Foliar application of zinc-glycine and zinc-sulfate differentially affects leaf biochemical attributes and fruit quality in Newhall orange trees

Abstract

Zinc (Zn) is an essential nutrient that impacts fruit quality, improves human health, and can help plants biochemically adapt to the adverse effects of climate change. This study evaluated the potential effects of foliar applications of two sources of Zn (Zn-glycine (Zn-G) and Zn-sulfate (Zn-S)), at three rates (150, 225, and 300 mg-Zn/L), on leaf biochemical attributes and fruit quality in Newhall orange over two consecutive years (2022–2023). The results showed that leaf biochemical and fruit quality traits increased dose-dependently for both Zn compounds. Most leaf biochemical traits showed a gradual increase with the increase in Zn rate, depending on the Zn source. Leaf biochemical attributes, in terms of chlorophyll a, chlorophyll b, soluble carbohydrate, starch, soluble protein, antioxidant capacity, and superoxide dismutase enzyme activity, were increased by 85, 75, 14, 62, 87, 42, and 14%, respectively, through superior treatment (G-300). Additionally, the findings revealed that the G-225 and G-300 treatments remarkably enhanced the colour intensity of Newhall peels, as evidenced by a notable increase in the colour index (6.10). Zn application increased the TSS/TA ratio in Zn-G and Zn-S treatments, especially with foliar application of Zn-S (8.7–10.2). The highest fruit antioxidant capacity and total phenolic contents were found in the foliar application of the G-300 treatment. These results suggest that the foliar application of Zn-G as a beneficial source, particularly in a concentration of 300 mg-Zn/L, can be considered an effective strategy to improve biochemical leaf traits and the external and internal quality of fruits in Zn-deficient soils.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-40539-x.

Introduction

Citrus cultivation is widespread in many countries, and Iran is the 10th-largest citrus producer in the world¹. Citrus fruits have a high nutritional value and are of interest to consumers because of their bioactive compounds, such as vitamin C, phenolic compounds, terpenoids, pectin, and high antioxidant activity². The Newhall nucellar navel orange is a relatively new variety whose cultivation is expanding in Iran.

Plant nutrition is one of the factors that affects fruit quality and can help prepare plants biochemically to bolster their resilience to climate change³. Among the essential nutrients for plants, Zn plays a crucial role in enhancing crop resistance to environmental stress by regulating various physiological and molecular mechanisms⁴. However, it is estimated that more than half of the agricultural soils are inherently deficient in Zn⁵. Additionally, approximately one-third of the global population suffers from Zn deficiency⁵. Biofortification, the biological enrichment of staple crops with essential micronutrients, is the most appropriate way to address microelement deficiencies⁶. Agronomic biofortification through fertilization (soil, or foliar) can be used to change agricultural systems in ways that will help to increase the Zn content of plants and combat Zn deficiency in human diets without changing their genetic structure⁷.

Zn deficiency is a common nutritional disorder in citrus trees, impacting flowering intensity, fruit set, yield, and production consistency⁸. Visual symptoms of Zn deficiency in citrus are characterized by irregular and chlorotic leaf spots, resulting in mottled leaves, small leaves on terminal growth, and severe dieback of twigs⁹. Inorganic Zn (i.e., Zn sulfate, Zn chloride, and Zn nitrate) and synthetic Zn-chelates (i.e., Zn-EDTA and Zn-DTPA) are common sources of Zn fertilizer for citrus^10–13. The best strategy for applying this nutrient, whether via soil or foliar spraying on trees, depends on the source of fertilizer used¹⁴. Compared to soil application, foliar fertilization is a fast and precise method of delivering minerals and bio-stimulants to plants and is also economical, ecological, and environmentally friendly¹⁵. In terms of Zn biofortification effects, synthetic Zn-chelates (i.e., Zn-EDTA) are less effective for foliar application in citrus when compared to Zn sources in the form of soluble salts¹⁴, due to a much larger size, higher points of deliquescence¹⁶, and the lower uptake efficacy by leaves¹⁴. Foliar application of Zn sources in the form of soluble salts, i.e., Zn sulfate, is a fast and effective method to improve the horticultural performance of trees¹⁴. Similarly, the positive effects of foliar Zn application on nutritional status¹⁰, and quality characteristics of citrus fruit¹⁷ have been reported in some studies. However, previous studies have shown that most commercial inorganic Zn fertilizers contain cadmium and other toxic heavy metals as impurities¹⁸. Also, long-term and excessive fertilizer application increases production costs and undermines soil health, and sustainable agriculture¹⁹. Another chelated Zn source is based on natural organic chelators, such as amino acids²⁰.

Today, it is crucial to enhance the sustainability of agricultural production systems through a significant reduction of synthetic agrochemicals, including pesticides and fertilizers. This can be achieved by adopting environmentally friendly innovations, including natural plant bio-stimulants and organic fertilizers. These practices are crucial for ensuring continuous productivity throughout the seasons, under both optimal and suboptimal conditions²¹. Plant nutrition can be improved by combining the bio-stimulant effects and nutrient supply in advanced fertilizers such as biochelates²². Amino chelates are among the new formulations of fertilizers that have a natural origin and have attracted the attention of researchers due to their great potential for use. They contain environmentally friendly chelating agents that improve soil health^23,24 and are biodegradable and non-toxic for humans and animals, with a reduced environmental footprint²². Therefore, they can be used in agriculture without the health and environmental concerns raised for synthetic chelates²². For example, Gil et al.²⁴ reported that amino acid-based biofertilizers derived from dairy waste have improved soil health, increased crop yields, decreased the need for synthetic fertilizers, and can contribute to sustainable agriculture. Some studies have indicated that the Zn-biofortified performance of Zn-G was better than that of ZnSO₄, while the foliar phytotoxicity of Zn-G was lower than excess Zn-S in plants²⁵. Also, the previous studies showed that the efficacy of Zn-based amino acid-fertilizer and Zn sulfate in promoting Zn delivery to plants was in a dose-dependent manner^26,27. Despite the great potential of biochelates as fertilizers, this technology is still slightly used in agriculture, and comparative performance analyses among Zn sources on citrus trees are scarce.

Despite the great importance of Zn in citrus nutrition, little information is available on the efficacy of foliar application of Zn, especially in the form complexed with amino acids. In Iran, citrus fruits are cultivated in the northern and southern provinces of the country. A study of the Zn distribution map in the soils of Iran showed that the Zn availability in most of these soils is sub-optimal⁸. Therefore, correcting Zn deficiency is essential to achieve higher yields and quality in citrus. Zn sulfate is the most commonly used Zn fertilizer. Nowadays, given the global trend towards using sustainable and low-input agricultural systems, as well as trends towards reducing the use of chemical fertilizers and moving towards organic and natural systems, the use of natural fertilizer sources for ensuring profitability, environmental health, and social and economic equity is inevitable. Applying amino acid-chelated Zn (amino chelates) as compatible compounds with less environmentally unfriendly agriculture can be a more sustainable approach to safer production than chemical fertilization. Therefore, the present study explores the possible advantages of the foliar application of Zn-glycine (Zn-G) as an innovative zinc fertilizer compared to traditional Zn-sulfate (Zn-S) fertilizers on leaf biochemical characteristics and fruit quality in Newhall orange. The specific objectives of the present study were: to identify an optimal Zn source and rate (1) to improve the external and internal quality of Newhall orange fruits, and (2) to stimulate leaf biochemical attributes to cope with environmental stress.

Results

Leaf biochemical attributes

The results showed that the effect of spray treatments (different Zn concentrations from various Zn sources including Zn-glycine at 150 (G-150), 225 (G-225), and 300 (G-300) mg-Zn/L rates and Zn-sulfate at 150 (S-150), 225 (S-225), and 300 (S-300) mg-Zn/L rates) on all studied traits, including the leaf Zn concentration, total phenolic, antioxidant capacity, super oxide dismutase (SOD) activity, soluble protein, total soluble carbohydrate, starch, and chlorophyll, was significant. Additionally, the studied year affected all traits except total soluble carbohydrates and starch content. In addition, the results showed that the effect of spray treatments on Zn concentration, soluble protein, and pigment contents of leaves depended on the year studied (Table 1).

Table 1.

Summary of two-factor analysis of variance (ANOVA) results (mean square values) for leaf biochemical attributes over two sampling years of Newhall trees sprayed with different Zn treatments (concentration and sources).

Source	Year	Error a	Zinc	Year*Zinc	Error b
df	1	6	6	6	36
Zinc	67*	11	1280*	33*	3.31
Chlorophyll a	41*	0.02	0.21*	0.16*	0.01
Chlorophyll b	1.33*	< 0.001	0.09*	0.06*	< 0.001
Carotenoids	0.002*	0.001	0.004*	0.007*	< 0.001
Protein	0.16*	< 0.001	0.08*	0.02*	0.01
Total soluble carbohydrate	0.01 n.s	0.09	0.17*	< 0.001 n.s	0.04
Starch	2.22 n.s	1.26	136*	0.77 n.s	1.65
Total phenolics	294,272*	297	3227*	239 n.s	412
Antioxidant capacity	762*	19	144*	13 n.s	6.50
Superoxide dismutase	0.014*	0.003	0.06*	0.002 n.s	0.003

*, P ≤ 0.05; ns, not significant.

Zn

The results indicated that in both years analyzed, applying Zn to the leaves increased the concentration of this element in the leaves. The highest and lowest Zn concentrations were found in the foliar application treatment of Zn-S at a 300 mg-Zn/L rate and the leaves of the control trees, respectively. The results showed that foliar application of 150, 225, and 300 mg-Zn/L of Zn-G source, on average, increased leaf Zn concentration by 103, 139, and 181%, respectively, while the same concentrations of the Zn-S source raised leaf Zn levels by 76, 165, and 267% (Fig. 1).

Fig. 1.

Effect of foliar Zn application (concentration (0, 150, 225, 300 mg-Zn/L) and source (Zn-Glycine and Zn sulfate)) on Zn content of Newhall orange leaves over two sampling years (2022–2023). Means with the same small letter are not significantly different at P ≤ 0.05.

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Pigments

The results showed that Zn foliar application promoted the leaf pigment content. In both years of the study, the highest levels of chlorophyll a and b were observed in the leaves sprayed with the Zn-G at a 300 mg-Zn/L rate, while the control leaves exhibited the lowest chlorophyll content (Fig. 2). Compared to Zn-S, the promotion effect of Zn-G on leaf chlorophyll was greater at all doses studied. Moreover, the results showed that only Zn-S application significantly increased leaf carotenoid contents (Fig. 2).

Fig. 2.

Effect of foliar Zn application (concentration (0, 150, 225, 300 mg-Zn/L) and source (Zn-Glycine and Zn sulfate)) on pigment content of Newhall orange leaves over two sampling years (2022–2023). For each attribute, Means with the same small letter are not significantly different at P ≤ 0.05.

Total soluble protein

The results showed that leaf protein content improved with the foliar application of Zn, especially at 225 and 300 mg-Zn/L rates. Furthermore, the results showed that the effect of Zn supply from the Zn-G source on leaf protein content was greater than that of Zn-S. Foliar application of 150, 225, and 300 mg‑Zn/L of Zn‑G, on average, increased leaf protein content by 30, 39, and 87%, respectively, while foliar application of 150, 225, and 300 mg‑Zn /L of Zn‑S raised leaf protein content by 15, 26, and 49%, respectively (Fig. 3).

Fig. 3.

Effect of foliar Zn application (concentration (0, 150, 225, 300 mg-Zn/L) and source (Zn-Glycine and Zn sulfate)) on total protein content of Newhall orange leaves over two sampling years (2022–2023). Means with the same small letter are not significantly different at P ≤ 0.05.

Carbohydrates and starch

The results showed that among the foliar application treatments studied, the soluble carbohydrate content of leaves was only significantly affected by the G-225 and G-300 treatments. The foliar application of G-225 and G-300 led to a 13 and 14% increase in the total soluble carbohydrates of leaves. Moreover, the results showed that the foliar application of G-150, G-225, G-300, and S-300 significantly improved the starch content of the leaves. The greatest increase in starch content, expressed as a percentage, after foliar zinc application was observed in G-225 (24.4 g/100 g F.W) and G-300 (24.7 g/100 g F.W) treatments. The results also showed that the promotion effects of Zn-G on carbohydrate content were slightly greater than the effects of Zn-S (Fig. 4).

Fig. 4.

Effect of foliar Zn application (concentration (0, 150, 225, 300 mg-Zn/L) and source (Zn-Glycine and Zn sulfate)) on total soluble carbohydrate (TSC) and starch content of Newhall orange leaves (Two-year average). For each attribute, Means with the same small letter are not significantly different at P ≤ 0.05.

Total phenolic content

The leaf phenolic content was significantly influenced by Zn foliar application. The highest total phenolic was observed in the G-300 and S-300 treatments, while the lowest was obtained in the control leaves. Foliar application of 150, 225, and 300 mg‑Zn/L of Zn‑G, on average, increased leaf phenolic compound by 2.9, 6.2 and 12%, respectively, while foliar application of 150, 225, and 300 mg‑Zn /L of Zn‑S raised phenolic compound by 3.5, 10, and 14%, respectively (Fig. 5A).

Fig. 5.

Effect of foliar Zn application (concentration (0, 150, 225, 300 mg-Zn/L) and source (Zn-Glycine and Zn sulfate)) on total phenolics (A), antioxidant capacity content (B), and SOD activity (C) in the Newhall orange leaves (Two-year average). For each attribute, Means with the same small letter are not significantly different at P ≤ 0.05.

Antioxidant capacity

The foliar application of Zn significantly enhanced the antioxidant capacity of the leaves, with the most notable improvements occurring in the glycine treatments (Fig. 5B). The results indicated that the foliar application of 150, 225, and 300 mg-Zn/L of Zn-G source, on average, increased leaf antioxidant capacity by 18, 29 and 42%, respectively, while the same concentrations of the Zn-S source raised leaf antioxidant capacity by 1.4, 17, and 28% (Fig. 5B).

SOD activity

The application of various concentrations of Zn-G and Zn-S significantly enhanced the SOD activity in the leaves. The highest SOD activity was observed in the G-300 treatment, whereas the control leaves exhibited the lowest activity. Additionally, the results indicated that the positive effects of Zn-G on SOD activity were more pronounced than those of Zn-S (Fig. 5C). The foliar application of 150, 225, and 300 mg-Zn/L of Zn-G source, on average, increased leaf SOD activity by 9.4, 12, and 14%, respectively, while the same concentrations of the Zn-S source raised leaf SOD activity by 6.0, 10, and 12% (Fig. 5C).

Fruit quality attributes

The results indicated significant main effects of Zn application and the year on various attributes, including TSS, TA, the TSS/TA ratio, peel thickness, citrus colour index (CCI), total phenolic content, and antioxidant capacity (Table 2). Additionally, the interaction effects of Zn treatments and the year on all the studied attributes, except for total phenolic content and peel thickness, were also found to be significant (Table 2

Table 2.

Summary of three-factor analysis of variance (ANOVA) results (mean square) for fruit quality attributes over two sampling years of Newhall trees sprayed with different Zn treatments (concentration and sources).

Source	Year	Error a	Zinc	year*zinc	Error b
df	1	6	6	6	36
Citrus colour index	70*	0.26	16*	1.09*	0.21
Peel thickness	29*	0.11	1.55*	0.10 n.s	0.13
Juice content	246 n.s	4.57	23 n.s	14 n.s	12
Total soluble solids	2.45*	0.03	0.58*	0.10*	0.04
Titratable acidity	0.04*	0.01	0.11*	0.02*	0.00
Total soluble solids to titratable acidity	0.02 n.s	0.63	8.97*	0.77*	0.22
Total phenolic	1259*	17	818	9.39 n.s	9.74
Antioxidant capacity	1307*	25	614*	29*	10

*, P ≤ 0.05; ns, not significant.

Peel colour

The interaction effects of Zn application × year on the CCI trait of fruit peel were significant (Table 2). In both years studied, the highest amount of CCI of fruit peel (year 1: 4.58; year 2: 7.63) was observed in the treatment of foliar application of G-300, and the lowest amount of this trait (year 1: 0.48; year 2: 3.57) was found in control fruits (Fig. 6). Compared to the control fruits, the peel CCI of the fruits picked up from trees treated with Zn-G and Zn-S exhibited a sharp and gradual increase, respectively. The results indicated that the G-225 and G-300 treatments remarkably accelerated and enhanced the colour intensity of Newhall peels, as indicated by the significant boost in CCI (implied as a* and b* in Supplemental Table S1), representing the promoted accumulation of red and yellow pigments in the peel following foliar application of Zn-G.

Fig. 6.

Effect of foliar Zn application (concentration (0, 150, 225, 300 mg-Zn/L) and source (Zn-Glycine and Zn sulfate)) on fruit peel colour over two sampling years (2022–2023). Means with the same small letter are not significantly different at P ≤ 0.05.

Peel thickness

The results showed that Zn spray increased the fruit peel’s thickness. The highest peel thickness (6.0 mm) was found in the G-300 and S-150 treatments, and the lowest peel thickness (4.9 mm) was found in the control fruits (Fig. 7A). In the Zn-G treatments, the peel thickness increased with Zn application rate, and the highest thickness was found in the G-300 treatment. However, in the Zn-S treatments, the peel thickness decreased slightly with the increase in Zn-S application rate, and fruits from trees sprayed with 150-S had the highest peel thickness.

Fig. 7.

Effect of foliar Zn application (concentration (0, 150, 225, 300 mg-Zn/L) and source (Zn-Glycine and Zn sulfate)) on peel thickness (A), total phenolics (B), and antioxidant capacity (C) in the Newhall fruits (Two-year average). For each attribute, Means with the same small letter are not significantly different at P ≤ 0.05.

Total phenolics

The results showed that the total phenolic content of fruits improved with the foliar application of Zn. The lowest total phenolic content of the fruit (39 mg-GAE/100 g) was found in the control fruit. The highest total phenolic content of the fruit (49 mg-GAE /100 g) was found in the G-300 treatment (Fig. 7B). Moreover, the results showed that the effect of Zn supply from Zn-G source on the total phenolic content of fruits was slightly greater than that of Zn-S (Fig. 7B). Relative to the control, the mean percent increase in total phenolics ranged between 12 and 26% and 12–18% in response to all Zn-G and Zn-S spraying rates, respectively.

Antioxidant capacity

In both years, the highest antioxidant capacity was found in the foliar application treatment of G-300. The lowest value of this trait was found in the control fruits (Fig. 7C). The results showed that foliar application of 150, 225, and 300 mg‑Zn/L of Zn-S, on average, increased fruit antioxidant capacity by 6, 21, and 44%, respectively, while foliar application of 150, 225, and 300 mg‑Zn /L of Zn‑G raised fruit antioxidant capacity by 35, 41, and 53%, respectively (Fig. 7C). In addition, the results also showed that at all Zn concentrations used, the effect of Zn-G on fruit antioxidant capacity was greater than the effect of Zn-S.

The ratio of total soluble solids to acidity

In both years studied, Zn application slightly increased TSS and moderately reduced TA, especially with foliar application of S-150 and S-225. Also, the highest TSS to TA was found in S-150 and S-225 treatments. Compared to Zn-G, the promotion effect of Zn-S on the TSS/TA of fruits was greater at all Zn concentrations studied. The results showed that foliar application of 150, 225, and 300 mg‑Zn/L of Zn-G, on average, increased fruit TSS/TA by 7, 16, and 14%, respectively, while foliar application of 150, 225, and 300 mg‑Zn /L of Zn‑S raised fruit TSS/TA by 43, 34, and 22%, respectively (Fig. 8).

Fig. 8.

Effect of foliar Zn application (concentration (0, 150, 225, 300 mg-Zn/L) and source (Zn-Glycine and Zn sulfate)) on total soluble solids (TSS) and titratable acidity (Ta) in the Newhall fruits over two sampling years (2022–2023). For each attribute, Means with the same small letter are not significantly different at P ≤ 0.05.

Panel test

The results of the sensory evaluation of Newhall oranges in terms of overall acceptability (eating quality) are shown in Fig. 9. As is apparent from Fig. 9, the fruit of trees sprayed with Zn had higher acceptance by the evaluation team regarding fruit peel and flesh colour, aroma, juiciness, sweetness, acidity, and general taste. Therefore, the panel test results also confirm the positive effect of applying Zn, especially G-300 treatment, during the fruit growth period on improving the quality of Newhall oranges at harvest time.

Fig. 9.

Effect of foliar Zn application (concentration (0, 150, 225, 300 mg-Zn/L) and source (Zn-Glycine and Zn sulfate)) on sensory evaluation of Newhall fruits.

Discussion

Zn deficiency is a pervasive issue in Iranian citrus groves, largely attributable to low soil Zn content, calcareous soil conditions, and high pH levels⁸. In the present study, both Zn compounds consistently exerted a positive effect on Zn concentration in the leaves, with a continuous increase observed as the Zn rate increased. Elevated Zn in treated trees implies potential benefits from supplemental Zn (sulfate or glycine). Across all foliar application rates, Zn concentration in leaves remained within the adequate range for citrus (25–100 mg/kg) (Fig. 1), and no symptoms of toxicity or leaf burning were observed in any of the treatments. Previous studies have documented positive effects of Zn-fertilizer application on promoting this nutrient element in the citrus trees^10,14,28,29. Furthermore, our results indicated that the effectiveness of both Zn-G and Zn-S fertilizers in facilitating Zn delivery to citrus was dose-dependent. Specifically, the Zn-G foliar application at a 150 mg-Zn/L rate (G-150 treatment) proved more efficient in promoting Zn uptake by leaves than the Zn-S treatment at the same rate (S-150). However, at higher concentrations (225 and 300 mg-Zn), Zn-S significantly outperformed Zn-G in increasing leaf Zn levels. This differential efficacy, where amino acid-based Zn is more effective at lower rates and Zn-S at higher rates, has been observed in other studies. For example, Bastam et al.³⁰ indicated that 0.2 Zn-g/L foliar application of Zn-amino acids was more effective than ZnSO₄ to enhance Zn concentrations of olive trees. In contrast, Najizadeh & Khoshgoftarmanesh²⁶ reported that 0.5% ZnSO₄ was more effective (12–35% higher) than 0.5% Zn-amino acid complexes, resulting in higher leaf Zn levels.

The leaf pigments showed a gradual increase with the increase in Zn rate, depending on the Zn source. This finding aligns with extensive literature demonstrating the beneficial impact of foliar Zn supplementation on chlorophyll levels across diverse plant species^10,17. It is widely recognized that inadequate Zn supply impairs both chlorophyll synthesis and overall photosynthetic efficiency in plants^10,31,32—a phenomenon also observed in the Zn-deficient control trees of the present investigation (Fig. 2). A decrease in chlorophyll level is possible because Zn deficiency resulted in photosynthetic apparatus damage, accompanied by subsequent chloroplast and grana disintegration³¹. In addition, the Zn cofactor is required for the normal development of pigment biosynthesis³³. Furthermore, Zn-G-treated leaves showed improved green recovery and higher chlorophyll levels, indicating superior corrective effects compared to Zn-S. Since glycine contains amine and carboxyl groups, the improved efficiency of Zn-G on leaf chlorophyll level compared to Zn-S may be associated with the status improvement of the nutrient elements such as nitrogen (N) and iron (Fe) that are involved in chlorophyll structure. The positive effects of amino acid application on improving the content of Fe²⁷, N⁶, and chlorophyll³⁴ in leaves have also been reported in other studies.

Foliar application of Zn significantly increases the protein content in the leaves, although these effects were dependent on the Zn sources (Fig. 3). Previous studies have also pointed to the positive influences of Zn application on the protein content in leaves³². Zn plays a crucial role in plant protein metabolism and is vital for RNA polymerase, which is essential for protein synthesis³⁵. Zn can also inhibit ribonuclease activity and prevent RNA decomposition. These two modes of action boost plant protein synthesis. In the present study, the improved efficiency of Zn-G on protein synthesis compared to Zn-S is expected because glycine is a reduced form of N that in leaves can be directly assimilated and accelerate protein biosynthesis. In addition, such improved effects in Zn-G treatments can be mainly due to higher chlorophyll biosynthesis (Fig. 2), probably higher photosynthesis rates, and enhanced protein biosynthesis (Fig. 3). From a nutritional perspective, higher leaf protein is beneficial for metabolic reactions and optimal photosynthesis³⁶. Similarly, previous studies have documented improved effects of amino acid application on protein biosynthesis^6,37.

The highest leaf carbohydrates were found in the Zn-G foliar application at 225 and 300 mg-Zn/L rates (G-225 and G-300 treatments). Additionally, the results indicated that exogenous Zn supplementation, especially Zn-G, increased starch biosynthesis in the citrus leaves (Fig. 4). Photosynthetically fixed carbon in leaves is initially allocated to sucrose; however, when photosynthetic activity increases and leaf sucrose concentration exceeds its storage capacity, the excess carbon is then allocated to starch³⁸. Leaf-synthesized starch from daytime photosynthates is termed transitory starch because it is degraded overnight to metabolism, energy generation, and biosynthesis in the absence of photosynthesis³⁹. Also, the results showed that the leaf content of TSC and starch in the glycine treatments was higher than in the sulfate treatments. As previously shown, the effectiveness of Zn-G in increasing leaf chlorophyll content was higher than that of Zn-S, which can lead to a significant increase in net photosynthetic rate. Previous studies suggested that the pigment content in leaves is closely related to the photosynthetic performance of peach leaves⁴⁰. In addition, the important role of N in synthesizing proteins and enzymes involved in the assimilation of CO₂⁴¹ could also be effective in the observed trend. Similarly, previous studies have indicated that chlorophyll contents and net photosynthetic rate seemed to decrease with reduced Zn contents in the navel orange³². Also, previous studies have shown positive effects of exogenous amino acid application in promoting photosynthetic production in the leaves³⁴. Plants allocate carbohydrates produced during photosynthesis to support various functions, including maintenance, growth, development, reproduction, and defense mechanisms⁴².

Compared to controls, exogenous Zn supplementation significantly increased the levels of most antioxidants (including total phenolics, carotenoids, and SOD enzyme) and antioxidant activity, suggesting that appropriate Zn supply, regardless of Zn supply sources, promotes antioxidant accumulation and activity (Fig. 5). The increased level of these beneficial compounds with Zn application enables the plant to cope with environmental stresses. The previous study has indicated that an adequate supply of Zn in plants promotes the activity of the antioxidant enzyme system and non-enzymatic antioxidants¹⁴. Field studies showed Zn supply reduced oxidative stress by modulating the antioxidant system in citrus¹⁴ and apple trees⁴³. Zn fertilizer stimulates the accumulation of carbohydrates, starch, which can provide a source of energy and carbon rings for polyphenol biosynthesis⁴⁴ proteins, and other defense and communication mechanisms. In the present study, the Zn-G treatments were more effective than Zn-S at stimulating SOD activity and antioxidant capacity. The important role of amino acids in synthesizing proteins and enzymes may also contribute to the observed trend. Similarly, Xing et al.³² reported that Zn-containing enzymes, including Cu/Zn superoxide dismutase (Cu/S-ZnOD), showed significantly lower activity in leaves with lower Zn content.

Citrus trees often face environmental stress in the field. The balanced nutritional status of plants with Zn supply could alleviate the effects of abiotic stress, mainly when trees were supplied with Zn, especially Zn-G via foliar applications (Figs. 1, 2, 3, 4 and 5). Hence, the findings of this study propose utilizing Zn-based amino acid-fertilizer can enhance citrus trees’ resilience and productivity under changing climate conditions. This is in line with the demonstrated role of Zn and amino acids in alleviating environmental stress by increasing the photosynthesis pigment and active oxygen scavenging substances²⁰.

In this work, Zn foliar application, especially in Zn-G form and at 225 and 300 mg-Zn/L rates, remarkably accelerated and enhanced the colour intensity of Newhall peels as indicated by the significant increase of CCI (Fig. 6), representing promoted red and yellow pigment accumulation in peel following foliar application of Zn-G. Therefore, supplying zinc, especially from the Zn-G source, not only improved the nutritional status of Zn in citrus trees but also led to improved fruit external quality and colour. This effect may result in a greater price in the market, since yield is directly proportional to the commodity’s price. In ripe citrus fruit, carotenoids are the chief pigments responsible for the characteristic red, orange, and yellow hues⁴⁵.

The results showed that foliar application of Zn increased fruit peel thickness (Fig. 7). Similarly, Arshad et al.⁴⁶ reported that Zn application can slightly increase peel thickness in citrus fruits. The results also showed that Zn supply from Zn-S moderated the increasing effects of Zn application on fruit peel thickness. However, the decrease of these parameters at the highest Zn-S application did not reach the level of control fruits. Also, the results showed that treatments containing glycine as a N supply source intensified the increasing effects of Zn on fruit peel thickness (Fig. 7). Other research has also indicated that the citrus fruit peel thickness can be increased by applying Zn²⁸ and N⁴⁷.

This study suggests that exogenous Zn supplementation, particularly in a concentration of 300 mg-Zn/L (Fig. 7), could be strategically utilized to enhance the accumulation of health-promoting compounds and antioxidant activity in Newhall fruit, potentially improving human nutrition. According to Song et al.⁴⁸, the increase in total phenolic content following Zn application could be due to an increased expression of genes responsible for phenolic compound biosynthesis. Similarly, an increase in the level of total polyphenols has been found in ʻKinnow’ mandarin²⁹, and grape berry⁴⁸ fruits with foliar application of Zn. Liaquat et al.⁴⁹ demonstrated that all the treatments of Zn sprays were significantly superior in improving the total antioxidants and non-enzymatic antioxidants of mandarin juice compared to the control.

In the present study, with foliar application of both studied sources, the fruit TSS to TA ratio was within the optimal range. Zn application slightly increased TSS and moderately reduced TA, especially with foliar application of Zn-S at 150 and 225 mg-Zn/L rates (S-150 and S-225 treatments; Fig. 8). The highest TSS to TA ratio was found in the 150-S and 225-S treatments. The increase in TSS as a result of Zn spray can be ascribed to an increase in photosynthesis and production of more assimilates due to auxin synthesis⁵⁰. Similar observations were recorded by the highest TSS in sweet orange⁵¹, mandarin⁴⁹, and Washington Navel Orange¹⁷ with foliar application of Zn. The leaves supply carbohydrates to the fruit through mass-flow translocation during maturation. The results indicated that exogenous Zn supplementation, particularly with Zn-S, enhanced the transport of photosynthetic products from leaves to fruits. In addition, it appears that sulfur in zinc sulfate treatments played an important role in regulating and accelerating fruit ripening time. As previously shown, following the foliar application of Zn-S, the increased percentage of protein, carbohydrate, and starch in the leaves was lower compared to foliar application of Zn-G, probably due to enhanced transport of photosynthetic products from leaves to fruits in the Zn-S treatments. Carbohydrates, comprising 75–80% of soluble citrus pulp components, are key to the juice’s sweetness⁵². The sourness of citrus fruits, determined by their acidity, is a key factor in consumer appeal and commercial viability, as its balance with sugar creates a desirable taste⁵². Similarly, El-Gioushy et al. ¹⁷ reported that foliar application of Zn-S resulted in increased TSS, decreased titratable acidity, and increased TSS to TA ratio in the fruit juice of Washington navel oranges. Apple trees treated with Zn showed improved firmness, higher TSS, lower TA, and a greater TSS to TA ratio⁵³. Shaaban & Abdel-Ati⁵¹ reported that the foliar application of amino acids and micronutrients led to an enhanced TSS to TA ratio in the fruit juice of sweet oranges (Citrus Sinensis).

Conclusion

Adequate zinc supply to citrus trees, either via glycine-Zn complex or Zn-sulfate salt, in Zn-deficient soils led to improved leaf biochemical characteristics and fruit quality in the Newhall trees. The findings demonstrated that the effectiveness of Zn-sulfate fertilizer, especially at 225 and 300 mg-Zn/L rates, in facilitating Zn delivery to citrus trees was higher than Zn-glycine. However, in terms of stimulating leaf biochemical attributes such as chlorophyll content, carbohydrate and starch levels, soluble protein, overall antioxidant capacity, and superoxide dismutase activity, zinc-glycine was more effective than zinc sulfate, probably due to the important role of glycine in the biosynthetic pathways of these compounds. Furthermore, the quality attributes of the fruit were notably influenced by Zn foliar application, depending on the source of fertilizer used. Zn-glycine, especially at 225 and 300 mg-Zn/L rates, markedly accelerated and intensified the colour development of Newhall orange peels. The effect of Zn application on TSS/TA ratios was particularly evident following foliar applications of Zn-sulfate at 150 and 225 mg-Zn/L rates. The study also revealed that the fruits’ antioxidant capacity and total phenolic content were enhanced through foliar application of Zn. Overall, the collective findings of this research suggested that Zn-glycine foliar application, particularly in a concentration of 300 mg-Zn/L, could be strategically utilized to prepare plants biochemically to mitigate the adverse effects of climate change, to improve external fruit quality, to enhance the accumulation of health-promoting compounds and antioxidant activity in Newhall fruit, potentially improving human nutrition. However, further studies should be conducted to evaluate the ecological effect of Zn-glycine applications in different types of soil, either via fertigation or foliar application, especially on the soil health and quality. Additionally, the molecular functions and signaling pathways of different zinc forms, such as synthetic chelates, soluble salts, and biochelates in plant-zinc interactions for promoting tree nutritional and biochemical status, remain unknown and require characterization.

Method and materials

The present study was conducted in a commercial orchard of 7-year-old Newhall orange trees (Citrus sinensis L.) grafted onto citrumelo rootstock (Poncirus trifoliata (L.) Raf.× Citrus paradisi Macf.). This orchard is located at the Citrus and Subtropical Fruit Research Center in Ramsar City, Mazandaran province, in northern Iran (36°54′11″N 50°39′30″E). The average annual rainfall and temperature at the site are around 1200 mm and 21 °C, respectively. Visual symptoms of Zn deficiency in the studied orchard were observed, and the results of the soil test (Table 3) and leaf analysis (Table 4) also confirmed zinc deficiency in the studied orchard.

Table 3.

Physicochemical soil properties of the studied orchard.

Depth	pH	Electrical conductivity	Sand	Clay	Organic carbon	Nitrogen	Phosphorus	Potassium	Zinc
cm	-	dS/m	%		%		mg/kg
0–30	7.28	252	51	17	0.85	0.091	28	150	1.62
30–60	7.16	218	49	20	0.73	0.074	13	105	0.43

Table 4.

The leaf analysis results of the studied Newhall organ trees.

Nitrogen	Potassium	Phosphorus	Zinc
%			mg/kg
2.5	1.57	0.22	15.23

The physical and chemical soil properties of the experimental site are displayed in Table 3. Pruning and irrigation were carried out using standard local agricultural methods. The study was conducted over two years (2022–2023). Fertilization of the orchard was performed using soil test (Table 3), leaf analysis data (Table 4), and the amount of fruit harvested based on the tree’s requirements according to the phonological stages.

Orchard treatments

Fifty-six trees were selected with normal cultural practices. The experiment was conducted as a randomized complete block design with seven treatments and four replicates (with two trees in each replicate) during 2022 and 2023. The experimental treatments consisted of two Zn sources, including ZnSO₄.7H₂O (Zn-S) (Zn: 23%; S: 11%) and Zn-Glycine (Zn-G) amino chelate (Zn: 26%; glycine: 30%), and different Zn concentrations (0, 150, 225, and 300 mg-Zn/L). All treatments, including various sources and concentrations of Zn, were applied by foliar spray at fruit set (12 June in the first year and 14 June in the second year) and one month later at the summer flush stage. In addition, control trees were sprayed with water and Tween 20%. Foliar sprays were applied during early morning, when the air temperature is relatively low and humidity is high, using a motorized sprayer.

Leaf analysis

The healthy, mature leaves from the middle of the 5-month-old spring flush were collected for biochemical measurements during mid-August 2022 and 2023. The minimum sample size was 40 leaves from a height of two meters for each of the trees. Some leaves in liquid nitrogen were immediately homogenized into a fine powder using a mortar and pestle and then stored at -80 °C for long-term preservation. These leaves were later used to measure pigments, total soluble carbohydrates, starch, total phenolics, soluble protein, superoxide dismutase enzyme activity, and antioxidant capacity. For Zn measurement, the remaining leaves were dried in an oven at 65 °C for 72 h and then ground in a mill to pass through a 30-mesh screen.

Pigments

The frozen leaf powder was immersed in 80% (v/v) aqueous acetone. Absorbance was measured using a spectrophotometer at wavelengths 646, 663, and 470 nm, and the concentration of chlorophyll a, b, and carotenoids was determined as described by Wellburn and Lichtenthaler⁵⁴.

Total soluble carbohydrate (TSC) and starch content

The frozen leaf powder was extracted with 80% ethanol at 70 °C as described by Raiesi and Moradi⁵⁵. The ethanol-soluble fractions were pooled. The ethanol-insoluble residue was subsequently used for starch extraction with 52% (v/v) perchloric acid⁵⁶. Then, the leaf contents of starch and TSC were estimated using the anthrone reagent⁵⁷.

Total phenolic (TPC) and antioxidant capacity

The frozen leaf powder was homogenized and extracted in acidified methanol for 1 h. The ability to scavenge free radicals, specifically 2,2-diphenyl-1-picrylhydrazyl (DPPH) hydrate, was assessed using the method described by Brand-Williams et al.⁵⁸. TPC was measured using the Folin-Ciocalteu method⁵⁹. The result was expressed as g of gallic acid equivalent (GAE) per 100 g of fresh weight.

Total soluble protein (TSP)

The frozen leaf powder was homogenized using a cold 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, and 4% (w/v) PVPP⁶⁰. The supernatants were collected after centrifugation (12,000 x g, 10 min, 4 °C). The Bradford method was used to estimate TSP⁶¹.

Superoxide dismutase enzyme activity (SOD)

Leaf SOD activity was determined using the method described by Dhindsa et al.⁶², which measures the enzyme’s ability to prevent the photoreduction of nitro blue tetrazolium (NBT). The absorbance of the reaction mixture was measured at a wavelength of 560 nm.

Zn

Oven-dried leaves were first dry ashing in muffle furnaces (T = 500 °C for 4 h) and then digested by 2 N HCl⁶³. Zn concentrations were measured using the atomic absorption spectroscopy method.

Fruit harvest

Mature and healthy fruits with uniform size (250 ± 20 g) at commercial maturity were harvested in the second week of November from a height of two meters outside the tree canopy each year. Twenty fruits were taken from each of the replications (trees). At harvest time, fruits were evaluated for peel colour, peel thickness, total soluble solids (TSS), titratable acidity (TA), juice percent (%), total phenolic content (TPC), and antioxidant capacity as described below.

Peel colour and thickness

The colour of the orange peel was assessed on the outer equatorial region of the peel of five fruits per replicate, utilizing a chromameter (Konica Minolta CR-400, Japan) to measure in the CIE L*, a*, and b* colour space. The Citrus Colour Index (CCI) was calculated using the formula proposed by Jiménez-Cuesta et al.⁶⁴, CCI = 1000 × (a*/L*× b*). Additionally, peel thickness was measured using a Vernier caliper.

Total soluble solids (TSS) and titratable acidity (TA)

The TSS content of orange juice was measured using a handheld refractometer (Atago ATC-20E, Japan). The TA was determined by titration with 0.1 N sodium hydroxide. The TA was expressed in terms of percentage of citric acid equivalents. Additionally, the fruit flavor index (TSS/TA) was calculated.

Total phenolic (TPC) and antioxidant capacity analysis

Fruit juice was extracted using chilled 80% ethanol for 24 h at 4 °C. The capacity to scavenge DPPH free radicals was evaluated following the method outlined by Brand-Williams et al.⁵⁸. The Folin-Ciocalteu method was used for TPC measurement⁵⁹. The result was expressed as mg of gallic acid equivalent (GAE) per 100 g of fresh weight.

Sensory evaluation

Sensory evaluations were conducted by a panel of trained eight judges using a 7-point hedonic scale to assess various characteristics. The scale ranges from 1 (dislike extremely) to 7 (like very much), encompassing the following parameters: peel and flesh colour, aroma, juice percentage, sweetness, acidity, and overall taste. The overall acceptability of the Newhall orange was determined based on the mean score from the judges’ evaluations.

Data analysis

Data were subjected to a combined analysis of variance (ANOVA) in SAS 9.4. Since the experiment repeated for two years and sampling was conducted during two years (2022–2023) to take into account annual variation, we used ANOVA procedure for a combined analysis of data^65,66. We determined appropriate F statistics and the subsequent significance levels for year, Zn fertilization treatments and their interaction by estimating variance components associated with each term in the model. If there were significant interaction effects between year and Zn fertilization treatments, we then considered testing only for the interaction term rather than the main effect of the Zn fertilization treatments. The Duncan’s post hoc test was used to separate the means of the leaf biochemical and fruit quality attributes at P ≤ 0.05.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

TR: designed and supervised the study. TR and MAS: performed experimental and data analyses and drafted the manuscript; TR, MAS, and HR: edited and critically revised the manuscript. All authors read the final version of the manuscript and approved the list of authors.

Data availability

All data used during the current study are available from the corresponding author (T.R) on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Statement of compliance

Permission to conduct orchard treatments and collect orange plant samples was obtained from the head of the Citrus and Subtropical Fruit Research Center before sampling. This study complies with relevant institutional, national, and international guidelines and legislation.