Mycorrhizal symbiosis-induced abiotic stress mitigation through phosphate transporters in Solanum lycopersicum L

Arbuscular mycorrhizal (AM) symbiosis and abiotic stress mitigation have intrigued researchers for more than a century, but how different phosphate transporters, such as members of the Pht1 gene family, are influenced during the combined presence of AM fungi and stress is not well known. In this study, the impact of AM fungi (Funneliformis mosseae) on tomato plants under water deficit and heat stress was investigated via observing the physiological changes and applying spectrophotometric and quantitative real-time PCR methods, with a focus on phosphate transporters (Pht1;1, Pht1;3, Pht1;4, Pht1;6, Pht1;7 and Pht1;8). Moreover, genes encoding heat-responsive proteins (HSFA2 and HSP70) and aquaporins (PIP2.5 and PIP2.7) were also studied. On the basis of our results, AM fungi seemingly mitigated heat and combined (heat and water deficit) stresses through the mediation of the expression of Pht1 family phosphate transporter genes. In addition to the Pht1;3 and Pht1;4 genes, Pht1;7 also seems to be an AM fungus-inducible phosphate transporter gene. The results of this study may provide insights into the behavior of phosphate transporter gene family members and a potential strategy to enhance the vigor of tomato plants through increased phosphorous uptake under heat stress, water deficit and heat and water deficit combined.


Introduction
Increases in average temperature, frequency of heat waves and duration of drought periods limit plant growth, reduce crop quality and threaten food security (Sun et al. 2019;Hari et al. 2020;AbdElgawad et al. 2022). Mitigating plant abiotic stress in an environmentally friendly way is a major challenge for agriculture (Pretty 2008). Thus, one of the main challenges involves using and expanding upon the already existing mechanisms in plants that have evolved to overcome these environmental issues (Ibrahim and El-Sawah 2021). The establishment of symbiotic relationships with arbuscular mycorrhizal (AM) fungi is one such option (Sheteiwy et al. 2021b). As obligate biotrophs, AM fungi acquire necessary photosynthetic products and lipids from their hosts (Pfeffer et al. 1999;Alotaibi et al. 2021;Sheteiwy et al. 2021a). Through direct and indirect mechanisms, plant-AM fungi interactions can help plants overcome abiotic stress (Dell'Amico et al. 2002;Begum et al. 2019). These direct mechanisms include the mediation of antioxidant systems, regulation of phytohormone biosynthesis pathways, and changes in the expression of stress-responsive genes (Rivero et al. 2015;Begum et al. 2019). Various abiotic stresses, such as water deficit and heat, can lead to the production of reactive oxygen species (ROS) (Dvorak et al. 2020); however, plants are able to eliminate these molecules by enzymatic (Zhao et al. 2018;Jahan et al. 2019) and nonenzymatic antioxidants, which include metabolites such as glutathione, ascorbate, carotenoids and flavonoids (Raja et al. 2020). Superoxide (O 2 − ) radicals actively generated through enzymatic pathways involved in stress-response signaling is rapidly converted to hydrogen peroxide (H 2 O 2 ) by superoxide dismutase (SOD), after which the H 2 O 2 is Communicated by Daolong Dou.
Viktor Szentpéteri and Zoltán Mayer have contributed equally to this work and share first authorship. neutralized by catalase (CAT) and peroxidase (POD) via catabolism into water and oxygen (Bienert and Chaumont 2014;Dvorak et al. 2020). In addition to the antioxidant system, mycorrhizal symbiosis modulates phytohormone pathway activity, and jasmonic acid, abscisic acid, auxin, ethylene and salicylic acid responses are triggered, thus reducing stress effects (Rivero et al. 2018;Chandrasekaran et al. 2021;Jajoo and Mathur 2021). Previous studies have shown that under water or high-temperature stress, mycorrhizal fungi are able to regulate the expression of heat shock factors, chaperones and aquaporins (Rivero et al. 2015;Duc et al. 2018;Recchia et al. 2018;He et al. 2019;Sharma et al. 2021;Malhi et al. 2021). The indirect increase in stress tolerance in plants is the result of increased water and essential macro-and micronutrient uptake ensured by the extension of the rhizosphere through the promotion of root growth by AM fungi or through the utilization of the fungal hyphal system (Smith et al. 2003;Vessey 2003;Aggarwal et al. 2011;Begum et al. 2019;Bhantana et al. 2021;Ngo et al. 2021). The physiological responses of roots under water deficit and heat stress, such as reduced root biomass, result in substantially reduced cation exchange capacity and water and nutrient uptake (Lukowska and Jozefaciuk 2013;Moles et al. 2018). All of these changes can be mitigated by AM fungi, thus ensuring optimal growth even under adverse conditions (Chandrasekaran et al. 2021).
Based on previous studies that have shown that genes belonging to the plant phosphate transporter 1 (Pht1) family might affect drought tolerance (Volpe et al. 2018;Li et al. 2019;Cao et al. 2020;Sun et al. 2021), we focused on changes in the phosphorous uptake of mycorrhizal plants under heat stress, water deficit and heat and water deficit combined. Proteins transcribed from this gene family are localized inside the plasma membrane, mostly in those of root cells, and act as Pi:H + symporters (Daram et al. 1998). One of the main inducers of their expression is phosphorous starvation; however, other factors can also affect them (Liu et al. 1998). AM fungi interact with Pht1 transporters Pht1;3, Pht1;4, and Pht1;5 in tomato, and these genes have been shown to be overexpressed in mycorrhizal roots (Harrison et al. 2002;Nagy et al. 2005). Moreover, some transporters act as necessary signal relays for the development of arbuscules. The loss of function of MtPT4, a Medicago truncatula phosphate transporter that essential for the delivery of Pi by AM fungi, leads to premature death of arbuscules, and thus, the symbiosis is terminated (Javot et al. 2007a). The expression of Pht1 phosphate transporters changes either through direct regulation by symbiosis or through changes in the phosphorous level in cells (Rausch et al. 2001, Nagy et al. 2005, 2006, Javot et al. 2007b, Xie et al. 2013). However, little is known about the importance of Pht1 transporters in mycorrhizal plants under heat and/ or water deficit stress. In this sense, we hypothesize that, by working together with Pht1 gene family members the AM fungus Funneliformis mosseae could alleviate the effects of heat and water deficit stress both individually and combined on tomato.

Production of AM fungal inocula
Funneliformis mosseae BEG12 (Glomerales, Glomeraceae) was propagated on maize (Zea mays L. 'Golda F1') growing on sterilized peat (Klasmann TS3, 100 mg L −1 P 2 O 5 ) and sand 1:3 (v/v) substrate for three successive propagation cycles, each lasting 5 months. The most probable number (MPN) of infective propagules (approximately 35 infective propagules g −1 ) was determined following the methods of Feldmann and Idczak (1992). For the calculation of the MPN, maize plants were cultivated on a tenfold dilution series generated from the original inoculant, with five replicates. Mycorrhizal structures were observed in all five replicates, and all dilution levels were characterized depending on the number of replicates with mycorrhizal structures (0-5). The least concentrated dilution in which all five root system replicates were colonized and the next two higher dilutions (10 and 100 times more diluted) were used in the calculations, and the resulting three digit number was multiplied by the appropriate dilution factor from the table, as demonstrated by Feldmann and Idczak (1992).

Plant material and treatments
Tomato (Solanum lycopersicum L. 'Kecskeméti F1') seeds were sterilized and allowed to germinate in the dark for three days in glass petri dishes. After germination, the seedlings were transplanted to plastic pots (one plant pot −1 ) whose bottoms were drilled out; the pots contained 570 g of a thrice-sterilized (121 °C for 30 min) peat (Klasmann TS3, 100 mg L −1 P 2 O 5 ):sand 1:4 (v/v) substrate. The treatment plants were inoculated with AM fungi; the inoculant consisted of 20 g soil containing spores and mycelia of F. mosseae (BEG12 (Glomerales, Glomeraceae). The control plants received the same amount of inoculant that was autoclaved. After potting, the seedlings were grown under a 16/8 h day/night photoperiod at a temperature of 26/20 °C (day/night), a photosynthetic photon flux density (PPFD) of 500 μmol m −2 s −1 and a relative humidity of 60% in a growth chamber (EKOCHL 1500) for 7 weeks. Afterward, the pots were placed on porous foam (Oasis IDEAL Floral Foam Maxlife brick) in plastic containers filled with water (water level of 22 cm) following the modified methods of Snow and Tingey (1985). The plants were irrigated weekly with 25 mL of modified Long Ashton (Hewitt 1966) nutrient solution consisting of 320 µM Pi. After cultivation, plants (mycorrhizal and nonmycorrhizal ones) were separated into four groups: a control group and three stress treatment groups. The control group was subjected to the same conditions as those applied during their growth. The plants in the treatment groups were exposed to different stress conditions (water deficit, heat and heat and water deficit combined). The water deficit-stressed plants were exposed to a gradual, two-week water deficit according to the methods described by Snow and Tingey (1985) modified by Fernández and Reynolds (2000) and further modified based on the results of our preliminary investigations. This was achieved by reducing the height of the water level to 1 cm (Fig. 1). The heat-stressed plants were exposed to a gradual daily temperature increase for a week. Starting with 26 °C between 4:00 and 8:00, the temperature was raised to 32 °C from 8:00 to 10:00. The heat stress peaked at 42 °C from 10:00 to 14:00 and then declined from 14:00 to 16:00 to 32 °C. From 16:00 to 20:00, the temperature was 26 °C, and plants were exposed to 20 °C overnight between 20:00 and 4:00 to simulate realistic conditions (Fig. 1). The plants exposed to combined stress were subjected to both conditions. Altogether, plants under eight different treatments replicated six times were included: mycorrhizal (CAM) and nonmycorrhizal control (C) plants, heat-stressed mycorrhizal (HAM) and nonmycorrhizal (H) plants, water deficit-stressed mycorrhizal (DAM) and nonmycorrhizal (D) plants, and combined (heat and water deficit)-stressed mycorrhizal (HDAM) and nonmycorrhizal (HD) plants. Directly after the stress treatments, the plant material was collected, frozen in liquid nitrogen and stored at -80 °C until use.

Measured parameters
Soil volumetric water content (VWC%) was monitored with a FieldScout TDR-350 Soil Moisture Meter (Spectrum Technologies, Illinois, USA). For the control and heat-stressed plants during the stress period, the VWC% was maintained at a level similar to that under field conditions (37.71 ± 1.70%) by continually maintaining the water level in the containers at 22 cm. In the water deficit and combined stress treatment groups in which the water level was reduced to 1 cm, the VWC% was reduced to 8.25 ± 0.55%. This percentage is already below the permanent wilting point according to Datta et al. (2017) and the FieldScout TDR-350 Soil Moisture Meter (Spectrum Technologies, Illinois, USA) user manual ( Figure S1).  gey (1985) water deficit method. Capillary irrigation is used to control the soil water content of potted plants, which are placed above a solid column with low water permeability. For porous foam (a, Oasis IDEAL Floral Foam Maxlife brick), the intensity of the water deficit is controlled by the water level in the plastic container (b). A Diagram of the water level under control (C, CAM) and heat stress (H, HAM) conditions. B Diagram of the water level under water deficit (D, DAM) and combined stress (HD, HDAM) conditions.; C Daily gradual temperature change during heat and heat and water deficit stress combined (generated with MS Office)

Assessment of tomato plant biomass and relative water content (RWC) of the leaves
The fresh and dry shoot and root biomass of each treatment was measured. For dry biomass weight determination, the shoots and roots were dried in a forced hot-air oven at 70 °C until constant weights were reached. The RWC of the leaves was determined according to the methods of Barrs and Weatherley (1962).

Assessment of root colonization
Root colonization was determined by visual inspection under a stereomicroscope at 100 × magnification. Before microscopic inspection, the roots were dyed. The root fragments were then cleaned with 10% KOH for 10 min, immersed in 2% hydrochloric acid, and stained with 0.05% trypan blue (1:1:1 proportion of water/glycerol/lactic acid) overnight (Trouvelot et al. 1986). A total of fifty 1-cm-long root fragments were randomly selected from each sample. Mycorrhizal colonization was estimated with the gridline intersection method by observing the presence or absence of mycorrhizal structures at the intersections between the root fragments and the gridlines (Giovannetti and Mosse 1980).

Phosphorus (P) content measurements
For P content analysis, dried shoots were ground in a mortar. Milled leaves were digested with 5 mL of 65 w/w% HNO 3 and 2 mL of 35 w/w% H 2 O 2 until complete dissolution occurred (40 min) in a CEM MARS 5 (Magne-Chem Ltd., Budapest, Hungary) device using the microwave pressure digestion method. A CP-OES spectrometer (HORIBA Jobin Yvon ACTIVA-M, Edison, NJ, USA) was used to quantify shoot P concentrations. The P content was estimated by multiplying the shoot dry weight and the measured P concentrations. The mycorrhizal P contribution was calculated in terms of the P content of the shoots of the nonmycorrhizal (NM) and mycorrhizal (AM) plants of the different treatments as follows: 100 × [(AM-NM)/NM] (Cavagnaro et al. 2003).

H 2 O 2 and MDA contents and ROS-scavenging enzyme activity measurements
H 2 O 2 content, malondialdehyde (MDA) content and ROSscavenging enzyme activity were measured in root samples of three biological replicates from each treatment. The H 2 O 2 content was determined according to the methods of Alexieva et al. (2001) on a UV-VIS spectrophotometer (U-2900, Hitachi, Japan) at 390 nm. The level of lipid peroxidation was determined by the methods of Heath and Packer (1968). The MDA amount was calculated based on the absorbance at 532 and 600 nm measured using a doublebeam spectrophotometer. The activity of POD (EC 1.11.1.7) was measured according to the methods of Rathmell and Sequeira (1974), and the change in absorbance was observed at 436 nm wavelength. The CAT (EC 1.11.1.6) enzyme activity was measured by the methods of Aebi (1984) based on the dissociation of H 2 O 2 ; the change in absorbance was observed at 240 nm for 5 min. Last, the SOD (EC 1.15.1.1) activity was determined according to the methods of Beyer and Fridovich (1987), with absorbance change measured at 560 nm.

RNA extraction and quantitative real-time PCR analysis
Total RNA was extracted from root samples of four biological replicates (the same three plants used for the enzyme measurements, plus an additional one) from each treatment using an EZNA Plant RNA Kit (Omega Bio-Tek, USA) following the manufacturer's protocol. DNase I (Thermo Scientific, USA) was applied to the samples. RNA integrity was evaluated according to the measurements of the 260/280 and 260/230 nm absorbance ratios measured by a Nanophotometer (IMPLEN GmbH, München, Germany). cDNA was synthesized from 0.5 µg of RNA by a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Before quantitative real-time PCR experiments, the cDNA was diluted 1:5. Quantitative real-time PCR amplification reactions were performed on 96-well plates on a Stratagene Mx3000P Real Time PCR System using ABsolute SYBR Green Low ROX qPCR Mix (Thermo Fisher Scientific, Inc., Vilnius, Lithuania), with three technical replications. The PCR mixtures consisted of 12.5 μL of ABsolute SYBR Green Low ROX qPCR Mix, 1 μL of each forward and reverse primer (10 mM), and 9.5 μL of nuclease-free water. Finally, 1 μL of diluted template cDNA (1:5) was added, resulting in a total volume of 25 μL PCR mixture −1 . The PCR cycling program was as follows: 15 min at 95 °C followed by 40 cycles of 15 s at 95 °C, 30 s at 59 °C, and 20 s at 72 °C. Melting curve cycling consisted of 60 s at 95 °C, 30 s at 59 °C, and 30 s at 95 °C. The relative transcript abundance was determined by normalization to the abundance of the internal control elongation factor 1-alpha (Ef1α) with use of the 2 −ΔΔCт method (Schmittgen and Livak 2008). The expression patterns of genes encoding phosphate transporters (Pht1;1, Pht1;3, Pht1;4,Pht1;6,Pht1;7,Pht1;8) and aquaporins (plasma intrinsic proteins (PIPs) PIP2.5; PIP2.7), heat stress-related genes (heat-inducible transcription factor (HSFA2) and heat shock protein 70 (HSP70)), and defense-related enzyme-coding genes (POD, SOD, CAT ) were measured. The sequences of the primers used are listed in Table S1.

Statistical analysis
All the data were statistically analyzed with R statistical software 4.0.2 (R Core Team 2020). Normality and homoscedasticity were checked via Kolmogorov-Smirnov and Levene tests, respectively. Two-way analysis of variance (ANOVA) was used considering mycorrhizal inoculation and stress treatment as factors, and the means were compared using Tukey's post-hoc test. A P value < 0.05 was considered to indicate statistical significance. Pearson correlation coefficients were used to determine relationships among plant root biomass, AM fungal colonization, stress markers, aquaporin genes, heat-inducible genes and phosphate transporter gene expression parameters via the function rcorr from the Hmisc R package. For graphic presentation of the heatmaps, Displayr (www. displ ayr. com) was used.

Effects of different treatments on tomato growth
Signs of AM fungal colonization were found on all the inoculated tomato plants but not on the plants in the nonmycorrhizal treatments (Table 1). There was no significant difference in root colonization by F. mosseae under the control and different stress conditions. AM fungal colonization significantly increased the shoot and root biomass under the control conditions. The stress treatments significantly reduced the biomass of the nonmycorrhizal plants compared to that of plants under the control conditions, reduced the biomass of the fresh shoots under water deficit and combined stress conditions, and reduced the biomass of the dry shoots under the combined stress. Mycorrhizal inoculation and stress had a significant influence on both the fresh and the dry biomass of the shoots and roots (Table 1). Significant differences in fresh shoot and root weights were observed for AM fungus-inoculated plants under control and stress conditions, respectively. Moreover, plants colonized with mycorrhizal fungi showed significantly higher fresh shoot and root biomass production than did the nonmycorrhizal plants under the heat and combined stress treatments. Overall, AM fungal inoculation increased the biomass production of the plants under the different treatments, and this effect was higher under heat stress and relatively minor under water deficit and the combined stress (Table 1).

RWC and P content
The RWC of the mycorrhizal plants did not change under control conditions. In comparison to that of the control plants, the RWC of nonmycorrhizal plants under the heat, water deficit and combined stresses significantly decreased. This decrease also occurred in the mycorrhizal plants; however, it was less severe. According to the pairwise comparisons of the mycorrhizal and nonmycorrhizal plants, a significantly higher RWC was found in the mycorrhizal plants under the water deficit (P < 0.05) and combined (P < 0.01) stresses (Table 1). Regarding the P content of the shoots, we found a general decrease caused by the stress treatments. Nevertheless, according to the pairwise comparisons, compared with their nonmycorrhizal counterparts, the mycorrhizal plants under all the stress treatments presented a higher P content (Table 1).

H 2 O 2 and MDA contents and the activity of antioxidant enzymes
AM fungal inoculation did not affect the level of H 2 O 2 under nonstress conditions. On the other hand, compared with the control treatment, the stress treatments resulted in significantly higher H 2 O 2 levels in the roots, while no differences were observed between the different inoculations (Table 2). Moreover, when comparing mycorrhizal to nonmycorrhizal plants, we found no significant differences in H 2 O 2 accumulation under different stress conditions (heat stress, water deficit and heat and water deficit combined). The levels of MDA did not change under the control treatment due to mycorrhizal inoculation. Under heat and water deficit stress, the levels of MDA significantly increased in the nonmycorrhizal plants. In contrast, according to the pairwise comparison of mycorrhizal and nonmycorrhizal plants under control and combined stress conditions, the detected MDA levels were not significantly different (Table 2). Similar to H 2 O 2 and MDA, colonization of AM fungi did not influence the activity of the measured defense-related enzymes under the control conditions. The activities of SOD, CAT and POD in the nonmycorrhizal plants were markedly increased under the stress conditions compared to the control conditions. The SOD activity was significantly (P < 0.05) lower in the mycorrhizal plants than in the nonmycorrhizal plants under heat, water deficit and combined stress conditions. Significantly lower SOD enzyme activities were detected in the control plants compared with the stressed plants, with the exception of the water deficit stress-treated mycorrhizal plants. SOD gene expression was affected by the stress treatment and mycorrhizal inoculation in the same way as the enzyme activity was. In general, the mycorrhizal plants did not show differences in CAT enzyme activity among the different stress treatments compared to the nonstress treatment. However, according to the pairwise comparison of mycorrhizal plants and nonmycorrhizal plants in the stress treatments, the mycorrhizal plants always showed significantly lower CAT enzyme activities. CAT gene expression appeared to consistently follow the same trend as that of Table 1 Biomass, relative water content (RWC), colonization, and phosphorous (P) content of plants under different treatments. P = phosphorous, DSW = dry shoot weight 1 Plants under control (C), control with mycorrhizal fungus (CAM), heat (H), heat with mycorrhizal fungus (HAM), water deficit (D), water deficit with mycorrhizal fungus (DAM), combined (heat + water deficit, HD) and combined with mycorrhizal fungus (heat + water deficit + AM, HDAM) stress conditions (9 weeks after transplanting, with water stress beginning during the 7th week and heat stress beginning during the 8th week) 2 The values are the mean ± SD. The values within a column followed by the same letter do not differ significantly according two-way analysis of variance (ANOVA) combined with Tukey's post-hoc test (P < 0.05) 3 *, significant at P < 0.05; **, significant at P < 0.01; ***, significant at P < 0.001; n.s. = not significant The values are the mean ± SD. The values within a column followed by the same letter do not differ significantly according two-way analysis of variance (ANOVA) combined with Tukey's post-hoc test (P < 0.05) 3 *, significant at P < 0.05; **, significant at P < 0.01; ***, significant at P < 0.001; n.s. = not significant  Table 2). The POD enzyme activity did not significantly differ between the mycorrhizal plants and the nonmycorrhizal plants under the control conditions. The stress treatments also increased the level of POD activity in the inoculated and noninoculated plants, with the highest change found in response to the combined stress. Independent of the stress treatment, the POD activity was significantly higher in the nonmycorrhizal plants than in the mycorrhizal plants (Table 2); this higher activity was especially prominent under heat stress (P < 0.01).
The POD gene expression showed the same trend, becoming upregulated under stress conditions; however, POD expression was significantly higher in the nonmycorrhizal plants than in the mycorrhizal plants under all the treatments (Table 2).

Relative expression of phosphate transporter, aquaporin, and heat stress-related genes
The root tissues of tomato plants showed slightly variable patterns of expression of the six transporter genes of the Pht1 family under the different conditions (Fig. 2). Under the control conditions, we detected a nearly 20-fold upregulation of the Pht1;7 gene. Under heat stress, three . The data shown are mean 2 −ΔΔCт values ± standard deviation (SD); the means followed by the same letter do not differ significantly according two-way analysis of variance (ANOVA) combined with Tukey post hoc test (P < 0.05);*, significant at P < 0.05; **, significant at P < 0.01; ***, significant at P < 0.001 genes (Pht1;1, Pht1;3, Pht1;4) showed higher expression (P < 0.01, P < 0.001 and P < 0.05, respectively), two genes were not significantly differentially expressed (Pht1;6, Pht1;7) and one gene (Pht1;8) was downregulated (P < 0.01) in the mycorrhizal plants compared to the nonmycorrhizal plants. Interestingly, the expression level of the Pht1;8 gene was threefold higher in the nonmycorrhizal plants than in the mycorrhizal plants under the same conditions. Pht1;1, Pht1;3 and Pht1;4 gene expression increased more than twofold, threefold and more than fourfold, respectively, in the mycorrhizal plants compared with the nonmycorrhizal plants (Fig. 2). Under water deficit, in contrast to the slightly higher Pht1;1 gene expression in the nonmycorrhizal plants, Pht1;3, Pht1;4, Pht1;7 and Pht1;8 were expressed to a greater degree in the mycorrhizal plants; nevertheless, the expression levels of these genes were statistically no different. Under the combined stress, in terms of their expression, Pht1;3 (P < 0.05) and Pht1;7 (P < 0.05) were significantly induced, Pht1;1 and Pht1;4 were slightly induced, Pht1;6 was significantly downregulated (P < 0.01), and Pht1;8 was slightly downregulated in the mycorrhizal plants (Fig. 2). The relative gene expression of the studied PIPs was highly variable in response to stress, as the expression of some of these genes was up-and downregulated in response to the different treatments, or there was no change in expression. Under water deficit, PIP2.5 showed significantly (P < 0.05) increased expression in the mycorrhizal plants, while water stress combined with heat stress reduced PIP2.5 expression in both the nonmycorrhizal and the mycorrhizal plants. The expression of PIP2.7 tended to be the same in the all treatments and decreased in the mycorrhizal plants. In response to combined heat and water stress, PIP2.7 expression significantly (P < 0.01) decreased in the mycorrhizal plants compared to the nonmycorrhizal plants (Fig. 3). Additionally, the expression of the heat stress-related genes HSP70 and HSFA2 were measured (Fig. 3). A marked upregulation in HSP70 and HSFA2 expression was observed in the case of heat stress alone (P < 0.05) and the combined stress (P < 0.01 and P < 0.05) in the nonmycorrhizal plants. The relative expression levels of the HSP70 and HSFA2 genes increased more than twofold under heat stress and one anda-half to fourfold under the combined stress; however, the expression differences were not significant under water deficit stress alone (Fig. 3). Mycorrhizal inoculation did reduce the expression of HSP70 in all the treatments; the difference was significant only under the heat and combined stresses.

Discussion
Water deficit is a major abiotic stress factor that affects plant growth, reducing yields and causing economic losses in, for example, tomato cultivation (Nemeskéri and Helyes 2019;Wu et al. 2021). Therefore, a thorough study of the mechanism of stress tolerance is the first step in finding a solution. Water stress has been simulated in many ways in previous studies (Zur et al. 1966;Steinberg and Henninger 1997;Bunce and Nasyrov 2012;Poorter et al. 2012;de Araújo Silva et al. 2021;Li et al. 2022). However, uniform 1 3 irrigation applied from the top might not mimic natural soil water deficit and does not consider the variation in physical parameters between individual plants. In our experiment, which was performed in accordance with the methods of Snow and Tingey (1985), the different water needs of different sized plants could be satisfy accordingly because the soil VWC% of the pots was maintained at a consistent level.
As expected, all applied stresses (heat stress, water deficit, and heat and water deficit combined) had negative effects on plant growth and physiology, such as restricted shoot and root growth, decreased RWC and decreased P content of the shoots. The highest reduction in plant biomass was detected under the combined heat and water deficit stress conditions, followed by water deficit and, lastly, heat stress (Table 1). The greater the effect of the stress were, the higher the level of the measured stress markers, such as H 2 O 2 and MDA contents, which in turn necessitated increased activity of enzymes and expression of genes involved in the antioxidant system (SOD, CAT, POD) ( Table 2). Fig. 3 Relative expression of two plasma intrinsic proteins (PIPs) (PIP2.5; PIP2.7) and heat stress-induced genes (HSP70, HSFA2) in the roots of tomato plants under control (C), control with mycorrhizal fungus (CAM), heat (H), heat with mycorrhizal fungus (HAM), water deficit (D), water deficit with mycorrhizal fungus (DAM), combined (heat + water deficit, HD) and combined with mycorrhizal fungus (heat + water deficit + AM, HDAM) treatments (generated with MS Office). The data shown are mean 2 −ΔΔCт values ± standard deviation (SD); the means followed by the same letter do not differ significantly according two-way analysis of variance (ANOVA) combined with Tukey post hoc test (P < 0.05); *, significant at P < 0.05; **, significant at P < 0.01; ***, significant at P < 0.001 AM fungi also promoted plant growth under heat and water deficit stress conditions Volpe et al. 2018;Yeasmin et al. 2019;Ronga et al. 2019). Regardless of the relatively low mycorrhizal colonization rate, like Cheng et al. (2021) did, we also found a positive effect of F. mosseae on tomato growth. Cheng et al. (2021) showed that, despite osmotic stress, by acting on plant aquaporin activities, F. mosseae increased shoot and root biomass through, as reflected by increased water permeability of the plasma membrane, increased water-use efficiency and enhanced resistance to stress. To varying degrees, in the present study, AM fungal inoculation increased plant biomass compared to that of the nonmycorrhizal plants in all the treatments ( Table 1). The increase in the biomass of the root system is crucial for plant survival (Chitarra et al. 2016a;Bona et al. 2017;Püschel et al. 2020). Growth-related parameters, such as RWC, are affected by mycorrhizal inoculation (Chandrasekaran et al. 2019), and plants with a lower RWC are more sensitive to drought stress (Zandalinas et al. 2017). In our study, F. mosseae positively affected the RWC of tomato plants under both water deficit and the combined stress (Table 1).
In addition to their effect on plant growth, the beneficial effect of mycorrhizal inoculation was realized via increased P contents of the shoots both under stress and under nonstress conditions. The calculated mycorrhizal P contribution was highest under the combined stress, followed by the water deficit and heat stress. This suggests that the mitigation of the stress effect causing the strongest biomass decrease could be achieved only with the help of stronger mycorrhizainduced processes. Rivero et al. (2018) also demonstrated that the positive effects of AM fungal inoculation are correlated with the severity of stress. The positive effect on root growth observed in our experiment is also the result of one of these AM fungus-induced processes. The increased root growth of the plants in response to AM fungi together with the lower level of antioxidants compared to those of nonmycorrhizal plants showed that the antioxidant system was not overloaded by the applied stress, as reported in other works (Porcel et al. 2003;Selim et al. 2021). The expression of HSFA2 and HSP70, which are heat-responsive genes, exhibited decreasing patterns in response to heat stress alone and the combined stress as a result of mycorrhizal colonization, also indicating that the mycorrhizal plants were not severely affected by the stress treatments.
The symbiotic effects of mycorrhizal fungi are especially important in terms of the P uptake of colonized plants, as it can be performed by over 80% through the mycorrhizal pathway (Smith et al. 2004). Through their external hyphal network, AM fungi provide an increased root surface area and offer different mechanisms (Kobae 2019). The up-and downregulation of some phosphate transporter genes under drought stress suggests that expression changes of members of the Pht1 gene family might also have a role in stress mitigation Zou et al. 2021). Eight members of this gene family have been identified in tomato (Nussaume et al. 2011;Chen et al. 2014;Inoue et al. 2014). In our study, we measured the expression changes of six (Pht1;1,Pht1;3,Pht1;4,Pht1;6,Pht1;7,Pht1;8) of these in response to the stress treatments. Like those of Nagy et al. (2005Nagy et al. ( , 2009, our results also support that Pht1;3 and Pht1;4 are mycorrhiza-inducible phosphate transporters. Furthermore, Volpe et al. (2018) showed that Pht1;2 and Pht1;4 play a major role in the water stress response. We found a similar upregulation of Pht1;4 in mycorrhizal water stress-treated plants, but the changes were not significant. This might be due to the different experimental designs or tomato cultivars used. In addition to the three phosphate transporter genes (Pht1;1, Pht1;3, Pht1;4) that were also studied by Volpe et al. (2018), we evaluated Pht1;6, Pht1;7 and Pht1;8; however, these genes did not seem to be influenced by water stress. Moreover, we evaluated the six Pht1 genes under heat stress and combined heat and water deficit stress. The upregulation of the Pht1;1, Pht1;3,Pht1;4,and Pht1;7 genes under the heat and combined stresses and the higher expression of the Pht1;6 gene during heat stress alone in the mycorrhizal plants compared to the nonmycorrhizal plants suggests that phosphate transporters play a role in heat stress mitigation. Moreover, on the basis of our results, it is suggested that the expression of the Pht1;7 gene might also be mycorrhizal fungus-inducible due to its 20-fold upregulation in the control nonstressed treatment group. However, Pht1;7 exhibited lower expression under the stress conditions compared to the control conditions; these findings contrast with those of Pht1;3 and Pht1;4, which showed higher expression during stress and lower expression under the control conditions in the mycorrhizal plants, suggesting that these genes have a main role under nonstress conditions. Differences in the expression of phosphate transporter genes under stress and nonstress conditions indicate that the functions of these genes are coordinated, as has been reported previously in response to drought and salt stresses (Yong et al. 2014;Zhang et al. 2016). Correlation analyses of the expressed genes highlighted that all phosphate transporters evaluated in the mycorrhizal plants under no stress conditions showed strong synergistic activity. In addition, during heat stress, phosphate transporters are more strongly activated, which is probably due to the higher temperature of the mycorrhizal plants compared with the nonmycorrhizal ones (Ma et al. 2022). Heat stress induced a strong simultaneous upregulation of these genes that was observable only in the mycorrhizal plants; in the nonmycorrhizal plants, only three phosphate transporters cooperated in the same way. Under water deficit stress, except for Pht1;1, each transporter gene continued to exhibit strong expression in the roots of the mycorrhizal plants. However, in the roots of the nonmycorrhizal plants, no more than two phosphate transporters showed active cooperation. The increased P content of the shoot is also in accordance with these findings for all the treatments, as described in previous studies. These differences between the individual and combined stresses show that the plants employ different mechanisms for stress mitigation. Begum et al. (2020) reported that the osmolyte content of the leaves of 45-day-old tobacco plants increased in response to AM fungal inoculation and P supplementation. Moreover, in vitro experiments have clearly shown that P has a role in the osmotic stress response (Sawwan et al. 2000; Abu-Romman and Suwwan 2011). On the basis of these works, it seems that, in the present study, the activity of phosphate transporters and excess P reduced the osmotic pressure and played a role in the increased growth of the mycorrhizal plants, which is in accordance with the results of Liu et al. (2016) and Volpe et al. (2018).
To obtain a more complete picture aside from the members of the Pht1 gene family, we also evaluated aquaporin genes, as they play a crucial role in water uptake and regulation in response to water deficit. Several studies have demonstrated that mycorrhizal symbiosis can modulate the expression of aquaporin genes under drought stress (Reuscher et al. 2013;Chitarra et al. 2016b;Liu et al. 2016;Hu et al. 2017;Jia-Dong et al. 2019;Sharma et al. 2021). We examined PIP2.5 and PIP2.7 aquaporin genes as strong participants in water deficit mitigation and water transport ). These mainly root-expressed genes have been shown to increase root hydraulic conductivity (Reuscher et al. 2013). The changes that we observed in PIP2.5 gene expression are in accordance with the findings of Reuscher et al. (2013), suggesting that PIP2.5 might be inducible only by exposure to a specific stimulus. Combined mycorrhizal inoculation and water deficit stress might be one of these specific stimuli. Moreover, with respect to the other studied aquaporin gene (PIP2.7), a tendency of downregulated expression was observed, which is in accordance with the findings of previous studies (Sánchez-Romera et al. 2016;Duc et al. 2018). PIP2.7 gene expression decreased significantly under the combined stress in the mycorrhizal plants; as a result, water transfer for respiration slowed, and thus, water loss was minimized . Through these changes, mycorrhizal plants are more resilient to both water deficit and heat stress.

Conclusion
Based on the physiological parameters, stress marker, enzyme activity and gene expression, the stress-alleviating effect of F. mosseae on tomato plants was observed under heat stress, water deficit and heat and water deficit combined. Mitigation of plant growth reduction was the most prominent under heat stress, while the most robust enhancement in P uptake was found under the combined stress. Our results suggest that mycorrhizal plants behave differently and use various strategies to alleviate the effects of individual and combined stresses. The changes in phosphate transporter gene expression in response to AM fungi and the increased P uptake through their activities could promote increased tomato plant vigor. Via this fine tuning, phosphate transporters contribute to the stress tolerance of mycorrhizal plants under water deficit, heat stress, and heat and water deficit combined. Moreover, in addition to Pht1;3 and Pht1;4, whose promoting effects have already been shown, Pht1;7 seems to be a potential AM fungus-inducible phosphate transporter gene.
Author contributions VSZ, ZM, KP planned and designed the research, and wrote the manuscript and organized the manuscript structure. VSZ and ZM collected and analyzed the data and applied funding to support the study. All authors reviewed and approved the final manuscript.
Funding Open access funding provided by Hungarian University of Agriculture and Life Sciences. This research was funded by "ÚNKP-20-4-II New National Excellence Program of the Ministry of Human Capacities", "ÚNKP-20-3-II New National Excellence Program of the Ministry of Human Capacities" and by Ministry of Innovation and Technology, grant number RRF-2.3.1-21-2022-00007 and the industrial research and development projects in Hungarian-Vietnamese cooperation, grant number 2019-2.1.12-TÉT_VN-2020-00001.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest
The authors have no relevant financial or non-financial interests to disclose.
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