Arbuscular Mycorrhizal Fungi Regulate MAPK Pathway Genes Expression and Enhance Drought Tolerance of Populus Simonii × P. Nigra Seedlings

Background: Arbuscular mycorrhizal fungi (AMF) form a symbiotic relationship with host plants, which can promote plants to absorb more water and nutrients, and thus improve the stress resistance of plants. Our study aimed to investigate the effects of Rhizophagus irregularis on Populus simonii × P. nigra seedlings under drought stress. Results: The experiment was a completely random design with two water conditions (well-watered or drought stress) and two AMF treatments (inoculated with or without R. irregularis). Our results showed that mycorrhizal seedlings performed less oxidative damage and stronger tolerance of drought, which recorded higher photosynthesis and less concentrations of Malondialdehyde (MDA), H 2 O 2 , and proline under drought stress versus non-mycorrhizal seedlings. Under drought stress, AMF inoculation reduced soluble sugar concentration in leaves but promoted its accumulation in roots. The superoxide dismutase (SOD) activity in leaves and roots, and catalase (CAT) activity in roots of mycorrhizal seedlings were lower than nonmycorrhizal seedlings, but CAT activity in leaves of mycorrhizal seedlings was higher than non-mycorrhizal seedlings under drought stress. Drought stress and AMF inoculation both induced the expressions of MAPKs of P. simonii × P. nigra, but the expression patterns of MAPKs under four treatments were obviously different. Conclusions: Overall, our results demonstrated that mycorrhizal seedlings had less oxidative damage and stronger tolerance to drought. MAPKs expressions of P. simonii×P. nigra (PsnMAPKs) were induced by drought stress and AMF inoculation, and the expression patterns of PsnMAPKs in response to drought stress were different between mycorrhizal and non-mycorrhizal seedlings. Non-mycorrhizal seedlings may be adapted to drought by up-regulating MAPKs expressions leading to stomatal closure. Drought stress decreased serval PsnMAPKs expressions induced by AMF inoculation, which may be associated with mycorrhizal colonization. H suggested had less and stronger tolerance to AMF inoculation promoted soluble sugar accumulated in roots under drought stress. In the present study, we conrmed that MAPKs expressions of P. simonii×P. nigra were induced by drought stress and AMF inoculation, and the expression patterns of PsnMAPKs in response to drought stress were different between mycorrhizal and nonmycorrhizal seedlings. Non-mycorrhizal seedlings may be adapted to drought by up-regulating PsnMAPKs expressions leading to stomatal closure. Drought stress decreased serval PsnMAPKs expressions induced by AMF inoculation, Furthermore, it PsnMAPKs.


Introduction
Plants in nature are constantly exposed to a variety of biotic and abiotic stresses. Among these abiotic stresses, drought stress is one of the most unfavorable factors for plants. Drought stress leads to the degradation of photosynthesis and the interruption of electron transport in thylakoids, and the closure of stomata leads to reduce carbon dioxide absorption and carbon ux [1], resulting in a decline in plant growth and productivity [2]. Meanwhile, drought stress causes a burst of reactive oxygen species (ROS) accumulation, disrupting cell metabolism [3]. Malondialdehyde (MDA) is an indicator to assess the level of lipid peroxidation in cell membranes. As one of the ROS, the decrease of H 2 O 2 degradation ability in leaves may be the root cause of oxidative damage and membrane lipid peroxidation in plants under drought stress [4]. Under adversity conditions, proline will accumulate in plants and plays an important role in the defense mechanism of plants [5]. Soluble sugar is of great help to osmotic regulation, and also acts as a source of carbon [6]. In addition, plants also turn on their antioxidant enzyme system to clear ROS under drought stress [7]. Moreover, some signal transduction molecules such as H 2 O 2 , can active signal pathways during plants acclimation to drought [4]. Arbuscular Mycorrhiza (AM) is the most common symbiotic structure between plants and soil microorganisms. Arbuscular Mycorrhizal Fungi (AMF) exist in most natural habitats, and they provide a series of important ecological functions, especially to improve plant nutrition, production, stress resistance, soil fertility and the restoration of degraded lands [8][9][10][11]. The poplar (Populus spp.) has important economic value, which plays an increasingly important role in the greening of the country, the construction of farmland shelterbelts, and the construction of industrial timber forests, especially in the context of global atmospheric changes, the carbon sequestration bene ts of poplars increasingly becoming the focus of attention [12].
As a main signal transducer, the mitogen-activated protein kinase (MAPK) cascade plays a vital role in drought stress [13]. In rice, the transcripts of OsMPK8, OsMPK7, OsMPK5, and OsMPK4 accumulated under drought stress [14,15]. It has been reported that GhMPK2 enhances the drought resistance of cotton through stomatal closure, which mainly responses to drought stress by reducing water loss and regulating intracellular osmotic pressure [16]. It is documented that OsDSM1 in rice may respond to drought stress mainly by regulating the ability of ROS scavenging [17]. Overexpression of GhMPK3 mainly enhances the drought tolerance of plants by regulating the activity of antioxidant enzymes [18]. Hence, the expressions of MAPKs are associated with drought tolerance of plants.
In arid and semi-arid areas, the water shortage has always been a key factor restricting agricultural and forestry production [19]. Therefore, improving drought resistance of woody plants is related to the forestry development in arid and semi-arid areas. In the past, the research on mycorrhiza to improve the drought tolerance of poplars mostly focused on root morphology, nutrient absorption, antioxidant system and photosynthetic system [20,21]. As far as we know, there is no report of mycorrhiza-induced MAPK genes of poplars. Our study aimed to investigate the effects of Rhizophagus irregularis on drought tolerance and MAPKs expressions of Populus simonii × P. nigra seedlings.

Effect of Drought and AMF inoculation on plant growth
Mycorrhizal colonization was not found in the roots of uninoculated seedlings. The mycorrhizal colonization rate of the AMF-inoculated seedlings was 81.56%±0.10% under WW treatment and 64.84%±0.12% under DS treatment, respectively. DS treatment signi cantly inhibited the growth of seedlings, but the AMF inoculation promoted plant growth, which was re ected in the height of the plant increased by 79.11%. In the case of WW treatment, AMF inoculation also increased the height of plants by 25.43% (Table 1). Under WW treatment, AMF inoculation signi cantly promoted the fresh weight of shoots, fresh weight of roots and total biomass, which increased by 45.59%, 29.96%, and 39.97%, respectively (Table 1). Under DS treatment, AMF inoculation dramatically increased fresh weight of roots by 99.56% but had no signi cant effect on fresh weight of shoots and total biomass. Notably, DS treatment increased the root-shoot ratio, but inoculation with AMF reduced the root-shoot ratio by 24.00%.  Figure 4).

Effect of Drought and AMF inoculation on Antioxidant Enzymes Activities
DS treatment signi cantly enhanced SOD activity in leaves, but SOD activity in leaves of AM seedlings was lower versus NM seedlings under both water conditions. Under WW treatment, there was no signi cant difference in SOD activity in leaves between NM and AM seedlings. DS treatment enhanced SOD activity in roots, but SOD activity of AM seedlings was also lower versus NM seedlings ( Figure 5A). Under WW treatment, there was no signi cant difference in CAT activity in leaves between NM and AM seedlings. DS treatment signi cantly enhanced CAT activity in leaves of AM seedlings but had no signi cant effect on NM seedlings. Notably, CAT activity in roots of AM seedlings was dramatically higher than NM seedlings under WW treatment. DS treatment signi cantly enhanced CAT activity in roots of NM seedlings but had no signi cant effect on AM seedlings ( Figure 5B).

Effect of Drought and AMF inoculation on expressions of MAPK genes
According to the homology with MAPKs of A. thaliana and rice, the MAPKs of P. simonii×P. nigra (PsnMAPKs) were divided into four groups: A, B, C, and D ( Figure 6). Phylogenetic analysis showed that PsnMAPK20-1 belonged to group A, PsnMAPK7-1 and PsnMAPK7-2 belonged to group C, and other PsnMAPKs were attributed to group D, without genes belonged to group B. The notable PsnMAPKs in leaves were PsnMAPK7-1 and PsnMAPK20-1, belonging to group C and A. The effects of DS treatment on the expressions of them were signi cantly different between NM and AM seedlings. The expressions of these two genes dramatically increased in NM seedling leaves under DS treatment, but PsnMAPK7-1 was down-regulated in AM seedling leaves. Moreover, DS treatment did not affect the expression of PsnMAPK20-1 in AM seedling leaves to a signi cant level. The PsnMAPKs of group D in NM and AM seedling leaves were down regulated by DS treatment. In addition, the effects of inoculation on the expression of PsnMAPK7-1 in leaves were also signi cantly different between both water treatment. Under WW treatment, AMF inoculation increased the expression of PsnMAPK7-1 in leaves, but under DS treatment, AMF inoculation decreased the expression of PsnMAPK7-1. However, the expression of PsnMAPK20-1 in leaves was down-regulated by AMF inoculation under both water treatments. Additionally, the expressions of several PsnMAPKs in group D increased dramatically after AMF inoculation under WW treatment, including PsnMAPK19-1, PsnMAPK20-2, PsnMAPK12, PsnMAPK17 ( Figure 7A).
In the present study, we observed that more PsnMAPKs were affected by DS treatment in roots versus leaves. DS treatment increased the expressions of all PsnMAPKs in roots of NM seedlings except PsnMAPK15-2 and PsnMAPK20-1. Among these MAPKs, PsnMAPK7-1 and PsnMAPK7-2 in group C performed different responses to drought between NM and AM roots. DS treatment increased the expressions of PsnMAPK7-1 and PsnMAPK7-2 in NM roots but decreased them in AM roots. The expressions of PsnMAPK15-2, PsnMAPK7-1, PsnMAPK3-2, PsnMAPK12 and PsnMAPK17 were dramatically increased by AMF inoculation under WW treatment. However, under DS treatment, except PsnMAPK15-2 and PsnMAPK3-2, the expressions of other MAPKs were decreased by AMF inoculation. Interestingly, the effect of DS treatment on the expressions of PsnMAPK15-2 were obviously different between NM and AM roots. DS treatment dramatically reduced the expression of PsnMAPK15-2 in AM roots but did not signi cantly affect the expression in NM roots ( Figure  7B).

Plant growth
Our study con rmed that soil drought decreases the mycorrhizal colonization rate, it may be because longterm drought stress inhibits mycorrhiza development [22]. Earlier studies suggested that drought stress strongly inhibits plant growth, while AM symbiosis considerably relieves the negative effects on plant growth [23]. In line with previous studies, our results showed that AM seedlings had a higher height than NM seedlings under two water conditions. Moreover, AMF inoculation under drought stress had a greater promotion on growth than well-watered conditions. Under well-watered conditions, AMF inoculation enhanced the fresh weight of shoots and roots and total biomass. In the present study, only the fresh weight of roots of AM seedlings was obviously higher versus NM seedlings. Drought stress causes changes in biomass allocation between shoots and roots of plants, and promotes photosynthates transfer to roots, resulting in an increase in root-shoot ratio [24], as shown in our study, root-shoot ratio signi cantly increased under drought conditions. Meanwhile, we noticed that root-shoot ratio of NM seedlings was higher than AM seedlings, which is consistent with the previous study [25]. In short, our results demonstrated that AMF has a positive effect on plant growth, especially on roots and under drought conditions.

Photosynthesis
Previous studies have reported that drought stress can reduce the Pn, gs, Tr [26,27]. Plant growth and carbohydrate accumulation depend on photosynthesis. A lot of studies have shown that the Pn of mycorrhizal plants is higher than that of non-mycorrhizal plants, and they also have higher gs [28]. In response to drought stress, stomatal closure is an rapid response to avoid water loss, although it leads to negative effects on photosynthesis [29]. Stomata regulate carbon assimilation rate and water loss, but drought stress reduces gs [30]. AM symbiosis can change the stomatal conductance of plants, which is a reason for the enhance of Pn. Our ndings are directly in line with previous ndings, we observed that AM seedlings had higher Pn, gs, and Tr versus NM seedlings under two water conditions. AMF inoculation improved gas exchange by increasing the stomatal conductance and transpiration in leaves, which may be related to the increase in nutrient and water uptake caused by AMF [31]. Meanwhile, we found that there was no signi cant difference in Ci, which is in accord with Chen et al. [32].

Oxidative damage
In previous studies, Pelargonium gravolens seedlings were inoculated with AMF under drought stress and then the accumulation of MDA was dramatically reduced [33]. The research on wheat also showed that AMF inoculation signi cantly reduced the MDA concentration under drought stress [34]. A same result was reached by our study, suggesting that oxidative damage of AM seedlings was slighter versus NM seedlings. Furthermore, this effect was more obvious in roots than leaves.  [40], and Poncirus trifoliata [41], after inoculation with AMF, the accumulation of proline was lower than nonmycorrhizal plants. A similar pattern of results was obtained in our study, AM seedlings recorded a lower proline concentration than NM seedlings under drought stress. This possibly because AM seedlings have better water status and growth than NM seedlings under drought conditions, so they are less stressed.
Yooyongwech et al. [42] found that the soluble sugar concentration in mycorrhizal Ipomoea batatas was increased, and the accumulation in roots was higher than leaves, indicating that there is a negative correlation between osmotic potential and soluble sugar accumulation in plants. In the present study, under well-watered conditions, AMF inoculation had no signi cant effect on soluble sugar concentration. A novel nding is that the accumulation of soluble sugar in the leaves and roots of AM seedlings showed different patterns. The concentration of soluble sugar in leaves of AM seedlings was lower than NM seedlings, but the accumulation in the AM roots was higher than NM roots. The accumulation of sugar in mycorrhizal plants is caused by the enhancement of photosynthesis capacity under osmotic stress [43]. AMF inoculation can promote the transportation of carbohydrate from leaves to roots under drought conditions. Meanwhile, drought stress can also limit sugar transport from the roots to extraradical hyphae, which leads to accumulation increased in AM roots.

Antioxidant enzyme activity
Plants have a physiological mechanism to respond to oxidative damage by inducing the production of antioxidant enzymes [7]. When plants exposed to drought stress, they mobilize antioxidant systems to remove ROS. The SOD activity in leaves was dramatically increased by drought stress. Under drought conditions, SOD activity in roots of AM seedlings was lower versus NM seedlings, which is inconsistent with the previous results of Zou et al. [44] and Gong et al. [45]. This may be related to a lower oxidative burst in AM seedlings, which is supported by the result of the accumulation of MDA.  [44], and Gong et al. [45]. Drought stress leads to accumulation of H 2 O 2 , which is cleared rely on CAT. CAT activity in the roots of NM seedlings was higher versus AM roots under drought stress, possibly because less H 2 O 2 was accumulated in AM roots.

Quantitative expressions of PsnMAPKs
Previous studies have showed that MAPK participate in response to drought stress [47,48]. We determined that drought stress can affect the expressions of MAPKs of P. simonii × P. nigra. In the present study, drought stress induced the expressions of PsnMAPK7-1 and PsnMAPK20-1 in leaves of NM seedlings, implying they are drought response genes in leaves. OsMAPK5 homologous to PsnMAPK20-1 has been con rmed to be activated by drought stress [14]. Hence, plants may adapt to drought stress by activating MAPK genes.
Interestingly, under drought stress, the expressions of PsnMAPK7-1 and PsnMAPK20-1 were down-regulated by AMF inoculation exactly. It has been reported that GhMPK2 regulates drought stress via stomatal closure [49]. As shown in our study, NM seedlings recorded a lower gs under drought conditions, as compared with AM seedlings. In addition, we observed that AMF inoculation activated several MAPKs unrelated to drought in leaves, which is an interesting phenomenon. Previous studies only analyzed on MAPKs in roots affected by AMF inoculation [50,51], further work should also analyze those MAPKs in leaves up-regulated by AMF inoculation.
Drought stress induced more MAPKs in roots than leaves, we observed that drought stress increased dramatically the expressions of all MAPKs in roots of NM seedlings except PsnMAPK15-2 and PsnMAPK20-

Conclusion
Overall, our results demonstrated an effective help of AMF on P. simonii×P. nigra seedlings under drought stress, re ecting in improvement of photosynthesis and a lower level of MDA and H 2 O 2 , which suggested mycorrhizal seedlings had less oxidative damage and stronger tolerance to drought. AMF inoculation promoted soluble sugar accumulated in roots under drought stress. In the present study, we con rmed that MAPKs expressions of P. simonii×P. nigra were induced by drought stress and AMF inoculation, and the expression patterns of PsnMAPKs in response to drought stress were different between mycorrhizal and nonmycorrhizal seedlings. Non-mycorrhizal seedlings may be adapted to drought by up-regulating PsnMAPKs expressions leading to stomatal closure. Drought stress decreased serval PsnMAPKs expressions induced by AMF inoculation, which may be associated with mycorrhizal colonization. Furthermore, it is a question of future research to investigate the functions of these induced PsnMAPKs.

Mycorrhizal fungal inoculum and plant material
In our study, the R. irregularis (BGC BJ09) obtained from the Beijing Academy of Agriculture and Forestry Sciences (Beijing, China) was propagated with Zea mays as its host for three months. At harvest time, roots and growth substrates were collected as the fungal inoculum, which contained the fungal spores (20 spores/g). Annual cuttings of P. simonii×P. nigra were obtained from a plant nursery in Lindian County, Daqing City, Hei Longjiang Province, China. After washing, the cuttings were cut into small pieces, each of which had only one bud and the same length. These pieces were inserted in the nursery pot, and their growth substrate was a mixture of soil, sand, and vermiculite (V: V: V=1:1:1). One month later, they germinated and were inserted into the same substrate to take roots. After rooting, seedlings were transferred into plots with 1kg mixture of soil and sand (V: V=1:1).

Experimental design
The experiment was a completely random design with two water conditions (WW: well-watered, DS: drought stress) and two AMF treatments (AM: inoculated with R. irregularis, NM: not inoculated with R. irregularis). Based on the previous experience, WW treatment was considered as 70%~75% of maximum water capacity of the sterilized soil/sand mixture, and DS treatment was considered as 30%~35% of the maximum water capacity of the sterilized soil/sand mixture. There were four treatments in the present experiment, each treatment had 24 pots, 96 pots in total.
Growth substrate and Plant culture The growth substrate was composed of autoclaved soil and sand. Before being put into the pots, the soil was sieved (2 mm) and autoclaved (121 ℃, 2 h) and the sand was washed and dried (180 ℃). The sterilized soil/sand mixture contained available nitrogen, 37.68 mg kg −1 ; available phosphorous, 11.28 mg kg −1 ; and available potassium, 139.10 mg kg −1 , and the pH value was 7.7. At the time of transferring the seedlings, 20 g mycorrhizal inoculums were applied to seedlings as the AM treatment. The NM treatment received 20 g autoclaved mycorrhizal inoculums. Before the drought treatment, keeping the sterilized soil/sand mixture moisture content at 70%~75% of maximum water capacity by weighing every day. After one week, half of the mycorrhizal and non-mycorrhizal plants were exposed to the 30%~35% of maximum water capacity for 12 weeks, and the other half was continued to grow under well-watered conditions for 12 weeks. Seedlings were placed in the greenhouse, where the temperature was 25~28 °C, and the light intensity was 3000 lx. During the experiment, 50 mL of Hoagland solution with 1/10 of the phosphate was weekly added into each pot for normal growth of seedlings.

Determination of mycorrhizal colonization
Roots at the same position on seedlings from four treatments were cut. After washing with tap water, the roots were decolorized with 5% KOH solution in a water bath at 90 °C for 20 min. After cooling, the roots were acidi ed with 2% HCl solution, and they were dyed with 1% Trypan at 90 ℃ for 15 min. Finally, the roots were decolorized with a mixture of lactic acid and glycerin (V: V=1:1) at room temperature. A compound microscope (Olympus BX43, Japan) was used to detect the AM colonization rate of each sample using the cross method [22].

Determination of Photosynthesis
Before harvest, the photosynthesis parameters of seedlings were determined using a Li-6400 Portable Photosynthesis System (Li-Cor Inc, Lincoln, NE, United States) on a sunny day from 9:00 to 11:00 am. Photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO 2 concentration (Ci), and transpiration rate (Tr) were measured after steady-state of gas exchange. Leaves on the same position were selected and leaves of each seedling used for measurement should be fully expanded, three leaves were selected for each seedling.

Determination of active substances
For MDA, 0.1g frozen leaves and roots samples ground in liquid nitrogen were exacted with a solution containing 0.3% 2-thiobarbituric acid (TBA) and 10% trichloroacetic acid (TCA). The MDA concentration was determined using the TBA reaction method, according to the method described by Hodges et al. [23]. For H 2 O 2 , 0.1g of frozen leaves and roots samples were ground with 1 mL of precooled acetone solution, and H 2 O 2 concentration was determined by the method described by Patterson et al. [24]. For proline, 0.1 g frozen leaves and roots samples ground in liquid nitrogen were exacted with 3% sulfosalicylic acid solution. The proline concentration was determined according to the method described by Bates et al. [25]. For soluble sugar, 0.1 g frozen leaves and roots samples were ground with 1 mL 75% methanol and determined following the method described by Shi et al. [26].
Determination of antioxidant enzyme activity 0.1 g frozen leaves and roots samples ground in liquid nitrogen were exacted with 1 mL 50 mM potassium phosphate buffer containing 1% polyvinylpyrrolidone (PVP). The homogenates were centrifuged at 12000×g for 20 min at 4 °C and the supernatants were used for the enzyme assays. The activity of SOD was measured by monitoring the inhibition of photochemical reduction of nitroblue tetrazole (NBT) [27]. CAT activity was determined by the method of Maehly and Chance [28].

MAPK genes fragment cloning and quantitative expression
After three months, the leaves and roots samples were ground completely in liquid nitrogen. Total RNA was extracted using a TIANGEN ® RNAprep pure Plant Kit (Tiangen, Beijing, China) following the manufacturer's protocol and then 1mg total RNA was used for reverse transcription following the manufacturer's protocol of HiScript ® II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). The sequences of MAPK family genes of P. simonii × P. nigra were obtained using the MAPKs of P. trichocarpa to perform a homology search in Blast of NCBI based on the transcriptome data of P. simonii × P. nigra (Database Number: SRP267437). The accession numbers of MAPKs of P. trichocarpa in NCBI used for blast were listed in Table 3. Primers used for cloning were designed using Primer Tool of NCBI were listed in Table 4. Phylogenetic tree was constructed with MAPKs of Arabidopsis thaliana, Populus trichocarpa, rice, cotton, and maize. Muscle was used for multisequence comparison, and maximum likelihood method was used for tree construction in Mega-X software.
For the qRT-PCR assay, the reaction system is 10 μL, which included 5 µL ChamQ SYBR ® qPCR Master Mix (Vazyme, Nanjing, China), 1 µL diluted cDNA template, 0.5 µL forward and reverse primers and 3 µL sterilized ddH 2 O. Primers used in our experiment for qRT-PCR were listed in Table 5, which were designed by Primer Tool of NCBI, and the speci city were checked using the Blast Tool of NCBI. The qRT-PCR ampli cation program was as follows: 95 ℃ for 30 s, 40 cycles at 95 ℃ for 10 s, annealing at the annealing temperature for 10 s and extending at 72 °C for 20 s, followed by heating from 65 °C to 95 °C at a rate of 0.5 °C every 5 s. Four biological replicates were used for all genetic analyses, CYP (coding peptidyl-prolyl cis-trans isomerase 1) and EF1α (coding elongation factor 1-alpha-like) were used as the endogenous control to calculate △Ct values.
The dissociation curves were used to detected the ampli cation speci city of primers and the 2 -△△Ct method was used to calculated the relative quanti cation value of genes. Heatmaps of MAPKs expressions in leaves and roots of P. simonii×P. nigra were drawn using ImageGP online website, and the data were standardized.  All data were analyzed statistically by using the software SPSS 16.0 (SPSS 16.0 for windows, USA). Independent-sample T-test and one-way analysis of variance (ANOVA) (P < 0.05) were used to compare the differences among treatments.

Declarations Authors' Contributions
Jing Tao: performed the experiments, analysed the data, and wrote the manuscript; Fengxin Dong and Yihan Wang: designed the experiment and provided experimental methods; Hui Chen: analysed the data; Ming Tang: provided nancial support and revised the manuscript.

Figure 1
Effects of R. intraradices on leaf and root MDA concentration of P. simonii×P. nigra seedlings subjected to wellwatered (WW) and drought stress (DS). NM: nonmycorrhizal seedling; AM: mycorrhizal seedlings. Data (means ± SD, n = 4) followed by different letters above the bars indicate signi cant differences (P < 0.05) between treatments.

Figure 2
Effects of R. intraradices on leaf and root H2O2 concentration of P. simonii×P. nigra seedlings subjected to well-watered (WW) and drought stress (DS). NM: nonmycorrhizal seedling; AM: mycorrhizal seedlings. Data (means ± SD, n = 4) followed by different letters above the bars indicate signi cant differences (P < 0.05) between treatments.

Figure 3
Effects of R. intraradices on leaf and root proline concentration of P. simonii×P. nigra seedlings subjected to well-watered (WW) and drought stress (DS). NM: nonmycorrhizal seedling; AM: mycorrhizal seedlings. Data (means ± SD, n = 4) followed by different letters above the bars indicate signi cant differences (P < 0.05) between treatments.

Figure 4
Effects of R. intraradices on leaf and root soluble sugar concentration of P. simonii×P. nigra seedlings subjected to well-watered (WW) and drought stress (DS). NM: nonmycorrhizal seedling; AM: mycorrhizal seedlings. Data (means ± SD, n = 4) followed by different letters above the bars indicate signi cant differences (P < 0.05) between treatments.

Figure 6
Phylogenetic tree of MAPKs of P P. simonii×P. nigra. Phylogenetic tree was constructed with MAPKs of Arabidopsis thaliana, Populus trichocarpa, rice, cotton, and maize. Muscle was used for multi-sequence comparison, and maximum likelihood method was used for tree construction in Mega-X software.