The Effects of Altitude on Fruit Characteristics, Nutrient Chemicals, and Biochemical Properties of Walnut Fruits (Juglans regia L.)

Abstract

This study aimed to investigate changes in fruit characteristics, total phenolics, total antioxidant capacity, organic acids, sugar content, and fatty acid composition in ten walnut genotypes and three cultivars grown at different altitudes under Mediterranean climate conditions. At altitudes of 500 m and 1200 m, total phenolics ranged between 237.51 and 412.96 mg GAE/100 g dw and 234.94 and 392.31 mg GAE/100 g dw, respectively. According to DPPH radical inhibition, the total antioxidant capacity varied between 47.65% and 64.99% at an altitude of 500 m and between 57.76% and 68.92% at an altitude of 1200 m. The oil content demonstrated variations between 53.44% and 76.17% at an elevation of 500 m and between 43.11% and 67.03% at an altitude of 1200 m. At both 500 m and 1200 m altitudes, linoleic acid emerged as the predominant fatty acid and ranged from 58.82% to 62.44% at 500 m altitude and from 57.33% to 59.38% at 1200 m altitude. Notably, malic acid was identified as the primary organic acid, with concentrations ranging from 1.35% to 7.33% at both altitudes. In conclusion, this study demonstrates that walnut seeds are abundant in oil, protein, total phenolics, antioxidants, and fatty acids, with variations influenced by the cultivar or genotype and notably affected by altitude.

1. Introduction

Pomology is one of the most important branches of horticulture. Fruits, which have been cultivated in many countries of the world for centuries and consumed both fresh and dried, have been one of the most important sources of commercial income for the producing countries. There are many studies on walnut, which has a wide range of uses around the world. Walnut (Juglans regia L.) belongs to the family Juglandaceae, and the most widely produced species is Juglans regia L., and is also known as Anatolian walnut or Iranian walnut [1,2]. In recent years, it has been known that there are 22 species belonging to the genus Juglans with a wide distribution area and production value [3]. Türkiye, which is the intersection point of three biodiversity centers in the world (Irano-Turanian, Mediterranean, and Euro–Siberian), is the homeland of walnuts and many fruit species due to its geographical location [4,5]. Therefore, Türkiye is one of the oldest walnut-producing countries in the world and has a deep-rooted fruit growing culture. World walnut production is around 3.5 million tons. Türkiye, which has an important location in walnut cultivation, ranks fourth in the world walnut production after China, the USA, and Iran. With an annual production of 325,000 tons, Türkiye provides 9.28% of the world’s production [6].

In fruit growing, genetic variation is a prerequisite for breeding programs. Identifying and investigating the source of genetic variation among genotypes and commercial varieties is always of great importance for plant breeders to carry out breeding programs in different fruit species. It has been reported in many studies that walnut trees in different parts of the world show high variability in different traits such as crown and trunk structure, leaf shapes, shell structure, kernel structures, fruit shape, and fruit size [7,8]. It is known that there are wild walnut trees adapted to the environment and growing conditions in every region of Anatolia and that these trees show significant differences in terms of vegetative growth and generative characteristics [3]. For this reason, as in other countries of the world, walnut has been one of the most widely selected fruit species among hard-shelled fruits in our country. This wide walnut genetic variation in our country is extremely important in terms of success in breeding programs in a short period of time and the use of these walnut genotypes and varieties with high nutritional content as parents in breeding studies. It is known that walnuts can be grown economically at altitudes of 600–1500 m. Out of these specified altitudes (low and high altitudes), problems are observed in yield and quality [9]. The morphological characteristics and biochemical contents of walnuts are affected by environmental factors [10]. It is important to know the growth performance, fat composition, and biochemical contents of walnut genotypes in different ecologies and at different altitudes to cultivate varieties suitable for theenvironment and consumer preferences.

Walnut selection studies have been conducted in different regions of Türkiye. There are many studies on the fruit quality characteristics and biochemical and mineral contents of walnut genotypes and varieties [11,12,13]. There are a few published reports on the fat, fatty acid, and other properties of walnuts, especially in the Mediterranean region (Kahramanmaraş), which is a temperate zone. However, no detailed study on the effect of altitude on biochemical compounds in the Mediterranean region has been published.

Today, there is an increasing trend toward the use of natural substances instead of synthetic substances. The use of many fruit varieties as a source of natural antioxidants is important for human health. Recent studies have demonstrated the important role of walnut fruit in human health and nutrition, and its benefits in the treatment of many diseases have been shown. It has been found to be beneficial in many diseases such as cardiovascular [14], some types of cancer [15,16], nervous system [17,18], diabetes [19], and obesity [20,21]. Walnut studies on antioxidant potential have shown that this fruit is a good source of antioxidant phenolic compounds [22]. Organic acids are extensively used for pharmacological purposes due to their antioxidant properties. The organic acid/sugar ratio is also an important criterion in the determination of fruit flavor [23]. Walnut fruit generally has a high fat content consisting of unsaturated fatty acids [24,25]. Walnut fat is rich in essential fatty acids, and its fatty acid composition is mostly composed of oleic, linoleic, and linolenic acids. Among hard-shelled fruits, walnuts have the highest total polyunsaturated fatty acids and linoleic acid levels (between 40% and 70%) [26,27].

Making great contributions to the national economy, walnut cultivation is spreading to wider areas, and production is increasing with each passing year. We believe that investigating and determining the relationship between the altitude at which the walnut plant is grown and the biochemical content of the fruits of walnut varieties in different regions can provide walnut cultivation in a more conscious way for producers and at the same time, this study can provide guiding information for researchers. Walnut breeders in the world and Türkiye face some problems in their breeding programs for these new walnut genetic variations due to the lack of research and data on the biochemical characteristics of genotypes from different regions. Therefore, we believe that this study may be a pioneering study for the identification and research of new genotypes with better biochemical properties. For this reason, the subject of our research is the comparison of ten walnut genotypes obtained by selection from Kahramanmaraş province and three walnut cultivars (Maraş-18, Chandler, and Franquatte) grown intensively in the region according to fruit characteristics, fat, fatty acid, total phenolics, total antioxidant, total protein, organic acid, and carbohydrate profiles depending on altitude (500 m and 1100 m) using spectrophotometric and chromatographic techniques. We hope that the data obtained from this study will serve as a resource for breeders, the food industry, and nutritionists. Hence, we are of the opinion that this study will serve as a novel reservoir of genetic diversity, thereby allowing breeders to cultivate fresh commercial varieties characterized by elevated biochemical content. These include, but are not limited to, total phenolics, total antioxidant capacity, organic acids, sugars, and fatty acid compositions.

2. Materials and Methods

2.1. Materials

2.1.1. Experimental Site Description

Kahramanmaraş is geographically situated at coordinates 37°43′ north latitude and 37°8′ east longitude, with an elevation of approximately 900 m above sea level. This province is located within the eastern Mediterranean region and possesses a climatic profile conducive to the successful cultivation of various fruits. Consequently, it stands as a significant region for fruit production.

Kahramanmaraş falls within the Mediterranean climate zone; however, it exhibits climatic characteristics that lie intermediate between the Mediterranean and southeastern Anatolia regions. The Mediterranean climate of Kahramanmaraş is characterized by warm and rainy winters, contrasted by dry and hot summers. Detailed weather data for both years are presented in Figure 1.

2.1.2. Plant Material

The materials used in this study comprised 10 prominent walnut genotypes selected from the Kahramanmaraş region. These genotypes were specifically chosen based on criteria such as fruit weight, productivity, cold tolerance, total oil, and total protein contents. They were sourced from walnut plantations associated with the ‘Walnut Selection Breeding’ project, administered by the General Directorate of Agricultural Research and Policies (TAGEM) in Kahramanmaraş province. This province is situated in the eastern Mediterranean region of Turkey. Additionally, three widely cultivated walnut varieties, namely Maraş-18, Chandler, and Franquette, are grown intensively in the region.

The study was conducted in two distinct districts of Kahramanmaraş province, Turkey, namely Çağlayancerit (at an elevation of 1200 m), and the Central district (at an elevation of 500 m), during the period spanning from 2021 to 2022. A total of 30 walnut samples were collected from each tree, encompassing 10 walnut genotypes and 3 distinct walnut cultivars, at the point of commercial maturity, which is characterized by fruits being at the stage of full maturity. In both study regions, fruits were harvested at the same maturity levels. Subsequently, these collected samples underwent prompt transportation to the Department of Horticulture Laboratory within the Faculty of Agriculture at Çukurova University. This transportation was meticulously executed within a cold chain transportation system to preserve the samples’ integrity. Cultural practices such as irrigation, fertilization, plant protection, and pruning are routinely carried out in the orchards. Irrigation and fertilization of the plants were carried out regularly. A fertilization program was applied to the plants with approximately 800 g net phosphorus and potassium and 1500 g net nitrogen per plant. Phosphorus and potassium were applied in a single dose in February, and nitrogen was applied 1/3 in three months, with one-month intervals, starting from March. Microelement application was applied to the leaves as of the end of April. Irrigation was carried out using the drip irrigation method at approximately 15-day intervals from the end of May to the end of September.

Upon reaching the laboratory, pomological analyses were conducted on the walnut samples. Following the completion of these pomological assessments, the remaining fruit samples were subjected to crushing in preparation for subsequent analyses.

2.2. Method

2.2.1. Pomological Analyzes

Pomological characteristics such as fruit weight (g), length (mm), width (mm), form index, kernel weight (g), shell thickness (mm), and kernel percentage (%) were determined in 25 walnut fruits from each genotype and cultivar. The fruit weight was measured with shell and kernel weight using a digital laboratory scale (precision of 0.01 g). The fruit width was measured with a shell along with the fruit length, the fruit height was measured with a shell, and the shell thickness was measured with a digital caliper.

2.2.2. Sample Preparation for Biochemical Properties

After the walnut samples were pulverized with the help of a grinder, 500 mg of walnut samples were placed in 15 mL tubes, and 2.5 mL of 80% methanol was added. After the samples were completely mixed, they were centrifuged at 4 °C and 4000 rpm for 10 min. After this step, the centrifuged samples were further processed for total phenolics and total antioxidant analyses.

Total Phenolics

Total phenolic content was determined using the modified Folin–Ciocalteu method described by Spanos and Wrolstad (1990) [28]. Total phenolic content was calculated from the absorbance values read at 765 nm wavelength in a spectrophotometer and calibration curve prepared with gallic acid. A 50 μL sample was taken from Falcon tubes and placed in 2 mL Eppendorf tubes. A total of 100 μL of Folin–Ciocalteu was added, and then 1500 μL of distilled water was added and kept for 10 min. A total of 50 μL of 20% Na₂CO₃ was added, and the samples were kept in the dark for 2 h. For the control, 50 μL ultrapure water, 100 μL folin, 1500 μL ultrapure water, and 50 μL Na₂CO₃ were prepared. Blank (blind reading) was prepared with 50 μL 80% methanol, 100 μL folin, 1500 μL ultrapure water, and 50 μL Na₂CO₃. A total of 250 μL of each control and blank and 250 μL of each sample were loaded onto the plate and read at a 760 nm wavelength on a Thermo Multi Scan Go spectrophotometer.

Total Antioxidant Capacity

For the determination of total antioxidant capacity, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity method of Brand-Williams et al. (1995) was used [29]. A 50 μL sample was taken from Falcon tubes and placed in 2 mL Eppendorf tubes. In the absence of light, 1950 μL of a 0.06 millimolar DPPH (2,2-diphenyl-1-picrylhydrazyl) solution was added. A total of 250 μL of each sample was loaded onto the plate. As the control, 50 μL of distilled water and 1950 μL of the DPPH solution were used. As a blank, 50 μL of 80% methanol and 1950 μL of the DPPH solution were used. Readings were taken at 515 nm wavelengths in the spectrophotometer (Shimadzu, Japan).

%DPPH INHIBITION = ((Control absorbance − (sample absorbance − blank absorbance))/control absorbance) × 100

(1)

%DPPH RADICAL SCAVENGING = ((Control absorbance − sample absorbance)/Control absorbance) × 100

(2)

2.2.3. Oil Content

Walnut seeds were manually separated from the shell. Fat extraction of seed powder (25 g) was carried out with an automatic Soxhlet device for 4 h, according to Bligh and Dyer (1959) [30]. Hexane was chosen as the organic solvent, and methylation was performed with boron trifluoride/methanol [31]. The fat content was calculated as a % based on the weight difference of the tubes before and after the experiment. The fat content was also used in the following fatty acid analysis.

2.2.4. Protein Content

The fruit samples were kept in an oven at 30 °C for 24 h for homogeneous drying. The protein content of the cultivars and genotypes in the study was determined according to the Kjehdahl method (Kacar, 1984) on 0.25 g of ground fruit samples [32]. The percentage of the protein content was indirectly quantified by determining the total N content obtained by the Kjeldahl method [31] using a nitrogen-to-protein conversion factor (Kc = 6.25; % protein = Kc* % total N) [31,33].

2.2.5. Analysis of Fatty Acids

Fatty acid analysis was performed on an Agilent GC with a flame ionization detector (Agilent, 7820A). An acid silicide salt tube SGE (30 m 0.32 mm ID 0.25 lm BP20 0.25 UM, USA) was used for fatty acid analysis. The analysis was started by keeping the oven temperature at 140 °C for 5 min. The oven temperature was increased to 200 °C at a rate of 4 °C/min and then to 220 °C at a rate of 1 °C/min. A mixture standard of fatty acid methyl esters (FAMEs) containing 37 components was used as a standard for the identification and proportioning of fatty acids. Results are given as percentages.

2.2.6. Analysis of Sugar Content

Sugar content analyses of walnut fruits were carried out using a modified version of the method described by Bernardez et al. (2004) [34]. Also, 0.2 g of fruit sample was mixed with 2.5 mL of distilled water and kept in an ultrasonic bath at 65 °C for 30 min. It was then centrifuged at 15,000 rpm for 20 min. After centrifugation, the upper liquid phase was removed, and this step was repeated once more. The supernatants were made up to 5 mL with distilled water and filtered with a 0.45 μm filter. After filtration, sugar analysis was performed on the samples transferred to the HPLC device. An HPLC VA 300/7.8 Nucleogel Sugar Pb column was used for sugar analysis. The temperature of the column was 80 °C. A total of 100% deionized water was used as a mobile phase, and an analysis was performed with a mobile flow rate of 0.3 mL/min. As a result of sugar analysis, glucose, fructose, sucrose, and total sugar contents of fruit samples were determined.

2.2.7. Analysis of Organic Acid Contents

One g of fresh fruit was mixed with 10 mL of deionized water and homogenized. The mixture was centrifuged at 1200 rpm for 60 min. The supernatants were then filtered through a 0.22 μm filter. The organic acid contents of the fruit samples were determined by HPLC on a Zorbax-Eclipse-AAA 4.6 * 250 mm, 5 μm column. The column temperature was set at 25 °C. A total of 25 mM potassium phosphate was used as the mobile phase, and the flow rate was set as 1 mL/1 min. Oxalic acid, citric acid, malic acid, and succinic acid were measured as organic acids in walnut fruits.

2.2.8. Statistical Assessments

The data acquired as part of this study were subjected to analysis of variance, and distinctions among the means were discerned through the Least Significant Difference (LSD) test. Furthermore, the study results underwent comprehensive evaluation through correlation analysis, principal component analysis (PCA), and heatmap analysis. For the statistical analysis of the data, the JMP Pro 14 software package was used (SAS Institute Inc., Cary, NC, USA).

3. Results and Discussion

3.1. Fruit Quality Parameters

Fruit pomological characteristics of the analyzed walnut genotypes and cultivars are shown in

Table 1. The effect of altitude on fruit quality and biochemical characteristics of walnut cultivars and genotypes (mean for 2021 and 2022).

Location	Genotypes	Fruit Weight (g)		Kernel Weight (mm)		Kernel Percentage (%)		Fruit Width (mm)		Fruit Length (mm)		Fruit Height (mm)
Low (500 m)	G-1	13.90 ± 0.11 kl	15.15 ± 1.93 a	8.00 ± 0.12 g	8.50 ± 1.45 a	57.54 ± 0.63 de	56.03 ± 6.05 a	43.42 ± 0.65 de	44.05 ± 4.39 a	35.07 ± 0.53 de	34.45 ± 2.01 a	38.18 ± 0.57 hi	38.72 ± 4.48 a
	G-2	13.69 ± 0.36 l		7.25 ± 0.11 h		52.99 ± 2.12 g–i		45.40 ± 0.68 c		34.49 ± 0.0 e–g		34.85 ± 0.52 l
	G-3	17.89 ± 0.21 a		9.10 ± 0.14 c		50.85 ± 1.34 g–i		43.05 ± 0.65 e		35.76 ± 0.52 cd		40.92 ± 0.61 ef
	G-4	15.28 ± 0.35 g–i		8.40 ± 0.13 ef		54.97 ± 0.73 ef		48.34 ± 0.73 b		36.33 ± 0.54 bc		39.22 ± 0.59 g
	G-7	17.15 ± 0.23 bc		9.00 ± 0.13 cd		52.47 ± 0.50 g–i		43.37 ± 0.65 de		36.30 ± 0.54 bc		40.29 ± 0.60 f
	G-10	17.34 ± 0.45 ab		9.90 ± 0.15 b		57.12 ± 2.20 de		47.31 ± 0.71 b		36.65 ± 0.55 bc		42.18 ± 0.63 d
	G-11	15.39 ± 0.52 f–h		11.00 ± 0.16 a		71.49 ± 2.14 a		44.46 ± 0.67 cd		35.80 ± 0.54 cd		48.68 ± 0.73 a
	G-12	16.01 ± 0.46 d–f		9.12 ± 0.14 c		56.98 ± 0.94 de		47.47 ± 0.71 b		38.98 ± 0.58 a		43.46 ± 0.65 c
	G-14	15.70 ± 0.46 e–g		9.90 ± 0.15 b		63.09 ± 2.73 b		44.67 ± 0.67 c		35.97 ± 0.54 cd		37.92 ± 0.57 ji
	G-16	14.70 ± 0.33 ij		8.40 ± 0.13 ef		57.16 ± 2.16 de		51.03 ± 0.77 a		36.45 ± 0.55 bc		37.85 ± 0.57 ji
	Maraş-18	16.50 ± 0.53 cd		8.74 ± 0.25 d		53.01 ± 2.48 g–i		42.58 ± 0.64 e		36.12 ± 0.0 bc		35.55 ± 0.53 kl
	Chandler	12.14 ± 0.22 m		6.13 ± 0.51 i		50.56 ± 4.86 ij		36.60 ± 0.55 h		30.87 ± 0.48 mn		33.34 ± 0.50 m
	Franquette	11.27 ± 0.53 n		5.64 ± 0.35 j		50.16 ± 5.15 h–j		34.30 ± 0.86 i		30.54 ± 1.04 n		30.93 ± 0.40 op
High (1200 m)	G-1	12.08 ± 0.77 m	14.05 ± 1.89 b	6.25 ± 0.09 i	7.48 ± 1.40 b	51.89 ± 3.95 g–i	53.11 ± 6.16 b	41.42 ± 0.62 fg	42.10 ± 4.09 b	34.07 ± 0.51 fg	33.10 ± 1.81 b	37.18 ± 0.56 j	36.59 ± 4.29 b
	G-2	12.08 ± 0.32 m		6.25 ± 0.09 i		49.26 ± 0.69 ij		42.40 ± 0.64 ef		31.49 ± 0.47 k–m		31.85 ± 0.48 on
	G-3	16.11 ± 0.20 de		8.10 ± 0.12 fg		50.29 ± 1.35 ij		41.05 ± 0.62 g		32.76 ± 0.49 h–j		38.92 ± 0.58 hg
	G-4	14.60 ± 0.38 ij		7.40 ± 0.11 h		50.68 ± 0.61 ij		45.34 ± 0.68 c		32.33 ± 0.48 i–k		37.22 ± 0.56 j
	G-7	17.09 ± 0.62 bc		8.00 ± 0.12 g		46.84 ± 2.15 j		41.37 ± 0.62 fg		31.30 ± 0.47 l–n		38.29 ± 0.57 hi
	G-10	14.94 ± 0.48 h–j		7.90 ± 0.12 g		52.90 ± 1.26 g–i		45.31 ± 0.68 c		33.65 ± 0.50 gh		40.18 ± 0.60 f
	G-11	15.29 ± 0.48 g–i		10.00 ± 0.15 b		65.43 ± 1.67 b		42.46 ± 0.64 ef		34.80 ± 0.52 ef		45.68 ± 0.69 b
	G-12	13.74 ± 0.39 l		8.12 ± 0.12 fg		59.12 ± 2.59 cd		45.47 ± 0.68 c		36.98 ± 0.55 b		41.46 ± 0.62 de
	G-14	14.45 ± 0.53 jk		8.90 ± 0.13 cd		61.64 ± 3.10 bc		42.67 ± 0.64 e		32.97 ± 0.49 hi		34.92 ± 0.52 l
	G-16	13.54 ± 0.15 l		7.40 ± 0.11 h		54.65 ± 1.03 e–g		50.03 ± 0.75 a		34.45 ± 0.52 e–g		35.85 ± 0.54 k
	Maraş-18	16.12 ± 0.08 de		8.70 ± 0.13 de		53.96 ± 0.68 e–h		40.58 ± 0.61 g		34.12 ± 0.51 fg		31.55 ± 0.47 on
	Chandler	11.35 ± 0.56 n		4.80 ± 0.07 k		42.31 ± 1.54 k		35.60 ± 0.53 h		31.87 ± 0.46 j–l		32.34 ± 0.49 n
	Franquette	10.74 ± 0.25 n		5.52 ± 0.34 j		51.49± 3.60 f–i		33.62 ± 0.85 i		31.17 ± 1.02 l–n		30.31 ± 0.39 p
LSD0.05		0.69 **	0.19 **	0.30 **	0.08 **	3.89 **	1.08 **	1.11 **	0.30 **	0.94 **	0.26	0.92 **	0.25 **

The differences among mean values shown on the same line with the same letter are not significant (p < 0.05). ** significant at p < 0.01.

. Fruit quality characteristics among cultivars and genotypes were statistically significant (p < 0.05). The highest fruit weight was 17.89 g (G-3) at 500 m altitude and 17.09 g (G-7) at 1200 m altitude. The lowest shell fruit weight was found in the Franquette cultivar at both elevations (11.27 g and 10.74 g, respectively). The highest inner fruit weight was found in genotype G-11 (11.00 g and 10.00 g, respectively) at both altitudes. The lowest kernel weight was found in Franquette (5.64 g) at 500 m altitude and Chandler (4.48 g) at 1200 m altitude. The kernel fruit ratio (yield), which is one of the important parameters in walnuts, was highest in the G-11 genotype (71.49% and 65.43%, respectively) at both elevations. Koyuncu et al. (2004) conducted a study with five local walnut cultivars (Bilecik, Sebin, Yalova-1, Yalova-3, and Yalova-4) at 300 m and 1200 m altitudes and reported that shelled and kernel weight and the kernel fruit ratio decreased with increasing altitude [35]. In the study conducted in the Kastamonu region, promising genotype selection was determined as 9.04 g −14.13 g fruit weight, 5.79–8.58 g kernel weight, and 53.00–65.38% kernel ratio [29]. Muradoğlu and Balta (2010) reported 9.91 g–15.22 g fruit weight, 5.00 g–6.24 g kernel weight, 40.9–52.3% kernel weight, and 40.9–52.3% kernel ratio as promising for 15 genotypes in the Ahlat region [36]. Şimşek (2010) found that the fruit length of walnut varieties obtained in the Diyarbakır region was between 33.10 mm and 42.50 mm, fruit width was between 28.90 mm and 35.4 mm, and fruit length was between 27.7 mm and 34.9 mm [37]. In a study conducted by Polat et al. (2015) in Bitlis, the amount of fruit weight, kernel weight, and kernel ratio for promising genotypes were determined as 10.42–14.25% g, 4.52 g–7.44 g, and 42.38–54.07%, respectively [38]. Bayazıt et al. (2020) investigated the performance of the Chandler walnut variety at 100 m, 400 m, 800 m, and 1100 m altitudes. The researchers reported that fruit weight decreased after 400 m, and the weight of the kernel and the ratio of the kernel decreased after 800 m altitude [9]. Büyüksolak et al. (2020) examined the fruit characteristics of the Chandler walnut cultivar at 650 m, 800 m, and 900 m altitude. They reported that the proportion of kernels decreased with higher altitudes [39]. Balcı (2002) reported that the higher altitude decreases the weight of shelled and kernel fruits in walnuts [40]. The values found in walnut morphological studies conducted in our country [38,41,42] are in accordance with the values in our study.

3.2. Total Phenolics and Total Antioxidant Capacity

The total phenolics and total antioxidant capacity of walnut cultivars and genotypes were found to be statistically significant (p < 0.05) (

Table 2. Total phenolics (mg GAE 100 g⁻¹ FW) and antioxidant capacity (% DPPH inhibition and %DPPH radical) in the fruits of ten walnut genotypes and cultivars in Türkiye (mean for 2021 and 2022).

Altitude	Genotype	Total Phenolics		DPPH Inhibition		DPPH Radical
Low (500 m)	G-1	412.96 ± 0.58 a	293.12 ± 53.24 A	71.63 ± 0.50 cd	63.43 ± 6.12 B	64.36 ± 0.50 cd	56.43 ± 6.12 A
	G-2	237.51 ± 0.91 s		64.43 ± 0.50 i		57.43 ± 0.50 i
	G-3	293.86 ± 0.31 i		56.89 ± 0.18 l		49.89 ± 0.18 l
	G-4	300.09 ± 0.70 g		57.85 ± 0.27 j		50.85 ± 0.27 k
	G-7	384.96 ± 0.28 c		68.43 ± 0.32 f		61.43 ± 0.32 f
	G-10	254.60 ± 0.52 p		64.84 ± 0.39 hi		57.84 ± 0.39 hi
	G-11	242.06 ± 0.60 r		65.52 ± 0.40 h		58.52 ± 0.40 h
	G-12	253.31 ± 0.48 p		70.94 ± 0.25 d		63.94 ± 0.25 d
	G-14	295.82 ± 0.78 h		71.99 ± 0.27 bc		64.99 ± 0.27 bc
	G-16	335.90 ± 0.89 f		55.07 ± 0.22 m		48.07 ± 0.22 m
	Maraş-18	292.03 ± 0.95 j		64.76 ± 0.19 i		57.76 ± 0.19 i
	Chandler	241.67 ± 0.94 r		57.57 ± 0.51 kl		50.57 ± 0.51 kl
	Franquette	265.75 ± 0.87 o		54.65 ± 0.52 m		47.65 ± 0.52 m
High (1200 m)	G-1	392.31 ± 0.94 b	298.70 ± 49.85 B	75.92 ± 0.53 a	70.40 ± 3.93 A	68.92 ± 0.53 a	63.04 ± 3.68 B
	G-2	290.45 ± 0.60 k		68.30 ± 0.53 fg		61.30 ± 0.53 f
	G-3	279.17 ± 0.30 m		72.62 ± 0.53 b		65.62 ± 0.53 b
	G-4	285.89 ± 0.59 l		61.32 ± 0.41 j		54.32 ± 0.41 j
	G-7	367.31 ± 0.92 d		72.53 ± 0.28 b		65.53 ± 0.28 b
	G-10	254.79 ± 0.80 p		72.09 ± 0.34 bc		65.09 ± 0.34 bc
	G-11	234.94 ± 0.61 t		69.45 ± 0.22 e		62.45 ± 0.22 e
	G-12	246.86 ± 0.74 q		75.19 ± 0.42 a		63.94 ± 0.42 d
	G-14	247.80 ± 0.62 q		72.31 ± 0.25 bc		64.99 ± 0.25 bc
	G-16	360.88 ± 0.49 e		71.93 ± 0.27 bc		64.93 ± 0.27 bc
	Maraş-18	295.16 ± 0.92 hi		71.17 ± 0.17 d		64.17 ± 0.17 d
	Chandler	242.06 ± 0.64 r		67.57 ± 0.19 g		60.57 ± 0.19 g
	Franquette	268.51 ± 0.73 n		64.76 ± 0.18 i		57.76 ± 0.18 i
LSD0.05		1.42 **	0.38 **	0.74 **	0.21 **	0.72 **	0.18 **

The differences among mean values shown on the same line with the same letter are not significant (p < 0.05). ** significant at p < 0.01.

). Total phenolic contents of walnut cultivars and genotypes ranged between 237.51 (G-2) and 412.96 (G-1) mg GAE/100 g dw at 500 m altitude and 234.94 (G-11) to 392.31 (G-1) mg GAE/100 g dw at 1200 m altitude. Total antioxidant capacity ranged between 54.65% (Franquette) and 71.99% (G-14) DPPH inhibition at 500 m altitude. DPPH radical 47 ranged from 65% (Franquette) to 64.99% (G-14); DPPH inhibition ranged between 64.76% (Franquette) and 75.92% (G-1); and DPPH radical ranged between 57.76% (Franquette) and 68.92% (G-1) at 1200 m altitude (Table 1). In terms of total phenolic content, G-1, G-7, and G-16 genotypes differed from the others for both altitudes. In addition, in terms of antioxidant activity, G-1 and G-14 genotypes stand out at 500 m and 1200 m altitudes. Genotypes and cultivars grown at 500 m altitude were found to have higher values than the others. Our results show similarities with some studies in terms of total phenolic content [43] (50 to 2499 mg.100 g⁻¹) and differences with some studies, such as [44] (1020 to 2052 mg.100 g⁻¹), [45] (954 to 1682 mg.100 g⁻¹), [27] (540.08 to 1067.81 mg.100 g⁻¹), and [46] (750.67 to 1245.64 mg.100 g⁻¹). The findings of the biochemical properties obtained in this study are in accordance with previous studies [13,46,47,48]. It is observed that the biochemical properties of walnuts decrease with increasing altitude. Studies related to the effect of elevation on biochemical properties in walnuts are insufficient. It has been reported that in addition to genetic structure, tree age, cultural practices, diseases and pests, ecological conditions, and the number of fruits on the plant are also effective on biochemical contents in fruits [9]. These factors collectively contribute to the nutritional physiology of the fruit. Furthermore, numerous researchers have asserted that temperature plays a significant role in shaping the composition of fruits. Consequently, it is evident that a multitude of factors exert an influence on the biochemical constituents of fruits.

3.3. Protein and Oil Contents

The fatty acid profile is one of the most important parameters for the classification of walnut genotypes and cultivars. Protein and oil contents of promising walnut genotypes and cultivars are given in

Table 3. Total protein and oil contents of the walnut cultivars and genotypes (%). (Mean for 2021 and 2022.)

Altitude	Genotype	Protein Content (%)		Oil Content (%)
Low (500 m)	G-1	15.41 ± 0.42 lm	17.63 ± 2.03 A	55.04 ± 1.49 lm	63.33 ± 7.68 A
	G-2	15.34 ± 0.37 m		54.78 ± 1.33 m
	G-3	16.35 ± 0.20 jk		58.42 ± 0.73 jk
	G-4	14.96 ± 0.24 m		53.44 ± 0.87 mn
	G-7	19.11 ± 0.18 de		68.28 ± 0.66 de
	G-10	19.36 ± 0.26 cd		69.14 ± 0.93 cd
	G-11	20.22 ± 0.42 b		72.22 ± 1.48 b
	G-12	19.78 ± 0.32 bc		70.67 ± 1.16 bc
	G-14	19.11 ± 0.26 de		68.25 ± 0.94 de
	G-16	21.32 ± 0.34 a		76.17 ± 1.23 a
	Maraş-18	18.10 ± 0.16 f		64.64 ± 0.58 f
	Chandler	13.71 ± 0.37 o		57.57 ± 0.51 k
	Franquette	16.47 ± 0.15 jk		54.65 ± 0.52 m
High (1200 m)	G-1	13.56 ± 0.37 o	15.65 ± 1.92 B	48.44 ± 1.31 o	55.43 ± 7.26 B
	G-2	13.49 ± 0.33 o		48.21 ± 1.17 p
	G-3	14.39 ± 0.18 n		51.41 ± 0.64 no
	G-4	13.16 ± 0.21 op		47.03 ± 0.77 p
	G-7	16.82 ± 0.16 ij		60.08 ± 0.58 ij
	G-10	17.03 ± 0.23 hi		60.84 ± 0.82 hi
	G-11	17.79 ± 0.37 fg		63.56 ± 1.30 fg
	G-12	17.41 ± 0.29 gh		62.19 ± 1.02 gh
	G-14	16.81 ± 0.23 ij		60.06 ± 0.83 ij
	G-16	18.76 ± 0.30 e		67.03 ± 1.08 e
	Maraş-18	15.92 ± 0.14 kl		56.88 ± 0.51 kl
	Chandler	12.07 ± 0.16 p		43.11 ± 1.16 p
	Franquette	14.49 ± 0.13 lm		51.76 ± 0.47 no
LSD0.05		0.54 **	0.14 **	1.94 **	0.54 **

The differences among mean values shown on the same line with the same letter are not significant (p < 0.05). ** significant at p < 0.01.

. The protein and oil contents of walnut varieties and genotypes were found to be statistically significant (p < 0.05). The protein content of these genotypes and cultivars ranged between 13.71% (Chandler) and 20.22% (G-11) at 500 m altitude and between 12.72% (Chandler) and 18.76% (G-16) at 1200 m altitude (Table 3). Oil content ranged between 53.44% (G-4) and 76.17% (G-16) at 500 m altitude and between 43.11% (Chandler) and 67.03% (G-16) at 1200 m altitude (Table 3). In the study conducted by Muradoglu et al. (2010), the average oil content of walnut genotypes was found as 58.2%. Kafkas et al. (2017) determined that the oil content was between 49.4% and 70.70% [13,36]. In another study, Kafkas et al. (2020) determined that the protein content was between 13.57% and 25.72% [27]. In another study, Okatan et al. (2021) reported that oil content was between 50.88 and 64.28% in different walnut genotypes [46]. It is observed that the studies conducted in previous years are in accordance with the findings obtained from our study. In addition, in our results, it was determined that with the increase in altitude, protein and oil contents decreased on average. However, in studies on the Chandler walnut variety, Bayazıt et al. (2020) reported that total protein and oil contents increased with altitude, while Büyüksolak et al. (2020) reported that oil content increased with altitude and protein content decreased [9,39]. It is known that fat and protein content is under the control of genetic structure. However, factors such as tree age, the number of fruits on the plant, cultural treatments applied, and diseases and pests affect the fat and protein content of a genotype [9].

3.4. Quantification of Fatty Acids

The palmitic, stearic, and myristic acid of walnut genotypes and cultivars were analyzed, and statistical differences between genotypes and altitude were found significant at p < 0.05 level (

Table 4. Comparison of palmitic, stearic, and myristic acids (%) of several walnuts (Juglans regia L.), cultivars, and genotypes grown in Türkiye (mean for 2021 and 2022).

Altitude	Genotypes	Palmitic Acid (%)		Stearic Acid (%)		Myristic Acid (%)		Other Fat Acids (%)		Σ Saturated FA
Low (500 m)	G-1	6.16 ± 0.12	7.24 ± 0.48	3.37 ± 0.43	3.50 ± 0.29 A	0.06 ± 0.01	0.05 ± 0.01	0.50 ± 0.68	0.98 ± 0.98	10.09
	G-2	7.39 ± 0.02		3.56 ± 0.06		0.05 ± 0.01		0.25 ± 0.17		11.25
	G-3	7.81 ± 0.05		3.13 ± 0.08		0.07 ± 0.02		0.89 ± 0.47		11.90
	G-4	7.33 ± 0.05		3.55 ± 0.05		0.05 ± 0.01		0.97 ± 1.07		11.90
	G-7	7.75 ± 0.04		3.87 ± 0.07		0.04 ± 0.01		0.18 ± 1.95		11.84
	G-10	6.91 ± 0.16		3.86 ± 0.00		0.06 ± 0.01		0.04 ± 0.58		10.84
	G-11	6.64 ± 0.50		3.90 ± 0.03		0.06 ± 0.01		0.01 ± 1.31		10.61
	G-12	7.19 ± 0.16		3.32 ± 0.29		0.05 ± 0.01		0.68 ± 0.74		11.24
	G-14	6.93 ± 0.13		3.23 ± 0.5		0.06 ± 0.01		0.31 ± 0.33		10.53
	G-16	7.26 ± 0.11		3.50 ± 0.03		0.03 ± 0.01		0.15 ± 0.43		10.94
	Maraş-18	6.83 ± 0.40		3.53 ± 0.09		0.05 ± 0.01		0.07 ± 0.69		10.48
	Chandler	7.49 ± 0.10		3.50 ± 0.17		0.06 ± 0.01		1.33 ± 0.67		12.38
	Franquette	7.48 ± 0.00		3.21 ± 0.02		0.05 ± 0.01		0.30 ± 0.94		11.04
High (1200 m)	G-1	6.81 ± 0.55	7.24 ± 0.41	2.93 ± 0.39	2.78 ± 0.24 B	0.05 ± 0.01	0.05 ± 0.01	0.01 ± 1.23	0.42 ± 1.25	9.80
	G-2	7.37 ± 0.08		2.74 ± 0.04		0.05 ± 0.01		0.78 ± 0.74		10.94
	G-3	7.78 ± 0.14		2.41 ± 0.06		0.06 ± 0.02		0.57 ± 0.60		10.82
	G-4	7.31 ± 0.14		2.73 ± 0.04		0.04 ± 0.01		0.75 ± 0.40		10.83
	G-7	7.73 ± 0.08		2.98 ± 0.05		0.04 ± 0.01		0.01 ± 1.46		10.76
	G-10	6.89 ± 0.11		2.97 ± 0.00		0.05 ± 0.01		1.63 ± 0.77		11.54
	G-11	6.63 ± 0.53		3.00 ± 0.02		0.06 ± 0.01		1.91 ± 0.88		11.60
	G-12	7.17 ± 0.24		2.56 ± 0.22		0.06 ± 0.01		0.48 ± 1.57		10.81
	G-14	6.91 ± 0.07		2.85 ± 0.30		0.03 ± 0.01		1.97 ± 1.46		11.76
	G-16	7.24 ± 0.20		2.70 ± 0.07		0.06 ± 0.01		1.73 ± 0.84		11.73
	Maraş-18	7.18 ± 0.09		2.79 ± 0.06		0.05 ± 0.01		1.90 ± 0.71		11.92
	Chandler	7.47 ± 0.07		2.94 ± 0.17		0.05 ± 0.01		0.42 ± 0.07		10.48
	Franquette	7.57 ± 0.02		2.53 ± 0.06		0.05 ± 0.01		0.75 ± 1.33		10.90
LSD0.05		N.S.	N.S.	N.S.	0.08 **	N.S.	N.S.	N.S.	N.S.

The differences among mean values shown on the same line with the same letter are not significant (p < 0.05). ** significant at p < 0.01 and N.S.: not significant.

). At 500 m altitude, walnut genotypes and cultivars contained 6.16% (G-1)-7.81% (G-3) palmitic acid, 3.13% (G-3)-3.90% (G-11) stearic acid, and 0.03% (G-16)-0.07% (G-3) myristic acid. At 1200 m altitude, walnut genotypes and cultivars contained 6.63% (G-11)-7.78% (G-3) palmitic acid, 2.41% (G-3)-3.00% (G-11) stearic acid, and 0.03% (G-14)-0.06% myristic acid. Doğan and Akgül (2005), in their study of some walnut (Juglans regia L.) varieties from eastern Anatolia, found that palmitic acid content ranged between 5.61% and 5.82%, while trace amounts of myristic acid (<0.1%) were detected in the samples [49]. Popa et al. (2011) reported 9.75% palmitic acid and 3.48% stearic acid in some walnut cultivars [50]. Özrenk et al. (2012) reported 4.98–6.77% palmitic acid, 0.050–0.12% palmitoleic acid, and 1.88–3.93% stearic acid in walnut genotypes [51]. In another study, Bayazıt and Sümbül (2012) found that palmitic acid values ranged between 6.98% and 8.77% and stearic acid values ranged between 3.22% and 4.99%, according to the results obtained from the genotypes they selected from the eastern Mediterranean region of Türkiye [11]. Bouabdallah et al. (2014) determined that palmitic acid ranged between 7.28% and 8.95%, while stearic acid ranged between 3.01% and 3.89% [52]. In another study, Kafkas et al. (2017) determined that palmitic acid content ranged between 5.61% and 5.82%, and myristic acid was a trace amount (<0.1%) in different walnut varieties [13]. In another study conducted by Kafkas et al. (2020), palmitic acid values ranged between 5.74% and 9.79% and stearic acid values ranged between 2.04% and 3.00% in different walnut varieties [27]. In another study conducted by Okatan et al. (2021), palmitic acid content ranged between 6.72% and 9.06%, stearic acid between 0.07% and 0.66%, and myristic acid between 0.12% and 0.74%, depending on the variety [46]. When the results of our study are compared with the findings of other studies conducted in previous years, it is seen that our results are consistent and compatible.

Among hard-shelled fruits, walnuts are rich in polyunsaturated (linoleic and linolenic acids) and monounsaturated fatty acids (oleic and palmitic acids). Hard-shelled fruits are especially important because of their high content of omega-3 and omega-6 fatty acids. In many studies, it has been mentioned that one of the most important components of walnut fruit is the fats it contains. The fact that these fats are proportionally rich in polyunsaturated fatty acids is more important in human health and nutrition [27]. Oleic, palmitoleic, linolenic, and linoleic acid contents of all walnut cultivars and genotypes in our study are given in

Table 5. Comparison of oleic, palmitoleic, linolenic, and linoleic acids (%) of several walnut (Juglans regia L.) cultivars and genotypes grown in Türkiye (mean for 2021 and 2022).

Altitude	Genotypes	Oleic Acid (%)		Palmitoleic Acid (%)		Total MUFA	Linolenic Acid (%)		Linoleic Acid (%)		Total PUFA
Low (500 m)	G-1	18.66 ± 0.65 i–l	7.24 ± 0.48	0.06 ± 0.01	0.05 ± 0.01	18.72	9.03 ± 0.06 f–k	9.92 ± 0.58 A	61.12 ± 1.15 b–d	60.62 ± 1.20 A	70.15
	G-2	18.35 ± 0.38 j–m		0.05 ± 0.01		18.40	9.47 ± 0.06 d–i		60.49 ± 0.18 c–f		69.96
	G-3	18.48 ± 0.43 j–l		0.06 ± 0.01		18.54	8.61 ± 0.35 kl		60.36 ± 0.15 d–g		68.97
	G-4	17.83 ± 0.27 lm		0.05 ± 0.00		17.88	9.63 ± 0.34 d–g		59.77 ± 1.14 e–h		69.40
	G-7	19.42 ± 0.68 d–i		0.04 ± 0.01		19.46	8.96 ± 0.71 g–k		58.82 ± 0.81 h–k		67.78
	G-10	17.82 ± 0.26 lm		0.05 ± 0.00		17.87	9.72 ± 0.32 d–f		60.97 ± 0.42 b–e		70.69
	G-11	17.47 ± 0.33 m		0.06 ± 0.00		17.53	8.74 ± 0.43 i–k		62.44 ± 0.74 a		71.18
	G-12	20.27 ± 0.78 b–d		0.02 ± 0.01		20.29	7.93 ± 0.43 l		59.69 ± 0.67 f–h		67.62
	G-14	18.81 ± 0.20 h–k		0.06 ± 0.00		18.87	9.12 ± 0.15 e–k		60.96 ± 0.43 b–e		70.08
	G-16	17.98 ± 0.19 k–m		0.03 ± 0.00		18.01	8.68 ± 0.24 k		62.00 ± 0.11 ab		70.68
	Maraş-18	18.51 ± 0.18 j–l		0.05 ± 0.00		18.56	8.66 ± 0.29 kl		61.67 ± 0.49 a–c		70.33
	Chandler	18.61 ± 0.20 i–l		0.05 ± 0.00		18.66	8.70 ± 0.42 jk		59.49 ± 0.77 f–i		68.19
	Franquette	19.17 ± 0.46 e–j		0.04 ± 0.00		19.21	8.88 ± 0.27 h–k		60.33 ± 0.39 d–g		69.21
High (1200 m)	G-1	19.78 ± 0.69 b–f	18.57 ± 0.89 B	0.06 ± 0.01	0.05 ± 0.01	19.84	10.02 ± 0.07 b–d	8.93 ± 0.64 B	59.38 ± 0.47 f–i	58.60 ± 0.83 B	69.40
	G-2	19.45± 0.40 c–i		0.05 ± 0.01		19.50	10.51 ± 0.07 a–c		58.54 ± 0.45 h–l		69.05
	G-3	19.59 ± 0.45 c–h		0.06 ± 0.01		19.65	9.56 ± 0.39 d–h		59.12 ± 0.77 g–k		68.68
	G-4	18.89 ± 0.28 f–j		0.05 ± 0.00		18.94	10.69 ± 0.38 ab		58.90 ± 042 h–k		69.59
	G-7	20.59 ± 0.72 b		0.04 ± 0.01		20.63	9.94 ± 0.79 cd		58.90 ± 0.55 h–k		68.84
	G-10	18.88 ± 0.28 g–j		0.05 ± 0.00		18.93	10.79 ± 0.36 a		58.10 ± 0.49 j–l		68.89
	G-11	18.52 ± 0.35 j–l		0.06 ± 0.00		18.58	9.70 ± 0.48 d–g		59.31 ± 0.40 g–j		69.01
	G-12	21.48 ± 0.83 a		0.02 ± 0.01		21.50	8.80 ± 0.48 i–k		58.43 ± 1.03 ij		67.23
	G-14	19.93 ± 0.21 b–e		0.05 ± 0.00		19.98	10.13 ± 0.16 a–d		57.33 0.91± l		67.46
	G-16	19.06 ± 0.21 e–j		0.03 ± 0.00		19.09	9.80 ± 0.15 c–e		58.95 ± 0.41 h–k		68.75
	Maraş-18	19.62 ± 0.19 c–h		0.05 ± 0.00		19.67	9.99 ± 0.21 b–d		57.90 ± 0.48 kl		67.89
	Chandler	19.73 ± 0.21 b–g		0.05 ± 0.00		19.78	9.58 ± 0.42 d–h		59.22 ± 0.10 g–j		68.80
	Franquette	20.32 ± 0.48 bc		0.04 ± 0.00		20.36	9.43 ± 0.37 d–j		58.70 ± 0.41 h–l		68.13
LSD0.05		0.88 **	0.24 **	N.S.	N.S.		0.74 **	0.20 **	1.24 **	0.34 **

The differences among mean values shown on the same line with the same letter are not significant (p < 0.05). ** significant at p < 0.01 and N.S.: not significant.

. At 500 m altitude, palmitoleic and oleic acids were 0.03–0.06% and 17.47–20.27%, respectively. At 1200 m altitude, palmitoleic and oleic acid values were 0.03–0.06% and 18.52–21.48%, respectively.

Among the genotypes and cultivars, the highest oleic acid content was observed in G-12, and the lowest was observed in G-11 genotypes at 500 m and 1200 m altitudes. Doğan and Akgül (2005), in their study of some walnut (Juglans regia L.) varieties from eastern Anatolia, determined that oleic acid contents ranged between 22.63% and 27.27% [49]. Kafkas et al. (2017) determined that the values of palmitoleic and oleic acid ranged between 0.11% and 14.36% and 0.13% and 27.57%, respectively [13]. In another study, Beyhan et al. (2017) found that oleic acid ranged between 14.73% and 24.17%, and palmitoleic acid ranged between 0.00% and 0.16% [53]. In another study conducted by Kafkas et al. (2020), oleic acid ranged between 10.85% and 20.65%, and palmitoleic acid between 0.06% and 0.46% [27]. As seen in Table 5, at 500 m altitude, the values of linolenic acid and linoleic acid ranged between 7.93% (G-12) and 9.72% (G-10) and 58.82% (G-7) and 62.44% (G-11), respectively. At 1200 m altitude, the values of linolenic acid and linoleic acid ranged between 8.80% (G-12) and 10.79% (G-10), 57.33% (G-14), and 59.38% (G-1), respectively. Doğan and Akgül (2005) found that oleic acid content varied between 22.63% and 27.27% in different walnut types, while linoleic acid and linolenic contents varied between 49.93% and 54.41% and 14.32% and 17.82%, respectively [49]. Ünver and Çelik (2005) found that linoleic acid ranged between 41.13 and 61.15% and oleic acid ranged between 22.39 and 49.12% in the walnut types they examined [54]. In another study, linoleic acid content in walnut fat was determined as 56.57%, and linolenic acid content was 12.09% [44]. In another study in 2011, Rabrenovic et al. reported linoleic acid as 57.2–65.1% and linolenic acid as 9.1–13.6% [55]. Beyhan et al. (2017) found that linoleic acid varied between 53.23% and 63.62%, linolenic acid between 10.75% and 15.24%, and oleic acid between 14.73% and 24.17%. Kafkas et al. (2017) determined that the values of linoleic acid and linolenic acid varied between 7.83% and 13.16% and 53.24% and 62.92%, respectively [13]. In another study conducted in 2020, Kafkas et al. (2020) determined that linoleic acid varied between 58.96% and 66.07%, and linolenic acid between 8.41% and 16.14% [27]. There are limited studies on the effect of altitude on fatty acid contents in walnut fruit. Yuemei et al. (2014) reported that altitude influenced the fat contents of walnuts, and there was a negative correlation between oleic and linoleic acid. They also reported a positive effect of altitude on oleic acid [56]. Fuentealba et al. (2017) reported that palmitic, oleic, and linoleic acid content showed an increasing rate with elevation, although it was not statistically significant [57]. Büyüksolak et al. (2020) reported that oleic and linolenic acid contents in walnuts increased with altitude [39]. Our results were in accordance with the previous studies.

3.5. Organic Acid Content

The amount of sugar composition in fruit species is one of the important compositions that affect the taste and may vary according to varieties, ecological conditions, and technical and cultural practices [23,58,59]. In this study, organic acid contents in the fruits of walnut genotypes and cultivars were investigated, and significant differences were found between genotypes (p < 0.05) (

Table 6. Oxalic acid, citric acid, tartaric acid, malic acid, succinic acid, and fumaric acid content (%) of walnut cultivars and genotypes in Türkiye (mean for 2021 and 2022).

Altitude	Genotypes	Oxalic Acid (%)		Citric Acid (%)		Malic Acid (%)		Succinic Acid (%)
Low (500 m)	G-1	0.27 ± 0.02 a	3.09 ± 0.05 A	1.59 ± 0.00 b	1.17 ± 0.45	4.99 ± 0.08 c	4.27 ± 1.48 A	1.38 ± 0.00 d–f	2.00 ± 1.17 B
	G-2	0.23 ± 0.02 ab		1.06 ± 0.11 cd		3.35 ± 0.11 f–h		0.78 ± 0.08 f
	G-3	0.22 ± 0.01 bc		2.08 ± 0.09 a		7.33 ± 0.04 a		2.86 ± 0.03 a–c
	G-4	0.19 ± 0.02 b–d		1.62 ± 0.03 b		6.57 ± 0.05 b		2.40 ± 0.04 c–e
	G-7	0.19 ± 0.00 b–d		1.71 ± 0.02 b		5.21 ± 0.02 c		2.75 ± 0.05 b–d
	G-10	0.16 ± 0.00 de		1.15 ± 0.03 cd		4.43 ± 0.06 d		0.93 ± 0.04 f
	G-11	0.09 ± 0.00 f		0.56 ± 0.05 f		1.39 ± 0.09 i		0.43 ± 0.05 f
	G-12	0.10 ± 0.00 f		0.71 ± 0.04 ef		4.33 ± 0.16 d		1.08 ± 0.01 ef
	G-14	0.10 ± 0.00 f		0.53 ± 0.04 f		3.28 ± 0.06 gh		1.03 ± 0.01 ef
	G-16	0.12 ± 0.05 ef		0.87 ± 0.09 de		3.51 ± 0.04 fg		1.00 ± 0.01 f
	Maras-18	0.18 ± 0.01 cd		1.22 ± 0.02 c		4.31 ± 0.11 de		4.03 ± 0.24 ab
	Chandler	0.22 ± 0.03 bc		1.04 ± 0.05 cd		2.97 ± 0.07 h		3.03 ± 0.63 a–c
	Franquette	0.15 ± 0.02 de		1.01 ± 0.05 cd		3.81 ± 0.19 ef		4.20 ± 0.03 a
High (1200 m)	G-1	0.22 ± 0.01 b	2.85 ± 0.07 B	1.30 ± 0.08 b–d	0.18 ± 0.39	4.20 ± 0.04 b–d	3.71 ± 1.09 B	5.97 ± 0.12 a	3.41 ± 1.61 A
	G-2	0.22 ± 0.01 b		1.72 ± 0.02 ab		4.25 ± 0.17 b–d		0.83 ± 0.08 f
	G-3	0.21 ± 0.01 bc		1.02 ± 0.03 de		5.78 ± 0.20 a		3.81 ± 0.22 c–e
	G-4	0.17 ± 0.01 cd		1.69 ± 0.00 a–c		3.07 ± 0.15 ef		3.70 ± 0.04 de
	G-7	0.21 ± 004 bc		0.39 ± 0.02 f		4.70 ± 0.07 ab		4.92 ± 0.22 b
	G-10	0.33 ± 0.02 a		1.85 ± 0.01 a		4.51 ± 0.00 bc		1.26 ± 0.04 f
	G-11	0.17 ± 0.00 cd		1.24 ± 0.22 cd		1.35 ± 0.03 g		3.06 ± 0.22 e
	G-12	0.15 ± 0.00 d		1.12 ± 0.05 de		3.25 ± 0.03 d–f		4.43 ± 0.04 b–d
	G-14	0.15 ± 0.02 d		0.90 ± 0.10 de		3.91 ± 0.12 b–e		0.89 ± 0.14 f
	G-16	0.00 ± 0.00 e		1.34 ± 0.04 b–d		2.27 ± 0.06 fg		1.25 ± 0.04 f
	Maras-18	0.16 ± 0.00 d		1.05 ± 0.13 de		3.70 ± 0.14 b–e		5.08 ± 0.12 ab
	Chandler	0.23 ± 0.02 b		0.93 ± 0.06 de		3.41 ± 0.09 c–e		4.70 ± 0.11 bc
	Franquette	0.15 ± 0.05 d		0.78 ± 0.03 ef		3.80 ± 0.26 b–e		4.40 ± 0.21 b–d
LSD0.05		0.44 **	0.01 **	0.38 **	N.S.	0.84 **	0.22 **	1.06 **	0.28 **

The differences among mean values shown on the same line with the same letter are not significant (p < 0.05). ** significant at p < 0.01 and N.S.: not significant.

). In the walnut cultivars and genotypes investigated, at 500 m altitude, oxalic acid content ranged between 0.09% (G-11) and 0.27% (G-1), citric acid content ranged between 0.53% (G-14) and 2.08% (G-3), malic acid content ranged between 1.39% (G-11) and 7.33% (G-3), and succinic acid content ranged between 0.43% (G-11) and 4.20% (Franquette). At 1200 m altitude, oxalic acid content ranged between 0.00% (G-16) and 0.33% (G-10), citric acid content ranged between 0.39% (G-7) and 1.85% (G-10), malic acid content ranged between 1.35% (G-11) and 5.78% (G-3), and succinic acid content ranged between 0.83% (G-2) and 5.97% (G-1). These results indicate that the most dominant and major organic acid in these walnut genotypes and cultivars is malic acid, followed by citric, succinic, and oxalic acid. There are not enough studies on the organic acid content of walnuts in the literature. In a study conducted by Koç et al. (2019) in different walnut genotypes, it was determined that oxalic, succinic, malic, and fumaric acid contents varied between 8.39 and 12.08 mg, 2.86 and 5.32 mg, 0.26 and 3.00 mg, and 0.26 and 0.58 mg per 100 g, respectively. The researcher reported that malic acid was the dominant acid [60]. Erdoğan et al. (2021) found that oxalic, malic, and fumaric acid contents ranged between 5.51 and 7.56%, succinic acid between 5.67 and 9.64%, and malic acid between 0.23 and 0.66% in different walnut genotypes [48]. In another study, Okatan et al. (2021), in their study on different walnut varieties, found that the highest organic acid was oxalic acid, followed by acetic acid and ascorbic acid [50]. These three acids were determined as 4.453–7.227%, 1.270–2.463%, and 3.163–4.017%, respectively. It is determined that our results are in accordance with the results determined by different researchers.

3.6. Carbohydrate (Sugars) Content

The energy required for metabolic events in the human body is provided by food. Carbohydrates, which are one of the basic nutrients and the main energy source of the human body, are the most important nutrients used to obtain energy. In fruits and vegetables, carbohydrates are the main flavor components and one of the main fruit quality parameters preferred by both producers and customers. Most of the carbohydrate in walnuts comes from dietary fiber. In this study, the contents of the essential sugar (sucrose, glucose, and fructose) contents in the fruits of different walnut genotypes and cultivars were analyzed, and the differences between genotypes were determined (

Table 7. The sugar content of walnut genotypes and cultivars (%) (mean for 2021 and 2022).

Altitude	Genotypes	Sucrose (%)		Glucose (%)		Fructose (%)		Total Sugar (%)
Low (500 m)	G-1	3.06 ± 0.02 a–c	2.38 ± 0.60 A	0.20 ± 0.02 k	0.35 ± 0.03	0.21 ± 0.03 ij	0.34 ± 0.10 A	3.48 ± 0.03 b–d	3.04 ± 0.03 A
	G-2	2.08 ± 0.01 e–g		0.22 ± 0.01 i–k		0.15 ± 0.00 jk		2.45 ± 0.00 h–k
	G-3	2.08 ± 0.08 e–g		0.40 ± 0.09 c–e		0.38 ± 0.04 b–e		2.78 ± 0.05 g–i
	G-4	3.12 ± 0.00 ab		0.24 ± 0.02 h–k		0.14 ± 0.00 k		3.51 ± 0.02 bc
	G-7	1.42 ± 0.02 jk		0.49 ± 0.01 a		0.43 ± 0.02 bc		2.35 ± 0.05 i–l
	G-10	2.42 ± 0.04 de		0.37 ± 0.08 d–f		0.32 ± 0.01 e–g		3.12 ± 0.13 d–f
	G-11	2.08 ± 0.00 e–g		0.33 ± 0.00 e–g		0.28 ± 0.00 gh		2.70 ± 0.01 g–i
	G-12	2.91 ± 0.01 a–c		0.48 ± 0.10 ab		0.40 ± 0.00 b–d		3.79 ± 0.09 ab
	G-14	1.34 ± 0.01 jk		0.40 ± 0.00 c–e		0.33 ± 0.01 e–g		2.07 ± 0.00 lm
	G-16	3.13 ± 0.01 a		0.43 ± 0.01 a–d		0.33 ± 0.01 e–g		3.89 ± 0.04 a
	Maras-18	2.75 ± 0.08 b–d		0.40 ± 0.08 b–e		0.33 ± 0.02 e–g		3.49 ± 0.02 bc
	Chandler	1.91 ± 0.08 gh		0.35 ± 0.04 e–g		0.25 ± 0.05 hi		2.51 ± 0.06 h–k
	Franquette	2.71 ± 0.13 cd		0.29 ± 0.03 g–i		0.32 ± 0.05 e–g		3.33 ± 0.06 c–e
High (1200 m)	G-1	3.03 ± 0.01 a–c	2.10 ± 0.65 B	0.21 ± 0.00 jk	0.35 ± 0.03	0.20 ± 0.03 i–k	0.30 ± 0.07 B	3.45 ± 0.07 c–e	2.80 ± 0.00 B
	G-2	1.16 ± 0.08 k		0.29 ± 0.00 g–j		0.37 ± 0.01 c–f		1.83 ± 0.09 m
	G-3	1.90 ± 0.04 gh		0.40 ± 0.01 c–e		0.37 ± 0.05 b–f		2.68 ± 0.00 g–j
	G-4	3.10 ± 0.06 ab		0.29 ± 0.02 f–h		0.24 ± 0.08 hi		3.65 ± 0.03 a–c
	G-7	1.47 ± 0.04 jk		0.43 ± 0.01 a–d		0.38 ± 0.03 b–e		2.29 ± 0.08 kl
	G-10	1.64 ± 0.07 h–j		0.44 ± 0.01 a–d		0.43 ± 0.01 b		2.52 ± 0.07 h–k
	G-11	1.69 ± 0.06 h–j		0.35 ± 0.02 e–g		0.29 ± 0.01 gh		2.33 ± 0.10 j–l
	G-12	2.92 ± 0.05 a–c		0.33 ± 0.01 e–g		0.36 ± 0.04 d–f		3.62 ± 0.02 a–c
	G-14	1.87 ± 0.00 g–i		0.41 ± 0.00 b–e		0.38 ± 0.01 b–f		2.66 ± 0.01 g–j
	G-16	1.53 ± 0.00 ij		0.46 ± 0.01 a–c		0.54 ± 0.00 a		2.54 ± 0.05 h–k
	Maras-18	2.33 ± 0.06 ef		0.34 ± 0.02 e–g		0.32 ± 0.01 fg		2.99 ± 0.08 e–g
	Chandler	1.90 ± 0.08 gh		0.23 ± 0.01 h–k		0.20 ± 0.01 i–k		2.34 ± 0.18 j–l
	Franquette	2.78 ± 0.11 a–c		0.34 ± 0.01 e–g		0.36 ± 0.03 d–f		3.49 ± 0.15 bc
LSD0.05		0.36 **	0.10 **	0.06 **	N.S.	0.06 **	0.02 **	0.34 **	0.98 **

The differences among mean values shown on the same line with the same letter are not significant (p < 0.05). ** significant at p < 0.01 and N.S.: not significant.

). All specific sugars and total sugars showed statistically significant differences between genotypes and cultivars (p < 0.05). Sugar contents of walnut seeds showed a wide variation depending on genotypes and cultivars. Among all genotypes and cultivars, sucrose and glucose were the main sugars at 500 m and 1200 m altitude. At 500 m altitude, sucrose, glucose–fructose, and total sugar contents ranged between 1.34% (G-14) and 3.13% (G-16), 0.20% (G-1), and 0.49% (G-7), 0.14% (G-4), 0.43% (G-7), 2.07% (G-14), and 3.89% (G-16), respectively. At 1200 m altitude, sucrose, glucose, fructose, and total sugar contents ranged between 1.16% (G-2) and 3.10 (G-4), 0.21% (G-1), 0.46 (G-16), 0.20% (G-1 and Chandler), 0.54 (G-16), 1.83% (G-2), and 3.62 (G-12), respectively. There are not enough studies on the sugar content of walnuts in the literature. On the other hand, the fructose, glucose, and sucrose contents of hard-shelled fruits vary depending on species, varieties, genotypes, cultivars, climate, and ecological factors. Kazankaya et al. (2008) determined that fructose and sucrose were the dominant and major sugars in different varieties of walnuts, almonds, and hazelnuts [58]. The same researchers determined 1.13% (Ohadi)-5.04% (Siirt) fructose, 1.01% (Siirt)-4.25% (red) glucose, 2.58% (Uzun)-4 74% (Buttum) sucrose, and 0.26 (red)-0.99 (Halebi) g/100 g maltose. The sugar contents of the seeds of walnut varieties were 0.35% (D-1)-2.67% (H-1) fructose, 0.13% (D-1)-6.26% (B-1) glucose, 1.76% (D-1)-4, 17% (V-1) sucrose, 0.80–4.00% fructose, and 1.52–0.86% glucose. There were 2.91–3.23% sucrose contents of hazelnut varieties, according to Tombul and E-1, respectively [58]. Studies on the effect of altitude on the sugar content of walnuts are limited. In this regard, Fuentealba et al. (2017) conducted a study in the coastal and mountainous regions and found that the sugar content of walnuts increased as the altitude increased from the coast to higher altitudes [57]. Based on the results of the study, we can conclude that the sugar content of walnuts is affected by climatic factors as well as genetic factors. There are not enough studies in the literature investigating the effect of altitude on the organic acid content of walnuts. Organic acids are found in plants as compounds such as salts, esters, and glycosides [59]. In addition, organic acids differ according to the nutritional status of plants because they provide cation balance in plants [61]. In our study, it was found that malic acid, succinic acid, and citric acid were found intensively in walnuts. This result supports the results of the study conducted by Koç et al. (2019) [60].

The graph of the correlation relationship between the chemical, biochemical, sugar, organic acid, and fatty acid properties examined within the scope of the study is given in Figure 2. There was no significant correlation between total phenolic matter and total antioxidant activity of walnuts and other properties. However, there was a significant positive correlation between total protein and total fat contents. It is seen that there is a significant positive correlation between total protein and total fat contents of the fruits, between linoleic and stearic acid among the fatty acids of the fruits, and between glucose and fructose contents among the sugar contents. However, it was determined that there was a negative relationship between total protein and total fat contents of the fruits, linoleic acid, and palmitoleic acid among the fatty acids of the fruits and organic acids. A strong negative relationship was found between oleic acid and linoleic acid, linolenic acid and stearic acid, and linolenic acid and stearic acid. On the other hand, a strong positive relationship was found between linoleic acid and stearic acid and between palmitoleic acid and myristic acid. Among the sugar contents of the fruits, there is a strong positive relationship between glucose and fructose, while there is a negative relationship between saccharose and glucose, as well as fructose. There is a positive relationship between the organic acid contents of the fruits. There is a strong positive relationship between oxalic acid and citric acid, as well as malic acid, and between citric acid and malic acid. The correlation findings obtained in this study are like the findings of Yuemei et al. (2014), Funtealba et al. (2017), and Rao et al. (2016) [56,57,61,62].

The results of the principal component analysis of 25 characteristics related to chemical, biochemical, sugar, organic acid, and fatty acid contents of walnut fruits are given in

Table 8. Principal component analysis of walnut genotypes based on chemical, biochemical, sugar, organic acid, and fatty acid data.

Properties	PCA 1	PCA 2	PCA 3	PCA 4	PCA 5	PCA 6	PCA 7
T.F	−0.048	−0.055	0.190	0.211	−0.121	0.493	0.399
T.A	−0.029	0.124	0.284	0.232	−0.335	−0.315	0.047
Protein	0.350	−0.057	−0.021	−0.019	−0.043	0.053	0.202
Oil	0.356	−0.036	−0.010	−0.039	−0.040	0.078	0.166
Oleic A.	−0.002	−0.391	−0.028	−0.016	0.228	0.181	−0.303
Linoleic A.	0.064	0.259	−0.256	0.173	−0.051	−0.223	0.363
Linolenic A.	−0.092	0.289	0.241	−0.030	−0.275	0.179	−0.035
Palmitic A.	−0.105	−0.270	0.267	−0.167	−0.094	−0.317	0.052
Palmitoleic A.	−0.160	0.328	−0.055	−0.137	0.268	−0.136	0.179
Stearic A.	0.089	0.228	0.030	−0.151	−0.138	0.534	0.090
Miristic A.	−0.160	0.330	−0.038	−0.135	0.272	−0.108	0.199
Sucrose	−0.074	−0.064	−0.117	0.520	0.100	0.031	0.030
Glucose	0.272	−0.178	0.201	−0.157	0.040	−0.055	0.241
Fructose	0.246	−0.220	0.161	−0.130	−0.012	−0.110	0.250
T. Sugar	−0.017	−0.131	−0.090	0.505	0.124	−0.001	0.116
Oxalic A.	−0.242	0.128	0.194	−0.102	0.212	0.264	−0.170
Citric A.	−0.144	0.200	0.353	0.241	0.016	−0.068	−0.029
Malic A.	−0.143	−0.084	0.418	0.065	0.282	−0.084	0.015
Succinic A.	−0.188	−0.193	−0.032	0.084	0.332	0.117	0.392
S.F.W	0.201	0.085	0.361	−0.052	0.284	−0.022	0.086
I.F.W	0.292	0.181	0.095	−0.027	0.250	−0.045	−0.013
I.R	0.265	0.199	−0.240	0.030	0.099	−0.048	−0.114
F.L	0.239	0.125	0.223	0.247	−0.174	−0.004	−0.197
F.W	0.280	0.036	0.080	0.258	0.199	0.053	−0.247
F.H	0.241	0.182	0.050	0.050	0.283	0.018	−0.157
Eigenvalue	7.41	4.67	3.26	3.10	2.03	1.47	1.00
Variations (%)	29.64	18.70	13.05	12.39	8.12	5.88	3.98
Total variation (%)	29.64	48.34	61.39	73.78	81.90	87.78	91.76

. According to the results of the principal component analysis, seven main components (eigenvalue ≥ 1.00) represented 91.76% of the total variation. In the principal component analysis, a cluster analysis is more reliable if 25% or more of the total variation can be explained by the first two or three components [63]. In our study, the first principal component is reliable as it represents 61.39% of the total variation. The first (PCA1), second (PCA2), and third (PCA3) principal components represent 29.64%, 18.70%, and 13.05% of the total variance, respectively. There are differences in the characteristics that make the most impact on the first three principal components. Total protein content and total fat content were most significant for the first principal component; oleic acid, palmitoleic acid, and myristic acid were most significant for the second principal component; and citric acid, malic acid, and fruit weight were most significant for the third principal component.

The heatmap graph based on Ward’s method, which evaluates walnut cultivars and genotypes together with their fruit characteristics, is given in Figure 3. The graph shows that walnut cultivars and genotypes are divided into two subgroups. Chandler and Franquette varieties and G-1, G-2, and G-4 genotypes were in the same group, while the other genotypes and Maraş-18 varieties were in the same group. The characteristics examined within the scope of the study were divided into two main groups, and these groups were again divided into subgroups within themselves. Among the fruit characteristics, pomological characteristics and total protein ratio, total fat ratio, glucose, and fructose contents of the fruits formed the first group. Other characteristics formed the second group. It is noteworthy that pomological characteristics were in the same group, while other characteristics showed different distributions.

4. Conclusions

In this study, the effects of altitude on the quality characteristics, oil, protein, total phenolics, total antioxidant capacity, fatty acid composition, organic acids, and sugar contents of walnut fruit in subtropical climate conditions were investigated. In our research, the differences between varieties and genotypes were effective in determining the biochemical content of walnut seeds, and when the results obtained for biochemical content were analyzed, it was determined that these differences were statistically significant (p < 0.05). Among the biochemical contents of the analyzed walnut genotypes and varieties, it was found that fat had the highest value and linoleic acid was the major fatty acid, followed by oleic and linolenic acids, respectively. In our study, the most dominant and major organic acid among all genotypes and cultivars at 500 m and 1200 m altitude was malic acid, followed by citric, succinic, and oxalic acid. Sucrose and glucose were found to be the most common major sugars in the genotypes and cultivars examined. In our study, it was also determined that altitude had significant effects on the biochemical contents of walnut genotypes and cultivars. It was determined that some contents increased and some contents decreased due to the increase in altitude. While our study results are similar to those of some other studies in the literature, there are differences in terms of some characteristics. The differences between the studies may be due to genetic factors, the effect of environment, and differences in methods.

In conclusion, the effect of altitude on walnut fruits was found to be significant. It was observed that there are a limited number of studies indicating the effect of altitude on walnut fruits. On the other hand, there are not enough studies on the organic acid and sugar content of walnuts in the literature. In this study, the results of the effects of altitude on fruit quality and biochemical properties of walnuts, which are missing in the literature, are seen. Temperature is the most important factor affecting fruit quality in walnuts. While high temperatures in summer cause inner darkening in walnuts, low temperatures and cold winds negatively affect vegetative development and the physical and chemical structure of the fruit. As altitude increases, climate change changes the yield and quality of walnuts. In addition, annual maintenance operations such as pruning, irrigation, and fertilization can also affect various characteristics of fruits. New studies should be designed by combining all these. We believe that these genotypes will serve as a source for future studies, can be used as parents in walnut breeding studies, and will help international walnut breeders and researchers interested in the development of varieties with superior and better qualities.