# Osmolyte Regulation as an Avocado Crop Management Strategy for Improving Productivity Under High Temperatures

Alberto San Bautista, Alba Agenjos-Moreno, Ana Martínez, Ana Isabel Escudero, Patricia Arizo-García, Rubén Simeón, Christian Meyer, Davie M Kadyampakeni

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Alberto San Bautista, Alba Agenjos-Moreno, Ana Martínez, Ana Isabel Escudero, Patricia Arizo-García, et al.. Osmolyte Regulation as an Avocado Crop Management Strategy for Improving Productivity Under High Temperatures. Horticulturae, 2025, 11 (3), pp.245. 10.3390/horticulturae11030245. [https://doi.org/10.3390/horticulturae11030245](https://doi.org/10.3390/horticulturae11030245)

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Article

# Osmolyte Regulation as an Avocado Crop Management Strategy for Improving Productivity Under High Temperatures

Alberto San Bautista <fnref n="1" />,* , Alba Agenjos-Moreno <fnref n="1" />, Ana Martínez <fnref n="1" />, Ana Isabel Escudero <fnref n="1" />, Patricia Arizo-García <fnref n="1" />, Rubén Simeón <fnref n="1" />, Christian Meyer <fnref n="2" /> and Davie M. Kadyampakeni <fnref n="3" />

**Abstract:** Climate change worsens abiotic stresses, primarily due to high temperatures, which have a negative impact on avocado productivity, leading to reduced crop yields, affecting fruit set and abscission. To tackle these challenges, antioxidants such as glycine, choline, and proline can enhance plant tolerance to these stressors and minimize plant cell damage. This work aimed to use these antioxidants to improve avocado commercial yield and quality under challenging environmental conditions. This study was conducted at the experimental farm of the Polytechnic University of Valencia, Spain, to evaluate the effects of glycine, choline, and proline on 'Hass' Persea americana plants. The research took place during the 2022-2023 and 2023-2024 seasons in a 2.0 ha orchard, using a randomized design with two treatments: one with antioxidants and the other without. Substances were applied at specific phenological phases, as the BBCH code indicated. Tree growth parameters, including trunk diameter, height, crown diameter, and tree canopy volume, were measured using geometric formulas. Leaf samples were collected to analyze the nutrient concentrations of N, P, K, Ca, Mg, S, B, Cu, Fe, Mn, and Zn using atomic emission spectrometry. Marketable fruit yield and quality parameters such as fat, fiber, and protein content were evaluated using the Association of Official Agricultural Chemists (AOAC) methods. The results showed that antioxidants did not significantly affect tree growth but altered leaf mineral nutrient composition. N and P concentrations were reduced, while K and Ca concentrations were increased. Mn and Zn levels were higher in the treated plants, whereas Cu levels were higher in the control plants. Productivity significantly improved, with a 49% increase in fruit yield, larger fruit size, and a 7% increase in fat content, though fiber and protein remained unchanged. These results show the selective benefits of antioxidants in optimizing avocado yield and quality under stress.

**Keywords:** choline; glycine; plant nutrition; *Persea americana*; productivity; proline

## 1. Introduction

Abiotic stress due to temperature, relative humidity, drought, or salinity can negatively influence food security through its impact on the agricultural system's productivity. High temperatures and prolonged droughts significantly reduce crop productivity; Knox et al. [1] reported that climate change could reduce yields in crops such as wheat, maize, rice, and others by 5 to 17% by 2050. Drought is another critical factor worldwide, limiting crop

<footnote n="1">¹ Departamento de Producción Vegetal, Universitat Politècnica de València, 46022 Valencia, Spain; alagmo@etsiamn.upv.es (A.A.-M.); anmarde4@etsiamn.upv.es (A.M.); anesiz@etsiamn.upv.es (A.I.E.); patarga1@etsiamn.upv.es (P.A.-G.); rusibro@etsiamn.upv.es (R.S.)</footnote>

<footnote n="2">² Institut Jean-Pierre Bourgin, INRAe, Université Paris-Saclay, 78026 Versailles, France; christian.meyer@inrae.fr</footnote>

<footnote n="3">³ Citrus Research and Education Center, Soil, Water and Ecosystem Sciences Department, University of Florida, 700 Experiment Station Rd., Lake Alfred, FL 33850, USA; dkadyampakeni@ufl.edu</footnote>

<footnote>* Correspondence: asanbau@prv.upv.es</footnote>
areas globally. Kim et al. [2] indicated that prolonged drought was responsible for direct economic losses in the agricultural industry. In addition, salinity stress, exacerbated by the lack of fresh water for irrigation, affects crop quality and quantity. Approximately 20% of the world's agricultural land is estimated to be affected by salinity [3]. These environmental constraints can affect the food supply and prices in world food markets, thus influencing global economic stability.

The avocado, often called "green gold" has become one of the most significant agricultural products in the global economy due to its increasing demand in international markets. Native to Central America and Mexico, this fruit has become essential in many diets because of its nutritional benefits linked to healthy lifestyles [4]. In 2023, global avocado consumption was projected to reach approximately 8 million tons, averaging about 1 kg per person per year worldwide. In the USA and Europe, annual avocado consumption ranged from 3.5 kg to 1.05 kg per capita [5]. The global avocado market was valued at roughly $15.5 billion, with a projected compound annual growth rate of 7.5% from 2023 to 2028 [6]. In emerging markets like China, although current consumption is lower at around 0.5 kg per person per year, it is growing at an impressive annual rate of over 15% [7,8]. In Europe, Spain is the largest producer of avocados, with a production of 117,000 tons in 2021, accounting for nearly 75% of the continent's total avocado yield [9]. The avocado has emerged as a vital product in the global economy due to its nutritional benefits and significant influence on producing countries' economies.

According to FAO [10], avocado is one of the most exported fruits, and its global market value is estimated at USD 16.5 billion annually. However, climate change significantly affects crop yields, which may reduce production and increase production costs. The yield of avocado crops is influenced by factors such as fruit set and abscission, which are governed by numerous physiological processes and environmental conditions, including temperature and humidity [11]. Regarding high temperatures over avocado trees, studies such as Tzatzani et al. [12] suggest that rising temperatures in key producing areas could reduce dry matter accumulation and so could affect productivity. Balikian et al. [13] reported that high temperatures can trigger stress in avocados. The assimilation of CO₂ was affected, as was limited photosynthetic efficiency by decreasing carbohydrate production. Under carbohydrate stress, plant growth and development were reduced, negatively affecting the fruit set [14]. However, flower abscission and fruit drop could also occur in low-temperature conditions [15,16]. Environmental conditions significantly limit avocado yields, as the fruit set in avocados is inherently low (<0.1%), even though the tree produces an abundance of flowers [17,18]. So, the initial fruit set is a limiting factor for profitable avocado production [19,20].

Osmolytes may play a crucial role in osmoregulation, helping to mitigate low productivity under high-temperature conditions. Compounds such as glycine, choline, and proline [21,22] are essential in this context. Among these, glycine betaine, which can be synthesized from glycine and choline [23], is particularly effective. These osmolytes enhance the ability of plants to withstand heat stress due to their osmoregulatory properties [24].

In addition, high temperatures can increase the production of reactive oxygen species (ROS) and generate oxidative stress. This process is particularly harmful to photosystem II (PSII), a key component in photosynthesis. When exposed to extreme heat, PSII's efficiency decreases, limiting the plant's photosynthesizing ability [25]. As a result, the plant struggles to produce sufficient energy for its metabolism, ultimately affecting its growth and productivity. Damage to the PSII may involve the inactivation of the D1 protein, one of the complex's critical components, and limit the overall photosynthetic efficiency of the plant [26]. Rising temperatures lead to the generation of various ROS, including hydrogen peroxide (H₂O₂), superoxide anions (O₂⁻), and hydroxyl radicals (OH⁻). In high
concentrations, these ROS can damage cell membranes, proteins, and deoxyribonucleic acid (DNA) [27]. This damage, called oxidative stress, reduces the antioxidant capacity of the plant, making it challenging to repair the physiological injury. To survive these difficult conditions, plants must develop more efficient antioxidant mechanisms to mitigate the adverse effects and preserve their viability.

Antioxidants in avocado cultivation can significantly enhance flowering, fruit set, and fruit ripening, particularly in abiotic stress factors like high temperatures and drought. Heat stress can decrease pollen viability during flowering and fruit set and negatively impact fertilization, reducing fruit formation [28]. Antioxidant protection helps minimize damage to photosystem II, which boosts photosynthetic efficiency and resource availability for fruit growth [27]. To mitigate these effects, plants activate antioxidant systems that include enzymes such as superoxide dismutase (SOD) and catalase (CAT), as well as non-enzymatic antioxidant compounds such as ascorbic acid and glutathione. These mechanisms help to neutralize ROS, protect the integrity of PSII, and reduce the adverse effects of oxidative stress [29,30]. Substances such as glycine, choline, or proline can be considered antioxidants [21].

Due to the anticipated low productivity, the expansion of avocado production in key production areas worldwide may be limited by climate change and high temperatures. To address these challenging conditions, this study hypothesizes that osmolytes are compounds that play an essential role in osmotic adjustment and protect cells from ROS. These can enhance the tolerance of avocados to abiotic stress, promoting flowering, fruit set, and ripening. The objective is to apply antioxidant substances such as glycine, choline, and proline to mitigate carbohydrate stress and reduce fruit drop, ultimately increasing avocados' commercial yield and quality under environmentally challenging conditions characterized by high temperatures.

## 2. Materials and Methods

This study was conducted at an experimental farm in Alicante (Spain) from the Polytechnic University of Valencia (Spain): 38°47′40.8″ N, 0°0′26.9″ W, at an altitude of 80 m. *Persea americana* plants of the 'Hass' cultivar were transplanted in May 2018 to a 5 × 6 m distribution of plants (plant to plant × row to 181-row distance). Trees were planted on soil classified as clay loam and were poorly drained with 44% sand, 28% silt, 28% clay, and 2.3% organic matter, pH = 7.4, and electric conductivity (EC) = 0.2 mhos cm⁻¹. The land has a natural slope of 0 to 2%. The soil weather was the AEMET (Agencia Española de Meteorología or the Meteorological Agency of Spain) climatic data registered in the location. Table 1 and Figure 1 show the most essential meteorological crop growth parameters that were collected during the study duration: air temperature (T), relative humidity (RH), and rainfall (R). The vapor pressure deficit was calculated according to Allen et al. [31].

A drip irrigation system was used in which four drippers with a discharge of 4 L h⁻¹ were installed per tree. Water requirements were determined according to crop evapotranspiration (ETc), following the methodology proposed by FAO [31]. The orchard plant mineral nutrient requirements, maintenance, and caretaking followed standard practices [32].

The experiment was conducted in a randomized design with two treatments and four replicates. The study was conducted for the 2022–2023 and 2023–2024 seasons. The overall experimental area was 2.0 ha. There were eight experimental units of equal size. The total number of trees in each replicate was 70, respectively. In each plot, one tree at each end of the bed was kept as a border tree. As a study factor, the influence of a treatment with antioxidant substances, glycine, choline, and proline, was evaluated in half of the plants.
The other half of the plants were not treated with these substances. Phenological stages were classified according to the BBCH scale [33]. The organic substances were applied at the phenological stages of avocados, according to BBCH codes 51, 59, 65, 71, 75, and 85, in a total of six applications. The trees were sprayed at the indicated phenological stages with a mixture of the three substances at the concentrations of glycine (75 mM), choline (50 mM), and proline (60 mM) according to previous research in other crops [34]. The volume applied per ha in each spraying was 2500 L.

<table><caption>**Table 1.** Average values of climatic variables in 2022, 2023, and 2012–2021 for all seasons and June–September.</caption><thead><tr><th>Climatic Variable</th><th>2022</th><th>2023</th><th>2012–2021</th><th>2022<br>Deviation * (%)</th><th>2023<br>Deviation * (%)</th></tr></thead><tbody><tr><td colspan="6"><strong>All Seasons</strong></td></tr><tr><td>T mean (°C)</td><td>18.58</td><td>18.30</td><td>17.76</td><td>+4.62</td><td>+3.02</td></tr><tr><td>T max (°C)</td><td>30.91</td><td>31.60</td><td>30.47</td><td>+1.44</td><td>+3.73</td></tr><tr><td>T min (°C)</td><td>7.77</td><td>6.34</td><td>6.35</td><td>+22.29</td><td>-0.11</td></tr><tr><td>RH mean</td><td>71.94</td><td>69.74</td><td>69.15</td><td>+4.04</td><td>+0.86</td></tr><tr><td>RH max (%)</td><td>99.59</td><td>99.84</td><td>98.72</td><td>+0.88</td><td>+1.13</td></tr><tr><td>RH min (%)</td><td>21.13</td><td>19.92</td><td>20.98</td><td>+0.74</td><td>-5.04</td></tr><tr><td>VPD (kPa)</td><td>1.89</td><td>1.94</td><td>1.83</td><td>+3.17</td><td>+5.86</td></tr><tr><td>Rainfall (mm)</td><td>894.84</td><td>373.1</td><td>637.741</td><td>+40.31</td><td>-41.50</td></tr><tr><td colspan="6"><strong>June–September</strong></td></tr><tr><td>T mean (°C)</td><td>26.19</td><td>25.26</td><td>24.54</td><td>+6.69</td><td>+2.93</td></tr><tr><td>T max (°C)</td><td>38.97</td><td>36.68</td><td>36.89</td><td>+5.66</td><td>-0.56</td></tr><tr><td>T min (°C)</td><td>15.24</td><td>15.08</td><td>13.88</td><td>+9.75</td><td>+8.60</td></tr><tr><td>RH mean</td><td>62.87</td><td>72.86</td><td>68.06</td><td>-7.63</td><td>+7.06</td></tr><tr><td>RH max (%)</td><td>98.78</td><td>99.53</td><td>97.73</td><td>+1.07</td><td>+1.84</td></tr><tr><td>RH min (%)</td><td>17.57</td><td>23.77</td><td>19.87</td><td>-11.55</td><td>+19.62</td></tr><tr><td>VPD (kPa)</td><td>2.90</td><td>2.40</td><td>2.56</td><td>+13.29</td><td>-6.32</td></tr><tr><td>Rainfall ** (mm)</td><td>499.60</td><td>128.20</td><td>148.95</td><td>+235.42</td><td>-13.93</td></tr></tbody></table>

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Monthly average values for vapor pressure deficit VPD (kPa) in 2022 (dotted line), 2023 (dashed line), and 2012–2021 (solid line).</figcaption></figure>

<footnote>* calculate according to the reference of 2012–2021 values; ** March–May. T mean—mean temperature, T max—maximum temperature, T min—minimum temperature, RH mean—mean relative humidity, RH max—maximum relative humidity, RH min—minimum relative humidity, and VPD—vapor pressure deficit.</footnote>
Tree growth measurements were taken before fruit harvesting in December. Ten trees from each plot were randomly selected, excluding border trees. A digital caliper measured the trunk diameter 8 cm above the graft union in a north-south direction. Tree height was measured perpendicularly from the soil surface to the highest leaf or branch. The canopy diameter was measured parallel (north-south direction) and perpendicular (east-west direction) to the tree row. Canopy volume was calculated using a geometric prolate spheroid formula as described by Obreza and Rouse [35]: [(4/3) (π) (tree height/2) (average canopy radius)²].

To determine the concentration of the elements in the leaves, they were taken in October. The sample consisted of 100 fully expanded leaves from the 3rd-4th position of the spring shoot [36] without fruit, avoiding immature, diseased, insect-damaged, mechanically injured, or dead leaves. Eight random trees were selected for leaf sampling, ensuring that border trees were excluded. The samples were kept in a cooler during the collection process and were acid-washed before analysis. They were then placed in an oven at 80 °C overnight to dry. Once dried, the samples from each plot were ground to pass through a 1 mm mesh screen. A five-gram portion of each leaf sample was analyzed using the dry ashing method and assessed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) to determine the concentrations of P, K, Ca, Mg, S, B, Cu, Fe, Mn, and Zn. The leaf N concentration was measured using macro dry combustion with a LECO analyzer (LECO Corporation, St. Joseph, MI, USA).

Furthermore, choline content was determined using high-performance liquid chromatography (HPLC). A 20 µL aliquot of a liquid sample obtained from avocado leaves was analyzed using a normal-phase silica column at 45 °C. The system employed a mobile phase composed of two buffers: acetonitrile, water, ethanol, acetic acid, and sodium acetate, while the second increased the system's polarity and ionic strength. The analysis was performed using a gradient system, achieving complete separation of the main water-soluble choline metabolites in 22 min with a flow rate of 2.7 mL/min [37].

To determine marketable yield (>150 g fruit⁻¹, undeformed, healthy pest and disease fruits, and no damaged peel), fruits from each plot were handpicked and weighed using a portable scale. The total commercial fruits of five trees per replicate were counted, and the fruit weight was determined. For fruit quality determination, 40 uniform-sized fruits of more than 150 g were selected. Quality parameters (fat, fiber, and protein) were determined according to AOAC [38]. Crude fat was determined by the Soxhet method, according to AOAC (920.39); crude fiber was determined by sequential acid-alkali digestion, following the AOAC method (962.09); and crude protein was determined by the standard Kjeldahl method as established by AOAC (984.13). All the analyses mentioned above were carried out in the laboratories of the Universitat Politècnica de València.

The influence of the factors' effect on fruit parameters was assessed using an analysis of variance [39]. The statistical effect of the main factor (year and antioxidant) and their interactions were partitioned. Mean separations were performed when appropriate using the least significant difference (LSD) test at *p* < 0.05.

### 3. Results

Trunk diameter and canopy volume were not significantly affected by year nor treatment with antioxidant substances (*p*-values = 0.821–0.921) (Table 2). In addition, the interaction between year and treatment was not statistically significant for these growth parameters (*p* = 0.821 and *p* = 0.887, respectively). Consequently, the plant treatments did not significantly influence plant morphology or tree growth each year.

According to the results obtained, it can be highlighted that the treatments with antioxidants have caused changes in the mineral composition of the leaves of avocado plants
compared to control plants. For the primary nutrients, the effect of the antioxidants was statistically significant (*p* < 0.01 and *p* < 0.05) in the N, P, K, and Ca concentrations (Table 3). Plants treated with antioxidants had lower N and P concentrations than control plants, showing statistically significant differences (*p* < 0.05). Control plant leaf concentrations were 13.9% and 18.3% higher in N and P, respectively. However, K and Ca concentrations in plants treated with antioxidants were higher (*p* < 0.05) than those obtained in control plants, with statistically significant differences. Potassium (K) and Ca concentrations were 32.5 and 14.8% higher in the leaves of treated plants. On the other hand, there were no statistically significant differences in Mg and S concentrations.

<table><caption>**Table 2.** The influence of antioxidant treatment on trunk diameter and tree canopy in two seasons.</caption><thead><tr><th>Factor</th><th>Trunk Diameter (cm)</th><th>Volume (m³)</th></tr></thead><tbody><tr><td>Year</td><td></td><td></td></tr><tr><td>2022</td><td>7.24</td><td>22.4</td></tr><tr><td>2023</td><td>7.28</td><td>22.2</td></tr><tr><td>Treatment</td><td></td><td></td></tr><tr><td>Control</td><td>7.25</td><td>22.1</td></tr><tr><td>Antioxidant</td><td>7.27</td><td>22.4</td></tr><tr><td colspan="3">Analysis of variance</td></tr><tr><td>Factor (degrees of freedom)</td><td colspan="2">p-value</td></tr><tr><td>Year (1)</td><td>ns</td><td>ns</td></tr><tr><td>Treatment (1)</td><td>ns</td><td>ns</td></tr><tr><td>Year × Treatment (1)</td><td>ns</td><td>ns</td></tr></tbody></table>

ns: indicates non-significant (*p* > 0.05).

<table><caption>**Table 3.** Leaf macronutrient concentration (%) as a function of antioxidant treatment.</caption><thead><tr><th>Factor</th><th>N</th><th>P</th><th>K</th><th>Ca</th><th>Mg</th><th>S</th></tr></thead><tbody><tr><td>Year</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>2022</td><td>1.79</td><td>0.17</td><td>1.05</td><td>2.5</td><td>0.36</td><td>0.29</td></tr><tr><td>2023</td><td>1.83</td><td>0.16</td><td>1.03</td><td>2.5</td><td>0.36</td><td>0.30</td></tr><tr><td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Control</td><td>1.93 a</td><td>0.18 a</td><td>0.89 b</td><td>2.3 b</td><td>0.38</td><td>0.31</td></tr><tr><td>Antioxidant</td><td>1.70 b</td><td>0.16 b</td><td>1.19 a</td><td>2.7 a</td><td>0.35</td><td>0.29</td></tr><tr><td colspan="7">Analysis of variance</td></tr><tr><td>Factor (degrees of freedom)</td><td colspan="6">p-value</td></tr><tr><td>Year (1)</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>Treatment (1)</td><td>*</td><td>**</td><td>**</td><td>*</td><td>ns</td><td>ns</td></tr><tr><td>Year × Treatment</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr></tbody></table>

\*, \*\*, and ns: indicate significant at *p* < 0.05 and *p* < 0.01, and non-significant. Different letters in the same column indicate statistically significant differences according to the LSD test at *p* < 0.05.

Finally, concerning the micronutrient concentration in the leaves of avocado plants, it should be noted that the treatment factor was statistically significant in the concentrations of Mn, Zn, and Cu but had no significant influence on the concentrations of Fe, B, or Mo (Table 4). Mn and Zn concentrations were higher in plants treated with organic substances than untreated plants, with statistically significant differences (*p* < 0.05). The concentrations of these two elements were much higher in the treated plants, Mn reaching more than double the concentration compared to the control plants, and 58.5% more in the control plants. In contrast, the leaves of the control plants had 13.8% more Cu, with statistically significant differences (*p* < 0.05). There were no statistically significant differences in the Fe, B, and Mo concentrations.
<table><caption>**Table 4.** Leaf macronutrient concentration (mg kg⁻¹) as a function of antioxidant treatment.</caption><thead><tr><th>Factor</th><th>Fe</th><th>Mn</th><th>Zn</th><th>Cu</th><th>B</th><th>Mo</th></tr></thead><tbody><tr><td>Year</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>2022</td><td>73.41</td><td>74.60</td><td>29.38</td><td>7.44</td><td>44.95</td><td>0.08</td></tr><tr><td>2023</td><td>72.02</td><td>73.03</td><td>28.97</td><td>7.28</td><td>44.00</td><td>0.07</td></tr><tr><td>Treatment</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Control</td><td>72.47</td><td>40.20 b</td><td>22.57 b</td><td>7.84 a</td><td>44.89</td><td>0.07</td></tr><tr><td>Antioxidant</td><td>72.97</td><td>107.42 a</td><td>35.78 a</td><td>6.89 b</td><td>44.06</td><td>0.08</td></tr></tbody></table><table><thead><tr><th colspan="7">Analysis of Variance</th></tr><tr><th colspan="7">Factor (degrees of freedom)</th></tr><tr><th colspan="7">p-value</th></tr></thead><tbody><tr><td>Year (1)</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>Treatment (1)</td><td>ns</td><td>**</td><td>**</td><td>*</td><td>ns</td><td>ns</td></tr><tr><td>Year × Treatment (1)</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr></tbody></table>

Regarding the statistical analysis of productive parameters, there were no interactions between year and antioxidant treatment (Table 5). Therefore, the study of the simple factors allowed us to determine their influence on yield (kg ha⁻¹), number of fruits per tree, and average weight of marketable fruits (g fruit⁻¹). The application of antioxidants to avocado trees had a statistically significant effect (*p* < 0.01; *p* < 0.05) on the three production indicators. However, the year effect only significantly influenced the number of fruits per tree and the average weight of marketable production (*p* < 0.05). Applying organic substances to plants with antioxidant attributes increased the number of fruits and the average weight of the commercial output and, consequently, increased the marketable yield per surface area (*p* < 0.05). The treatment resulted in the highest fruit yield per tree. The higher avocado fruit yield on treated plants (+49%) was due to a significant increase in fruit number (+38%) and fruit size (+8%). The year's study indicated that this factor did not show a statistically significant influence on the marketable yield of avocado fruit. However, statistically significant differences (*p* < 0.05) were found in the number of fruits per tree and the average weight of commercial fruits between the two years studied (2022 and 2023). A higher mean fruit weight accompanied the lower fruit set in 2023; the opposite occurred in 2022. According to these results, there was no effect of year or treatment interaction on tree alternate bearing.

<table><caption>**Table 5.** The influence of antioxidant treatment on marketable agronomic parameters: fruit yield, fruit size, and fruit set in two seasons (2022 and 2023).</caption><thead><tr><th>Factor</th><th>Fruit Yield (kg ha<sup>-1</sup>)</th><th>Fruit Number (per Plant)</th><th>Fruit Weight (g fruit<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Year</td><td></td><td></td><td></td></tr><tr><td>2022</td><td>10,343.83</td><td>152.91 a</td><td>202.20 b</td></tr><tr><td>2023</td><td>10,200.50</td><td>138.60 b</td><td>219.83 a</td></tr><tr><td>Treatment</td><td></td><td></td><td></td></tr><tr><td>Control</td><td>8245.83 b</td><td>122.58 b</td><td>202.67 b</td></tr><tr><td>Antioxidant</td><td>12,298.5 a</td><td>168.93 a</td><td>219.37 a</td></tr></tbody></table><table><thead><tr><th colspan="4">Analysis of variance</th></tr><tr><th colspan="4">Factor (degrees of freedom)</th></tr><tr><th colspan="4">p-value</th></tr></thead><tbody><tr><td>Year (1)</td><td>ns</td><td>*</td><td>*</td></tr><tr><td>Treatment (1)</td><td>**</td><td>*</td><td>*</td></tr><tr><td>Year × Treatment</td><td>ns</td><td>ns</td><td>ns</td></tr></tbody></table>

<footnote>\*, \*\*, and ns: indicate significant at *p* < 0.05 and *p* < 0.01, and non-significant. Different letters in the same column indicate statistically significant differences according to the LSD test at *p* < 0.05.</footnote>

<footnote>\*, \*\*, and ns: indicate significance at *p* < 0.05 and *p* < 0.01, and non-significant. Different letters in the same column indicate statistically significant differences according to the LSD test at *p* < 0.05.</footnote>
Regarding the study of the influence of treatment with antioxidants in avocado plants on the quality of commercial fruits, a statistically significant influence (*p* < 0.05) was shown in the fat content (Table 6). The application of these substances resulted in a higher fat content in the fruits with statistically significant differences (*p* < 0.05) concerning the fruits of the control plants. Fat content was 7% higher in plants compared to control plants. No statistically significant differences were found between treated and control plants for crude fiber and protein content. The effect of the year did not show a statistically significant influence on fat, crude fiber, or protein content. No significant differences were found between the two years studied. The interaction between year and treatment was statistically significant for none of the fruit quality parameters analyzed.

<table><caption>**Table 6.** The influence of antioxidant treatment on fruit quality parameters: fat, fiber, and protein (%).</caption><thead><tr><th>Factor</th><th></th><th>Fat</th><th>Fiber</th><th>Protein</th></tr></thead><tbody><tr><td>Year</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>2022</td><td>18.56</td><td>12.97</td><td>1.93</td></tr><tr><td></td><td>2023</td><td>18.55</td><td>13.02</td><td>1.90</td></tr><tr><td>Treatment</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>Control</td><td>17.95 b</td><td>12.72</td><td>1.77</td></tr><tr><td></td><td>Antioxidant</td><td>19.17 a</td><td>13.22</td><td>2.08</td></tr><tr><td>Analysis of variance</td><td></td><td></td><td><i>p</i>-value</td><td></td></tr><tr><td>Factor (degrees of freedom)</td><td></td><td></td><td></td><td></td></tr><tr><td>Year (1)</td><td></td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>Treatment (1)</td><td></td><td>*</td><td>ns</td><td>ns</td></tr><tr><td>Year × Treatment</td><td></td><td>ns</td><td>ns</td><td>ns</td></tr></tbody></table>

## 4. Discussion

The influence of the antioxidants on oxidative control showed a positive effect on the treated avocado plants compared to the control plants, mainly due to a positive, productive response. Other indicators of avocado crop performance were not affected, including the growth of avocado trees. The diameter of the trunk and the tree canopy volume did not change due to the foliar application of these organic substances. This lack of influence on plant growth can be understood by the direct impact of antioxidants in plant photosynthesis on eliminating ROS and protecting the D1 protein of PSII [31,40], resulting in a significant accumulation of dry matter (biomass) in the plant. However, following the partitioning reported by Loomis and Connor, it did not translate into an increase in the canopy size due to a distribution of photoassimilates toward the fruits [41].

According to Lovatt's recommendations, the N concentration in leaves reached values close to optimum levels of 1.85% [42]. However, the leaves of the treated plants showed a N concentration 8.8% lower than the standard value. This reduction in N concentration did not prevent a better yield response in the antioxidant-treated plants. Only when the N concentration was below the critical nutrient range was the correlation between N concentration and yield positive and significant [43]. Other authors confirmed that N concentration is not closely related to yield for N values within a range considered normal to high [44]. Moreover, these differences in N concentration are irrelevant since there is no close relationship between N inputs, leaf N concentration, and yield in avocado plants [45]. Similarly, the response to P could be considered, but P concentration in leaves can be expected [46].

On the other hand, K and Ca concentration was higher in avocado plants treated with antioxidants, which confers a better performance to plants against physiological disorders

<footnote>*, and ns: indicate significance at *p* < 0.05 and non-significant. Different letters in the same column indicate statistically significant differences according to the LSD test at *p* < 0.05.</footnote>
and fruit quality [47,48], and fruit ripening was not affected [49,50]. Similar results were found with seaweed extract supply that mitigated the effects of abiotic stress early on and enhanced K and Ca concentrations in the leaves [51]. The higher Mg concentration in the control plants could be explained by the higher K and Ca concentrations in the treated plants [43].

Concerning the influence of micronutrients in avocado plants, a higher concentration of Mn and Zn was observed, which resulted in higher productivity since these nutrients are closely related to fruit quality [52,53]. Fruits can suffer significant deformations that depreciate their commercial value. Regarding B concentration, no influence of antioxidant treatment was noted. Nevertheless, the productive response of the treated plants was superior to that of the control plants. Boron was not a determinant or critical element to achieve a higher commercial yield in avocado plants, even though it is essential for pollination and fertilization, impacting fruit set and yield, and applying the recommended amount of B results in a good yield of high-quality avocado fruit [54].

Results on B have been conflicting, and their response is variable, as reported by Boldingh et al. [55]. They noted that B content was notably higher in flowering styles that set successfully in New Zealand; however, no significant differences were found in the boron content of styles from Spain. In addition, the relevance of B on avocado plant nutrition has been cited by the literature scientists for suggesting that fruit is an important competing sink for B in harvest years, which could result in less B available to support flower and early fruit development the following spring [56].

Due to the plant partitioning produced by the antioxidant positive action, a distribu-
tion of carbohydrates toward the newly set fruits was made, resulting in more commercial
fruits and a higher marketable yield in the two years studied in the experiment. Aroli
et al. [57] conducted experiments on avocados in Australia using seaweed extract (Durvil-
laea potatorum and Ascophyllum nodosum). They reported an increase in yield and the
number of fruits setting at 38% and 42%, values of the same magnitude as those obtained
in this work. Similar effects were reported by Rojas-Rodriguez et al. [58] on avocado crop
productivity after applications of biostimulants formulated with free amino acids.

Different researchers have reported that osmoprotectants and antioxidants positively affect plants when they are subjected to abiotic stress, such as high temperatures. Os-molytes like glycine betaine and proline betaine serve as osmotic agents [21], and their osmoprotective effects may significantly enhance the ability of plants to tolerate heat stress. The substances utilized in this experiment—glycine, proline, and choline—positively con-tribute to the synthesis of these osmoprotectants, specifically glycine betaine and proline betaine [23,59]. Typically, osmolyte levels increase during stress due to enhanced biosyn-thesis, reduced degradation, or a combination of both factors [60–62].

Anti-stress effects are also reported, with protective compounds found in seaweed
extracts, such as antioxidants and regulators of endogenous stress-responsive genes, poten-
tially playing a role [63]. In treated plants, seaweed extract also increases the endogenous
concentrations of stress-related molecules, such as cytokinins, proline, and antioxidants [64].
Other authors reported similar results when these substances were applied to crops such
as rice [34]. A similar response was obtained in the plant concerning the number of fruit
sets. The difficulty of increasing the production of flowers and fruit sets is evident in the
avocado crop due to high temperatures and high relative humidity values [65]. Under
high-temperature conditions, food production and security are threatened [66].

The study of the evolution of the vapor pressure deficit during 2022, 2023, and the
period 2012–2021 showed that avocado plants grew under stressful conditions during each
year of the study. In 2022, the vapor pressure deficit was higher than the average value
of the 2012–2021 period during the spring months (a critical period for flowering). On
the other hand, in 2023, the most stressful period for avocado plants occurred during the summer months, in the middle of the fruit growth phase. Therefore, it was evident that during the years of the experiment, there were abiotic stress conditions due to limiting environmental conditions, resulting in a higher average temperature in 2022 and 2023 (7.2 and 6.3%, respectively) over the study's average value of the last 10 years. These values are related to reporting by Ameem et al. [67] regarding the escalation of climate change, which has worsened these issues and adversely affected plant growth, fruit yield, and overall quality. High temperatures during the summer months can significantly impact avocado development, leading to a reduced concentration of dry matter, as explored by Tzatzani et al. [12]. Elevated temperature stress inhibits PSII repair during photoinhibition by interfering with D1 protein synthesis [68]. The action of the antioxidants and the oxidative control exerted on the reactive oxygen substances [69,70] increased the yield by 49% compared to that obtained in control plants and by 54% to the average yield value in avocado plantations in Spain [71].

In addition, a higher average fruit weight was obtained in these temperature-limiting conditions with the application of these substances when high temperatures limited the accumulation of carbohydrates in the sink organs as a source of carbohydrates [72]. The higher dry matter accumulation in avocado fruits derived from the oxidative control was manifested by a higher fat content in avocado fruits (+6.7%). Other indicators of fruit quality, such as protein and fiber, were not altered due to the application of these antioxidant substances. So, treatments with antioxidant substances could be considered improvers of marketable fruits.

In general, the application of organic substances with osmoregulatory and antioxidant effects—either directly or through the synthesis of other osmolytes—has a positive impact on the efficiency of nutrient uptake, particularly potassium (K) and calcium (Ca²⁺), as shown in the research by Chen and Murata [73]. Furthermore, these authors also report a beneficial effect on the plant photosystem II (PSII) due to the antioxidant properties of these substances, which leads to increased photosynthetic activity and improved energy balance. Therefore, future work should be directed toward a deeper understanding of stress-related genes and their overexpression at different phenological stages under these conditions. In this way, the substances responsible for the physiological response and the concentrations and doses of application could be better adjusted.

**5. Conclusions**

Antioxidant treatments applied to avocado plants positively affected productivity, particularly under stress conditions. While no significant changes were noted in tree growth—such as trunk diameter or canopy size—antioxidants contributed to a more substantial accumulation of dry matter, primarily directed toward the fruits. The better distribution of photoassimilates results in more marketable fruit and higher yields than control trees. Although the nitrogen concentration in the leaves of treated plants was somewhat lower than the recommended values, this did not hinder their productive response. Additionally, potassium (K) and calcium (Ca) levels were higher in treated plants, enhancing their resistance to physiological disorders and improving fruit quality. This increase in cation concentration may be linked to greater plasma membrane permeability and more efficient nutrient utilization. This study also found that, despite challenging environmental conditions such as high temperatures and humidity, antioxidant treatments helped alleviate the effects of abiotic stress, resulting in better fruit yield and quality. In summary, antioxidants improved avocado yield and quality by increasing dry matter accumulation without negatively impacting growth or nutritional quality.
**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11030245/s1, Figure S1: Fruit development in the different phenological stages.

**Author Contributions:** Conceptualization, A.S.B., C.M. and D.M.K.; Methodology, A.S.B., A.A.-M., A.M., P.A.-G., R.S., C.M. and D.M.K.; Software, A.S.B., A.A.-M., A.I.E., P.A.-G., R.S. and D.M.K.; Validation, A.S.B., A.M., A.I.E., R.S., C.M. and D.M.K.; Formal analysis, A.S.B., A.A.-M., A.M., A.I.E., P.A.-G., R.S., C.M. and D.M.K.; Investigation, A.S.B., A.A.-M., A.I.E., P.A.-G., R.S., C.M. and D.M.K.; Resources, A.S.B.; Data curation, A.S.B., A.A.-M., A.M., A.I.E., P.A.-G., R.S., C.M. and D.M.K.; Writing—original draft, A.S.B., A.A.-M., A.M., P.A.-G., R.S., C.M. and D.M.K.; Writing—review & editing, A.S.B., A.M., A.I.E., R.S., C.M. and D.M.K.; Visualization, A.S.B., A.M., A.I.E., P.A.-G. and D.M.K.; Supervision, A.S.B., A.A.-M. and D.M.K.; Project administration, A.S.B.; Funding acquisition, A.S.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Data is contained within the article and supplementary material.

**Conflicts of Interest:** The authors declare no conflict of interest.

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