PEER-REVIEW ARTICLE PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Al-Saif et al. (2024). “Amino acids as biostimulant,” BioResources 19(3), 5978-5993. 5978 Amino Acids as Safe Biostimulants to Improve the Vegetative Growth, Yield, and Fruit Quality of Peach Adel M. Al-Saif,a,* Lidia Sas-Paszt,b Ragab. M. Saad,c and Walid F. A. Mosa c The influence of exogenous application of the amino acids Glutamic acid (Glu), Methionine (Met), L-Tryptophan (L-Try), and Lysine (Lys) at concentrations of 250 and 500 ppm was studied relative to the growth of peach trees. The trees were sprayed three times; before flowering, during full bloom, and one month later by 250 ppm Glu + 250 ppm Met + 250 ppm L-Try + 250 ppm Lys (combination 1) and 500 ppm Glu + 500 ppm Met + 500 ppm L-Try + 500 ppm Lys (combination 2), in comparison to trees that were not sprayed (control). A randomized complete block design was used. The individual application of four amino acids positively improved the shoot diameter, leaf chlorophyll, leaf area, and productivity as opposed to not spraying the trees. Additionally, the applied amino acids increased the fruit weight, size, firmness, length, and diameter, and the fruit content from the percentages of total soluble solids (TSS), TSS-acid, and anthocyanin contents, in contrast to the control. They also improved the fruit content from total, reduced, and non-reduced sugars as well as vitamin C and the leaf nutritional content from NPK. The application of combination 2, over the two seasons, was more beneficial. DOI: 10.15376/biores.19.3.5978-5993 Keywords: Fruit yield; Fruit quality; Amino acids; Sustainable production Contact information: a: Department of Plant Production, College of Food and Agriculture Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia; b: The National Institute of Horticultural Research, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland; c: Plant Production Department (Horticulture-Pomology), Faculty of Agriculture, Saba Basha, Alexandria University, Alexandria 21531, Egypt; *Corresponding author: adelsaif@ksu.edu.sa INTRODUCTION Peach (Prunus persica L.) is a deciduous tree from the Rosaceae family, with a cultivated area of 1,505 hectares globally, yielding 25 tons. In Egypt, the cultivated area is 13,76 hectares, producing 244 tons (FAO 2021). Peaches are valued as a fresh and versatile functional food, which is suitable for both direct consumption and various applications in the food industry. The consumable parts of the peach, such as the flesh, are abundant in carbohydrates, including the soluble sugars sucrose, glucose, fructose, and sorbitol. Additionally, they contain organic acids, fats, proteins, dietary fiber, minerals, and vitamins, as well as volatile and bioactive compounds. Notably, the peach seed is also a noteworthy source of nutritional and bioactive elements (Reig et al. 2023). Peach, such as the cultivated variety ‘Florida Prince’, is a delicious and juicy early- season cultivar. Its tolerance limit for coldness is about 150 hours at or below 7.2 °C. Its fruit needs 78 days for development, from fruit set to maturity. The fruit takes 78 days to develop from the time it sets until it matures and has good resistance to heat stress. It is a beautiful, aromatic fruit with 80% of its skin displaying a red blush and faint dark red PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Al-Saif et al. (2024). “Amino acids as biostimulant,” BioResources 19(3), 5978-5993. 5979 stripes, which cover most of the surface, with a yellow/orange background splash. The fruit is slightly smaller than a typical peach, featuring uniformly firm yellow flesh and semi- clingstone pits (Olmstead et al. 2016). Utilizing amino acids as biostimulants is a viable strategy in horticultural crops to mitigate the adverse effects caused by environmental stresses. These compounds serve as hormone precursors, play a role in regulating carbon and nitrogen metabolisms, and facilitate nitrogen assimilation (Colla and Rouphael 2015; Bulgari et al. 2019). Amino acids, being natural stimulants for plant growth, are widely employed to enhance growth patterns (Mohammadi and Khoshgoftarmanesh 2014; Romero et al. 2014). They also contribute to stimulating root growth in plants, thereby improving both water and nutrient absorption capabilities, ultimately leading to increased yield productivity (Souri et al. 2017; Khan et al. 2019; Noroozlo et al. 2019). Furthermore, the external spraying of amino acids greatly improved the uptake of nutrients and leaf mineral content, including essential elements such as iron and zinc (Pranckietienė et al. 2015). Moreover, the external application of amino acids facilitates growth, enhances nutrient absorption, triggers leaf pigmentation, promotes chlorophyll biosynthesis, and mitigates chlorophyll decomposition in various crops and this results in increased efficiency in photosynthesis and influences stomatal movement (Souri et al. 2017; Mohammadipour and Souri 2019). Spraying of amino acids optimizes processes such as nutrient uptake, translocation, and metabolism. It also contributes to the biosynthesis of vitamins, and biostimulation of growth, and it also raises the resistance to environmental stresses like drought, salinity, and coldness (Alfosea- Simón et al. 2020b; Matysiak et al. 2020). Applying Glu has demonstrated positive effects even in stressful conditions by mitigating physiological damage. This is achieved through the promotion of protein formation and the activation of antioxidant enzymes (Okumoto et al. 2016; Teixeira et al. 2017). Additionally, Glu application has been associated with improved yield and enhanced berry quality in grape (Stino et al. 2017; González-Santamaría et al. 2018). Met is a crucial amino acid that plays a significant role in various physiological functions within plants. The restriction of this amino acid can jeopardize plant survival, as it serves as a potent regulator in the growth and development of plants experiencing water scarcity (Mehak et al. 2021). Being a fundamental amino acid, Met actively contributes to a range of physiological functions and effectively regulates the development of plants under conditions of water deficit (You et al. 2019). L-Try acts as a signal-transducing molecule and facilitates the preparation of nutrients by plants (Teixeira et al. 2017). Lys is an amino acid that participates in various responses to abiotic and biotic stresses through the saccharine pathway (Arruda and Barreto 2020). Hence, the current research was undertaken to explore the beneficial impact of applying Glu, Met, Lys, and Try as eco-friendly biostimulants, which can improve the performance of peach trees to produce good yields with high fruit quality characteristics. EXPERIMENTAL Materials and Methods Applied treatments, location, and experimental design The experiment, conducted in 2022 and 2023, involved 10-year-old ‘Florida Prince’ peach trees grafted onto Nemaguard peach rootstock. These trees were planted with PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Al-Saif et al. (2024). “Amino acids as biostimulant,” BioResources 19(3), 5978-5993. 5980 a spacing of 3×4 meters in sandy soil within a private orchard under drip irrigation, situated in the Rashid region of El-Beheira governorate, Egypt. The physicochemical analysis of the experimental soil followed the protocol outlined by Sparks et al. (2020) and is exhibited in Table 1. Sixty-six peach trees, chosen for uniform vigor, were selected for the study, and subjected to identical agricultural practices throughout the two seasons. Table 1. Analysis of the Experimental Soil Sand Silt Clay Textural Class Organic Matter pH CaCO3 EC (dS/m) (Soil Extraction 1:5) 93.5% 3.5% 3% Sandy 0.3% 7.9 4.8% 0.810 Soluble Anions (meq/L) Soluble Cations (meq/L) Available Nutrients (mg/kg) HCO3 - CL- SO4 -- Ca++ Mg++ Na+ K+ N P K 2.1 2.50 4.18 2.20 3.46 2.12 1.22 115 17.8 307 The trees were exogenously sprayed three times: before flowering, during full bloom, and one month later using various treatments: water (control), Glu, Met, L-Try, and Lys amino acids at the concentration of 250 and 500 ppm, and by their combinations: 250 ppm Glu + 250 ppm Met + 250 ppm L-Try + 250 ppm Lys (combination 1) and 500 ppm Glu + 500 ppm Met + 500 ppm L-Try + 500 ppm Lys (combination 2). The experimental design followed a randomized complete block design, with each treatment consisting of six replicates (six trees). Vegetative Parameters At the end of the growing seasons, the diameter for four shoots from each side on each tree/replicate was measured in cm by using a Digital Vernier Caliper in both seasons. The leaf total chlorophyll was measured using a digital chlorophyll meter (SPAD 502 Plus, Konica Minolta, Inc., Tokyo, Jap) by taking 10 reads from the mature leaves from each replicate/tree. The average leaf area (cm²) was determined using Eq. 1 (Mosa et al. 2021). LA = −0.5 + (0.23 × L /W) + (0.67 × L × W) (1) where LA is the leaf area (cm2), L is the leaf length (cm), and W is the leaf width (cm). Fruit Yield Yield was estimated for the period May 2022 to 2023 in the units of kg per tree and in ton per hectare. Fruit Quality, Fruit Physical Characteristics After harvest, 30 fruits were chosen randomly from each replicate, and their weight (g), seed weight (g), length (cm), and diameter (cm) were measured. Fruit firmness (lb/inch²) was determined using a Magness and Taylor pressure tester equipped with a 7/18-inch plunger. Fruit size (cm³) was assessed by measuring the volume of displaced water after immersing the fruit. Total soluble solids (TSS%) were measured using a hand refractometer (ATAGO Co. LTD., Tokyo, Japan). PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Al-Saif et al. (2024). “Amino acids as biostimulant,” BioResources 19(3), 5978-5993. 5981 Fruit Chemical Characteristics Anthocyanin content was determined during the coloration stage (mg/100 g fresh weight peel) following the method outlined by Nangle et al. (2015). The ascorbic acid content in the juice was assessed through titration with 2,6-dichloro phenol-indo-phenol and expressed in milligrams in mg/100 mL of juice. Total and reducing sugars contents were quantified calorimetrically using the Neilsen arsenate-molybdate colorimetric method (Nielsen 2010). Fruit acidity, expressed as a percentage and measured in terms of malic acid content, was determined in fruit juice using a titration method with 0.1 N sodium hydroxide, employing phenolphthalein as an indicator (AOAC 2005). The TSS-acid ratio was computed by dividing the TSS value by the titratable acidity value. Mineral Content in Leaves In June 2022 to 2023, 30 leaves were collected from each tree (Arrobas et al. 2018) for the determination of macronutrient mineral contents. The leaves were first washed in tap water and then rinsed with distilled water. Subsequently, they were dried in an oven at 70 °C until a consistent weight was achieved, followed by crushing. The samples underwent digestion with H2SO4 and H2O2. Nitrogen content was quantified using the micro-Kjeldahl method (Wang et al. 2016), phosphorus was determined using the vanadomolybdate method (Weiwei et al. 2017), and potassium was measured utilizing a flame photometer (SKZ International Co., Ltd., Jinan Shandong, China) (Chapman 2021). Statistical Analysis Results were analyzed by using a one-way analysis of variance analysis (ANOVA) for a Randomized Complete Block Design (RCBD) design. The least significant difference at 0.05 was used to compare the means of the treatments as described by Snedecor and Cochran (2021). RESULTS Vegetative Growth Parameters The results presented in Table 2 indicate a substantial increment in shoot thickness, leaf area, and total chlorophyll content with the foliar application of Glu, Try, Met, Lys, and their combinations, compared to untreated trees, during both experimental seasons. Notably, the impact of 500 ppm from Try surpassed the effect of Glu at the same concentration in both seasons. Furthermore, the most effective treatment that gave the highest increments in shoot thickness, and leaf area was combination 2 when compared with the utilized treatments in 2022 to 2023. The increments in leaf total chlorophyll were (26.0 and 28.3%) by the spraying of combination 2 comparing with not treated trees in the first and the second season. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Al-Saif et al. (2024). “Amino acids as biostimulant,” BioResources 19(3), 5978-5993. 5982 Table 2. Spraying Effect of Glu, Try, Met, and Lys Amino Acids and their Combinations on Shoot Thickness, Leaf Area and Leaf Total Chlorophyll of Peach During 2022 to 2023 Treatment Shoot Thickness (mm) Leaf Area (cm2) Total Chlorophyll (SPAD) 2022 2023 2022 2023 2022 2023 Control 0.30e 0.32f 0.32c 0.33e 32.33d 34.67c Glu 250 ppm 0.32cde 0.33ef 0.35c 0.35d 33.00d 35.00c 500 ppm 0.33cde 0.36cd 0.35bc 0.38cd 39.33b 43.00b Try 250 ppm 0.33cde 0.34def 0.35c 0.37cd 36.33c 41.67b 500 ppm 0.37b 0.41b 0.40ab 0.41b 43.67a 42.33b Met 250 ppm 0.33cde 0.33f 0.34c 0.36d 32.67d 37.33c 500 ppm 0.36bc 0.39bc 0.36bc 0.40bc 40.00b 42.67b Lys 250 ppm 0.31de 0.32f 0.34c 0.35d 32.33d 35.00c 500 ppm 0.34bcd 0.39bc 0.36bc 0.38cd 40.33b 43.33b Combination 1 0.33cde 0.36de 0.35bc 0.38cd 38.67bc 41.67b 2 0.43a 0.44a 0.42a 0.44a 43.67a 48.33a LSD0.05 0.03 0.03 0.04 0.02 2.61 2.95 The same letters in the same column indicate no significant differences between treatments. Fruit Yield The results shown in Table 3 indicated that the spraying of Glu, Try, Met, Lys, and their combinations improved the fruit weight and fruit yields in the two seasons. Additionally, the application of 500 ppm from each one of Try, Met, and Lys was more effective than the application of 250 ppm from Glu, Try, Met, and Lys in their effects in enhancing the fruit weight and fruit yields. Combination 2 gave the highest and notable results in fruit weight (29.2 and 27.5%), and fruit yields in kg or in ton per hectare by (14.8 and 15.9%) in contrast to not treated trees in the first and second seasons respectively. The concentration of 500 ppm from each one of the sprayed amino acids was more effective than the application of 250 ppm. Table 3. Spraying Effect of Glu, Try, Met, and Lys Amino Acids and their Combinations on Fruit Weight and Fruit Yields of Peach During 2022 to 2023 Treatment Fruit Weight (g) Fruit Yield (kg) Fruit Yield (ton/h) 2022 2023 2022 2023 2022 2023 Control 0 55.00d 59.67e 141.69d 143.55d 113.35d 114.84d Glu 250 ppm 55.67d 62.00de 140.97d 144.14d 112.78d 115.31d 500 ppm 68.67b 77.00b 150.08c 159.25bc 120.06c 127.40bc Try 250 ppm 63.00c 71.33c 145.96cd 155.95c 116.77cd 124.76c 500 ppm 73.67a 76.67b 156.64b 163.95b 125.31b 131.16b Met 250 ppm 57.67d 65.33d 145.69cd 155.71c 116.55cd 124.57c 500 ppm 75.33a 78.00b 157.51b 160.13bc 126.01b 128.11bc Lys 250 ppm 57.33d 61.67de 143.02d 147.75d 114.42d 118.20d 500 ppm 75.00a 77.67b 151.35bc 161.16bc 121.08bc 128.93bc Combination 1 66.67bc 75.33bc 149.99c 156.18c 119.99c 124.95c 2 77.67a 82.33a 166.39a 170.74a 133.11a 136.59a LSD0.05 3.95 4.18 5.98 6.17 4.78 4.93 The same letters in the same column indicate no significant differences between treatments. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Al-Saif et al. (2024). “Amino acids as biostimulant,” BioResources 19(3), 5978-5993. 5983 Physical Fruit Characteristics The data presented in Table 4 indicates the positive impact of spraying Glu, Try, Met, and Lys amino acids, as well as their combinations on the enhancement of fruit weight, seed weight, and fruit firmness compared to untreated trees in both experimental seasons. The highest effect of Glu, Try, Met, and Lys amino acids at 500 ppm gave more increases in flesh fruit weight, seed weight and fruit firmness than those at 250 ppm in the two seasons. Additionally, the most notable results were obtained by the use of combination 2 in contrast to the individual application of the amino acids in the two seasons. Table 4. Spraying Effect of Glu, Try, Met, and Lys Amino Acids and their Combinations on Flesh Fruit Weight, Seed Weight and Fruit Firmness of Peach During 2022 to 2023 Treatment Flesh Fruit Weight (g) Seed Weight (g) Fruit Firmness (Ib/inch2) 2022 2023 2022 2023 2022 2023 Control 43.67d 49.00d 11.33c 10.67ef 10.17g 10.03f Glu 250 ppm 45.33d 50.33d 10.33c 11.67def 10.17g 10.07f 500 ppm 55.00b 59.67bc 13.67ab 17.33a 10.90cde 10.93cd Try 250 ppm 50.67c 58.00c 12.33bc 13.33b-e 10.58ef 10.53de 500 ppm 60.00a 62.00b 13.67ab 14.67abc 11.30b 11.33bc Met 250 ppm 47.33cd 52.33d 10.33c 13.00cde 10.33fg 10.43ef 500 ppm 61.33a 62.33b 14.00ab 15.67abc 11.23bc 11.50b Lys 250 ppm 46.33d 51.67d 11.00c 10.00f 10.43fg 10.07f 500 ppm 59.33a 62.00b 15.67a 15.67abc 11.03bcd 11.30bc Combination 1 54.40b 61.00bc 12.27bc 14.33bcd 10.80de 10.60de 2 63.00a 66.33a 14.67a 16.00ab 11.70a 11.93a LSD at 0.05 3.40 3.50 2.08 2.48 0.33 0.40 The same letters in the same column indicate no significant differences between treatments. Table 5. Spraying Effect of Glu, Try, Met, and Lys Amino Acids and their Combinations on Fruit Volume, Length and Diameter of Peach During 2022 to 2023 Treatment Fruit Volume (cm3) Fruit Length (cm) Fruit Diameter (cm) 2022 2023 2022 2023 2022 2023 Control 68.33e 73.33e 3.43g 3.73e 3.50f 3.83f Glu 250 ppm 69.67e 74.00e 3.50fg 4.03e 3.47f 4.17de 500 ppm 80.67c 88.67bc 4.40c 4.77bcd 4.63ab 4.47bcd Try 250 ppm 75.33d 85.33 c 4.07d 4.47d 4.23cd 4.33cd 500 ppm 87.33ab 89.33b 4.80b 5.00b 4.70a 4.70b Met 250 ppm 70.00e 78.33d 3.87de 4.70bcd 3.97de 4.40bcd 500 ppm 85.00b 87.33bc 4.70b 4.93b 4.73a 4.60bc Lys 250 ppm 70.00e 75.33de 3.77ef 4.67bcd 3.87e 4.00ef 500 ppm 86.33b 89.67b 4.57bc 4.87bc 4.70a 4.53bc Combination 1 80.67c 89.33b 4.37c 4.53cd 4.40bc 4.43bcd 2 90.67a 94.33a 5.33a 5.63a 4.83a 5.10a LSD0.05 3.83 3.56 0.28 0.31 0.27 0.28 The same letters in the same column indicate no significant differences between treatments. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Al-Saif et al. (2024). “Amino acids as biostimulant,” BioResources 19(3), 5978-5993. 5984 The results presented in Table 5 demonstrate the positive impact of foliar application of exogenous amino acids, including Glu, Try, Met, and Lys, as well as their combinations, on fruit volume, length, and diameter in comparison to untreated trees during the 2022-2023 period. It was seen that the exogenous application of Glu, Try, Met, and Lys at 500 ppm was more effective in their effect in increasing these parameters than the application of 250 ppm in the two seasons. Additionally, the most effective treatment was the combination 2 in the two seasons. Fruit Chemical Characteristics The results in Table 6 illustrate that the use of Glu, Try, Met, and Lys increased the fruit content from soluble solids, and TSS-acid ratio, as opposed to untreated trees. In addition, they diminished the fruit content from acidity in the two seasons. The most significant increments were associated with the application of 500 ppm from Try, Met and Lys rather than the application of 250 ppm from them, while they lowered the fruit acidity. The results showed that the combination 2 was the superior treatment where it remarkably increased TSS percentages (20.4 and 20.3%), anthocyanin content (17.9 and 23.2%) and TSS-acidity (39.8 and 38.4%). Meanwhile, it minimized the fruit acidity percentages (32.3 and 29.7%) in the first and in the second seasons over not treated trees. Table 6. Spraying Effect Spraying of Glu, Try, Met, and Lys Amino Acids and their Combinations on Fruit Content from TSS, Acidity, TSS-Acid ratio and Anthocyanin During 2022 to 2023 Treatment TSS (%) Acidity (%) TSS-Acid Ratio Anthocyanin (mg/100 g) 2022 2023 2022 2023 2022 2023 2022 2023 Control 8.83e 8.77e 0.82a 0.83a 10.73h 10.53e 0.55d 0.53e Glu 250 ppm 9.17e 9.00de 0.79ab 0.80ab 11.66g 11.20de 0.54d 0.55de 500 ppm 9.27e 10.17b 0.65d 0.74c 14.26cd 13.80c 0.58bcd 0.60bc Try 250 ppm 9.17e 9.43cd 0.71c 0.76bc 12.85ef 12.36d 0.57cd 0.58cde 500 ppm 10.53b 10.37b 0.66d 0.65d 16.06b 16.09ab 0.637ab 0.63b Met 250 ppm 9.30de 9.48cd 0.74c 0.80ab 12.52fg 11.87de 0.59bcd 0.55de 500 ppm 10.17bc 10.23b 0.65d 0.65d 15.66b 15.76b 0.65a 0.63bc Lys 250 ppm 8.87e 8.83e 0.75bc 0.83a 11.78g 10.60e 0.56d 0.59bcd 500 ppm 9.80cd 10.27b 0.67d 0.72c 14.70c 14.35c 0.63abc 0.58cde Combination 1 9.00e 9.83bc 0.66d 0.71c 13.64de 13.81c 0.57d 0.61bc 2 11.10a 11.00a 0.62d 0.64d 17.81a 17.10a 0.67a 0.69a LSD 0.05 0.50 0.53 0.04 0.05 0.91 1.25 0.05 0.05 The same letters in the same column indicate no significant differences between treatments. The results presented in Table 7 revealed that the foliar application of 500 ppm from Try, Met, and Lys led to an increase in fruit content from total, reduced, and vitamin C (VC) rather than the exogenous spraying of 250 ppm from each one of them in the two seasons. Regarding the fruit content from non-reduced sugars, the results showed that they were significantly increased by the spraying of 500 ppm from Met in the first season and Lys or Glu in the second season. Additionally, the highest increases in fruit content from total and reducing sugars and VC have resulted from the exogenous application of combination 2 rather than the other sprayed treatments. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Al-Saif et al. (2024). “Amino acids as biostimulant,” BioResources 19(3), 5978-5993. 5985 Table 7. Spraying effect of Glu, Try, Met, and Lys Amino Acids and their Combinations on Fruit Content from Total, Reduced, and Non-Reduced Sugars, and Vitamin C of peach 2022 to 2023 Treatment Total Sugars (%) Reducing Sugars (%) Non- Reduce Sugars (%) Vitamin C (mg/100 mL) 2022 2023 2022 2023 2022 2023 2022 2023 Control 7.80c 7.67e 3.23f 3.62c 4.57abc 4.04bcd 7.43d 7.47e Glu 250 ppm 7.90c 7.67e 3.29f 3.76c 4.61abc 3.91cd 7.62cd 7.87de 500 ppm 8.07c 8.73bc 4.02bc 4.00c 4.04c 4.73a 7.93cd 8.63a-c Try 250 ppm 7.97c 8.13d 3.67de 3.82c 4.29bc 4.31a-d 7.90cd 8.23b-d 500 ppm 8.87b 8.97b 4.16b 4.63a 4.71ab 4.33a-d 8.63ab 8.70a-c Met 250 ppm 7.80c 8.23d 3.47ef 3.69c 4.33bc 4.54abc 7.77cd 7.80de 500 ppm 8.87b 8.77bc 3.94b-d 4.47ab 4.93a 4.30a-d 8.20bc 8.03c-e Lys 250 ppm 7.83c 7.57e 3.28f 3.79c 4.55abc 3.77d 7.73cd 7.67de 500 ppm 8.63b 8.83b 4.15bc 4.05bc 4.49a-c 4.78a 8.10b-d 8.17b-e Combination 1 7.87c 8.37cd 3.82cd 3.76c 4.05c 4.61ab 7.83cd 8.80ab 2 9.43a 9.53a 4.82a 4.90a 4.62abc 4.63ab 9.03a 9.07a LSD0.05 0.42 0.42 0.31 0.43 0.52 0.56 0.64 0.66 The same letters in the same column indicate no significant differences between treatments. Nutritional Status The foliar application of amino acids Glu, Try, Met, and Lys and their combinations resulted in a positive impact on improving the mineral contents of peach leaves from nitrogen, phosphorus, and potassium, compared to untreated trees (Table 8). Moreover, the spraying of 500 ppm from Try, and Met were effective in their effect in improving the leaf mineral content from N, P and K from the spraying of 250 and 500 ppm from Glu and Lys or from also 250 ppm from Try or Met in the two seasons. Table 8. Spraying Effect of Glu, Try, Met, and Lys Amino Acids and their Combinations on the Leaf Mineral Content of Peach from Nitrogen, Phosphorous and Potassium During 2022 to 2023 Treatment N (%) P (%) K (%) 2022 2023 2022 2023 2022 2023 Control 2.04d 2.01e 0.43e 0.45d 2.20f 2.28f Glu 250 ppm 2.05d 2.05de 0.44de 0.46d 2.20f 2.28f 500 ppm 2.36c 2.43b 0.50c 0.55c 2.28e 2.44bc Try 250 ppm 2.04d 2.22cd 0.46d 0.47d 2.22f 2.42bcd 500 ppm 2.77a 2.67a 0.57b 0.62b 2.44b 2.43bcd Met 250 ppm 2.06d 2.11de 0.44de 0.47d 2.23f 2.35e 500 ppm 2.64b 2.70a 0.56b 0.60b 2.39c 2.43bcd Lys 250 ppm 2.04d 2.06de 0.45de 0.45d 2.21f 2.38cde 500 ppm 2.54b 2.48b 0.52c 0.56c 2.35d 2.46b Combination 1 2.28c 2.32bc 0.47d 0.54c 2.21f 2.38de 2 2.88a 2.80a 0.61a 0.66a 2.49a 2.53a LSD 0.05 0.11 0.16 0.03 0.04 0.04 0.05 The same letters in the same column indicate no significant differences between treatments. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Al-Saif et al. (2024). “Amino acids as biostimulant,” BioResources 19(3), 5978-5993. 5986 Additionally, the most obvious enhancements in leaf mineral content from N (29.17 and 28.21%), P (29.51 and 31.82%) and K (11.65 and 2.88%) were observed with the application of combination 2 compared to control in the first and the second season. DISCUSSION The application of Glu, Met, Try, and Lys individually or in combinations through foliar spraying significantly enhanced the growth, yield, and fruit quality of peach as opposed to untreated trees. These results are in the same trend as the findings of Cao et al. (2011), who emphasized the importance of Glu in nitrogen metabolism, highlighting its role in nitrogen assimilation in plants and aminotransferase reactions. The application of Glu increased photosynthetic activity and chlorophyll fluorescence measurements (Fabbrin et al. 2013; Lee et al. 2017; Röder et al. 2018), and this enhancement is attributed to the connection between photosynthetic capacity and leaf nitrogen concentration. Besides, Glu improved the protein and sugar content and productivity (Haghighi and Teixeira Da Silva 2013). Glu is the amino acid that most notably influences the root growth in numerous plant species (Forde 2014). In ‘Flame Seedless’ grapevines, the external spraying of Glu at a rate of 500 mg resulted in a 10%, 15%, and 22% increase in leaf mineral contents from N, K and P, respectively, with respect to the control treatment (Belal et al. 2016). Glu can organize the growth of the roots and defense responses in plants (Toyota et al. 2018; Goto et al. 2020). Glu influences the processes of pollination and fruit set and encourages the production of secondary metabolites (El-Shiekh and Umaharan 2014), as well as the activation of genes linked to stress and defence mechanisms (Li et al. 2019a,b). Additionally, Glu significantly improved the quality and yield of ‘Thompson Seedless’ grapevines, (Abou-Zaid and Eissa 2019). The application of Glu has been reported to stimulate the sprouting of both vegetative and reproductive buds, increase chlorophyll concentration, and enhance fruit quality. Improvements include increased fruit weight, size, firmness, and higher citric acid concentration (Soberanes-Pérez et al. 2020). Almutairi et al. (2022) reported that the foliar application of Glu at concentrations of 500 or 1000 ppm on guava trees resulted in increased shoot length and diameter, elevated leaf chlorophyll levels, improved fruit set, enhanced fruit yield, increased fruit firmness, and elevated content of total soluble solids, vitamin C, and total sugars. Furthermore, the leaf mineral content showed higher concentrations of N, K, and P in contrast to not sprayed trees. The application of exogenous Met through foliar spray is a practical approach that has a positive effect on the integrity of photosynthetic pigments, the accumulation of compatible osmolytes, the mitigation of reactive oxygen species, and the enhancement of cowpea growth and yield, as demonstrated by Merwad et al. (2018). The application of Met to plants can enhance the absorption of nitrogen and sulfur, as indicated by Santi et al. (2017), ultimately contributing to the growth and developmental processes of plants. Met plays an important role in the biosynthesis of auxins, cytokinins, and Brassinosteroids (Yong et al. 2014). Additionally, Met functions as a precursor for various biomolecules, including cofactors, polyamines, and vitamins, and serves as an essential component in the synthesis of antioxidants such as glutathione. These antioxidants, in turn, contribute to the production of defense compounds and participate in cellular redox homeostasis, as highlighted by Paungfoo-Lonhienne et al. (2008). Met plays a crucial role in regulating PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Al-Saif et al. (2024). “Amino acids as biostimulant,” BioResources 19(3), 5978-5993. 5987 chlorophyll biosynthetic processes and preventing chlorophyll degradation by enhancing the scavenging system of reactive oxygen species, as suggested by Abdelhamid et al. (2013). The application of Met can influence the synthesis and regulation of various genes and enzymes involved in the biosynthesis and signaling of other amino acids. This, in turn, contributes to maintaining redox homeostasis, resulting in elevated amino acid contents and improved salinity tolerance in plants (Zhang et al. 2017). Met organizes transpiration, protein synthesis, and the process of photosynthesis, and preserves membrane firmness, and relative water content in conditions of water deficit (Merwad et al. 2018; Mehak et al. 2021). Additionally, it effectively regulates plant growth and development under conditions of water scarcity (You et al. 2019). L-Try, a precursor to IAA, naturally occurs in the root exudates of plants (Quiroz- Villareal et al. 2012). Besides, it serves as a precursor for the production of indole-3-acetic acid, a compound with a structure similar to that of melatonin (Arnao and Hernandez-Ruiz 2019), and it markedly increases the levels of free IAA (Wójcik et al. 2016). Besides, the application of exogenous L-Try at concentrations of 125, 250, and 375 ppm was studied for its effects on various growth parameters of lettuce plants exposed to salt stress. The researchers observed that exogenous spraying with higher concentrations significantly influenced leaf number, salt tolerance, fresh leaf, and root weights, as well as the surface area of lettuce plants under salinity conditions at 200 mM (Hancı and Tuncer 2020). Applying L-Try to “Anna” apple trees at concentrations of 25, 50, and 100 ppm led to notable improvements in several growth and yield parameters. These enhancements included increased shoot length and diameter, expanded leaf area, higher total chlorophyll levels, greater percentages of fruit set and yield, and positive changes in both the physical and chemical advantages of the fruit. The leaf mineral composition, encompassing macronutrients such as N, P, K, and Ca, and micronutrients such as Fe, Zn, Mn, and B, also exhibited noteworthy improvements. Furthermore, the treatment resulted in a depression in the fruit drop percentages in comparison to the control trees for both seasons studied. Notably, concentrations of 50 and 100 ppm demonstrated superior effectiveness in enhancing these parameters compared to the 25 ppm concentration, as reported by Mosa et al. (2021). The spraying of Lys at a concentration of 15 mM on tomatoes proved to be advantageous for the growth of the aerial part, net assimilation of CO2, and water utilization efficiency, as highlighted by Alfosea-Simón et al. (2020a). Lys metabolism is implicated in various forms of plant stress responses. It is primarily catabolized through the saccharine pathway that notably raised the resistance to both abiotic and biotic stresses (Bernsdorff et al. 2016; Arruda and Barreto 2020). Lys serves as an essential component in the construction of proteins, playing a fundamental role in this process. Additionally, it functions as a precursor for Glu, an important signaling amino acid that plays a pivotal role in regulating plant growth and responses to environmental stimuli (Galili 2002). Applying Lys at a concentration of 100 mg/L through spray applications has been demonstrated to improve the plant height, leaf number, lateral branches, leaf area, chlorophyll content, yield, tuber length and diameter, dry matter, and the tubers-shoots ratio. Furthermore, it positively influenced the chemical composition of starch, total carbohydrates, nitrogen, phosphorus, and potassium (Hassan et al. 2020). From the discussed results, the application of four amino acids has been found to be an efficient tool to improve the vegetative growth, yield, fruit quality and nutritional status of peach trees. The amino acids could be used to reduce the usage of chemical PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Al-Saif et al. (2024). “Amino acids as biostimulant,” BioResources 19(3), 5978-5993. 5988 fertilizers to avoid their undesirable effects of the fruit quality characteristics of peach as well as their effective role in alleviating the environmental stresses with their high absorption rate via leaves. CONCLUSIONS 1. The results showed that the application of amino acids is an effective way to improve the growth, yield, and fruit quality of peach. 2. The application of 500 ppm from Glu, Met, L-Try and Lys was more beneficial than the application of 250 ppm. 3. Notably, the most effective treatment was combination 2 (500 ppm Glu + 500 ppm Met + 500 ppm L-Try + 500 ppm Lys) in increasing the growth, yield, fruit quality characteristics, as well as nutritional content from N, P and K as opposed to not spraying the trees. 4. The surface application of amino acids could be used as an eco-friendly and environmentally friendly way to improve the growth and productivity of peach trees without any undesirable effects on the environment. Funding This research was funded by Researchers Supporting Project number (RSP2024R334), King Saud University, Riyadh, Saudi Arabia. ACKNOWLEDGMENTS The authors extend their appreciation to the Researchers Supporting Project number (RSP2024R334), King Saud University, Riyadh, Saudi Arabia. REFERENCES CITED Abdelhamid, M. T., Rady, M. M., Osman, A. S., and Abdalla, M. A. 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