Improving quality and water tolerance in strawberry through combined application of Nano Zinc and Brassinosteroids

Improving quality and water tolerance in strawberry through combined application of Nano Zinc and Brassinosteroids | 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 Improving quality and water tolerance in strawberry through combined application of Nano Zinc and Brassinosteroids WarqaaMuhammed ShariffAlSheikh, Heidar Meftahizade, Neda Tariverdizadeh, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7378337/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Strawberry ( Fragaria × ananassa Duch.), are vulnerable to water limitations due to their root structure and high-water content. The experiment was carried out as a factorial experiment in a randomized complete block design with three replications. water deficiency at 4 levels (100% of FC, 75% of FC, 50% of FC, and 25% of FC), brassinosteroid at 4 levels (control, 100, 200, and 400 ml/liter), and zinc nanoparticles (control, 1, 2, and 3 g/liter) in Sabrina cultivar were treated. The results showed, the highest fruit weight (20.53 g) was observed under 75% FC and 400 ml L − 1 Br in combination with 3 g L − 1 nano zinc. the interaction between Br and nano zinc (1.79 N) resulted in the lowest firmness in the treatment with 25% drought stress and 1 g L − 1 of nano zinc. Also, the application of Br (200 ml L − 1 ) and nano zinc (2 g L − 1 ) under FC (25%) enhanced the accumulation of TSS (11.6%) in the fruit. content of TS had the highest value (1.137 gr − 1 FW) in fruit subjected to mild FC (75%) with the application of 400 ml L − 1 Br combined with 2 g L − 1 nano Zn. The highest Fo was observed in fruits treated with 75% FC and 400 ml L − 1 Br in combination with 3 g L − 1 nano zinc. The lowest Fo was recorded under moderate drought stress and Br in combination with 3 g L − 1 nano zinc. The lowest Fv/Fm was found after 50% FC and 100 ml L − 1 Br treatment in combination with 2 g L − 1 nano zinc. Thus, foliar application of nano zinc and brassinosteroids under drought stress conditions can be considered an effective strategy to enhance growth, quality, and stress tolerance in the ’Sabrina’ strawberry cultivar. Foliar Nutrition Photosystem II Efficiency (Fv/Fm) Phenolic Compound Sabrina Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Strawberry ( Fragaria × ananassa Duch.), belonging to the genus Fragaria and the Rosaceae family, and often referred to as the Queen of Fruits, is rich in vitamin C and a wide range of antioxidants and phenolic compounds that contribute to cardiovascular health and the regulation of blood glucose levels [ 1 – 3 ]. Drought is a growing environmental challenge that is known to decrease crop yield, and strawberries [ 4 , 5 ] are no exception. Strawberries are particularly vulnerable to water limitations due to their root structure, large leaf area, and high-water content [ 6 – 8 ]. Ultimately, the resultant stress from the drought leads to decreased growth, chlorophyll loss, photosynthesis reduction, and oxidative stress [ 6 – 8 ]. The reduction of fruit yield and quality due to drought will depend on cultivar tolerance or sensitivity and the definition of stress: nature, intensity and duration [ 9 ]. Ultimately, the duration of drought stress leads to an excess generation of reactive oxygen species (ROS) or and unequal generation between ROS and the antioxidant systems. The ROS over generation leads to potentially oxidative damage to the photosynthetic apparatus [ 8 , 10 ]. Drought and other stressors in combination will create a negative physiological response of the plant and cause various limitations to the uptake of nutrients, photosynthesis, growth, and consequently yields [ 7 , 11 and 12 ]. Tolerant plants cope with these effects by regulating photosynthesis, accumulating osmolytes and inducing genes encoding antioxidants, closed their stomata and increased stomatal resistance to decrease transpiration [ 8 ]. Reactive oxygen species (ROS) that develop under stress result in cellular damage, including lipid peroxidation, protein degradation, and enzyme inactivation. Proline not only scavenges free radicals but its role in oxidative stress marks it as critical in conferring stress tolerance as an osmolyte. Drought-stressed plants manage oxidative damage with the stimulation of antioxidant enzymes [ 13 ]. In a study conducted by Zahedi et al. [ 8 ], the physiological and biochemical responses of two strawberry cultivars were evaluated under four different drought stress levels (100%, 75%, 50% and 25%) of field capacity. The resulted of this research indicated that drought stress led to a significant decrease in chlorophyll content, carotenoids, and phenolic compounds in both cultivars. In the study, three strawberry cultivars (California, Earlibrite, and Sweet Charlie) were evaluated under four different irrigation levels corresponding to 100%, 75%, 50%, and 25% of field capacity. The results showed that increasing drought severity significantly reduced growth parameters such as plant height, leaf area, fruit weight, as well as leaf gas exchange and relative water content. On the other hand, drought stress led to an increase in total soluble solids (TSS), malondialdehyde (MDA), anthocyanins, and proline, indicating the activation of the plant's defensive responses to oxidative and osmotic stress [ 11 ]. Previous studies have shown that the combined application of zinc oxide nanoparticles (ZnO NPs) and melatonin (MT) can improve growth and drought tolerance in strawberry plants. This combination enhances nutrient uptake, increases chlorophyll content, and boosts antioxidant enzyme activities, thereby reducing oxidative damage caused by drought stress. Experimental results demonstrated increased shoot and root length, fruit biomass, and bud number in plants treated with ZnO NPs and MT. These findings highlight the potential of this approach to enhance strawberry resilience under drought conditions [ 14 ]. At this time, multiple technologies have been reported to enhance plant tolerance or resistance to environmental stresses (i.e., elicitors and nanotechnologies) which boost crop yield and quality [ 12 , 15 ]. Use of plant growth stimulators, such as brassinosteroids (BRs), is an important factor for the improvement of crop quality and yield. Not only do BRs regulate physiological processes like plant growth, photosynthesis, flowering, and fruit set, they are also able to enhance plant tolerance or resistance to a wide range of abiotic stress factors including drought and oxidative stress [ 16 – 18 ]. MARTÍNEZ-PÉREZ et al. [ 19 ] explained that BRs promote vegetative growth by modifying the mechanical properties of the cell wall, increasing its plastic extensibility. Studies have shown that BRs significantly increase yields in various crops such as tomato [ 20 ], strawberry [ 21 ]. BR levels are crucial for promoting plant growth and productivity, especially under environmental stresses like drought [ 7 ]. Moreover, modulation of endogenous BR levels has been reported to improve crop quality and performance under stress conditions [ 22 ]. Khatoon et al. [ 18 ] assessed the plant growth regulator brassinosteroids to pea foliar on strawberry, brassinosteroids 0.2 ppm were applied during three phenological stages, vegetative, flowering, and fruiting we found this application the most response in the yield potential of the strawberry plant. The result of the study indicates that brassinosteroids offering great potential in improving growth parameters that improve fruit yield and increase growth rate through to the produce. Nanotechnology coming up with different options to minimize abiotic stress on production. The unique properties of zinc oxide nanoparticles to modulate antioxidant enzyme processes, promote nutrient uptake, and enhance the photosynthetic process [ 23 , 24 ], have provided keen interest for their use in the field. Nano zinc has a prominent role in potential plant alleviate abiotic stresses through regulating plant water balance and stabilizing cellular osmotic levels to improve stress tolerance from cold, drought, and salinity [ 25 ]. The two most common methods of applying nano zinc to plants are root application and foliar application methods. In foliar application, zinc oxide nanoparticles are sprayed onto the plant leaf surface, when the nanoparticles are absorbed in through the stomata and cuticle, and once inside the plant, the nanoparticles can be translocated through the plant through the phloem sieve tubes resulting in systemic dispersal [ 25 , 26 ]. There has been significant research on the effects of brassinosteroids and nano zinc individually, but nothing has been published on the effects they may have together on alleviating drought stress. Keeping in mind the need to improve strawberry production, this study is looking to examine the individual and combined influences of drought stress, BRs, and nano zinc on the physiological, biochemical and growth characteristics of strawberry plants. Drought stress is known to negatively impact the growth and physiological performance of strawberries, leading to reduced yield and water use efficiency. However, the application of brassinosteroids (BRs) and nano zinc, either individually or in combination, has the potential to mitigate these adverse effects by enhancing antioxidant enzyme activities and improving plant stress tolerance. A better understanding of these effects could facilitate the development of effective approaches to improve crop tolerance and maximize water use efficiency. 2. Materials and Methods The treatments were carried out at the research greenhouse of Ilam University on 2024-2025. The strawberry's ‘Sabrina” cultivar was provided from the Royal Green Agricultural Company of Kurdistan, Soil with sandy loam texture (suitable for strawberry cultivation based on soil test data according to Table 1 ) was used as culture medium. at the end of September, well-rooted strawberry daughter plants were selected for similar size. Then, the seedling was stored in a cold storage room at 4°C for 240 hours to determine the need for cold storage. Table 1. The soil physical and chemical characteristics of the experimental field Texture Clay (%) Silt (%) Sand (%) K (mg/kg) P (mg/kg) N (%) T.N.V (%) pH EC (dS/m) Sandy- Loamy 15 25 60 342 9.2 0.11 3.8 7.62 1.23 To conduct this experiment, 64 plastic pots (4×4×4) with a volume of 50 kg were filled with a mixture of 60% cocopeat and 40% perlite, and three strawberry plants of the Sabrina cultivar were planted inside each pot (Fig 7). The pots were divided into four rows as four experimental blocks. Initially, in order to establish the plants, all pots were fed with Hoagland nutrient solution for one week. The experiment was carried out as a factorial experiment in a randomized complete block design with three replications. The first factor included water deficiency at 4 levels (100% of FC, 75% of FC, 50% of FC, and 25% of FC), the second factor included brassinosteroid at 4 levels (control, 100, 200, and 400 ml/liter), and zinc nanoparticles at 4 levels (control, 1, 2, and 3 g/liter) were treated. The nanoparticles in this study were obtained from Nanosani Company, the characteristics of which are given in Table 2 . In this study, ZnO (Zinc Oxide) was used to synthesize zinc nanoparticles. Spraying solutions of various concentrations of zinc nanoparticles, after the initial pruning, from the fourteenth to the eighteenth week, once a week (5 times) is performed. Table 2. Structural properties of zinc nanoparticles Structural properties of zinc nanoparticles zinc nanoparticles Thermogravimetric analysis (TGA) 3.2±0.04 Inductively coupled plasma (ICP) 2.8±0.03 size 20-31 Purity percentage 96 Active surface (g/m 2 ) 50-135 24-epibrassinolide EBL (Sigma, USA) solution was readied by ethanol. To increase regulators uptake, Tween-20 (0.05%) was used as a surfactant [27]. It was sprayed during experimental periods. According to Equation (1), the soil moisture content was measured at 100% of FC based on the difference between the soil weight after drainage (FCW), and soil weight after drying (DW). Finally, fresh seedlings were planted. When the weight of the pot containing soil and plants were lower than a certain level (measurements were performed daily on all pots based on the FC assigned for each treatment) irrigation was repeated. Equation 1: Field capacity (FC): soil weight in field capacity (FCW)−weight of dry soil (DW)/ weight of dry soil (DW). Measurement of physiological, biochemical, and morphological traits Fruit weight, fruit length, and fresh weight of vegetative tissues measurement At the end of the experiment, the fruit weight was measured using a digital scale with an accuracy of 0.001 grams, the fruit length was measured using a digital caliper (150-1108). Fruit firmness measurement It was evaluated with a penetrometer® Fruit Pressure Tested Wagner FT 327, made in the USA with a 2.3 mm thick tip at the height of the equatorial diameter. TSS (Brix) and total Sugar content measurement TSS content of fruit juice was measured using an Abbe refractometer (model: CETI, Belgium). The TSS values were expressed as % Brix, following the procedure described by Shiukhy [28]. TS were quantified using the anthrone method, as described by Mirshekari et al. [29]. For this analysis, 0.2 mL of concentrated fruit extract was mixed with 3 mL of anthrone reagent, which was prepared by dissolving 150 mg of anthrone in 100 mL of 13 M sulfuric acid. The mixture was then incubated in a water bath at 100 °C for 20 minutes. After cooling to room temperature, the absorbance was measured at 620 nm using a spectrophotometer. The total soluble sugar content was calculated using a glucose standard calibration curve and expressed as mg glucose per gram of fresh weight (Micro gr/gr FW). Titratable acidity (TA) measurement The titratable acidity was determined in triplicate with the method of Horwitz and Latimer [30]. 10 mL of strawberry juice per sample were taken. The sample was transferred to an Erlenmeyer flask and 4 drops of phenolphthalein 1% solution were added (0.5 g of phenolphthalein plus 70 mL of ethyl alcohol, calibrated to 100 mL with distilled water). The sample was titrated with a 0.1 N NaOH solution until the purplish color change was maintained for one minute. The titratable acidity was expressed as a percentage of citric acid and was calculated using the following formula: % acid = V NaOH × N NaOH × meq acido /V × 100. Total chlorophyll content measurement Photosynthetic pigments were determined following the method of Tariverdizadeh et al. [31]. Fresh leaf samples (0.5 g) were ground and extracted in 100 ml of 80% acetone. The mixture was then centrifuged at 13,552 × g for 10 minutes at 4°C, and the supernatant was collected for analysis. Absorbance readings were taken at wavelengths of 663, 646, and 470 nm using a spectrophotometer. Chlorophyll a, chlorophyll b, and carotenoid contents were calculated using the following equations, and results were expressed as mg per gram of fresh weight (mg g⁻¹ FW): Chla (mg ml -1 ) =12.25A663-2.79A646 Chlb (mg ml -1 ) =21.50A646-5.10A663 TChl=Chla + CHlb Total phenol content (TPC) and total flavonoid content (TFC) measurement The total phenols were determined according to the Folin- Ciocalteu’s procedure [32]. The absorption of these metabolites was determined at 725 nm. Flavonoids levels were spectrophotometrically determined by modified method of Upadhyay and Maier [33]. The fruits samples were prepared with 2 mL acidic methanol (80% methanol with 1% HCl) in the mortar at room temperature for 2 h. The extracts were centrifuged at 1000 × g for 15 min and the absorption recorded at 560 nm. Proline content measurement Proline content in leaves was determined using a colorimetric method based on the protocol of Bates et al. [34]. Approximately 0.5 g of fresh leaf samples were weighed and homogenized in 10 mL of 3% sulfosalicylic acid. The homogenate was centrifuged at 6000 × g for 5 minutes to separate the supernatant. Then, 2 mL of the supernatant was mixed with 2 mL of glacial acetic acid and 2 mL of ninhydrin reagent, and the mixture was incubated in a water bath at 100°C for 60 minutes. After incubation, the samples were cooled, and 4 mL of toluene was added and thoroughly mixed. The toluene phase was separated, and its absorbance was measured at 520 nm using a UV–Vis spectrophotometer (Model: UV-1800, Shimadzu, Japan). Proline concentration was quantified using a standard proline calibration curve and expressed as milligrams of proline per gram of fresh weight. Measurement of chlorophyll fluorescence parameters (Fo, Fm) and calculation of maximum quantum efficiency (Fv/Fm) The photosynthesis system equipped with a chlorophyll fluorescence system (LI-6200) was used to measure chlorophyll fluorescence parameters of the second fully expanded leaf. After 30 min of dark treatment, the minimum fluorescence (Fo), the saturated maximum fluorescence (Fm) of dark-adapted were determined under normal light, and each treatment was repeated three times. The maximum quantum yield of photosystem II (FV/FM) was estimated using the OJIP protocol as described by Habibi et al. [35]. Young mature leaves were carefully selected for each experimental treatment. After 20 min in the dark-adapted condition, chlorophyll fluorescence parameters were recorded using a portable fluorometer (Fluorpen FP 100-MAX; Photon Systems Instruments, Drasov, Czech Republic). Data analysis Data analysis was conducted in R software. A variance analysis (ANOVA) using the F-test was conducted to evaluate the effects of treatments at each sampling interval, with means compared using Tukey’s test at the 5% significance level. Principal component analysis (PCA) was applied to interpret the response patterns. Additionally, in this study, the relationships among the evaluated traits were analyzed using the Pearson correlation coefficient were employed to identify relationships between parameters with the ggplot2 package in R. Prior to combined analysis, Levene’s test for homogeneity of variances was conducted. 3. Results 3.1. Effect of Br and nano Zinc on yield parameters of strawberry fruit Fruit length The application of drought stress, Br and nano zinc caused a statistically significant difference in FL ( P < 0.01). maximum FL in fruits treated with both Br (400 ml L -1 ) and nano zinc (3 g L -1 ) at under 75% FC significantly shown (6 cm). In contrast, under 75% FC, the lowest FL (3.50 cm) was obtained in the plants treated with 100 ml L -1 Br and 1 g L -1 nano zinc (Fig 1and Fig 7). fruit weight The lowest fruit weight of strawberry (13.63 g) was observed under 25% FC combined with 200 ml L -1 Br and 2 g L -1 nano zinc, followed by 25% FC combined with 100 ml L -1 Br and 1 g L -1 nano zinc (13.70 g). Whereas, the highest fruit weight (20.53 g) was observed under 75% FC and 400 ml L -1 Br in combination with 3 g L -1 nano zinc (Fig 1). Fresh weight of vegetative The fresh weight of vegetative tissues significantly increased under all applied treatments ( P < 0.01). Comparison of the mean FW. V showed that the highest value (95.3 g) was obtained under 75% FC combined with 400 g L -1 Br and 2 g L -1 nano zinc, whereas the lowest value (74.83 g) was recorded under 25% FC in combination with the lowest concentrations of Br and nano zinc (Fig 1 and Fig 7). fruit firmness The interaction among the three factors was not statistically significant. However, the interaction between nano zinc and irrigation limitation, as well as the interaction between Br and nano zinc, indicated that the most fruit firmness obtained under the application of 3 g L -1 nan zinc and 100 g L -1 Br in combination with 75% FC and 1 g L -1 nan zinc and 200 g L -1 Br. The least fruit firmness under the interaction between drought stress and nano zinc (1.72 N) was observed in the treatment with 25% FC and 1 g L -1 of nano zinc. Similarly, the interaction between Br and nano zinc (1.79 N) resulted in the lowest firmness in the treatment with 25% drought stress and 1 g L -1 of nano zinc (Fig 1). 3.2. Effect of Br and nano Zinc on yield parameters of strawberry fruit TSS. Brix The application of Br (200 ml L -1 ) and nano zinc (2 g L -1 ) under FC (25%) enhanced the accumulation of TSS (11.6 %) in the fruit. However, under 75% drought stress conditions, foliar application of 100 ml L -1 Br and 1 g L -1 of nano zinc led to decreases in TSS equal to 6.1% in the fruit (Fig 2). Titratable Acidity The results indicated that the fruits treated with 75% FC, 100 ml L -1 Br and 2 g L -1 nano zinc demonstrated a significant increase in TA (1.31%). In contrast, the lowest TA (0.8%) was observed in fruits subjected to 75% FC, 100 ml L -1 Br and 3 g L -1 nano zinc (Fig 2). Total sugar The results revealed that the content of TS had the highest value (1.137 gr gr -1 FW) in fruit subjected to mild FC (75%) with the application of 400 ml L -1 Br combined with 2 g L -1 nano Zn. The application of 200 ml L -1 Br and 1 g L -1 nano zinc under severe FC (25%) reduced the TS by 0.5 % (Fig 2). Total Chlorophyll content In this study, all treatments increased the total chlorophyll content compared to the control (Fig 2). Interestingly, the highest total chlorophyll content under drought stress conditions was observed in the treatment with 75% drought stress and 1 g L -1 of nano zinc combined with 200 ml L -1 Br (316.33 μg cm -2 ). while the lowest content was found in the control (213.33 μg cm -2 ). TPC (Phenol) and TFC (flavonoids) The highest levels of TPC (7.20 mg g -1 FW) and TFC (32.33 mg g -1 FW) were observed under 50% FC and 25% FC combined with 200 ml L -1 Br and the lowest concentrations of nano zinc. The lowest TFC (14.80 mg g -1 FW) was recorded under mild drought stress and nano zinc combined with 200 ml L -1 Br, followed by mild drought stress and Br combined with 3 g L -1 nano zinc. In contrast, the lowest TPC (2.50 mg g -1 FW) was obtained in the control (Fig 2). Proline content As shown in Fig. 2, the proline content increased under the 25% FC treatment combined with the concentrations of 2 ml L -1 Br and 2 g L -1 nano zinc (20 mg g -1 FW), while the lowest proline content (15.46 mg g- 1 FW) was observed in the control treatment (Fig 2). Photosystem II Efficiency (Fo, Fm, Fv/Fm) Fig 2 shows the Fo in fruits under different treatments. The highest Fo was observed in fruits treated with 75% FC and 400 ml L -1 Br in combination with 3 g L -1 nano zinc. The lowest Fo was recorded under moderate drought stress and Br in combination with 3 g L -1 nano zinc. The lowest Fo was shown in moderate concentrations of drought stress and Br in combination with 3 g L -1 nano zinc. Interestingly, this treatment exhibited the highest value for Fm and Fv/Fm. The lowest Fm was recorded across all treatments with moderate concentrations of drought stress, Br, and nano zinc. The lowest Fv/Fm was found after 50% FC and 100 ml L -1 Br treatment in combination with 2 g L -1 nano zinc. Correlation analysis In order to investigate the interrelationships between physiological, biochemical, and phytochemical traits, as well investigated treatments, Pearson’s correlation coefficients were calculated, and the results are presented (Fig 3 and Fig 4). The strongest positive correlation was observed between TPC and Pro with a correlation coefficient of r = 0.90, indicating a very strong association between these two traits. This strong relationship suggests that proline accumulation under stress conditions may be accompanied by an increased biosynthesis of phenolic compounds, both of which play key roles in plant defense mechanisms. Additionally, significant positive correlations were found between TPC and TFC (r = 0.86) and as well as between TFC and Pro (r = 0.82), indicating that these secondary metabolites may exhibit similar responses to environmental stress. A moderately strong positive correlation was found between fruit weight and FWV (r = 0.65). Furthermore, both traits exhibited strong positive correlations with TS (r = 0.70), indicating that increased fruit biomass is associated with higher accumulation of soluble compounds such as sugars. These results imply that fruit size and composition are closely linked and can serve as important indicators of fruit quality. One of the most striking findings was the very strong negative correlation between Fo and the Fv/Fm ratio (r = -0.94). Since the Fv/Fm ratio represents the maximum quantum efficiency of photosystem II (PSII), its decrease, accompanied by an increase in Fo, implies possible damage to PSII or reduced photosynthetic efficiency . This correlation suggests that Fo and Fv/Fm are highly responsive to environmental stress. PCA Biplot To explore the overall variation among the samples and identify patterns based on measured traits, a Principal Component Analysis (PCA) biplot was constructed (Fig 4). The first two principal components, PC1 and PC2, accounted for 39.9% and 25.6% of the total variation, respectively, cumulatively explaining 65.5% of the observed variance. The PCA revealed clear differentiation among the 27 samples, which were grouped into nine distinct clusters. This clustering reflects the similarity in their biochemical and morphological traits. Cluster 1 lies to the right and exhibits partial alignment with FF, Fv/Fm and Fm. Cluster 2 are positioned in the upper left quadrant and appear moderately associated with TA and Fo, indicating possible increases in TA and FF. These traits may contribute to extended shelf life and improved textural quality under the corresponding treatments. Samples from Cluster 3 are grouped in the lower central part of the plot and show a weak association with the trait vectors. Their positioning suggests a neutral or minimal influence on most of the measured traits. Cluster 4 are grouped in the top central part of the plot. Their greater distance from most trait vectors indicates comparatively lower levels of the measured traits than those in other clusters. On the other hand, Cluster 5, located on the right-hand side of the plot, showed a strong association with traits such as FW, FWV, and TS, suggesting enhanced growth and sugar accumulation under the corresponding treatments. Cluster 6 is located in the upper left quadrant, slightly below Cluster 2, and shows some association with TA and Fo. Cluster 7 are located near the center of the biplot with a slight orientation toward Fm, suggesting moderate photosynthetic efficiency or chlorophyll-related responses. This single sample lies in the upper-right region and is closely associated with F.F (fruit firmness), indicating a marked increase in fruit texture quality. This suggests that the treatment applied had a unique and strong effect on fruit firmness. Samples in Cluster 9 are positioned to the left of the origin and below the horizontal axis, showing a strong association with TPC, Pro, and TSS.Brix. This indicates a greater accumulation of these biochemical compounds in the samples . The length of the vectors implies the magnitude of their influence. For instance, traits like TFC, TSS, and Pro have longer vectors, indicating a stronger influence on the differentiation of the samples. Boxplot Analysis Chlorophyll content showed significant variation across treatments. Treatments with low stress had higher median values with narrow interquartile ranges (IQR), indicating stable plant responses under optimal conditions (Fig 6). However, with increasing drought stress, data dispersion increased, reflecting variable plant responses to stress. The control treatment showed the lowest median and the least variability. Fruit firmness exhibited minimal variation across treatments (Fig 5). Median values consistently remained around 2 N, with narrow interquartile ranges (IQRs) and low data dispersion, suggesting that treatments had little effect on this trait and that plant responses were stable. An increase in treatment intensity, particularly with higher BR and nano zinc levels, was associated with a significant rise in fruit length (Fig 5). Treatments under low stress exhibited narrower interquartile ranges (IQRs), whereas those under moderate to high stress showed wider IQRs, indicating greater variability in fruit length responses. Control plants had the lowest median and exhibited limited variability. Median fruit weight was highest in treatments with elevated BR and nano zinc levels. These treatments also exhibited moderate to high data dispersion, reflecting diverse fruit weight responses among plants. Flavonoid content increased with treatment intensity, with the highest median values observed in late-stage treatments. These treatments exhibited wide interquartile ranges (IQRs) and the presence of outliers, indicating substantial variation in plant oxidative stress responses. Control plants showed lower median values and minimal variability. Treatments with low to moderate drought stress showed higher median Fm values, indicating better PSII efficiency. Data dispersion was greater in low-stress treatments and reduced under high stress and control, reflecting the negative impact of stress on photosynthetic electron transport. The Fv/Fm ratio decreased with increasing stress intensity. Medians were higher in low-stress treatments and lowest in the control. Mid-stress treatments showed wider IQRs and some outliers, indicating stress-induced damage to photosystem II. Highest median total plant weight was observed in low-stress treatments. Significant reductions occurred under high stress with increased data dispersion, suggesting heterogeneous plant growth responses to stress. Total phenol content increased under high stress treatments with broad IQRs, indicating enhanced antioxidant defense responses and variability. Control plants showed moderate median values with less dispersion. Proline exhibited the greatest variability among the measured traits. Mid-stress treatments had the highest median values and the widest interquartile ranges (IQRs). Control plants showed the lowest median and least variability, highlighting proline accumulation as a key stress response. The median TA was highest in high-stress treatments. These treatments also showed greater range and variability, indicating diverse plant responses. Low-stress and control treatments had lower median TA and less variability. Ts content had higher median values under low-stress treatments and decreased with increasing stress. Intermediate treatments exhibited more variability, while the control showed relatively high median with limited range. Median TSS was highest in late-stage treatments, showing an increasing trend across treatments. Data dispersion was generally low with narrow interquartile ranges (IQRs), reflecting a uniform plant response in this osmotic parameter. Discussion Water limitation is known to influence plant functions, significantly disrupting membrane stabilization and reducing PS-II efficiency. The main effects of drought stress include excessive production of reactive oxygen species (ROS), leading to oxidative stress and cellular damage [ 36 , 37 ]. To alleviate damage, many studies show that zinc nanoparticles (Zn-NPs) are beneficial in improving plant stress tolerance. Stability and great potential to improve crop production have been shown with Zn-NPs under abiotic stress [ 37 , 38 ]. Nano zinc preserved the integrity of a membrane and reduced the loss of vital osmolytes through the suppression of MDA and H₂O₂ accumulation [ 39 , 40 ]. Nano zinc has demonstrated a potential protective role to cellular components, but there is also indication that it can enhance phenolic compound biosynthesis when the plant is under drought stress. Exogenous applications of nano zinc at concentrations of 25 and 50 mg/L led to a remarkable increase in accumulated phenolic compounds under drought stress via exogenous Nano Zinc [ 40 ]. Flavonoids and phenolic compounds are secondary metabolites that are important and part of the phenylpropanoid pathway. The phenylpropanoid pathway exists as a parallel pathway to the true phenolic and flavonoid biosynthetic pathways. The phenylpropanoid pathway operates by processing phenylalanine, which then travels to phenylalanine ammonia-lyase to trans-cinnamic acid. Again, both of things (i.e., trans-cinnamic acid and phenylalanine) are documented in the above sections and have lucrative antioxidant activity and inherent ability to scavenge reactive oxygen species (ROS), can complex with metals, and the mechanisms help the plant with abiotic tolerances [ 23 , 41 ]. In publication by Yaqoob et al. [ 42 ] they outline additional secondary anti-ROS detoxifications mechanisms that secondary metabolites (i.e.: flavonoids and phenolic compounds) interact with to complete ROS scavenging. Antioxidant phytochemicals such as flavonoids and phenolic compounds with deteriorate oxidative stress and subsequently magnify plant tolerance [ 41 ]. In addition, congruent with the present study findings, extraneous studies have demonstrated that BRs increase flavonoids accumulation in Glycine max [ 43 ], Camellia sinensis [ 44 ], Vitis vinifera [ 45 ], and Oryza sativa [ 41 ]. Thus, various studies have argued that BRs have enhanced plant stress tolerance through modification of the phenylpropanoid metabolic pathway [ 23 ]. Foliar application of Br and nano zinc has been widely demonstrated as an effective approach to enhance plant morphological traits and physiological responses under abiotic stress conditions [ 46 ]. The highest fruit weight and length under high concentrations of Br and nano zinc during moderate drought indicate that these treatments stimulate fruit development via promotion of cell division and expansion. Nano zinc reinforces these processes by participating as a cofactor in the enzymatic reactions that are involved in energy metabolism and protein synthesis [ 47 , 48 ]. Severe drought is associated with elevated oxidative stress and depressed photosynthetic efficiency that hinders growth in plants by reducing the amounts of assimilates for developing fruits [ 49 , 50 ]. Development of greater fresh weight of vegetative tissues occurred when exposed to a 75% FC factors with moderate Br and low nano zinc indicating protection and growth stimulation. Nano zinc’s enhances uptake and mobility within the plant, supporting key metabolic processes essential for cell division and expansion. Brs regulate gene expression linked to cell proliferation, increasing biomass production even under stress conditions. This synergistic interaction improves osmotic adjustment and maintains cell turgor, enabling growth despite water limitation [ 51 – 53 ]. The highest fruit firmness observed under 3 g L⁻¹ nano zinc combined with moderate FC (50%) can be attributed to the essential role of zinc in maintaining cell wall integrity. Zinc acts as a cofactor for enzymes such as pectin methylesterase, which help stabilize and strengthen cell wall structure [ 54 ]. Brs enhance this effect by activating biosynthetic pathways related to cellulose and lignin deposition, further reinforcing cell walls [ 55 ]. Collectively, these compounds alleviate drought-induced oxidative stresses, a common cause of cell wall degradation, and loss of fruit firmness [ 56 ]. There was the least firmness in the severe drought + low nano-zinc treatment, likely due to inadequate enzymatic protection against oxidative injury [ 57 ]. The variation in photosystem II photochemical efficiency with nano-zinc and brassinosteroids indicates that at least some protection against drought-induced oxidative injury was provided to the photosynthetic apparatus. Among other functions, brassinosteroids can be proantioxidant and stimulate antioxidant defense mechanisms, like the enzymes that scavenge reactive oxygen species (ROS), such as superoxide dismutase (SOD) and catalase (CAT), and affect chloroplast stability [ 58 ], while the nano-zinc treatment was able to improve chloroplast-linked enzyme activities and preserve chlorophyll content [ 49 ]. The higher chlorophyll content in drought-stressed plants receiving nano-zinc and brassinosteroids also suggested that pigment stability/synthesis rates were improved, and were also likely necessary for maintaining photosynthesis during the stress [ 54 ]. The higher TSS content is likely due to the accumulation of osmolytes, which help maintain cellular osmotic balance during drought [ 59 ]. Previous studies on strawberries have similarly documented increased sugar accumulation and improved fruit quality under drought conditions following Zn and brassinosteroid (Br) treatments [ 60 ]. Variations in titratable acidity suggest shifts in organic acid metabolism modulated by treatments influencing Krebs cycle enzymes [ 61 ]. Proline accumulation increased significantly in untreated or low-treatment plants, consistent with its role in osmoprotection and ROS scavenging [ 62 ]. To investigate the interrelationships among physiological, biochemical, and phytochemical traits, Pearson’s correlation coefficients were calculated and visualized through a correlation heat map. The strongest positive correlation was found between total phenolic content (TPC) and proline (Pro) with r = 0.90, indicating a very strong association. This suggests that under stress conditions, proline accumulation might be linked to increased biosynthesis of phenolic compounds, both of which are crucial components of plant defense mechanisms [ 63 ]. Additionally, significant positive correlations were observed between TPC and total flavonoid content (TFC) (r = 0.86), as well as between TFC and Pro (r = 0.82). These results imply that these secondary metabolites may respond similarly to environmental stresses, highlighting their potential coordinated roles in stress adaptation. A moderately strong positive correlation between fruit weight and fruit water volume (FWV) (r = 0.65) was noted. Both traits also showed strong positive correlations with total soluble solids (TS) (r = 0.70), suggesting that larger fruits tend to accumulate higher levels of soluble compounds like sugars. This close relationship underscores the link between fruit size and composition, which could be critical indicators of fruit quality [ 64 ]. One of the most notable findings was the very strong negative correlation between initial fluorescence (Fo) and the maximum quantum efficiency of photosystem II (Fv/Fm) (r = -0.94). Since Fv/Fm reflects PSII efficiency, an increase in Fo alongside a decrease in Fv/Fm likely indicates damage to PSII or a reduction in photosynthetic performance, marking these parameters as sensitive indicators of environmental stress [ 65 ]. Conclusion The results of this work illustrate the high potential of nano zinc and Br in enhancing the physiological performance, stress tolerance and fruit quality of strawberry plants under drought stress conditions. Under moderate drought stress, the combination treatments of nano zinc and Br improved cell wall integrity, osmotic adjustments and photosynthetic efficiency in strawberry plants. These treatments were able to modulate fundamental metabolic pathways, antioxidant capacity, mitigate oxidative stress and allow physiological activities to function continuously. Correlation analysis showed significant correlation relationships among measured phytochemical and physiological traits. Strong positive correlations noted for proline, phenolics and flavonoids suggested a synergistic action in stress tolerance and protection. In addition, many traits based on Fo and Fv/Fm are reliable for PSII functioning and drought-stress damage. These results are consistent with a long list of tagging the positive aspects of the combined effects of nano zinc with plant hormones such as brassinosteroids to improve crop resilience and drought stress tolerance. Declarations Authors’ contributions Heidar Meftahizade conceived the original idea, managed project and all results, and contributed to the final version. WarqaaMuhammed ShariffAl-Sheikh carried out the experiments. Mohamed Baqer Hussine Almosawi carried out the experiments. Masoumeh Eskandariperformed data analysis and Neda Tariverdizadehwrote the primary manuscript. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. 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15:42:51","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":173104,"visible":true,"origin":"","legend":"","description":"","filename":"fc9d9ca391f643f9bc85fd1aeb28126a1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7378337/v1/da10f2dd300ce5b0fab506a7.xml"},{"id":93608026,"identity":"bf49959d-74be-4297-be44-2a198e08fac3","added_by":"auto","created_at":"2025-10-15 15:50:51","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":184045,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7378337/v1/4d38d015f4eb9e32aef50754.html"},{"id":93607717,"identity":"617707af-cac1-4df9-8de7-3a5613ca0504","added_by":"auto","created_at":"2025-10-15 15:42:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":102115,"visible":true,"origin":"","legend":"\u003cp\u003eyield traits of strawberry under combined treatments of irrigation level, nano zinc, and brassinosteroid.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7378337/v1/65cedbb712a62a08df43b113.png"},{"id":93607721,"identity":"9ba50541-5f98-40da-a124-45e2a08d9e57","added_by":"auto","created_at":"2025-10-15 15:42:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":226352,"visible":true,"origin":"","legend":"\u003cp\u003ephysiological and biochemical traits of strawberry under combined treatments of irrigation level, nano zinc, and brassinosteroid.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7378337/v1/053b9d6486aff6c46c24a2f6.png"},{"id":93608012,"identity":"dddb38ab-8e17-49cd-ba75-ababad8af27b","added_by":"auto","created_at":"2025-10-15 15:50:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":121434,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation heat map of Morphological, Physiological, and Biochemical Traits in Strawberry under Combined Irrigation level, Nano Zinc, and Brassinosteroid Treatments.Color scale represents correlation coefficients from −1 to +1.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7378337/v1/e4cd46f12a5177398445e76e.png"},{"id":93608014,"identity":"f60f3218-04fd-4e79-8f44-ea16f9482064","added_by":"auto","created_at":"2025-10-15 15:50:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67677,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of Morphological, Physiological, and Biochemical Traits in Strawberry under Combined Irrigation level, Nano Zinc, and Brassinosteroid Treatments.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7378337/v1/f478c7af24125cd5b632adc5.png"},{"id":93609167,"identity":"e69fc2c7-0ff6-4d4c-9f6a-5a2f5ee90678","added_by":"auto","created_at":"2025-10-15 15:58:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":135836,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 4\u003c/strong\u003e. Principal Component Analysis (PCA) Biplot of Strawberry Treatments Grouped by Cluster Analysis Based on Morphological, Physiological, and Biochemical Traits\u003c/p\u003e","description":"","filename":"04.png","url":"https://assets-eu.researchsquare.com/files/rs-7378337/v1/8fc01f5dc90806a1d9bdf1ef.png"},{"id":93607722,"identity":"677fcdf0-05c9-48eb-bc63-69e352272fc6","added_by":"auto","created_at":"2025-10-15 15:42:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":134752,"visible":true,"origin":"","legend":"\u003cp\u003eFig 5. Box Plot of Yield traits of strawberry under combined treatments of drought stress, nano zinc, and brassinosteroid.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7378337/v1/eb12d6e214b0b17911e10325.png"},{"id":93607725,"identity":"21ec476b-4a6c-4eb0-9e34-ca5bdb8e898f","added_by":"auto","created_at":"2025-10-15 15:42:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":298112,"visible":true,"origin":"","legend":"\u003cp\u003eFig 6. Box Plot of physiological and biochemical traits of strawberry under combined treatments of drought stress, nano zinc, and brassinosteroid.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7378337/v1/34be0c2cb9e031925b0175e5.png"},{"id":93607727,"identity":"cdd1633d-cf30-48d0-8618-8721d4fb9e93","added_by":"auto","created_at":"2025-10-15 15:42:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":529283,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 7\u003c/strong\u003e. Samples of pots and Stages of strawberry fruit formation under brassinosteroid and zinc nanoparticles treatments\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7378337/v1/b08ff2b8b3bb9057be03c97a.png"},{"id":94641045,"identity":"4064dac3-5d21-42ec-962b-1b8523075f82","added_by":"auto","created_at":"2025-10-29 07:50:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2564801,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7378337/v1/5a367515-3c03-4bbc-8f09-220bce426d10.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improving quality and water tolerance in strawberry through combined application of Nano Zinc and Brassinosteroids","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eStrawberry (\u003cem\u003eFragaria\u003c/em\u003e \u0026times; \u003cem\u003eananassa\u003c/em\u003e Duch.), belonging to the genus \u003cem\u003eFragaria\u003c/em\u003e and the Rosaceae family, and often referred to as the Queen of Fruits, is rich in vitamin C and a wide range of antioxidants and phenolic compounds that contribute to cardiovascular health and the regulation of blood glucose levels [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDrought is a growing environmental challenge that is known to decrease crop yield, and strawberries [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] are no exception. Strawberries are particularly vulnerable to water limitations due to their root structure, large leaf area, and high-water content [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Ultimately, the resultant stress from the drought leads to decreased growth, chlorophyll loss, photosynthesis reduction, and oxidative stress [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The reduction of fruit yield and quality due to drought will depend on cultivar tolerance or sensitivity and the definition of stress: nature, intensity and duration [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Ultimately, the duration of drought stress leads to an excess generation of reactive oxygen species (ROS) or and unequal generation between ROS and the antioxidant systems. The ROS over generation leads to potentially oxidative damage to the photosynthetic apparatus [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Drought and other stressors in combination will create a negative physiological response of the plant and cause various limitations to the uptake of nutrients, photosynthesis, growth, and consequently yields [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e and \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Tolerant plants cope with these effects by regulating photosynthesis, accumulating osmolytes and inducing genes encoding antioxidants, closed their stomata and increased stomatal resistance to decrease transpiration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Reactive oxygen species (ROS) that develop under stress result in cellular damage, including lipid peroxidation, protein degradation, and enzyme inactivation. Proline not only scavenges free radicals but its role in oxidative stress marks it as critical in conferring stress tolerance as an osmolyte. Drought-stressed plants manage oxidative damage with the stimulation of antioxidant enzymes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn a study conducted by Zahedi et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the physiological and biochemical responses of two strawberry cultivars were evaluated under four different drought stress levels (100%, 75%, 50% and 25%) of field capacity. The resulted of this research indicated that drought stress led to a significant decrease in chlorophyll content, carotenoids, and phenolic compounds in both cultivars. In the study, three strawberry cultivars (California, Earlibrite, and Sweet Charlie) were evaluated under four different irrigation levels corresponding to 100%, 75%, 50%, and 25% of field capacity. The results showed that increasing drought severity significantly reduced growth parameters such as plant height, leaf area, fruit weight, as well as leaf gas exchange and relative water content. On the other hand, drought stress led to an increase in total soluble solids (TSS), malondialdehyde (MDA), anthocyanins, and proline, indicating the activation of the plant's defensive responses to oxidative and osmotic stress [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious studies have shown that the combined application of zinc oxide nanoparticles (ZnO NPs) and melatonin (MT) can improve growth and drought tolerance in strawberry plants. This combination enhances nutrient uptake, increases chlorophyll content, and boosts antioxidant enzyme activities, thereby reducing oxidative damage caused by drought stress. Experimental results demonstrated increased shoot and root length, fruit biomass, and bud number in plants treated with ZnO NPs and MT. These findings highlight the potential of this approach to enhance strawberry resilience under drought conditions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. At this time, multiple technologies have been reported to enhance plant tolerance or resistance to environmental stresses (i.e., elicitors and nanotechnologies) which boost crop yield and quality [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Use of plant growth stimulators, such as brassinosteroids (BRs), is an important factor for the improvement of crop quality and yield. Not only do BRs regulate physiological processes like plant growth, photosynthesis, flowering, and fruit set, they are also able to enhance plant tolerance or resistance to a wide range of abiotic stress factors including drought and oxidative stress [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. MART\u0026Iacute;NEZ-P\u0026Eacute;REZ et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] explained that BRs promote vegetative growth by modifying the mechanical properties of the cell wall, increasing its plastic extensibility. Studies have shown that BRs significantly increase yields in various crops such as tomato [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], strawberry [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. BR levels are crucial for promoting plant growth and productivity, especially under environmental stresses like drought [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, modulation of endogenous BR levels has been reported to improve crop quality and performance under stress conditions [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Khatoon et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] assessed the plant growth regulator brassinosteroids to pea foliar on strawberry, brassinosteroids 0.2 ppm were applied during three phenological stages, vegetative, flowering, and fruiting we found this application the most response in the yield potential of the strawberry plant. The result of the study indicates that brassinosteroids offering great potential in improving growth parameters that improve fruit yield and increase growth rate through to the produce. Nanotechnology coming up with different options to minimize abiotic stress on production. The unique properties of zinc oxide nanoparticles to modulate antioxidant enzyme processes, promote nutrient uptake, and enhance the photosynthetic process [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], have provided keen interest for their use in the field. Nano zinc has a prominent role in potential plant alleviate abiotic stresses through regulating plant water balance and stabilizing cellular osmotic levels to improve stress tolerance from cold, drought, and salinity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The two most common methods of applying nano zinc to plants are root application and foliar application methods. In foliar application, zinc oxide nanoparticles are sprayed onto the plant leaf surface, when the nanoparticles are absorbed in through the stomata and cuticle, and once inside the plant, the nanoparticles can be translocated through the plant through the phloem sieve tubes resulting in systemic dispersal [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThere has been significant research on the effects of brassinosteroids and nano zinc individually, but nothing has been published on the effects they may have together on alleviating drought stress. Keeping in mind the need to improve strawberry production, this study is looking to examine the individual and combined influences of drought stress, BRs, and nano zinc on the physiological, biochemical and growth characteristics of strawberry plants. Drought stress is known to negatively impact the growth and physiological performance of strawberries, leading to reduced yield and water use efficiency. However, the application of brassinosteroids (BRs) and nano zinc, either individually or in combination, has the potential to mitigate these adverse effects by enhancing antioxidant enzyme activities and improving plant stress tolerance. A better understanding of these effects could facilitate the development of effective approaches to improve crop tolerance and maximize water use efficiency.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eThe treatments were carried out at the research greenhouse\u0026ensp;of Ilam University on 2024-2025. The strawberry\u0026apos;s \u0026lsquo;Sabrina\u0026rdquo; cultivar was provided from the Royal Green Agricultural Company of Kurdistan, Soil with sandy loam texture (suitable for strawberry cultivation based on soil test data according to \u003cstrong\u003eTable 1\u003c/strong\u003e) was used as culture medium. at the end of September, well-rooted strawberry daughter plants were selected for similar size. Then, the seedling was stored in a cold storage room at 4\u0026deg;C for 240 hours to determine the need for cold storage.\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\" valign=\"top\" style=\"width: 624px;\"\u003e\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eThe soil physical and chemical characteristics of the experimental field\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003eTexture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003eClay (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49px;\"\u003e\n \u003cp\u003eSilt\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003eSand (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eK (mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003eP (mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003eN\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003eT.N.V (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003eEC (dS/m)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003eSandy- Loamy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e342\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e9.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 59px;\"\u003e\n \u003cp\u003e7.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e1.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eTo conduct this experiment, 64 plastic pots (4\u0026times;4\u0026times;4) with a volume of 50 kg were filled with a mixture of 60% cocopeat and 40% perlite, and three strawberry plants of the Sabrina cultivar were planted inside each pot (Fig 7). The pots were divided into four rows as four experimental blocks. Initially, in order to establish the plants, all pots were fed with Hoagland nutrient solution for one week. \u0026nbsp;The experiment was carried out as a factorial experiment in a randomized complete block design with three replications. The first factor included water deficiency at 4 levels (100% of FC, 75% of FC, 50% of FC, and 25% of FC), the second factor included brassinosteroid at 4 levels (control, 100, 200, and 400 ml/liter), and zinc nanoparticles at 4 levels (control, 1, 2, and 3 g/liter) were treated. The nanoparticles in this study were obtained from Nanosani Company, the characteristics of which are given in \u003cstrong\u003eTable 2\u003c/strong\u003e. In this study, ZnO (Zinc Oxide) was used to synthesize zinc nanoparticles. Spraying solutions of various concentrations of zinc nanoparticles, after the initial pruning, from the fourteenth to the eighteenth week, once a week (5 times) is performed.\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Structural properties of zinc nanoparticles\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eStructural properties of zinc nanoparticles \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ezinc nanoparticles\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eThermogravimetric analysis (TGA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.2\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eInductively coupled plasma (ICP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.8\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003esize\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e20-31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePurity percentage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eActive surface (g/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e50-135\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e24-epibrassinolide EBL (Sigma, USA) solution was readied by ethanol. To increase regulators uptake, Tween-20 (0.05%) was used as a surfactant [27]. It was sprayed during experimental periods.\u003c/p\u003e\n\u003cp\u003eAccording to \u003cstrong\u003eEquation (1),\u003c/strong\u003e the soil moisture content was measured at 100% of FC based on the difference between the soil weight after drainage (FCW), and soil weight after drying (DW). Finally, fresh seedlings were planted. When the weight of the pot containing soil and plants were lower than a certain level (measurements were performed daily on all pots based on the FC assigned for each treatment) irrigation was repeated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEquation\u0026nbsp;\u003c/strong\u003e1: Field capacity (FC): \u0026nbsp;soil weight in field capacity (FCW)\u0026minus;weight of dry soil (DW)/ weight of dry soil (DW).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of physiological, biochemical, and morphological traits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFruit weight, fruit length, and fresh weight of vegetative tissues measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the experiment, the fruit weight was measured using a digital scale with an accuracy of 0.001 grams, the fruit length was measured using a digital caliper (150-1108).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFruit firmness measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eIt was evaluated with a penetrometer\u0026reg; Fruit Pressure Tested Wagner FT 327, made in the USA with a 2.3 mm thick tip at the height of the equatorial diameter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTSS (Brix) and total Sugar content measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTSS content of fruit juice was measured using an Abbe refractometer (model: CETI, Belgium). The TSS values were expressed as % Brix, following the procedure described by Shiukhy [28].\u003c/p\u003e\n\u003cp\u003eTS were quantified using the anthrone method, as described by Mirshekari et al. [29]. For this analysis, 0.2 mL of concentrated fruit extract was mixed with 3 mL of anthrone reagent, which was prepared by dissolving 150 mg of anthrone in 100 mL of 13 M sulfuric acid. The mixture was then incubated in a water bath at 100 \u0026deg;C for 20 minutes. After cooling to room temperature, the absorbance was measured at 620 nm using a spectrophotometer. The total soluble sugar content was calculated using a glucose standard calibration curve and expressed as mg glucose per gram of fresh weight (Micro gr/gr FW).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTitratable acidity (TA) measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe titratable acidity was determined in triplicate with the method of Horwitz and Latimer [30]. 10 mL of strawberry juice per sample were taken. The sample was transferred to an Erlenmeyer flask and 4 drops of phenolphthalein 1% solution were added (0.5 g of phenolphthalein plus 70 mL of ethyl alcohol, calibrated to 100 mL with distilled water). The sample was titrated with a 0.1 N NaOH solution until the purplish color change was maintained for one minute. The titratable acidity was expressed as a percentage of citric acid and was calculated using the following formula:\u003c/p\u003e\n\u003cp\u003e% acid = V NaOH \u0026times; N NaOH \u0026times; meq acido /V \u0026times; 100.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal chlorophyll content measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhotosynthetic pigments were determined following the method of Tariverdizadeh et al. [31]. Fresh leaf samples (0.5 g) were ground and extracted in 100 ml of 80% acetone. The mixture was then centrifuged at 13,552 \u0026times; g for 10 minutes at 4\u0026deg;C, and the supernatant was collected for analysis. Absorbance readings were taken at wavelengths of 663, 646, and 470 nm using a spectrophotometer. Chlorophyll a, chlorophyll b, and carotenoid contents were calculated using the following equations, and results were expressed as mg per gram of fresh weight (mg g⁻\u0026sup1; FW):\u003c/p\u003e\n\u003cp\u003eChla (mg\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eml\u003csup\u003e-1\u003c/sup\u003e) =12.25A663-2.79A646\u003c/p\u003e\n\u003cp\u003eChlb (mg\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eml\u003csup\u003e-1\u003c/sup\u003e) =21.50A646-5.10A663\u003c/p\u003e\n\u003cp\u003eTChl=Chla + CHlb\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal phenol content (TPC) and total flavonoid content (TFC) measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total phenols were determined according to the Folin- Ciocalteu\u0026rsquo;s procedure [32]. The absorption of these metabolites was determined at 725 nm. Flavonoids levels were spectrophotometrically determined by modified method of Upadhyay and Maier [33]. The fruits samples were prepared with 2 mL acidic methanol (80% methanol with 1% HCl) in the mortar at room temperature for 2 h. The extracts were centrifuged at 1000 \u0026times; g for 15 min and the absorption recorded at 560 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProline content measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProline content in leaves was determined using a colorimetric method based on the protocol of Bates et al. [34]. Approximately 0.5 g of fresh leaf samples were weighed and homogenized in 10 mL of 3% sulfosalicylic acid. The homogenate was centrifuged at 6000 \u0026times; g for 5 minutes to separate the supernatant.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eThen, 2 mL of the supernatant was mixed with 2 mL of glacial acetic acid and 2 mL of ninhydrin reagent, and the mixture was incubated in a water bath at 100\u0026deg;C for 60 minutes. After incubation, the samples were cooled, and 4 mL of toluene was added and thoroughly mixed. The toluene phase was separated, and its absorbance was measured at 520 nm using a UV\u0026ndash;Vis spectrophotometer (Model: UV-1800, Shimadzu, Japan).\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eProline concentration was quantified using a standard proline calibration curve and expressed as milligrams of proline per gram of fresh weight.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of chlorophyll fluorescence parameters (Fo, Fm) and calculation of maximum quantum efficiency (Fv/Fm)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe photosynthesis system equipped with a chlorophyll fluorescence system (LI-6200) was used to measure chlorophyll fluorescence parameters of the second fully expanded leaf. After 30 min of dark treatment, the minimum fluorescence (Fo), the saturated maximum fluorescence (Fm) of dark-adapted were determined under normal light, and each treatment was repeated three times. The maximum quantum yield of photosystem II (FV/FM) was estimated using the OJIP protocol as described by Habibi et\u0026nbsp;al.\u0026nbsp;[35]. Young mature leaves were carefully selected for each experimental treatment. After 20 min in the dark-adapted condition, chlorophyll fluorescence parameters were recorded using a portable fluorometer (Fluorpen FP 100-MAX; Photon Systems Instruments, Drasov, Czech Republic).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData analysis was conducted in R software.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eA variance analysis (ANOVA) using the F-test was conducted to evaluate the effects of treatments at each sampling interval, with means compared using Tukey\u0026rsquo;s test at the 5% significance level. Principal component analysis (PCA) was applied to interpret the response patterns. Additionally, in this study, the relationships among the evaluated traits were analyzed using the Pearson correlation coefficient were employed to identify relationships between parameters with the ggplot2 package in R. Prior to combined analysis, Levene\u0026rsquo;s test for homogeneity of variances was conducted.\u0026nbsp;\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Effect of Br and nano Zinc on yield parameters of strawberry fruit\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFruit length\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe application of drought stress, Br and nano zinc caused a statistically significant difference in FL (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). maximum FL in fruits treated with both Br (400 ml L\u003csup\u003e-1\u003c/sup\u003e) and nano zinc (3 g L\u003csup\u003e-1\u003c/sup\u003e) at under 75% FC significantly shown (6 cm). In contrast, under 75% FC, the lowest FL (3.50 cm) was obtained in the plants treated with 100 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr and 1 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc (Fig 1and Fig 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003efruit weight\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe lowest fruit weight of strawberry (13.63 g) was observed under 25% FC combined with 200 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr and 2 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc, followed by 25% FC combined with 100 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr and 1 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc (13.70 g). Whereas, the highest fruit weight (20.53 g) was observed under 75% FC and 400 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr in combination with 3 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc (Fig 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFresh weight of vegetative\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fresh weight of vegetative tissues significantly increased under all applied treatments (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Comparison of the mean FW. V showed that the highest value (95.3 g) was obtained under 75% FC combined with 400 g L\u003csup\u003e-1\u003c/sup\u003e Br and 2 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc, whereas the lowest value (74.83 g) was recorded under 25% FC in combination with the lowest concentrations of Br and nano zinc (Fig 1 and Fig 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003efruit firmness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe interaction among the three factors was not statistically significant. However, the interaction between nano zinc and irrigation limitation, as well as the interaction between Br and nano zinc, indicated that the most fruit firmness obtained under the application of 3 g L\u003csup\u003e-1\u003c/sup\u003e nan\u0026nbsp;zinc and 100 g L\u003csup\u003e-1\u003c/sup\u003e Br in combination with 75% FC and\u0026nbsp;1 g L\u003csup\u003e-1\u003c/sup\u003e nan\u0026nbsp;zinc and 200 g L\u003csup\u003e-1\u003c/sup\u003e Br. The least fruit firmness under the interaction between drought stress and nano zinc (1.72 N) was observed in the treatment with 25% FC and 1 g L\u003csup\u003e-1\u003c/sup\u003e of nano zinc. Similarly, the interaction between Br and nano zinc (1.79 N) resulted in the lowest firmness in the treatment with 25% drought stress and 1 g L\u003csup\u003e-1\u003c/sup\u003e of nano zinc (Fig 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Effect of Br and nano Zinc on yield parameters of strawberry fruit\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTSS.\u0026nbsp;Brix\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe application of Br (200 ml L\u003csup\u003e-1\u003c/sup\u003e) and nano zinc (2 g L\u003csup\u003e-1\u003c/sup\u003e) under FC (25%) enhanced the accumulation of TSS (11.6 %) in the fruit. However, under 75% drought stress conditions, foliar application of 100 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr and 1 g L\u003csup\u003e-1\u003c/sup\u003e of nano zinc led to decreases in TSS equal to 6.1% in the fruit (Fig 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTitratable Acidity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results indicated that the fruits treated with 75% FC, 100 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr and 2 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc demonstrated a significant increase in TA (1.31%). In contrast, the lowest TA (0.8%) was observed in fruits subjected to 75% FC, 100 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr and 3 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc (Fig 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal sugar\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results revealed that the content of TS had the highest value (1.137 gr gr\u003csup\u003e-1\u003c/sup\u003e FW) in fruit\u0026nbsp;subjected to mild FC (75%) with the application of 400 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr combined with 2 g L\u003csup\u003e-1\u003c/sup\u003e nano Zn.\u0026nbsp;The application of 200 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr and 1 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc under severe FC (25%) reduced the TS by 0.5 % (Fig 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal Chlorophyll content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, all treatments increased the total chlorophyll content compared to the control (Fig 2). Interestingly, the highest total chlorophyll content under drought stress conditions was observed in the treatment with 75% drought stress and 1 g L\u003csup\u003e-1\u003c/sup\u003eof nano zinc combined with 200 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr (316.33 \u0026mu;g cm\u003csup\u003e-2\u003c/sup\u003e). while the lowest content was found in the control (213.33 \u0026mu;g cm\u003csup\u003e-2\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTPC (Phenol) and TFC (flavonoids)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe highest levels of TPC (7.20 mg g\u003csup\u003e-1\u003c/sup\u003e FW) and TFC (32.33 mg g\u003csup\u003e-1\u003c/sup\u003e FW) were observed under 50% FC and 25% FC combined with 200 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr and the lowest concentrations of nano zinc. The lowest TFC (14.80 mg g\u003csup\u003e-1\u003c/sup\u003e FW) was recorded under mild drought stress and nano zinc combined with 200 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr, followed by mild drought stress and Br combined with 3 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc. In contrast, the lowest TPC (2.50 mg g\u003csup\u003e-1\u003c/sup\u003e FW) was obtained in the control (Fig 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProline content\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 2, the proline content increased under the 25% FC treatment combined with the concentrations of 2 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr and 2 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc (20 mg g\u003csup\u003e-1\u003c/sup\u003e FW), while the lowest proline content (15.46 mg g-\u003csup\u003e1\u003c/sup\u003e FW) was observed in the control treatment (Fig 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhotosystem II Efficiency (Fo, Fm, Fv/Fm)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig 2 shows the Fo in fruits under different treatments. The highest Fo was observed in fruits treated with 75% FC and 400 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr in combination with 3 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc. The lowest Fo was recorded under moderate drought stress and Br in combination with 3 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc. The lowest Fo was shown in moderate concentrations of drought stress and Br in combination with 3 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc. Interestingly, this treatment exhibited the highest value for Fm and Fv/Fm. The lowest Fm was recorded across all treatments with moderate concentrations of drought stress, Br, and nano zinc. The lowest Fv/Fm was found after 50% FC and 100 ml L\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eBr treatment in combination with 2 g L\u003csup\u003e-1\u003c/sup\u003e nano zinc.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eanalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to investigate the interrelationships between physiological, biochemical, and phytochemical traits, as well investigated treatments, Pearson\u0026rsquo;s correlation coefficients were calculated, and the results are presented (Fig 3 and Fig 4). The strongest positive correlation was observed between TPC and Pro with a correlation coefficient of r = 0.90, indicating a very strong association between these two traits. This strong relationship suggests that proline accumulation under stress conditions\u0026nbsp;may be accompanied by an increased biosynthesis of phenolic compounds, both of which play key roles in plant defense mechanisms.\u0026nbsp;Additionally, significant positive correlations were found between TPC and TFC (r = 0.86) and as well as between TFC and Pro (r = 0.82), indicating that these secondary metabolites may exhibit similar responses to environmental stress. A moderately strong positive correlation was found between fruit weight and FWV (r = 0.65). Furthermore, both traits exhibited strong positive correlations with TS (r = 0.70), indicating that increased fruit biomass is associated with higher accumulation of soluble compounds such as sugars. These results imply that fruit size and composition are closely linked and can serve as important indicators of fruit quality. One of the most striking findings was the very strong negative correlation between Fo and the Fv/Fm ratio (r = -0.94). Since the Fv/Fm ratio represents the maximum quantum efficiency of photosystem II (PSII), its decrease, accompanied by an increase in Fo, implies possible damage to PSII or reduced photosynthetic efficiency\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e This correlation suggests that Fo and Fv/Fm are highly responsive to environmental stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCA Biplot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the overall variation among the samples and identify patterns based on measured traits, a Principal Component Analysis (PCA) biplot was constructed (Fig 4). The first two principal components, PC1 and PC2, accounted for 39.9% and 25.6% of the total variation, respectively, cumulatively explaining 65.5% of the observed variance. The PCA revealed clear differentiation among the 27 samples, which were grouped into nine distinct clusters. This clustering reflects the similarity in their biochemical and morphological traits.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eCluster 1 lies to the right and exhibits partial alignment with FF, Fv/Fm and Fm. Cluster 2 are positioned in the upper left quadrant and appear moderately associated with TA and Fo, indicating possible increases in TA and FF. These traits may contribute to extended shelf life and improved textural quality under the corresponding treatments. Samples from Cluster 3 are grouped in the lower central part of the plot and show a weak association with the trait vectors. Their positioning suggests a neutral or minimal influence on most of the measured traits. Cluster 4 are grouped in the top central part of the plot. Their greater distance from most trait vectors indicates comparatively lower levels of the measured traits than those in other clusters. On the other hand, Cluster 5, located on the right-hand side of the plot, showed a strong association with traits such as FW, FWV, and TS, suggesting enhanced growth and sugar accumulation under the corresponding treatments.\u0026nbsp;Cluster 6 is located in the upper left quadrant, slightly below Cluster 2, and shows some association with TA and Fo. Cluster 7 are located near the center of the biplot with a slight orientation toward Fm, suggesting moderate photosynthetic efficiency or chlorophyll-related responses. This single sample lies in the upper-right region and is closely associated with F.F (fruit firmness), indicating a marked increase in fruit texture quality. This suggests that the treatment applied had a unique and strong effect on fruit firmness. Samples in Cluster 9 are positioned to the left of the origin and below the horizontal axis, showing a strong association with TPC, Pro, and TSS.Brix. This indicates a greater accumulation of these biochemical compounds in the samples\u003cspan dir=\"RTL\"\u003e.\u0026nbsp;\u003c/span\u003eThe length of the vectors implies the magnitude of their influence. For instance, traits like TFC, TSS, and Pro have longer vectors, indicating a stronger influence on the differentiation of the samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBoxplot Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChlorophyll content showed significant variation across treatments. Treatments with low stress had higher median values with narrow interquartile ranges (IQR), indicating stable plant responses under optimal conditions (Fig 6). However, with increasing drought stress, data dispersion increased, reflecting variable plant responses to stress. The control treatment showed the lowest median and the least variability.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eFruit firmness exhibited minimal variation across treatments (Fig 5). Median values consistently remained around 2 N, with narrow interquartile ranges (IQRs) and low data dispersion, suggesting that treatments had little effect on this trait and that plant responses were stable.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eAn increase in treatment intensity, particularly with higher BR and nano zinc levels, was associated with a significant rise in fruit length (Fig 5). Treatments under low stress exhibited narrower interquartile ranges (IQRs), whereas those under moderate to high stress showed wider IQRs, indicating greater variability in fruit length responses. Control plants had the lowest median and exhibited limited variability.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eMedian fruit weight was highest in treatments with elevated BR and nano zinc levels. These treatments also exhibited moderate to high data dispersion, reflecting diverse fruit weight responses among plants.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eFlavonoid content increased with treatment intensity, with the highest median values observed in late-stage treatments. These treatments exhibited wide interquartile ranges (IQRs) and the presence of outliers, indicating substantial variation in plant oxidative stress responses. Control plants showed lower median values and minimal variability.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eTreatments with low to moderate drought stress showed higher median Fm values, indicating better PSII efficiency. Data dispersion was greater in low-stress treatments and reduced under high stress and control, reflecting the negative impact of stress on photosynthetic electron transport.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eThe Fv/Fm ratio decreased with increasing stress intensity. Medians were higher in low-stress treatments and lowest in the control. Mid-stress treatments showed wider IQRs and some outliers, indicating stress-induced damage to photosystem II.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eHighest median total plant weight was observed in low-stress treatments. Significant reductions occurred under high stress with increased data dispersion, suggesting heterogeneous plant growth responses to stress.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eTotal phenol content increased under high stress treatments with broad IQRs, indicating enhanced antioxidant defense responses and variability. Control plants showed moderate median values with less dispersion.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eProline exhibited the greatest variability among the measured traits. Mid-stress treatments had the highest median values and the widest interquartile ranges (IQRs). Control plants showed the lowest median and least variability, highlighting proline accumulation as a key stress response. The median TA was highest in high-stress treatments. These treatments also showed greater range and variability, indicating diverse plant responses. Low-stress and control treatments had lower median TA and less variability. Ts content had higher median values under low-stress treatments and decreased with increasing stress. Intermediate treatments exhibited more variability, while the control showed relatively high median with limited range. Median TSS was highest in late-stage treatments, showing an increasing trend across treatments. Data dispersion was generally low with narrow interquartile ranges (IQRs), reflecting a uniform plant response in this osmotic parameter.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWater limitation is known to influence plant functions, significantly disrupting membrane stabilization and reducing PS-II efficiency. The main effects of drought stress include excessive production of reactive oxygen species (ROS), leading to oxidative stress and cellular damage [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. To alleviate damage, many studies show that zinc nanoparticles (Zn-NPs) are beneficial in improving plant stress tolerance. Stability and great potential to improve crop production have been shown with Zn-NPs under abiotic stress [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Nano zinc preserved the integrity of a membrane and reduced the loss of vital osmolytes through the suppression of MDA and H₂O₂ accumulation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Nano zinc has demonstrated a potential protective role to cellular components, but there is also indication that it can enhance phenolic compound biosynthesis when the plant is under drought stress. Exogenous applications of nano zinc at concentrations of 25 and 50 mg/L led to a remarkable increase in accumulated phenolic compounds under drought stress via exogenous Nano Zinc [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Flavonoids and phenolic compounds are secondary metabolites that are important and part of the phenylpropanoid pathway. The phenylpropanoid pathway exists as a parallel pathway to the true phenolic and flavonoid biosynthetic pathways. The phenylpropanoid pathway operates by processing phenylalanine, which then travels to phenylalanine ammonia-lyase to trans-cinnamic acid. Again, both of things (i.e., trans-cinnamic acid and phenylalanine) are documented in the above sections and have lucrative antioxidant activity and inherent ability to scavenge reactive oxygen species (ROS), can complex with metals, and the mechanisms help the plant with abiotic tolerances [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In publication by Yaqoob et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] they outline additional secondary anti-ROS detoxifications mechanisms that secondary metabolites (i.e.: flavonoids and phenolic compounds) interact with to complete ROS scavenging. Antioxidant phytochemicals such as flavonoids and phenolic compounds with deteriorate oxidative stress and subsequently magnify plant tolerance [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In addition, congruent with the present study findings, extraneous studies have demonstrated that BRs increase flavonoids accumulation in \u003cem\u003eGlycine max\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], \u003cem\u003eCamellia sinensis\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], Vitis vinifera [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], and \u003cem\u003eOryza sativa\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Thus, various studies have argued that BRs have enhanced plant stress tolerance through modification of the phenylpropanoid metabolic pathway [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFoliar application of Br and nano zinc has been widely demonstrated as an effective approach to enhance plant morphological traits and physiological responses under abiotic stress conditions [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The highest fruit weight and length under high concentrations of Br and nano zinc during moderate drought indicate that these treatments stimulate fruit development via promotion of cell division and expansion. Nano zinc reinforces these processes by participating as a cofactor in the enzymatic reactions that are involved in energy metabolism and protein synthesis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Severe drought is associated with elevated oxidative stress and depressed photosynthetic efficiency that hinders growth in plants by reducing the amounts of assimilates for developing fruits [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Development of greater fresh weight of vegetative tissues occurred when exposed to a 75% FC factors with moderate Br and low nano zinc indicating protection and growth stimulation.\u003c/p\u003e\u003cp\u003eNano zinc\u0026rsquo;s enhances uptake and mobility within the plant, supporting key metabolic processes essential for cell division and expansion. Brs regulate gene expression linked to cell proliferation, increasing biomass production even under stress conditions. This synergistic interaction improves osmotic adjustment and maintains cell turgor, enabling growth despite water limitation [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The highest fruit firmness observed under 3 g L⁻\u0026sup1; nano zinc combined with moderate FC (50%) can be attributed to the essential role of zinc in maintaining cell wall integrity. Zinc acts as a cofactor for enzymes such as pectin methylesterase, which help stabilize and strengthen cell wall structure [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Brs enhance this effect by activating biosynthetic pathways related to cellulose and lignin deposition, further reinforcing cell walls [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Collectively, these compounds alleviate drought-induced oxidative stresses, a common cause of cell wall degradation, and loss of fruit firmness [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. There was the least firmness in the severe drought\u0026thinsp;+\u0026thinsp;low nano-zinc treatment, likely due to inadequate enzymatic protection against oxidative injury [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The variation in photosystem II photochemical efficiency with nano-zinc and brassinosteroids indicates that at least some protection against drought-induced oxidative injury was provided to the photosynthetic apparatus. Among other functions, brassinosteroids can be proantioxidant and stimulate antioxidant defense mechanisms, like the enzymes that scavenge reactive oxygen species (ROS), such as superoxide dismutase (SOD) and catalase (CAT), and affect chloroplast stability [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], while the nano-zinc treatment was able to improve chloroplast-linked enzyme activities and preserve chlorophyll content [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The higher chlorophyll content in drought-stressed plants receiving nano-zinc and brassinosteroids also suggested that pigment stability/synthesis rates were improved, and were also likely necessary for maintaining photosynthesis during the stress [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe higher TSS content is likely due to the accumulation of osmolytes, which help maintain cellular osmotic balance during drought [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Previous studies on strawberries have similarly documented increased sugar accumulation and improved fruit quality under drought conditions following Zn and brassinosteroid (Br) treatments [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Variations in titratable acidity suggest shifts in organic acid metabolism modulated by treatments influencing Krebs cycle enzymes [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Proline accumulation increased significantly in untreated or low-treatment plants, consistent with its role in osmoprotection and ROS scavenging [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo investigate the interrelationships among physiological, biochemical, and phytochemical traits, Pearson\u0026rsquo;s correlation coefficients were calculated and visualized through a correlation heat map. The strongest positive correlation was found between total phenolic content (TPC) and proline (Pro) with r\u0026thinsp;=\u0026thinsp;0.90, indicating a very strong association. This suggests that under stress conditions, proline accumulation might be linked to increased biosynthesis of phenolic compounds, both of which are crucial components of plant defense mechanisms [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Additionally, significant positive correlations were observed between TPC and total flavonoid content (TFC) (r\u0026thinsp;=\u0026thinsp;0.86), as well as between TFC and Pro (r\u0026thinsp;=\u0026thinsp;0.82). These results imply that these secondary metabolites may respond similarly to environmental stresses, highlighting their potential coordinated roles in stress adaptation. A moderately strong positive correlation between fruit weight and fruit water volume (FWV) (r\u0026thinsp;=\u0026thinsp;0.65) was noted. Both traits also showed strong positive correlations with total soluble solids (TS) (r\u0026thinsp;=\u0026thinsp;0.70), suggesting that larger fruits tend to accumulate higher levels of soluble compounds like sugars. This close relationship underscores the link between fruit size and composition, which could be critical indicators of fruit quality [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. One of the most notable findings was the very strong negative correlation between initial fluorescence (Fo) and the maximum quantum efficiency of photosystem II (Fv/Fm) (r = -0.94). Since Fv/Fm reflects PSII efficiency, an increase in Fo alongside a decrease in Fv/Fm likely indicates damage to PSII or a reduction in photosynthetic performance, marking these parameters as sensitive indicators of environmental stress [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results of this work illustrate the high potential of nano zinc and Br in enhancing the physiological performance, stress tolerance and fruit quality of strawberry plants under drought stress conditions. Under moderate drought stress, the combination treatments of nano zinc and Br improved cell wall integrity, osmotic adjustments and photosynthetic efficiency in strawberry plants. These treatments were able to modulate fundamental metabolic pathways, antioxidant capacity, mitigate oxidative stress and allow physiological activities to function continuously. Correlation analysis showed significant correlation relationships among measured phytochemical and physiological traits. Strong positive correlations noted for proline, phenolics and flavonoids suggested a synergistic action in stress tolerance and protection. In addition, many traits based on Fo and Fv/Fm are reliable for PSII functioning and drought-stress damage. These results are consistent with a long list of tagging the positive aspects of the combined effects of nano zinc with plant hormones such as brassinosteroids to improve crop resilience and drought stress tolerance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeidar Meftahizade conceived the original idea, managed project and all results, and contributed to the final version. WarqaaMuhammed ShariffAl-Sheikh carried out the experiments. Mohamed Baqer Hussine Almosawi carried out the experiments. Masoumeh Eskandariperformed data analysis and Neda Tariverdizadehwrote the primary manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo specific financial credit was used in this experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares they have no financial interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated during this study are included in the article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors conducted the experiments in collaboration and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eŞener, S., Sayğı, H., \u0026amp; Duran, C. 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G., Han, E., Li, X., Rosenqvist, E., \u0026amp; Liu, F. (2023). The chlorophyll fluorescence parameter Fv/Fm correlates with loss of grain yield after severe drought in three wheat genotypes grown at two CO2 concentrations. \u003cem\u003ePlants, 12\u003c/em\u003e(3), 436. https://doi.org/10.3390/plants12030436\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Foliar Nutrition, Photosystem II Efficiency (Fv/Fm), Phenolic Compound, Sabrina","lastPublishedDoi":"10.21203/rs.3.rs-7378337/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7378337/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eStrawberry (\u003cem\u003eFragaria\u003c/em\u003e \u0026times; \u003cem\u003eananassa\u003c/em\u003e Duch.), are vulnerable to water limitations due to their root structure and high-water content. The experiment was carried out as a factorial experiment in a randomized complete block design with three replications. water deficiency at 4 levels (100% of FC, 75% of FC, 50% of FC, and 25% of FC), brassinosteroid at 4 levels (control, 100, 200, and 400 ml/liter), and zinc nanoparticles (control, 1, 2, and 3 g/liter) in Sabrina cultivar were treated. The results showed, the highest fruit weight (20.53 g) was observed under 75% FC and 400 ml L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Br in combination with 3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nano zinc. the interaction between Br and nano zinc (1.79 N) resulted in the lowest firmness in the treatment with 25% drought stress and 1 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of nano zinc. Also, the application of Br (200 ml L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and nano zinc (2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) under FC (25%) enhanced the accumulation of TSS (11.6%) in the fruit. content of TS had the highest value (1.137 gr\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW) in fruit subjected to mild FC (75%) with the application of 400 ml L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Br combined with 2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nano Zn. The highest Fo was observed in fruits treated with 75% FC and 400 ml L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Br in combination with 3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nano zinc. The lowest Fo was recorded under moderate drought stress and Br in combination with 3 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nano zinc. The lowest Fv/Fm was found after 50% FC and 100 ml L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Br treatment in combination with 2 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e nano zinc. Thus, foliar application of nano zinc and brassinosteroids under drought stress conditions can be considered an effective strategy to enhance growth, quality, and stress tolerance in the \u0026rsquo;Sabrina\u0026rsquo; strawberry cultivar.\u003c/p\u003e","manuscriptTitle":"Improving quality and water tolerance in strawberry through combined application of Nano Zinc and Brassinosteroids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 15:42:46","doi":"10.21203/rs.3.rs-7378337/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"70760b8e-7f25-4a83-9afa-c8584a8e0cc4","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-29T06:54:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 15:42:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7378337","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7378337","identity":"rs-7378337","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}