Funneliformis mosseae Modulates Root 14‐3‐3 Gene Expression to Enhance Drought Tolerance in Trifoliate Orange: A Molecular‐Physiological Integration

ABSTRACT

The 14‐3‐3 family proteins are involved in plant metabolism and stress signal transduction, while whether and how they contribute to the enhancement of host drought tolerance by arbuscular mycorrhizal (AM) fungi remains unclear. This study aimed to identify the 14‐3‐3 gene family (GRF) in trifoliate orange ( Poncirus trifoliata ) and analyze their responses in roots to drought and inoculation with the AM fungus Funneliformis mosseae, integrating molecular data with physiological assessments. Despite drought‐inhibited AM fungal colonization and soil mycelium length, AM inoculation markedly increased plant biomass, root growth, soluble sugars, antioxidant enzyme activities, and phytohormone levels under drought. Fifteen PtGRF members were identified with variable gene structures and abscisic acid/stress‐related promoter elements. Drought stress predominantly suppressed the expression of most PtGRF genes in non‐AM plants, while it triggered the upregulation of a specific subset (PtGRF3, 6, 9, 13, 14) in AM plants. Inoculation with AM fungi under drought conditions dramatically modulated the host's transcriptional response, significantly upregulating 10 of the 13 detected PtGRFs, most notably PtGRF13. Principal component and correlation analyses delineated distinct functional associations among PtGRF members: PtGRF1, PtGRF2, PtGRF5, and PtGRF15 were closely linked to soluble sugar accumulation and antioxidant defense, while PtGRF3, PtGRF6, PtGRF8, PtGRF9, and PtGRF13 showed stronger integration with abscisic acid and isopentenyl adenine. In summary, inoculation with AM fungi enhanced drought tolerance in trifoliate orange by modulating the expression of drought‐responsive specific PtGRF genes, which functioned as integrated signaling modules to coordinate sugars, antioxidant defense, and abscisic acid levels.

1. Introduction

Water serves as a fundamental solvent for substance transport and metabolic reactions in plant cells, while also constituting the basis for sustaining plant growth, development, and productivity (Moshelion et al. 2015; Solhi et al. 2023). However, soil drought frequently occurs in natural environments due to uneven precipitation patterns and intensified evaporation, subjecting plants to persistent or periodic drought stress (Bai et al. 2019). To combat drought stress, 14‐3‐3 family proteins contribute to establishing multi‐level defense systems by directly regulating physiological processes through modification of target protein activities (Jiang et al. 2023) or indirectly enhancing water and nutrient acquisition via symbiotic associations with arbuscular mycorrhizal (AM) fungi (An et al. 2025; Ma et al. 2021).

AM fungi form symbiotic associations with over 80% of terrestrial plant species, exchanging carbon (primarily as lipids) from the host for improved water and nutrient uptake, thereby aiding plant adaptation to environmental stresses like drought (Chandrasekaran 2022; Luginbuehl et al. 2017; Zheng et al. 2026; Zou et al. 2021). AM fungi extend extraradical hyphae into the soil, increasing the effective root surface area and enabling plants to access water and nutrients (especially phosphorus and nitrogen) beyond the depletion zone of roots (Abdalla et al. 2023; Jiang et al. 2026; Liu et al. 2026; Püschel et al. 2020). AM symbiosis boosts the accumulation of osmolytes (soluble sugars and proline) and enhances antioxidant enzyme activities, thus reducing oxidative damage from drought‐induced reactive oxygen species (ROS) (Chandrasekaran and Paramasivan 2022; Luo et al. 2024; Zou et al. 2021). AM fungi modulate plant hormone levels (e.g., abscisic acid, cytokinins, and strigolactones) and upregulate drought‐responsive genes, including those involved in aquaporin expression, flavonoids, MAPK signaling, and polyamine metabolism, further enhancing drought resilience (Huang et al. 2020; Luo et al. 2024; Meng et al. 2026; Zhang et al. 2020, 2026). Based on existing evidence, AM fungi enhance plant drought tolerance through multi‐layered hormonal regulation, antioxidant activity, and soluble sugar metabolism, although the precise mechanisms remain incompletely understood.

14‐3‐3 proteins, ubiquitous eukaryotic regulators also termed General Regulatory Factors (GRFs), function by binding phosphorylated motifs on client proteins to modulate plant metabolism, development, and stress responses (Sheikh et al. 2024; Sedlov and Sluchanko 2025; Zhang et al. 2024). Early studies identified 22 genes encoding 14‐3‐3 proteins in soybean ( Glycine max ), which were phylogenetically classified into ε and non‐ε groups and shown to respond to various environmental stresses (Wang et al. 2023). Arabidopsis AtGRF9‐overexpressing plants showed improved growth under osmotic stress (He et al. 2015), while transgenic tobacco overexpressing MsGRF2 exhibited enhanced stress tolerance through elevated antioxidant enzyme activity and osmoprotectant (Chai et al. 2024). The 14‐3‐3 family represents a widely conserved and functionally diverse group of regulatory proteins, with specific members conferring drought tolerance by enhancing key physiological processes. Nevertheless, a genome‐wide study of the entire 14‐3‐3 family in citrus, a globally important fruit crop, is still lacking.

Crucially, AM symbiosis upregulates 14‐3‐3 gene expression in plants, which appears to strengthen drought resilience. In maize, symbiosis with AM fungi significantly upregulated the expression of 14‐3‐3 genes and activated the antioxidant system, which aids in scavenging ROS accumulation during drought stress (Wang et al. 2023). AM fungi also enhanced abscisic acid (ABA) signaling efficiency in tomato by regulating 14‐3‐3 genes within the ABA signaling pathway, thereby inducing stomatal closure under drought stress to reduce water transpiration (Xu et al. 2018). In poplar, the mycorrhiza‐induced expression level of a 14‐3‐3 gene under drought stress was positively correlated with nearly all leaf sugar metabolism indicators (Han et al. 2023). Thus, AM fungi enhance plant drought tolerance partly through the regulation of 14‐3‐3 proteins, which integrate antioxidant defense, hormone signaling, and metabolic adjustment. In addition, emerging evidence demonstrates that AM fungal‐derived 14‐3‐3 proteins, localized in hyphae, spores, and arbuscules, are essential for mediating drought tolerance in host plants by regulating symbiotic development, as confirmed by the identification and host‐induced silencing of specific genes like Ri14‐3‐3 and RiBMH2 in Rhizophagus irregularis, which impairs arbuscule formation (Porcel et al. 2006; Sun et al. 2018; An et al. 2025). However, the coordinated regulation of 14‐3‐3 genes under the combined influence of AM colonization and drought stress remains unexplored in citrus. Likewise, the links between specific PtGRF family members and drought‐resistant physiological traits have yet to be established.

Citrus is a key global fruit crop facing significant challenges from drought stress in arid and semi‐arid regions, leading to suppressed growth and reduced photosynthetic efficiency that limit industry sustainability (Peng et al. 2020; Ziogas et al. 2021). Early studies have confirmed that AM fungi can enhance drought resistance in citrus (Meng et al. 2026; Zhang et al. 2020), while the molecular mechanisms, particularly the systematic response of the entire 14‐3‐3 gene family, remain poorly understood. To address this knowledge gap, we hypothesized that AM fungi enhance drought tolerance in citrus by modulating the expression of the conserved 14‐3‐3 gene family, thereby coordinately activating a suite of physiological defense responses. To test this hypothesis, the citrus rootstock trifoliate orange ( Poncirus trifoliata L. Raf.) was used to (1) examine key physiological traits (plant growth, root architecture, osmolytes, and antioxidant enzyme activity) under AM fungal inoculation and drought stress, (2) perform genome‐wide identification and characterization of the 14‐3‐3 gene family, and (3) elucidate the links between AM fungus‐mediated PtGRF gene expression and key drought‐resistant physiological traits.

2. Materials and Methods

2.1. Plant Culture

The seeds of trifoliate orange ( Poncirus trifoliata ) were first treated with 1 mol/L NaOH for 10 min to remove surface pectin, followed by thorough rinsing with distilled water. Subsequently, the seeds were surface‐disinfected with 75% ethanol for 5 min and then washed clean with distilled water before sowing. The seedlings were sown in autoclaved (121°C, 0.1 MPa, 2 h) river sand within a constant‐temperature incubator for a 30‐day period. On April 7, 2023, uniform and robust 5‐leaf‐old seedlings were selected and transplanted into a growth substrate consisting of an autoclaved soil‐to‐river sand mixture at a 3:1 volume ratio. The AM fungal inoculum used in the experiment was Funneliformis mosseae (BGC XZ02A), acquired from the Bank of Glomerales in China (BGC). This inoculum was propagated using white clover ( Trifolium repens L.) as a host plant for 10 weeks, and the resulting mixture of root fragments, growth substrate, and spores, with a density of approximately 19 spores per gram, was applied as the fungal inoculant. The plants were grown in plastic pots with an upper diameter of 16.5 cm, a bottom diameter of 10.5 cm, and a height of 14.5 cm.

2.2. Experimental Design

This experiment consisted of a 2 × 2 two‐factor design, with the first factor being water treatment, comprising ample‐watered conditions (AT, maintained at 70%–75% of field maximum water holding capacity) and drought‐stressed conditions (DT, maintained at 50%–55% of field maximum water holding capacity). The second factor is inoculation with F. mosseae (+Fmo) or non‐F. mosseae inoculation (−Fmo). During transplantation, the +Fmo group received 120 g of the fungal inoculum per pot, while the −Fmo group received an equivalent amount (120 g) of autoclaved inoculum, supplemented with a filtrate (30 μm) of the fungal inoculum. The drought level (50%–55% of field maximum water holding capacity) was selected based on preliminary experiments, which showed that this severity significantly inhibited the biomass of non‐mycorrhizal plants without causing mortality, making it suitable for evaluating the potential mitigating effects of mycorrhizal inoculation (Meng et al. 2026). Eight weeks after establishment, half of the mycorrhizal (+Fmo) and half of the non‐mycorrhizal (−Fmo) trifoliate orange seedlings were subjected to DT, with the remaining plants continuing under AT. Soil moisture content was monitored daily at 18:00 using the weighing method, and water was replenished accordingly to maintain the target moisture levels. The drought treatment lasted for 7 weeks. The experiment included seven replicates per treatment, with three seedlings per replicate, totaling 28 pots and 84 seedlings, all arranged randomly.

2.3. Determinations of Growth and Physiological Variables

Following the 7‐week drought treatment period, plants were harvested for subsequent analysis. At harvest, the biomass of roots, stems, and leaves was measured. Roots were scanned using an Epson Perfection V700 Photo Dual Lens System scanner (Model J221A), and the resulting images were analyzed with WinRHIZO software (Version 2007b) to quantify root morphological parameters, including total root length, surface area, volume, projected area, and average diameter.

Plant samples were first deactivated at 110°C for 15 min and then dried at 70°C until a constant weight was achieved. The dried tissues were ground, and 50 mg aliquots were used for soluble sugar extraction. Each sample was mixed with 4 mL of 80% ethanol and incubated in a water bath at 80°C for 40 min with continuous stirring. After centrifugation at 2500×g for 6 min, the supernatant was collected. The residue was re‐extracted with 80% ethanol, and the combined supernatant was treated with 10 g of activated carbon for decolorization at 80°C for 30 min. The filtered supernatant was used as the test solution for sucrose, glucose, and fructose quantification according to the method described by Wu et al. (2015).

Phytohormone contents, including ABA, N⁶‐isopentenyl adenine (IP), indole‐3‐acetic acid (IAA), indole‐3‐butyric acid (IBA), dihydrozeatin (DZ), and trans‐zeatin (TZ), in leaves and roots were measured using Enzyme‐Linked Immunosorbent Assay (ELISA) kits (Shanghai Yuanju Biotechnology Co. Ltd.), following the manufacturer's instructions. The kit catalog numbers were as follows: ABA (cat. no. X0110), IP (cat. no. X0965), IAA (cat. no. X0950), IBA (cat. no. X0955), DZ (cat. no. X2070), and TZ (cat. no. X2620).

For antioxidant enzyme assays, 0.3 g of fresh plant tissue was homogenized in 5 mL of ice‐cold 0.05 mol/L phosphate buffer. The homogenate was centrifuged at 4000×g/min for 10 min, and the resulting supernatant served as the crude enzyme extract. Superoxide dismutase (SOD, EC 1.15.1.1) activity in leaves and roots was determined using the colorimetric method outlined by Wu et al. (2015), while peroxidase (POD, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) activities were measured according to He et al. (2020). Malondialdehyde (MDA) content was measured as an indicator of lipid peroxidation. Briefly, 0.3 g of fresh tissue was homogenized in 5 mL of 5% trichloroacetic acid and centrifuged at 4000×g/min for 10 min. Then, 2 mL of the supernatant was mixed with an equal volume of 0.67% thiobarbituric acid, heated in a boiling water bath for 30 min, cooled, and centrifuged again. The absorbance of the supernatant was measured at 450, 532, and 600 nm, and the MDA concentration was calculated based on the method of Sudhakar et al. (2001).

2.4. Determinations of Mycorrhizal Status

Root colonization by the AM fungus was assessed using approximately 1‐cm‐long root segments stained with trypan blue according to the method of Phillips and Hayman (1970), and root AM fungal colonization was observed microscopically. The mycorrhizal colonization rate was expressed as the percentage of root segment length colonized by AM fungi relative to the total root length examined. Soil hyphal length was analyzed based on the procedure of Bethlenfalvay and Ames (1987).

2.5. Whole‐Genome Identification and Characterization of the 14‐3‐3 Gene Family in Trifoliate Orange

Based on the 14‐3‐3 gene sequences obtained from the Arabidopsis Genome Database (TAIR), putative 14‐3‐3 genes in Poncirus trifoliata were identified by BLAST analysis against the Citrus Pan‐genome to Breeding Database using TBtools software. The resulting sequences were subjected to domain validation via SMART and Pfam databases, and those lacking the characteristic 14‐3‐3 domains were excluded. The confirmed 14‐3‐3 genes were systematically named according to their chromosomal locations. Basic physicochemical properties of the encoded proteins, including molecular weight and theoretical isoelectric point, were predicted using the ExPASy online server. Conserved motifs within the protein sequences were analyzed with MEME, while conserved domains were examined via NCBI CDD. Visualization of protein motifs, gene structures, and chromosomal distribution was performed using TBtools. To elucidate phylogenetic relationships, 14‐3‐3 protein sequences from Arabidopsis thaliana , tomato ( Solanum lycopersicum ), and poplar ( Populus trichocarpa ) were retrieved from TAIR and NCBI. A neighbor‐joining phylogenetic tree was constructed with MEGA software based on the aligned amino acid sequences from Poncirus trifoliata , Arabidopsis thaliana , tomato, and rice ( Oryza sativa ), and the resulting tree was visualized and refined using the iTOL online platform.

2.6. Analysis of 14‐3‐3 Gene Expression via qRT‐PCR

Total RNA was extracted from frozen root and leaf samples ground in liquid nitrogen using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme Biotech Co. Ltd). The integrity of the extracted RNA was verified by agarose gel electrophoresis, and its purity was assessed spectrophotometrically. First‐strand cDNA was synthesized from the RNA using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme). Gene‐specific primers for the identified 14‐3‐3 gene family members were designed with Primer Premier 5.0 (Table S1) for subsequent qRT‐PCR analysis. To ensure specificity, all primer pairs were verified using NCBI Primer‐BLAST, and their specificity was confirmed by melting curve analysis and agarose gel electrophoresis of PCR products. Quantitative analysis was performed on a CFX96 Real‐Time PCR Detection System (BIO‐RAD) with a reaction mixture containing 10 μL of SYBR GREEN PCR Master Mix, 8.5 μL of sterile water, 0.5 μL of cDNA, and 0.5 μL each of forward and reverse primers. The amplification protocol consisted of initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and a final dissociation step. All procedures were conducted according to the manufacturer's instructions. The relative expression levels of the target genes were calculated using the 2^−ΔΔCt method (Livak and Schmittgen 2001), where β‐actin was taken as the reference gene (Zou et al. 2025; Pu et al. 2025), with normalization to the AT‐Fmo treatment group as the control.

2.7. Statistical Analysis and Data Visualization

All statistical analyses were performed using IBM SPSS Statistics 27 (IBM Corp.). A two‐way analysis of variance (ANOVA) was employed to assess the significance of differences among treatment groups, followed by Tukey's test for multiple comparisons, with a significance threshold set at p < 0.05. The heatmap of Pearson's correlation coefficients was plotted using Microsoft Excel 2019. Principal component analysis (PCA) was performed using GraphPad Prism 10.1.2 (GraphPad Software).

3. Results

3.1. Effects of Drought Stress on Mycorrhizal Status

Under both AT and DT conditions, extensive extraradical hyphae of F. mosseae were observed surrounding the roots of trifoliate orange (Figure 1a–b). The mycorrhizal colonization rate in inoculated plants ranged from 29.50% to 57.75% (Figure 1d), and soil mycelium length varied from 14.50 to 28.67 cm/g (Figure 1e). However, DT treatment significantly reduced both mycorrhizal colonization and soil mycelium length by 48.92% and 49.43%, respectively, compared with the AT treatment (Figure 1d–e).

FIGURE 1.

Mycorrhizal colonization in Poncirus trifoliata roots visualized by stereomicroscopy (a), biological microscopy (b), and the corresponding seedling growth phenotype (c), respectively, alongside the effects of Funneliformis mosseae inoculation on root colonization rate (d), soil mycelium length (e), as well as leaf biomass (f), stem biomass (g), root biomass (h), root total length (i), root projected area (j), root surface area (k), root average diameter (l), and root total volume (m) under ample‐watered and drought‐stressed conditions. Data (means ± SD, n = 4) followed by different letters above the bars among four treatments indicate significant (p < 0.05) differences. Abbreviations: AT, ample‐water treatment; DT, drought‐stressed treatment; +Fmo, inoculation with F. mosseae; −Fmo, inoculation without F. mosseae.

3.2. Effects of Inoculation With F. mosseae and Drought Treatment on Biomass Production

Compared to the AT treatment, the DT treatment significantly reduced the leaf biomass of −Fmo plants by 70.73%, and also significantly decreased the root, stem, and leaf biomass of +Fmo plants by 48.26%, 30.11%, and 63.31%, respectively (Figure 1f–h). Inoculation with F. mosseae (+Fmo) significantly enhanced biomass under both water regimes: under AT conditions, root, stem, and leaf biomass increased by 67.10%, 64.52%, and 70.50%, respectively, while under DT conditions, the corresponding increases were 66.40%, 80.77%, and 76.47%, compared with −Fmo plants. Two‐way ANOVA revealed a significantly interactive effect of water treatment and AM fungal inoculation on leaf biomass (p < 0.001) and root biomass (p < 0.05) (Table 1). Root biomass, stem biomass, and leaf biomass showed significantly positive correlations with root mycorrhizal colonization and soil mycelium length (Figure 2). Among the PtGRF genes, PtGRF1, PtGRF2, PtGRF3, PtGRF5, PtGRF13, and PtGRF15 were significantly positively correlated with stem biomass (Figure 2).

TABLE 1.

The probability levels of arbuscular mycorrhizal (AM) fungi and drought stress treatment (DT) and their interactions in all the variables tested.

Variables	Drought stress	AM fungi	Drought stress×AM fungi
Root biomass	***	***	*
Steam biomass	***	***	0.50
Leaf biomass	***	***	***
Root total length	***	***	***
Root projected area	***	***	*
Root surface area	***	***	***
Root average diameter	***	***	0.52
Root total volume	***	***	*
Sucrose	*	***	*
Fructose	***	***	*
Glucose	*	***	**
DZ	***	0.10	**
TZ	***	***	***
IP	0.47	***	***
IBA	***	0.15	**
IAA	***	***	***
ABA	0.39	***	***
SOD	**	***	*
POD	***	***	0.83
CAT	*	***	0.37
MDA	***	*	0.87
PtGRF1	***	***	*
PtGRF2	***	***	0.74
PtGRF3	0.07	***	***
PtGRF5	***	***	0.49
PtGRF6	***	***	***
PtGRF7	***	0.56	***
PtGRF8	***	**	***
PtGRF9	***	***	***
PtGRF10	***	***	***
PtGRF12	*	**	***
PtGRF13	***	***	***
PtGRF14	***	***	*
PtGRF15	***	***	0.09

Note: *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

Heat map of correlation between root colonization, physiological variables, and expression levels of the PtGRFs gene family in Poncirus trifoliata roots. The lower triangle (bottom‐left section) displays the correlation coefficients (r values), while the upper triangle (top‐right section) displays the significance asterisks.

3.3. Effects of Inoculation With F. mosseae and Drought Treatment on Root Morphological Variables

In −Fmo plants, the DT treatment significantly reduced total root length, projected area, surface area, average diameter, and volume by 34.21%, 33.70%, 8.07%, 10.91%, and 44.57%, respectively, compared to the AT treatment; similarly, these parameters were suppressed by 14.83%, 20.16%, 3.20%, 7.69%, and 17.91% in +Fmo plants (Figure 1i–m). Inoculation with F. mosseae (+Fmo) consistently enhanced all root morphological traits under both water regimes. Under AT conditions, +Fmo treatment increased total root length, projected area, surface area, average diameter, and total volume by 19.77%, 20.16%, 9.05%, 15.38%, and 24.38%, respectively, compared with −Fmo treatment. Under DT conditions, the enhancements were even more pronounced, with increases of 38.02%, 17.04%, 13.62%, 18.33%, and 48.94%, respectively. Two‐way ANOVA indicated a significantly interactive effect of drought treatment and AM fungal inoculation on total root length (p < 0.001), total surface area (p < 0.001), total projected area (p < 0.05), and total root volume (p < 0.05) (Table 1). Root morphological parameters showed significantly positive correlations with both root mycorrhizal colonization and soil mycelium length (Figure 2). Among the PtGRF genes, PtGRF1, PtGRF2, PtGRF3, PtGRF5, PtGRF13, and PtGRF15 were significantly positively correlated with all measured root morphological traits (Figure 2).

3.4. Effects of Inoculation With F. mosseae and Drought Treatment on Root Sugar Profiles

The DT treatment significantly reduced root sucrose, fructose, and glucose contents in −Fmo plants by 15.08%, 13.05%, and 39.29%, respectively, compared with the AT treatment (Figure 3a–c). In contrast, in +Fmo plants, the DT treatment did not significantly affect root sucrose and glucose levels but led to a 15.24% significant decrease in root fructose levels. On the other hand, +Fmo treatment significantly enhanced sucrose, fructose, and glucose levels by 46.11%, 39.80%, and 41.68% under AT conditions and by 54.44%, 38.25%, and 66.09% under DT conditions, relative to −Fmo treatment. Two‐way ANOVA revealed significantly interactive effects between drought treatment and AM fungal inoculation on glucose (p < 0.01), fructose (p < 0.05), and sucrose (p < 0.05) levels (Table 1). Soluble sugars (sucrose, glucose, and fructose) were positively correlated with PtGRF2, PtGRF5, and PtGRF15 (Figure 2).

FIGURE 3.

Effects of inoculation with Funneliformis mosseae on sucrose (a), fructose (b), and glucose (c) levels in Poncirus trifoliata roots exposed to ample‐watered and drought‐stressed treatments. Data (means ± SD, n = 4) followed by different letters above the bars among four treatments indicate significant (p < 0.05) differences. The abbreviation was shown in Figure 1.

3.5. Effects of Inoculation With F. mosseae on Antioxidant Enzyme Activities and Malondialdehyde Levels in Roots

Under −Fmo conditions, DT treatment significantly reduced POD activity by 13.12%, compared with AT treatment; in +Fmo plants, DT notably decreased SOD and POD activities by 15.76% and 11.01%, respectively (Figure 4a,b). Nevertheless, under both AT and DT conditions, +Fmo inoculation significantly increased the activities of SOD (by 57.05% and 64.49%) and POD (by 24.11% and 25.91%). Additionally, +Fmo markedly increased CAT activity by 65.73% under DT conditions (Figure 4c). The DT treatment significantly elevated MDA content in both −Fmo and +Fmo plants by 33.11% and 43.84%, respectively (Figure 4d). Despite a consistent trend of lower MDA levels in +Fmo plants compared to −Fmo plants, the difference was not statistically significant. Drought treatment and AM fungal inoculation significantly (p < 0.05) interacted on only SOD activity (Table 1). The activities of SOD, POD, and CAT were significantly positively correlated with root mycorrhizal colonization, soil mycelium length, and soluble sugars (sucrose, fructose, and glucose) (Figure 2). Additionally, SOD, POD, and CAT were significantly positively correlated with PtGRF1, PtGRF2, PtGRF3, PtGRF5, PtGRF13, and PtGRF15, and SOD also showed significant positive correlations with PtGRF9 and PtGRF14.

FIGURE 4.

Effects of inoculation with Funneliformis mosseae on SOD activity (a), POD activity (b), CAT activity (c), and MDA content (d) in Poncirus trifoliata roots exposed to ample‐watered and drought‐stressed treatments. Data (means ± SD, n = 4) followed by different letters above the bars among four treatments indicate significant (p < 0.05) differences. The abbreviation was shown in Figure 1.

3.6. Effects of Inoculation With F. mosseae on Endogenous Hormone Levels in Roots

Compared to the AT treatment, the DT treatment significantly reduced IP, IAA, and ABA levels in −Fmo plants by 13.49%, 4.50%, and 5.74%, respectively, while increasing IBA level by 3.55% (Figure 5c–f). In +Fmo plants, however, the DT treatment significantly elevated DZ, TZ, IP, IBA, IAA, and ABA levels by 5.80%, 12.29%, 16.05%, 8.08%, 20.38%, and 4.07%, respectively (Figure 5a–f). Compared with −Fmo inoculatioin, +Fmo inoculation significantly suppressed DZ, TZ, IP, IBA, and IAA levels under AT conditions by 3.97%, 11.32%, 11.76%, 3.08%, and 17.36%, respectively, while it significantly enhanced IP, IAA, and ABA levels under DT conditions by 16.69%, 6.90%, and 9.87%, along with no significant effects on DZ, TZ, and IBA levels under DT conditions. Two‐way ANOVA revealed a significantly interactive effect on TZ (p < 0.001), IAA (p < 0.001), ABA (p < 0.001), DZ (p < 0.01), and IBA (p < 0.01) (Table 1). Correlation analysis showed that DZ, TZ, and IAA were negatively correlated with mycorrhizal indicators (Figure 2). ABA positively correlated with root traits and PtGRF2, PtGRF3, PtGRF5, PtGRF6, PtGRF9, and PtGRF13 but negatively with PtGRF10. IP positively correlated with PtGRF3, PtGRF6, PtGRF8, PtGRF9, and PtGRF13 but negatively with PtGRF7, PtGRF10, and PtGRF12. IBA positively correlated with PtGRF14, and IAA negatively correlated with PtGRF12. In PCA, ABA was positively correlated with PtGRF3, PtGRF6, PtGRF8, PtGRF9, PtGRF13, and PtGRF14, and these genes together with PtGRF14 were positively associated with ABA and IP (Figure 6).

FIGURE 5.

Effects of inoculation with Funneliformis mosseae on dihydrozeatin (DZ) (a), trans‐zeatin (TZ) (b), N⁶‐isopentenyladenine (IP) (c), indole‐3‐butyric acid (IBA) (d), indole‐3‐acetic acid (IAA) (e), and abscisic acid (ABA) (f) in Poncirus trifoliata roots exposed to ample‐watered and drought‐stressed treatments. Data (means ± SD, n = 4) followed by different letters above the bars among four treatments indicate significant (p < 0.05) differences. The abbreviation is shown in Figure 1.

FIGURE 6.

The principal component analysis (PCA) of physiological parameters and gene expression levels in roots of trifoliate orange seedlings.

3.7. Identification and Bioinformatic Analysis of PtGRF Gene Family

The 14‐3‐3 gene family in trifoliate orange was identified in this study through a genome‐wide analysis. A total of 15 members containing intact open reading frames were confirmed and designated PtGRF1 to PtGRF15 according to their chromosomal locations (Table 2). As summarized in Table 2, the deduced PtGRF proteins vary in length from 92 (PtGRF3) to 265 (PtGRF8) amino acids, with molecular weights ranging from 9862.31 Da (PtGRF3) to 29,786.53 Da (PtGRF5). Their theoretical isoelectric points span from 4.68 (PtGRF2) to 9.20 (PtGRF11). Subcellular localization predictions indicate that most PtGRF proteins are localized to the nucleus and cytoplasm.

TABLE 2.

Physicochemical properties and subcellular localization of the 14‐3‐3 gene (PtGRF) family in Poncirus trifoliata .

Gene name	Original ID	Length (aa)	MW (Da)	pI	Subcellular localization
PtGRF1	Pt2g005460.1	261	29400.93	4.73	Nucleus
PtGRF2	Pt2g024660.1	258	29419.01	4.68	Nucleus
PtGRF3	Pt2g027810.1	92	9862.31	6.03	Cytoplasm
PtGRF4	Pt3g033830.1	176	19473.22	8.98	Cytoplasm
PtGRF5	Pt4g007220.1	261	29786.53	5.14	Nucleus
PtGRF6	Pt4g009890.1	81	9526.93	9.03	Cytoplasm
PtGRF7	Pt5g009710.1	253	28861.31	4.92	Nucleus
PtGRF8	Pt5g009920.1	265	29758.30	4.69	Nucleus
PtGRF9	Pt5g010400.1	252	28526.33	4.80	Nucleus
PtGRF10	Pt6g000630.1	263	29726.30	4.74	Nucleus
PtGRF11	Pt6g017810.1	196	21804.27	9.20	Cytoplasm
PtGRF12	Pt7g014770.1	245	27688.44	4.87	Nucleus
PtGRF13	Pt8g005080.1	123	13690.90	5.66	Nucleus
PtGRF14	PtUn009450.1	144	15680.70	6.89	Cytoplasm
PtGRF15	PtUn010900.1	261	29442.15	4.84	Nucleus

Chromosomal localization analysis revealed that the 15 identified PtGRFs were randomly distributed across eight chromosomes (Figure 7a). To investigate the evolutionary relationships of these genes, a phylogenetic tree was constructed using the neighbor‐joining method in MEGA‐X, comprising 15 PtGRFs along with 13 Arabidopsis, 13 tomato, 14 poplar, and 8 rice 14‐3‐3 homologs (Figure 7b). The phylogenetic analysis classified the PtGRFs into six distinct subgroups. Subgroup I primarily contained five poplar and five PtGRFs members. Subgroup II was mainly composed of five tomato and four poplar GRFs. Subgroup III included one PtGRF and one Arabidopsis GRF, while Subgroup IV contained three PtGRFs together with two GRF members each from tomato, poplar, rice, and Arabidopsis. Subgroup V consisted of two tomato GRFs and one PtGRF. Interestingly, Subgroup VI was predominantly populated by rice GRF members, accompanied by four PtGRFs.

FIGURE 7.

Chromosomal localization (a) of the PtGRFs and phylogenetic analysis (b) of GRF proteins from Poncirus trifoliata and other plant species.

To elucidate the functional characteristics of the PtGRFs gene family, their phylogenetic relationships (Figure 7b), conserved motifs (Figure 8a), protein domains (Figure 8b), and gene structures (Figure 8c) were visualized using TBtools. A total of 10 conserved motifs, designated Motif 1 to Motif 10, were identified among the PtGRF members (Figure 8a). Analysis revealed that Motif 1 is present in 14 out of the 15 members, while Motifs 3 and 4 were each detected in 10 PtGRF members. Additionally, Motifs 2, 5, and 6 were each found in 9 PtGRF members. All PtGRFs were confirmed to contain either a 14‐3‐3 domain or a 14‐3‐3‐like superfamily domain (Figure 8b). Gene structure analysis showed that the number of exons in PtGRF genes ranges from 1 to 7, with PtGRF6 possessing the fewest and PtGRF5 the most (Figure 8c).

FIGURE 8.

The phylogenetic tree of GRFs predicted in Poncirus trifoliata (a) and their motif analysis (b), conserved domain systematic analysis (c), and gene structure.

An analysis of cis‐acting elements in the promoter regions of PtGRFs revealed a variety of transcription factor binding sites associated with stress responses, development, light responsiveness, and plant hormones (Figure 9a). Specifically, 50 binding sites were identified as stress‐responsive elements, 119 as hormone‐responsive elements, 23 as growth and development‐related elements, and 79 as light‐responsive elements (Figure 9b). Among the hormone‐responsive elements, ABRE (ABA‐responsive element) and CGTCA motifs were the most abundant. Additionally, the promoter regions contained numerous G‐box light‐responsive elements, with PtGRF4 notably containing 10 G‐boxes. It is worth noting that 10 PtGRF members (PtGRF1, PtGRF3, PtGRF5, PtGRF6, PtGRF8, PtGRF9, PtGRF10, PtGRF11, PtGRF12, and PtGRF15) were found to contain MBS (MYB binding site involved in drought‐inducibility) elements.

FIGURE 9.

Cis‐acting elements in the promoters of the 15 PtGRFs genes. The distribution of cis‐acting elements with similar functions marked with the same colors and the promoter length indicated by the black line (a). Visualization of the number of cis‐acting elements related to stress responsive, phytohormone responsive, growth and development and light responsive in 15 PtGRFs gene promoters (b).

3.8. AM‐ and Drought‐Responsive Expression Patterns of PtGRF Genes

Analysis of the relative expression levels of PtGRF genes in trifoliate orange roots under both AT and DT conditions, with or without F. mosseae inoculation (+Fmo/−Fmo), revealed that PtGRF4 and PtGRF11 genes were not detected in any root samples under the conditions tested; whether they are expressed in other organs (e.g., leaves and stems) or under different developmental stages requires further investigation (Figure 10a–m). Under −Fmo conditions, the DT treatment significantly downregulated the expression of most PtGRFs compared to the AT treatment, with reductions ranging from 0.34 to 3.71 folds, except for PtGRF7, PtGRF10, PtGRF12, and PtGRF14. In contrast, under +Fmo conditions, the DT application significantly suppressed the expression of only PtGRF1, PtGRF2, PtGRF5, PtGRF7, PtGRF12, and PtGRF15, by 0.20‐, 0.26‐, 0.19‐, 0.25‐, 0.31‐, and 0.41‐fold, respectively, while it distinctly upregulated the expression of PtGRF3, PtGRF6, PtGRF9, PtGRF13, and PtGRF14 by 0.44‐, 0.43‐, 1.18‐, 0.22‐, and 0.70‐fold, respectively.

FIGURE 10.

Effects of inoculation with Funneliformis mosseae on the expression of 13 PtGRF genes in Poncirus trifoliata roots exposed to ample‐watered and drought‐stressed treatments. Data (means ± SD, n = 4) followed by different letters above the bars among four treatments indicate significant (p < 0.05) differences. The abbreviation was shown in Figure 1.

Notably, under AT conditions, +Fmo inoculation significantly induced the expression of most PtGRFs compared to −Fmo inoculation. Specifically, PtGRF1, PtGRF2, PtGRF3, PtGRF5, PtGRF7, PtGRF10, PtGRF12, PtGRF14, and PtGRF15 were upregulated by 0.69‐, 0.55‐, 0.17‐, 0.65‐, 0.70‐, 0.49‐, 0.55‐, 0.30‐, and 0.64‐fold, respectively, whereas the expression of PtGRF6 and PtGRF8 was downregulated by 0.28‐fold. Under DT conditions, +Fmo treatment significantly reduced the expression of PtGRF7, PtGRF10, and PtGRF12 by 0.87‐, 2.82‐, and 0.40‐fold, respectively, while it distinctly enhanced the expression of other PtGRFs by 0.36–0.99‐fold. Among these, PtGRF13 showed the most pronounced upregulation, increasing by 0.99‐fold. Two‐way ANOVA indicated a significantly interactive effect on PtGRF1 (p < 0.05), PtGRF3 (p < 0.001), PtGRF6‐10 (p < 0.001), PtGRF12 (p < 0.001), PtGRF13 (p < 0.001), and PtGRF14 (p < 0.05) (Table 1).

4. Discussion

4.1. Mycorrhizal Colonization Is Suppressed by Drought Stress

Our results demonstrate that the DT treatment significantly suppressed both mycorrhizal colonization rate and soil mycelium density in trifoliate orange roots compared to the AT treatment. Similar drought‐induced suppression of mycorrhizal development has also been documented in other plant species such as melon, Panicum virgatum , potato, and tobacco (Basyal and Walker 2023; Begum et al. 2020; Huang et al. 2010; Yooyongwech et al. 2016). This cross‐species consistent response indicates a universal adaptive strategy of AM fungi‐plant symbionts under drought stress, rather than a species‐specific phenotypic change, which further confirms the high sensitivity of mycorrhizal symbiosis to soil water availability. The reduction in mycorrhizal status under DT versus AT is an outcome of multiple intertwined factors, such as suppressed host photosynthesis, inhibited spore germination and hyphal elongation, and limited carbon resources (Zou et al. 2025).

4.2. Genome‐Wide Identification Reveals Conserved Structural Features of the PtGRF Family

The 14‐3‐3 gene family has been identified in multiple species (Liu et al. 2016; Qin et al. 2016; Sheikh et al. 2024; Xia et al. 2022), while the information regarding the 14‐3‐3 gene family in trifoliate orange remains unknown. In this study, a total of 15 14‐3‐3 family genes (PtGRF1–PtGRF15) were identified in the trifoliate orange genome using bioinformatic approaches. Based on evolutionary homology with GRF gene families from Arabidopsis thaliana , tomato ( Solanum lycopersicum ), rice ( Oryza sativa ), and poplar ( Populus trichocarpa ), these 15 PtGRFs were classified into six subfamilies. Notably, PtGRF12 and AtGRF5 were grouped into a separate subfamily, indicating a high degree of homology between them. Given that AtGRF5 plays an important role in leaf primordium cell proliferation (Horiguchi et al. 2005), this homologous clustering suggests that PtGRF12 may retain a conserved function in regulating morphological development in trifoliate orange, implying functional conservation of 14‐3‐3 genes across woody and herbaceous plants. Furthermore, PtGRF5 also exhibits high homology with SlTFT4 and SlTFT6. SlTFT4 has been identified as a key gene responding to salt stress in tomato; silencing this gene significantly increased cellular damage and reduced tomato's tolerance to salt stress (Jia et al. 2022). Liang et al. (2023) also found that SlTFT6 plays an important role in enhancing thermo tolerance in tomato plants. Therefore, it is hypothesized that PtGRF5 may possess functions similar to those of SlTFT4 and SlTFT6. Beyond these individual gene predictions, the PCA and correlation analyses revealed broader functional differentiation among PtGRF members at the subfamily level. Notably, these functional clusters partially correspond to phylogenetic subgroups: subgroup I/II members (PtGRF1/2/5) and subgroup IV member (PtGRF15) mainly correlated with sugar metabolism and antioxidant defense, whereas subgroup VI members (PtGRF3/6/9/13) were more closely linked to hormone signaling (ABA and IP). This subgroup‐specific functional differentiation suggests that evolutionary divergence of PtGRF genes may have driven a functional specialization, allowing distinct subfamily members to participate in different physiological processes, including energy metabolism, stress defense, and hormone signal transduction. These findings provide a potential mechanistic explanation for the broad involvement of the 14‐3‐3 gene family in multiple physiological processes in perennial woody citrus plants and offer insight into the subfunctionalization of plant 14‐3‐3 gene families.

4.3. Promoter Cis‐Elements Suggest Stress and Hormone Responsiveness of PtGRFs

Cis‐acting elements in gene promoter regions precisely regulate the gene transcription level and spatiotemporal specificity by binding to proteins such as transcription factors (Ezeh and Yamamoto 2024; Kong et al. 2018). In this study, all PtGRF promoters were found to contain MYB and MYC elements. Every member of the PtGRF gene family harbored at least one stress response‐related cis‐element. This conservative feature suggests that the PtGRF gene family has retained the core regulatory function of responding to environmental stress throughout evolution, implying its broad participation in the stress adaptation process of citrus and potentially other plant species. Furthermore, the promoters of all PtGRFs, with the exception of PtGRF3 and PtGRF13, were enriched with multiple hormone‐ and stress‐responsive elements, including CGTCA‐motif and ABRE (ABA‐responsive element). This indicates that various hormonal and stress stimuli may differentially regulate these promoters. ABRE plays a crucial role in ABA signal transduction (Barman et al. 2023), which aligns with the significant correlation observed between the expression of most PtGRF family members and ABA levels in this study. Among the 15 identified PtGRF gene family members, the conserved motifs exhibited varying degrees of absence but maintained similar distribution patterns, particularly among members within the same subfamily. The number of introns and exons also varied significantly across different PtGRF subfamilies. During the early stages of gene expansion, introns may be lost over time as genes evolve to adapt to their environment (Roy and Gilbert 2006).

4.4. AM Symbiosis Selectively Modulates Drought‐Responsive Expression of PtGRF Genes

The expression profiling of PtGRF genes under varying water availability and mycorrhizal status provides critical insights into the molecular interplay between AM symbiosis and drought adaptation in trifoliate orange. The absence of PtGRF4 and PtGRF11 transcripts across all root samples highlights the spatial and contextual specificity of 14‐3‐3 gene regulation. This lack of detectable expression in roots may have several possible explanations. One plausible explanation is tissue‐ or developmental‐stage‐specific expression, as individual plant 14‐3‐3 isoforms can exhibit distinct spatial and temporal expression patterns; for example, some Arabidopsis 14‐3‐3 isoforms show highly restricted expression in reproductive tissues rather than roots (Keicher et al. 2017; Paul et al. 2012). Alternatively, the lack of root expression may reflect functional redundancy among PtGRF paralogs, whereby other co‐expressed 14‐3‐3 isoforms perform overlapping regulatory functions. Such redundancy, together with isoform‐specific expression and target recognition, has been well documented in plant 14‐3‐3 families (Paul et al. 2012; Pallucca et al. 2014). Additionally, these genes might be expressed under specific developmental stages or environmental cues not tested in this study, such as during floral or fruit development, or in response to other abiotic stresses.

Under non‐mycorrhizal (−Fmo) conditions, DT significantly repressed the expression of most PtGRFs in trifoliate orange roots, indicating a general suppression of growth‐related processes mediated by GRF genes under drought in the absence of fungal symbiosis. GRF genes are known to play crucial roles in plant growth and development, including cell proliferation and expansion, by regulating cell cycle genes (Lee et al. 2022). The downregulation of these genes under drought stress without AM fungal inoculation aligns with the observed growth depression in our experiment under DT versus AT. However, the contrasting response pattern observed in +Fmo plants, where drought significantly upregulated PtGRF3, PtGRF6, PtGRF9, PtGRF13, and PtGRF14, implies that AM colonization modulates the host's transcriptional landscape toward stress acclimation. This modulation is not a general activation but a selective process, suggesting a sophisticated mechanism by which the host tailors its response to the combined stress of drought and symbiosis. This highlights a potential mechanism by which F. mosseae enhances drought tolerance in trifoliate orange, possibly by selectively activating GRF genes involved in stress adaptation or by promoting growth under stress conditions. In Solanum tuberosum, members of the StGRF family in potato also exhibit tissue specificity: StGRF14h is predominantly highly expressed in roots, whereas StGRF14a, StGRF14b, StGRF14g, StGRF14i, and StGRF14k show higher expression levels in stems compared to leaves and roots (Hajibarat et al. 2022). Similarly, members of the papaya CpGRF gene family display both tissue‐specific expression patterns and temporal dynamics (Li et al. 2021). Importantly, given that the 14‐3‐3 gene family also exists in fungi, the functional interplay between plant PtGRFs and fungal 14‐3‐3 homologs warrants further exploration. Sun et al. (2018) demonstrated that AM fungal 14‐3‐3 genes (Fm201, Ri14‐3‐3, and RiBMH2) were significantly upregulated under drought stress in both mycorrhizal roots and extraradical hyphae, and that silencing these genes impaired arbuscule formation, underscoring their essential roles in symbiotic stress responses. The interplay between plant and fungal 14‐3‐3 proteins could be a critical aspect of the symbiosis. Future investigations should therefore include the quantification of fungal 14‐3‐3 gene expression alongside the host's to fully elucidate the bipartite regulatory network underlying mycorrhiza‐induced drought tolerance. In contrast to the tissue‐ and temporal‐specific expression of GRFs driven primarily by endogenous developmental programs in the above‐mentioned species, the differential expression of PtGRFs in trifoliate orange observed here is largely associated with mycorrhizal symbiosis and drought stress interaction. This contrast suggests that woody plants such as citrus may have the capacity to remodel their conserved GRF regulatory network to cope with abiotic stress through symbiotic associations with microorganisms. Under AT conditions, AM fungal inoculation generally induced the expression of most PtGRFs compared to non‐AM fungal treatment, indicating that even in the absence of stress, AM fungal inoculation can prime the plant's growth and developmental machinery, potentially contributing to overall plant vigor. However, PtGRF6 and PtGRF8 were downregulated, suggesting a nuanced regulatory role for AM fungi that can also involve selective repression of certain GRF genes. This indicates that the symbiosis is not simply an indiscriminate activator but a fine‐tuner of gene expression.

Conversely, all PtGRFs, with the exception of PtGRF7, PtGRF10, and PtGRF12, showed enhanced expression under DT conditions, with PtGRF13 exhibiting the most pronounced upregulation (0.99 folds). The specific upregulation of PtGRF13 suggests that it might play a key regulatory node in AM‐mediated drought tolerance, possibly through influencing ABA signaling or ROS‐scavenging pathways (Ren et al. 2025), as evidenced by a significantly positive correlation between PtGRF13 and SOD, POD, CAT activities, and ABA levels. After inoculating Populus cathayana roots with AM fungi, it was also found that most GRF members showed a significant positive correlation with antioxidant enzymes (SOD and POD) (Han et al. 2022), further highlighting the regulatory role of the PtGRF gene family in modulating antioxidant enzyme defense. The downregulation of PtGRF7, PtGRF10, and PtGRF12 in +Fmo plants versus −Fmo plants under DT suggests functional diversification among 14‐3‐3 isoforms, where certain members are selectively suppressed to fine‐tune stress responses under symbiotic conditions. This highlights the complexity of the GRF gene family, where individual members can have distinct or even opposing roles under different environmental cues (Li et al. 2024). In conclusion, these expression dynamics reinforce that AM symbiosis does not merely amplify the host's inherent drought response but actively reshapes it through selective transcriptional regulation of 14‐3‐3 genes. This selective regulation may contribute to the enhanced stress resilience observed in mycorrhizal plants. This study thus provides a set of candidate PtGRF genes, particularly PtGRF13, for future functional validation to establish their direct roles in AM‐mediated drought tolerance in woody plants.

4.5. AM Fungi Promote Biomass Production, Root Growth and Sugar Accumulation in Association With PtGRF ‐Mediated Regulatory Networks

The reduction in soil moisture can limit the expression of key genes such as TUBULIN and CYCLIN, thereby impairing cell division and proliferation, and ultimately inhibiting plant growth (Settler and Flannigan 2001). AM fungi have been demonstrated to significantly enhance biomass production of host plants under drought conditions, as evidenced in tomato ( Solanum lycopersicum ) and Bauhinia faberi var. microphylla (Alam et al. 2023). In the present study, AM fungal inoculation significantly increased root, stem, and leaf biomass production under both AT and DT conditions, thus alleviating the inhibitory effect of DT on biomass accumulation. In addition, biomass of roots, stems, and leaves showed highly significant positive correlations with mycorrhizal colonization rate and soil mycelium length, which aligns with the findings reported by Erice et al. (2024). This result further confirmed that efficient mycorrhizal symbiosis establishment is a prerequisite for AM fungi to exert growth‐promoting effects under drought stress at the physiological level. The biomass of roots, stems, and leaves was significantly positively correlated with PtGRF1, PtGRF2, PtGRF5, PtGRF13, and PtGRF15, further supporting the potential key regulatory role of the 14‐3‐3 gene family in AM fungal‐mediated growth promotion. As ubiquitous signaling regulators, 14‐3‐3 proteins can interact with a wide array of phosphorylated target proteins to modulate critical physiological processes, including cell cycle progression, carbon and nitrogen metabolism, ROS scavenging, and ABA signaling (Huang et al. 2022; Zhao et al. 2021). In the present study, the expression levels of PtGRF members were highly coordinated with plant biomass accumulation and the extent of mycorrhizal colonization, suggesting that AM fungi may enhance growth under drought stress by upregulating specific PtGRF genes to orchestrate downstream metabolic and defense pathways, thereby promoting overall plant growth (An et al. 2025).

Roots are fundamental for water and nutrient acquisition, and their morphology is a critical determinant of plant resilience to environmental challenges (Su et al. 2024). Our findings indicated that DT application significantly inhibited the root morphology of both mycorrhizal and non‐mycorrhizal trifoliate orange plants. However, inoculation with AM fungi significantly promoted root development, irrespective of soil moisture, enabling host plants to explore the soil more efficiently (Khan et al. 2022; Zhu et al. 2025). The promotive effect of AM fungi on root morphology has also been confirmed in kenaf ( Hibiscus cannabinus ) (Xu et al. 2024). Correlation analysis further indicated that total root length, root projected area, surface area, average diameter, and volume were all positively and significantly correlated with mycorrhizal colonization rate and soil hyphal length. The promotion of root morphology under mycorrhization is due to the balance of auxins, cytokinins, polyamines, and nutrients (Liang et al. 2025; Liu et al. 2023). Integrating our correlation analyses, it proposes that AM fungi orchestrate a synergistic regulatory axis linking mycorrhizal colonization, hormone homeostasis, and nutrient supply, which together alleviate drought‐induced root damage and promote favorable root morphology in trifoliate orange. In addition, PtGRF1, PtGRF2, PtGRF3, PtGRF5, PtGRF8, PtGRF13, and PtGRF15 were significantly positively correlated with all measured root morphological traits. In Arabidopsis, the 14‐3‐3 gene GRF9 enhanced whole‐plant growth and root growth under polyethylene glycol (PEG)‐induced stress conditions (He et al. 2015). Similarly, a soybean 14‐3‐3 gene, GsGF14o, contributed to drought tolerance in Arabidopsis, also impacting root hair development (Sun et al. 2014). These findings highlight the direct involvement of specific 14‐3‐3 isoforms in modulating root morphological responses to DT.

Plants regulate their sugar metabolism network in response to environmental stress, where soluble sugars serve not only as energy donors but also function as osmoregulatory substances and signaling molecules involved in stress responses (Wang et al. 2018; Zhu et al. 2024). This drought‐induced depletion of root soluble sugars in uninoculated plants represents a typical physiological deficit under drought: it not only limits cellular energy metabolism but also reduces intracellular osmotic potential, thereby weakening root water retention capacity and overall plant drought tolerance (Tsuji et al. 2022). However, the response of sugar content in trifoliate orange roots to F. mosseae inoculation differed significantly under varying water conditions. Inoculation with F. mosseae significantly increased the soluble sugar content under both water regimes. Notably, the promotive effect on sucrose and glucose was more pronounced under DT versus AT. This finding aligns with studies on tomato under cold stress conditions and on Saccharum arundinaceum under DT conditions (Mirshad and Puthur 2016). AM fungi can enhance the transport and allocation of sugars to roots, upregulate the expression of SWEET family genes to facilitate their long‐distance transport from shoots, and maintain sugar homeostasis by modulating the activity of key enzymes such as sucrose synthase and acid invertase, thereby reducing futile consumption of soluble sugars (He et al. 2025; Wang et al. 2019; Tao et al. 2024). Correlation analysis also indicated that sucrose, glucose, and fructose levels were all significantly positively correlated with mycorrhizal colonization, mycelium length, and ABA. Carbon sources (e.g., fructose, glucose, and sucrose) can both amplify the beneficial effects and increase the AM fungal colonization rates (Zheng et al. 2024). The positive correlation between ABA and sugar levels suggests a mechanism by which ABA might indirectly support mycorrhizal growth by increasing the availability of sugars in the roots (Jing et al. 2025). Additionally, PtGRF1, PtGRF2, PtGRF3, PtGRF5, PtGRF13, and PtGRF15 showed significantly positive correlations with sucrose, glucose, and fructose levels in roots. This correlation provides a mechanistic link, suggesting that the observed upregulation of some 14‐3‐3 genes (e.g., PtGRF1, PtGRF2, etc.) by AM fungi underlies the enhancement of the host antioxidant system, potentially via the well‐established role of 14‐3‐3 proteins in modulating the activity of stress‐responsive transcription factors and enzymes (Huang et al. 2022; Zhao et al. 2021). Consistent with these findings, the observed positive correlations suggest that under DT, AM fungi upregulated PtGRF genes (e.g., PtGRF1, PtGRF2, PtGRF3, PtGRF5, PtGRF13, and PtGRF15), which potentially promotes root sugar accumulation, a process that both supports fungal colonization and synergizes with ABA to improve stress adaptation (An et al. 2025; Jing et al. 2025). Notably, these correlative observations do not establish causation; functional experiments are required to verify the proposed roles of specific PtGRFs in mediating sugar accumulation and ABA signaling under mycorrhizal drought conditions.

4.6. PtGRFs Correlate With Antioxidant Defense and Phytohormone Signaling Under Drought

In this study, SOD, POD, and CAT activities in roots were significantly enhanced by AM fungi under DT conditions. This indicates that AM fungi elevate the antioxidant enzyme defense capacity in host plants to more effectively scavenge ROS accumulated due to DT, aligning with the findings of Abdi et al. (2023) in wheat, Kavatagi and Lakshman (2023) in tomatoes, and Adavi et al. (2020) in potatoes. 14‐3‐3 proteins play a significant role in modulating antioxidant defense system (Li et al. 2007). For example, overexpression of 14‐3‐3 genes in potato tubers led to a 45% increase in antioxidant activity compared with control plants, while repression of 14‐3‐3 genes had the opposite effect (Łukaszewicz et al. 2002). In mycorrhizal Populus, AM‐induced 14‐3‐3 expression correlated with higher SOD and POD activities and improved drought resistance, suggesting 14‐3‐3s mediate symbiosis‐enhanced antioxidant defenses (Han et al. 2022). Our study also revealed a significantly positive correlation between PtGRF1, PtGRF2, PtGRF3, PtGRF5, PtGRF13, PtGRF15 and SOD, POD, and CAT activities. These findings suggest a potential link between the observed upregulation of some 14‐3‐3 genes (e.g., PtGRF1, PtGRF2, etc.) by AM fungi and the enhancement of the host's antioxidant system, thereby potentially contributing to improved drought resistance, though further experimental validation of this mechanistic link is required. The lack of a statistically significant reduction in MDA levels in roots by AM fungi, despite higher antioxidant enzyme activities, may be due to temporal sampling (e.g., measuring at a single time point may not capture the peak of oxidative damage or the full recovery phase) or tissue heterogeneity within the roots (Shi et al. 2026). Nevertheless, the elevated enzyme activities indicate a substantially enhanced capacity to scavenge ROS, which is a key component of the AM‐mediated drought tolerance mechanism.

Phytohormones play key roles in plant growth, development, and stress responses, and AM fungi can influence plant responses to drought stress by modulating endogenous hormone levels (Liu et al. 2023; Quiroga et al. 2020). In this study, AM fungal inoculation under AT conditions reduced the concentrations of DZ, TZ, IP, IAA, and IBA. In contrast, under DT conditions, the inoculation significantly increased the concentrations of ABA, IP, and IAA. Under DT, AM fungi may promote root growth by enhancing IAA synthesis or reducing its degradation rate, as evidenced by AM plants exhibiting superior root development and IAA levels under DT, given that IAA is fundamental to both root development and plant tolerance to stress (Ma et al. 2022). This IAA‐mediated root morphological improvement provides a structural basis for enhanced water and nutrient uptake under drought stress, representing a key morphological adaptation mechanism of mycorrhizal plants to drought. Root ABA levels were upregulated by AM fungi exclusively under DT conditions, revealing a demand‐activated regulatory strategy of the mycorrhizal symbiosis in influencing ABA homeostasis. Unlike the constitutive regulation of hormones in non‐stress environments, the drought‐specific induction of ABA by AM fungi represents a stress‐targeted regulatory strategy that enhances the plant's intrinsic stress response capacity. This implies that AM fungi enhance drought resistance by amplifying the plant's stress alarm signal (ABA), thereby strengthening a series of downstream physiological responses, such as osmotic solute accumulation and antioxidant defense (Aroca et al. 2009).

Notably, the expression of PtGRF1, PtGRF2, PtGRF3, PtGRF5, PtGRF6, PtGRF9, and PtGRF13 was significantly positively correlated with ABA levels. In tomato ( Solanum lycopersicum ), the AM symbiont could enhance plant drought tolerance by regulating 14‐3‐3 genes within the ABA signaling pathway (Xu et al. 2018). Research on poplar further confirmed that AM fungal inoculation under drought stress upregulated the expression of 14‐3‐3 family protein genes, particularly specific PtGRF genes (e.g., PtGRF1, PtGRF2, PtGRF3, PtGRF5, PtGRF6, PtGRF9, and PtGRF13) whose expression is significantly positively correlated with ABA levels (Han et al. 2022). This suggests that ABA, as an upstream stress signal, may enhance drought adaptation by upregulating specific 14‐3‐3 proteins, which then broadly regulate downstream physiological processes like antioxidant defense and sugar metabolism. In future studies, functional verification of key 14‐3‐3 family members will be performed via gene overexpression and silencing, combined with exogenous ABA application, to directly confirm whether ABA acts as an upstream stress signal mediating the regulation of these genes in response to drought stress. Such genetic manipulation and pharmacological experiments will help clarify the causal regulatory relationship between ABA signaling and 14‐3‐3 proteins, rather than relying solely on correlational evidence.

5. Conclusions

In this study, 15 members of the 14‐3‐3 gene family (PtGRF1–PtGRF15) were identified in trifoliate orange. These genes exhibit conserved protein domains and structures; yet, they vary in exon count and promoter cis‐elements. Their promoter regions are enriched in binding sites related to stress responses, phytohormones (particularly ABA), and light signaling. Of these, expression of 13 PtGRFs was detected in roots, while PtGRF4 and PtGRF11 were not expressed in roots under the conditions examined, possibly due to tissue‐specific expression, functional redundancy, or expression under untested developmental stages or environmental cues. Under drought, AM fungi specifically up‐regulated the expression of 10 of 13 root‐expressed PtGRF genes. These induced PtGRF genes appear to function as key molecular interfaces linking mycorrhizal symbiosis with host physiological adaptation, showing significant positive correlations with root architecture, sugar accumulation, antioxidant enzyme activities, and ABA levels (Figure 11). Although these correlations suggest potential regulatory roles, functional experiments (e.g., gene silencing or overexpression) are required to confirm the direct involvement of specific PtGRFs, especially PtGRF13, in AM‐mediated drought tolerance.

FIGURE 11.

A schematic diagram displaying the role of AM fungi in drought resistance. Here, ↑meant significant increase in this variable after inoculating AM fungi. ABA, abscisic acid; AM, arbuscular mycorrhizal; CAT, catalase; DZ, dihydrozeatin; Fru, fructose; Glc, glucose; IAA, indole‐3‐acetic acid; IBA, indole‐3‐butyric acid; IP, N⁶‐isopentenyladenine; POD, peroxidase; SOD, superoxide dismutase; Suc, sucrose; TZ, trans‐zeatin.

Author Contributions

Feng‐Ling Zheng: conceptualization, data curation, methodology, formal analysis, writing – original draft. Ke‐Xing Yan: methodology, formal analysis. Zhen Liu: methodology, formal analysis. Ying‐Ning Zou: review and editing, supervision. Funding acquisition. Qiang‐Sheng Wu: review and editing, methodology. Qing‐Ping Yi: review and editing, supervision. Mashael Daghash Alqahtani: methodology, funding acquisition.

Funding

This work is supported by the Opening Project of China Agricultural Valley Development Research Center (grant no. 2025zgng01). The authors would like to extend their sincere appreciation to the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R355), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Supporting information

Table S1: Specific primer sequences of genes in qRT‐PCR.

Zheng, F.‐L. , Yan K.‐X., Liu Z., et al. 2026. “ Funneliformis mosseae Modulates Root 14‐3‐3 Gene Expression to Enhance Drought Tolerance in Trifoliate Orange: A Molecular‐Physiological Integration.” Physiologia Plantarum 178, no. 5: e71110. 10.1111/ppl.71110.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.