per plant to ensure accuracy, and arithmetic means of these measurements used for all subsequent analyses. Measurements were conducted during the late morning to early afternoon (10 AM to 12 PM), a period when gas exchange is in stable form [31, 32]. Efforts were directed towards maintaining stable environmental conditions during the measurement.\u0026nbsp;\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Growth responses to withholding irrigation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. Shoot dry weight (SDW) and Root dry weight (RDW)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrowth parameters (SDW and RDW) were assessed during water withholding and recovery. Significant differences in the interaction effect of irrigation treatments and clonal hybrid were observed (\u003cem\u003eP\u0026le;0.01\u003c/em\u003e) during water withholding. Under control conditions, C1 and C4-2\u0026nbsp;had\u0026nbsp;the highest SDW. Water\u0026nbsp;stress\u0026nbsp;reduced SDW in all clones, with the lowest values in C4-2 and C8-3. C1\u0026nbsp;showed minimal reduction (Fig.\u0026nbsp;2A).\u003c/p\u003e\n\u003cp\u003eDrought stress\u0026nbsp;responses in RDW varied. UCB1 and C2 increased RDW by 96.3% and 50%, respectively. Other clones showed a decrease in RDW, with C16-1 having the\u0026nbsp;highest RDW under\u0026nbsp;\u0026nbsp;control and\u0026nbsp;C1\u0026nbsp;the lowest during drought (Fig.\u0026nbsp;2B).\u003c/p\u003e\n\u003cp\u003e30 days after recovery, SDW and RDW were significantly influenced by treatment interactions (\u003cem\u003eP\u0026le;0.01\u003c/em\u003e). C4-2 and C1 exhibited strong regrowth, while C8-3 showed the lowest SDW, indicating limited recovery (Fig. 2C). RDW showed no significant differences between rehydration and control treatments for most clones, except UCB1, C9-4 and C16-1, which had higher RDW in control (Fig. 2D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRoot to shoot ratio, growth and recovery rate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe root-to-shoot ratio, growth rate, and recovery rate were significantly influenced by the interaction between irrigation and clonal hybrids (\u003cem\u003eP\u0026le;0.01\u003c/em\u003e). Under stress conditions, the root-to-shoot ratio increased in the C2, C8-3, UCB1 and C4-2 clones compared to the control group. However, for the other clones, no significant differences were observed between the stress treatment and the control. Following the recovery period, the root-to-shoot ratio showed no significant differences between the stressed clones and their respective controls across all clones (Fig. 3A,B).\u003c/p\u003e\n\u003cp\u003eOur findings revealed that the C1 and C4-2 clones exhibited the highest growth rates under control conditions. However, under water stress, growth was nearly completely suppressed across all clones, with no significant differences observed among them in this condition (Fig. 3C).\u003c/p\u003e\n\u003cp\u003eThe C4-2, identified as sensitive cline in this study, exhibited the highest recovery rate, surpassing even the resistant clones, C9-4 and C1. Although both C4-2 and C8-3 were categorized as sensitive, C4-2 demonstrated rapid recovery, while C8-3 had the lowest recovery rate. The UCB1, C2 and C9-4 showed no significant differences in recovery rate, with their performance being statistically similar (Fig. 3D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eLeaf area\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the water withholding period, leaf area decreased significantly (\u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026lt;0.01\u003c/em\u003e) compared to\u0026nbsp;controls.\u0026nbsp;In the irrigated group,\u0026nbsp;the C1 and C2 displayed larger leaf areas compared to the other clones. Under water stress, C1\u0026nbsp;and C2\u0026nbsp;experienced only\u0026nbsp;slight reductions\u0026nbsp;in leaf area, whereas C8-3 and C4-2 were the most\u0026nbsp;severely impacted (Fig. 4A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt the end of the rehydration period, significant differences in leaf area responses were observed (\u003cem\u003eP\u0026le;0.01\u003c/em\u003e). C1 and C9-4 had larger leaf areas in the rehydration treatment compared to controls, while C8-3 and C4-2 had the smallest leaf areas under recovery (Fig. 4B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Water relations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. Relative water content (RWC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRWC was significantly influenced by the interaction effect between irrigation and clonal hybrids (\u003cem\u003eP\u0026le;0.01\u003c/em\u003e) across all stages: mild stress, severe stress, and recovery. Under mild stress, no significant differences were found in RWC for C9-4, C16-1, and C1 between drought and well-watered treatments, but RWC in C2, C4-2, C8-3 and UCB1 significantly decreased. RWC was 79.5% for UCB1 and 70% for C8-3, with C9-4 showing the highest RWC and C8-3 the lowest (Fig. 5A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter 30 days of\u0026nbsp;water\u0026nbsp;withholding (severe stress), RWC decreased significantly in all clones compared to the controls. C8-3 showed the greatest decrease (88%), followed by C4-2 (76.5%) and C16-1 (59.3%). C9-4 and C1 had minimal changes (6% and 13.48%) (Fig. 5B).\u0026nbsp;These results indicate that C8-3\u0026nbsp;was most\u0026nbsp;affected by drought, while\u0026nbsp;C9-4 and C1 were more resilient.\u003c/p\u003e\n\u003cp\u003eIn the recovery stage, RWC did not differ significantly between control and recovery conditions for C1, C16-1, C2, and C9-4, suggesting their strong ability to recover. C8-3 had the lowest recovery RWC (50.5%), and UCB1 showed a 20% reduction in RWC compared to its control, indicating a significant decrease in recovery relative to other samples (Fig. 5C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. Leaf water potential\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificant differences in leaf water potential (LWP) were observed between hybrids and irrigation treatments (\u003cem\u003eP\u0026le;0.01\u003c/em\u003e) across all stages: mild stress, severe stress and recovery. Fifteen days after mild drought stress, LWP decreased, with most clones showing no significant changes, except for C8-3, which had a notably lower LWP. Under drought, LWP ranged from -0.43 MPa (C2) to -1.73 MPa (C8-3). (Fig. 6A).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;End of the drought stress period, LWP significantly decreased in C8-3, C4-2, C16-1, and UCB1. The lowest LWP was recorded in C8-3 (-4.43 MPa), followed by C4-2 (-3.19 MPa), C16-1 (-2.95 MPa) and UCB1 (-1.5 MPa) (Fig. 6B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt the end of the rehydration period, C4-2, C9-4 and UCB1 showed no significant differences with controls, indicating a quick return to stable conditions. The highest LWP was observed in C1 (-0.15 MPa), while the lowest was in C8-3 (-1.26 MPa) (Fig. 6C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. Leaf and root osmotic potential\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeaf and root samples were collected at the end of drought and rehydration periods. Significant differences in leaf osmotic potential (LOP) and root osmotic potential (ROP) were observed due to the interaction of clonal hybrids and irrigation (\u003cem\u003eP\u0026le;0.01\u003c/em\u003e). After 30-day drought\u0026nbsp;stress, LOP\u0026nbsp;significantly\u0026nbsp;decreased, with the most notable\u0026nbsp;decline\u0026nbsp;in\u0026nbsp;clone\u0026nbsp;C9-4 (-1.61 MPa), indicating higher osmolyte accumulation under stress\u0026nbsp;(Fig. 7A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn\u0026nbsp;roots, ROP significantly decreased in C1 under drought stress, with the lowest ROP recorded in C1 (-0.61 MPa). No significant effect on ROP was found in other clones. Overall, osmolyte accumulation in C1 roots increased significantly under drought stress (Fig. 7B).\u003c/p\u003e\n\u003cp\u003eThere were no significant differences in LOP between recovery and control treatments for any clone. The lowest LOP was observed in UCB1 (-1.12 MPa), which did not differ significantly from C16-1 and C2 under stress (Fig. 7C). For ROP, only UCB1, with the lowest root osmotic potential (-0.5 MPa), showed a significant difference compared to C16-1 under recovery. No significant differences were found in other treatments (Fig. 7D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Osmoregulators\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. Glycine betaine concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eleaf and root glycine betaine content was significantly influenced by the interaction effect of irrigation and clonal hybrids (\u003cem\u003eP\u0026le;0.01\u003c/em\u003e) under the drought \u0026nbsp;stress and recovery.\u0026nbsp;Under stress, glycine betaine levels increased in all clones compared to the control, but the increase was significant only in C1, C9-4 and C2. In the commercial UCB1 clone, there was no increase in glycine betaine levels. Glycine betaine increased by 49.4% in C9-4, 47% in C1 and 22.5% in C2 (Fig. 8A).\u003c/p\u003e\n\u003cp\u003eA similar trend was found in the roots, with higher accumulation in the leaves than in the roots. The highest glycine betaine content was 236 \u0026micro;mol\u0026middot;g⁻\u0026sup1; DW in the leaves and 188 \u0026micro;mol\u0026middot;g⁻\u0026sup1; DW in the roots of C9-4 under drought stress (Fig. 8B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDuring recovery, glycine betaine levels decreased in both leaves and roots compared to the stress phase. C9-4, C1 and C2 clones maintained the highest glycine betaine levels in both organs (Fig. 8C,D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. Proline concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProline concentration in both leaves and roots increased in all clones under water withholding, except for the UCB1 clone (Fig. 9A,B). The highest proline content was found in C9-4 and C1, which were the most drought-tolerant rootstocks in this study. The highest proline concentrations were 83 \u0026micro;mol\u0026middot;g⁻\u0026sup1; FW in leaves and 60 \u0026micro;mol\u0026middot;g⁻\u0026sup1; FW in roots. In C9-4 clones, proline increased by 29.5% in leaves and 41.5% in roots compared to their controls under drought stress.\u003c/p\u003e\n\u003cp\u003eAt the end of rehydration period, significant differences in leaf and root proline, were observed among the hybrids (\u003cem\u003eP\u0026le;0.01\u003c/em\u003e), but no significant for irrigation treatments and their interaction. The highest proline levels were found in UCB1 clones, with 73 \u0026micro;mol\u0026middot;g⁻\u0026sup1; FW in leaves and 66 \u0026micro;mol\u0026middot;g⁻\u0026sup1; FW in roots, significantly different from the other clones. No significant differences in proline levels were observed in the leaves and roots of the other clones (Fig. 10A,B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. Total soluble carbohydrate content (TSC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder drought stress, all clones showed an increase in TSC in both leaf and root tissues. The increase was significant in C1, C2 and C9-4 compared to controls (Fig. 11A,B). C9-4 and C1 had the highest TSC levels, with increases of 97.3% in leaves and 129.3% in roots. After recovery, TSC levels decreased in both leaves and roots, but C9-4, C2 and C1 still had higher TSC than their controls (Fig. 11C,D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Stomatal traits and leaf temperature\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStomata morphological parameters (stomatal density, stomatal length, stomatal width, pore aperture, pore length) was assessed in three stage (mild stress, severe stress and revovery stage) (Fig. 12). Effect of clonal hybrid, irrigation and their interaction for stomata parameters and leaf temperature was not significant at any of the stages (Supplementary table 1, 2 and 3). The results of this study indicated that stomatal regulation does not play a role in the development of drought stress resistance in our clones; it is likely that other mechanisms are involved in conferring resistance in the resistant clones.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHeatmap and cluster analyses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study used cluster heat map analysis to classify pistachio clonal hybrids based on their response to water stress, evaluating traits like water status, stomatal characteristics, osmotic adjustment and growth. Seven hybrids were categorized into three groups: tolerant (I), moderately tolerant (II) and sensitive (III). The tolerant group (C9-4, C1 and C2) showed high levels of RWC, LGb, rGb, proline, and other stress-related traits, alongside low LOP and ROP indicating increased solutes for stress tolerance. In contrast, the sensitive group exhibited reduced growth traits such as leaf area, SDW, RDW and LWP, with minimal reduction in the tolerant group. Stomatal parameters and leaf temperature were largely unaffected across groups (Fig. 13).\u003c/p\u003e\n\u003cp\u003eUnder re-watering condition, pistachio hybrid were classified into the \u0026ldquo;strong (I)\u0026rdquo;, \u0026ldquo;weak (II)\u0026rdquo; and \u0026ldquo;moderate (III)\u0026rdquo; recovery groups including (C1), (C8-3), (UCB1, C2, C16-1, C9-4 and C4-2) respectively. Groups I and III \u0026nbsp;showed rapid recovery in LWP and ROP, with quick growth recovery in C1 and C4-2. Group II showed minimal recovery, with most hybrids recovering well except those in the weak group (Fig. 14,15).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCorrelation and principal component analyses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificant correlations were found between the drought stress index (DSI) and various physiological and growth traits under water stress (Fig. 16). LWP showed a negative correlation with DSI of ROP (r =\u0026nbsp;-0.47\u003csup\u003e*\u003c/sup\u003e), and positive correlations with DSI of SDW, RWC, LTSC, rTSC, rGb, and LGb (r = 0.45\u003csup\u003e*\u003c/sup\u003e, r = 0.906\u003csup\u003e***\u003c/sup\u003e, r = 0.75\u003csup\u003e***\u003c/sup\u003e, r = 0.64\u003csup\u003e**\u003c/sup\u003e, r = 0.5\u003csup\u003e**\u003c/sup\u003e, r = 0.64\u003csup\u003e*\u003c/sup\u003e, respectively). RWC was positively correlated with SDW, LTSC, rTSC, rGb, LGb, Lproline and rproline\u0026nbsp;(r = 0.56\u003csup\u003e**\u003c/sup\u003e, r = 0.86\u003csup\u003e**\u003c/sup\u003e, r = 0.76\u003csup\u003e***\u003c/sup\u003e, r = 0.69\u003csup\u003e***\u003c/sup\u003e, r = 0.79\u003csup\u003e***\u003c/sup\u003e,\u0026nbsp;r = 0.57\u003csup\u003e**\u003c/sup\u003e, r = 0.6\u003csup\u003e**\u003c/sup\u003e, respectively), and negatively correlated with the DSI of ROP (r = -0.55\u003csup\u003e**\u003c/sup\u003e) and LOP (r\u0026nbsp;= -0.57\u003csup\u003e**\u003c/sup\u003e). A strong positive correlation was found between LArea and SDW. LTSC, rTSC, LGb, rGb, rproline, and Lproline all showed significant positive correlations with each other, while negatively correlating with LOP, ROP, and RDW (Fig. 16).\u003c/p\u003e\n\u003cp\u003ePrincipal Component Analysis (PCA) was applied to all physiological and growth traits of the seven pistachio clonal hybrids under water stress to identify key parameters and relationships among traits. PCA using DSI (value of trait under stressed conditions / value under well-watered conditions \u0026times; 100) identified 18 significant principal components (PCs), with the first six components explaining over 88% of the total variation under severe drought stress. The first principal component (PC1), explaining more than 45% of the variations, was negatively correlated with LGb (96%), rGb (91%), LTSC (94%), rTSC (91%), rproline (89%), Lproline (85%), RWC (86%), and LWP (71%), making these traits the most sensitive indicators of drought effects on pistachio hybrids. The second principal component (PC2), explaining over 16% of the variation, was negatively correlated with LArea (74%), RDW (66%) and SDW (63%) (Fig 17).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDrought stress significantly affects pistachio (\u003cem\u003ePistacia vera\u003c/em\u003e L.), reducing its growth, yield, and quality [33].\u0026nbsp;Plants respond to drought through various morphological, physiological and molecular adaptations,\u0026nbsp;which vary among species and genotypes\u0026nbsp;[34].\u0026nbsp;Understanding drought resistance mechanisms is crucial for improving crop resilience, ensuring food security, and promoting sustainable agriculture under water scarcity conditions\u0026nbsp;[35].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we used nine-month saplings of seven clonal interspecific hybrids of \u003cem\u003ePistacia atlantica\u0026nbsp;\u003c/em\u003eDscf.\u003cem\u003e× Pistacia integerrima\u003c/em\u003e Stewart labeled C1, C2, C16-1, C8-3, C4-2, C9-4 and UCB1. These clones were examined to assess their responses to drought stress, focusing on osmotic regulation in their leaves and roots, as well as stomatal indices. The decrease in shoot dry weight (SDW) under drought stress is mainly due to limited water, impaired photosynthesis, stomatal closure and survival-oriented physiological responses, leading to lower shoot biomass. [36–38].\u0026nbsp;Our results showed that under drought stress conditions, SDW decreased in all clones, but this decrease was minimal in C1 and maximal in C8-3.\u0026nbsp;Additionally, the growth rate analysis indicated that under control conditions, C1 and C4-2 had the highest growth rates, which is consistent with the findings of Akbari\u0026nbsp;[18]\u0026nbsp;who reported the superiority of C1 over the commercial clone UCB1 in terms of growth.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;According to Blum [39], drought stress reduces root dry weight (RDW) by limiting water availability and reallocating resources to survival, impairing root growth, and reducing nutrient absorption.\u0026nbsp;In our experiment, different clones showed varying responses to water withholding, with UCB1 and C2 showing increases in RDW by 96.3% and 50%, respectively, compared to controls. In contrast, the other clones experienced a decrease in RDW. The increased root biomass observed in UCB1 under drought conditions reflects a strategy to enhance water access. However, this adaptation likely comes with elevated metabolic costs, potentially delaying shoot recovery\u0026nbsp;[40].\u0026nbsp;However, as noted by Sánchez-Blanco\u0026nbsp;[41], root systems do not consistently respond to drought stress by undergoing changes. For instance, a study on the drought-resistant pistachio variety 'Sarakhs' reported no increase in root biomass under water stress conditions\u0026nbsp;[42]. Similarly, our findings align with these observations, as the most resistant clones (C9-4 and C1) also showed no increase in root biomass.\u003c/p\u003e\n\u003cp\u003eRecent studies suggest that plants need extended periods of stress exposure to adjust their root\u0026nbsp;systems. For example, Vives-Peris reported that changes in root morphology, such as increased depth and biomass, require prolonged stress exposure [43]. Bhargava and Sawant, [44] noted that during short-term drought, plants prioritize physiological changes over morphological ones, using stored resources to maintain metabolic functions. In our experiment, the 30-day water withholding period might not have allowed sufficient time for morphological changes in roots, so\u0026nbsp;saplings responded mainly with physiological adaptations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing the recovery period, SDW increased across all clones, with the sensitive clone C4-2 exhibiting the most vigorous growth and achieving the highest recovery rate. Rapid recovery is typically associated with efficient stress management mechanisms, such as osmotic adjustment, photosynthesis, and stomatal control, while weaker genotypes struggle due to limitations in these traits [45]. Certain drought-sensitive genotypes may exhibit notable growth improvements following rehydration, utilizing recovery mechanisms that enable them to capitalize on the renewed availability of water [46].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, our results showed that C9-4 (the most resistant clone) had higher RDW values in the control state, while the other clones exhibited no significant difference in RDW between the rehydration treatment and their respective controls. According to Flexas and Medrano [38], some resistant genotypes may fail to significantly increase root dry weight after recovery due to physiological and biochemical limitations. Drought stress may cause lasting effects on root architecture and function, preventing full recovery even in typically resistant genotypes.\u003c/p\u003e\n\u003cp\u003eThe increase in the root-to-shoot ratio under stress likely indicates a plant's effort to optimize resource allocation [47], particularly in sensitive clones such as C8-3 and C4-2. However, this adjustment did not align with enhanced resistance, suggesting that other mechanisms, such as osmotic regulation or antioxidant responses, may contribute to stress tolerance. In contrast, resistant clones exhibited no significant changes in this ratio, indicating their reliance on alternative strategies, such as metabolic stability, for enduring stress. Following the recovery phase, no notable differences were observed between recovery and control, likely because vegetative growth resumed quickly, emphasizing the temporary nature of stress-induced changes and the critical role of recovery mechanisms in restoring normal conditions [41].\u003c/p\u003e\n\u003cp\u003eOur results also revealed a negative relationship between drought stress and leaf area, although\u0026nbsp;responses varied among clones. C1 and C2 showed slight reductions in leaf area, while C8-3 and C4-2\u0026nbsp;exhibited the most significant reductions. These findings are consistent with reports by Khoyerdi [2] and Kasmani [48], on pistachio. Reducing leaf area is a typical response to water scarcity, primarily due to reduced cell division [49]. As water potential decreases, cell expansion becomes the most sensitive process affected by water deficits [50]. During drought stress, plants limit leaf expansion to reduce water loss through transpiration, conserving water and supporting survival. This reduction in leaf area helps balance metabolic needs with available water, enabling more efficient resource allocation [51–53].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter the recovery period, only the C1 and C9-4 clones exhibited a greater leaf area during the rehydration period compared to their controls. Some clones may increase leaf area after recovery due to better growth and photosynthesis, while others maintain reduced leaf area due to impaired recovery\u0026nbsp;mechanisms following drought stress [54, 55]. Rapid regrowth during recovery highlights an adaptive trade-off, where the short-term cost of osmotic adjustment is offset by long-term benefits in growth and yield. This underscores the importance of selecting efficient osmotic adjustment mechanisms in breeding programs to enhance drought resilience [39, 56, 57].\u003c/p\u003e\n\u003cp\u003eTo evaluate plant water status, assessing leaf water potential and relative water content (RWC) is essential for understanding physiological responses to drought stress [2]. Literature suggests that RWC is higher in drought-resistant plants, indicating a strong correlation with the amount of water absorbed or retained by the leaves [58].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our study, under\u0026nbsp;mild\u0026nbsp;drought stress, no significant differences in RWC were observed between drought-stressed and well-watered treatments for clones C9-4, C16-1 and C1. However, for clones C2, C4-2, C8-3 and UCB1, RWC significantly declined compared to their controls. After 30 days of withheld irrigation (severe stress), RWC significantly decreased for all clones compared to controls. This reduction was less severe in resistant clones, with C9-4 and C1 showing decreases of 6% and 13%, respectively. In contrast, the sensitive clones C8-3 and C4-2 experienced much larger reductions of 88% and 76%, respectively.\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that, similar to drought stress, salinity stress also reduces RWC in pistachio rootstocks, with a more significant decrease observed in sensitive varieties [59]. Our findings align with studies by Liu\u0026nbsp;[60] and Fathi [61], which reported that while mild stress only slightly affects RWC, severe drought stress leads to a significant reduction in RWC across all genotypes.\u003c/p\u003e\n\u003cp\u003eWater potential is widely recognized as a key indicator of water status and essential for irrigation scheduling in fruit and nut trees [62]. A decline in leaf water potential (LWP) typically signals increased water stress, which can negatively impact photosynthesis, growth, and overall plant health. Moderate and severe water stress have distinct effects on LWP and plant physiology\u0026nbsp;[63].\u0026nbsp;Early signs of moderate stress can be detected through changes in root osmotic potential, increased root turgor pressure, or a difference between root and leaf turgor pressure\u0026nbsp;[64].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In our experiment, LWP decreased under drought stress, with the reduction being more pronounced under severe stress. The C8-3 and C4-2 clones were more affected, showing a significant decline in LWP. In contrast, resistant clones exhibited a smaller decline in water potential overall. At the end of the rehydration period, LWP increased in all clones, approaching control levels. However, C8-3 still had the lowest value, indicating its limited ability to resume growth after recovery. According to \u0026nbsp;Pita\u0026nbsp; [65] ensitive clones typically experience a greater decline in LWP than resistant ones under drought stress. This is due to their inability to maintain turgor pressure and regulate water loss effectively, resulting in more severe physiological stress that impacts growth and development [66, 67].\u003c/p\u003e\n\u003cp\u003eOsmotic adjustment is a net increase in solute content per cell that is independent of the volume changes that result from loss of water. It’s a crucial mechanism that helps plants adapt to drought stress by reducing their osmotic potential [68]. This process primarily involves the accumulation of compatible solutes. The increased accumulation of these solutes lowers osmotic potential, improving the plant's tolerance to drought stress [69]. In our experiments, under drought stress conditions, the C9-4 and C2 clones accumulated more osmolytes in their leaves compared to other clones. Additionally, osmolyte accumulation in the roots of the C1 clone significantly increased with water withholding. However, in other clones, drought stress did not significantly affect root osmotic potential (ROP).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;After rehydration, there were no significant differences in leaf osmotic potential (LOP) or ROP between the recovery and control plants for any of the clones. Some studies have also found no significant differences in leaf and root osmotic potential between recovered and control plants [70]. These findings suggest that, while osmotic adjustment is an important drought response mechanism, its effects may not persist after rehydration in certain cases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProline and glycine betaine are key organic osmolytes that help plants tolerate drought by maintaining water potential and protecting cellular structures [71]. Total soluble carbohydrates (TSC) also play a vital role in drought tolerance, with drought-tolerant genotypes accumulating higher levels of TSC than sensitive ones [72, 73]. Our research demonstrated that, under stress, levels of glycine betaine, proline, and TSC increased in all clones, with resistant clones showing a significantly greater increase. Glycine betaine accumulation was three times higher than proline, and this trend was similar in the roots, though with higher accumulation in the leaves.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;After recovery, levels of compatible solutes (glycine betaine, proline, TSC) decreased in both leaves and roots compared to the drought stress stage. However, C9-4, C2, and C1 still had higher levels of these solutes compared to their control plants. Naser reported that proline, glycine betaine, and TSC accumulation is particularly notable in response to drought [74]. Our findings align with those of Behzadi Rad, who showed that high levels of proline and TSC in \u003cem\u003ePistacia vera\u003c/em\u003e L. ‘Ghazvini’ rootstock may explain its higher tolerance to salinity compared to other rootstocks [59]. Our results are consistent with previous research on pistachio [75, 76].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDrought stress significantly impacts stomatal morphology in many crops. Studies show that water deficit increases stomatal density while reducing their size (length and width) [77, 78]. These changes negatively affect photosynthesis and transpiration but improve water use efficiency [77]. Under water stress, stomatal apertures tend to close. Interestingly, stomatal density and size vary across growth stages, with later stages showing higher density [78]. Drought-tolerant varieties typically exhibit lower stomatal density and smaller stomata compared to susceptible ones [77].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur findings indicate that stomatal parameters (density, length, width, pore aperture and pore length) and leaf temperature were not significantly affected by drought stress or recovery treatments. This suggests that stomatal regulation may not play a key role in dehydration tolerance of pistachio in our clones, and other mechanisms could be at play. Previous research on isohydric and anisohydric plants indicates that pistachios are likely categorized as anisohydric. These plants do not close their stomata during water stress; instead, they utilize strategies like enhanced photosynthesis and osmotic adjustment to improve their stress tolerance [79]. Studies have demonstrated that pistachios and almonds maintain high photosynthetic rates under drought conditions [80], supporting the notion that enhanced photosynthesis and osmotic adjustments are critical for drought tolerance in these species [80–82]. Similarly, our findings suggest that pistachio's drought tolerance might rely on mechanisms beyond stomatal regulation, potentially involving osmotic adjustments. This indicates that the importance of stomatal regulation in drought resistance may vary between species and could be less significant in species like pistachio [82].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Studies also suggest that while stomatal regulation helps control water loss and gas exchange, it may not be the sole factor in drought resistance. Plants often use a combination of strategies, like osmotic adjustment and root architecture changes, to cope with water stress. [83]. Since leaf temperature in our study remained unaffected, it further confirms that stomatal regulation is not crucial in drought resistance for our clones.\u003c/p\u003e\n\u003cp\u003eThe cluster heat map, PCA and correlation analyses in this study provide valuable insights into the phenotypic responses of pistachio clones under drought stress. These analyses grouped the hybrids into three categories based on drought resistance, highlighting important traits like leaf glycine betaine, RWC, and leaf water potential. These traits are strongly linked to drought sensitivity, suggesting that they should be prioritized in breeding programs. It is also noteworthy that plants typically adopt various strategies to manage drought stress, including escape, avoidance, and tolerance [34]. In this study, the clonal hybrids showed an increase in osmolytes, such as glycine betaine, which clearly indicates a dehydration tolerance strategy [84]. This finding suggests that the hybrids rely on preserving cellular functions and maintaining osmotic balance under water-deficit conditions, rather than employing escape or avoidance mechanisms. The accumulation of osmolytes underscores the tolerance strategy and highlights the critical role of osmotic adjustment in improving drought resistance.\u003c/p\u003e\n\u003cp name=\"removable\"\u003e\u003cstrong\u003e5. Conclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp name=\"removable\"\u003eOur study highlights the complexities of drought stress responses in pistachio clonal hybrids, revealing significant differences in mechanisms underlying drought resistance. While drought stress adversely affected shoot and root dry weights across all clones, the C1 demonstrated superior tolerance with minimal reductions. Notably, the resistant clones, such as C9-4 and C1, exhibited less pronounced declines in relative water content and osmotic potential, suggesting effective osmotic regulation and other adaptive strategies. The accumulation of compatible solutes, particularly glycine betaine and proline, was significantly higher in resistant clones, reinforcing their ability to withstand drought conditions. Furthermore, the lack of significant changes in stomatal parameters indicates that stomatal regulation may not be a primary mechanism for drought tolerance in the pistachio hybrids. Instead, the traits such as osmotic adjustment, likely play a more critical role. These findings emphasize the importance of identifying and utilizing resilient genotypes in agricultural industry to enhance pistachio productivity in the face of increasing water scarcity. These findings also offer valuable insights for breeding drought-resistant cultivars and selecting appropriate rootstocks for drought-prone regions. With the growing adoption of clonal rootstocks due to their benefits in disease resistance, stress tolerance and overall performance, utilizing resistant clonal rootstocks could be an effective strategy for areas facing water scarcity.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur study highlights the complexities of drought stress responses in pistachio clonal hybrids, revealing significant differences in mechanisms underlying drought resistance. While drought stress adversely affected shoot and root dry weights across all clones, the C1 demonstrated superior tolerance with minimal reductions. Notably, the resistant clones, such as C9-4 and C1, exhibited less pronounced declines in relative water content and osmotic potential, suggesting effective osmotic regulation and other adaptive strategies. The accumulation of compatible solutes, particularly glycine betaine and proline, was significantly higher in resistant clones, reinforcing their ability to withstand drought conditions. Furthermore, the lack of significant changes in stomatal parameters indicates that stomatal regulation may not be a primary mechanism for drought tolerance in the pistachio hybrids. Instead, the traits such as osmotic adjustment, likely play a more critical role. These findings emphasize the importance of identifying and utilizing resilient genotypes in agricultural industry to enhance pistachio productivity in the face of increasing water scarcity. These findings also offer valuable insights for breeding drought-resistant cultivars and selecting appropriate rootstocks for drought-prone regions. With the growing adoption of clonal rootstocks due to their benefits in disease resistance, stress tolerance and overall performance, utilizing resistant clonal rootstocks could be an effective strategy for areas facing water scarcity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their gratitude to the University of Tehran and the Iran National Science Foundation (Project No: 4014915) for their valuable support in the completion of this study. We would also like to extend our sincere appreciation to the research Center of Royeshe Sabze Farda (Pistat) which provided us plant materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: M.R.R and S.S; methodology: M.O and M.M.A; software: M.O; validation: M.R.R; K.V; M.M.A; investigation: M.M.R;\u0026nbsp;M.M.A; resources: M.R.R; M.M.A; data curation: M.O; writing original draft preparation: M.O; writing review and editing: M.O; M.R.R; M.M.A;\u0026nbsp;K.V; visualization: M.O; supervision: M.R.R; S.S.; project administration: M.R.R; funding acquisition: M.R.R.\u0026nbsp;and S.S.\u0026nbsp;All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u0026nbsp;The financial support provided by the Iran National Science Foundation (Project No: 4014915) and the grant of the University of Tehran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e\u0026nbsp;The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate: \u003c/strong\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u0026nbsp;Not Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e\u0026nbsp;All data generated or analyzed during this study are included this published paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e1. Cola\u0026ccedil;o AF, Molin JP, Rosell-Polo JR, Escol\u0026agrave; A. Application of light detection and ranging and ultrasonic sensors to high-throughput phenotyping and precision horticulture: Current status and challenges. Hortic Res. 2018;5. https://doi.org/10.1038/s41438-018-0043-0.\u003c/li\u003e\n \u003cli\u003e2. Khoyerdi FF, Shamshiri MH, Estaji A. Changes in some physiological and osmotic parameters of several pistachio genotypes under drought stress. Sci Hortic (Amsterdam). 2016;198:44\u0026ndash;51. https://doi.org/10.1016/j.scienta.2015.11.028.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e3. Spiegel-Roy P, Mazigh D, Evenari M. Response of Pistachio to Low Soil Moisture Conditions1. J Am Soc Hortic Sci. 2022;102:470\u0026ndash;3.\u0026nbsp;https://doi.org/10.21273/jashs.102.4.470.\u003c/li\u003e\n \u003cli\u003e4. Miranda MT, Pires GS, Pereira L, de Lima RF, da Silva SF, Mayer JLS, et al. Rootstocks affect the vulnerability to embolism and pit membrane thickness in Citrus scions. Plant Cell Environ. 2024;47:3063\u0026ndash;75. https://doi.org/10.1111/pce.14924.\u003c/li\u003e\n \u003cli\u003e5. Han Q, Guo Q, Korpelainen H, Niinemets \u0026Uuml;, Li C. Rootstock determines the drought resistance of poplar grafting combinations. Tree Physiol. 2019;39:1855\u0026ndash;66. https://doi.org/10.1093/treephys/tpz102.\u003c/li\u003e\n \u003cli\u003e6. Chen Y, Fei Y, Howell K, Chen D, Clingeleffer P, Zhang P. Rootstocks for Grapevines Now and into the Future: Selection of Rootstocks Based on Drought Tolerance, Soil Nutrient Availability, and Soil pH. Aust J Grape Wine Res. 2024;2024. https://doi.org/10.1155/2024/6704238.\u003c/li\u003e\n \u003cli\u003e7. Liu Q, Wu C, Gao L, Ying L. Coordination between non-structure carbohydrates and hydraulic function under different drought duration, intensity, and post-drought recovery. Environ Exp Bot. 2023;215:105485. https://doi.org/10.1016/j.envexpbot.2023.105485.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e8. Cui M, Lin Y, Zu Y, Efferth T, Li D, Tang Z. Ethylene increases accumulation of compatible solutes and decreases oxidative stress to improve plant tolerance to water stress in Arabidopsis. J Plant Biol. 2015;58:193\u0026ndash;201. https://doi.org/10.1007/s12374-014-0302-z.\u003c/li\u003e\n \u003cli\u003e9. Blum A. Osmotic adjustment is a prime drought stress adaptive engine in support of plant production. Plant Cell Environ. 2017;40:4\u0026ndash;10. https://doi.org/10.1111/pce.12800.\u003c/li\u003e\n \u003cli\u003e10. Guizani A, Askri H, Amenta ML, Defez R, Babay E, Bianco C, et al. Drought responsiveness in six wheat genotypes: identification of stress resistance indicators. Front Plant Sci. 2023;14 September:1\u0026ndash;17. https://doi.org/10.3389/fpls.2023.1232583.\u003c/li\u003e\n \u003cli\u003e11. Condorell GE, Newcomb M, Groli EL, Maccaferri M, Forestan C, Babaeian E, et al. Genome Wide Association Study Uncovers the QTLome for Osmotic Adjustment and Related Drought Adaptive Traits in Durum Wheat. Genes (Basel). 2022;13:1\u0026ndash;19. ttps://doi.org/10.3390/genes13020293.\u003c/li\u003e\n \u003cli\u003e12. Ratzmann G, Zakharova L, Tietjen B. Optimal leaf water status regulation of plants in drylands. Sci Rep. 2019;9:1\u0026ndash;9.\u0026nbsp;https://doi.org/10.1038/s41598-019-40448-2.\u003c/li\u003e\n \u003cli\u003e13. Merga D, Beksisa L. Mechanisms of Drought Tolerance in Coffee (\u0026amp;lt;i\u0026amp;gt;Coffea arabica\u0026amp;lt;/i\u0026amp;gt; L.): Implication for Genetic Improvement Program: Review. Am J Biosci. 2023;11:63\u0026ndash;70. https://doi.org/10.11648/j.ajbio.20231103.12.\u003c/li\u003e\n \u003cli\u003e14. Matkowski H, Daszkowska-Golec A. Update on stomata development and action under abiotic stress. Front Plant Sci. 2023;14 October:1\u0026ndash;14. https://doi.org/10.3389/fpls.2023.1270180.\u003c/li\u003e\n \u003cli\u003e15. Marchin RM, Ossola A, Leishman MR, Ellsworth DS. A Simple Method for Simulating Drought Effects on Plants. Front Plant Sci. 2020;10 January:1\u0026ndash;14. https://doi.org/10.3389/fpls.2019.01715.\u003c/li\u003e\n \u003cli\u003e16. Daszkowska-Golec A, Szarejko I. Open or close the gate - Stomata action under the control of phytohormones in drought stress conditions. Front Plant Sci. 2013;4 MAY:1\u0026ndash;16. https://doi.org/10.3389/fpls.2013.00138.\u003c/li\u003e\n \u003cli\u003e17. Ferguson L, Sanden B, Grattan S, Epstein L, Krueger B. Pistachio Rootstocks. Pist Prod Man. 2005;:67\u0026ndash;73. https://doi.org/10.21273/hortsci.30.4.855g.\u003c/li\u003e\n \u003cli\u003e18. Akbari M, Hokmabadi H, Heydari M, Ghorbani A. \u0026lsquo;Arota\u0026rsquo;: A New Interspecific Hybrid Pistachio Rootstock. HortScience. 2020;55:965\u0026ndash;6.\u0026nbsp;https://doi.org/10.21273/HORTSCI14845-20.\u003c/li\u003e\n \u003cli\u003e19. Hoagland DR, Arnon DI. The water culture method for growing plants without soil. Miscellaneous Publications n\u003csup\u003eo\u003c/sup\u003e 354. Circular of California Agricultural Experimental Station. 1941;347:461.\u0026nbsp;https://doi.org/10.5962/bhl.title.60957.\u003c/li\u003e\n \u003cli\u003e20. Benedicto Ottoni Filho T, Vasconcelos Ottoni M, Batista de Oliveira M, Ronaldo de Macedo J, Reichardt K, Redentor Av Zulamith F. Theophilo Benedicto Ottoni Filho et al. REVISITING FIELD CAPACITY (FC): VARIATION OF DEFINITION OF FC AND ITS ESTIMATION FROM PEDOTRANSFER FUNCTIONS (1). 2014;38:1750\u0026ndash;64. https://doi.org/10.1590/S0100-06832014000600010.\u003c/li\u003e\n \u003cli\u003e21. Dghim F, Abdellaoui R, Boukhris M, Neffati M, Chaieb M. Physiological and biochemical changes in Periploca angustifolia plants under withholding irrigation and rewatering conditions. South African J Bot. 2018;114:241\u0026ndash;9. https://doi.org/10.1016/j.sajb.2017.11.007.\u003c/li\u003e\n \u003cli\u003e22. Arab MM, Askari H, Aliniaeifard S, Mokhtassi-Bidgoli A, Estaji A, Sadat-Hosseini M, et al. Natural variation in photosynthesis and water use efficiency of locally adapted Persian walnut populations under drought stress and recovery. Plant Physiol Biochem. 2023;201 January:107859. https://doi.org/10.1016/j.plaphy.2023.107859.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e23. Abid M, Tian Z, Ata-Ul-Karim ST, Wang F, Liu Y, Zahoor R, et al. Adaptation to and recovery from drought stress at vegetative stages in wheat (Triticum aestivum) cultivars. Funct Plant Biol. 2016;43:1159\u0026ndash;69. https://doi.org/10.1071/FP16150.\u003c/li\u003e\n \u003cli\u003e24. Zhang W. Digital image processing method for estimating leaf length and width tested using kiwifruit leaves (Actinidia chinensis Planch). PLoS One. 2020;15:1\u0026ndash;14. \u0026nbsp;https://doi.org/10.1371/journal.pone.0235499.\u003c/li\u003e\n \u003cli\u003e25. Mart\u0026igrave;nez JP, Lutts S, Schanck A, Bajji M, Kinet JM. Is osmotic adjustment required for water stress resistance in the Mediterranean shrub Atriplex halimus L? J Plant Physiol. 2004;161:1041\u0026ndash;51. https://doi.org/10.1016/j.jplph.2003.12.009.\u003c/li\u003e\n \u003cli\u003e26. da Silva AA, Silva CO, do Rosario Rosa V, Santos MFS, Kuki KN, Dal-Bianco M, et al. Metabolic adjustment and regulation of gene expression are essential for increased resistance to severe water deficit and resilience post-stress in soybean. PeerJ. 2022. https://doi.org/10.7717/peerj.13118. https://doi.org/10.7717/peerj.13118.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e27. Sithtisarn S, Harinasut P, Pornbunlualap S, Cha-Um S, Carillo P, Gibon Y. PROTOCOL : Extraction and determination of glycine betaine Initiating Author Name : Kasetsart J - Nat Sci. 2009;43 5 SUPPL.:146\u0026ndash;52.\u0026nbsp;https://doi.org/10.1002/aris.2009.1440430113.\u003c/li\u003e\n \u003cli\u003e28. Claussen W. Proline as a measure of stress in tomato plants. Plant Sci. 2005;168:241\u0026ndash;8. https://doi.org/10.1016/j.plantsci.2004.07.039.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e29. Irigoyen JJ, Einerich DW, S\u0026aacute;nchez‐D\u0026iacute;az M. Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativd) plants. Physiol Plant. 1992;84:55\u0026ndash;60. https://doi.org/10.1111/j.1399-3054.1992.tb08764.x.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e30. Aliniaeifard S, Van Meeteren U. Stomatal characteristics and desiccation response of leaves of cut chrysanthemum (Chrysanthemum morifolium) flowers grown at high air humidity. Sci Hortic (Amsterdam). 2016;205:84\u0026ndash;9. https://doi.org/10.1016/j.scienta.2016.04.025.\u003c/li\u003e\n \u003cli\u003e31. Kathpalia R, Bhatla SC. Plant Water Relations. 2018.\u0026nbsp;https://doi.org/10.1007/978-981-13-2023-1_1.\u003c/li\u003e\n \u003cli\u003e32. Radiation I. 2.1 Introduction. 2017.\u0026nbsp;https://doi.org/10.1016/j.semradonc.2017.05.001.\u003c/li\u003e\n \u003cli\u003e33. Raoufi A, Salehi H, Rahemi M, Shekafandeh A, Khalili S. In vitro screening: The best method for salt tolerance selection among pistachio rootstocks. J Saudi Soc Agric Sci. 2021;20:146\u0026ndash;54.\u0026nbsp;https://doi.org/10.1016/j.jssas.2020.12.010.\u003c/li\u003e\n \u003cli\u003e34. Fang Y, Xiong L. General mechanisms of drought response and their application in drought resistance improvement in plants. Cell Mol Life Sci. 2015;72:673\u0026ndash;89.\u0026nbsp;https://doi.org/10.1007/s00018-014-1767-0.\u003c/li\u003e\n \u003cli\u003e35. Venkateswarlu B, Shanker AK, Shanker C, Maheswari M. Crop stress and its management: Perspectives and strategies. 2012. https://doi.org/10.1007/978-94-007-2220-0.\u003c/li\u003e\n \u003cli\u003e36. Aroca R. Front Matter: Plant responses to drought stress. Plant Responses to Drought Stress. 2012.\u0026nbsp;https://doi.org/10.1007/978-3-642-32653-0.\u003c/li\u003e\n \u003cli\u003e37. Chaves MM, Pereira JS, Maroco J, Rodrigues ML, Ricardo CPP, Os\u0026oacute;rio ML, et al. How plants cope with water stress in the field. Photosynthesis and growth. Ann Bot. 2002;89 SPEC. ISS.:907\u0026ndash;16. https://doi.org/10.1093/aob/mcf105.\u003c/li\u003e\n \u003cli\u003e38. Flexas J, Medrano H. Drought-inhibition of photosynthesis in C3 plants: Stomatal and non-stomatal limitations revisited. Ann Bot. 2002;89:183\u0026ndash;9. https://doi.org/10.1093/aob/mcf027. https://doi.org/10.1093/aob/mcf027.\u003c/li\u003e\n \u003cli\u003e39. Blum A. Drought resistance, water-use efficiency, and yield potential - Are they compatible, dissonant, or mutually exclusive? Aust J Agric Res. 2005;56:1159\u0026ndash;68. https://doi.org/10.1071/AR05069.\u003c/li\u003e\n \u003cli\u003e40. Sutka MR, Manzur ME, Vitali VA, Micheletto S, Amodeo G. Evidence for the involvement of hydraulic root or shoot adjustments as mechanisms underlying water deficit tolerance in two Sorghum bicolor genotypes. J Plant Physiol. 2016;192:13\u0026ndash;20. https://doi.org/10.1016/j.jplph.2016.01.002.\u003c/li\u003e\n \u003cli\u003e41. S\u0026aacute;nchez-Blanco MJ, \u0026Aacute;lvarez S, Ortu\u0026ntilde;o MF, Ruiz-S\u0026aacute;nchez MC. Root System Response to Drought and Salinity: Root Distribution and Water Transport. 2014;:325\u0026ndash;52.\u0026nbsp;https://doi.org/10.1007/978-3-642-54276-3_15.\u003c/li\u003e\n \u003cli\u003e42. Saadatmand AR, Banihashemi Z, Maftoun M, Sepaskhah AR. Interactive effect of soil salinity and water stress on growth and chemical compositions of pistachio nut tree. J Plant Nutr. 2007;30:2037\u0026ndash;50. https://doi.org/10.1080/01904160701700483.\u003c/li\u003e\n \u003cli\u003e43. Vives-Peris V, L\u0026oacute;pez-Climent MF, P\u0026eacute;rez-Clemente RM, G\u0026oacute;mez-Cadenas A. Root involvement in plant responses to adverse environmental conditions. Agronomy. 2020;10. https://doi.org/10.3390/agronomy10070942.\u003c/li\u003e\n \u003cli\u003e44. Bhargava S, Sawant K. Chapter Plant Responses to Drought Stress: Morphological, Physiological, Molecular Approaches, and Drought Resistance and regulation of gene expression. Plant Breed. 2013;132:21\u0026ndash;32.\u0026nbsp;https://doi.org/10.1111/pbr.12004.\u003c/li\u003e\n \u003cli\u003e45. Liu Y, He Z, Xie Y, Su L, Zhang R, Wang H, et al. Drought resistance mechanisms of Phedimus aizoon L. Sci Rep. 2021;11:13600.\u0026nbsp;https://doi.org/10.1038/s41598-021-93118-7.\u003c/li\u003e\n \u003cli\u003e46. Deka D, Singh AK, Singh AK. Effect of Drought Stress on Crop Plants with Special Reference to Drought Avoidance and Tolerance Mechanisms: A Review. Int J Curr Microbiol Appl Sci. 2018;7:2703\u0026ndash;21.\u0026nbsp;https://doi.org/10.20546/ijcmas.2018.709.336.\u003c/li\u003e\n \u003cli\u003e47. Nwaogu, EN. Soil fertility changes and their effects on ginger (Zingiber officinale Rosc.) yield response in an ultisol under different pigeon pea hedgerow alley management in South Eastern Nigeria. African J Agric Res. 2014;9:2158\u0026ndash;66.\u0026nbsp;https://doi.org/10.5897/ajar2013.7291.\u003c/li\u003e\n \u003cli\u003e48. Kasmani RM, Andrews GE, Phylaktou HN. Experimental study on vented gas explosion in a cylindrical vessel with a vent duct. Process Saf Environ Prot. 2013;91:245\u0026ndash;52.\u0026nbsp;https://doi.org/10.1016/j.psep.2012.05.006.\u003c/li\u003e\n \u003cli\u003e49. Koch G, Rolland G, Dauzat M, B\u0026eacute;di\u0026eacute;e A, Baldazzi V, Bertin N, et al. Leaf production and expansion: A generalized response to drought stresses from cells to whole leaf biomass\u0026mdash;a case study in the tomato compound leaf. Plants. 2019;8:1\u0026ndash;17.\u0026nbsp;https://doi.org/10.3390/plants8100409.\u003c/li\u003e\n \u003cli\u003e50. Biglouei MH, Assimi MH, Akbarzadeh A. Effect of water stress at different growth stages on quantity and quality traits of Virginia (flue-cured) tobacco type. Plant, Soil Environ. 2010;56:67\u0026ndash;75.\u0026nbsp;https://doi.org/10.17221/163/2009-pse.\u003c/li\u003e\n \u003cli\u003e51. Farquharson KL. Fine-tuning plant growth in the face of drought. Plant Cell. 2017;29:4.\u0026nbsp;https://doi.org/10.1105/tpc.17.00038.\u003c/li\u003e\n \u003cli\u003e52. Claeys H, Inz\u0026eacute; D. of choice: How plants balance growth and survival under water-limiting conditions. Plant Physiol. 2013;162:1768\u0026ndash;79.\u0026nbsp;https://doi.org/10.1104/pp.113.220921.\u003c/li\u003e\n \u003cli\u003e53. Zhao Y, Gao J, Kim JI, Chen K, Bressan RA, Zhu JK. Control of plant water use by ABA induction of senescence and dormancy: An overlooked lesson from evolution. Plant Cell Physiol. 2017;58:1319\u0026ndash;27.\u0026nbsp;https://doi.org/10.1093/pcp/pcx086.\u003c/li\u003e\n \u003cli\u003e54. Zhang YJ, Xie ZK, Wang YJ, Su PX, An LP, Gao H. Effect of water stress on leaf photosynthesis, chlorophyll content, and growth of oriental lily. Russ J Plant Physiol. 2011;58:844\u0026ndash;50.\u0026nbsp;https://doi.org/10.1134/s1021443711050268.\u003c/li\u003e\n \u003cli\u003e55. Marron N, Dreyer E, Boudouresque E, Delay D, Petit JM, Delmotte FM, et al. Impact of successive drought and re-watering cycles on growth and specific leaf area of two Populus x canadensis (Moench) clones, \u0026ldquo;Dorskamp\u0026rdquo; and \u0026ldquo;Luisa_Avanzo.\u0026rdquo; Tree Physiol. 2003;23:1225\u0026ndash;35.\u0026nbsp;https://doi.org/10.1093/treephys/23.18.1225.\u003c/li\u003e\n \u003cli\u003e56. Chaves MM, Oliveira MM. Mechanisms underlying plant resilience to water deficits: Prospects for water-saving agriculture. J Exp Bot. 2004;55:2365\u0026ndash;84.\u0026nbsp;https://doi.org/10.1093/jxb/erh269.\u003c/li\u003e\n \u003cli\u003e57. Farooq M, A.Wahid, Kobayashi N, Fujita D, Basra SMA. Review article Plant drought stress : e ff ects , mechanisms and management. Agron Sustain Dev. 2009;29:185\u0026ndash;212.\u0026nbsp;https://doi.org/10.1051/agro:2008021.\u003c/li\u003e\n \u003cli\u003e58. Afrousheh M, Javanshah A. The effect of humic acid on the growth and physiological indices of pistachio seedling (Pistacia vera) under drought stress. J Nuts. 2020;11:1\u0026ndash;12.\u0026nbsp;https://doi.org/10.22034/jon.2020.1879490.1069\u003c/li\u003e\n \u003cli\u003e59. Behzadi Rad P, Roozban MR, Karimi S, Ghahremani R, Vahdati K. Osmolyte accumulation and sodium compartmentation has a key role in salinity tolerance of pistachios rootstocks. Agric. 2021;11.\u0026nbsp;https://doi.org/10.3390/agriculture11080708.\u003c/li\u003e\n \u003cli\u003e60. Wan T, Feng Y, Liang C, Pan L, He L, Cai Y. Metabolomics and transcriptomics analyses of two contrasting cherry rootstocks in response to drought stress. Biology (Basel). 2021;10:1\u0026ndash;20.\u0026nbsp;https://doi.org/10.3390/biology10030201.\u003c/li\u003e\n \u003cli\u003e61. Fathi H, Esmailamiri M, Imani A, Hajilou J, Nikbakht J. PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES IN SOME ALMOND (PRUNUS AMYGDALUS BATSCH.) GENOTYPES (GRAFTED ON/GN15) SUBMITTED TO DROUGHT STRESS. 2018.\u003c/li\u003e\n \u003cli\u003e62. Memmi H, Couceiro JF, Gij\u0026oacute;n C, P\u0026eacute;rez-L\u0026oacute;pez D. Impacts of water stress, environment and rootstock on the diurnal behaviour of stem water potential and leaf conductance in pistachio (Pistacia vera L.). Spanish J Agric Res. 2016;14.\u0026nbsp;https://doi.org/10.5424/sjar/2016142-8207.\u003c/li\u003e\n \u003cli\u003e63. Sharkey TD, Seemann JR. Mild Water Stress Effects on Carbon-Reduction-Cycle Intermediates, Ribulose Bisphosphate Carboxylase Activity, and Spatial Homogeneity of Photosynthesis in Intact Leaves. Plant Physiol. 1989;89:1060\u0026ndash;5.\u0026nbsp;https://doi.org/10.1104/pp.89.4.1060.\u003c/li\u003e\n \u003cli\u003e64. Setum P, Des T, Mcpherson BHG. Sensitivity of root and leaf water status in maize ( Zea mays ) subjected to mild soil dryness. 2017; January 1998. https://doi.org/10.1071/PP97047.\u0026nbsp;https://doi.org/10.1071/pp97047.\u003c/li\u003e\n \u003cli\u003e65. Pita P, Gasc\u0026oacute; A, Pardos JA. Xylem cavitation, leaf growth and leaf water potential in Eucalyptus globulus clones under well-watered and drought conditions. Funct Plant Biol. 2003;30:891\u0026ndash;9.\u0026nbsp;https://doi.org/10.1071/fp03055.\u003c/li\u003e\n \u003cli\u003e66. Costa E Silva F, Shvaleva A, Maroco JP, Almeida MH, Chaves MM, Pereira JS. Responses to water stress in two Eucalyptus globulus clones differing in drought tolerance. Tree Physiol. 2004;24:1165\u0026ndash;72.\u0026nbsp;https://doi.org/10.1093/treephys/24.10.1165.\u003c/li\u003e\n \u003cli\u003e67. Luiz Ferraresso Conti Junior J, Jos\u0026eacute; de Araujo M, Cesar de Paula R, Barroso Queiroz T, Eiji Hakamada R, Hubbard RM. Quantifying turgor loss point and leaf water potential across contrasting Eucalyptus clones and sites within the TECHS research platform. For Ecol Manage. 2020;475 June:118454.\u0026nbsp;https://doi.org/10.1016/j.foreco.2020.118454.\u003c/li\u003e\n \u003cli\u003e68. Shabala S, Shabala L. Ion transport and osmotic adjustment in plants and bacteria. Biomol Concepts. 2011;2:407\u0026ndash;19.\u0026nbsp;https://doi.org/10.1515/bmc.2011.032.\u003c/li\u003e\n \u003cli\u003e69. Hsiao TC, Otoole JC, Yambao EB, Turner NC. Influence of Osmotic Adjustment. 1984;:338\u0026ndash;41.\u0026nbsp;https://doi.org/10.1104/pp.75.2.338.\u003c/li\u003e\n \u003cli\u003e70. Mokotedi MEO. Water relations of Eucalyptus nitens \u0026times; Eucalyptus grandis: Is there interclonal variation in response to experimentally imposed water stress? South For. 2013;75:213\u0026ndash;20.\u0026nbsp;https://doi.org/10.2989/20702620.2013.858212.\u003c/li\u003e\n \u003cli\u003e71. Rana V, Ram S, Nehra K. Review Proline Biosynthesis and its Role in Abiotic Stress. Int J Agric Innov Res. 2017;6:2319\u0026ndash;1473.\u0026nbsp;https://doi.org/10.1556/0806.44.2016.009.\u003c/li\u003e\n \u003cli\u003e72. Kerepesi I, Galiba G, B\u0026aacute;nyai \u0026Eacute;. Osmotic and Salt Stresses Induced Differential Alteration in Water-Soluble Carbohydrate Content in Wheat Seedlings. J Agric Food Chem. 1998;46:5347\u0026ndash;54.\u0026nbsp;https://doi.org/10.1021/jf980455w.\u003c/li\u003e\n \u003cli\u003e73. Kerepesi,I. and Galiba G. Osmotic and Salt Stress-Induced Alteration in Soluble Carbohydrate Content in Wheat Seedlings \u0026acute;. Crop Sci J. 2014;40:482\u0026ndash;7.\u0026nbsp;https://doi.org/10.2135/cropsci2000.402482x.\u003c/li\u003e\n \u003cli\u003e74. Escalante-Maga\u0026ntilde;a C, Aguilar-Caamal LF, Echevarr\u0026iacute;a-Machado I, Medina-Lara F, Cach LS, Mart\u0026iacute;nez-Est\u0026eacute;vez M. Contribution of glycine betaine and proline to water deficit tolerance in pepper plants. HortScience. 2019;54:1044\u0026ndash;54.\u0026nbsp;https://doi.org/10.21273/hortsci13955-19.\u003c/li\u003e\n \u003cli\u003e75. Reyhani Haghighi S, Hosseininaveh V, Maali-Amiri R, Talebi K, Irani S. Improving the Drought Tolerance in Pistachio (Pistacia vera) Seedlings by Foliar Application of Salicylic Acid. Gesunde Pflanz. 2021;73:495\u0026ndash;507.\u0026nbsp;https://doi.org/10.1007/s10343-021-00569-z.\u003c/li\u003e\n \u003cli\u003e76. Jamshidi Goharrizi K, Amirmahani F, Salehi F. Assessment of changes in physiological and biochemical traits in four pistachio rootstocks under drought, salinity and drought + salinity stresses. Physiol Plant. 2020;168:973\u0026ndash;89.\u0026nbsp;https://doi.org/10.1111/ppl.13042.\u003c/li\u003e\n \u003cli\u003e77. Zhao W, Sun Y, Kjelgren R, Liu X. Response of stomatal density and bound gas exchange in leaves of maize to soil water deficit. Acta Physiol Plant. 2015;37.\u0026nbsp;https://doi.org/10.1007/s11738-014-1704-8.\u003c/li\u003e\n \u003cli\u003e78. Mehri N, Fotovat R, Saba J, Jabbari F. Variation of stomata dimensions and densities in tolerant and susceptible wheat cultivars under drought stress. J Food, Agric Environ. 2009;7:167\u0026ndash;70.\u0026nbsp;http://www.world-food.net/.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e79. Sade N, Gebremedhin A, Moshelion M. Risk-taking plants. Plant Signal Behav. 2012;7:767\u0026ndash;70.\u0026nbsp;https://doi.org/10.4161/psb.20505.\u003c/li\u003e\n \u003cli\u003e80. Sedaghati N, Hokmabadi H. Optimizing pistachio irrigation management using the relationship between Echo-Physiological characteristics and water stress. J Agric Sci Technol. 2015;17:189\u0026ndash;200.\u0026nbsp;http://dorl.net/dor/20.1001.1.16807073.2015.17.1.8.9.\u003c/li\u003e\n \u003cli\u003e81. Dur\u0026aacute;n-Zuazo VH, Rodriguez BC, Guti\u0026eacute;rrez-Gordillo S, Bilbao M, Sacrist\u0026aacute;n PC, P\u0026eacute;rez Parra J, et al. Rethinking irrigated almond and pistachio intensification: a shift towards a more sustainable water management paradigm. Rev Ci\u0026ecirc;ncias Agr\u0026aacute;rias. 2020;43 Especial 2:24\u0026ndash;49.\u0026nbsp;https://doi.org/10.19084/rca.19651\u003c/li\u003e\n \u003cli\u003e82. Bellvert J, Adeline K, Baram S, Pierce L, Sanden BL, Smart DR. Monitoring Crop Evapotranspiration and Crop Coefficients Over an Almond and Pistachio Orchard Throughout Remote Sensing. Remote Sens. 2018;10.\u0026nbsp;https://doi.org/10.3390/rs10122001.\u003c/li\u003e\n \u003cli\u003e83. Neumann PM. Coping mechanisms for crop plants in drought-prone environments. Ann Bot. 2008;101:901\u0026ndash;7.\u0026nbsp;https://doi.org/10.1093/aob/mcn018.\u003c/li\u003e\n \u003cli\u003e84. Li N, Gao Y, pu K, Zhang M, Wang T, Li J, et al. Glycine betaine enhances tolerance of low temperature combined with low light in pepper (Capsicum annuum L.) by improving the antioxidant capacity and regulating GB metabolism. Plant Physiol Biochem. 2025;222:109705. https://doi.org/10.1016/j.plaphy.2025.109705.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Osmolyte accumulation, stomatal parameters, water withholding, rootstock, UCB1","lastPublishedDoi":"10.21203/rs.3.rs-5905176/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5905176/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePistachio (\u003cem\u003ePistacia vera\u003c/em\u003e L.) growth, yield and quality are affected by abiotic stress especially drought. Understanding the strategies that improve dehydration tolerance is essential for developing resistant pistachio rootstocks. In the experiment, nine-month-old saplings of seven clonal interspecies hybrids of \u003cem\u003ePistacia atlantica\u003c/em\u003e \u0026times; \u003cem\u003eP. integerrima\u003c/em\u003e (C1, C2, C16-1, C8-3, C4-2, C9-4 and UCB1) were assessed for growth and physiological responses to water withholding and recovery.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e \u003cp\u003eWater deficit negatively impacted growth parameters, including shoot dry weight, root dry weight and leaf area, in all hybrids; however, the C1 demonstrated relatively minor reductions compared to the other hybrids. Glycine betaine content in leaves increased by 49.4% in C9-4 and 47% in C1, while only 7% and 11% increases were found in the most sensitive clones, C8-3 and C4-2. Notably, C9-4, identified as the most tolerant clone, displayed the highest proline levels, with increases of 29.5% in leaves and 41.5% in roots, in contrast to C8-3, which showed minimal increases of 6% and 11% in leaves and roots, respectively. Clones with higher compatible solutes maintained higher relative water content (RWC), lower osmotic potential and smaller reductions in leaf water potential. RWC declined by just 6% in C9-4, whereas it dropped by 88% in C8-3. Osmotic potentials in C9-4 were \u0026minus;\u0026thinsp;1.61 MPa in leaves and \u0026minus;\u0026thinsp;0.271 MPa in roots, while in C8-3, they were \u0026minus;\u0026thinsp;0.93 MPa and \u0026minus;\u0026thinsp;0.11 MPa in leaves and roots, respectively. Following recovery, evaluations of growth, physiological traits and visual observations indicated that C8-3 had poor recovery ability. Heatmap and PCA analyses categorized the clones into three groups: \"tolerant\" (C9-4, C1 and C2), \"moderately tolerant\" (UCB1) and \"sensitive\" (C8-3, C4-2 and C16-1).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe results of this study underscore the significance of osmotic adjustment as a more critical trait compared to growth and stomatal parameters in effectively differentiating tolerant clones from sensitive ones.\u003c/p\u003e","manuscriptTitle":"Revealing Drought Tolerance Strategies in Pistachio Clonal Hybrids: Role of Osmotic Adjustment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 09:27:37","doi":"10.21203/rs.3.rs-5905176/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-15T11:51:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-13T02:54:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-08T15:29:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20584995085888481393328723142341473014","date":"2025-04-03T04:57:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218230597879130191954200425426492625478","date":"2025-03-30T21:45:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-28T08:26:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-27T09:33:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-03-26T18:31:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"84277d2f-6aa1-4adf-a9f8-2848858bccab","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-05T16:00:21+00:00","versionOfRecord":{"articleIdentity":"rs-5905176","link":"https://doi.org/10.1186/s12870-025-06583-x","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-05-02 15:57:24","publishedOnDateReadable":"May 2nd, 2025"},"versionCreatedAt":"2025-03-31 09:27:37","video":"","vorDoi":"10.1186/s12870-025-06583-x","vorDoiUrl":"https://doi.org/10.1186/s12870-025-06583-x","workflowStages":[]},"version":"v1","identity":"rs-5905176","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5905176","identity":"rs-5905176","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}