# Polyploidy modulates the adaptation to water deficit in citrus scion/rootstock associations evaluated under controlled pot condition and relates to specific changes in root and leaf transcriptome

Lény Calvez, Alexis Dereeper, Bénédicte Favreau, Gary Sivager, Pierre Mournet, Saturnin Bruyère, Marie Bonnin, Hélène Vignes, Guillaume Marti, Gerardo Cebrián-Torrejón, et al.

► To cite this version:

Lény Calvez, Alexis Dereeper, Bénédicte Favreau, Gary Sivager, Pierre Mournet, et al. Polyploidy modulates the adaptation to water deficit in citrus scion/rootstock associations evaluated under controlled pot condition and relates to specific changes in root and leaf transcriptome. Plant stress, 2026, 19, pp.101166. 10.1016/j.stress.2025.101166. hal-05449667

HAL Id: hal-05449667

https://hal.science/hal-05449667v1

Submitted on 9 Jan 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# Polyploidy modulates the adaptation to water deficit in citrus scion/rootstock associations evaluated under controlled pot condition and relates to specific changes in root and leaf transcriptome

Leny Calvez<sup>a,f</sup>, Alexis Dereeper<sup>a,b</sup>, Benedicte Favreau<sup>a</sup>, Gary Sivager<sup>a</sup>, Pierre Mournet<sup>b</sup>, Saturnin Bruyere<sup>a</sup>, Marie Bonnin<sup>a</sup>, Hélène Vignes<sup>a</sup>, Guillaume Marti<sup>c</sup>, Gerardo Cebrian-Torrejon<sup>d</sup>, Antonio Doménech-Carbó<sup>e</sup>, Barbara Hufnagel<sup>a</sup>, Patrick Ollitrault<sup>a</sup>, Raphaël Morillon<sup>a,*</sup>

<sup>a</sup> Unité Mixte de Recherche Amélioration Génétique et Adaptation des Plantes méditerranéennes et tropicales (UMR AGAP) Institut, Université de Montpellier, Centre de coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Institut Agro, Montpellier, France

<sup>b</sup> Unité Mixte de Recherche Plant Health Institute of Montpellier (UMR PHIM), Institut de Recherche pour le Développement (IRD), F-34394 Montpellier, France

<sup>c</sup> Metatoul-AgromiX Platform, Laboratoire de Recherche en Sciences Végétales (LRSV), Université de Toulouse, CNRS, INP, Toulouse, France

<sup>d</sup> COVACHIM-M2E Laboratory EA 3592, UFR des Sciences Exactes et Naturelles, Université des Antilles, BP 592 - 97159 Pointe-à-Pitre, Guadeloupe, France

<sup>e</sup> Departament de Química Ananítica, Facultat de Química, Universitat de València, Spain

<sup>f</sup> Fundo de Defesa da Citicultura (Fundecitrus), Araraquara, SP, Brazil

## ARTICLE INFO

**Keywords:**

Citrus

Polyploidy

Scion/rootstock interaction

Tetraploid

Transcriptomic

Triploid

Water deficit

## ABSTRACT

Citrus, one of the world's most important crops, is facing significant challenges due to drought events. Previous studies have demonstrated that tetraploid rootstocks may exhibit greater tolerance to abiotic stresses than their diploid counterparts. The effects of combining a tetraploid rootstock with a triploid scion under water deficit conditions have not been thoroughly explored. A water deficit experiment was conducted under controlled pot conditions using four citrus scion/rootstock combinations: diploid and tetraploid Swingle citrumelo rootstocks grafted with diploid Mexican lime and triploid Persian lime. Physiological, biochemical, and transcriptomic analyses under controlled pot condition revealed that tetraploid rootstocks exhibited significantly improved performance under drought stress, with an even greater effect when the scion was the triploid Persian lime. In that condition, the improved resilience was associated with reduced water consumption, higher photosynthesis, increased stomatal conductance and transpiration under water stress conditions. Elevated abscisic acid levels and stronger antioxidant activity in polyploid rootstocks further contributed to the stress response. Transcriptomic data revealed distinct gene expression changes in roots and leaves, influenced by organ ploidy and rootstock-scion interactions. Taken together our results provide insights into drought adaptation mechanisms including osmotic adjustment, oxidative stress protection, sustained photosynthesis, antioxidant enzyme activity and enhanced synthesis of protective barriers. These findings underscore ploidy's role at both rootstock and scion levels in shaping the plant's response to water deficit, revealing useful interactions between rootstock and scion influencing drought resilience.

## 1. Introduction

Drought is considered the most widespread and devastating abiotic stress, especially in the context of climate change and rising food demand (Bray, 2007; Gupta et al., 2020). The risk of droughts on cropland is predicted to rise globally, with the most significant increases expected

in North America and Europe (Christian et al., 2023). In plants, water deficit primarily affects various key physiological processes at both cellular and molecular levels, this includes a cascade of events that compromise plant viability. Water deficit reduces transpiration activity, which in turn disrupts water flow and consequently nutrient uptake and transport from the roots to the shoot, compromising overall metabolism

<footnote>* Corresponding author.
E-mail address: raphael.morillon@cirad.fr (R. Morillon).</footnote>
including photosynthesis and cell expansion. Water deficit initially restricts growth and ultimately accelerates leaf senescence while prolonged drought can lead to severe tissue damage or even plant death (Bray, 1997). These effects are particularly critical in woody perennials crop where long-term water balance is essential for productivity and fruit quality.

To mitigate drought damage, plants have developed a wide range of adaptive strategies, including stomatal regulation, osmotic adjustment, hormonal signaling, antioxidant defense, epigenetic and gene expression regulation (Basu et al., 2016; Bonnin et al., 2025). The root system, senses drying soil and sends signals to the leaves (Christmann et al., 2013). Abscisic acid (ABA) is a crucial hormonal signal and, during a progressive water stress its concentration in leaves increases rapidly inducing in turn stomatal closure, reducing water loss through transpiration and thus enhancing water conservation (Saradadevi et al., 2017; Takahashi et al., 2020). As the stress persists, ABA levels remain elevated to sustain stress acclimation responses and coordinate longer-term drought tolerance mechanisms (Ren et al., 2007), contributing to the plant's overall drought response with the implementation of hormonal crosstalk involving other hormone including jasmonic acid (JA), salicylic acid (SA), ethylene (ET), cytokinin (CK), gibberellin (GA), brassinosteroids (BR) and auxin (Khan, 2025).

While effective for reducing water loss, stomatal closure restricts gas exchange and carbon assimilation, thereby decreasing photosynthesis rates and altering plant metabolism (Chaves et al., 2009). Photosynthesis is directly impacted by stomatal closure, which limits assimilation, reducing energy production and potentially leading to photoinhibition and oxidative stress due to an overproduction of reactive oxygen species (ROS) that cause severe cellular and molecular damages (Gill and Tuteja, 2010). Plants counteract this oxidative stress through enzymatic (e.g. superoxide dismutase, catalase and ascorbate peroxidase) and nonenzymatic (e.g. ascorbic acid, glutathione, polyphenol and flavonoids) antioxidant defenses to reduce ROS accumulation (Schneider et al., 2019).

In citriculture, the use of rootstocks is essential, as they enhance tree adaptation to pedoclimatic constraints, increase resistance to diseases and pests, and improve key agronomic traits (Bowman and Joubert, 2020). The citrus relative Poncirus trifoliata (L.) Raf. is an important resource of rootstock, providing cold hardiness (Wang et al., 2019), good adaptation to clay and wet soil (Bowman and Joubert, 2020), and resistance or tolerance of several major citrus diseases and pests (Peng et al., 2020), including Citrus tristeza virus (Bowman and Joubert, 2020), nematodes (Talon and Gmitter, 2008), Phytophthora (Aparicio-Durán et al., 2021), and Huanglongbing (HLB) disease (Rawat et al., 2017). Genotypes resulting from intergeneric hybridization between Poncirus and Citrus, such as Swingle citrumelo (Citrus paradisi Macf. x P. trifoliata), citrandarin (C. reticulata Blanco x P. trifoliata), and citrange (C. sinensis Osb. x P. trifoliata), possess valuable agronomic traits and are widely used in the citrus industry (Bowman and Joubert, 2020). The influence of rootstocks on scion adaptation arises from bidirectional communication between the root and aerial parts through xylem and phloem vessels. This exchange involves water, nutrients, hormones, metabolites, peptides, small organic molecules, and nucleic acids (Albacete et al., 2015; Dogan et al., 2025b, 2025a). Rootstock-scion hormonal signalling, particularly involving auxins, CK, GA, ET, JA and ABA, act as mobile signal molecules, that regulate growth, development, abscission, and the plant's responses to biotic and abiotic stresses (Albacete et al., 2015; Habibi et al., 2022; Bolat et al., 2024). Work in grapevine has shown that rootstock-scion compatibility significantly influences physiological responses to multiple stresses, including lime stress and drought (Daler et al., 2024, 2025). Recent advancements in biotechnologies, such as transcriptomics, have revealed that rootstocks can also alter the expression of specific genes in the scion (Cookson and Ollat, 2013; Cookson et al., 2014). Additionally, molecules such as mRNA, small RNA, and proteins transported through the conducting vessels play a significant role in scion-rootstock interactions (Rasool et al., 2020).

In many crops, polyploid varieties exhibit significant advantages in counteracting both biotic and abiotic stresses (Tossi et al., 2022; Li et al., 2024). Most citrus are diploid (2×), (2n=2×=18) (Krug, 1943), but natural polyploids have been identified in the citrus germplasm such as the triploid (3×) Persian lime (C. latifolia Tan.) (Jackson and Sherman, 1976) and tetraploid (4x) Hong Kong Kumquat (Fortunella hindsii (Champ.) Swing.) (Longley, 1925). Tetraploids may result from chromosome doubling within a species (autotetraploids) or from interspecific hybridization (allotetraploids), while triploids can be obtained from 2× × 2× crosses involving unreduced gametes or from 2× × 4× hybridizations (Chen, 2007; Comai, 2005; Navarro et al., 2015; Ollitrault et al., 2008). Triploid is mainly used for scion diversification, as 3× confer male and female sterility, allowing the production of seedless fruits, a highly desirable trait for consumers. Polyploidization leads to significant anatomical and morphological changes (Ræbild et al., 2024), notably characterized by increased cell size, which in turn leads to bigger organs (leaves, fruits, seeds, roots, etc.) as well as thicker leaves (Allario et al., 2011; Padoan et al., 2013; Guerra et al., 2014; Ruiz et al., 2020).

Polyploid roots typically have larger cells and xylem elements but often fewer vessels per unit cross-section changing in turn the vessel conductivity and vulnerability to cavitation and resistance at the graft union (Losada et al., 2023). Also, root system architecture (thicker roots, deeper rooting) often changes with ploidy too, affecting whole-plant water uptake (Ruiz et al., 2020) even if specific (per-area) conductance changes. Studies of grape rootstocks show rootstock-dependent differences in root hydraulic conductance and associated changes in scion transpiration/stomatal behaviour (Losada et al., 2023). At cellular level, ploidy can alter expression of root aquaporins (PIP5), affecting radial water flow into xylem modulating in turn the hydraulic conductance (Losada et al., 2023; Perrone et al., 2012). Nutrient transport may also be affected since root morphology and cell size can alter root surface area and root hair density, directly influencing ion uptake rates. Because nutrient movement to the canopy is coupled to xylem water flow, any ploidy-driven change in hydraulic conductance feeds into mass flow of mineral nutrients (e.g., nitrate, K⁺, Ca²⁺) (Rossdeutsch et al., 2021). At cellular level, ploidy often modifies gene expression levels globally regulating in turn ion transporter abundance (NRTs, K⁺ channels, H⁺-ATPases) that can be up- or down-regulated in polyploids, changing uptake kinetics. Rootstock genotype controls xylem hormone composition and thereby scion stomatal and growth responses; several grapevine studies link rootstock differences to variation in xylem ABA and cytokinin and to scion gene expression changes (Labarga et al., 2023). The implication of ploidy regarding the drought tolerance via modified water use patterns and anatomy is still discussed (da Silva Costa et al., 2025a; 2025b). Indeed, some polyploid rootstocks may improve drought resilience and nutrient supply to the scion, others may change vulnerability to cavitation or shift nutrient balances unfavourably (Losada et al., 2023). If investigations were performed in 2x to unravel rootstock × scion interaction (Alabacete et al., 2015), more work is required in the polypod context. At the cellular level, increased cell size in polyploids is often associated with larger vacuole volumes, which enhance cellular water storage capacity and contribute significantly to maintaining turgor and osmotic adjustment under water deficit (Sankar et al., 2025). Additionally, modifications in cell wall properties, such as increased thickness and altered composition, in thicker leaves of polyploids can improve structural rigidity and reduce water loss (Corneillie et al., 2019).

Tetraploids exhibited a dwarfing effect, resulting in shorter plants with thicker roots and larger fruits. These phenotypic modifications are frequently associated with physiological, biochemical and gene expression changes (Ruiz et al., 2020; Oustric et al., 2023). A field trial evaluating the performance of “Valencia” orange demonstrated that several 4× rootstocks effectively controlled tree size, enhanced yield efficiency, and improved fruit quality compared to conventional 2x
rootstocks (Kunwar et al., 2023). In addition, in pot condition, 4× citrus have been described to be more tolerant than 2× to various abiotic stresses, including salinity (Saleh et al., 2008; Mouhaya et al., 2008; Podda et al., 2013; Ruiz et al., 2016; Khalid et al., 2020; Mahmoud et al., 2023; Bonnin et al., 2023; 2024), nutrient deficiency (Oustric et al., 2019a; 2019b; 2021), cold (Oustric et al., 2017; Lourkisti et al., 2020; Mahmoud et al., 2024), chromium toxicity (Balal et al., 2017), drought (Allario et al., 2013; Dutra de Souza et al., 2017; Oustric et al., 2020; Hussain et al., 2023) and HLB (Sivager et al., 2022). In a previous study, Allario et al. (2013) suggested that sweet orange grafted onto a 4× Rangpur lime rootstock conferred a better tolerance to water deficit than those grafted onto its respective 2× counterpart. Another work showed that 3× hybrid varieties (“Fortune” mandarin x “Ellendale” tangor) grafted onto 2× C-35 citrange were more tolerant to natural chilling condition (Lourkisti et al., 2020) and water deficit (Lourkisti et al., 2021; 2022) than 2x hybrids.

A more recent study contradicts previous finding, showing that when Sunki “Tropical” 2× and 4× rootstocks were grown in competition within the same pot under similar soil moisture conditions, the 4× rootstock does not provide any advantage over the 2x one (da Silva Costa et al., 2025a; 2025b). Whereas the benefits of polyloids in the evolution of angiosperms and plant adaptation to environmental stresses are well recognized (Van de Peer et al., 2017), the mechanisms related to polyploid adaptation remain poorly understood. Transcriptome studies allow for comparative gene expression analyses between 2× and polyploid plant to identify genes linked to drought adaptation. For example, Allario et al. (2013) conducted a microarray analysis to study gene expression in sweet oranges grafted onto 2× and 4× Rangpur lime rootstocks, revealing a greater expression of drought-responsive genes in the 4x rootstock. RT-qPCR analyses was also conducted to compare gene expression in 2× and 4× Carrizo citrange, highlighting differential expression of genes involved in ABA biosynthesis (NCED3), ABA signaling regulation (ABI1), and catalase enzyme production (CAT2) (Oliveira et al., 2017). More recently, RNA-seq-based global transcriptome analyses have uncovered distinct gene expression profiles, such as upregulation of antioxidant activity genes in 4× P. trifoliata (Wei et al., 2019), and enhanced regulation of photosynthesis, phosphorylation, and plant hormone signaling in 4× C. wilsonii compared to its 2× counterpart (Jiang et al., 2022). In other models, transcriptomic studies have revealed that 4× cassava exhibits greater drought tolerance than 2× cassava. The identification of long noncoding RNA (lncRNA) in 4× cassava, which are more commonly expressed under drought conditions, suggests that lncRNA may play a role in enhancing drought tolerance (Xiao et al., 2022). Similarly, 4× daylily plants demonstrate faster and more effective adaptation to drought stress by activating alternative signaling pathways (Misiukevičius et al., 2023; 2024).

Most of the experiments showing apparent better adaptation of 4× to water deficit have been carried out in controlled pots condition. However, under field conditions, the dynamics seems to be different, as compared to 4×, 2× rootstocks have a more developed root system, with greater root growth capacity in the deeper layers and then a better capacity to absorb water particularly during drought period (Girardi et al., 2021). Nevertheless, 4× rootstocks offer valuable agronomic traits, including dwarfing effects and improved fruit quality, making them promising candidates for high-density orchards with potentially competitive yields compared with traditional 2× rootstocks. 4× rootstock represents also an important parental resource in breeding program for the development of new generation of 4× rootstock hybrid using the “Tetrazyg” breeding strategy (Grosser et al., 2003, 2007; 2015; Calvez et al., 2020, 2023). In this context, a 4× hybrid population was generated through sexual hybridization between the 4× Volkamer lemon and the 4× Swingle citrumelo (Calvez et al., 2020). Given the importance of roots traits for drought adaptation, one objective of the study was to identify hybrids with vigorous root systems. Phenotyping of root growth of the new hybrids led to the identification of a very promising 4x individual with extensive root development (Calvez et al.,

2023) which is currently under field evaluation.

In the present study, we investigated the response of four scion/rootstock combinations with various ploidy levels under water deficit conditions. Diploid Swingle citrumelo seedlings and their homologous 4× counterpart were grafted with 3× Persian Lime (C. Latifolia Tan.) and 2× Mexican Lime (C. aurantiifolia (Christm.) Swing.). The objective of this study was to decipher in pot condition, the adaptive trait to water resulting from the association of rootstocks and scions with different polyploidy levels. To achieve this, we conducted a comprehensive analysis of drought tolerance traits, including phenotypic, physiological, biochemical, electrochemical, and transcriptomic assessments, to unravel the impact of polyploidy through scion/rootstock interactions.

## 2. Materials and methods

### 2.1. Plant materials, growth conditions, and water deficit treatment

Two rootstocks, Swingle citrumelo 4475 (C. sinensis × P. trifoliata SRA 928) diploid (2×) and its homologous doubled diploid (4×), were grafted with two commercial scions, the 2× Mexican lime (C. aurantiifolia, SRA 140) and the 3× Persian lime (C. latifolia, SRA 58). 2× and 4× Swingle citrumelo 4475 seeds were obtained from the biological resource center collection “CRB Citrus” INRAE-CIRAD of San Giuliano in Corsica, France (Luro et al., 2017). The ploidy status of the 2× and 4× Swingle citrumelo seedlings was checked and confirmed using flow cytometry (CyFlow Cube 6, Sysmex Partec, Gorlitz, Germany), following Froelicher et al. (2007). The seedling’s genetic conformity was confirmed using single nucleotide polymorphism (SNP) markers developed by Bruyère et al. (2016). Bud grafting was performed with two cultivars on eight-month-old Swingle citrumelo 2× and 4× seedlings. The four scion/rootstock combinations obtained are presented in Table 1 and were evaluated for their response to water deficit. Thirty-two-year-old plants (eight biological replications per combination) were then transplanted into 40-L pots with the same substrate amount (containing 50 % volcanic rock and 50 % peat). The plants were grown up for 2 years in an insect-proof greenhouse with a natural photoperiod, day/night temperatures of 25–32 °C and 22–28 °C, and relative humidity between 70 % and 98 %. Daily irrigation and regular NPK fertilization were conducted. One month before the experiment, trees were pruned to homogenize tree size and canopy. Before applying the water deficit, pot weight was equilibrated to 37.5 kg by addition of substrate. All pots were then watered at field capacity and covered with plastic sheeting to avoid water loss due to soil evaporation. The 32 trees were split into two group treatments as follows: (i) control-plant set with 12 trees under regular irrigation and (ii) and a drought stress set with 20 trees subjected to water deficit by stopping the irrigation. After 5 days of

<table><caption>**Table 1**

Scion-rootstock combinations.</caption><thead><tr><th>Number of combination</th><th>Scion</th><th>Rootstock</th><th>Combination abbreviation</th></tr></thead><tbody><tr><td>1</td><td>Mexican Lime (2x)<br/>C. aurantiifolia</td><td>Swingle citrumelo 4475 (2×)<br/>C. paradisi x Poncirus trifoliata</td><td>ML/2x</td></tr><tr><td>2</td><td>Mexican Lime (2x)<br/>C. aurantiifolia</td><td>Swingle citrumelo 4475 (4×)<br/>C. paradisi x Poncirus trifoliata</td><td>ML/4x</td></tr><tr><td>3</td><td>Persian Lime (3x)<br/>C. latifolia</td><td>Swingle citrumelo 4475 (2×)<br/>C. paradisi x Poncirus trifoliata</td><td>PL/2x</td></tr><tr><td>4</td><td>Persian Lime (3x)<br/>C. latifolia</td><td>Swingle citrumelo 4475 (4×)<br/>C. paradisi x Poncirus trifoliata</td><td>PL/4x</td></tr></tbody></table>
water deficit, some genotypes presented strong leaf wilting and drop leaves. From the day-5 to day-9, water was added daily to the trees with the highest consumption to maintain viability and standardize all pots to a final weight of 30.5 kg. By the end of the experiment (day-9), all pots reached similar water loss levels with comparable soil water potential, minimizing bias in future analyses for drought response characterization. To characterize the scion/rootstock combinations' water deficit tolerance, physiological, biochemical, hormonal and transcriptomic studies were conducted.

## 2.2. Leaf physiological measurements, leaf anatomical traits, and plant water consumption

Leaf photosynthetic parameters, such as leaf stomata conductance ($g_s$, mol·m⁻²·s⁻¹), leaf net photosynthetic rate ($P_{net}$, µmol·m⁻²·s⁻¹), and transpiration ($E$, mmol·H₂O·m⁻²·s⁻¹), were measured daily. Measurements ($A$, $g_s$, $E$) were taken using a gas exchange analyzer from LCpro-SD Portable Photosynthesis Systems (ADC BioScientific Ltd., Hoddeston, United Kingdom) with photosynthetically active radiation of 1400 µmol photons·m⁻²·s⁻¹ and ambient leaf temperature, air humidity, and carbon dioxide ($CO_2$) concentration. Measurements were taken in the morning between 7 and 10 am on three mature leaves of each plant that had been previously selected and marked to use the same leaf for each measurement and avoid bias due to a change in individual leaf metabolism.

Ten leaves from each tree were used to determine relative water content (RWC). Leaves were collected at 9:00 a.m. The RWC (%) was calculated as $= RWC [(FW-DW) / (TW-DW)] \times 100$, where FW is the fresh mass, TW is the turgid mass after rehydration, and DW is the dry mass after oven-drying. The RWC reflects the proportion of water in the leaf relative to its maximum water-holding capacity (Barrs and Weath-erley, 1962).

Leaf greenness was assessed with a SPAD meter (Minolta SPAD-502, Japan). Leaf thickness and area were measured using a micrometer (IP65, Mitutoyo) and by scanning samples analyzed in ImageJ software (http://rsbweb.nih.gov/ij/) before drying. Slides for stomatal analysis (stomatal size and density) were prepared following the protocol of Morillon and Chrispeels (2001). Sixty stomata were measured from three independent slide preparations, each from mature leaves.

To measure each plant's water consumption, all the pots were weighed daily during the stress, and the difference with the initial weight was the amount of water loss. Soil water potential ($Ψ_{soil}$) was recorded at the start and end of the experiment for control plants and at various time points during the stress treatment. For measurement, 50 g of substrate was collected from pots, placed into plastic bags, and analysed using a dew point potential meter (WP4, Decagon, USA).

Leaf area from each variety/rootstock combination was measured using a planimeter (LICOR LI-31OOC, Lincoln, United States). At the end of the experiment, roots system, branches, and leaves were weighed to determine the fresh weight, and the plant material was then dried at 60 °C for 72 h to determine the dry weight.

## 2.3. Biochemical, hormonal, and transcriptomic analysis

Fresh leaves and roots samples were also collected at day 9 and immersed in liquid nitrogen to avoid any sample degradation and then stored at -80 °C. Among the eight trees per combination, leaves and roots from seven individuals were sampled (3 control and 4 stressed), for a total of 56 samples (28 samples per organ). Samples were then ground under liquid nitrogen using a mortar and pestle to obtain a homogeneous powder, which was conserved at -80 °C until further analysis.

### 2.3.1. Electrochemistry by cyclic voltammetry (CV)

The electrochemical methodology, specifically cyclic voltammetry (CV), is an efficient way to evaluate the antioxidant potential in plants (Keffous et al., 2016; Idris et al., 2019; Alam et al., 2022). Plant material preparation and electrochemistry analysis was conducted, following

Sivager et al. (2022). 100 mg of frozen root and leaf powder was mixed with 10 mL of methanol and incubated for 72 h at room temperature. The extracts were then filtered and 5 mg of tetrabutylammonium hydrogen sulfate 97 % (Sigma Aldrich) were added to 1.5 mL of filtrate and were used for electrochemical analyses, following Palmeira-Mello et al. (2021). Cyclic voltammograms were recorded at room temperature, at 298 ± 1 K in a conventional three-electrode system including a glassy carbon working electrode (BAS MF 4012, geometrical area 0.071 cm²), a platinum wire auxiliary electrode, and an Ag/AgCl (3 M NaCl) reference electrode. Measurements were taken with a Potentiostat electronic device (SP-300, Bio-Logic, France) operated with EC-lab® software (V 10.40). A 0.10-M potassium phosphate buffer at pH 7.0 was used as a supporting electrolyte. The working electrode was prepared by evaporating 50 µL of methanol from leaf and root filtrate extract under air. To mimic the natural environment, no degasification of the electrolyte was performed. Measurements were taken from -1.5 V to 1.5 V (red/ox), 1.5 V to -1.5 V (ox/red), and 0 to 1.5 V (short ox).

### 2.3.2. Hormonal measurements

Hormonal identification and quantification were performed at the MetaToul-Agromix platform (University of Toulouse-France) for all the 56 samples. About 100 mg of frozen leaf or root powder was extracted with 1 mL of cold modified Bieleski's extraction solvent (methanol/water/formic acid 75/20/5, v/v/v) (Hoyerová et al., 2006) and 4 ng of labelled internal standard ABA D6 (Olchemim). The samples were ground using FastPrep (MP Biomedical) and maintained at -20 °C overnight. Samples were then centrifuged, and the supernatant was recovered for analysis. An extraction and purification step on SPE OASIS HLB cartridges (30 mg, Waters, Milford, MA, USA) was performed (Pons et al., 2020). Eluates were dried under nitrogen and then taken up in 80 µl of acidified water (1 M formic acid) before analysis by UHPLC-MS. ABA detection and quantification in leaves and roots were performed using QTrap 5500 mass spectrometer (Applied Biosystem Sciex) coupled with an UHPLC (Dionex Ultimate 3000, Thermo Scientific) equipped with a Kinetex Polar C18 chromatographic column (100 × 2.1 mm, 2.6 µm, 100 Å, Phenomenex) as described in Pons et al. (2020). The column was heated at 45 °C. Five-µL samples were injected. Separation was performed at a constant flow rate of 0.3 mL.min⁻¹, in a gradient of solvent A (water + 0.1 % formic acid) and B (acetonitrile + 0.1 % formic acid): 1 min 5 % B; 11 min 5 % to 100 % B; 2 min 100 % B, and re-equilibration to the initial conditions in 4 min. The Q-Trap 5500 mass spectrometer was equipped with an electrospray source. Curtain gas was set to 30 psi, nebulizer to 40 psi and turbo gas to 60 psi. Capillary voltage was set to -4.5 kV (negative mode) on Electrospray Ionization (ESI) source (400 °C). Samples were monitored in negative modes for ABA detection, and the analyses were performed in MRM mode (multiple reaction monitoring). MRM parameters were as follows: Quantification transition was set to parent ion at *m/z* 263 and product ion at *m/z* 153. A confirmation transition was used between parent ion at *m/z* 263 and product ion at *m/z* 219. Data quantification was performed using Skyline software (20.1 version) (Henderson et al., 2018). ABA was quantified using external calibration curves established from ABA standard prepared at different known concentrations. The limit of detection was calculated at 7E-10 M. Other hormones were also quantified in the samples following the methodology of Pons et al. (2020). This included jasmonic acid (JA), salicylic acid (SA), and auxins such as indole-3-butyric acid (IBA) and N-(3-indolylacetyl)-l-aspartate (IAA-Asp). Cytokinins (e.g., isopentenyl adenosine, IPR), the ABA conjugate ABA-glucose ester (ABA-GE), and gibberellins (GA₃, GA₄) were also measured.

### 2.3.3. Total RNA isolation, cDNA library preparation, and sequencing

Total RNA was extracted from 100 mg of leaf and root frozen powder using the RNeasy® Plant Mini kit (Qiagen, Hilden, Germany) per the manufacturer's instructions. RNA quality and quantity were determined using a mySPEC microvolume spectrophotometer (VWR, Radnor, PA,
USA). High-purity RNA samples (OD<sub>260</sub>/OD<sub>280</sub> values at 1.8–2.0) were used for library construction and sequencing. For each RNA sample, cDNA libraries were prepared for sequencing from 1 µg of total RNA using the Truseq Stranded mRNA kit (Illumina, San Diego, CA, USA), following the manufacturer's protocol. Among the four banks for each organ that were prepared in stress condition, the three with the best quality control were selected for sequencing. The GENEWIZ company (https://www.genewiz.com, South Plainfield, USA) then sequenced the 48 libraries on Illumina HiSeq2000 (Illumina, San Diego, CA, USA), and reads were generated in 150-bp pair-end format.

### 2.3.4. RNA-seq data processing

First, RNA-seq reads were evaluated with FastQC software for quality control (Andrews, 2010). Then, raw sequencing read cleaning was performed using trimmomatic software (Bolger et al., 2014) to remove low-quality bases and adapters and to maintain at least a 36-bp read length. The filtered reads were then aligned to the reference genome of *C. clementina* 182 v1.0 and its gene annotation, available at https://phytozome.jgi.doe.gov (Wu et al., 2014), using the MapSplice-gapped alignment algorithm (Wang et al., 2010). Samples were then processed to obtain count reads using the HTSeq-count Python tool (Anders et al., 2015). Expression values were reported as reads per kilo base per million mapped reads (RPKM) value. To evaluate the quality of the filtered RNA-seq data and to highlight sample relationships, partial least square discriminant analysis (PLSDA) was conducted using the {mixOmics} R package (Rohart et al., 2017).

### 2.3.5. Differential expression analysis and functional categorization

Gene expression analysis was conducted using the {DESeq2} R package (Love et al., 2014). First, multifactorial analysis was conducted using the following model for leaf samples (Scion genotype + Treatment + Scion genotype x Treatment) and root samples (Rootstock ploidy + Treatment + Rootstock ploidy x Treatment). The objective is to identify lists of genes whose expression is affected by treatment (stress: WD vs. control: Ctrl), scion genotype (Persian lime: PL vs. Mexican lime: ML), rootstock ploidy (tetraploid: 4x vs. diploid: 2x), and interactions term, in both leaf and root tissues.

Then, more detailed pairwise comparisons were performed on each group to refine our understanding and highlight the effect of water deficit (ML/2x WD versus ML/2x Ctrl, PL/2x WD versus PL/2x Ctrl, ML/4x WD versus ML/4x Ctrl and PL/4x WD versus PL/4x Ctrl), scion genotype (PL/2x Ctrl versus ML/2x Ctrl; PL/4x Ctrl versus ML/4x Ctrl; PL/2x WD versus ML/2x WD and PL/4x WD versus ML/4x WD) and rootstock ploidy (ML/4x Ctrl versus ML/2x Ctrl, PL/4x Ctrl versus PL/2x Ctrl, ML/4x WD versus ML/2x WD and PL/4x WD versus PL/2x WD). Genes were considered as differentially expressed genes (DEG) at false discovery rate (FDR) < 0.05 and the absolute value of log2 fold change (|log2FC|) ≥ 1. DEGs GO enrichment analysis was performed on Cytoscape software applications (version 3.10.2 available at www.cytoscape.org). Biological Network Gene Ontology (BINGO) application (version 3.0.5) (Maere et al., 2005) was used to identify overrepresented gene ontology (GO) terms by applying a hypergeometric test and a multiple test correction using the Benjamini and Hochberg False Discovery Rate (FDR) correction with a significance level of 0.05. Enriched GO terms visualization was performed using the EnrichmentMap application (version 3.4.0) (Merico et al., 2010) with an FDR q-value <0.05. GO terms were clustered and annotated using the AutoAnnotate application (version 1.5.1) (Kucera et al., 2016). R package {ClusterProfiler} (version 4.12.6) (Yu et al., 2012) was applied to perform gene set enrichment analysis (GSEA) using the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway (Kanehisa and Goto, 2000) with p-value <0.05.

## 3. Results

### 3.1. Plant water consumption and leaf photosynthesis parameter

Water deficit was applied for nine days, after five days, leaf wilting symptoms appeared, revealing contrasting phenotypical responses among the different scion/rootstock combinations (Fig. 1A & B). PL/2x exhibited severe wilting, ML/2x showed the same symptom but less pronounced. In contrast, no visible leaf wilting was observed in PL/4x and ML/4x. Scions grafted onto 2x rootstocks (PL/2x and ML/2x) were more affected than those grafted onto 4x rootstocks (PL/4x and ML/4x). On day nine, at the end of the water deficit treatment, all associations showed advanced wilting symptoms.

During the water deficit stress, daily water consumption was evaluated by weighing the pots, revealing significant differences in the dynamics of water loss between scion/rootstock combinations (Fig. 1C). PL/2x lost water much faster than other combinations, consuming 7 kg of water in only 4 days. Other combinations reached the same amount of water loss in seven days for ML/2x, 8 days for PL/4x, and 9 days for ML/4x. To keep all the trees alive and at the same soil water potential ($Ψ_{soil}$), the pot weight of the different scion/rootstock associations was maintained at 30.4 kg (7 kg of water loss; -1.35MPas) by adding water every subsequent day until day 9 (Fig. 1C & G). By the end of the experiment, all trees had lost comparable amount of water and were in a similar state of stress.

Different leaf parameters such as the leaf area, leaf thickness, RWC, epidermal cell area and leaf stomatal size and density were measured the week prior the stress application in Mexican and Persian limes grafted respectively onto 2x and 4x Swingle citrumelo (Table 2). In that condition, 3x genotypes presented largest leaf area, leaf thickness and leaf RWC when compared to the 2x. Epidermal cell and stomatal areas where were higher in 3x compared to 2x. As expected, the stomatal density was higher in 2x compared to 3x. No significant change was induced by the ploidy of the rootstock compared to 2x. During the experiment, daily physiological measurements of stomatal conductance (gs), photosynthesis rate (A), and transpiration (E) were carried out on the different scion/rootstock combinations. For stomatal conductance (Fig. 1D), a substantial decrease was observed for ML/2x and ML/4x combinations from day 4 and reached a very low value (approximately 0.05 mmol H<sub>2</sub>O.m<sup>-2</sup>.s<sup>-1</sup>). PL/2x reached the lowest value on day 5. However, PL/4x combination maintained relatively high stomatal conductance values (approximately 0.2 mmol H<sub>2</sub>O.m<sup>-2</sup>.s<sup>-1</sup>) throughout the stress, with a noticeable decline after 7 days, reaching levels comparable to the other combinations on day 8. Regarding the photosynthesis rate (Fig. 1E), combinations with 2x rootstocks (PL/2x and ML/2x) showed a substantial decrease after day 4, reaching a lower-level plateau at day 5 (values around 2 µmol CO<sub>2</sub>.m<sup>-2</sup>.s<sup>-1</sup>). ML/4x maintained a relatively small photosynthetic activity during the stress (4 µmol CO<sub>2</sub>.m<sup>-2</sup>.s<sup>-1</sup>), while the PL/4x combination maintained higher photosynthesis values until day 7 (about 10 µmol CO<sub>2</sub>.m<sup>-2</sup>.s<sup>-1</sup>). Regarding transpiration (Fig. 1F), PL/2x presented the fastest decrease with a very low value on day 5 (0.5 mol H<sub>2</sub>O.m<sup>-2</sup>.s<sup>-1</sup>), followed by ML/2x and ML/4x with a higher value on the same day (approximately 1 and 2.5 mol.H<sub>2</sub>O. m<sup>-2</sup>.s<sup>-1</sup>respectively). PL/4x maintained a high transpiration value until day 6 (about 3 mol H<sub>2</sub>O.m<sup>-2</sup>.s<sup>-1</sup>) before dropping drastically. At the end of the water deficit treatment, on day nine, no significant difference was observed between combinations for the three physiological parameters measured.

Fresh and dry matter from leaves, branches, and roots were weighed at the end of the experiment. Significantly higher fresh matter in leaves and branches for PL/2x was observed when analyzing the different combinations. In addition, fresh matter roots were heavier in PL/2x and PL/4x than in ML/2x and ML/4x. Dry matter variations in leaves, branches, and roots were not significantly different. To conclude, fresh and dry matter's root/scion ratio was higher in PL than in ML combination (Supplementary Fig. S1).
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Water deficit impact investigated on the four scion/rootstock combinations (PL/2×; PL/4×; ML/2×; ML/4×). (A, B) Photographs showing the water deficit impact on the phenotype of the four different scion/rootstock combinations after five days of water deficit condition. (A) Mexican Lime grafted onto Swingle citrumelo 2× (ML/2×) and Swingle citrumelo 4× (ML/4×) and (B) Persian Lime grafted onto Swingle citrumelo 2× (PL/2×) and Swingle citrumelo 4× (PL/4×). (C, D, E, F) Physiological parameters related to water deficit during 9 days. Graphic showing (C) the relative pot water loss; (D) stomatal conductance "gs", (E) photosynthesis rate "A"; (F) Transpiration "E"; and (G) Soil water potential "ψsoil" along the 9 days of water deficit treatment. The irrigation was stopped on day-1 and parameters were measured daily. From the day-5 (indicated by the arrow), water was added each day to keep the trees alive and bring all the pots at a final weight of 30,5 kg. On the day-9, all the pots reached the same water loss and all the plants presented the same water potential. Filled circle: PL/2×; open circle: PL/4×; filled downward triangle: PL/2×; open downward triangle: PL/2×. Results are presented with mean value ± SE (n = 5). Error bars with different letters represent a statistical difference (One way ANOVA followed by Tukey's post hoc test, P ≤ 0.05).</figcaption></figure>

## 3.2. Hormonal analysis: ABA measurements

To highlight the implication of phytohormones in plant response to water deficit, we quantified ABA, JA, SA, and auxins, including IBA) and IAA-Asp. We also measured cytokinins such as IPR, the ABA conjugate ABA-GE, and gibberellins (GA₃, GA₄). Measurements were assessed in the leaves and roots of stressed and control plants (Fig. 2). ABA concentration analysis showed generally a higher ABA content in leaves than in roots (Anova *p*-value=0.002374). A significant increase was observed for all combinations under water deficit conditions compared to controls. ABA concentration showed no significant difference between combinations under control conditions in both leaves and root organs. However, PL/4x showed a higher ABA content in leaves under stress conditions than PL/2x. In addition, an increase was also observed in stressed roots, the concentration was significantly higher in 4x

rootstocks (ML/4x and PL/4x) than in 2x rootstocks (ML/2x and PL/2x). However, for the other hormones detected, no significant differences were identified between scion-rootstock combinations and between stressed and control samples.

## 3.3. Electrochemistry

Cyclic voltammograms (CV) of leaves and roots from the four scion/rootstock combinations (ML/2x, PL/2x, ML/4x, and PL/4x) were studied with samples collected at the end of the experiment. All voltammograms (Figs. 3& 4) are dominated by a well-defined cathodic peak ca. -1.0 V (C) and a more or less prominent rising current near 1.0 V, corresponding to the oxygen evolution reaction (OER). The peak C is representative of the reduction of dissolved oxygen (oxygen reduction reaction, ORR) and can be taken as an internal standard to measure peak
<caption>**Table 2**</caption>

Leaf physiological characterization of Mexican and Persian limes grafted onto 2× and 4× Swingle citrumelo.

<table><thead><tr><th rowspan="2">Scion<br>Rootstock</th><th colspan="2">Mexican Lime</th><th colspan="2">Persian Lime</th></tr><tr><th>Cit 2x</th><th>Cit 4x</th><th>Cit 2x</th><th>Cit 4x</th></tr></thead><tbody><tr><td>Leaf area (mm²)</td><td>1,660 ± 291 a</td><td>1,642 ± 320 a</td><td>3,185 ± 272 b</td><td>3,241 ± 447 b</td></tr><tr><td>Leaf thickness (mm)</td><td>0.31 ± 0.01 a</td><td>0.32 ± 0.02 a</td><td>0.37 ± 0.08 b</td><td>0.39 ± 0.07 b</td></tr><tr><td>RCW (%)</td><td>79.9 ± 2.1 a</td><td>80.8 ± 1.3 a</td><td>84.1 ± 0.6 b</td><td>83.4 ± 1.1b</td></tr><tr><td>Epidermal cell area<br>(µm²)</td><td>179 ± 8 a</td><td>187 ± 6 a</td><td>215 ± 9 b</td><td>219 ± 7 b</td></tr><tr><td>Stomatal area (µm²)</td><td>340 ± 5 a</td><td>335 ± 3.0 a</td><td>377 ± 6 b</td><td>372 ± 7 b</td></tr><tr><td>Stomatal density<br>/mm²</td><td>437 ± 6 a</td><td>447 ± 8 a</td><td>378 ± 9 b</td><td>387 ± 5 b</td></tr></tbody></table>

Leaf area, leaf thickness, relative water content (RWC), epidermal cell area and leaf stomatal size and density, measured in the control Mexican and Persian limes grafted respectively onto Swingle citrumelo 2x (Cit 2x) and 4x (Cit 4x). Results are expressed as mean ± SE (n = 6 to 10 for the leaf area, leaf thickness and RWC; n = 100 for the analysis of the epidermal cell area, stomatal area, and stomatal density). ANOVA tests were performed to determine if the ploidy of the scion or the rootstock led to significant differences. For each given datum, different letters indicate a statistical difference between genotypes (one-way ANOVA followed by Tukey's post hoc test, P 0.05).

currents and peak areas. The former were measured from the horizontal base line appearing in the voltammograms as marked by arrows in Figs. 3 and 4. Peak areas can be measured upon integration of the current/potential curves from the above baseline. The relevant aspect to underline is that in the region of positive potentials vs. Ag/AgCl, the studied leaf samples yield different additional signals. Under both control and stressed conditions ML/2x displayed two weak anodic waves at ca. 0.5 (A1) and 0.9 V (A2) (Fig. 3A), while of PL/2x (Fig. 3B) displayed for both conditions two anodic signals at ca. 0.75 V (A3) and ca. 1 V (A4). In the case of ML/4x anodic signals A1 and A2 were accompanied by a new signal at ca. 1 V (A5) in both control and stressed conditions (Fig. 3C). The voltammograms of PL/4x displayed peaks A2 and A5, the former being the most intense in both control and stressed conditions. The most relevant feature is that the peak A5 appears as a potential marker of 4x rootstock.

The A1-A5 signals can be assigned to the oxidation of polyphenolic components, basically, lignins and flavonoids (Doménech-Carbó et al., 2013; 2015). Although, given the enormous complexity of the leaf composition, there is no possibility to assign it to concrete plant components, the position and intensity of the voltammetric peaks evidence significant compositional differences between the different specimens. In addition, as being such signals representative of the antioxidant

capacity of the system, the intensity of signals A1-A5 relative to the intensity of peak C1 ($I_A/I_{C1}$) can be taken as representative of that property. Given the strong overlapping of the A signals, the $I_A/I_{C1}$ ratio can be approximated by the ratio between the peak areas or by the ratio between the maximum peak current of the A-peaks and the peak current of C1, $I_{Amax}/I_{C1}$. Remarkably, the $I_{Amax}/I_{C1}$ ratio becomes essentially insensitive to changes in stress condition for both ML/2x (Ctrl: 0.18; WD: 0.2) and ML/4x samples (Ctrl: 0.4; WD: 0.5), while increases from control to stressed samples in PL/2x (Ctrl: 0.05; WD: 0.5) and PL/4x samples (Ctrl: 0.4; WD: 0.3). This result can be interpreted in terms of variations in the antioxidant capacity associated with the plant response to hydric stress. This stress does not affect significantly the response of ML samples, but results in an increased plant response in PL samples which, apparently, enhance their antioxidant capacity upon stress.

The voltammetry of root samples is less conclusive, only PL/2x and PL/4x displaying an A-signal ca. 1.0 V (A6) (Fig. 4). As occurring in leaf samples, upon stress, the $I_{Amax}/I_{C1}$ ratio increases.

### 3.4. Transcriptomic analysis

Transcriptomic analysis was conducted to investigate the mechanisms activated in rootstock and scion during water deficit and try identifying the genes triggered by polyploidy leading to the observed phenotype. In order to evaluate rootstock/association in the same water deficit condition, samples were collected at day-9 when all the genotypes reached the same pot water loss and a comparable soil water potential (Fig. 1C & G). In such conditions, the more sensitive associations may have expertise a stress for a longer time, triggering in turn earliest plant stress response. Earlier samplings points would have provided more global vision into stress response dynamics. However, in this condition, the different tree scion/rootstocks associations would have expertise differences into the soil and leaf water potentials and would have in turn make more difficult the interpretation of the results at molecular level. Also, it was in our opinion, the better way to make the experiment easier to be reproducible. Two count matrices were generated, covering 24,534 genes across 28 leaf and root organ samples. Three sample (two leaves and one root) from the 56 RNA were discarded for further analysis due to very low read quality and a poor percentage of read mapping. Genes with expression levels below 10 counts (RPKM < 10) were filtered out for the following analysis, leaving a matrix of 21,328 genes from leaves samples and 22,367 genes from roots samples. Data supporting the results of this article are available in the National Center for Biotechnology Information archive (NCBI) (https://www.ncbi.nlm.nih.gov) under the project accession number (GSE255759).

First, PLS-DA analysis assessed data quality and distinguished sample clustering based on gene expression profiles. Then, DEGs were identified in overall comparison using multifactorial model to highlight impact of

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Abscisic acid content in response to water deficit. ABA content in (A) leaves and (B) roots of Mexican Lime and Persian Lime grafted onto 2× and 4× Swingle citrumelo rootstocks (ML/2×, ML/4×, PL/2×, PL/4×) after 9 days of water deficit. Values are ng/g fresh weight. Black bars represent well-watered controls, and white bars represent water-deficit treated plants. Data are means ± SE (n = 3 for controls, n = 4 for stressed samples). Different letters indicate significant differences by one-way ANOVA followed by Tukey's post hoc test (P ≤ 0.05).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig. 3. Cyclic voltammograms of leaf samples under water deficit. Cyclic voltammogram profiles of Mexican Lime and Persian Lime grafted onto 2× and 4× Swingle citrumelo rootstocks (ML/2×, ML/4×, PL/2×, PL/4×) under control (green curve) and water deficit (red curve) conditions: (A) ML/2×, (B) PL/2×, (C) ML/4×, and (D) PL/4×. Potential scan initiated at 0.0 V in the negative direction; scan rate 10 mV/s. Consistent profiles were observed across biological replicates (n = 3 for control, n = 4 for stressed samples). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 4. Cyclic voltammograms of root samples under water deficit. Cyclic voltammogram profiles of Mexican Lime and Persian Lime grafted onto 2× and 4× Swingle citrumelo rootstocks (ML/2×, ML/4×, PL/2×, PL/4×) under control (green curve) and water deficit (red curve) conditions: (A) ML/2x, (B) PL/2×, (C) ML/4x, and (D) PL/4x. Potential scan initiated at 0.0 V in the negative direction; scan rate 10 mV/s. Consistent profiles were observed across biological replicates (n = 3 for control, n = 4 for stressed samples). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).</figcaption></figure>

treatment (stress: WD vs. control: Ctrl), scion genotype (Persian lime: PL vs. Mexican lime: ML) and rootstock ploidy (4× vs. 2×) interaction terms on scion and rootstock transcriptome. Subsequently, pairwise comparisons were also employed to examine more precisely gene expression variation within each scion/rootstock combinations. Finally, GO and KEGG pathway enrichment of DEG were performed (Supplementary Fig. S2).

### 3.4.1. *Change in leaf transcriptome under water deficit*

In leaves, PLS-DA revealed a pronounced discrimination in transcriptome profiles between control (Ctrl) and water deficit (WD) samples, explaining 33 % of the variance in total (27 % of variance explained by latent variable 1 and 6 % of variance explained by latent variable 2) (Fig. 5A).

Multifactorial analysis was conducted to assess the impact of treatment on leaf gene expression by comparing Ctrl samples with WD samples. A list of 2517 genes were identified, including 1286 up-
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Global changes in leaf gene expression in response to water deficit treatment. (A) PLS-DA plot showing the effect of water deficit treatment (Controlled: Ctrl vs. Stressed: WD samples) on leaf transcriptome profiles in Mexican Lime and Persian Lime grafted onto 2× and 4× Swingle citrumelo rootstocks (ML/2×, ML/4×, PL/2×, PL/4×); (B) Histogram presented the number of DEGs upregulated (black bar) and downregulated (white bar) induced by water deficit (FDR <0.05, |log2FC| ≥ 1); (C) Overrepresented gene ontology (GO) enrichment map of water-responsive DEGs (Ctrl vs. WD) upregulated (blue node) and downregulated (red node). Nodes size is proportional to the number of genes. Additionally, edges thickness is proportional to the similarity between nodes (q-value<0.05); (D) GSEA dot plot showing KEGG pathway enrichment activated and suppressed in response to water deficit (FDR<0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).</figcaption></figure>

**Table 3**

Number of genes in leaves and roots tissues influenced by the effect of treatment (WD vs. Ctrl), scion genotype (PL vs. ML), rootstock ploidy (4x vs. 2x) and the Interactions term Scion genotype x Treatment and Rootstock ploidy x Treatment.

<table><thead><tr><th rowspan="2">Organ</th><th rowspan="2">Treatment</th><th colspan="2">Scion Genotype</th><th rowspan="2">Rootstock Ploidy</th><th>Scion Genotype</th><th>Rootstock Ploidy</th></tr><tr><th></th><th></th><th>x</th><th>x</th></tr></thead><tbody><tr><td>Leaves</td><td>2517</td><td>✓ 1286<br/>✓ 1231</td><td>1703</td><td>✓ 929<br/>✓ 774</td><td>-</td><td>✓ 30<br/>✓ 69</td><td>-</td></tr><tr><td>Roots</td><td>5189</td><td>✓ 1262<br/>✓ 3927</td><td>-</td><td>687</td><td>✓ 174<br/>✓ 513</td><td>-</td><td>✓ 68<br/>✓ 6</td></tr></tbody></table>

Wald test (FDR <0.05, |log2FC| ≥ 1); ✓: up-regulated genes; \: down-regulated genes.
regulated genes associated with transferase activity, carbohydrate metabolic process, hydrolase chitinase activity, polyketide metabolic process cellular response to stress, RNA modification, oxidoreductase activity, and endopeptidase regulator activity. Conversely, 1231 down-regulated genes were linked to oxidoreductase activity, photosynthesis, chlorophyll metabolic process, transmembrane transport, phloem xylem histogenesis, cell-cell junction, amine lyase activity, oxylipin metabolic process, fructose phosphatase activity and methyltransferase activity (Table 3, Fig. 5C, Supplementary Dataset S1).

Four pairwise comparisons were performed to highlight the water
deficit impact on the four scion/rootstock associations: ML/2x WD vs.
ML/2x Ctrl, PL/2x WD vs. PL/2x Ctrl, ML/4x WD vs. ML/4x Ctrl, and
PL/4x WD vs. PL/4x Ctrl. The water deficit in the ML/2x combination
led to 2514 DEGs (1259 up-regulated and 1255 down-regulated). For
PL/2x, fewer DEGs were observed, totaling 1353 (1010 up-regulated
and 343 down-regulated). ML/4x comparison revealed 591 DEGs (335
up-regulated and 256 down-regulated), and PL/4x combination showed
the highest number of DEGs, with 4109 DEGs (1862 up-regulated and
2247 down-regulated). PL/4x stands out from the other associations,
with more down-regulated genes (Fig. 5B, Supplementary Dataset S1).

According to GSEA KEGG enrichment analysis, the galactose meta-
bolism pathway was significantly activated in all comparisons. Protein
processing in the endoplasmic reticulum was notably activated in mul-
tiple comparisons (e.g., ML/2x WD vs. ML/2x Ctrl, ML/4x WD vs. ML/4x
Ctrl and PL/4x WD vs. PL/4x Ctrl). Protein export, glycerolipid meta-
bolism, cutin, suberin, and wax biosynthesis were also activated in ML/
2x WD vs. ML/2x Ctrl. In PL/4x WD vs. PL/4x Ctrl. Various pathways
were activated, including DEGs related to glutathione metabolism, ABC
transporters, MAPK signaling pathway, two oxocarboxylic acid meta-
bolism, endocytosis, biosynthesis of amino acid, valine, leucine and
isoleucine biosynthesis, plant-pathogen interaction, amino sugar and
nucleotide sugar metabolism. Other pathways were significantly sup-
pressed in ML/2x WD vs. ML/2x Ctrl and PL/4x WD vs. PL/4x Ctrl, such
as photosynthesis-related pathways and photosynthesis antenna pro-
teins. Nitrogen cycle, pentose, and glucuronate interconversions were
suppressed in ML/2x WD vs. ML/2x Ctrl. The ribosome pathway was
primarily activated in ML/4x WD vs. ML/4x Ctrl and PL/2x WD vs. PL/
2x Ctrl comparisons. To end, glutathione metabolism was suppressed in
PL/2x WD vs. PL/2x Ctrl comparisons (Fig. 5D, Supplementary Dataset
S1).

3.4.2. Contrasted leaf gene expression between ML and PL scion genotype

PLS-DA analysis revealed a clear differentiation in gene expression based on the scion genotype. ML and PL samples were projected into two distinct clusters, explaining 32 % of the variance in total (19 % of variance explained by latent variable 1 and 13 % explained by latent variable 2) (Fig. 6A).

The identification of 1703 genes further highlighted the effect of the
scion genotype on the leaf transcriptome (Table 3). Among these, 929
genes were up-regulated, associated with processes such as structural
constituent, ROS, metabolic process glycolate, nucleosome organiza-
tion, endoplasmic reticulum chaperone, sodium ion transport, tetra-
pyrrole heme binding cytochrome complex, chitinase activity.
Conversely, 774 genes were down-regulated, related to response to
external stimulus, response to oxidative stress, oxylipin metabolic pro-
cess, glucan synthase activity, and inflorescence development (Fig. 6C,
Supplementary Dataset S1).

Four pairwise comparisons were performed to identify specific DEGs
in response to scion genotype: PL/2x Ctrl vs. ML/2x Ctrl; PL/4x Ctrl vs.
ML/4x Ctrl; PL/2x WD vs. ML/2x WD and PL/4x WD vs. ML/4x WD.
Under control conditions, 849 DEGs were identified between PL/2x and
ML/2x (441 up-regulated and 408 down-regulated), while 531 DEGs
were identified between PL/4x and ML/4x (404 up-regulated and 127
down-regulated). Under water deficit conditions, 1066 DEGs were found
between PL/2x and ML/2x (655 up-regulated and 411 down-regulated),
and 1621 DEGs between PL/4x and ML/4x (1052 up-regulated and 569

A

<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

C

<figure><img src="image_9.png" /></figure>

D

<figure><img src="image_10.png" /></figure>

E

<figure><img src="image_11.png" /></figure>

**Fig. 6.** Global changes in leaf gene expression induce by scion genotype change. (A) PLS-DA plot showing the scion genotype effect (Persian lime: PL vs. Mexican lime: ML samples) on leaf transcriptome profiles in Mexican lime and Persian lime grafted onto 2x and 4x Swingle citrumelo rootstocks (ML/2x, ML/ 4x, PL/2x, PL/4x); (B) Histogram presented the number of DEGs upregulated (black bar) and downregulated (white bar) induced by scion genotype change (FDR <0.05, ||log2FC| ≥ 1); (C) Overrepresented gene ontology (GO) enrich- ment map of scion genotype-responsive DEGs (PL vs. ML); (D) GO enrichment map of DEGs from the interaction multifactorial model "Sion genotype x Treatment". Upregulated (blue node) and downregulated (red node) DEGs. Nodes size is proportional to the number of genes. Additionally, edges thickness is proportional to the similarity between nodes (q-value<0.05); (E) GSEA dot plot showing KEGG pathway enrichment activated and suppressed in response to scion genotype change (FDR<0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

down-regulated) (Fig. 6B, Supplementary Dataset S1).

The GSEA pathway analysis revealed that in PL/2x Ctrl vs. ML/2x
Ctrl, the pathway significantly activated protein processing in the
endoplasmic reticulum and the metabolism of amino and nucleotide
sugar. Protein export and brassinosteroid biosynthesis pathways were
activated in PL/4x Ctrl vs. ML/4x Ctrl. Nucleotide metabolism was
activated in PL/2x WD vs. ML/2x WD. In PL/4x WD vs. ML/4x WD
DEGs, zeatin biosynthesis and mRNA surveillance pathways were activated.

Glucosinolate biosynthesis and 2-oxocarboxylic acid metabolism pathway were suppressed in PL/4x Ctrl vs. ML/4x Ctrl and PL/2x WD vs. ML/2x WD. In PL/4x Ctrl vs. ML/4x Ctrl, pathways such as cyanoamino acid metabolism, MAPK signaling pathway, and plant-pathogen interaction were suppressed. We also found an inactivation of nucleocytoplasmic transport in PL/2x Ctrl vs. ML/2x Ctrl and a suppression of galactose and riboflavin metabolism pathway in PL/2x WD vs. ML/2x WD. PL/4x WD vs. ML/4x WD exhibited activation of monoterpenoid biosynthesis, flavonoid biosynthesis, and plants circadian rhythm pathways (Fig. 6E).

### 3.4.3. Contrasted leaf gene expression between PL and ML scion genotype under water deficit condition

In the multifactorial analysis, the interaction term between treatment and scion genotype revealed 99 genes (Table 3). Of these, 30 were up-regulated, and 69 were down-regulated. Although no enrichment was observed in the up-regulated gene set, notable DEGs included those encoding for a nine-cis-epoxycarotenoid dioxygenase 3 and a lip-oxygenase 3. Involved in ABA and JA biosynthesis were overexpressed. In down-regulated DEGs were enriched in chloroplast nucleoid and

serine-type endopeptidase (Fig. 6D, Supplementary Dataset S1).

### 3.4.4. Change in leaf gene expression due to rootstock ploidy level

Discriminant analysis also revealed a light effect of rootstock ploidy on leaf gene expression, explaining 26 % of the total variance (16 % of variance explained by latent variable 1 and 10 % explained by latent variable 2) (Fig. 7A).

The factorial analysis comparing 2x and 4x rootstock samples did not reveal any significant genes; however, DEGs were detected through pairwise comparisons. The following comparisons were conducted: ML/4x Ctrl vs. ML/2x Ctrl, PL/4x Ctrl vs. PL/2x Ctrl, ML/4x WD vs. ML/2x WD, and PL/4x WD vs. PL/2x WD. No significant difference was observed between ML/4x Ctrl and ML/2x Ctrl. But, in PL/4x Ctrl vs. PL/2x-Ctrl, one down-regulated DEG coding for a respiratory burst oxidase homolog protein B. Under water deficit conditions, 12 DEGs (1 up-regulated and 11 down-regulated) were found between ML/4x WD and ML/2x WD, and 961 genes (710 up-regulated and 261 down-regulated) were differentially expressed between PL/4x WD vs. PL/2x WD (Fig. 7B, Supplementary Dataset S1). Enriched pathways were identified in PL/4x WD vs. PL/2x WD DEGs with activation in pathways related to valine, leucine, and isoleucine degradation, and pantothenate and CoA biosynthesis, phenylpropanoid, and glucosinolate biosynthesis.

<figure><img src="image_12.png" /><figcaption>**Fig. 7.** Global changes in leaf gene expression in response to rootstock ploidy modification. (A) PLS-DA plot showing the effect of rootstock ploidy change (4× vs. 2×) on leaf transcriptome profiles in Mexican lime and Persian lime grafted onto 2× and 4× Swingle citrumelo rootstocks (ML/2×, ML/4×, PL/2×, PL/4×); (B) Histogram presented the number of DEGs upregulated (black bar) and downregulated (white bar) induced by rootstock ploidy change (FDR <0.05, |log2FC| ≥ 1); (C) GSEA dot plot showing KEGG pathway enrichment activated and suppressed in response to ploidy rootstock modification. FDR<0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).</figcaption></figure>
In contrast, several other pathways were suppressed, including photo-synthesis, fatty acid degradation, tyrosine metabolism, ubiquinone and other terpenoid-quinone biosynthesis, pentose phosphate pathway, fructose and mannose metabolism, and photosynthesis antenna proteins (Fig. 7C).

### 3.4.5. Change in roots gene expression under water deficit

In the roots part, samples were tightly separated according to the water deficit treatment, explaining 40 % of the total variance (34 % of variance explained by latent variable 1 and 6 % explained by latent variable 2) (Fig. 8A).

A list of 5195 genes was identified between Ctrl and WD conditions (Table 3). On this number, 1241 genes were up-regulated, mainly associated with transmembrane transporter activity, response to cellular

stimulus, starch glycogen biosynthesis, glycolate process primary, mitochondrial respiratory chain, spindle disassembly autophagosome, galactinol sucrose galactosyltransferase. The other 3954 down-regulated genes were involved in various processes, including enzyme activity, microtubule cytoskeleton, cell wall organization, response stimulus cellular, enzyme inhibitor activity, cell cycle, pectin catabolic process, external pectin cell wall, phloem xylem histogenesis, nitrate assimilation, diphenols related substances, cell junction and DNA replication (Fig. 8C, Supplementary Dataset S2).

Similar pairwise comparisons used for leaf DEG analyses were applied to assess the impact of water deficit on root transcriptome for each scion-rootstock combination. The highest number of DEGs was observed in the ML/2x combination, with 5863 DEGs (1564 up-regulated and 4299 down-regulated), followed by PL/4x with 3147

<figure><img src="image_13.png" /><figcaption>**Fig. 8.** Global changes in root gene expression in response to water deficit treatment. (A) PLS-DA plot showing the effect of water deficit treatment (Controlled: Ctrl vs. Stressed: WD samples) on root transcriptome profiles in Mexican Lime and Persian Lime grafted onto 2× and 4× Swingle citrumelo rootstocks (ML/2×, ML/4×, PL/2×, PL/4×); (B) Histogram presented the number of DEGs upregulated (black bar) and downregulated (white bar) induced by water deficit (FDR <0.05, |log2FC| ≥ 1); (C) Overrepresented gene ontology (GO) enrichment map of water-responsive DEGs (Ctrl vs. WD) upregulated (blue node) and downregulated (red node). Nodes size is proportional to the number of genes. Additionally, edges thickness is proportional to the similarity between nodes (q-value<0.05); (D) GSEA dot plot showing KEGG pathway enrichment activated and suppressed in response to water deficit (FDR<0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).</figcaption></figure>
DEGs (1109 up-regulated and 2038 down-regulated) and ML/4x with 2471 DEGs (927 up-regulated and 1544 down-regulated). The PL/2x combination had the fewest DEGs, with 1170 (482 up-regulated and 688 down-regulated) (Fig. 8B, Supplementary Dataset S2).

KEGG analysis revealed significant pathway enrichment among DEGs between controlled and stressed conditions. In all scion-rootstock combinations, we observed an activation of the galactose metabolism and a suppression of the steroid, sesquiterpenoid, triterpenoid, and phenylpropanoid biosynthesis pathways.

Ribosome, glycerolipid metabolism, and fatty acid elongation pathways were activated in ML/2x, ML/4x, and PL/4x. In ML/2x and PL/4x, the carbon metabolism pathway was activated, and pentose and glucuronate interconversions and mismatch repair were suppressed. In ML/2x WD vs. ML/2x Ctrl, pathways such as protein processing in the endoplasmic reticulum, spliceosome, ribosome biogenesis, and nucleocytoplasmic transport pathways were activated. In PL/2x WD vs. PL/2x Ctrl, genes involved in biosynthesis cofactors were up-regulated, and genes involved in linoleic acid metabolism were down-regulated. Starch and sucrose metabolism pathways were activated in PL/2x WD vs. PL/2x Ctrl and ML/4x WD vs. ML/4x Ctrl. Cutin, suberin, and wax biosynthesis were activated in stressed ML/4x and PL/4x samples. Tryptophan metabolism was suppressed in stressed ML/2x and ML/4x samples.

To end, DEGs involved in alpha-linoleic acid metabolism pathways were down-regulated in stressed PL/2x, ML/4x, and PL/4x samples (Fig. 8D).

### 3.4.6. Contrasted roots gene expression between PL and ML scion genotype

PLSDA analysis in roots transcriptome showed no clear separation between ML and PL samples (Fig. 9A). This observation was supported by the absence gene identified with the multifactorial design. However, some DEGs were found in group pairwise comparisons in stressed conditions. PL/2x WD vs. ML/2x WD showed 4 DEGs: 1 up-regulated coding for CLAVATA3/ESR-RELATED and three down-regulated coding for a hypothetical protein, senescence-related gene one and cyclic nucleotide-gated ion channel 2. PL/4x WD vs. ML/4x WD displayed 4 DEGs up-regulated, 3 of them are coding for caffeic acid 3-O-methyltransferase-caffeic acid and one for receptor-like serine/threonine-protein kinase SD1-6 (Fig. 9B, Supplementary Dataset S2).

### 3.4.7. Change in roots gene expression due rootstock ploidy level

PLS-DA analysis revealed distinct differences in gene expression related to rootstock ploidy, explaining 42 % of the total variance (16 % of variance explained by latent variable 1 and 23 % explained by latent variable 2) (Fig. 10A).

The comparison between 2x and 4x rootstocks allowed us to identify 687 genes (Table 3). On the one hand, 174 genes were up-regulated and

involved in response to stress, transcription regulator activity, cell wall, and protein folding chaperone. On the other hand, 513 genes were down-regulated and associated with transferase kinase activity, thiosulfate transmembrane transporter, cell wall metabolic process, and regulation signaling pathway (Fig. 10C, Supplementary Dataset S2).

To assess the impact of rootstock ploidy on drought tolerance, DEGs were analyzed across four pairwise comparisons: ML/4x Ctrl vs. ML/2x Ctrl, PL/4x Ctrl vs. PL/2x Ctrl, ML/4x WD vs. ML/2x WD, and PL/4x WD vs. PL/2x WD. Under control conditions, 682 DEGs (160 up-regulated and 522 down-regulated) were found between ML/4x and ML/2x, while only 8 DEGs (4 up-regulated and four down-regulated) were observed between PL/4x and PL/2x. Under water deficit conditions, 70 DEGs (58 up-regulated and 12 down-regulated) were identified between ML/4x and ML/2x, and 245 DEGs (87 up-regulated and 158 down-regulated) were found between PL/4x WD and PL/2x WD (Fig. 10B, Supplementary Dataset S2).

KEGG enrichment analysis revealed pathways activated in protein processing in the endoplasmic reticulum and sulfur metabolism for DEGs between ML/4x Ctrl and ML/2x Ctrl. Plant hormone signal transduction pathway was suppressed in ML/4x Ctrl vs. ML/2x Ctrl. In addition, cutin, suberin, and wax biosynthesis pathways were activated while biosynthesis of various plant secondary metabolites was suppressed in PL/4x WD vs. PL/2x WD (Fig. 10E).

### 3.4.8. Change in roots gene expression between 2x and 4x water-deficient rootstocks

An interaction effect between treatment and rootstock ploidy was identified with 74 genes (Table 3). Among these, 68 genes were up-regulated, with GO enrichment in apoplast ion binding and organic biosynthetic processes. Interested DEGs coding for a jasmonate-zim-domain protein 1, acyl-CoA oxidase 4 and a RmlC-like cupins super-family protein were upregulated. Six genes were down-regulated, involved in enzyme regulator activity (Fig. 10D, Supplementary Dataset S2).

## 4. Discussion

In the present work, we studied the response to water deficit in controlled pot condition of 2x Mexican Lime (ML) and 3x Persian Lime (PL) grafted onto 2x and 4x Swingle citrumelo rootstocks to better understand the respective impact of rootstock ploidy and scion genotype on the tolerance/sensitivity to drought (Table 1). During the water deficit, the estimation of water consumption trees showed that the PL/2x combination lost water much faster than the others (day 4), followed by ML/2x (day 7). PL/4x lost the same amount of water on day eight and ML/4x on day nine (Fig. 1C). Furthermore, after five days of water

<figure><img src="image_14.png" /><figcaption>**Fig. 9.** Global changes in root gene expression induce by scion genotype change. (A) PLS-DA plot showing the scion genotype effect (Persian lime: PL vs. Mexican lime: ML samples) on root transcriptome profiles in Mexican lime and Persian lime grafted onto 2x and 4x Swingle citrumelo rootstocks (ML/2x, ML/4x, PL/2x, PL/4x); (B) Histogram presented the number of DEGs upregulated (black bar) and downregulated (white bar) induced by scion genotype change (FDR <0.05, |log2FC|≥1).</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Fig. 10.** Global changes in root gene expression in response to rootstock ploidy modification. (A) PLS-DA plot showing the effect of rootstock ploidy change (4× vs. 2×) on root transcriptome profiles in Mexican lime and Persian lime grafted onto 2x and 4x Swingle citrumelo rootstocks (ML/2×, ML/4×, PL/2×, PL/4×); (B) Histogram presented the number of DEGs upregulated (black bar) and downregulated (white bar) induced by rootstock ploidy change (FDR <0.05, |log2FC| ≥ 1); (C) Overrepresented gene ontology (GO) enrichment map of rootstock ploidy differentiation DEGs; (D) GO enrichment map of DEGs from the interaction "rootstock ploidy x Treatment". Upregulated (blue node) and downregulated (red node). Nodes size is proportional to the number of genes. Additionally, edges thickness is proportional to the similarity between nodes (q-value<0.05); (E) GSEA dot plot showing KEGG pathway enrichment activated and suppressed in response to ploidy rootstock modification. FDR<0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

deficit, associations grafted on 2x rootstock showed a strong leaf wilting phenotype, while trees grafted on 4x Swingle citrumelo did not showed any symptoms of water deprivation (Fig. 1A & B). This first observation confirmed that 4x rootstocks conferred a more limited water consumption than their 2x counterparts, as proposed by Allario et al. (2013). This is in agreement with previous studies also performed in pot condition, suggesting that tetraploidization provides better adaptation to various abiotic stresses, including water deficit, as observed in doubled-diploid Carrizo Citrange rootstocks (Oliveira et al., 2017), trifoliate orange (Wei et al., 2019), "Zhique" (C. wilsonii) (Jiang et al., 2022) and sour orange (Hussain et al., 2023).

## 4.1. Morphological and physiological changes induced by polyploidy result in different capacities to absorb water

Physiological parameter measurements, including stomatal conductance (gs), transpiration (E), and photosynthesis (A), showed that under controlled pot condition, PL/2x association was the most sensitive to water deficit, followed by ML/2x. ML/4x and PL/4x associations were the most tolerant genotypes since they lost water much less quickly (Fig. 1D–F). Investigation of leaf parameters measured in control condition prior the stress showed that polyploidy of the scion led to large changes regarding leaf surface, leaf thickness, leaf RWC, epidermal cell size and stomata size and density (Table 2) as previously reported by several studies (Romero-Aranda et al., 1997; Allario et al., 2011; Padoan
et al., 2013; Guerra et al., 2014; Oliveira et al., 2017; Ruiz et al., 2020; Oustric et al., 2023). In previous works we identified a sizeable phenotypic differentiation at the scion level between PL and ML (Sivager et al., 2021) and at the rootstock level, between 4x and 2x Swingle citrumelo in control condition, and under Huanglongbing infection (Sivager et al., 2022). Interestingly, in the present study in control condition, the ploidy of the rootstock did not induce any change in the 2x and 3x leaf parameters (Table 2). Also, since measurements were performed just prior the stress and because of the short duration of the stress, it is expected that no anatomical change was induced by the water stress. At the scion level, the leaf area was higher in PL than in ML, and a greater weight of fresh leaf material was measured for the PL/2x association (Supplementary Fig. S1). This could explain why, for the different parameters studied, PL/2x was shown to be the most sensitive association. Indeed, a larger leaf area would considerably increase the transpiration area and typically leads to greater water loss, making genotype with a smaller leaf area better adapted to drought. Conversely, the thicker leaves we observed in PL compared to ML (Table 2; Sivager et al., 2021), may enhance water storage and reduce transpiration losses (Wei et al., 2019; Jiang et al., 2022). Polyploidy also impact tissue storage capacity by increasing cell size and vacuole volume, improving the ability of tissues to store more resources, such as nutrients and water. This increased storage capacity in polyploid can be advantageous in environments with limited resources availability (Pacey et al., 2022). In addition, 3x PL exhibits larger stomata but at lower density (Sivager et al., 2021), this may lead to distinct transpiration stream regulation compared to conventional 2x scions. Nevertheless, these adaptive traits in 3x scions when coupled with 2x rootstock were insufficient to offset the greater water loss associated with their larger transpiring leaf surface.

While PL/4x and ML/4x maintained higher stomatal conductance and transpiration rates for longer periods under stress compared to 2x combinations (Fig. 1D–F), they paradoxically showed lower water loss during the initial phase of stress and resisted to wilting more effectively (Fig. 1A–C). In plants, water flow is closely related to the roots’ hydraulic conductivity and the leaves’ transpiration (Xiong et al., 2006; Christmann et al., 2007). At the rootstock levels, 2x Swingle citrumelo exhibits finer and more numerous secondary roots than in its 4x counterpart. It suggests that 2x roots have a larger exchange surface in the soil and would have the capacity to absorb more water and faster than 4x roots (Syvertsen et al., 2000; Allario et al., 2013; Oliveira et al., 2017). Investigation of the hydraulic architecture in absence of stress in different 2x and 4x citrus genotypes (Poncirus and Citrandarin) did not reveal significant difference (Dutra de Souza et al., 2016). However, investigation performed in 2x and 4x Carrizo citrange in control and salt stress condition revealed that strong anatomical differentiation between both genotypes reduced hydraulic conductance in 4x compared to 2x (Ruiz et al., 2016), thereby limiting water uptake and transport and consequently modulating transpiration dynamics in the scion. Such trait would represent a significant advantage under water deficit for trees grown in pot, as 4x rootstocks exhibit slower water uptake and therefore consume less than 2x. The better performance of PL/4x under drought stress, maintaining higher stomatal conductance, photosynthesis, and transpiration rate, can be attributed by anatomical trait variation. Since the hydraulic conductivity is closely related to vessel diameter (Martre et al., 2001), the larger xylem and phloem vessels observed in 3x PL and 4x Swingle citrumelo compared with 2x ML and 2x Swingle citrumelo (Sivager et al., 2021; 2022) likely enhance water transport capacity. Diploid ML may thus require higher water conductivity to supply water to the leaves due to smaller vessels. In contrast, the larger xylem vessels in PL offer less resistance, facilitating water transport to the leaves and supporting the maintenance of leaf gas exchange and overall metabolism under stress.

When stomata are closed under water deficit condition, the ratio of transcellular to apoplastic water flow in the plant tissues becomes more important compared to when stomata are open and the overall water

flux is high and is mostly apoplastic (Steudle, 2000; Morillon and Chrispeels, 2001). Assuming the lipidic plasma membrane's hydraulic conductivity remains similar between 2x and polyploid plants, larger cell size in polyploid plants would favor cell-to-cell water movement, since the main constraint is the number of membranes water must cross (Morillon et al., 2001).

Therefore, under water deficit in controlled pot condition, the hydraulic architecture of the 4x rootstocks appears to actively limit water fluxes from the soil to the leaves, representing an adaptive mechanism for water conservation (Meland et al., 2007; Trifilò et al., 2007; Zach et al., 2010; Tombesi et al., 2010; Ruiz, Quiñones, et al., 2016; Shafqat et al., 2021). Thus, the more limited capacity of 4x to extract water from the soil compared to 2x would make this genotype more sensitive to water deficit as proposed by da Silva Costa et al. (2025a, 2025b). However, in water deficit condition, limited information do exist regarding the biochemical responses and genome expression of that rootstock genotypes associated with 2x and 3x scions. Moreover, the differences in leaf area, stomatal size, and stomatal density between polyploid and diploid scions, and their consequences on plant water flux and potential involvement in drought tolerance, remain poorly documented. The integration of the number and the size of stomata for a single leaf suggests that there is almost a double surface of stomata in PL leaf compared to ML leaf (Table 2). Taking account that the leaf area in PL is approximately doubled compares to ML and that the total leaf weights measured for the different scion/rootstock associations were similar (Supplementary Fig. S1), this means that the number of leaves was increased in ML (ca. doubled) compared to PL making difficult to figure out the impact of the stomatal / size density on the total water flux. Another important issue deals with the speed of the stomatal opening/closure in polyploid compared to 2x that have to our knowledge not yet investigated. Finally, studies integrating key parameters, such as root surface area, rooting depth capacity, rootstock–scion hydraulic conductivity, and canopy transpiration across different scion/rootstock combinations and ploidy levels are still lacking. Such integrative approaches are essential to accurately predict functional hydraulic behaviour in relation to water-deficit adaptation. Indeed, strong anatomical differences do not always predict functional hydraulic behavior (Daler et al., 2025).

## 4.2. Transcriptomic, hormonal and biochemical changes in response to water deficit

Water deficit triggers a cascade of complex coordinated physiological and molecular responses in plants. In this study we integrated hormonal profiling, biochemical assays, and RNA-seq analyses to investigate the drought-responsive molecular determinant of four scion/rootstock combinations. In response to water deficit, DEGs were approximately two times more numerous in roots than in leaves, highlighting the central role of roots in drought stress perception and responses. Roots, being the first organ exposed to drying soil, are responsible for water uptake and undergo great physiological and developmental changes during water deficit. This requires the activation of a large set of genes involved in hormone signaling, nutrient and water transport, and stress regulation (Kalra et al., 2024). In our study, GO enrichment analysis revealed that photosynthesis related genes were predominantly down-regulated in stressed plants. This result supports our physiological results, which showed a decrease in the photosynthetic activity during the water deficit treatment (Fig. 1). Other studies corroborate this, demonstrating that photosynthetic inhibition is one of the earliest responses to drought stress, as plants reallocate resources toward survival mechanisms (Chaves et al., 2009).

Phytohormones play a key role in plant response to biotic and abiotic stresses (Wei et al., 2020). ABA is involved in growth regulation and root-to-shoot signaling during environmental stress response (Takahashi et al., 2020). During drought, ABA is primarily synthesized in roots and transported to leaves, where it induces stomatal closure to thereby
reduce transpirational water loss. While stomatal closure conserves water, it concurrently reduces CO₂ uptake, impacting photosynthesis and overall plant metabolism (Chen et al., 2020). ABA accumulation also triggers extensive transcriptional reprogramming through its signaling pathway (Lee and Luan, 2012; Saradadevi et al., 2017; Ali et al., 2020).

In our experiment, increased ABA concentrations in both stressed scions and rootstocks (Fig. 2) were consistent with the up-regulation of key genes involved in hormonal biosynthesis and signaling. Hormone-related pathways, particularly those associated with ABA signaling, were prominently enriched in both organs. For example, genes encoding protein phosphatase 2C (PP2C; CICLE_v10005200mg) and a highly ABA-induced PP2C gene 2 (CICLE_v10028495mg), major components of ABA signaling cascades, were induced (Lee and Luan, 2012; Rao et al., 2020).

Other hormone-related gene were also differentially expressed, notably, ethylene biosynthesis genes (ACC synthase 1 and ethylene-forming enzyme; CICLE_v10019970mg, CICLE_v10015962mg) and JA biosynthesis genes such as allene oxide synthase (CICLE_v10028333mg) were up-regulated.

In addition, during a water deficit, plants enhance their antioxidant system to mitigate oxidative stress caused by increased ROS levels. This adaptive response involves the upregulation of antioxidant enzymes like superoxide dismutase and catalase, which help to maintain cellular integrity under drought conditions.

Most studies that have investigated the adaptation of polyploid to abiotic stresses have shown that 4x citrus have a more efficient antioxidant system compared to the 2x (Allario et al., 2013; Oliveira et al., 2017; Wei et al., 2019; Khalid et al., 2020; Lourkisti et al., 2020; 2021; 2022; Bonnin et al., 2023; 2024). Similar results were found in *Populus davidiana* (Wang et al., 2025a, 2025b). Various DEGs related to stress response have been identified. Genes involved in response to oxidative stress and oxidoreductase activity were significantly enriched in both roots and leaves organs, including those encoding purple acid phosphatase (CICLE_v10013402m), peroxidase superfamily protein (CICLE_v10021010m, CICLE_v10012179m, CICLE_v10008925m, CICLE_v10020412m), which are implicated in hydrogen peroxide detoxification (Panda et al., 2024). These results are consistent with our electrochemical data, with the presence of signals related to the activation of the antioxidant defense system in drought-stressed samples, suggesting an induction of mechanisms to counteract the oxidative stress caused by the accumulation of ROS (Jara-Palacios et al., 2024).

Additionally, carbohydrate metabolic processes, including galactose metabolism, were up-regulated in stressed scion and rootstock. Under water deficit conditions, plants often accumulate sugar soluble to reduce cells' water potential and improve plants' ability to absorb and retain water (Ke et al., 2025). Osmotic activity was also highlighted, with genes involved in osmolyte pathway upregulated in stressed leaves and roots including stachyose synthase (CICLE_v10006437mg, CICLE_v100 18822mg) and galactinol synthase 1 (CICLE_v10001308mg). These processes are essential for regulating osmotic balance, maintaining energy levels, and protecting cellular structures during water scarcity (Yang et al., 2021a, 2021b).

In addition, several noteworthy transcription factors were identified, involved in hormonal signalisation, stress regulation cellular protection and antioxidant such as No Apical Meristem (NAC) and NAC-like proteins activated by AP3/PI (CICLE_v10008812mg, CICLE_v100323 04mg), a WRKY family transcription factor (CICLE_v10005165mg), and an AP2-EREBP factor (CICLE_v10021265mg) (Supplementary Dataset S1 & S2). These regulators are known to orchestrate transcriptional reprogramming under stress.

### 4.3. Contrasted gene regulation between Persian and Mexican limes

A wide divergence in leaf gene expression was observed between PL and ML scions. At this stage, this variation cannot be definitively attributed to the scion polyploidy, unlike Swingle citrumelo rootstock,

because the two scion genotypes being compared are genetically distinct (Rouiss et al., 2018). Thus, the observed variation in gene expression is likely due to inherent changes in the varietal genetic background rather than being exclusively explained by scion polyploidy.

Pairwise comparisons were performed to identify DEGs between PL and ML under both controlled and stress conditions. The analysis revealed that water deficit stress amplifies transcriptional reprogramming in both genotypes, but with distinct molecular patterns. The higher number of DEGs detected between PL and ML under stress compared to controlled conditions indicates that drought accentuates gene expression variation between the two scions, thereby enhancing their contrasting regulatory and adaptive responses (Fig. 6B). Under stress condition comparison between PL and ML revealed an up-regulation of several heat shock protein (HSP; CICLE_v10029824mg, CICLE_v10030032mg, CICLE_v10016190mg, CICLE_v10016190mg) typically regulated during stress to protect, maintain essential cellular functions. DEGs between PL4x and ML4x revealed genes associated with functional categories relevant to drought tolerance, which may help explain the superior physiological performance observed in PL/4x. Among the up-regulated genes, we identified several encoding transcription factor including C-repeat-binding factor 4 (CICLE_v1 0021923mg, CICLE_v10013766mg) and AP2-EREBP (CICLE_v100 26032mg), as well as stress responsive proteins such as dehydrins (CICLE_v10029285mg, CICLE_v10029285mg), LEA proteins (CICLE_v10013625mg), jasmonate-zim-domain protein (CICLE_v10017198mg, CICLE_v10026376mg, CICLE_v10032858mg, CICLE_v10026527mg) and aquaporin-like superfamily protein (CICLE_v10023306mg, CICLE_v10023306mg) (Supplementary Dataset S1). Together, these transcriptional changes highlight coordinated regulatory, protective, and hydraulic adjustments that may underlie the improved drought tolerance of PL/4x.

DEGs identified through the multifactorial model testing the interaction between scion genotype and treatment revealed 99 genes differentially expressed between PL and ML under water deficit, in leaves. Downregulated genes were enriched in chloroplast-related functions and serine-type endopeptidase activity, explaining the differences response between PL and ML in stressed conditions with contrasted photosynthetic and enzymatic activity (Chaves et al., 2009; D'Ippólito et al., 2021; Moloi and Ngara, 2023). While no GO enrichment was detected in up-regulated genes, several of them exhibit relevant functions. For instance, some are involved in hormone biosynthesis pathway such as gene coding a nine-cis-epoxcarotenoid dioxygenase 3 (CICLE_v10019364mg) associated with ABA biosynthesis and another gene coding a lipoxygenase 3 protein (CICLE_v10024819mg) implicated in JA biosynthesis. Both ABA and JA acid are well known to play a key role in plant water deficit response (Kuromori et al., 2018; Dahro et al., 2023; Rao et al., 2024). In addition, the transcription factor WRKY DNA-binding protein 28 (CICLE_v10032012mg) was also up-regulated (Supplementary Dataset S1).

The influence of the scion genotype on root gene expression condition was not detected under controlled condition. However, under stress conditions pairwise combinations aimed to detect a slight variation of the rootstock transcriptome. When comparing PL/2x and ML/2x, four DEGs were identified: one up-regulated gene coding for CLAVATA3/ESR-RELATED and 3 down-regulated coding for a hypothetical protein, a senescence-related gene 1 and a cyclic nucleotide-gated ion channel 2. Similar comparison between water-deficient PL/4x and ML4x displayed 4 DEGs up-regulated. Three of them are coding for caffeic acid 3-O-methyltransferase caffeic acid, and one for receptor-like serine/threonine-protein kinase SD1-6 (Supplementary Dataset S2). Although limited in number, these DEGs are involved in various signaling pathways related to development and stress responses (Ren et al., 2023; Bashyal et al., 2024).
### 4.4. Rootstock polyploidy modulates gene expression in roots and leaves under water deficit conditions

The comparison of 2× and 4× Swingle citrumelo transcriptome revealed an effect of rootstock ploidy level on gene expression, in both roots and leaves. In the root part, significant differential expression (687 DEGs) was identified between the 4× and 2× rootstock, with distinct and significant enrichment of genes mainly related to response to stress stimulus (Fig. 10C), consistent with previous studies that found that gene expression in 4× were closely associated with stress response, when compared to 2× (Deng et al., 2012; Tan et al., 2015; Wei et al., 2019). Tetraploid may enhance rootstock's ability to manage stress by regulating more efficiently stress responsive genes.

Moreover, genes related to cell walls organization and modification were overrepresented in the 4× vs. 2× rootstock comparison. Polyploid plants exhibited thicker cell walls compared to 2× (Allario et al., 2011; Ruiz et al., 2020; Huang et al., 2025). This modification is crucial for regulating cell wall strength and extensibility, which plays a significant role in the plant's response to environmental stresses. Specifically, the increased elasticity and structural integrity of cell walls in polyploids suggests that they may have a superior ability to regulate osmotic potential, aiding in drought stress avoidance (Verslues et al., 2006; Diallo et al., 2016).

To evaluate how polyploidy modulates the transcriptional response to water deficit, we applied a multifactorial model testing the effect of Rootstock ploidy × Treatment interaction, which revealed 74 DEGs (Table 3). Complementary pairwise comparisons were then conducted to identify DEGs between 2× and 4× genotypes under stress conditions, detecting 70 DEGs between ML/4x and ML/2x, and 245 DEGs between PL/4x WD and PL/2x WD (Fig. 10B). These results provide additional insights into the molecular mechanisms underlying the differential drought response observed in tetraploid rootstocks.

The ABA measurements in stress condition were performed at day-9, when the trees were at the same soil water potential. At this stage, ABA concentrations were higher in 4x rootstocks than in 2×. This difference may reflect the fact that ABA in scions grafted onto 2× rootstocks had already begun to be degraded due to advanced stress progression, whereas ML/4x and PL/4x combinations were still actively responding to the deficit (Fig. 2). The significantly higher ABA content observed in 4× rootstocks under stress is also consistent with our transcriptomic results, with the notable overexpression in "rootstock ploidy × treatment" interaction of gene coding an ABI five binding protein (CICLE_v10011883mg). This gene is well known for its central role in ABA signaling (Collin et al., 1996; Vittozzi et al., 2024) suggesting a stronger ABA-dependent stress signaling cascade in 4x Swingle citrumelo rootstock in stress condition. In addition, in pairwise comparisons under water deficit condition (ML4x vs. ML2x and PL4x vs. PL2x), other genes associated with ABA signaling and stress response were upregulated. These included a highly ABA-induced PP2C gene 2 (CICLE_v10023343mg), an HPT phosphotransmitter (CICLE_v100 27101mg), an early dehydration-responsive protein (CICLE_v1 0014407mg), and late embryogenesis abundant (LEA) proteins (CICLE_v10012748mg, CICLE_v10016351mg) (Supplementary Dataset S2). The coordinated induction of these genes suggests the activation of ABA-dependent protective mechanisms in 4x rootstocks under water deficit.

Similarly, in Liu et al. (2021) triploid mulberry exhibited higher ABA content associated with an up-regulated expression of genes related to ABA signaling, leading to better tolerance under severe drought stress compared to the 2× genotype. Additionally, in 4× rootstocks, several genes associated with other hormonal signaling pathways were upregulated under water deficit conditions. Among them, jasmonate-zim-domain protein 1 (CICLE_v10026376mg) and acyl-CoA oxidase 4 (CICLE_v10013617mg), both key components of the JA biosynthetic pathway, suggest an enhanced activation of jasmonate-mediated responses. This likely contributes to the

reinforcement of antioxidant defenses and osmotic adjustment mechanisms. Similarly, the upregulation of indole-3-acetic acid inducible 29 (CICLE_v10008935mg) points to the involvement of auxin-dependent signaling in maintaining root growth and cell expansion under stress. Altogether, these results indicate a coordinated activation of ABA, JA, and auxin responsive pathways in tetraploid rootstocks, reflecting an integrated hormonal network that supports stress adaptation.

A RmlC-like cupins superfamily protein (CICLE_v10022118mg) was also upregulated in 4x rootstock, this protein is known to support drought adaptation by regulating carbohydrate and secondary metabolism, enhancing ROS detoxification, and contributing to hormone-mediated stress signaling (Chen et al., 2025), further could contributing to their more efficient antioxidant system of 4x rootstock. A DEG coding a voltage dependant anion channel 3 (VDAC 3) was up-regulated in 4x rootstock under stress condition, compared to 2× rootstock. VDACs are key transport proteins embedded in the mitochondrial outer membrane, where they mediate the exchange of metabolites between mitochondria and the cytoplasm. They have already been reported to confer drought tolerance in transgenic tobacco (Yang et al., 2021a, 2021b) and wheat (Yu et al., 2022), as well as to modulate plant pathogen interactions in Arabidopsis (Singh et al., 2024).

Additionally, gene encoding a chalcone and stilbene synthase family protein (CICLE_v10028605mg) involved in flavonoid biosynthesis, and a dehydroquinate dehydratase (CICLE_v10000874mg), involved in chorismate biosynthesis, were identified. These genes participate in the biosynthesis of secondary metabolites and are described as playing an important role in drought response (Hu et al., 2019; Jayaraman et al., 2021; Hou et al., 2022; Liu et al., 2022).

Other GO terms, such as oxidoreductase activity, were up-regulated in 4× rootstock. Electrochemical analyses reveal a common signal (A5) specific to the 4× rootstock (Fig. 3), which is likely associated with antioxidant compounds present in 4× rootstock under stress condition. This finding supports the notion that polyploids' better tolerance to stresses is usually associated with a more effective enzymatic and non-enzymatic antioxidant system, which enables them to limit ROS accumulation and mitigate oxidative damage (Deng et al., 2012; Allario et al., 2013).

A particularly crucial finding for drought tolerance was the upregulation of genes involved in cutin, suberin and wax biosynthesis pathways in 4x Swingle citrumelo rootstock under drought condition (Fig. 10E). Notably encoding cytochrome P450 (CICLE_v10030097mg and CICLE_v10019616mg) previously linked to this same biosynthesis pathways (Höfer et al., 2008) were activated.

Other genes involved in suberin biosynthesis were up-regulated in 4x rootstock, such as 4-coumarate CoA ligase 2 (CICLE_v10025280mg), PHE ammonia-lyase 1 (CICLE_v10011175mg) or O-methyltransferase 1 (CICLE_v10012087mg) (Supplementary Dataset S2). These hydrophobic compounds are critical in reducing water loss by forming protective barriers in plant tissues (Harman-Ware et al., 2021). Suberin, in particular, is deposited around the casparian strip in the root endodermis, limiting water and ion loss while maintaining selective uptake. Under water deficit, enhanced suberization of root cells reduces root hydraulic conductivity, helping to conserve soil water (Calvo-Polanco et al., 2021). Polyploid plants, including citrus, exhibit thicker root cortices and increased suberin deposition, contributing to improved drought tolerance by lowering water loss and enhancing water use efficiency (Ruiz et al., 2020).

In leaves, pairwise comparisons between scions grafted onto 4x versus 2x rootstocks were performed to identify gene expression changes in the scion specifically driven by rootstock ploidy. In controlled leaves, no variation in gene expression was detected between ML/4x and ML/2x, and only one DEGs was identified in PL/4× vs. PL/2× comparison (Fig. 7B). Conversely, under water deficit conditions, only a few DEGs were detected in scion for the ML/4× WD vs. ML/2x WD comparison (12 genes), including one upregulated gene encoding a VDAC3 (CICLE_v10021785mg). Interestingly, the same VDAC3 was also
upregulated in tetraploid rootstocks and a VDAC1 (CICLE_v10032366mg) in the scion of PL4x vs. PL2x comparisons under water deficit, suggesting a conserved role of this mitochondrial channel in stress adaptation across genotypes and tissues.

Gene expression variation in scion due to rootstock ploidy was mainly detected under stress conditions, when comparing PL/4x to PL/ 2x (971) (Fig. 7, Supplementary Dataset S1). This enhanced gene expression correlates with our physiological observations of sustained higher stomatal conductance, photosynthetic activity, and transpiration rates in PL/4x during the water deficit treatment (Fig. 1). Consistently, genes associated with hormonal signaling were also differentially expressed. In the JA pathway, jasmonate ZIM-domain proteins (CICLE_v10017198mg, CICLE_v10026376mg, CICLE_v10032858mg, CICLE_v10026527mg) and allene oxide cyclase (CICLE_v10012528mg, CICLE_v10012552mg, CICLE_v10032510mg), as well as allene oxide synthase (CICLE_v10000825mg), were upregulated, suggesting an enhanced JA biosynthesis and signaling under stress.

Regarding the ABA pathway, upregulation of cytochrome P450 (CICLE_v10011655mg), protein phosphatase 2C (PP2C; CICLE_v10028590mg, CICLE_v10005200mg), and a MYB-domain tran- scription factor (CICLE_v10001979mg) indicates activation of ABA- dependent signaling cascades. In addition, genes involved in ethylene biosynthesis, including ethylene-forming enzyme (CICLE_v100 15965mg, CICLE_v10011912mg, CICLE_v10015962mg), were also induced, reflecting complex hormonal crosstalk in response to water deficit in PL/4x scions. This result could be also linked with the upre- gulation of aquaporin-encoding genes in the PL/4x scion, suggesting improved regulation of transcellular water flux in leaf tissues. DEGs analysis, revealed the up-regulation of aquaporin encoding genes, including a tonoplast intrinsic protein (TIP; CICLE_v10029134mg) and a Nodulin 26-like Intrinsic Proteins (NIP; CICLE_v10016171mg). Inter- estingly, the higher up regulation of aquaporins under stress conditions is expected to enhance transcellular water flux, thereby contributing to osmoregulation in leaf tissues. These findings further suggest that water flux regulation in leaves may also be modulated at the rootstock level. In addition, DEGs encoding dehydrin family proteins (CICLE_ v10002349mg) and a dehydrin LEA protein (CICLE_v10029285mg) were upregulated. Dehydrins are late embryogenesis abundant proteins that play a critical role in protecting cellular structures during water deficit by stabilizing membranes and proteins, thereby contributing to drought tolerance.

Our physiological measurements revealed that, under pot conditions, the PL/4x combination performed exceptionally well under drought stress, maintaining metabolic activity and displaying strong transcrip- tional responses to water deficit. This ability to maintain water flux helps to preserve essential cellular functions and activate adaptive pathways, ultimately conferring improved drought tolerance compared with other scion/rootstock combinations. These findings suggest that triploid scions such as PL interact more effectively with 4x rootstocks under water deficit condition, thereby enhancing the translocation of drought-regulating signals. Although our study did not directly track mobile molecules, previous studies have highlighted the importance of rootstock–scion signaling, particularly the long-distance movement of hormones, metabolites, proteins, and small RNA molecules who can move across the graft union and modulate gene expression in both or- gans (Albacete et al., 2015; Rasool et al., 2020; Wang et al., 2025a, 2025b). The simultaneous regulation of drought-responsive pathways such as ABA metabolism, ROS detoxification, osmoprotection, and cell wall remodeling in both leaves and roots in our dataset is therefore consistent with a coordinated, bidirectional signaling network. Main- taining sap flow under water deficit is critical to ensure this signaling continuity through molecular dialogue supporting coordinated stress responses and efficient redistribution of water and minerals. In this context, polyploid rootstocks appear especially well-suited to sustain signaling and resource allocation under drought, thereby facilitating scion adaptation and growth despite environmental constraints

(Cookson and Ollat, 2013).

5. Conclusion

Plant response to drought is a complex process, leading to vast modifications at the physiological, biochemical and molecular levels. In this study, we investigated drought adaptation mechanisms under controlled pot conditions in four scion-rootstock combinations with different ploidy levels: 2× ML and 3× PL grafted onto 2× and 4× Swingle citrumelo rootstocks (Fig. 11).

Our results highlight that under controlled pot condition, 4x root-
stocks were more effective at regulating water loss and displayed a
delayed wilting phenotype. In addition, in 4x rootstock combination, 3x
PL seems to have better behaviour than 2x ML, maintaining a higher
photosynthetic activity and metabolic performance despite water stress.
Hydraulic conductivity is a key factor in water loss regulation, and
differential roots anatomy between 2× and 4× rootstocks appear to
contribute to the distinct water uptake and consumption patterns
observed.

At the biochemical levels, polyploid combinations displayed several adaptive advantages, including enhanced ABA accumulation in 4x rootstocks under stress supported by transcriptomic data with DEGs involved in hormonal biosynthesis and signalling. Integration of electrochemical profile, with gene expression data, further revealed a more efficient activation of antioxidant defence systems in 4x rootstock combinations, particularly when paired with PL, suggesting a superior capacity to mitigate oxidative stress.

Transcriptomic analysis aimed to identified water deficit responsive DEGs in roots and leaves, influenced by rootstock ploidy and scion genotype. Notably the up-regulation of genes involved in drought adaptation such as those responsible for synthesis of protective barriers (cutin, suberin, and wax) in 4x rootstocks may contributes to their superior ability to limit water loss. Additionally, genes associated with general stress response, hormone biosynthesis and cross-talk (e.g. ABA, JA, ethylene), specialized protein activity related to water transport, osmotic adjustment (e.g. aquaporins, dehydrins) and stress protective proteins (HSP), as well as key transcriptional regulators were differentially expressed, reflecting the broader reprogramming of stress response networks in polyploid combinations. Together, these molecular responses demonstrate that polyploidy, at both the rootstock and scion levels, could modulate citrus's adaptive response to water deficit in controlled pot condition.

Future studies incorporated a dedicated temporal sampling design will be essential to fully resolve the dynamic transcriptional trajectories underlying differential water deficit responses between diploid and polyploid plants. In addition, deeper exploration of molecular mechanisms using other genomic tools, such as advanced epigenetic, sRNA studies or other multi-omics strategies, could provide a more comprehensive understanding of molecular determinants contributing to drought-tolerance in polyploids citrus genotype.

The superior performance of 4x rootstocks under controlled pot condition is particularly advantageous in greenhouse cultivation as they require less irrigation, allow better growth control due to their reduced vigor, and exhibit a more efficient defense system against various stresses. In field conditions, the use of 4x rootstocks seems to be less suited to drought than 2x rootstocks, due to their shallower root systems, which limit water access, particularly during prolonged water deficit periods. However, 4x rootstock use remain promising for high-density orchards, thanks to their dwarfing effect, which facilitates harvesting and contributes also to good fruit quality and yield. Future trials in field environments are necessary to assess the full agronomic relevance of 4x rootstocks. Tetraploid citrus represents also a valuable parental resource for breeding programs, as its hybridization with 2x or 4x parents can generate novel 3x or 4x hybrids with improved stress adaptation and horticultural traits.
<figure><img src="image_16.png" /><figcaption>**Fig. 11.** Schematic model summarizing the water deficit impact on the four scion/rootstock combination. Mexican lime (ML) and Persian lime (PL) grafted onto 2× and 4× Swingle citrumelo rootstocks (ML/2×, ML/4×, PL/2×, PL/4×). ABA: abscisic acid content; gs: stomatal conductance; E: transpiration; A: photosynthesis rate; DEGs: differentially expressed genes.</figcaption></figure>

## Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

## Funding

Leny Calvez's doctoral fellowship was supported by the “Région Guadeloupe”. Investigations were supported by the FEDER-Guadeloupe region “Cavalbio” project.

## Data availability

The RNA-seq data underlying this article are available in Gene Expression Omnibus (GEO) database at [https://www.ncbi.nlm.nih.gov/geo/], and can be accessed with the project accession number: GSE255759.

## CRediT authorship contribution statement

**Leny Calvez:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation. **Alexis Dereeper:** Software, Methodology, Formal analysis. **Benedicte Favreau:** Writing – review & editing, Methodology, Formal analysis. **Gary Sivager:** Investigation, Formal analysis. **Pierre Mournet:** Data curation. **Saturnin Bruyere:** Resources, Conceptualization. **Marie Bonnin:** Investigation, Formal analysis. **Hélène Vignes:** Data curation. **Guillaume Marti:** Formal analysis. **Gerardo Cebrian-Torrejon:** Methodology, Formal analysis. **Antonio Doménech-Carbó:** Methodology. **Barbara Hufnagel:** Formal analysis, Data curation. **Patrick Ollitrault:** Supervision, Funding acquisition. **Raphaël Morillon:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, 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.

## Acknowledgements

Sylvie Fournier from the MetaToul-AgromiX Platform at the Toulouse University is thanked for her valuable help with the hormonal analysis. The authors would like to express their gratitude to all those who contributed, directly or indirectly, to the completion of this work. Their support, input, and efforts have been greatly appreciated throughout the course of this project.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.stress.2025.101166.

## Data availability

Data will be made available on request.

## References

Alam, M.W., Najeeb, J., Naeem, S., Usman, S.M., Nahvi, I., Alismail, F., Abuzir, A., Farhan, M., Nawaz, A., 2022. Electrochemical methodologies for investigating the antioxidant potential of plant and fruit extracts: a review. Antioxid. (Basel Switz.) 11 (6). https://doi.org/10.3390/antiox11061205. Article 6.

Albacete, A., Martínez-Andújar, C., Martínez-Pérez, A., Thompson, A.J., Dodd, I.C., Pérez-Alfocea, F., 2015. Unravelling rootstock × scion interactions to improve food security. J. Exp. Bot. 66 (8). https://doi.org/10.1093/jxb/erv027. Article 8.

Ali, S., Hayat, K., Iqbal, A., Xie, L., 2020. Implications of abscisic acid in the drought stress tolerance of plants. Agronomy 10 (9). https://doi.org/10.3390/agronomy10091323. Article 9.

Allario, T., Brumos, J., Colmenero-Flores, J.M., Iglesias, D.J., Pina, J.A., Navarro, L., Talon, M., Ollitrault, P., Morillon, R., 2013. Tetraploid Rangpur lime rootstock increases drought tolerance via enhanced constitutive root abscisic acid production. Plant Cell Env. 36 (4). https://doi.org/10.1111/pce.12021. Article 4.

Allario, T., Brumos, J., Colmenero-Flores, J.M., Tadeo, F., Froelicher, Y., Talon, M., Navarro, L., Ollitrault, P., Morillon, R., 2011. Large changes in anatomy and physiology between diploid Rangpur lime (Citrus limonia) and its autotetraploid are not associated with large changes in leaf gene expression. J. Exp. Bot. 62 (8). https://doi.org/10.1093/jxb/erq467. Article 8.

Anders, S., Pyl, P.T., Huber, W., 2015. HTSeq—A Python framework to work with high-throughput sequencing data. Bioinformatics 31 (2). https://doi.org/10.1093/bioinformatics/btu638. Article 2.

Andrews, S., 2010. FastQC A quality control tool for high throughput sequence data. Babraham Bioinformatics. https://www.bioinformatics.babraham.ac.uk/projects/fa_stqc/.

Aparicio-Durán, L., Arjona-López, J.M., Hervalejo, A., Calero-Velázquez, R., Arenas-Arenas, F.J., 2021. Preliminary findings of new citrus rootstocks potentially tolerant to foot Rot caused by phytophthora. Horticulturae 7 (10). https://doi.org/10.3390/horticulturae7100389. Article 10.
Balal, R.M., Shahid, M.A., Vincent, C., Zotarelli, L., Liu, G., Mattson, N.S.,
Rathinasabapathi, B., Martínez-Nicolas, J.J., Garcia-Sanchez, F., 2017. Kinnow
mandarin plants grafted on tetraploid rootstocks are more tolerant to Cr-toxicity
than those grafted on its diploids one. Env. Exp. Bot. 140, 8–18. https://doi.org/
10.1016/j.envexpb.2017.05.011.

Barrs, H., Weatherley, P., 1962. A Re-Examination of the Relative Turgidity Technique for Estimating Water Deficits in Leaves. Australian J. Biologi. Sci. 15, 413–428. https://doi.org/10.1071/BIJ9620413

Bashyal, S., Gautam, C.K., Müller, L.M., 2024. CLAVATA signaling in plant-environment interactions. Plant Physiol. 194 (3), 1336–1357. https://doi.org/10.1093/plphys kiad591.

Basu, S., Ramegowda, V., Kumar, A., Pereira, A., 2016. Plant adaptation to drought stress. F1000Res. 5, 1554. https://doi.org/10.12688/f1000research.7678.1.

Bolat, I., Korkmaz, K., Dogan, M., Turan, M., Kaya, C., Seyed Hajizadeh, H., Kaya, O., 2024. Enhancing drought, heat shock, and combined stress tolerance in Myrobalan 29C rootstocks with foliar application of potassium nitrate. BMC Plant Biol. 24 (1), 140. https://doi.org/10.1186/s12870-024-04811-4.

Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30 (15). https://doi.org/10.1093/bioinformatics/btu170. Article 15.

Bonnin, M., Diop, K., Cavelier, G., Crastes, M., Groenewald, R., Nguyen, H.T., Morillon, R., Pontvianne, F., 2025. Physiolog, genetic and epigenetic determinants of water deficit tolerance in fruit plants. Plants 14 (12). https://doi.org/10.3390/plants14121769. Article 12.

Bonnin, M., Favreau, B., Soriano, A., Leonhardt, N., Oustric, J., Lourkisti, R., Ollitrault, P., Morillon, R., Berti, L., Santini, J., 2023. Insight into physiological and biochemical determinants of salt stress tolerance in tetraploid citrus. Antioxidants 12 (8). https://doi.org/10.3390/antiox12081640. Article 8.

Bonnin, M., Soriano, A., Favreau, B., Lourkisti, R., Miranda, M., Ollitrault, P., Oustric, J., Berti, L., Santini, J., Morillon, R., 2024. Comparative transcriptomic analyses of diploid and tetraploid citrus reveal how ploidy level influences salt stress tolerance. Front. Plant Sci. 15, 1469115. https://doi.org/10.3389/fpls.2024.1469115.

Bowman, K.D., Joubert, J., 2020. Chapter 6—Citrus rootstocks: In: Talon, M., Caruso, M., Gmitter, F.G. (Eds.), The Genus Citrus. Woodhead Publishing, pp. 105–127. https://doi.org/10.1016/B978-0-12-812163-4.00006-1.

Bray, E.A., 1997. Plant responses to water deficit. Trends. Plant Sci. 2 (2). https://doi.org/10.1016/S1360-1385(97)82562-9. Article 2.

Bray, E.A., 2007. Plant response to water-deficit stress. Encyclopedia of Life Sciences. John Wiley & Sons, Ltd (Ed.). https://doi.org/10.1002/9780470015902.a0001298.pub2 (p. a0001298.pub2). John Wiley & Sons, Ltd.

Bruyère, S., Luro, F., Froelicher, Y., Morillon, R., Ollitrault, P., 2016. *Poncirus phylogenetic diagnostic SNPs markers are useful to analyse zygotic rates in diploid and tetraploid *Citrus x Poncirus* rootstock seedlings [Conference_item]. Abstract Book Sustainable Citriculture: The Role of Applied Knowledge; IAPAR. https://agritrop.cirad.fr/583551/.

Calvez, L., Dereeper, A., Mournet, P., Froelicher, Y., Bruyère, S., Morillon, R., Ollitrault, P., 2020. Intermediate inheritance with disomic tendency in tetraploid intergeneric *Citrus × Poncirus* hybrids enhances the efficiency of citrus rootstock breeding. Agron. (Basel). https://doi.org/10.3390/agronomy10121961.

Calvez, L., Dereeper, A., Perdereau, A., Mournet, P., Miranda, M., Bruyère, S., Hufnagel, B., Froelicher, Y., Lemainque, A., Morillon, R., Ollitrault, P., 2023. Meiotic behaviors of allotetraploid citrus drive the interspecific recombination landscape, the genetic structures, and traits inheritance in tetrazy progenies aiming to select new rootstocks. Plants (Basel Switz.) 12 (8), 1630. https://doi.org/10.3390/plants12081630.

Calvo-Polanco, M., Ribeyre, Z., Dauzat, M., Reyt, G., Hidalgo-Shrestha, C., Diehl, P., Frenger, M., Simonneau, T., Muller, B., Salt, D.E., Franke, R.B., Maurel, C., Boursiac, Y., 2021. Physiological roles of Caspian strips and suberin in the transport of water and solutes. New Phytol. 232 (6), 2295–2307. https://doi.org/10.1111/nph.17765.

Chaves, M.M., Flexas, J., Pinheiro, C., 2009. Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann. Bot. 103 (4). https://doi.org/10.1093/aob/mcn125. Article 4.

Chen, H., Xiao, M., Shang, W., Wang, X., Gao, H., Zheng, W., Ma, Z., 2025. Genome-wide identification, molecular evolution, and abiotic stress-responsive regulation of Cupin superfamily genes in rice (Oryza sativa L.). Agronomy 15 (8), 1925. https://doi.org/10.3390/agronomy15081925.

Chen, K., Li, G.-J., Bressan, R.A., Song, C.-P., Zhu, J.-K., Zhao, Y., 2020. Abscisc acid dynamics, signaling, and functions in plants. J. Integr. Plant Biol. 62 (1). https://doi.org/10.1111/jipb.12899. Article 1.

Chen, Z.J., 2007. Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids. Annu. Rev. Plant Biol. 58, 377–406. https://doi.org/10.1146/annurev-arplant.58.032806.103835.

Christian, J.I., Martin, E.R., Basara, J.B., Furtado, J.C., Otkin, J.A., Lowman, L.E.L., Hunt, E.D., Mishra, V., Xiao, X., 2023. Global projections of flash drought show increased risk in a warming climate. Commun. Earth Environ. 4 (1), 1–10. https://doi.org/10.1038/s43247-023-00826-1.

Christmann, Op., Grill, E., Huang, J., 2013. Hydraulic signals in long-distance signaling. Curr. Opin. Plant Biol. 16 (3). https://doi.org/10.1016/j.pbi.2013.02.011. Article 3.

Christmann, A., Weiler, E.W., Steudle, E., Grill, E., 2007. A hydraulic signal in root-to-shoot signalling of water shortage. Plant J. 52 (1). https://doi.org/10.1111/j.1365-313X.2007.03234.x. Article 1.

Collin, A., Daszkowska-Golec, A., Szarejko, I., 1996. Updates on the Role of ABSCISIC ACID INSENSITIVE 5 (ABI5) and ABSCISIC ACID-RESPONSIVE ELEMENT BINDING FACTORS (ABFs) in ABA Signaling in Different Developmental Stages in Plants. Cells 10 (8). https://doi.org/10.3390/cells10081996.

Comai, L., 2005. The advantages and disadvantages of being polyploid. Nat. Rev. Genet. 6 (11), 836–846. https://doi.org/10.1038/nrg1711.

Cookson, S.J., Clemente Moreno, M.J., Hevin, C., Nyamba Mendome, L.Z., Delrot, S., Magnin, N., Trossat-Magnin, C., Ollat, N., 2014. Heterografting with nonself rootstocks induces genes involved in stress responses at the graft interface when compared with autografted controls. J. Exp. Bot. 65 (9). https://doi.org/10.1093/jxb/eru145. Article 9.

Cookson, S.J., Ollat, N., 2013. Grafting with rootstocks induces extensive transcriptional re-programming in the shoot apical meristem of grapevine. BMC Plant Biol. 13 (1). https://doi.org/10.1186/1471-2229-13-147. Article 1.

Corneillie, S., De Storme, N., Van Acker, R., Fangel, J.U., De Bruyne, M., De Rycke, R., Geelen, D., Willats, W.G.T., Vanholme, B., Boerjan, W., 2019. Polyploidy affects plant growth and alters cell wall composition. Plant Physiol. 179 (1), 74–87. https://doi.org/10.1104/pp.18.00967.

da Silva Costa, L., Coelho Filho, M.A., Araújo da Silva, M.A., Moreira, A.S., Dos S., Soares Filho, W., Freschi, L., da Silva Gesteira, A., 2025a. Revisiting citrus rootstocks polyploidy as a means to improve drought resilience: sometimes less is more. Plant Cell Env. 48 (1), 149–163. https://doi.org/10.1111>pce.15126.

da Silva Costa, L., Freschi, L., Coelho Filho, M.A., Araújo da Silva, M.A., Dos Santos Nascimento, F., da Silva Gesteira, A., 2025b. Reassessing drought tolerance in citrus tetraploid rootstocks: myth or reality? Physiol. Plant 177 (2), e70199. https://doi.org/10.1111/plp.70199.

Dahro, B., Li, C., Liu, J.-H., 2023. Overlapping responses to multiple abiotic stresses in citrus: from mechanism understanding to genetic improvement. Hortic. Adv. 1 (1), 4. https://doi.org/10.1007/s44281-023-00007-2.

Daler, S., Kaya, O., Kılıç, T., Hajizadeh, H.S., Bay, H., Ates, F., Yilmaz, T., Hatterman-Valenti, H., 2025. Exogenous putrescine enhances lime stress tolerance in grapevine rootstock-scion combinations. BMC Plant Biol. 25 (1), 854. https://doi.org/10.1186/s12870-025-06873-4.

Daler, S., Korkmaz, N., Kılıç, T., Hatterman-Valenti, H., Karadağ, A., Kaya, O., 2024. Modulatory effects of selenium nanoparticles against drought stress in some grapevine rootstock/scion combinations. Chem. Biol. Technol. Agric. 11 (1), 108. https://doi.org/10.1186/s40538-024-00609-6.

Deng, B., Du, W., Liu, C., Sun, W., Tian, S., Dong, H., 2012. Antioxidant response to drought, cold and nutrient stress in two ploidy levels of tobacco plants: low resource requirement confers polytolerance in polyploids? Plant Regul. 66 (1). https://doi.org/10.1007/s10725-011-9626-2. Article 1.

Diallo, A.M., Nielsen, L.R., Kjær, E.D., Petersen, K.K., Ræbild, A., 2016. Polyploidy can confer superiority to West African Acacia senegal (L.) Willd. Trees. Front. Plant Sci. 7, 821. https://doi.org/10.3389/fpls.2016.00821.

D'Ippolito, S., Rey-Burusco, M.F., Feingold, S.E., Guevara, M.G., 2021. Role of proteases in the response of plants to drought. Plant Physiol. Biochem. 168, 1–9. https://doi.org/10.1016/j.plaphy.2021.09.038.

Dogan, M., Bolat, I., Turan, M., Kaya, O., 2025a. Differential rootstock-mediated regulation of physiological and hormonal responses enhances apricot resilience to combined drought and heat stress. Physiol. Plant 177 (4), e70423. https://doi.org/10.1111/plp.70423.

Dogan, M., Bolat, I., Turan, M., Kaya, O., 2025b. Physiological and biochemical responses of "Divadona" peach on Rootpac 20 and Rootpac 40 under drought and heat stress adaptation and its recovery mechanisms. Physiol. Plant 177 (3), e70250. https://doi.org/10.1111/plp.70250.

Domènech-Carbó, A., Cebrian-Torrejón, G., Lopes-Souto, A., Martins-de-Moraes, M., Jorge-Kato, M., Fechine-Tavares, J., Barbosa-Filho, J.M., 2015. Electrochemical ecology: VIMP monitoring of plant defense against external stressors. RSC Adv. 5 (75). https://doi.org/10.1039/C5RA11336A. Article 75.

Domènech-Carbó, A., Martini, M., de Carvalho, L.M., Viana, C., Domènech-Carbó, M.T., Silva, M., 2013. Screening of pharmacologic adulterant classes in herbal formulations using voltammetry of microparticles. J. Pharm. Biomed. Anal. 74, 194–204. https://doi.org/10.1016/j.jpba.2012.10.031.

Dutra de Souza, J., de Andrade Silva, E.M., Coelho Filho, M.A., Morillon, R., Bonatto, D., Micheli, F., da Silva Gesteira, A., 2017. Different adaptation strategies of two citrus scion/rootstock combinations in response to drought stress. PLoS One 12 (5). https://doi.org/10.1371/journal.pone.0177993. Article 5.

Dutra de Souza, J., Rodrigues, V., Barigah, T.S., Ollitrault, P., Da Silva Gesteira, A., Morillon, R., 2016. Whole genome expression and hydraulic architecture of diploid and doubled diploid citrus seedlings in intra and interspecific contexts. Citrus Res. Techno. 37 (2), 206–210. https://doi.org/10.4322/crtICC054.

Froelicher, Y., Bassene, J.-B., Jedidi-Neji, E., Dambier, D., Morillon, R., Bernardini, G., Costantino, G., Ollitrault, P., 2007. Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets. Plant Cell Rep. 26 (7). https://doi.org/10.1007/s00299-007-0314-y. Article 7.

Gill, S.S., Tuteja, N., 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48 (12). https://doi.org/10.1016/j.plaphy.2010.08.016. Article 12.

Girardi, E.A., Ayres, A.J., Girotto, L.F., Peña, L., 2021. Tree growth and production of rainfed Valencia sweet orange grafted onto trifoliate orange hybrid rootstocks under aw climate. Agronomy 11 (12). https://doi.org/10.3390/agronomy11122533. Article 12.

Grosser, J.W., Barthe, G.A., Castle, B., Gmitter, F.G.J., Lee, O., 2015. The development of improved tetraploid citrus rootstocks to facilitate advanced production systems and sustainable citriculture in Florida. "Acta horticulturae" https://pubag.nal.usda.gov/catalog/1239541.

Grosser, J.W., Graham, J.H., Hoyte, A., Rubio, H.M., Bright, D.B., Gmitter, J., Chen, C.X., Gmitter, F.G., 2007. Continued development of rootstocks tolerant of the Phytophthora-Diaprepes complex via greenhouse screening. Proc. Fla. State Hortic. Soc. 120, 103–109.
Grosser, J.W., Graham, J.H., McCoy, C.W., Hoyte, A., Rubio, H.M., Bright, D.B.,
Chandler, J.L., 2003. Development of "Tetrazy" rootstocks tolerant of
the Diaprepes/phytophthora complex under greenhouse conditions. Proc. Fla. State
Hortic. Soc. 116, 263-267.

Guerra, D., Wittmann, M.T.S., Schwarz, S.F., Souza, P.V.D.de, Gonzatto, M.P., Weiler, R.
L., 2014. Comparison between diploid and tetraploid citrus rootstocks:
morphological characterization and growth evaluation. Braganția 73 (1). https://
doi.org/10.1590/brag.2014.007. Article 1.

Gupta, A., Rico-Medina, A., Caño-Delgado, A.I., 2020. The physiology of plant responses to drought. Science 368 (6488), 266-269. https://doi.org/10.1126/science.aaz7614.

Habibi, F., Liu, T., Folta, K., Sarkhosh, A., 2022. Physiological, biochemical, and molecular aspects of grafting in fruit trees. Hortic. Res. 9, uhac032. https://doi.org/10.1093/hr/uhac032.

Harman-Ware, A.E., Sparks, S., Addison, B., Kalluri, U.C., 2021. Importance of suberin biopolymer in plant function, contributions to soil organic carbon and in the production of bio-derived energy and materials. Biotechnol. Biofuels 14, 75. https://doi.org/10.1186/s13068-021-01892-3.

Henderson, C.M., Shulman, N.J., MacLean, B., MacCoss, M.J., Hoofnagle, A.N., 2018.
Skyline performs as well as vendor software in the quantitative analysis of serum 25-
hydroxy vitamin D and vitamin D binding globulin. Clin. Chem. 64 (2), 408-410.
https://doi.org/10.1374/clinchem.2017.282293.

Höfer, R., Briesen, I., Beck, M., Pinot, F., Schreiber, L., Franke, R., 2008. The Arabidopsis
cytochrome P450 CYP86A1 encodes a fatty acid omega-hydroxylase involved in
suberin monomer biosynthesis. J. Exp. Bot. 59 (9), 2347-2360. https://doi.org/
10.1093/jxb/ern101.

Hou, Q., Li, S., Shang, C., Wen, Z., Cai, X., Hong, Y., Qiao, G., 2022. Genome-wide characterization of chalcone synthase genes in sweet cherry and functional characterization of CpCHS1 under drought stress. Front. Plant Sci. 13, 989959. https://doi.org/10.3389/fpls.2022.989959.

Hoyerová, K., Gaudinová, A., Malbeck, J., Dobrev, P.I., Kocábek, T., Šolcová, B.,
Trávníková, A., Kamínek, M., 2006. Efficiency of different methods of extraction
and purification of cytokinins. Phytochemistry 67 (11), 1151-1159. https://doi.org/
10.1016/j.phytochem.2006.03.010.

Hu, B., Yao, H., Gao, Y., Wang, R., Li, F., Guo, J., Li, K., Zhao, M., & Jin, L. (2019). *Overexpression of chalcone synthase gene improves flavonoid accumulation and drought tolerance in tobacco*. https://doi.org/10.21203/rs.2.18297.v1.

Huang, P., Xu, T., Wang, G., Zhang, L., Yao, Y., Zhang, M., Zhang, C., 2025.
Morphological and metabolic changes in Changshan Huyou (Citrus changshan-
huyou) following natural tetraploidization. BMC Plant Biol. 25 (1), 301. https://doi.
org/10.1186/s12870-025-06293-4.

Hussain, S., Sohail, H., Noor, I., Ahmad, S., Ejaz, S., Ali, M.A., Haider, S., Muhammad, S.,
Jaffer, H., Ercisli, S., Morillon, R., Khalid, M., 2023. Physiological and biochemical
determinants of drought tolerance in tetraploid vs diploid sour orange citrus
rootstock. J. Hortic. Sci. Biotechnol. https://doi.org/10.1080/
14620316.2023.2192230.

Idris, N.A., Yasin, H.M., Usman, A., 2019. Voltammetric and spectroscopic determination of polyphenols and antioxidants in ginger (Zingiber officinale Roscoe). Heliyon 5 (5). https://doi.org/10.1016/j.heliyon.2019.061717. Article 5.

Jackson, L.K., Sherman, W.B., 1976. Chromosome counts in "Tahiti" lime. Proc. Fla.
State Hortic. Soc. 88, 458-459.

Jara-Palacios, M.J., Begines, E., Heredia, F.J., Escudero-Gilete, M.L., Hernanz, D., 2024.
Effectiveness of cyclic voltammetry in evaluation of the synergistic effect of phenolic
and amino acids compounds on antioxidant activity: optimization of electrochemical
parameters. Foods 13 (6), 906. https://doi.org/10.3390/foods13060906.

Jayaraman, K., K, V.R., Sevanthi, A.M., S, S, r., Gayatri, C., Mohapatra, V., T, Mandal, P.K.,
2021. Stress-inducible expression of chalcone isomerase2 gene improves
accumulation of flavonoids and imparts enhanced abiotic stress tolerance to rice.
Env. Exp. Bot. 190, 104582. https://doi.org/10.1016/j.envexpbot.2021.104582.

Jiang, J., Yang, N., Li, L., Qin, G., Ren, K., Wang, H., Deng, J., Ding, D., 2022. Tetraploidy
in Citrus wilsonii enhances drought tolerance via synergistic regulation of
photosynthesis, phosphorylation, and hormonal changes. Front. Plant Sci. 13,
875011. https://doi.org/10.3389/fpls.2022.875011.

Kalra, A., Goel, S., Elias, A.A., 2024. Understanding role of roots in plant response to drought: way forward to climate-resilient crops. Plant Genome 17 (1), e20395. https://doi.org/10.1002/tpg2.20395.

Kanehisa, M., Goto, S., 2000. KEGG: kyoto encyclopedia of genes and genomes. Nucleic
Acids Res. 28 (1), 27-30.

Ke, X., Yao, J., Jiang, Z., Gu, X., Xu, P., 2025. Recover and surpass: the mechanisms of
plants transition upon rehydration from drought. Plant Stress 15, 100782. https://
doi.org/10.1016/j.stress.2025.100782.

Keffous, F., Belboukhari, N., Sekkoum, K., Djeradi, H., Cheriti, A., Aboul-Enein, H.Y.,
2016. Determination of the antioxidant activity of limoniastrum feeti aqueous extract
by chemical and electrochemical methods. Cogent Chem. 2 (1). https://doi.org/
10.1080/23312009.2016.1186141. Article 1.

Khalid, M.F., Hussain, S., Anjum, M.A., Ahmad, S., Ali, M.A., Ejaz, S., Morillon, R., 2020.
Better salinity tolerance in tetraploid vs diploid volkamer lemon seedlings is
associated with robust antioxidant and osmotic adjustment mechanisms. J. Plant
Physiol. 244, 153071. https://doi.org/10.1016/j.jplph.2019.153071.

Khan, N., 2025. Molecular insights into ABA-mediated regulation of stress tolerance and development in plants. Int. J. Mol. Sci. 26 (16), 7872. https://doi.org/10.3390/
ijms26167872.

Krug, C.A., 1943. Chromosome numbers in the subfamily auriantioideae with special reference to the genus citrus. Bot. Gaz. 104 (4). Article 4.

Kucera, M., Isserlin, R., Arkhangorodsky, A., Bader, G.D., 2016. AutoAnnotate: a
Cytoscape app for summarizing networks with semantic annotations. F1000Res.
5, 1717. https://doi.org/10.12688/f1000research.90090.1.

Kunwar, S., Meyering, B., Grosser, J., Gmitter, F.G., Castle, W.S., Albrecht, U., 2023.
Field performance of 'Valencia' orange trees on diploid and tetraploid rootstocks in
different huanglongbing-endemic growing environments. Sci. Hortic. 309, 111635.
https://doi.org/10.1016/j.scienta.2022.111635.

Kuromori, T., Seo, M., Shinozaki, K., 2018. ABA transport and plant water stress responses. Trends Plant Sci. 23 (6). https://doi.org/10.1016/j.tplants.2018.04.001. Article 6.

Labarga, D., Mairata, A., Puelles, M., Martín, I., Albacete, A., García-Escudero, E.,
Pou, A., 2023. The rootstock genotypes determine drought tolerance by regulating
aquaporin expression at the transcript level and phytohormone balance. Plants 12
(4), 718. https://doi.org/10.3390/plants12040718.

Lee, S.C., Luan, S., 2012. ABA signal transduction at the crossroad of biotic and abiotic stress responses. Plant Cell Env. 35 (1). https://doi.org/10.1111/j.1365-3040.2011.02426.x. Article 1.

Li, X., Zhang, L., Wei, X., Datta, T., Wei, F., Xie, Z., 2024. Polyploidization: a biological force that enhances stress resistance. Int. J. Mol. Sci. 25 (4), 1957. https://doi.org/10.3390/ijms25041957.

Liu, H., Sun, H., Bao, L., Han, S., Hui, T., Zhang, R., Zhang, M., Su, C., Qian, Y., Jiao, F.,
2021. Secondary metabolism and hormone response reveal the molecular
mechanism of triploid mulberry (Morus Alba L.) trees against drought. Front. Plant
Sci. 12, 720452. https://doi.org/10.3389/fpls.2021.720452.

Liu, Z., Cheng, S., Liu, X.-Q., Kuca, K., Hashem, A., Al-Arjani, A.-B.F., Almutairi, K.F.,
Abd>Allah, E.F., Wu, Q.-S., Zou, Y.-N., 2022. Cloning of a CHS gene of poncirus
trifoliata and its expression in response to soil water deficit and arbuscular
mycorrhizal fungi. Front. Plant Sci. 13. https://doi.org/10.3389/fpls.2022.1101212.

Longley, A.E., 1925. Polycary, polyspory, and polyploidy in citrus and citrus relatives.
J. Wash. Acad. Sci. 15 (14). Article 14.

Losada, J.M., Blanco-Moure, N., Fonollá, A., Martínez-Ferrí, E., Hormaza, J.I., 2023.
Hydraulic tradeoffs underlie enhanced performance of polyploid trees under soil
water deficit. Plant Physiol. 192 (3), 1821-1835. https://doi.org/10.1093/plphys/
kiad204.

Lourkisti, R., Froelicher, Y., Herbette, S., Morillon, R., Giannettini, J., Berti, L.,
Santini, J., 2021. Triploidy in citrus genotypes improves leaf gas exchange and
antioxidant recovery from water deficit. Front. Plant Sci. 11, 615335. https://doi/
org/10.3389/fpls.2020.615335.

Lourkisti, R., Froelicher, Y., Herbette, S., Morillon, R., Tomi, F., Gibernau, M.,
Giannettini, J., Berti, L., Santini, J., 2020. Triploid citrus genotypes have a better
tolerance to natural chilling conditions of photosynthetic capacities and specific leaf
volatile organic compounds. Front. Plant Sci. 11, 330. https://doi.org/10.3389/
fpls.2020.00330.

Lourkisti, R., Froelicher, Y., Morillon, R., Berti, L., Santini, J., 2022. Enhanced
photosynthetic capacity, osmotic adjustment and antioxidant defenses contribute to
improve tolerance to moderate water deficit and recovery of triploid citrus
genotypes. Antioxid. (Basel Switz.) 11 (3). https://doi.org/10.3390/
antiox11030562. Article 3.

Love, M.I., Huber, W., Anders, S., 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15 (12), 550. https://doi.org/10.1186/s13059-014-0550-8.

Luro, F., Bloquel, E., Tomu, B., Costantino, G., Tur, I., Riolacci, S., Varamo, F.,
Ollitrault, P., Froelicher, Y., Curk, F., Pailly, O., 2017. The INRA-CIRAD citrus
germplasm collection of San Giuliano, Corsica. In: Zech-Matterne, V., Fiorentino, G.
(Eds.), AGRUMED: archaeology and History of Citrus Fruit in the Mediterranean:
Acclimatization, Diversifications, Uses. Publications du Centre Jérard. https://
doi.org/10.4000/books.pcbj.2232.

Maere, S., Heymans, K., Kuiper, M., 2005. BiNGO: a Cytoscape plugin to assess
overrepresentation of gene ontology categories in Biological networks.
Bioinformatics 21 (16), 3448-3449. https://doi.org/10.1093/bioinformatics/
bti551.

Mahmoud, L.M., Killiny, N., Holden, P., Gmitter, F.G., Grosser, J.W., Dutt, M., 2023.
Physiological and biochemical evaluation of salt stress tolerance in a citrus
tetraploid somatic hybrid. Horticulturea 9 (11). https://doi.org/10.3390/
horticulturea9111215. Article 11.

Mahmoud, L., Quirico, M., Grosser, J., Killiny, N., Dutt, M., 2024. Phenotypic and physiological traits of autotetraploid 'UF SunLime' finger lime hybrid plants. Plant Cell Tissue Organ Cult. (PCTOC) 158. https://doi.org/10.1007/s11240-024-02827-0.

Martre, P., Cochard, H., Durand, J.-L., 2001. Hydraulic architecture and water flow in
growing grass tillers (Festuca arundinacea Schreb.). Plant Cell Env. 24 (1). https://
doi.org/10.1046/j.1365-3040.2001.00657.x. Article 1.

Meland, M., Moe, M.E., Frøynes, O., 2007. Differences in growth and development of
functional xylem of grafted and budded sweet cherry trees. Acta Hortic. 732. https://
doi.org/10.17660/ActaHortic.2007.732.47. Article 732.

Merico, D., Isserlin, R., Stueker, O., Emili, A., Bader, G.D., 2010. Enrichment map: a
network-based method for gene-set enrichment visualization and interpretation.
PLoS One 5 (11), e13984. https://doi.org/10.1371/journal.pone.0013984.

Misiukevičius, E., Mažeikienė, I., Gossard, J., Starkus, A., Stanys, V., 2023.
Transcriptome analysis of diploid and autotetraploid hemerocallis response to
drought stress. Horticulturea 9 (11), 1194. https://doi.org/10.3390/
horticulturea9111194.

Misiukevičius, E., Mažeikienė, I., Stanys, V., 2024. Plody's role in daily plant resilience to drought stress challenges. Biology 13 (5), 289. https://doi.org/10.3390/
biology13050289.
Moloi, S.J., Ngara, R., 2023. The roles of plant proteases and protease inhibitors in drought response: a review. Front. Plant Sci. 14. https://doi.org/10.3389/fps.2023.1165845.

Morillon, R., Chrispeels, M.J., 2001. The role of ABA and the transpiration stream in the regulation of the osmotic water permeability of leaf cells. Proc. Natl. Acad. Sci. 98 (24), 14138-14143. https://doi.org/10.1073/pnas.231471998.

Morillon, R., Liénard, D., Chrispeels, M.J., Lassalles, J.P., 2001. Rapid movements of plants organs require solute-water cotransporters or contractile proteins. Plant Physiol. 127 (3), 720-723.

Mouhaya, W., Allario, T., Costantino, G., Andres, F., Talon, M., Luro, F., Ollitrault, P., Morillon, R., 2008. *Citrus tetraploid rootstocks are more tolerant to salt stress than diploid* [Conference item]. In: Modern Variety Breeding for Present and Future Needs. 18th EUCARPIA General Congress, 9-12 September 2008, Valencia, Spain; n. https://agritrop.cirad.fr/547588/.

Navarro, L., Aleza, P., Cuenca, J., Juarez, J., Pina, J.A., Ortega, C., Navarro, A., Ortega, V., 2015. The mandarin triploid breeding program in Spain. Acta Hortic. 1065. Article 1065.

Oliveira, T.M., Yahmed, J.B., Dutra, J., Maserti, B.E., Talon, M., Navarro, L., Ollitrault, P., da, S., Gesteira, A., Morillon, R., 2017. Better tolerance to water deficit in doubled diploid 'Carrizo citrange' compared to diploid seedlings is associated with more limited water consumption. Acta Physiol. Plant. 39 (9). https://doi.org/10.1007/s11738-017-2497-3. Article 9.

Ollitrault, P., Dambier, D., Luro, F., Froelicher, Y., 2008. Ploidy manipulation for breeding seedless triploid citrus. In: Janick, J. (Ed.), Plant Breeding Reviews. John Wiley & Sons, Inc, pp. 323-352. https://doi.org/10.1002/9780470380130.ch7.

Oustric, J., Herbette, S., Quilichini, Y., Morillon, R., Giannettini, J., Berti, L., Santini, J., 2021. Tetraploid Citruelo 4475 rootstocks improve diploid common clementine tolerance to long-term nutrient deficiency. Sci. Rep. 11 (1). https://doi.org/10.1038/s41598-021-8838-3. Article 1.

Oustric, J., Lourkisti, R., Herbette, S., Morillon, R., Paolacci, G., Gonzalez, N., Berti, L., Santini, J., 2020. Effect of propagation method and ploidy level of various rootstocks on the response of the common clementine (Citrus clementina Hort. Ext Tan) to a mild water deficit. Agriculture 10 (8). https://doi.org/10.3390/agriculture10080321. Article 8.

Oustric, J., Morillon, R., Hussain, S., Santini, J., Ruiz, M., 2023. *Challenges and Advances in Citrus breeding: Polyploidy manipulation for Better Production* [Book section]. Citrus Production: Technological Advancements and Adaptation to Changing Climate. CRC Press. https://doi.org/10.1201/9781003119852-4.

Oustric, J., Morillon, R., Luro, F., Herbette, S., Lourkisti, R., Giannettini, J., Berti, L., Santini, J., 2017. Tetraploid carrizo citrange rootstock (Citrus sinensis Osb. X Poncirus trifoliata L. Raf.) enhances natural chilling stress tolerance of common clementine (Citrus clementina Hort. Ext Tan). J. Plant Physiol. https://doi.org/10.1016/j.jplph.2017.04.014.

Oustric, J., Morillon, R., Luro, F., Herbette, S., Martin, P., Giannettini, J., Berti, L., Santini, J., 2019a. Nutrient deficiency tolerance in citrus is dependent on genotype or ploidy level. Front. Plant Sci. https://doi.org/10.3389/fpls.2019.00127.

Oustric, J., Quilichini, Y., Morillon, R., Herbette, S., Luro, F., Giannettini, J., Berti, L., Santini, J., 2019b. Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones. Plant Physiol. Biochem. 135, 372-384. https://doi.org/10.1016/j.plaphy.2018.12.020.

Pacey, E.K., Maherali, H., Husband, B.C., 2022. Polyploidy increases storage but decreases structural stability in *Arabidopsis thaliana*. Curr. Biol. 32 (18), 4057-4063. https://doi.org/10.1016/j.cub.2022.07.019 e3.

Padoan, D., Mossad, A., Chiancone, B., Germana, M.A., Khan, P.S.S.V., 2013. Ploidy levels in Citrus clementine affects leaf morphology, stomatal density and water content. Theor. Exp. Plant Physiol. 25 (4). https://doi.org/10.1590/S2197-00252013000400006.

Palmeira-Mello, M.V., Souza, J.L., Pérez, A.F., Cavalcanti, A.dos S., Kahn, S.A., Passe-Coutrin, N., Guerra, I.R., Doménech-Carbó, A., Jauregui-Haza, U.J., de Souza, A.M. T., Lanznaster, M., Cebrián-Torrejón, G., 2021. Insights of Tris(2-pyridylmethyl) amine as anti-tumor agent for osteosarcoma: experimental and in silico studies. J. Mol. Struct. 1228, 129773. https://doi.org/10.1016/j.molstruc.2020.129773.

Panda, S.K., Gupta, D., Patel, M., Vyver, C.V.D., Koyama, H., 2024. Functionality of reactive oxygen species (ROS) in plants: toxicity and control in poaceae crops exposed to abiotic stress. Plants 13 (15). https://doi.org/10.3390/plants13152071. Article 15.

Peng, Z., Bredeson, J.V., Wu, G.A., Shu, S., Rawat, N., Du, D., Parajuli, S., Yu, Q., You, Q., Rohsar, D.S., Gmitter, F.G., Deng, Z., 2020. A chromosome-scale reference genome of trifoliate orange (Poncirus trifoliata) provides insights into disease resistance, cold tolerance and genome evolution in Citrus. Plant J. 104 (5). https://doi.org/10.1111/tpj.14993. Article 5.

Perrone, I., Gambino, G., Chitarra, W., Vitali, M., Pagliarani, C., Riccomagno, N., Balestrini, R., Kaldenhoff, R., Uehlein, N., Gribauldo, I., Schubert, A., Lovisolo, C., 2012. The grapevine root-specific aquaporin VvPIP2;4 controls root hydraulic conductance and leaf gas exchange under well-watered conditions but not under water stress. Plant Physiol. 160 (2), 965-977. https://doi.org/10.1104/pp.112.203455.

Podda, A., Cecucci้s, G., Mouhaya, W., Centeno, D., Rofidal, V., Del Carratore, R., Luro, F., Morillon, R., Ollitrault, P., Maserti, B.E., 2013. Salt-stress induced changes in the leaf proteome of diploid and tetraploid mandarins with contrasting Na+ and Cl- accumulation behaviour. J. Plant Physiol. 170 (12). https://doi.org/10.1016/j.jplph.2013.03.006. Article 12.

Pons, S., Fournier, S., Chervin, C., Bécard, G., Rochange, S., Frey, N.F.D., Pagès, V.P., 2020. Phytohormone production by the arbuscular mycorrhizal fungus Rhizophagus

irregularis. PLoS One 15 (10), e0240886. https://doi.org/10.1371/journal.pone.0240886.

Ræbild, A., Ananamawhat-Jónsson, K., Egertsdotter, U., Immanen, J., Jensen, A.M., Koutouleas, A., Martens, H.J., Nieminen, K., Olofsson, J.K., Röper, A.-C., Salojarvi, J., Strömvik, M., Vatanparast, M., Vivian-Smith, A., 2024. Polyploidy – A tool in adapting trees to future climate changes? A review of polyploidy in trees. For. Ecol. Manag. 560, 121767. https://doi.org/10.1016/j.foreco.2024.121767.

Rao, M.J., Duan, M., Eman, M., Yuan, H., Sharma, A., Zheng, B., 2024. Comparative analysis of citrus species' Flavonoid metabolism, gene expression profiling, and their antioxidant capacity under drought stress. Antioxid. (Basel Switz.) 13 (9), 1149. https://doi.org/10.3390/antiox13091149.

Rao, S., Tian, Y., Xia, X., Li, Y., Chen, J., 2020. Chromosome doubling mediates superior drought tolerance in Lycium ruthenicum via abscisic acid signaling. Hortic. Res. 7 (1). https://doi.org/10.1038/s41438-020-0260-1. Article 1.

Rasool, A., Mansoor, S., Bhat, K.M., Hassan, G.I., Baba, T.R., Alyemeni, M.N., Alshali, A. A., El-Serehy, H.A., Paray, B.A., Ahmad, P., 2020. Mechanisms underlying graft union formation and rootstock scion interaction in horticultural plants. Front. Plant Sci. 11, 1778. https://doi.org/10.3389/fpls.2020.590847.

Rawat, N., Kumar, B., Albrecht, U., Du, D., Huang, M., Yu, Q., Zhang, Y., Duan, Y.-P., Bowman, K.D., Gmitter, F.G., Deng, Z., 2017. Genome resequencing and transcriptome profiling reveal structural diversity and expression patterns of constitutive disease resistance genes in Huanglongbing-tolerant poncirus trifoliata and its hybrids. Hortic. Res. 4 (1). https://doi.org/10.1038/hortres.2017.64. Article 1.

Ren, H., Gao, Z., Chen, L., Wei, K., Liu, J., Fan, Y., Davies, W.J., Jia, W., Zhang, J., 2007. Dynamic analysis of ABA accumulation in relation to the rate of ABA catabolism in maize tissues under water deficit. J. Exp, Bot 58 (2), 211-219. https://doi.org/10.1093/jxb/erl117.

Ren, X., Chen, J., Chen, S., Zhang, H., Li, L., 2023. Genome-wide identification and characterization of CLAVATA3/EMBRYO SURROUNDING REGION (CLE) gene Family in foxtail millet (Setaria italica L.). Genes 14 (11), 2046. https://doi.org/10.3390/genes14112046.

Rohart, F., Gautier, B., Singh, A., Lé Cao, K.-A., 2017. mixOmics: an R package for 'omics feature selection and multiple data integration. PLoS Comput. Biol. 13 (11). https://doi.org/10.1371/journal.pcbi.1005752. Article 11.

Romero-Aranda, R., Bondada, B.R., Syvertsen, J.P., Grosser, J.W., 1997. Leaf characteristics and net gas exchange of diploid and autotetraploid citrus. Ann. Bot. 79 (2), 153-160. https://doi.org/10.1006/anbo.1996.0326.

Rossdeutsch, L., Schreiner, R.P., Skinkis, P.A., Deluc, L., 2021. Nitrate uptake and transport properties of two grapevine rootstocks with varying vigor. Front. Plant Sci. 11. https://doi.org/10.3389/fpls.2020.608813.

Rouissi, H., Bakry, F., Froelicher, Y., Navarro, L., Aleza, P., Ollitrault, P., 2018. Origin of C. latifolia and C. aurantiifolia triploid limes: the preferential disomic inheritance of doubled-diploid 'Mexican' lime is consistent with an interplay hybridization hypothesis. Ann. Bot. 121 (3), 571-585. https://doi.org/10.1093/aob/mcx179.

Ruiz, M., Oustric, J., Santini, J., Morillon, R., 2020. Synthetic polyploidy in certified crops. Front. Plant Sci. 11, 540894. https://doi.org/10.3389/fpls.2020.540894.

Ruiz, M., Quinones, A., Martínez-Alcántara, B., Aleza, P., Morillon, R., Navarro, L., Primo-Millo, E., Martínez-Cuenca, M.-R., 2016a. Effects of salinity on diploid (2x) and doubled diploid (4x) Citrus macrophylla genotypes. Sci. Hortic. https://doi.org/10.1016/j.scienta.2016.05.007.

Ruiz, M., Quinones, A., Martínez-Cuenca, M.R., Aleza, P., Morillon, R., Navarro, L., Primo-Millo, E., Martínez-Alcántara, B., 2016b. Tetraploidy enhances the ability to exclude chloride from leaves in carrizo citrange seedlings. J. Plant Physiol. 205, 1-10. https://doi.org/10.1016/j.jplph.2016.08.002.

Saleh, B., Allario, L., Dambier, D., Ollitrault, P., Morillon, R., 2008. Tetraploid citrus rootstocks are more tolerant to salt stress than diploid. C. R. Biol. 331 (9). https://doi.org/10.1016/j.crvi.2008.06.007. Article 9.

Sankar, C., Rajangam, J., Kavino, M., Auxcilia, J., Premalakshmi, V., Rajamanickam, C., Sundarrajan, R.V., Naveen, R., 2025. Impacts of polyploidy on phenotypic expression in Musa spp. Progenies. Discov. Appl. Sci. 7 (9), 948. https://doi.org/10.1007/s42452-025-07417-z.

Saradadevi, R., Palta, J.A., Siddique, K.H.M., 2017. ABA-mediated stomatal response in regulating water use during the development of terminal drought in wheat. Front. Plant. Sci. 8, 1251. https://doi.org/10.3389/fpls.2017.01251.

Schneider, J.R., Caverzan, A., Chavarria, G., 2019. Water deficit stress, ROS involvement, and plant performance. Arch. Agron. Soil Sci. 65 (8). https://doi.org/10.1080/03650340.2018.1556789. Article 8.

Shafqat, W., Mazrou, Y.S.A., Sami-ur-Rehman, Nehela, Ikram, Y., Bibi, S., Naqvi, S., A, S., Hameed, M., Jaskani, M.J, 2021. Effect of three water regimes on the physiological and anatomical structure of stem and leaves of different citrus rootstocks with distinct degrees of tolerance to drought stress. Horticulturae 7 (12). https://doi.org/10.3390/horticulturae7120554. Article 12.

Singh, N., Ravi, B., Saini, L.K., Pandey, G.K., 2024. Voltage-dependent anion channel 3 (VDAC3) mediates *P. syringae* induced ABA-SA signaling crosstalk in *Arabidopsis thaliana*. Plant Physiol. Biochem. 206, 108237. https://doi.org/10.1016/j.plaphy.2023.108237.

Sivager, G., Calvez, L., Bruyere, S., Boisne-Noc, R., Brat, P., Gros, O., Ollitrault, P., Morillon, R., 2021. Specific physiological and anatomical traits associated with polyploidy and better detoxification processes contribute to improved huanglongbing tolerance of the Persian lime compared with the Mexican lime. Front. Plant Sci. 12, 1343. https://doi.org/10.3389/fpls.2021.685679.

Sivager, G., Calvez, L., Bruyere, S., Boisne-Noc, R., Hufnagel, B., Cebrian-Torrejon, G., Doménech-Carbó, A., Gros, O., Ollitrault, P., Morillon, R., 2022. Better tolerance to Huanglongbing is conferred by tetraploid Swingle citrumelo rootstock and is
influenced by the ploidy of the scion. Front. Plant Sci. 13. https://www.frontiersin.org/articles/10.3389/fpls.2022.1030862.

Steudle, E., 2000. Water uptake by plant roots: an integration of views. Plant Soil 226, 45–56. https://doi.org/10.1023/A:1026439226716.

Syvertsen, J.P., Lee, L.S., Grosser, J.W., 2000. Limitations on growth and net gas exchange of diploid and tetraploid citrus rootstock cultivars grown at elevated CO2. J. Am. Soc. Hortic. Sci. 125. https://doi.org/10.21273/JASHS.125.2.228.

Takahashi, F., Kuromori, T., Urano, K., Yamaguchi-Shinozaki, K., Shinozaki, K., 2020. Drought stress responses and resistance in plants: from cellular responses to long-distance intercellular communication. Front. Plant Sci. 11, 1407. https://doi.org/10.3389/fpls.2020.556972.

Talon, M., Gmitter, F.G., 2008. Citrus genomics. Int. J. Plant Genom. 2008, 1–17. https://doi.org/10.1155/2008/528361.

Tan, F.-Q., Tu, H., Liang, W.-J., Long, J.-M., Wu, X.-M., Zhang, H.-Y., Guo, W.-W., 2015. Comparative metabolic and transcriptional analysis of a doubled diploid and its diploid citrus rootstock (C. junos cv. Ziyang xiangcheng) suggests its potential value for stress resistance improvement. BMC Plant Biol. 15, 89. https://doi.org/10.1186/s12870-015-0450-4.

Tombesi, S., Johnson, R.S., Day, K.R., DeJong, T.M., 2010. Relationships between xylem vessel characteristics, calculated axial hydraulic conductance and size-controlling capacity of peach rootstocks. Ann. Bot. 105 (2). https://doi.org/10.1093/aob/mcp281. Article 2.

Tossi, V.E., Martínez Tosar, L.J., Laino, L.E., Iannicelli, J., Regalado, J.J., Escandón, A.S., Baroli, I., Causin, H.F., Pitta-Álvarez, S.I., 2022. Impact of polyploidy on plant tolerance to abiotic and biotic stresses. Front. Plant Sci. 13. https://doi.org/10.3389/fpls.2022.869423.

Trifilò, P., Lo Gullo, M.A., Nardini, A., Pernice, F., Salleo, S., 2007. Rootstock effects on xylem conduit dimensions and vulnerability to cavitation of Olea europaea L. Trees 21 (5). https://doi.org/10.1007/s00468-007-0148-9. Article 5.

Van de Peer, Y., Mizrachi, E., Marchal, K., 2017. The evolutionary significance of polyploidy. Nat. Rev. Genet. 18 (7). https://doi.org/10.1038/nrg.2017.26. Article 7.

Verslues, P.E., Agarwal, M., Katiyar-Agarwal, S., Zhu, J., Zhu, J.-K., 2006. Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. Plant J.: Cell Mol. Biol. 45 (4), 523–539. https://doi.org/10.1111/j.1365-313X.2005.02593.x.

Wang, K., Singh, D., Zeng, Z., Coleman, S.J., Huang, Y., Savich, G.L., He, X., Mieczkowski, P., Grimm, S.A., Perou, C.M., MacLeod, J.N., Chiang, D.Y., Prins, J.F., Liu, J., 2010. MapSplice: accurate mapping of RNA-seq reads for splice junction discovery. Nucleic Acids Res. 38 (18). https://doi.org/10.1093/nar/gkq622. Article 18.

Vittozzi, Y, Krüger, T, Majee, A, Née, G, Wenkel, S, 2024. ABI5 binding proteins: key players in coordinating plant growth and development. Trends Plant Sci. 29 (9), 1006–1017. https://doi.org/10.1016/j.tplants.2024.03.009.

Wang, M., Dai, W., Du, J., Ming, R., Dahro, B., Liu, J.-H., 2019. ERF109 of trifoliate orange (Poncirus trifoliata (L.) Raf.) Contributes to cold tolerance by directly regulating expression of Prx1 involved in antioxidative process. Plant Biotechnol. J. 17 (7). https://doi.org/10.1111/pbi.13056. Article 7.

Wang, P., Liu, F., Sun, Y., Liu, X., Jin, L., 2025a. Physiological and molecular insights into citrus rootstock–Scion interactions: compatibility, signaling, and impact on growth, fruit quality and stress responses. Horticulturae 11 (9), 1110. https://doi.org/10.3390/horticulturae11091110.

Wang, X., Lu, M., Liu, Y., Li, S., Yu, Y., Chen, S., Meng, N., Li, K., Liu, W., Chen, su., 2025b. Generation and characterization of a tetraploid Populus davidiana. Plant Cell Tissue Organ Cult. (PCTOC) 160. https://doi.org/10.1007/s11240-024-02953-9.

Wei, T., Wang, Y., Liu, J.-H., 2020. Comparative transcriptome analysis reveals synergistic and disparate defense pathways in the leaves and roots of trifoliate orange (Poncirus trifoliata) autotetraploids with enhanced salt tolerance. Hortic. Res. 7 (1). https://doi.org/10.1038/s41438-020-0311-7. Article 1.

Wei, T., Wang, Y., Xie, Z., Guo, D., Chen, C., Fan, Q., Deng, X., Liu, J., 2019. Enhanced ROS scavenging and sugar accumulation contribute to drought tolerance of naturally occurring autotetraploids in Poncirus trifoliata. Plant Biotechnol. J. 17 (7). https://doi.org/10.1111/pbi.13064. Article 7.

Wu, G.A., Prochnik, S., Jenkins, J., Salse, J., Hellsten, U., Murat, F., Perrier, X., Ruiz, M., Scalabrin, S., Terol, J., Takita, M.A., Labadie, K., Poulain, J., Couloux, A., Jabbari, K., Cattonaro, F., Del Fabbro, C., Pinosio, S., Zuccolo, A., Rokhsar, D., 2014. Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication. Nat. Biotechnol. 32 (7). https://doi.org/10.1038/nbt.2906. Article 7.

Xiao, L., Lu, L., Zeng, W., Shang, X., Cao, S., Yan, H., 2022. DNA methylome and LncRNAome analysis provide insights into mechanisms of genome-dosage effects in autotetraploid cassava. Front. Plant Sci. 13. https://doi.org/10.3389/fpls.2022.915056.

Xiong, Y.-C., Li, F.-M., Zhang, T., 2006. Performance of wheat crops with different chromosome ploidy: root-sourced signals, drought tolerance, and yield performance. Planta 224 (3). https://doi.org/10.1007/s00425-006-0252-x. Article 3.

Yang, M., Duan, X., Wang, Z., Yin, H., Zang, J., Zhu, K., Wang, Y., Zhang, P., 2021a. Overexpression of a voltage-dependent anion-selective channel (VDAC) protein-encoding gene, MsVDAC, from Medicago sativa confers cold and drought tolerance to transgenic tobacco. Genes 12 (11), 1706. https://doi.org/10.3390/genes12111706.

Yang, X., Lu, M., Wang, Y., Wang, Y., Liu, Z., Chen, S., 2021b. Response mechanism of plants to drought stress. Horticulturae 7 (3). https://doi.org/10.3390/horticulturae7030050. Article 3.

Yu, G., Wang, L.-G., Han, Y., He, Q.-Y., 2012. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS: J. Integr. Biol. 16 (5), 284. https://doi.org/10.1089/omi.2011.0118.

Yu, M., Yu, Y., Song, T., Zhang, Y., Wei, F., Cheng, J., Zhang, B., Zhang, X., 2022. Characterization of the voltage-dependent anion channel (VDAC) gene family in wheat (Triticum aestivum L.) and its potential mechanism in response to drought and salinity stresses. Gene 809, 146031. https://doi.org/10.1016/j.gene.2021.146031.

Zach, A., Schuldt, B., Brix, S., Horna, V., Culmsee, H., Leuschner, C., 2010. Vessel diameter and xylem hydraulic conductivity increase with tree height in tropical rainforest trees in Sulawesi, Indonesia. *Flora - morphology, distribution*. Funct. Ecol. Plants 205 (8). https://doi.org/10.1016/j.flora.2009.12.008. Article 8.