Morphological, physiological, and biochemical responses of some olive tree cultivars to low temperature stress

Al-Saif, A. M., Abdel-Aziz, H. F., El-khamissi, H., Abd El-Hakim, A. F., Abd El-wahed, A. E.- wahed N., Elnaggar, I. A., Farouk, M. H., Hamdy, A. E., and Hammad, E. M. (2024). "Morphological, physiological, and biochemical responses of some olive tree cultivars to low temperature stress," BioResources 19(4), 9582–9605.

Abstract

This study evaluated the impact of night frost incidents on the biochemical, physiological, and reproductive functions of the olive varieties Manzanillo, Coratina, Koroneiki, and Picual. Certain cultivars were more suited to moderate cold night stress than others, based on the changes in the performance of the stressed plants, including vegetative growth, tree yield, fruit physical characteristics, and fruit chemical characteristics. Compared to other tested cultivars, the biochemical responses of the plants in terms of photosynthetic pigments, relative water content (RWC), total phenolic compounds, total flavonoid, and antioxidant enzyme accumulation demonstrated that some cultivars could withstand the applied stress. The conclusion that some cultivars responded differently to cold stress than others was supported by the plant phenology. This research could be a game-changer for farmers. By understanding how olive trees adapt to cold snaps, a common stressor in open fields, they can make informed decisions about breeding and choosing the best cultivars, ultimately leading to more resilient crops. The results showed that all tested olive tree cultivars differ significantly regarding cold stress conditions. Coratina and Koroneiki were the most resistant tested cultivars in terms of biochemical, physiological, and reproductive functions, followed in ascending order by Manzanello and Picual.

Download PDF

Full Article

Morphological, Physiological, and Biochemical Responses of Some Olive Tree Cultivars to Low Temperature Stress

Adel M. Al-Saif,^a,* Hosny F. Abdel-Aziz,^b Haitham El-khamissi,^cAhmed F. Abd El-Hakim,^c Abd El-wahed N. Abd El-wahed,^b Ibrahim A. Elnaggar,^b Mohammed H. Farouk,^d Ashraf E. Hamdy,^b,* and Eman M. Hammad ^e

DOI: 10.15376/biores.19.4.9582-9605

Keywords: Frost; Climate change; Injury; Chlorophyll; Plant phenotype; Olea europaea L.

Contact information: a: Department of Plant Production, College of Food and Agriculture Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia; b: Department of Horticulture, Faculty of Agriculture, Al-Azhar University, Cairo, 11884, Egypt; hosny_fathy86@azhar.edu.eg (H.F.A); c: Department of Agriculture Biochemistry, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt; d: Key Laboratory of Product Quality and Security, Ministry of Education, Jilin Agricultural University, Changchun, PR China; e: Department of Food Science and Technology, Faculty of Agriculture (for Girls), Al-Azhar University, 11884 Cairo, Egypt;

* Corresponding authors: adelsaif@ksu.edu.sa; ashrafezat@azhar.edu.eg

GRAPHICAL ABSTRACT

INTRODUCTION

Olive trees and bushes, scientifically referred to as Olea europaea L., thrive in subtropical and temperate regions with temperatures ranging from 30 to 45 °C in each hemisphere (Petruccelli et al. 2022). They are a sizeable economic motive force in the Mediterranean Basin, with olive oil extracted from their fruit being a primary contributor, alongside desk olives being a famous food item (Boskou 2015). Olive trees are affected by wintry weather frost, which leads to reduced productivity (Fraga et al. 2020). To address this issue, researchers have explored numerous techniques, along with selecting naturally frost-resistant olive types and comparing cultivars’ overall performance at some point of real frost occasions. Another approach includes checking the resilience of various olive genotypes under managed freezing conditions in laboratories.

Low temperatures have a widespread effect on the distribution of woody flora and can lead to reduced crop yields because of freezing damage (Medda et al. 2022). Optimal plant increase takes place within a selected temperature range, with growth being hindered outside of this range. Factors influencing plant survival in low temperatures consist of cultivars, organ kinds, extent of temperature reduction, and length of exposure to low temperatures (Wahid et al. 2007). The freezing tolerance of leaves and stems is closely linked to bloodless acclimation, initiated through shorter day lengths, and decreasing temperatures in autumn, ensuing in expanded freezing tolerance in deciduous woody flora. This process entails adjustments in gene expression, consisting of stress protein encoding, alterations in photosynthesis, antioxidant compounds, and lipid and protein composition. Cold acclimation also includes the accumulation of cytoprotective compounds such as soluble carbohydrates, proline, and proteins, which form ice crystals, facilitate osmotic adjustment, preserve cell turgor, and decorate plant resilience to dehydration stresses (Ershadi et al. 2016). Mete et al. (2023) evaluated frost tolerance in olive cultivars with the aid of measuring ion leakage in leaves exposed to exclusive temperatures. The tolerance of cultivars varies between seasons. Butko, Memeli, and GemLik confirmed higher tolerance, and at the same time, Edincik and Sinop No. 6 were less tolerant. While adapting to colder temperatures improved olive trees’ tolerance, this effect wasn’t permanent. The Istarska bjelica variety stood out for its high levels of beneficial plant compounds, particularly oleuropein, which remained consistently elevated. Interestingly, lower air temperature seemed to boost oleuropein levels and antioxidant activity in other varieties. In Egypt, olive growers cultivate various types for different purposes. Toffahi and Egazy are popular for table olives, while Picual and Manzanillo serve as table olives and for oil extraction. Finally, Koroneiki and ChemLali are specifically chosen for their high-quality olive oil production. In this respect, Koroneiki had higher oil content than the Picual olive cultivar (Samra et al. 2009). Research by López-Bernal et al. (2015) indicates that low temperatures, rather than the length of daylight, are the key environmental cue for olive trees to halt vegetative growth and enter a state of cold acclimation.

The growing demand for olive oil has brought about the growth of olive plantations beyond the traditional Mediterranean region into better-range areas (Gómez et al. 2014). This shift has had positive and negative consequences. In chillier areas, dropping winter and early spring temperatures can result in freezing or frost damage to the plants because of ice crystal formation and fast temperature fluctuations. On the other hand, cool autumns can improve the excellence of olive oil through more desirable enzyme activities within the lipoxygenase pathway, leading to advanced flowering within the following spring (Ozturk et al. 2019). While olive timber displays mild resistance to freezing temperatures, subzero situations can cause extensive harm. Studies imply that maximum olive cultivars enjoy leaf drop and twig desiccation at temperatures below -7 °C, with temperatures below -12 °C resulting in extensive reductions in productiveness and tree lifespan (León et al. 2016). The variability in freezing tolerance among olive cultivars underscores the importance of understanding the biochemical and physiological mechanisms underlying cold tolerance (Lodolini et al. 2022). This knowledge can be a resource in figuring out precise genes or new cultivars with greater bloodless resistance, using improvements in genetic research. Low temperatures control these physiological and biochemical changes by causing major changes in gene expression (Yurtsever and Vural Korkut 2019). More recently, the transcriptome alterations brought on by low temperatures in olives were monitored using RNA-Seq analysis (Guerra et al. 2015). Both RNA-seq experiments showed upregulation of genes encoding enzymes involved to modifications in the lipid composition of membranes, as well as stress-related genes such as cold-regulating genes and dehydrin, and downregulation of genes involved in photosynthesis, and activation of carbohydrate metabolism (Petruccelli et al. 2022). Several biochemical and physiological factors play an important function in choosing freezing-tolerant olive sorts, which include stomatal density, stomatal length, phenolic compounds, ion leakage, soluble sugars, general soluble proteins, and antioxidant enzymes. However, counting on a single parameter is inadequate for pinpointing precise cultivars due to the quantitative nature of cold tolerance. The main aim of the present investigation was to assess olive physiological, biochemical and reproductive responses under the imposition of low- temperature stress, imitating night frost incidents. A multidimensional approach was used to study visual changes shoot length (cm), leaf area index, and canopy volume (m³). Tests also included the initial fruit set, final fruit set, fruit weight, and fruit yield of all cultivars considered, physiological performance, chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids. Other tests included relative water content and proline, fruit oil yield/ tree, 7otal soluble solids (%) (TSS) %, total acidity %, and T.S.S acid ratio %. Also measured were the total phenolics and total flavonoids, and biochemical response (antioxidant enzymes) towards the comprehension of olive capacity to cope with unfavorable environmental conditions. Manzanillo, Coratina, Koroneiki, and Picual olive cultivars were chosen because of their importance in new and existing olive plantations.

EXPERIMENTAL

Experimental Site

This study was conducted for two years and examined four olive cultivars (Manzanillo, Coratina, Koroneiki, and Picual trees) during the 2022 and 2023 seasons at a private orchard, Al-Salehia district in El-Sharkia Governorate (30°39′14″N, 31°52′ 28″E, and 30.653862°N 31.874371°E). Weather Data of Egypt- Al-Salehia, El-Sharkia -Daily-2022-2023 is represented in Table 1. The trees were planted using an artesian water drip irrigation system at 4 m × 6 m (1250 trees/ha) in sandy soil. Every 50 cm on the hose, there were two JR hoses for every tree. A 2 L per hour discharging point was present. The Manzanillo, Coratina, Koroneiki, and Picual trees were planted in 2010 Owen rooted. The care practices for the orchard involved consistent watering and nutrient application, ensuring the trees were free of pests and any signs of nutrient deficiencies. The impact of low temperature on the aboveground portions of all trees was evaluated during May 2022 and 2023. Mature leaves were collected from the study trees’ final autumn cycle (four shoots in each of the four origin directions around the tree canopy, namely North, East, South, and West) per tree in May of each season to perform various measurements on them.

Field and Laboratory Determinations

Vegetative growth

The number of damaged leaves per shoot was determined by counting the total number of leaves per shoot (four shoots in each of the four origin directions around the tree canopy, namely North, East, South, and West) and recording this information in early May of each year. To measure growth metrics such as shoot length (in cm), a random sample of nine shoots/trees from each of the two seasons was selected and labeled on each tree. Also, we estimated the height of trees at the end of growth in September in each season. In additionally, a sample of nine mature leaves per tree was abscised in May, and the leaf area (cm²) was calculated using Eq. 1 (Ahmed and Morsy 1999),

Leaf area = 0.53 (L × W) -1.66 = ……cm² (1)

where L is maximum leaf length and W is width, respectively.

Equation (2) was used to estimate the volume of the olive tree canopy, using the field measurements (Sola-Guirado et al. 2017),

Ground Volume = 1/6π𝐷₁×𝐷₂×(𝐻_𝑡−𝐻_𝑠) (2)

where D₁ and D₂ are the diameters of the tree crown measured in two different directions, H_t is the height of the tree, and H_s is the average distance of the lowest point of the tree crown from the ground, measured in the same positions as the diameter.

Table 1. Daily Weather Data of Egypt- Al-Salehia, El-Sharkia (2022-2023)

Photosynthetic pigments

From fresh leaves of four different olive cultivars, chlorophyll A, B, total chlorophyll, and total carotenoids were extracted. This was accomplished by homogenizing fresh leaves (0.2 g) with 10 mL of acetone (80% v/v) and then centrifuging the resulting mixture at 12,000 × g for 10 min. Three mL of the liquid extract was used for spectrophotometric measurements (Wellburn 1994; Brito et al. 2011).

Determination relative water content (RWC)

The Yamasaki and Dillenburg (1999) method was used to calculate the relative water content (R.W.C.) of leaves,

RWC (%) = [(FM-DM) (TM –DM)] × 100. (3)

In each repetition, two leaves were randomly selected from the middle sections of the plants. Initially, the fresh masses (FM) of the leaves were determined after they were detached from the stems. To determine the saturation mass (TM), the samples were sealed in distilled water for a full day at 22 °C to ensure they reached their maximum saturation mass. Following this, the samples were weighed. After, leaves were kept in an electrical oven with an air temperature of 80 °C for 48 h, during which time their dry mass (DM) was determined. Every measurement was completed.

Determination proline content

A rapid colorimetric method was used to determine proline content (Bates et al. 1973). Ninhydrin (1.25 g) was dissolved in a solution of glacial acetic acid (30 mL) and phosphoric acid (6 M, 20 mL). This mixture was heated for clarity, cooled, and stored at 4 °C. Following homogenization in sulfosalicylic acid (0.5 g explant material in 10 mL, 3% aqueous), the mixture was filtered. The filtrate (2 mL) was then treated with glacial acetic acid (2 mL) and freshly prepared ninhydrin solution (2 mL). The reaction mixture was heated to 100 °C for 1 h, then cooled on ice. Toluene (4 mL) was added with vigorous shaking (15 to 20 s), allowing for phase separation. Proline concentration was determined by measuring the chromophore’s absorbance at 520 nm with a spectrophotometer using toluene as a blank. The standard curve was used to calculate proline concentration based on the fresh weight of the sample.

Determination of antioxidant enzymes

To extract peroxidase activity (POX), polyphenol oxidase (PPO), and catalase (CAT) enzyme, fresh leaves (0.5 g) of four different olive cultivars were mashed in a mortar with 5 mL of 0.1 M cold phosphate buffer (pH 7.1), and the mixture was centrifuged at 15,000 × g for 20 min at 4 °C. The supernatant was used in an enzyme activity experiment (Esfandiari et al. 2007). POX activity was measured using an approach that was based on Amako et al.’s study (1994). PPO activity was measured in compliance with Kavrayan and Aydemir (2001). The activity of the CAT enzyme was measured in compliance with Aebi (1984).

Physical characteristics of fruits

The study characterized a detailed flower and fruit phenotyping process for different tree cultivars. Researchers recorded flowering dates, average panicles per tree, and flower counts at full bloom (including perfect and male hermaphrodite flowers) from five representative panicles. Similarly, at harvest, fruits were collected in a bag and then transferred to the chemical analytical laboratories of the horticulture and biochemistry departments at the Faculty of Agriculture in Cairo, Al-Azhar University. Samples of twenty-seven fruits of each tree were replicated three times and devoted to determining the following parameters: fruit number and weight (average from five fruits) were assessed at harvest. This approach aligns with Ferguson and Gratten’s study (2005), which counted fruits on designated shoots before harvest.

The yield per tree (kg) was calculated using fruit weight. According to Khattab et al. (2021), the four tested olive oil cultivars were harvested on September 15^th in both seasons. There was a yield recorded (kg/tree). The percentage was calculated using Eq. 4 (Hamdy et al. 2022):

(4)

Chemical characteristics of fruits

A digital refractometer (force-Gouge ModelIGV-O.SA to FGV-100A. Shimpo instruments) was used to measure the total solids (TSS%) in fruit oil. Titration was used to determine total acidity, which was then expressed as citric acid in accordance with AOAC (McKie and McCleary 2016). The values of total soluble solids divided by the values of total acids and oil content percentage were used to compute the total soluble solids/acid ratio (McKie and McCleary 2016).

Total phenolic compounds and total flavonoid

According to Singleton et al. (1999), the total phenolic content of four olive tree leaves measured using the Folin–Ciocalteu colorimetric method. The results were expressed as (mg/g) using gallic acid as a standard. According to Chang et al. (2002), aluminum chloride (AlCl₃) colorimetric method was used to evaluate the total flavonoid in fresh leaves of the four olive cultivars. Rutin was used as a standard and the results were represented as mg/g.

Statistical Analysis

The design of the present study was a complete randomized block design. The analysis of variance as one-way ANOVA was used through Costat software Snedecor and Cochran (1980), and means of different treatments were compared using the Duncan test (p ≤ 0.05).

RESULTS

Vegetative Growth of Olive Cultivars

The results in Fig. 1 indicate that there were significant differences between all tested olive cultivars regarding the vegetative growth parameters such as shoot length (cm), leaf area index, and canopy volume (m³) during the two studied seasons (2022 and 2023) under low temperature conditions. In the same time, there were significant differences between the two tested seasons (2022 and 2023) regarding the vegetative growth parameters in all tested olive cultivars, where that data is consistent with an impact of low temperature stress on vegetative growth parameters in all tested olive cultivars in the first season was higher than the impact in the second season under the same condition, as a result of the decrease in temperature in the first season from the second season.

Fig. 1. The shoot length (cm), leaf area index, canopy volume, and tree height of all tested cultivars (Manzanillo, Coratina, Koroneiki, and Picual) under low-temperature conditions for three months. Different letters depict statistically significant changes (p ≤ 0.05, LSD test) and error bars depict standard error.

It was clear that Picual and Coratina trees were also superior to those of Manzanello and Koroneiki with respect to tree height under the same conditions. Also, the results in Fig. 1A showed that Coratina trees had the longest shoots, followed by Manzanello, Koroneiki, and then Picual. Similarly, the leaf area index data in Fig.1B follows the same trend. Coratina trees had the largest leaf area, followed by Manzanello. Koroneiki and Picual trees had the smallest leaf area in two tested seasons. The data in Fig. 1C indicated that Coratina trees had the largest canopy volume, followed by Koroneiki and then Manzanello. On the other hand, Picual trees had the smallest canopy volume among the cultivars studied. According to Fig. 1D, Picual and Coratina trees were higher than both Koroneiki and Manzanello. Olive trees halt their vegetative growth and enter a dormant stage in the autumn when the temperature drops below the ideal range for growth (5 to 10 °C) and the photoperiod shortens.

It can be concluded that in terms of all vegetative growth parameters, including shoot length, leaf area, canopy volume, and tree height, Coratina trees outperformed the other olive cultivars.

Leaf Photosynthetic Pigments

Differences between olive cultivars in the values of plant pigments, whether chlorophyll a, b, and carotenoids under cold conditions are shown in Figs. 2 (A to D).

Fig. 2. Chlorophyll a, chlorophyll b, a total of chlorophyll, and carotenoids of all tested cultivars (Manzanillo, Coratina, Koroneiki, and Picual) under low-temperature conditions for three months. Different letters depict statistically significant changes (p ≤ 0.05, LSD test) and error bars depict standard error.

Koroneiki cv. had the highest values in chlorophyll (a (0.567), b (0.256), and a + b (0.823)), followed by Coratina cv. which shared the highest values in chlorophyll (b (0.262)), and carotenoids (0.266 mg/g F. wt.). On the other hand, Picual cv. had the lowest values of plant pigments; chlorophyll (a (0.192), b (0.100)), and carotenoids (0.192) compared to other cultivars.

Leaf chemical characteristics

Figure 3 (A and B) show the effect of low-temperature conditions on relative water content and proline accumulation in the leaf of the four tested olive varieties. It was established that the relative water content in the leaves of Manzanello, Coratina, Koroneiki, and Picual was decreased under cold conditions. There were significant differences between all tested olive tree cultivars in relation to cold stress conditions, where Coratina gained the highest relative water content followed in ascending order by Manzanello, Koroneiki, and Picual. On the other hand, growing all tested olive tree varieties under low-temperature conditions caused an increase in the proline content of all tested olive tree cultivars (Fig. 3). There were no significant differences between all tested varieties in relation to the effect of cold stress on leaf proline content where Coratina gained the highest proline content followed in ascending order by Manzanello, Koroneiki, and Picual.

Fig. 3. Relative water content and proline of all tested cultivars (Manzanillo, Coratina, Koroneiki, and Picual) under low-temperature conditions for three months. Different letters depict statistically significant changes (p ≤ 0.05, LSD test) and error bars depict standard error.

Antioxidant enzymes activities

The enzymatic activities of POX, PPO, and CAT in leaves of four olive cultivars were measured under frost during the 2022 and 2023 seasons (Fig. 4 A to C). The highest POX activity was recorded in Coratina cv. (25.14 U/mg F.wt.), followed by Manzanillo (17.41 U/mg F. wt.), and Koroneiki (17.25 U/mg F. wt.), whereas the lowest POX activity was assayed in cv. Picual (8.14 U/ mg F. wt.) (Fig. 3A). The highest PPO activity was found in Coratina cv. (56.86 U/mg F. wt.), followed by Manzanillo (49.02 U/mg F. wt.), whereas the lowest PPO activity was observed in cv. Koroneiki (33.41 U/ mg F. wt.) (Fig. 4B). The highest and the lowest CAT activities were detected in Manzanillo (8.19 U/mg F. wt.) and Koroneiki (3.77 U/ mg F. wt.) cultivars, respectively (Fig. 4C).

Fig. 4. Peroxidase (POX), polyphenol oxidase (PPO), and catalase (CAT) of all tested cultivars (Manzanillo, Coratina, Koroneiki, and Picual) under low-temperature conditions for three months. Different letters depict statistically significant changes (p ≤ 0.05, LSD test) and error bars depict standard error.

Fruit physical characteristics at harvest

Figure 5 shows significant differences between fruit physical characteristics among the four studied olive cultivars under frost conditions. Coratina trees were superior to those of Manzanello, Koroneiki, and Picual varieties in terms of initial and final fruit set percentage during the two studied seasons (2022 and 2023). On the other hand, the growth of olive trees under frost conditions through 2022 and 2023 led to a reduction in the fruit weight and yield of the four tested olive tree cultivars, where the Manzanello olive cultivar was superior to the other tested cultivars, followed by the descending order of Picual, Coratina, and Koroneiki.

Fig. 5. Initial fruit set, final fruit set, fruit weight, and fruit yield of all tested cultivars (Manzanillo, Coratina, Koroneiki, and Picual) under low-temperature conditions for three months. Different letters depict statistically significant changes (p ≤ 0.05, LSD test) and error bars depict standard error.

Fruit chemical characteristics at harvest

Figure 6 shows the effect of cold stress on fruit chemical characteristics such as fruit oil yield/tree, T.S.S %, total acidity %, and T.S.S acid ratio % of the four tested olive cultivars as follows, Manzanello, Coratina, Koroneiki, and Picual. There were significant differences between all tested olive tree cultivars regarding the effect of low temperature on fruit chemical characteristics of all tested cultivars where the Coratina olive tree cultivar possessed the highest values of fruit chemical characteristics such as fruit oil yield/tree, T.S.S %, Total acidity %, and T.S.S acid ratio %, followed in ascending order by those of Manzanello, Koroneiki, and Picual varieties. Picual olive tree cultivars gained the highest value of total acidity followed in ascending order by Koroneiki, Manzanello, and Coratina.

Fig. 6. Fruit oil yield/ tree, T.S.S %, Total acidity %, and T.S.S acid ratio % of all tested cultivars (Manzanillo, Coratina, Koroneiki, and Picual) under low-temperature conditions for three months. Different letters depict statistically significant changes (p ≤ 0.05, LSD test) and error bars depict standard error.

Fig. 7. Total phenolic and total flavonoid of all tested cultivars (Manzanillo, Coratina, Koroneiki, and Picual) under low-temperature conditions for three months. Different letters depict statistically significant changes (p ≤ 0.05, LSD test) and error bars depict standard error.

Total phenolic and total flavonoids

Figure 7 (A and B) illustrates significant differences noted in leaf total phenolic and total flavonoid contents among olive cultivars. Koroneiki cv. was revealed to have the highest values of phenolics (20.82 mg/g F. wt.), whereas the lowest content was found in Coratina cv. (11.05 mg/g F. wt.) (Fig. 7A). According to Fig. 7B, the maximum and minimum contents of total flavonoids were detected in Coratina and Picual (11.60 and 5.75 mg/g FW), respectively.

DISCUSSION

Low temperatures are one of the main environmental conditions that restrict the dispersion and productivity of plants (Macek et al. 2012). Frost-related leaf damage that occurs in the spring and autumn slows down a tree’s overall growth and has a big effect on plant development and yields (Zouari et al. 2016). The main cause of frost damage is ice breaking down cell structures physically and causing dehydration (Yadu et al. 2017). This is because the interior of a cell has a higher water potential than the area where ice crystals form (Ashraf and Foolad 2007). Olive trees are susceptible to severe damage from frost, especially in their early planting years. Shoot tip necrosis, leaf fall, and bark wounding are typical symptoms. Olive trees may completely die in temperatures below -12 °C. Plants that are susceptible to cold temperatures have been shown to respond by down regulating their photosynthetic efficiency in response to lowered air temperatures (Allen and Ort 2001; Larcher 2000). In the present work, the results of morphological, biochemical, and enzyme activity in leaves or fruits of all stressed tested olive cultivars Manzanillo, Coratina, Koroneiki, and Picual reveal only minor effects of cold stress. However, it should be mentioned that Coratina and Koroneiki stressed plants exhibited higher morphological, biochemical parameters values than Manzanillo and Picual stressed plants, suggesting that Coratina and Koroneiki trees were able to cope better with night-frost incidents. The results showed that in terms of all vegetative growth parameters, including shoot length, leaf area, canopy volume, and tree height, Coratina trees outperformed other olive cultivars (Fig.1). The phenological stages of the plant and the season in which low temperatures occur determine the temperature thresholds that cause frost damage to olive trees (early frost, winter frost, and late frost). The younger parts of the tree (leaves, one-year shoots) are usually injured at minimum temperatures of 0 °C/−3 °C (slight damage), showing a reduction in growth and production; almost all of the plant’s organs are damaged at minimum temperatures of −6 °C/−7 °C (moderate damage); and the entire plant may be compromised at temperatures below −12 °C/−18 °C (severe damage) ( Rahemi et al. 2016). Low temperatures (<12 °C) can be harmful to all stages of plant development in plants that are susceptible to chilling, such as subtropical and tropical plants. This can result in phenotypic symptoms such as reduced plant growth, shorter leaf area, reduction in root length, reduced leaf expansion, loss of leaves, chlorosis, and necrosis, as well as damage to reproductive organs such as decreased pollen production and viability, reduced pollen tube growth, and increased flower abortion (Lukatkin et al. 2012). Strong vegetative growth may be linked to good fruit yields, suggesting that tree growth may reflect the ability to adapt to new environments (Rallo et al. 2018). The interplay between cultivar genotype and the particular environment had an impact on growth of olive trees as height and trunk diameter growth (Kassout et al. 2023). Since all of the olive trees in this study were grown at the same location with the same soil, climate, and cultivation practices, genotype was the primary factor influencing the variations in height and diameter growth among cultivars. As a result, olive trees can thrive and endure. Different climates, soil types, and farming practices may be the cause of regional variations in growth (Arenas-Castro et al. 2020). According to the present study, olive trees recover quickly from short-term stressors like drought and high temperatures, suggesting that they are very resilient and adaptive to mild stressors (Araújo et al. 2019). However, long-term exposure or extreme stress caused irreversible harm to the physiological functioning of the leaves (Trabelsi et al. 2019). In the present results, Coratina and Koroneiki trees were able to cope better with night-frost incidents. Thus, the present results are in line with those of other researchers. For example, Saadati et al. (2019) found that “Koroneiki” plants were also able to cope with mild frost incidents, despite the less extended gene expression alteration. “Koroneiki” has been classified as moderately tolerant in a previous experiment based on electrolyte leakage in leaves.

The primary pigments in leaf tissue that absorb light energy are chlorophyll and carotenoids. Also, plant photosynthesis strength is represented by the levels of chlorophyll (Zhao et al. 2020). The process of photosynthesis is sensitive to low temperatures. Exposure to (frost) low temperatures leads to the disruption of all the main components of the process, such as inhibiting electron transfer in photophosphorylation and reducing the efficiency of photosystem II (PSII). PSII is one of the most vulnerable components of photosynthesis (Banerjee and Roychoudhury 2019). Low temperatures cause a decrease in the rate of net photosynthesis, transpiration, stomatal conduction, and damage to photosystem II in olive cultivars (Saadati et al. 2019). In this study, photosynthetic pigments in Koroneiki and Coratina cultivars were considered the least affected under frost conditions (Fig 2.). The physiological process of photosynthesis can be significantly impacted by abiotic stressors (Gururani et al. 2015). On the other hand, previous studies on olive trees have shown that their photosynthetic system is tolerant of a variety of abiotic stresses, such as drought and high UV radiation (Koubouris et al. 2010). These findings concur with those of Mohajeri et al. (2022), who examined how cold stress affected the photosynthetic pigments in the leaves of two different cultivars of olive seedlings. According to a report, when two tested olive seedling cultivars’ leaves were cooled from 10 to -10 ° C, their levels of chlorophyll a, b, total chlorophyll, and carotenoids were lower than those of the control group.

Low temperatures are known to cause cell dehydration, and RWC is an easy method to gauge this phenomenon (Ghosh et al. 2016). The capacity of plants to preserve cellular water by raising the relative moisture content is one of the most crucial elements in ensuring their survival under cold stress (Mohajeri et al. 2022). As shown in Fig. 3A, the relative water content in the leaves of Manzanello, Coratina, Koroneiki, and Picual was decreased under cold conditions. There are significant differences between all tested olive tree cultivars in relation to cold stress conditions where Coratina gained the highest relative water content. The leaves’ water content decreased due to cold adaptation, which also increased the concentration of solutes in the cells, increasing the cell’s capacity. The ability of the leaf to retain more water under stress is, in theory, indicated by an increase in the relative moisture content of the leaf. This is accomplished by osmotic regulation or the root’s capacity to absorb water (Mishra et al. 2023). In a similar way, Li et al. (2018) on tea plant (Camellia sinensis) reported that the RWC under freezing temperature decreased significantly, even in relation to the duration of the treatment (from 3 h to 12 h). Because of an imbalance between the amount of water absorbed by roots and transpiration, low temperatures can also cause dehydration symptoms in shoots and leaves (Centeno et al. 2018).

As the main osmotic regulator in plant cells, proline is important in stabilizing the

cell structure and inhibiting the production of reactive oxygen species. The most significant basic physiological marker for abiotic stress reflection is proline (Chen et al. 2018). In a similar vein, transgenic plants that overexpressed the proline or glycine betaine biosynthesis genes also displayed decreased levels of lipid peroxidation in the presence of abiotic stress (Khan et al. 2015). The molecules share the ability to accumulate at high levels without disrupting intracellular biochemistry. These compatible solutes primarily include proline and glycine betaine. Complementary solute accumulation in plant cells is positively correlated with increased stress tolerance. This is because (i) the plant cell’s potential water decreases, increasing water absorption from the rhizosphere; (ii) free radical scavenging; (iii) activation of enzymatic and non-enzymatic antioxidant systems; and (iv) protection of the photosynthetic apparatus from damage (Dawood et al. 2014; Zouari et al. 2016; Yadu et al. 2017; Youssef et al. 2018). Ashraf and Foolad (2007) state that proline and glycine betaine can function as antioxidants directly or indirectly by activating antioxidant responses and thereby reduce the damage caused by oxidative stress. Zhou et al. (2022) reported that in maize, there is a close correlation between proline accumulation and improved cold tolerance. Many reports show a dramatic relationship between proline accumulation and tolerance in plants under freezing stress (Sarikhani et al. 2014; Ershadi et al. 2016).

Low temperatures cause oxidative stress due to excessive production of reactive oxygen species (ROS): hydrogen peroxide (H₂O₂), superoxide radical (O₂⁻) and hydroxyl radical (OH⁻) (Nievola et al. 2017). According to Jamshidi Goharrizi et al. (2021), H₂O₂ accumulated as cold stress increased. Similar to this, Fagopyrum tataricum and Arabidopsis thaliana have shown a considerable increase in H₂O₂ and O₂^– accumulation in response to increased cold stress (Yao et al. 2018). The ability of several antioxidant enzymes to function simultaneously is essential for shielding plant cells from ROS toxicity (Das and Roychoudhury 2014). Plant species that are tolerant to freezing-induced oxidative stress tend to exhibit greater resilience against such stressors. This resilience can also be reinforced by enhancement of antioxidant enzyme activities (Luo et al. 2007; Cansev et al. 2011). Catalase and POX convert free radicals such as hydrogen peroxide (H₂O₂) into water and oxygen (Møller et al. 2007). In the current study, Coratina cv. had the highest activity of POX and PPO enzymes under low temperatures, and this may be related to frost tolerance (Fig. 4A to B). Higher values of antioxidant enzymes such as APX, PPO, and CAT were reported in olive cultivars under low temperatures in other studies (Cansev et al. 2011; Ortega-García and Peragón 2009). Hashempour et al. (2014) reported that there is a substantial correlation between olive trees’ ability to frost tolerance and their APX, CAT, and POX activities. According to Ortega-García and Peragon (2009), the activities of PPO and phenylalanine ammonia-lyase (PAL) are crucial in plants’ defense against freezing stress because they can prevent major oxidative damage brought on by freezing.

In this study, Coratina cv were superior in terms of initial and final fruit set percentage, while Manzanello olive cultivar was superior in the fruit weight and yield under frost conditions during the two studied seasons (2022 and 2023) (Fig. 5). A temperature drop (from −0.4 to −3 °C) can cause drupe wilting and dehydration, surfaces with brownish blisters and spots, fully frozen drupes, internal browning around the pit, skin browning and mild flesh discoloration, and a decrease in oil accumulation (Bheemanahalli et al. 2019). Research conducted on different plant species has demonstrated that cold stress results in a decrease in pollen tube length and germination rate (Geßler et al. 2006). In general, the climate has a significant impact on the phenology and adaptation of trees (Zhu et al. 2013). Springtime temperatures have a significant impact on the development of olive flowers, pollen germination, fruit set and development, and panicle number of flowers (Ben-Ari et al. 2021; Koubouris et al. 2010). Furthermore, flowers and flower buds may sustain severe damage at temperatures as low as 0 °C (Kudo and Ida 2013). Long-term low temperatures have been shown to have a negative impact on fruit set, flowering, and pollination (Francini and Sebastiani 2019). Pollen germination may be hampered by low temperatures during flowering, which could compromise fruit set (Koubouris et al. 2010).

In the present study it was shown that Coratina olive tree cultivar possessed the highest values of fruit chemical characteristics such as fruit oil yield/tree, T.S.S %, Total acidity %, and T.S.S acid ratio %, followed in ascending order by those of Manzanello, Koroneiki, and Picual varieties (Fig. 6). Olive yield and quality are known to be significantly impacted by ecological factors and cultivation conditions (Calvo-Polanco et al. 2016). The differences in oil content in olive fruits are also related to fruit size, which is influenced by exogenous and endogenous factors. Frost, or low air temperatures of 0 ~C or below, can harm olive fruits by causing the water in their pulp to freeze, lowering enzymatic and biochemical reactions, and suppressing microbial activity (Bubola et al. 2020). According to Mafrica et al. (2021) high altitude-typical low temperatures and heavy rainfalls postpone fruit development and ripening while improving the quality of olive oil. On the other hand, differences in the tolerance of olive cultivars to cold stress were associated by chemical indicators such as the amount of proline, soluble sugars, as well as the content of soluble carbohydrates and phospholipids (Gulen et al. 2009). Abiotic stresses affect different cellular processes such as growth, photosynthesis, carbohydrate and lipid metabolism, osmotic homeostasis, protein synthesis and gene expression. Soluble sugars may either act directly as negative signals or as modulators of plant sensitivity and thus, they can also play important roles in cell responses to stress-induced remote signals (Rosa et al. 2004). Soluble sugars actively protect plant cells from damage caused by cold stress. In addition, accumulation of TSS, and thus enhanced the cold tolerance of Chinese olive fruit (Lin et al. 2016). According to Martínez et al. (2004), the buildup of compatible solutes in plants is believed to help stressed cells either by serving as cytoplasmic osmolytes to aid in water uptake and retention or by shielding macromolecules (such as proteins, membranes and carbohydrates) and their structural integrity from stress-induced damage.

Titratable acidity (TA) was used as an indicator to evaluate the quality of fresh produces under abiotic stress (Lin et al. 2020). As the primary components of oil production, oil content and fruit production are the most crucial economic factors for characterizing olive cultivars (Gómez-del-Campo 2013). Mediterranean-produced olive oil has generally been identified by its acidity, fatty acid composition, peroxide levels, and polyphenol content (Fernández-Espinosa 2016). The present results indicated that the difference in total acidity values between different olive cultivars is due to a response to stress resulting from low temperature. Additionally, plants use fatty acids as part of their defense mechanism against biotic and abiotic stressors (Okazaki and Saito 2014). Biomembranes are the first to suffer damage when plants are stressed by low temperatures. Plant cold resistance is directly correlated with membrane fluidity and stability. In general, unsaturated fatty acids are thought to be advantageous for preserving membrane integrity (Upchurch 2008). Consequently, in low-temperature, increasing the amount of unsaturated fatty acids in membrane lipids can improve cold resistance (Wang et al. 2019).

At low temperatures, the accumulation of secondary metabolites such as phenolic compounds and anthocyanins occur (Francini and Sebastiani 2019). Cold stress leads to increased cellular damage caused by increased production of ROS. Therefore, the development of the antioxidant defense system, which includes antioxidants such as phenolic and flavonoids, may be partly related to cold stress resistance. The most abundant secondary metabolites in plants, phenolic compounds, have a potent ability to donate electrons and hydrogen atoms, which allows them to neutralize ROS and stop lipid peroxidation (Huang et al. 2019). In this study, the high amounts of phenolic and flavonoids in the Koroneiki and Coratina cultivars, respectively, may have a direct relationship to the resistance of these cultivars to frost. The values of total phenolic and total flavonoids can be used as biomarkers to distinguish olive varieties under frost stress. These results agree with Ortega‐García and Peragón’s study (2009). They found changes in total phenolic and proline content in different olive cultivars under cold stress.

CONCLUSIONS

In summary, the findings demonstrated that the tested olive cultivars responded differently to the growing region’s environmental conditions. The analysis of the many different parameters considered in this article revealed that low temperatures below the limits of adaptation have a significant impact on morphology, physiology, biochemical, and productivity of olive trees. Based on the findings of this study on olive cultivar behavior, the length of the event, the genotypes, the stage at which the plant is growing, and the interactions with the environment can determine differences in how different cultivars respond to the cold acclimation process, thereby influencing their intrinsic tolerance to cold stress. There were significant variations in olive tree cultivar responses to cold stress. Coratina and Koroneiki emerged as the most resilient cultivars, demonstrating superior performance in biochemical, physiological, and reproductive functions compared to Manzanillo and Picual. These findings empower farmers to make informed decisions regarding olive cultivar selection and breeding programs, ultimately promoting improved adaptation to low-temperature events frequently encountered in open plantations.

ACKNOWLEDGMENTS

The authors extend their appreciation to the Researchers Supporting Project number (RSP2024R334), King Saud University, Riyadh, Saudi Arabia.

Funding

This research was funded by Researchers Supporting Project number (RSP2024R334), King Saud University, Riyadh, Saudi Arabia.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Conceptualization, H.F.A and A.E.H.; methodology H.F.A;IAE;A.N.A;H.K.;A.F. and E.M.H software, A.E.H. and A.N.A; validation, H.F.A and A.F; formal analysis, H.F.A and A.E.H.; H.K.; E.M.H and A.F.A. investigation, H.F.A.; ANA , E.M.H and I.A.F resources, A.N.A.; data curation, I.A.E.; writing—H.F.A.; writing—review and editing, M.H.F. and E.M.H visualization, A.E.H.; supervision, A.E.H. and M.H.F.; project administration, A.M.S.; funding acquisition, A.M.S. All authors have read and agreed to the published version of the manuscript.

REFERENCES CITED

Aebi, H. (1984). “Catalase in vitro,” Methods in Enzymology 105, 121-126. DOI: 10.1016/S0076-6879(84)05016-3

Ahmed, F. F., and Morsy, M. H. (1999). “A new method for measuring leaf area in different fruit species,” Minia J. Agricultural Research and Development 19, 97-105.

Allen, D. J., and Ort, D .R. (2001). “Impacts of chilling temperatures on photosynthesis in warm-climate plants,” Trends in Plant Science 6(1), 36-42. DOI: 10.1016/S1360-1385(00)01808-2

Amako, K., Chen, G.-X., and Asada, K. (1994). “Separate assays specific for ascorbate peroxidase and guaiacol peroxidase and for the chloroplastic and cytosolic isozymes of ascorbate peroxidase in plants,” Plant and Cell Physiology 35(3), 497-504. DOI: 10.1093/oxfordjournals.pcp.a078621

Araújo, M., De Oliveira, J. M. P. F., Santos, C.V., Moutinho-Pereira, J. M., Correia, C., and Dias, M. C. (2019). “Responses of olive plants exposed to different irrigation treatments in combination with heat shock: Physiological and molecular mechanisms during exposure and recovery,” Planta 249(5), 1583-1598. DOI: 10.1007/s00425-019-03109-2.

Arenas-Castro, S., Gonçalves, J. F., Moreno, M., and Villar, R., (2020). “Projected climate changes are expected to decrease the suitability and production of olive varieties in southern Spain,” Science of the Total Environment 709, article 136161. Doi:10.1016/j.scitotenv.2019.136161.

Ashraf, M., and Foolad, M. R. (2007). “Roles of glycine betaine and proline in improving plant abiotic stress resistance,” Environmental and Experimental Botany 59(2), 206-216. DOI: 10.1016/j.envexpbot.2005.12.006

Banerjee, A., and Roychoudhury, A. (2019). “Cold stress and photosynthesis. Photosynthesis, Productivity and Environmental Stress; 4: pp. 27-37. DOI: 10.1002/9781119501800.ch2

Bates, L. S., Waldren, R. P., and Teare, I. D. (1973). “Rapid determination of free proline for water-stress studies,” Plant and Soil 39, 205-207. DOI: 10.1007/BF00018060

Ben-Ari, G., Biton, I., Many, Y., Namdar, D., and Samach, A. (2021). “Elevated temperatures negatively affect olive productive cycle and oil quality,” Agronomy 11(8), article 1492. DOI: 10.3390/agronomy11081492

Bheemanahalli, R., Sunoj, V. S. J., Saripalli, G., Prasad, P. V. V., Balyan, H. S., Gupta, P. K., Grant, N., Gill, K. S., and Jagadish, S. V. K. (2019). “Quantifying the impact of heat stress on pollen germination, seed set, and grain filling in spring wheat,” Crop Science 59(2), 684-696. DOI: 10.2135/cropsci2018.05.0292

Boskou, D. (2015). “Olive fruit, table olives, and olive oil bioactive constituents,” in: Olive and Olive Oil Bioactive Constituents, AOCS Press, pp. 1-30.

Brito, G. G., Sofiatti, V., Brandão, Z. N., Silva, V. B., Silva, F. M., and Silva, D. A. (2011). “Non-destructive analysis of photosynthetic pigments in cotton plants,” Acta Scientiarum Agronomy 33(4), 671-678. DOI: 10.4025/actasciagron.v33i4.10926

Bubola, K. B., Lukić, M., Novoselić, A., Krapać, M., and Lukić, I. (2020). “Olive fruit refrigeration during prolonged storage preserves the quality of virgin olive oil extracted therefrom,” Foods 9(10), article 1445. DOI:10.3390/foods9101445

Calvo-Polanco, M., Sánchez-Castro, I., Cantos, M., García, J. L., Azcón, R., Ruiz-Lozano, J. M., Beuzón, C. R., and Aroca, R. (2016). “Effects of different Arbuscular mycorrhizal fungal backgrounds and soils on olive plants growth and water relation properties under well-watered and drought conditions,” Plant, Cell and Environment 39(11), 2498-2514. DOI: 10.1111/pce.12807

Cansev, A., Gulen, H., and Eris, A. (2011). “The activities of catalase and ascorbate peroxidase in olive (Olea europaea L. cv. GemLik) under low temperature stress,” Horticulture, Environ. Biotechnol. 52, 113-120. DOI: 10.1007/s13580-011-0126-4

Centeno, A., Memmi, H., Moreno, M. M., Moreno, C., and Pérez-López, D. (2018). “Water relations in olive trees under cold conditions,” Sci. Hortic. 235, 1-8. DOI: 10.1016/j.scienta.2018.02.070.

Chang, C.-C., Yang, M.-H., Wen, H.-M., and Chern, J.-C. (2002). “Estimation of total flavonoid content in propolis by two complementary colorimetric methods,” Journal of Food and Drug Analysis 10(3), 3. DOI: 10.38212/2224-6614.2748

Chen, J., Cui, Y., Yan, J., Jiang, J., Cao, X., and Gao, J. (2018). “Molecular characterization of elongase of very long-chain fatty acids 6 (elovl6) genes in Misgurnus anguillicaudatus and their potential roles in adaptation to cold temperature,” Gene 666, 134-144. DOI: 10.1016/j.gene.2018.05.019

Das, K., and Roychoudhury, A. (2014). “Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants,” Frontiers in Environmental Science 2. DOI: 10.3389/fenvs.2014.00053

Dawood, M. G., Taie, H. A. A., Nassar, R. M. A., Abdelhamid, M. T., and Schmidhalter, U. (2014). “The changes induced in the physiological, biochemical and anatomical characteristics of Vicia faba by the exogenous application of proline under seawater stress,” South African Journal of Botany 93, 54-63. DOI: 10.1016/j.sajb.2014.03.002

Ershadi, A., Karimi, R., and Mahdei, K. N. (2016). “Freezing tolerance and its relationship with soluble carbohydrates, proline and water content in 12 grapevine cultivars,” Acta Physiologiae Plantarum 38, 2. DOI: 10.1007/s11738-015-2021-6

Esfandiari, E.-O., Shakiba, M. R., Mahboob, S. A., Alyari, H., and Toorchi, M. (2007). “Water stress, antioxidant enzyme activity and lipid peroxidation in wheat seedling,” 5(1), 149-153.

Ferguson, L., and Grattan, S. R. (2005). “How salinity damages citrus: Osmotic effects and specific ion toxicities,” HortTechnology 15, 95-99. DOI: 10.21273/HORTTECH.15.1.0095

Fernández-Espinosa, A. J. (2016). “Combining PLS regression with portable NIR spectroscopy to on-line monitor quality parameters in intact olives for determining optimal harvesting time,” Talanta 148, 216-228. DOI: 10.1016/j.talanta.2015.10.084.

Fraga, H., Moriondo, M., Leolini, L., and Santos, J. A. (2020). “Mediterranean olive orchards under climate change: A review of future impacts and adaptation strategies,” Agronomy 11(1), article 56. DOI: 10.3390/agronomy11010056

Francini, A., and Sebastiani, L. (2019). “Abiotic stress effects on performance of horticultural crops,” Horticulturae 5(4), article 67. DOI: 10.3390/horticulturae5040067

Geßler, A., Keitel, C., Kreuzwieser, J., Matyssek, R., Seiler, W., and Rennenberg, H. (2006). “Potential risks for European beech (Fagus sylvatica L.) in a changing climate,” Trees 21, 1-11. DOI: 10.1007/s00468-006-0107-x

Ghosh, T., Rai, M., Tyagi, W., and Challam, C. (2016). “Seedling stage low temperature response in tolerant and susceptible rice genotypes suggests role of relative water content and members of OsSNAC gene family,” Plant Signal. Behav. 11(5), article e1138192. DOI:10.1080/15592324.2016.1138192.

Gómez, J., Infante-Amate, J., De Molina, M., Vanwalleghem, T., Taguas, E., and Lorite, I. (2014). “Olive cultivation, its impact on soil erosion and its progression into yield impacts in southern Spain in the past as a key to a future of increasing climate uncertainty,” Agriculture 4(2), 170-198. DOI: 10.3390/agriculture4020170.

Gómez-del-Campo, M., (2013). “Summer deficit-irrigation strategies in a hedgerow olive orchard cv. ‘Arbequina’: Effect on fruit characteristics and yield,” Irrigation Sci. 31(3), 259-269. DOI 10.1007/s00271-011-0299-8

Gulen, H., Cansev, A., and Eris, A. (2009). “Cold hardiness of olive (Olea europaea L.) cultivars in cold-acclimated and non-acclimated stages: Seasonal alteration of soluble sugars and phospholipids.” J. Agric. Sci. 147(4), 459-467. DOI: 10.1017/S0021859609008600

Guerra, D., Lamontanara, A., Bagnaresi, P., Orrù, L.; Rizza, F., Zelasco, S., Beghè, D., Ganino, T., Pagani, D., and Cattivelli, L. (2015). “Transcriptome changes associated with cold acclimation in leaves of olive tree (Olea europaea L.),” Tree Genet. Genomes 11(113), 1-24. DOI: 10.1007/s11295-015-0939-x

Gururani, M. A., Venkatesh, J., and Tran, L. S. P. (2015). “Regulation of photosynthesis during abiotic stress-induced photoinhibition,” Molecular Plant 8(9), 1304-1320. DOI: 10.1016/j.molp.2015.05.005

Hamdy, A. E., Abdel-Aziz, H. F., El-khamissi, H., AlJwaizea, N. I., El-Yazied, A. A., Selim, S., Tawfik, M. M., AlHarbi, K., Ali, M. S. M., and Elkelish, A. (2022). “Kaolin improves photosynthetic pigments, and antioxidant content, and decreases sunburn of mangoes: Field study,” Agronomy 12(7), article 1535. DOI: 10.3390/agronomy12071535

Hashempour, A., Ghasemnezhad, M., Fotouhi Ghazvini, R., and Sohani, M. M. (2014). “Olive (Olea europaea L.) freezing tolerance related to antioxidant enzymes activity during cold acclimation and non acclimation,” Acta Physiologiae Plantarum 36, 3231-3241. DOI: 10.1007/s11738-014-1689-3

Huang, H., Ullah, F., Zhou, D.-X., Yi, M., and Zhao, Y. (2019). “Mechanisms of ROS regulation of plant development and stress responses,” Frontiers in Plant Science 10, article 800. DOI: 10.3389/fpls.2019.00800

Jamshidi Goharrizi, K., Meru, G., Ghotbzadeh Kermani, S., Heidarinezhad, A., and Salehi, F. (2021). “Short-term cold stress affects physiological and biochemical traits of pistachio rootstocks,” South African Journal of Botany 141, 90-98. DOI: 10.1016/j.sajb.2021.04.029

Kassout, J., Terral, J.F., Azenzem, R., El Ouahrani, A., Sahli, A., Houssni, M., Chakkour, S., Kadaoui, K., and Ater, M., (2023). “Olea europaea and stressful conditions,” in: Medicinal Plant Responses to Stressful Conditions, CRC Press, pp. 243-272. DOI: 10.1201/9781003242963-13

Kavrayan, D., and Aydemir, T. (2001). “Partial purification and characterization of polyphenoloxidase from peppermint (Mentha piperita),” Food Chemistry 74(2), 147-154. DOI: 10.1016/S0308-8146(01)00106-6

Khan, M. S., Ahmad, D., and Khan, M. A. (2015). “Utilization of genes encoding osmoprotectants in transgenic plants for enhanced abiotic stress tolerance,” Electronic Journal of Biotechnology 18(4), 257-266. DOI: 10.1016/j.ejbt.2015.04.002

Khattab, M. M., Hamed, H. H., Awad, N. A., and ElKorashy, H. A. (2021). “Prolonging the shelf life and maintaining fruit quality of Naomi mango cultivar,” Plant Archives 21(1), 1667-1675. DOI: 10.51470/PLANTARCHIVES.2021.v21.no1.229

Koubouris, G. C., Metzidakis, I. T., and Vasilakakis, M. D. (2010). “Influence of cross-pollination on the development of parthenocarpic olive (Olea europaea) fruits (shotberries),” Exper. Agriculture 46(1), 67-76. DOI: 10.1017/S0014479709990500

Kudo, G., and Ida, T. Y. (2013). “Early onset of spring increases the phenological mismatch between plants and pollinators,” Ecology 94(10), 2311-2320. DOI: 10.1890/12-2003.1

Larcher, W. (2000). “Temperature stress and survival ability of Mediterranean sclerophyllous plants,” Plant Biosystems – An International Journal Dealing with all Aspects of Plant Biology 134(3), 279-295. DOI: 10.1080/11263500012331350455

León, L., Arias-Calderón, R., De La Rosa, R., Khadari, B., and Costes, E. (2016). “Optimal spatial and temporal replications for reducing environmental variation for oil content components and fruit morphology traits in olive breeding,” Euphytica 207, 675-684. DOI: 10.1007/s10681-015-1569-y

Li, X., Ahammed, G. J., Li, Z. X., Zhang, L., Wei, J. P., Yan, P., Zhang, L. P., and Han, W. Y. (2018). “Freezing stress deteriorates tea quality of new flush by inducing photosynthetic inhibition and oxidative stress in mature leaves,” Scientia Horticulturae 230, pp. 155-160. DOI: 10.1016/j.scienta.2017.12.001

Lin, Y., Li, N., Lin, H., Lin, M., Chen, Y., Wang, H., Ritenour, M. A. and Lin, Y. (2020). “Effects of chitosan treatment on the storability and quality properties of longan fruit during storage,” Food Chemistry 306, article 125627. DOI: 10.1016/j.foodchem.2019.125627

Lin, Y., Lin, H., Lin, Y., Zhang, S., Chen, Y., and Jiang, X., (2016). “The roles of metabolism of membrane lipids and phenolics in hydrogen peroxide-induced pericarp browning of harvested longan fruit,” Postharvest Biology and Technology 111, 53-61. DOI: 10.1016/j.postharvbio.2015.07.030

Lodolini, E. M., Alfei, B., Cioccolanti, T., Zucchini, M., and Neri, D. (2022). “Comparison of frost damages in eleven olive cultivars after two freezing events in central Italy,” Acta Horticulturae 1346, 161-168. DOI: 10.17660/ActaHortic.2022.1346.21

López-Bernal, Á., García-Tejera, O., Testi, L., Orgaz, F., and Villalobos, F. J. (2015). “Low winter temperatures induce a disturbance of water relations in field olive trees,” Trees 29, 1247-1257. DOI: 10.1007/s00468-015-1204-5

Lukatkin, A. S., Brazaitytė, A., Bobinas, Č., and Duchovskis, P. (2012). “Chilling injury in chilling-sensitive plants: A review,” Žemdirbystė=Agriculture 99(2), 111-124. UDK 634.1:581.17:576.3

Luo, L., Lin, S., Zheng, H., Lei, Y., Zhang, Q., and Zhang, Z. (2007). “The role of antioxidant system in freezing acclimation-induced freezing resistance of Populus suaveolens cuttings,” Forestry Studies in China 9, 107-113. DOI: 10.1007/s11632-007-0016-0

Macek, P., Klimeš, L., Adamec, L., Doležal, J., Chlumská, Z., de Bello, F., and Řeháková, K. (2012). “Plant nutrient content does not simply increase with elevation under the extreme environmental conditions of Ladakh NW Himalaya,” Arct. Antarct. Alp. Res. 44(1), 62-66. DOI: 10.1657/1938-4246-44.1.62.

Mafrica, R., Piscopo, A., de Bruno, A., and Poiana, M. (2021). “Effects of climate on fruit growth and development on olive oil quality in cultivar carolea,” Agriculture 11(2), article 147. DOI: 10.3390/agriculture11020147

Martínez, J. P., Lutts, S., Schanck, A., Bajji, M., and Kinet, J. M. (2004). “Is osmotic adjustment required for water stress resistance in the Mediterranean shrub Atriplex halimus L.?” J. Plant Physiol. 161(9), 1041-1051. DOI: 10.1016/j. jplph.2003.12.009

McKie, V. A., and McCleary, B. V. (2016). “A novel and rapid colorimetric method for measuring total phosphorus and phytic acid in foods and animal feeds,” Journal of AOAC International 99(3), 738-743. DOI: 10.5740/jaoacint.16-0029

Medda, S., Fadda, A., and Mulas, M. (2022). “Influence of climate change on metabolism and biological characteristics in perennial woody fruit crops in the Mediterranean environment,” Horticulturae 8(4), 273. DOI: 10.3390/horticulturae8040273

Mete, N., Gülen, H., Çeti̇n, Ö., Hakan, M., Güloğlu, U., Kaya, H., and Uluçay, N. (2023). “Frost tolerances of Turkish olive (Olea europaea L.) cultivars,” Tekirdağ Ziraat Fakültesi Dergisi 20(2), 293-305. DOI: 10.33462/jotaf.1081561

Mishra, N., Jiang, C., Chen, L., Paul, A., Chatterjee, A., and Shen, G., (2023). “Achieving abiotic stress tolerance in plants through antioxidative defense mechanisms,” Frontiers in Plant Science 14, article 1110622. DOI: 10.3389/fpls.2023.1110622

Mohajeri, K., Tabari, M., Sadati, E., Javanmard, Z., Guidi, L., and Vicente, O. (2022). “Effect of cold stress on water relations, photosynthetic pigments and antioxidant enzymes in olive seedlings,” European Journal of Horticultural Science 87(2), 1-9. DOI: 10.17660/eJHS.2022/021

Møller, I. M., Jensen, P. E., and Hansson, A. (2007). “Oxidative modifications to cellular components in plants,” Annual Review of Plant Biology 58, 459-481. DOI: 10.1146/annurev.arplant.58.032806.103946

Nievola, C. C., Carvalho, C. P., Carvalho, V., and Rodrigues, E. (2017). “Rapid responses of plants to temperature changes,” Temperature 4(4), 371-405. DOI: 10.1080/23328940.2017.1377812

Okazaki, Y., and Saito, K. (2014). “Roles of lipids as signaling molecules and mitigators during stress response in plants,” The Plant J. 79 (4), 584-596. DOI: 10.1111/tpj.12556

Ortega-García, F., and Peragón, J. (2009). “The response of phenylalanine ammonia- lyase, polyphenol oxidase and phenols to cold stress in the olive tree (Olea europaea L. cv. Picual),” Journal of the Science of Food and Agriculture 89, 1565-1573. DOI: 10.1002/jsfa.3625

Ozturk, B., Aglar, E., Karakaya, O., Saracoglu, O., and Gun, S. (2019). “Effects of preharvest GA3, CaCl₂ and modified atmosphere packaging treatments on specific phenolic compounds of sweet cherry,” Turkish Journal of Food and Agriculture Sciences 1(2), 44-56. DOI: 10.14744/turkjfas.2019.009

Petruccelli, R., Bartolini, G., Ganino, T., Zelasco, S., Lombardo, L., Perri, E., Durante, M., and Bernardi, R. (2022). “Cold stress, freezing adaptation, varietal susceptibility of Olea europaea L.: A review,” Plants 11(10), article 1367. DOI: 10.3390/plants11101367

Rahemi M., Yazdani F., and Sedaghat, S. (2016). “Evaluation of freezing tolerance in olive cultivars by stomatal density and freezing stress,” Int. J. Hortic. Sci. Technol. 3, 145-153. DOI: 10.22059/IJHST.2016.62914.

Rallo, L., Barranco, D., Díez, C. M., Rallo, P., Suárez, M. P., Trapero, C., and Pliego-Alfaro, F. (2018). “Strategies for olive (Olea europaea L.) breeding: Cultivated genetic resources and crossbreeding,” Advances in Plant Breeding Strategies: Fruits, Vol. 3, pp. 535-600. DOI: 10.1007/978-3-319-91944-7_14

Rosa, M., Hilal, M., González, J. A., and Prado, F. E. (2004). “Changes in soluble carbohydrates and related enzymes induced by low temperature during early developmental stages of quinoa (Chenopodium quinoa) seedlings,” J. Plant Physiol. 161(6), 683-689. DOI: 10.1078/0176-1617-01257

Saadati, S., Baninasab, B., Mobli, M., and Gholami, M. (2019). “Measurements of freezing tolerance and their relationship with some biochemical and physiological parameters in seven olive cultivars,” Acta Physiol. Plant. 41, 1-11. DOI: 10.1007/s11738-019-2843-8

Samra, N., El-Agamy, S., and Samra, B. (2009). “Evaluation of some olive cultivars grown under Egyptian conditions,” Journal of Plant Production 34(6), 6741-6748. DOI: 10.21608/jpp.2009.118657

Sarikhani, H., Haghi, H., Ershadi, A., Esna-Ashari, M., and Pouya, M. (2014). “Foliar application of potassium sulphate enhances the cold-hardiness of grapevine (Vitis vinifera L.),” The Journal of Horticultural Science and Biotechnology 89(2), 141-146. DOI: 10.1080/14620316.2014.11513060

Singleton, V. L., Orthofer, R., and Lamuela-Raventós, R. M. (1999). “Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent,” Methods in Enzymology 299, 152-178. DOI: 10.1016/S0076-6879(99)99017-1

Snedecor, G. W., and Cochran, W. G. (1980). Statistical Methods, 7^th Edition, Iowa State University, Press, Ames, Iowa.

Sola-Guirado, R., Castillo-Ruiz, F. J., Jiménez-Jiménez, F., Blanco-Roldan, G. L., Castro-García, S., and Gil-Ribes, J. A. (2017). “Olive actual ‘on Year’ yield forecast tool based on the tree canopy geometry using UAS imagery,” Sensors 17(8), article 1743. DOI:10.3390/s17081743

Trabelsi, L., Gargouri, K., Hassena, B. A., Mbadra, C., Ghrab, M., Ncube, B., Amoo, S. O., and Gargouri, R. (2019). “Impact of drought and salinity on olive water status and physiological performance in an arid climate,” Agric. Water Manag. 213, 749-759. DOI: 10.1016/j.agwat.2018.11.025

Upchurch, R. G. (2008). “Fatty acid unsaturation, mobilization, and regulation in the response of plants to stress,” Biotechnol. Lett. 30, 967-977. DOI 10.1007/s10529-008-9639-z

Wahid, A., Gelani, S., Ashraf, M., and Foolad, M. (2007). “Heat tolerance in plants: An overview,” Environmental and Experimental Botany 61(3), 199-223. DOI: 10.1016/j.envexpbot.2007.05.011

Wang, X., Yu, C., Liu, Y., Yang, L., Li, Y., Yao, W., Cai, Y., Yan, X., Li, S., and Cai, Y. (2019). “GmFAD3A, a ω-3 fatty acid desaturase gene, enhances cold tolerance and seed germination rate under low temperature in rice,” Int. J. Mol. Sci. 20(15), article 3796. DOI:10.3390/ijms20153796

Wellburn, A. R. (1994). “The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution,” Journal of Plant Physiology 144(3), 307-313. DOI: 10.1016/S0176-1617(11)81192-2

Yadu, B., Chandrakar, V., Meena, R. K., and Keshavkant, S. (2017). “Glycinebetaine reduces oxidative injury and enhances fluoride stress tolerance via improving antioxidant enzymes, proline and genomic template stability in Cajanus cajan L.,” South African Journal of Botany 111, 68-75. DOI: 10.1016/j.sajb.2017.03.023

Yamasaki, S., and Dillenburg, L. R. (1999). “Measurements of leaf relative water content in Araucaria Angustifolia,” Revista Brasileira de Fisiologia Vegetal 11, 69-75.

Yao, P., Sun, Z., Li, C., Zhao, X., Li, M., Deng, R., Huang, Y., Zhao, H., Chen, H., and Wu, Q. (2018). “Overexpression of Fagopyrum tataricum FtbHLH2 enhances tolerance to cold stress in transgenic Arabidopsis,” Plant Physiology and Biochemistry 125, 85-94. DOI: 10.1016/j.plaphy.2018.01.028

Youssef, S. M., Abd Elhady, S. A., Aref, R. M., and Riad, G. S. (2018). “Salicylic acid attenuates the adverse effects of salinity on growth and yield and enhances peroxidase isozymes expression more competently than proline and glycine betaine in cucumber plants,” Gesunde Pflanzen 70, 75-90. DOI: 10.1007/s10343-017-0413-9

Yurtsever, M., and Vural Korkut, S. (2019). “Cloning of lipase from Olea europaea cv. Gemlik leaves and expression analysis in response to cold stress,” Turk. J. Bot. 43(3), 290-297. DOI: 10.3906/bot-1810-7

Zhao, X., Han, L., Xiao, J., Wang, L., Liang, T., and Liao, X. (2020). “A comparative study of the physiological and biochemical properties of tomato (Lycopersicon esculentum M.) and maize (Zea mays L.) under palladium stress,” Sci. of the Total Environ. 705, article 135938. DOI: 10.1016/j.scitotenv.2019.135938

Zhou, X., Muhammad, I., Lan, H., and Xia, C. (2022). “Recent advances in the analysis of cold tolerance in maize,” Frontiers in Plant Science 13, article 866034. DOI: 10.3389/fpls.2022.866034

Zhu, W., Zhou, P., Xie, J., Zhao, G., and Wei, Z. (2013). “Advances in the pollination biology of olive (Olea europaea L.),” Acta Ecologica Sinica 33(2), 64-71. DOI: 10.1016/j.chnaes.2013.01.001

Zouari, M., Ben Ahmed, C., Elloumi, N., Bellassoued, K., Delmail, D., Labrousse, P., Ben Abdallah, F., Ben Rouina, B. (2016). “Impact of proline application on cadmium accumulation, mineral nutrition and enzymatic antioxidant defense system of Olea europaea L. cv Chemlali exposed to cadmium stress,” Ecotoxicology and Environmental Safety 128, 195-205. DOI: 10.1016/j.ecoenv.2016.02.024

Article submitted: July 26, 2024; Peer review completed: October 5, 2024; Revised version received and accepted: October 15, 2024; Published: October 28, 2024.

DOI: 10.15376/biores.19.4.9582-9605