# Drought-stress memory confers cold hardiness in grapefruit (Citrus paradisi) through modulations in antioxidant system, osmolyte production and carbohydrate metabolism

Shahid Iqbal, Carlos Eduardo Aucique-Perez, Sajjad Hussain, Rashad Balal, Guillaume Charrier, Matthew Mattia, John Chater, Muhammad Shahid

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Shahid Iqbal, Carlos Eduardo Aucique-Perez, Sajjad Hussain, Rashad Balal, Guillaume Charrier, et al. Drought-stress memory confers cold hardiness in grapefruit (Citrus paradisi) through modulations in antioxidant system, osmolyte production and carbohydrate metabolism. Plant stress, 2025, 15, pp.100801. 10.1016/j.stress.2025.100801. hal-05030303

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# Drought-stress memory confers cold hardiness in grapefruit (Citrus paradisi) through modulations in antioxidant system, osmolyte production and carbohydrate metabolism

Shahid Iqbal<sup>a</sup>, Carlos Eduardo Aucique-Perez<sup>a</sup>, Sajjad Hussain<sup>b</sup>, Rashad M. Balal<sup>c</sup>, Guillaume Charrier<sup>d</sup>, Matthew Mattia<sup>e</sup>, John M. Chater<sup>f</sup>, Muhammad A. Shahid<sup>a,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

### Keywords:

Abiotic stress

Enzymatic activities

Field capacity

Priming

Sugar metabolism

## ABSTRACT

Priming is a phenomenon in which plants are initially exposed to mild stress to develop resilience to subsequent or severe stress. There is a lack of scientific knowledge regarding the drought-priming (DP) effect on the freeze tolerance mechanism in cold-sensitive grapefruit (Citrus paradisi). Therefore, the present study was conducted to gain insight into how plants perform under freezing stress (FS) when given short-term drought stress (DS) as a priming event. This study investigated the potential of DP to enhance freeze tolerance in cold-sensitive grape-fruit. The plants were subjected to drought-priming at 50 % and 75 % field capacity (FC), with control (non-stress- 100 % FC) for three weeks before being subjected to freezing stress (-6 °C). DP exhibited enhanced freezing tolerance through an improved antioxidant system, osmolyte production, and carbohydrate metabolism. It also triggered the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione peroxidase (GPX), and ascorbate peroxidase (APX), mitigating oxidative damage caused by reactive oxygen species (ROS). The accumulation of osmolytes, including proline, glycine betaine (GB) and soluble sugars, was also induced, stabilizing cellular structures and maintaining osmotic balance. Additionally, carbohydrate metabolism shifted towards sugar biosynthesis, with reduced starch reserves providing energy for stress adaptation. It is concluded that drought-stress memory as a primer acclimated the plants to freezing stress by alleviating oxidative damage to membranes and maintained high energy production through enhanced antioxidant activities, osmolyte accumulation, and regulating carbohydrate metabolism. This study highlights the practical potential of drought-priming as a cost-effective and non-invasive strategy to improve cold hardiness in citrus and related species. By integrating drought-priming into management practices, growers can enhance the resilience of young grapefruit plants to freezing conditions, contributing to sustainable citrus production in regions prone to cold stress or unexpected freeze events in winter or late summer.

## 1. Introduction

Abiotic stress such as drought, cold, heat, and salinity pose severe threats and cause adverse effects on plant growth and development, significantly reducing yield and exacerbating environmental

degradation. Through the stress conditions, the plant suffers a series of morphological, biochemical, physiological, and molecular changes that affect the plant's normal growth and productivity (Wang et al., 2000). While various stresses impact plant growth, the internal mechanisms triggered by drought and freezing stress predominantly hinder and

<footnote><sup>a</sup> Horticultural Science Department, North Florida Research and Education Center, University of Florida/IFAS, Quincy, FL, 32351, USA</footnote>

<footnote><sup>b</sup> Citrus Center, Texas A&M University Kingsville, Weslaco TX 78599, USA</footnote>

<footnote><sup>c</sup> Department of Horticulture, College of Agriculture, University of Sargodha, Sargodha 40100, Pakistan</footnote>

<footnote><sup>d</sup> UMR 547 PIAF, INRAE, Université Clermont Auvergne, 63000 Clermont-Ferrand, France</footnote>

<footnote><sup>e</sup> United States Horticultural Research Laboratory, United States Department of Agriculture, Agricultural Research Service, 2001 South Rock Road, Ft. Pierce, FL 34945, USA</footnote>

<footnote><sup>f</sup> Horticultural Science Department, Citrus Research and Education Center, University of Florida/IFAS, Lake Alfred, FL, 33850, USA</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail addresses: shahidiqbal@ufl.edu (S. Iqbal), c.auciqueperez@ufl.edu (C.E. Aucique-Perez), sajjad.hussain@tamuk.edu (S. Hussain), guillaume.charrier@inrae.fr (G. Charrier), matthew.mattia@usda.gov (M. Mattia), jchater@ufl.edu (J.M. Chater), mshahid@ufl.edu (M.A. Shahid).</footnote>
restrict plant growth (Bohnert et al., 1995; S. Iqbal et al., 2024; Ahmad et al., 2024). The combined drought and freezing stress is unique and causes more severe damage to the plant than the individual stress (Mittler, 2006; Prasch and Sonnewald, 2013). Many studies have reported plant-adaptive responses to individual stress, combined stress, and primed or parental stress (Basu et al., 2016; Guo et al., 2022; Hayat et al., 2023; Hussain et al., 2018). The combined (drought and freezing) stress can induce significant changes in plant morphology, such as browning, wilting, and senescence of leaves, stem and root growth inhibition and dryness, and severely reducing crop yield (Guo et al., 2022; Handa et al., 2019). However, understanding the plant response to priming stress is necessary to enhance plant adaptation under field conditions (Pandey et al., 2015). Generally, low temperatures (< 10 °C) and water shortage affect photosynthetic activity, nutrient uptake, and internal cell structure, which ultimately slow down the plant metabolic process (Rizhsky et al., 2002; Shi et al., 2018). Therefore, reducing net photosynthetic rate and sugar metabolism lowers CO₂ assimilation and enzymatic activity in vital metabolic processes, eventually affecting the membrane lipids (Chaves et al., 2003). In citrus, especially grapefruit, little is known about the drought-priming effect on freezing stress and whether the plant response to these stresses is unique, which is still unclear.

Drought and freezing stresses are major abiotic factors that can occur simultaneously or sequentially, influencing plant responses synergistically and antagonistically (Mushtaq et al., 2022; Zandalinas and Mittler, 2022). Synergistically, both stresses often induce oxidative and osmotic stress, accumulating compatible solutes like proline and sugars, which help stabilize cellular structures and improve water retention. This shared response can prime plants to better tolerate subsequent stress events, a phenomenon observed in DP plants showing enhanced freezing tolerance through osmoprotectants and antioxidant systems (Mahajan and Tuteja, 2005). However, antagonistic interactions also occur, particularly in hormonal regulation with higher regulation during drought, promoting stomatal closure to reduce water loss, but its role in freezing stress is more complex, and excessive hormone signaling can inhibit cold-induced processes (Verma et al., 2016; Zhao et al., 2021). Additionally, drought reduces water content, making tissues more prone to freezing damage, highlighting a delicate balance between these stress responses (Iqbal et al., 2020; Seleiman et al., 2021).

In plants, oxidative stress is critical to drought and freezing stress-affected plants, producing large amounts of reactive oxygen species (ROS) that impair the plant's internal mechanisms (Naseer et al., 2022). Excessive production and accumulation of ROS, such as superoxide ($O_2^\cdot$) and hydrogen peroxide ($H_2O_2$), impair plant function and cause DNA degradation, protein denaturation, and enzymatic inhibition. Therefore, the upregulation of scavenging activities that inhibit the production of highly reactive molecules is important for plant stress tolerance, and this can only be done by the overexpression of antioxidant enzymes (Halliwell, 2006; Logan, 2005). Several studies have shown that oxidative damage can only be mitigated by activating an antioxidant defense system that enhances plant resistance against stress. Thus, the plant possesses a highly effective and sophisticated antioxidant defense system that helps the plant control the overproduction of ROS (Guo et al., 2021; Hayat et al., 2021; Hussain et al., 2016). In this situation, the plant activates enzymatic and non-enzymatic antioxidant systems to maintain its normal growth and metabolism during stress. Among them, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX) can effectively regulate the metabolic homeostasis in plants and are associated with the tolerance of plants against abiotic stress (Gill and Tuteja, 2010; Hayat et al., 2022; Zhao et al., 2022). SOD is the primary scavenger of $O_2^\cdot$ and acts as a first line of defense against ROS, which converts excessive $O_2^\cdot$ into $H_2O_2$ and $O_2$, through which $H_2O_2$ is further converted into $H_2O$ through POD. Ascorbate peroxidase also plays a vital role in catalyzing $H_2O_2$, which is further converted into ascorbic acid (AsA) (Chaves et al., 2003; Chinnusamy et al., 2007), while the content of malondialdehyde

(MDA) determines the lipid peroxidation (LPO) and strength of the plant to withstand adverse abiotic stress condition (Ensminger et al., 2006; Gimeno et al., 2009).

Together with antioxidants, the accumulation of different osmolytes such as proline, glycine betaine (GB), sugars, and carbohydrates also plays a vital role in stress tolerance by maintaining a solvation layer around macromolecules to keep them functional (Anjum et al., 2017). Osmoregulation/osmotic adjustment helps plants maintain cellular homeostasis, allowing metabolic processes such as photosynthetic activity and optimum moisture status under stressful conditions (Subbarao et al., 2000). Osmotic adjustment is directly related to the accumulation of osmolytes in different concentrations, mainly depending on stress type and severity. Drought and freezing stress have been reported to disrupt osmotic balance and cause dehydration under these conditions (Farooq et al., 2009). Higher accumulation of osmolytes in leaves is generally linked with drought tolerance due to its facilitation in maintaining leaf turgor to acquire maximum CO₂ by improving stomatal conductance and promoting water uptake (Blum, 2005; Chimenti et al., 2006; Kiani et al., 2007), while freezing stress also decreases the protein content in stressed plants, which reduces the synthesis process and conversely increases the level of free amino acids particularly, the accumulation of proline contents (Mercado et al., 1997). Proline mainly accumulates from the young leaves and is considered an essential solute against abiotic stress (Pérez-Pérez et al., 2009); thus, prime stress uniquely affects the osmolyte level in stressed plants.

Priming has been known to potentially enhance plant stress tolerance through prior exposure to any biotic or abiotic stress (Bruce et al., 2007). In wheat, priming of drought stress (Li et al., 2014; Wang et al., 2014) at the early stage of reproductive development helped the plants to tolerate the same or other stress at a later stage, and that is because priming stress enhances stress tolerance through a more effective antioxidant defense system and its photosynthetic electron transport system. In grapes, pre-treatment of drought helps the plants to adapt to freezing stress by improving its osmoregulatory system with a higher concentration of proline and sucrose, which indicates that DP has a positive effect on cold hardiness of grapes with higher freezing tolerance and less damage to plants (Babajamali et al., 2022). In chickpeas, DP elevates membrane damage and chlorophyll degradation with a higher accumulation of cryoprotectants during chilling stress, which helps the plants improve their growth and yield (Saini et al., 2022).

Citrus is one of the most important perennial fruit crops grown widely throughout the tropical and subtropical regions of the globe. Citrus, mainly grapefruit, is more sensitive to drought and freezing stress, the major limiting factors affecting citrus production. Freezing in winter and dehydration in the hot season cause significant losses to citrus production. Therefore, tolerance to drought and freezing stress has been the main objective of citrus breeders in recent years. There is a lack of scientific knowledge regarding the DP effect on the physiological and biochemical attributes of cold-sensitive citrus cultivars, such as grapefruit. Considering the aforementioned scenario, the present study was designed to understand how cold-sensitive grapefruit (Citrus paradisi) responds to DP effects under FS and what morphological, physiological, and biochemical modulation occurs. We anticipate that the findings of this study will enhance our understanding of DP-induced cold hardiness at molecular and transcriptional levels, thereby contributing to ecosystem improvement and promoting sustainable agriculture.

## 2. Material and methods

### 2.1. Plant material and growing conditions

Two-year-old grapefruit (Citrus paradisi) cv. Flame plants grafted onto US-942 rootstock propagated by a certified citrus nursery (Brite Leaf Citrus Nursery, Lake Panasoffkee, Florida, USA) with uniform height and canopy size were used for this study. The experiment was performed at the greenhouse facility of the Horticultural Sciences
Department at North Florida Research and Education Center, University of Florida, Quincy, FL (30°32'44"N 84°35'38"W 75 m). The plants were grown in 11.35 L plastic pots filled with coconut fiber growing media. The plants were grown in a controlled greenhouse, maintaining an average day and night temperature of 25 ± 2 °C, with 80 % relative humidity and a photoperiod of 12 h for one month to ensure proper growth. Plants were allowed to grow undisturbed for two weeks in greenhouse conditions after delivery from the citrus nursery to mitigate any transport shock before exposure to drought-priming and freeze treatments.

## 2.2. Drought-priming, freeze treatments, and experimental design

Drought-stress (DS) as priming was induced based on the field capacity (FC) of the potting media. Plants were divided into three groups by maintaining different FC percentages, i.e., 100 % (non-stressed [NS]-control), 75 % (medium drought-stress), and 50 % (severely drought-stress). The FC was determined using the gravimetric method described by Medeiros (Medeiros et al., 2012), which involves calculating the difference in weight between wet soil after saturation and free drainage, and dry soil. For accurate water measurement, the pots were weighed daily, and any water loss was determined using a count scale (Uline, H-1651, USA) with a capacity of up to 25 kg. The plants were maintained under these conditions for 21 days; afterward, plant tissue samples were collected for fresh and dry biomass and biochemical analysis. Immediately after drought treatments, the plants (non-stressed and stressed) were subjected to freezing temperature (−6 °C) in a programmed controlled environment cold chamber (Conviron, CMP 6050, Canada) with a photoperiod of 12 h and 96 % relative humidity. The target freezing temperature was achieved by gradually decreasing the cold chamber temperature by 2 °C per hour, starting from 4 °C until it reached −6 °C and staying the plants there for one and a half hours. This freezing temperature was selected in this study because it is the lowest freezing temperature in the southeastern USA during winter, particularly in north Florida, causing damage to citrus plants. After the cold treatment, leaf samples were collected for fresh and dry biomass and biochemical and stomatal analysis.

## 2.3. Leaf greenness, biomass content and stomatal morphology

The leaf greenness was determined using a Soil Plant Analysis Development (SPAD) chlorophyll meter (SPAD-502 Plus, Konica Minolta, INC, Japan). The SPAD measurements were taken from 15 fully matured leaves per plant per treatment around all sides of the plant canopy. To measure the fresh and dry biomass contents, leaves and stems were carefully separated from the plants and cleaned with tissue paper to remove dust. The fresh weight of leaves and stems was recorded and then oven-dried at 72 °C for three days (Fisher, Isotemp Oven, Model 230F), and the average dry weight was determined.

To study stomatal morphology, leaves from drought-stress (DS) and drought-primed freezing stress (DP-FS) plants were collected and promptly analyzed. Transparent glue was applied to the leaves' abaxial surface, covering 2–3 cm² to create epidermal impressions, and left for 10 min to dry properly. Clear adhesive tape was used to remove the impressions from the leaf surface and stick them to the microscope slide (Thomas Scientific). All samples were collected interveinal, at the midrib. The stomatal diameter was measured, and the image of the stomatal behavior was acquired using the microscope (Zeiss, LEICA DFC 3000 G), facility available at FAMU viticulture center, Tallahassee, FL (30°28'36"N 84°10'21"W 26 m). Three biological repetitions were employed in this study.

## 2.4. Reactive oxygen species and lipid peroxidation

Superoxide ($O_2^•$) was determined following the method described by Elstner and Heupel (1976). In brief, 0.5 g of the leaf sample was

grounded in liquid nitrogen, incubated into the 5 mL of cold phosphate buffer (50 mM, pH 7.5), centrifuged at 12,000 × g for 15 min at 4 °C, and collected the supernatant for analysis. Then, prepare the reaction setup (1 mL) using 0.1 % NBT in phosphate buffer, add 50–100 µL of the supernatant solution, and incubate at room temperature for 15–30 min in the dark; then, measure the absorbance at 540 nm. The content of hydrogen peroxide ($H_2O_2$) and MDA was determined using reagent kits (Elabscience, Houston, TX, USA). A total of 20 mg of leaf sample was collected and washed the tissue in cold PBS (0.01 M, pH 7.4), and then homogenized in 180 µL PBS (0.01 M, pH 7.4) and then centrifuged at 10,000 × g for 10–15 min to remove insoluble material. Collect supernatant and keep it on ice for detection. Meanwhile, determine the protein concentration of supernatant for $O_2^•$ and $H_2O_2$ according to the reagents protocol. After the operational steps by manufacturer's protocol, measure the OD value for $H_2O_2$ at 405 nm. The contents of $O_2^•$ and $H_2O_2$ were expressed in mmol per gram of protein (nmol g⁻¹ prot). For LPO, the OD value was determined at 532 nm using a spectrophotometer (Spectronic Unicam-Genesys 8, England), and was expressed as nmol g⁻¹ of MDA content.

## 2.5. Antioxidant enzymes and osmolytes

The activity of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and dehydroascorbate reductase (DHAR) were measured using the enzyme extraction kits (Elabscience, Houston, TX, USA) following the manufacturer protocol. In detail, 20 mg of fresh tissue samples for each enzymatic activity was collected and washed with PBS (0.01 M, pH 7.4) at 4 °C, and absorb the extra water with filter paper and weigh. After that, the samples were homogenized in 180 µL PBS (0.01 M, pH 7.4) and kept at 4 °C, then centrifuged at 10,000 × g for 10 min at 4 °C to remove insoluble material. Collect supernatant and keep it on ice for detection. Meanwhile, determine the protein concentration of supernatant (Catalog No 23,227). The absorbance of SOD, POD, CAT, APX, GPX and DHAR was recorded at 550, 420, 405, 290, 412, and 265 nm by using a spectrophotometer (Spectronic Unicam-Genesys 8, England), respectively. The activities of the antioxidant enzymes were quantified as units per milligram of protein (U mg⁻¹ prot) using their standard absorbance curves. One unit was defined as the enzyme amount that 1 mg of substrate catalyzed by the 1 mg of protein extract per minute at 37 °C.

The proline and glycine betaine (GB) content was determined using the assay kit's protocol by Elabscience Biotechnology Inc. A fresh leaf sample of 600 mg was collected and washed in cold PBS (0.01 M, pH 7.4), and placed on ice. Then the samples were homogenized in 6 mL extracting solution provided within it and kept at 4 °C to remain cold. After that, the mixture was centrifuged at 10,000 × g for 15 min to remove insoluble material. Collect the supernatant and keep it on ice for detection. The absorbance of proline and GB was recorded at 520 and 365 nm using a spectrophotometer (Spectronic Unicam-Genesys 8, England). The unit mg g⁻¹ of fresh weight (FW) for proline and GB quantification represents milligrams of proline and GB per gram of FW in plant tissue, respectively.

## 2.6. Enzyme related to carbon metabolism (extraction and activity assay)

The enzymatic activity was measured using the protocol outlined in a previous study by this group (S. Iqbal et al., 2024). To determine the activities of enzymes involved in carbohydrate metabolism, 0.5 g of leaf tissue was taken and homogenized in a solution containing 50 mM Tris-hydrochloric acid, along with magnesium chloride (5 mM), β-mercaptoethanol (5 mM), ethylenediaminetetraacetic acid (1 mM), ethylene glycol-bis-β-aminoethyl ether (1 mM), 2-hydroxyl-1-ethanethiol (0.1 mM), and glycerol (15 %). Following homogenization, the mixture was centrifuged at 17,000 × g for 15 min at 4 °C. The resulting supernatant was then used to determine the enzymatic activities.
For the measurement of total acid invertase (AI), a reaction mixture containing 0.2 mL of the extract, sodium acetate (0.1 M, pH 4.8), and sucrose (0.1 M) was prepared. In the case of neutral invertase (NI), the reaction mixture comprised dipotassium phosphate (0.1 M), sodium citrate (0.1 M), and sucrose (0.1 M). For AI and NI reaction mixtures were supplemented with 0.2 mL of the enzyme extract and incubated at 37 °C in the dark for 40 min. The reaction was stopped by adding dinitro-salicylic acid (1 mL). The activities of AI and NI were determined using a spectrophotometer (Spectronic Unicam- Genesys 8, England) at a wavelength of 540 nm. Sucrose synthase (SuSy) and sucrose phosphate synthase (SPS) enzymatic activity was acquired through methodologies earlier described by Rufty Jr and Huber (1983). The activities of the enzymes were quantified as µmol sucrose min⁻¹ mg⁻¹ of the fresh weight (FW). One unit (U) is defined as the amount of enzyme that hydrolyzes 1 µmol of sucrose per minute per milligram at 37 °C.

Fructokinase (FK) was examined using the method established by Zrenner et al. (1995). The reaction mixture consisted of magnesium chloride (5 mM), Tris-hydrochloric acid (0.1 M, pH 8.5), nicotinamide adenine dinucleotide (0.5 M), fructose (10 mM), glucose-6-phosphate dehydrogenase (2 U), and adenosine triphosphate (2 mM). The activities of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) were determined using enzyme kits (Sigma-Aldrich, USA) following the manufacturer protocols and employing a spectrophotometer at a wavelength of 340 nm. The activities were quantified as µmol min⁻¹ mg⁻¹ of the fresh weight. One unit (U) is defined as enzyme activity as the number of micromoles of a specific substrate that is converted into a product per minute per milligram of fresh plant tissue.

## 2.7. Determination of sugar and starch contents

The sugars and starch contents were determined using a BioTek Synergy Neo2 Hybrid Multimode Reader (Agilent, CA, United States). The determination method for each soluble sugar content was employed using the extraction kit and the method provided by the manufacturer. In detail, 10 mg of the sample was used for each analysis and grounded in liquid nitrogen to make fine powder. Preparing and mixing of the reagents and samples was done according to the manufacturer's protocol and transferred ~200 µl in each well of 96 wells plate. The following kits and wavelengths (nm) were used to determine the absorbance (OD) values, i.e. glucose at 570 nm (Sigma-Aldrich-MAK476), starch at 570 nm (Sigma-Aldrich-MAK368), fructose at 340 nm (Sigma-FA20), and sucrose at 340 nm (Sigma-Aldrich-SCA20). Sugars and starch contents are expressed as mg g⁻¹ of dry weight (DW), indicating the milligrams of sugars or starch present per gram of oven-dried plant tissue, providing a standardized measure independent of water content.

## 2.8. Statistical analysis

The study was conducted using a randomized complete block design (RCBD). Each treatment was replicated four times, with five plants per replicate. The collected data were analyzed using Analysis of variance (ANOVA) and Tukey's HSD (honestly significant difference) test for significant differences, using Statistix 10.0 software (Analytical Software, USA). The graphs were created using SigmaPlot software (v 15.0). Besides, principal component analysis (PCA) and cluster analysis were carried out using R software (v 4.3.3).

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Changes in leaf morphology, biomass contents, leaf greenness, stomatal behavior and size of stomata of the grapefruit plants under drought stress (DS) and drought-prime freezing stress (DP-FS) with different filed capacity (FC) are shown. Leaf morphology of DS and DP-FS leaves (non-stressed [NS] and stressed plants) are shown as (A and B) NS-control; (C and D) 75 % FC; (E and F) 50 % FC plants, respectively. Biomass contents were determined and shown as (G and H) leaf fresh and dry biomass content; (I and J) stem fresh and dry biomass content. Leaf chlorophyll content was determined and shown as (K) leaf greenness value by the SPAD (Soil Plant Analysis Development) meter. The stomatal behavior refers to the opening and closing of stomata, and the stomata size of DS and DP-FS plants and shown as (L) shows the size of the opened stomata of both DS and DP-FS plants under NS and stress conditions. The visualization of opened stomata is shown as (M and N) NS; (O and P) 75 % FC; (Q and R) 50 % FC of DS and DP-FS plants. The 100 % FC plants showed as control (non-stressed [NS] plants), while 75 % and 50 % FC showed the medium and severely stressed plants of DS and DP-FS, respectively. The lower-case letters on graphs represent honestly significant difference (HSD) according to Tukey's test (*p* < 0.05), while vertical bars represent the mean standard error (n = 4); *g* represents a unit in grams; *DS* = drought stress; *DP-FS* = drought-prime freezing stress; *NS* = non-stress.</figcaption></figure>
# 3. Results

## 3.1. Biomass content, leaf and stomatal morphology

Substantial morphological changes occurred in the leaves of non-stressed (NS) and stressed plants under DS and DP-FS conditions (Fig 1A-F). During priming, NS plants showed maximum leaf greenness (75.57 SPAD), while DS plants maintained at 75 % FC showed leaves with some yellowish spots (58.3 SPAD), and plants at 50 % FC showed maximum yellowing and minimum leaf greenness (53.8 SPAD) in their leaves (Fig 1A, C, E and K). On exposure to freezing-stress (FS), the NS plants showed maximum freezing droplets (water-soaked patches) in leaf tissues with leaf greenness of 53.87. In comparison, 75 % and 50 % FC showed fewer freezing droplets with a greenness index of 44.57 and 32.35, respectively (Fig 1B, D, F and K). Overall, drought-primed (DP) plants with 75 % FC showed fewer freezing droplets and maximum freeze tolerance with less progression in leaf yellowing, indicating priming-induced improvement in cold hardiness.

The fresh and dry biomass were measured at two stages: one after DS and one after DP-FS conditions. We observed a decrease in the leaf and stem fresh and dry biomass contents among the DS plants. In terms of leaves, the maximum fresh weight was recorded in NS plants (77.05 g), followed by 75 % (56.42 g) and 50 % FC (40.97 g); the same case was observed in dry biomass with maximum dry contents recorded in NS plants (33.9 g), followed by 75 % (21.9 g) and 50 % FC (15.32 g). After exposure of these DP plants to FS, we found a slight reduction in the leaf fresh and dry biomass contents among treatments (63.3, 46.1, 33.2 g and 29.1, 18.3 g), respectively. A similar observation was recorded in stem fresh and dry biomass content. Thus, these results showed that drought-priming helped plants maintain their growth and biomass under severe freezing stress (Fig 1G, H, I and J).

The stomatal morphology was also altered during DS and DP-FS conditions (Fig 1M-R). The NS plants showed the maximum stomata size (21.06 mm) with maximum opened stomata, followed by 75 % FC with 18.21 mm stomata size. The plants with 50 % FC had minimum open stomata with a size of 17.36 mm (Fig M, O, Q and L). After exposed to freezing conditions, DP plants maintained almost similar stomatal size (Fig 1N, P, R, and L). Stomatal closure at lower FC is a critical adaptive

mechanism to minimize water loss and might affect photosynthesis. Generally, mitigated reductions in biomass and stomatal size under DP-FS highlight the protective effect of drought priming.

## 3.2. Reactive oxygen species (ROS) and lipid peroxidation

Plants showed increased reactive oxygen species ($O_2^\cdot$ and $H_2O_2$) formation in response to DS effect. DS plants with 75 % and 50 % FC showed higher $O_2^\cdot$ and $H_2O_2$ levels than NS plants (Fig 2A and B). However, this trend was reversed when these DP plants were exposed to FS, limiting ROS formation. Under FS, the higher level of $O_2^\cdot$ and $H_2O_2$ (0.13 and 0.67 nmol g$^{-1}$ protein) was found in NS plants, while the minimum level (0.10 and 0.44 nmol g$^{-1}$ protein) was recorded in DP plants at 75 % FC, followed by 50 % FC (0.12 and 0.47 nmol g$^{-1}$ protein) (Fig 2A and B). So, DP-FS showed a significant reduction in $O_2^\cdot$ and $H_2O_2$ production compared to DS at 75 % and 50 % FC, indicating better control of oxidative stress due to priming and an enhanced antioxidant response.

The concentration of LPO was increased in response to drought-priming. Under DS, the highest LPO rate of 1.48 nmol g$^{-1}$ of MDA content was recorded in plants maintained at 50 % FC, followed by 75 % FC plants (1.03 nmol g$^{-1}$ of MDA content), while the minimum concentration was recorded in the NS plants. However, this trend was reversed when these DP plants were subjected to freezing temperature, with a maximum rate of LPO recorded in (1.79 nmol g$^{-1}$ of MDA content) rate of LPO recorded in NS, followed by 75 % (0.92 nmol g$^{-1}$ of MDA content) and 50 % (0.87 nmol g$^{-1}$ of MDA content) (Fig 2C). LPO increased progressively with decreasing FC, indicating membrane damage under DS, while DP-FS plants showed a decrease in LPO across all FC levels, especially under severe stress, implying better membrane protection.

## 3.3. Antioxidant enzyme activity

In this study, the activities of antioxidant enzymes such as SOD, POD, CAT, APX, GPX, and DHAR increased as FC level decreased with highest activity at 50 % FC. The lowest activities of antioxidant enzymes were recorded in NS plants, i.e., SOD, POD, CAT, APX, GPX and DHAR (1.21, 0.11, 0.10, 1.42, 0.41 and 1.16 U mg$^{-1}$ protein), respectively (Fig 3A-F).

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Effect of drought stress (DS) and drought-primed freezing stress on the reactive oxygen species (ROS) enzymatic activity of non-stressed (NS) and stressed plants. (A) superoxide anion ($O_2^\cdot$), (B) hydrogen peroxide ($H_2O_2$), and (C) lipid peroxidation (LPO) activity of the plants maintained at 100 %, 75 %, and 50 % field capacity. The 100 % FC plants showed as control (non-stressed [NS] plants) while 75 % and 50 % FC showed the medium and severe stressed plants of DS and DP-FS, respectively. The lower-case letters on graphs represent honestly significant difference (HSD) according to Tukey's test ($p < 0.05$), while vertical bars represent the mean standard error ($n = 4$); DS=drought stress; DP-FS=drought-primed freezing stress; NS=non-stress.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Effect of drought stress (DS) and drought-primed freezing stress (DP-FS) on the antioxidant enzymatic activity of the non-stressed (NS) and stressed plants. The antioxidant enzymatic activities are shown as (A) superoxide dismutase (SOD); (B) peroxidase (POD); (C) catalase (CAT); (D) ascorbate peroxidase (APX); (E) glutathione peroxidase (GPX); and (F) dehydroascorbate reductase (DHAR) activity of the plants under NS, and 75 % and 50 % FC conditions. The 100 % FC plants showed as control (non-stressed [NS] plants), while 75 % and 50 % FC showed the medium and severely stressed plants of DS and DP-FS, respectively. The lower-case letters on graphs represent honestly significant difference (HSD) according to Tukey's test ($p < 0.05$), while vertical bars represent the mean standard error ($n = 4$); DS= drought stress; DP-FS= drought-primed freezing stress; NS= non-stress.</figcaption></figure>

The exposure of these non-stressed (NS) and drought-stressed (DS) plants to freezing temperatures caused further improvement in the enzymatic activities of antioxidants. DP plants with 75 % FC showed the highest SOD, POD, CAT and APX (2.15, 0.22 and 0.17 U mg⁻¹ protein, respectively), followed by those DP at 50 % FC (1.64, 0.18 and 0.15 U mg⁻¹ protein). The NS plants showed the lowest antioxidant activities compared to DS plants in response to freezing stress (Fig 3A-D). In the case of GPX and DHAR, the DP plants maintained higher antioxidant activities than NS plants. Likewise, the lower level of GPX and DHAR was recorded in NS plants (0.51 and 1.35 U mg⁻¹ protein), while the maximum level was recorded in DS plants maintained at 50 % FC (0.98 and 2.03 U mg⁻¹ protein), followed by 75 % FC (0.82 and 1.79 U mg⁻¹ protein) under FS (Fig 3E-F). DP-FS consistently enhances antioxidant enzyme activities compared to DS plants, indicating that drought priming prepares the plants for more efficient ROS detoxification, protecting cellular structures and mitigating oxidative damage during freezing stress.

### 3.4. Osmolyte accumulation

The proline and glycine betaine (GB) contents were increased with the decrease in FC during DP, indicating an adaptive stress response. The lowest contents of proline and GB were recorded in NS plants (0.17 and 0.25 mg g⁻¹ FW), followed by 75 % FC (0.32 and 0.42 mg g⁻¹ FW), and reached a maximum level at 50 % FC (0.52 and 0.59 mg g⁻¹ FW). We noticed a significant difference in osmolyte accumulation among different FC levels when these plants were exposed to freezing stress. The highest content of proline and GB was found in plants having DP at 75 % FC (0.91 and 0.98 mg g⁻¹ FW), followed by those having DP at 50 % FC (0.66 and 0.70 mg g⁻¹ FW), while the lowest level (0.40 and 0.35 mg g⁻¹ FW) of proline and GB was found in NS plants (Fig 4A-B). Thus, the stronger metabolic response of proline and GB in DP-FS plants suggests their prominent role in stabilizing cellular membranes to prevent damage.
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Effect of drought stress (DS) and drought-primed freezing stress (DP-FS) on the cryoprotectant activity of the non-stressed (NS) and stressed plants. (A) proline and (B) glycine betaine (GB) of the plants under NS, and 75 %, and 50 % FC conditions. The 100 % FC plants showed as control (non-stressed [NS] plants), while 75 % and 50 % FC showed the medium and severely stressed plants of DS and DP-FS, respectively. The lower-case letters on graphs represent honestly significant difference (HSD) according to Tukey's test (*p* < 0.05), while vertical bars represent the mean standard error (n = 4); FW = fresh weight; DS = drought stress; DP-FS = drought-primed freezing stress; NS = non-stress.</figcaption></figure>

### 3.5. Carbohydrate metabolism

#### 3.5.1. Enzymes of glycolysis metabolism

The enzymatic activities of fructokinase (FK), hexokinase (HK), phospho-fructokinase (PFK), and pyruvate kinase (PK) were decreased in DS plants (75 % and 50 % FC) as compared to NS (100 % FC) plants (Fig 5). The maximum activity of these enzymes was noted in NS plants, i.e., 12.07, 1.87, 5.53 and 9.92 µmol min⁻¹ mg⁻¹ FW, followed by the DP plants maintained at 75 % FC, i.e., 8.73, 1.65, 3.47 and 7.22 µmol min⁻¹ mg⁻¹ FW. In contrast, the lowest (8.04, 1.39, 2.05, and 6.04 µmol min⁻¹ mg⁻¹ FW, respectively) activity was recorded in plants maintained at 50 % FC.

On exposure to freezing stress, the DP plants showed increases in FK, HK, PFK and PK activities compared to NS plants. Higher FK and HK activities were recorded in DP plants maintained at 75 % FC (10.46 and

1.77 µmol min⁻¹ mg⁻¹ FW), followed by 50 % FC (10.17 and 1.65 µmol min⁻¹ mg⁻¹ FW). The NS plants showed minimum FK and HK activity (9.26 and 1.36 µmol min⁻¹ mg⁻¹ FW) compared to DP at 50 % and 75 % FC levels (Fig 5A-B). Likewise, PFK and PK activities were lowest in NS plants (2.19 and 5.40 µmol min⁻¹ mg⁻¹ FW), followed by DP plants maintained at 75 % FC (3.68 and 8.08 µmol min⁻¹ mg⁻¹ FW) and 50 % FC (4.81 and 8.55 µmol min⁻¹ mg⁻¹ FW) (Fig 5C-D). So, DP plants showed higher FK, HK, PFK, and PK activities due to their exposure to FS.

#### 3.5.2. Enzymes of sucrose metabolism

Drought-stress caused a reduction in acid invertase (AI) activities. NS plants exhibited higher AI activities of 0.87 µmol sucrose min⁻¹ mg⁻¹ FW, followed by DP plants maintained at 75 % (0.72 µmol sucrose min⁻¹ mg⁻¹ FW) and 50 % FC (0.53 µmol sucrose min⁻¹ mg⁻¹ FW). However, this trend was reversed when these DP plants were exposed to freezing

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Effect of drought stress (DS) and drought-primed freezing stress (DP-FS) on the carbohydrate metabolic activity of the non-stressed (NS) and stressed plants. The enzymes of glycolysis metabolism are shown as (A) fructokinase (FK); (B) hexokinase (HK); (C) phosphofructokinase (PFK); and (D) pyruvate kinase (PK) of the plants under NS and 75 %, and 50 % FC conditions. The 100 % FC plants showed as control (non-stressed [NS] plants), while 75 % and 50 % FC showed the medium and severely stressed plants of DS and DP-FS, respectively. The lower-case letters on graphs represent honestly significant difference (HSD) according to Tukey's test (*p* < 0.05), while vertical bars represent the mean standard error (n = 4); FW = fresh weight; DS = drought stress; DP-FS = drought-primed freezing stress; NS = non-stress.</figcaption></figure>
temperatures, as plants maintained at 50 % and 75 % FC showed higher AI activities than NS plants. Plants with medium DS (75 % FC) maintained the maximum AI activities under FS (Fig 6A). The enzymatic activity of neutral invertase (NI) was increased with the decrease in FC with maximum (0.22 µmol sucrose min⁻¹ mg⁻¹ FW) NI activities in plants at 50 % FC. NS plants had minimum NI activity (0.05 µmol sucrose min⁻¹ mg⁻¹ FW) compared to DP-FS plants. Likewise, DP plants also showed higher NI activity under FS than NS plants. On exposure to FS, plants maintained at 50 % FC had maximum (0.25 µmol sucrose min⁻¹ mg⁻¹ FW) NI activity, followed by 75 % FC (0.23 µmol sucrose min⁻¹ mg⁻¹ FW) (Fig 6B).

DS caused an increase in the activity of sucrose synthase (SuSy) and sucrose phosphate synthase (SPS). Under DS, plants at 50 % FC showed the maximum activity of SS and SPS (0.29 and 0.21 µmol sucrose min⁻¹ mg⁻¹ FW, respectively), followed by 75 % FC (0.25 and 0.15 µmol sucrose min⁻¹ mg⁻¹ FW). While under FS, these DP plants at 50 % FC further increased the activity of SuSy and SPS with maximum activity (0.43 and 0.31 µmol sucrose min⁻¹ mg⁻¹ FW), followed by 75 % FC (0.38 and 0.21 µmol sucrose min⁻¹ mg⁻¹ FW) (Fig 6C-D). So, DS prioritizes the breakdown of sucrose via AI to sustain osmotic balance, while FS prioritizes sucrose biosynthesis via SPS and stabilizes SS and NI to protect against freezing damage.

### 3.5.3. Soluble sugars and starch contents

Drought-stressed plants exhibited an increase in sugars while exhibiting a decrease in starch contents. The lowest concentration of glucose, fructose, sucrose, and total soluble sugars (TSS) was recorded in NS plants (6.51, 4.37, 8.60, and 20.97 mg g⁻¹ DW, respectively), followed by DS plants maintained at 75 % FC (9.66, 6.12, 14.05, 29.66 mg g⁻¹ DW), and was highest in plants at 50 % FC (11.38, 7.58, 19.26, and 50.17 mg g⁻¹ DW). Under FS, these DP plants further improved glucose, fructose, sucrose, and total soluble sugar concentration. The NS plants showed lower levels (12.58, 6.60, 19.85, and 53.90 mg g⁻¹ DW) for all soluble sugars (glucose, fructose, sucrose, and TSS) compared to DP plants maintained at 75 % and 50 % FC. It was observed that higher glucose and fructose (22.04 and 8.98 mg g⁻¹ DW) were recorded in

plants having DP at 75 % FC, while sucrose and TSS (24.25 and 138.05 mg g⁻¹ DW) were in plants at 50 % FC (Fig 7A-C, and E).

DS caused a reduction in starch contents with the maximum in NS plants, i.e., 106.91 mg g⁻¹ DW, followed by those subjected to DS at 75 % FC (77.84 mg g⁻¹ DW), and 50 % FC levels (63.79 mg g⁻¹ DW). However, when these DP plants were exposed to FS, the effect was reversed. The DP plants with 75 % FC showed maximum starch contents (93.12 mg g⁻¹ DW), followed by 50 % FC (83.81 mg g⁻¹ DW), while the lowest starch contents were recorded in NS plants (60.94 mg g⁻¹ DW) (Fig 7D). So, DP enhances the sugar levels, indicating that prior DS improved its internal tolerance mechanism against severe stress. In the case of starch, the DP enables the plants to balance starch degradation and soluble sugar accumulation more effectively.

### 3.6. Multivariate analysis of physiological and biochemical parameters

Principal component analysis (PCA) was conducted to identify uncorrelated variables and detect factor patterns in datasets with high dimensions. The PCA represented a clear separation between the two groups, i.e., DS and DP-FS. In this study, PC1 (58.5 %) explains most of the variation, while PC2 (25.5 %) contributes to the additional variability. DS and DP-FS variables are strongly differentiated along PC1, with DP-FS variables showing greater variability (Fig 8A). The heatmap shows the hierarchal clustering of traits and treatments across DS and DP-FS conditions (Fig 8B). The cluster was divided into two groups, i.e., DS and DP-FS, and each group contained all three treatments, i.e., non-stressed (100 % FC) and stressed (75 and 50 % FC). The DP-FS consistently exhibits a distinct pattern of up-regulation for stress-adoptive traits. Traits related to osmolytes and metabolic energy pathways are clustered closely, suggesting coordinated metabolic regulation under stress. In contrast, the ROS traits cluster is distinct from others, reflecting their association with damage under DS. The cluster analysis grouped all the parameters with higher (pink color) and lower (green color) expression patterns.

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Effect of drought stress (DS) and drought-primed freezing stress (DP-FS) on the carbohydrate metabolic activity of the non-stressed (NS) and stressed plants. The enzymes of sucrose metabolism are shown as (A) acid invertase (AI); (B) neutral invertase (NI); (C) sucrose synthase (SuSy); and (D) sucrose phosphate synthase (SPS) of the plants under NS and 75 %, and 50 % FC conditions. The 100 % FC plants showed as control (non-stressed [NS] plants), while 75 % and 50 % FC showed the medium and severely stressed plants of DS and DP-FS, respectively. The lower-case letters on graphs represent honestly significant difference (HSD) according to Tukey's test (*p* < 0.05), while vertical bars represent the mean standard error (n = 4); FW= fresh weight; DS= drought stress; DP-FS= drought-primed freezing stress; NS= non-stress.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Effect of drought stress (DS) and drought-primed freezing stress (DP-FS) on the soluble sugar content (SSC) of the non-stressed (NS) and stressed plants. The soluble sugar contents are shown as (A) glucose; (B) fructose; (C) sucrose; (D) starch; and (E) total soluble sugars (TSS) of the plants under NS and 75 %, and 50 % FC conditions. The 100 % FC plants showed as control (non-stressed [NS] plants) while 75 % and 50 % FC showed the medium and severely stressed plants of DS and DP-FS, respectively. The lower-case letters on graphs represent honestly significant difference (HSD) according to Tukey's test (*p* < 0.05), while vertical bars represent the mean standard error (n = 4); DW= dry weight; DS= drought stress; DP-FS= drought-primed freezing stress; NS= non-stress.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Observation plots of (A) Principal component analysis (PCA) and (B) clustering of the variables showing grouping, correlation, and average expression between the variables and traits of the study under DS and DP-FS. The principal component (PC) analysis was performed among biomass contents, concentration of enzymatic activities, and concentration of carbohydrates. The percentage of total variance represented by PC1 and PC2 is shown in parentheses. In the cluster graph, the grouping is shown in the color strips on the top left side of the graph, and the expression bar is shown on the top of the graph, as pink shows a higher expression while green shows a lower expression. DS= drought stress; DP-FS= drought-primed freezing stress.</figcaption></figure>

**4. Discussion**

Unexpected freeze events during winter and late summer are the

most limiting factor for citrus production in the southeastern United
States. The changes in plant external morphology are the first sign of any
plant stress that directly reflects the effect of stress and can be used as an
index of plant stress resistance (Azhar et al., 2011; Xu et al., 2015). Stress-priming is an integrated approach that helps plants withstand freezing temperatures and enhances their adaptability to fluctuating environmental conditions, contributing to more robust and resilient agricultural systems.

In the present study, substantial changes were observed in plants exposed to drought DS, followed by FS, showing that short-term DS, as priming events, triggers various physiological, biochemical and molecular changes in plants, thereby acclimating them to upcoming severe stress conditions. Leaf stomata are considered a pivotal entry point controlling CO₂ influx and water status, influenced by environmental factors such as light, temperature and water status (Xu and Zhou, 2008). So, depending upon the various intensifications, the limitation in photosynthesis may impact stomata. Our results showed that DP-FS increased the stomata size, positively affecting the plants in regulating photosynthesis to withstand freezing stress. In addition, DP-FS helps the plants maintain their biomass accumulation compared to NS plants, indicating that DP helps the plants enhance their tolerance under FS, consistent with previous studies (Li et al., 2014). It was observed that plants experiencing short-term exposure to one type of stress factor hardened them through various physiological and biochemical changes to a different kind of extreme stress. These changes include the formation of various compatible solutes, accelerating antioxidant activity, and regulating the glycolysis pathway, helping plants retain stress memory for upcoming severe stress events (Tabassum et al., 2018). Therefore, this study examined a unique approach to how DP enhances cross-stress tolerance to freezing conditions in young cold-sensitive grapefruit plants.

Carbon balance is critical in managing plant energy resources and their survival (Smith and Stitt, 2007). DP can lead to adaptive changes in carbon allocation and increase the accumulation of osmoprotectants during FS, providing membrane structure stability and improving metabolic efficiency and cellular respiration under stress conditions (Liu et al., 2022). The over-production of ROS in plants due to biotic or abiotic stress factors severely disrupts the balance between cell organelles, resulting in oxidative damage inhibiting plant growth and development (Gill and Tuteja, 2010; Sharma et al., 2012). However, the plants have developed a complex antioxidant defense system that helps them overcome excessive ROS production (Hussain et al., 2016), and maintain the balance between ROS generation and their detoxification through enhanced antioxidants under stress conditions (Ruelland et al., 2009). In this study, DP plants had reduced ROS formation because DP strengthened the plant antioxidant defense system by triggering the activities of various antioxidant enzymes for effective and efficient ROS scavenging, consistent with the findings of the study reported in olive (Abdallah et al., 2017). The overproduction of ROS in stressed plants leads to an increase in the lipid peroxidation rate indicated by higher MDA content, which is the secondary product of the oxidation of polyunsaturated fatty acids by ROS (Lee et al., 2004). Reports showed that plant oxidative response to multiple interacting stress differs from a single stressor (Des Marais et al., 2017). MDA is an indicator of LPO linked to oxidative stress and redox signaling. The increase in MDA may increase the susceptibility of oxidative damage to the plant cell membrane system, particularly under conditions where stress has a detrimental impact on plants (Davey et al., 2005). DP alleviated the freezing-induced oxidative damage by limiting ROS formation and reducing the LPO rate (Martinez-Medina et al., 2016). In this case, short-term exposure of plants to one stress factor induces resistance to the same or different stress factor, termed the priming effect. In this study, DS significantly increases the production of ROS and LPO, increasing from non-stressed to stressed plants, while DP has a significantly decreasing effect on ROS production under severe freezing stress, indicating that DP helps the plants in alleviating the oxidative damage through accelerated antioxidant system in grapefruit plants, thus making them more resilient to freezing temperatures.

Abiotic stress conditions cause oxidative damage to the cells due to

the higher formation of ROS; plants cope with this situation by activating their antioxidant defense system (Chongling et al., 1997). In this study, DP accelerated the antioxidant activities of SOD, POD, CAT, APX, GPX, and DHAR as compared to NS plants, which were further improved when plants were exposed to FS, confirming the findings of previous studies reported in eggplant (Hannachi et al., 2022) and Coffea spp. (Ramalho et al., 2018). Thus, drought-stress memory triggered antioxidant enzyme activities, improving the ROS-eliminating effect under freezing temperature, resulting in limited oxidative damage under FS. The ROS level has a dual role within plant tissues; on the one hand, they cause oxidative damage to the cells, while on the other, they serve as signaling molecules, like H₂O₂. The increased ROS contents in DP grapefruit plants are because the priming effect activated the formation of ROS, which act as a caution for the antioxidant defense system, due to their nature as a signaling molecule for better effective and efficient response to further stress, i.e., freezing stress in this study. Therefore, the DP plants had lower concentrations of ROS than the NS plants, resulting in lower lipid peroxidation (oxidative damage) under the FS, which clearly explains that pre-freezing exposure to drought conferred freezing tolerance by alleviating freeze-induced deleterious effects on membrane stability and integrity in leaf tissues of grapefruit plants. This improved cold hardiness in response to DP is associated with low formation of ROS, explaining that the priming effect would have prepared the plants to better scavenge the ROS due to enhanced antioxidant activities in the leaves.

The reduction in intracellular water due to osmotic stress also limits plant growth under stress conditions (Salehi-Lisar and Bakhshayeshan-Agdam, 2016). The accumulation of osmolytes helps the plants to alleviate the osmotic stress for their survival under stressful conditions. Osmoprotectants play a crucial role in facilitating the absorption and retention of cellular water, restoring it to normal osm regulation, and maintaining protein structure, thus preserving normal cellular function (Wood et al., 2001). The synthesis of osmolytes is closely linked to starch hydrolysis in plants as a part of their response to environmental stress. When the plant encounters any stress conditions, it activates enzymes such as amylase to break down starch to produce glucose and other sugars, which act as a precursor for osmolytes that accumulate in the cytosol to protect the cellular structure and maintain osmotic balance (Liang et al., 2013; Yancey, 2005). Compatible solutes such as proline and GB also mitigate ROS-induced oxidative damage by enhancing their scavenging capacity directly or indirectly through enhancing antioxidant activities. Therefore, the accumulation and synthesis of osmolytes are considered a defense mechanism against oxidative damage and osmotic stress caused by biotic or abiotic stressors in plants (Ghosh et al., 2021). In this study, DP plants had a higher accumulation of proline and GB than NS plants under freezing conditions, indicating that DP-FS positively affects osmotic stress tolerance and regulates cellular metabolism. Our findings are consistent with the study reported in oil palm, indicating an increase in proline concentrations under drought and low-temperature stresses (Cao et al., 2011). Osmoprotectants are crucial in regulating cell turgor through osmotic adjustment and supporting redox metabolism to reduce ROS (Krasensky and Jonak, 2012). DP plants could have maintained better cellular osmotic balance and tolerance to oxidative damage due to the higher accumulation of proline and GB under FS.

Glycolysis is the primary metabolic pathway that involves the breakdown of glucose to produce energy. It provides energy to different plant parts by involving different enzymes like fructokinase (FK), hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK). In the present investigation, DS resulted in decreased activities of FK, HK, PFK, and PK enzymes, but DP plants showed an increase in the activities of these enzymes on exposure to FS. It can be explained that DP may have activated the plants by adjusting their metabolic and regulatory networks, enabling an immediate response to subsequent FS. Earlier studies have reported the important role of glycolysis enzymes in sugar metabolism, which helps the plant to regulate its normal growth (Dhal
and Pal, 2022; Vega et al., 2016). Soluble carbohydrates are essential in plant metabolism, osmoregulation, signaling, and can be identified under DS or FS conditions (Welling and Palva, 2006). Carbohydrates also play an important role in cellular ROS elimination mechanisms, particularly in scavenging hydroxyl radicals (Van den Ende and Valluru, 2009). In plants, sugars act as a protective signaling molecule under biotic or abiotic stress, while sucrose maintains a balance between sucrose production and its translocation and is the main component in carbohydrate metabolism (Choudhury et al., 2010). Soluble sugars such as glucose, fructose and sucrose are the immediate energy source for plant metabolism, and their reduction leads to the imbalance of plant growth (Smeekens and Hellmann, 2014). The sugar formation and degradation process are important in cryoprotection and alleviating osmotic damage. The present study recorded a higher accumulation of glucose, fructose, sucrose, and total soluble solids in DP plants, while starch content was reduced in response to DS. However, glucose, fructose and sucrose were improved when DP plants were exposed to FS. This indicates that drought-priming activated the carbohydrate metabolism, resulting in further optimum accumulation of sugars in the leaves of DP plants when exposed to freezing stress. The modulation in carbohydrate metabolism in DP grapefruit plants under FS is associated with increased activities of enzymes of carbohydrate metabolism, conferring improved cold hardiness due to direct association with the concentration of sugars (Larsson, 1994).

Acid invertase (AI) and neutral invertase (NI) are important enzymes of sugar metabolism that catalyze the hydrolysis of sucrose into glucose and fructose (Roitsch et al., 2000; Sturm, 1999). Under stressed conditions, plants often enhance sucrose accumulation as an adaptive strategy to maintain energy reserve and osmotic balance through its hydrolysis into glucose and fructose by AI and NI. In the present study, DP plants maintained higher AI and NI activities under freezing stress than NS plants, indicating that priming helped plants tolerate freezing temperatures by facilitating sucrose hydrolysis, enhancing energy availability, and helping in osmotic adjustment. These sugars serve as osmoprotectants and help safeguard cellular membranes by interacting with the lipid bilayer, aiding in adaptation to DS or FS (Strauss and Hauser, 1986). Our results align with the previous studies reported by Chen and Michalak (Chen et al., 2022; Michalak, 2006) that an increase in the metabolic activity of these enzymes reduces oxidative damage and enhances stress resistance. Also, the activity of sucrose synthase (SuSy) and sucrose phosphate synthase (SPS) increased in DP plants under freezing stress. Therefore, the higher sucrose accumulation is directly linked to higher SPS activity, while higher glucose and sucrose activity is directly associated with higher SuSy activity. Our findings support prior studies

describing that increasing metabolic activities (sugars and carbohydrates) could help the plant enhance stress resilience under adverse environmental conditions (Gupta and Kaur, 2005). The potential agricultural application of drought-priming technique, particularly in citrus, offers promising avenues for enhancing crop resilience and ensuring sustainable production. DP could help citrus growers in regions prone to unexpected freezing events (e.g. southern United States) by pre-conditioning trees to tolerate colder temperatures, which could reduce crop loss and improve yield. Pairing this DP technique with a soil moisture sensor or other irrigation technology can optimize its practical application. So, the implementation of the DP technique has the potential to revolutionize citrus farming by providing a practical tool for enhancing resilience in the face of climate variability. Moreover, the response mechanism of antioxidant enzymes, osmolytes and carbohydrate metabolism in DP grapefruit plants under FS is illustrated in Fig 9.

## 5. Conclusion

Metabolic flexibility in plants allows acclimation to different conditions. So, a priming event induces metabolic flexibility, and its result can be associated with activating tolerance mechanisms. In grapefruit plants, drought-priming improved cold hardiness by stabilizing cellular structures and safeguarding against oxidative damage by accelerating the ROS scavenging processes. DP triggered the accumulation and biosynthesis of compatible solutes, i.e., proline, GB and sugars in leaves, helping plants maintain cellular turgor and function under freezing temperatures. DP grapefruit plants have a strong antioxidant defense system, neutralizing freeze-induced ROS formation. Short-term exposure to drought stress prepared the grapefruit plants with improved oxidative defense, thus making them more freeze-resilient. When exposed to freezing conditions, the priming effectively triggered the adaptive metabolic responses that optimized carbohydrate allocation and utilization. Thus, it ensured the optimum energy reserves for crucial physiological and biochemical functions in DP plants, making them more cold hardy. Priming-induced physiological and biochemical adaptations helped grapefruit plants improve freeze tolerance and recover from freezing stress. It is suggested that DP improves freeze tolerance in grapefruit plants by initiating adaptive stress responses, making them resilient to freezing temperatures. This pre-conditioning of plants through priming triggered physiological and molecular modulations that better prepared the plants for subsequent FS. Finally, understanding DP can lead to developing more cold-resilient citrus cultivars, improving agricultural sustainability and food security in the face of unexpected freeze events. There is a need for multi-omics to study proteomics,

<figure><img src="image_12.png" /><figcaption>**Fig. 9.** Response mechanism of antioxidant enzymes, osmolytes, and carbohydrate metabolism to drought-primed freezing stress (DP-FS) in grapefruit plants.</figcaption></figure>
metabolomics and transcriptomics to understand better the mechanism and molecular basis of priming-induced cold hardiness in grapefruit plants.

## Abbreviations

AI, Acid Invertase; APX, Ascorbate Peroxidase; ASA, Ascorbic Acid; CAT, Catalase; DS, Drought Stress; DP, Drought Priming; P-FS, Drought-priming Freezing Stress; FC, Field Capacity; FK, Fructokinase; GPX, Glutathione Peroxidase; GB, Glycine Betaine; HK, Hexokinase; LPO, Lipid Peroxidation; NI, Neutral Invertase; NS, Non-Stressed; POD, Peroxidase; PFK, Phosphofructokinase; PCA, Principal Component Analysis; PK, Pyruvate Kinase; RCBD, Randomized Complete Block Design; ROS, Reactive Oxygen Species; SPS, Sucrose Phosphate Synthase; SuSy, Sucrose Synthase; SOD, Superoxide Dismutase; TSS, Total Soluble Sugars

## Funding

We are grateful to Hatch project FLA-NFC-006,212 for supporting this study.

## CRediT authorship contribution statement

**Shahid Iqbal:** Writing – original draft, Visualization, Methodology, Investigation, Data curation. **Carlos Eduardo Aucique-Perez:** Formal analysis. **Sajjad Hussain:** Formal analysis. **Rashad M. Balal:** Writing – review & editing, Resources. **Guillaume Charrier:** Writing – review & editing. **Matthew Mattia:** Writing – review & editing, Resources. **John M. Chater:** Writing – review & editing. **Muhammad A. Shahid:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgment

The author acknowledges all participants of this study for their outstanding contributions.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.stress.2025.100801.

## Data availability

All data used in this article is presented

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