Drought stress has a negative impact on plant growth and production. Arbuscular mycorrhizal (AM) fungi, which establish symbioses with most terrestrial vascular plant species, play important roles in improving host plant mineral nutrient acquisition and resistance to drought. However, the physiological and molecular regulation mechanisms occurring in mycorrhizal Eucalyptus grandis coping with drought stress remain unclear. Here, we studied the physiological changes and mitogen-activated protein kinase (MAPK) cascade gene expression profiles of E. grandis associated with AM fungi under drought stress. The results showed that colonization by AM fungi significantly enhanced plant growth, with higher plant biomass, shoot height, root length, and relative water content (RWC) under drought conditions. Mycorrhizal plants had lower levels of accumulation of proline, malondialdehyde (MDA), H2O2, and O2·- than seedlings not colonized with AM fungi. In addition, mycorrhizal E. grandis also had higher peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities under drought conditions, improving the antioxidant system response. Eighteen MAPK cascade genes were isolated from E. grandis, and the expression levels of the MAPK cascade genes were positively induced by symbiosis with AM fungi, which was correlated with changes in the proline, MDA, H2O2, and O2·- contents and POD, SOD, and CAT activities. In summary, our results showed that AM symbiosis enhances E. grandis drought tolerance by regulating plant antioxidation abilities and MAPK cascade gene expression. IMPORTANCE Arbuscular mycorrhizal (AM) fungi play an important role in improving plant growth and development under drought stress. The MAPK cascade may regulate many physiological and biochemical processes in plants in response to drought stress. Previous studies have shown that there is a complex regulatory network between the plant MAPK cascade and drought stress. However, the relationship between the E. grandis MAPK cascade and AM symbiosis in coping with drought remains to be investigated. Our results suggest that AM fungi could improve plant drought tolerance mainly by improving the antioxidant ability to protect plants from reactive oxygen species (ROS) and alleviate oxidative stress damage. The expression of the MAPK cascade genes was induced in mycorrhizal E. grandis seedlings under drought stress. This study revealed that MAPK cascade regulation is of special significance for improving the drought tolerance of E. grandis. This study provides a reference for improving mycorrhizal seedling cultivation under stress.

Zinc (Zn) is one of the most essential micronutrients for plant growth and metabolism, but Zn excess can impair many basic metabolic processes in plant cells. In agriculture, crops often experience low phosphate (Pi) and high Zn double nutrient stresses because of inordinate agro-industrial activities, while the dual benefit of arbuscular mycorrhizal (AM) fungi protects plants from experiencing both deficient and toxic nutrient stresses. Although crosstalk between Pi and Zn nutrients in plants have been extensively studied at the physiological level, the molecular basis of how Pi starvation triggers Zn over-accumulation in plants and how AM plants coordinately modulate the Pi and Zn nutrient homeostasis remains to be elucidated. Here, we report that a novel AsZIP2 gene, a Chinese milk vetch (Astragalus sinicus) member of the ZIP gene family, participates in the interaction between Pi and Zn nutrient homeostasis in plants. Phylogenetic analysis revealed that this AsZIP2 protein was closely related to the orthologous Medicago MtZIP2 and Arabidopsis AtZIP2 transporters. Gene expression analysis indicated that AsZIP2 was highly induced in roots by Pi starvation or Zn excess yet attenuated by arbuscular mycorrhization in a Pi-dependent manner. Subcellular localization and heterologous expression experiments further showed that AsZIP2 encoded a functional plasma membrane-localized transporter that mediated Zn uptake in yeast. Moreover, overexpression of AsZIP2 in A. sinicus resulted in the over-accumulation of Zn concentration in roots at low Pi or excessive Zn concentrations, whereas AsZIP2 silencing lines displayed an even more reduced Zn concentration than control lines under such conditions. Our results reveal that the AsZIP2 transporter functioned in Zn over-accumulation in roots during Pi starvation or high Zn supply but was repressed by AM symbiosis in a Pi-dependent manner. These findings also provide new insights into the AsZIP2 gene acting in the regulation of Zn homeostasis in mycorrhizal plants through Pi signal.

Melatonin is a new kind of plant growth regulator. The aim of this study was to figure out the effect of melatonin on arbuscular mycorrhizal (AM) symbiosis and heavy metal tolerance. A three-factor experiment was conducted to determine the effect of melatonin application on the growth, AM symbiosis, and stress tolerance of Medicago truncatula. A two-factor (AM inoculation and Pb stress) experiment was conducted to determine the effect of AM fungus on melatonin accumulation under Pb stress. AM plants under Pb stress had a higher melatonin accumulation than non-mycorrhizal (NM) plants under Pb stress. Acetylserotonin methyltransferase (ASMT) is the enzymatic reaction of the last step in melatonin synthesis. The accumulation of melatonin may be related to the expression of MtASMT. Melatonin application increased the relative expression of MtPT4 and AM colonization in AM plants. Melatonin application decreased Pb uptake with and without AM inoculation. Both melatonin application and AM inoculation improved M. truncatula growth and increased antioxidant response with Pb stress. These results indicated that melatonin application has positive effects on AM symbiosis and Pb stress tolerance under Pb stress. AM inoculation improve melatonin synthesis capacity under Pb stress. Melatonin application may improve AM plant growth by enhancing AM symbiosis, stimulating antioxidant response, and inhibiting Pb uptake.

Soil salinization and the associated land degradation are major and growing ecological problems. Excess salt in soil impedes plant photosynthetic processes and root uptake of water and nutrients such as K+. Arbuscular mycorrhizal (AM) fungi can mitigate salt stress in host plants. Although, numerous studies demonstrate that photosynthesis and water status are improved by mycorrhizae, the molecular mechanisms involved have received little research attention. In the present study, we analyzed the effects of AM symbiosis and salt stress on photosynthesis, water status, concentrations of Na+ and K+, and the expression of several genes associated with photosynthesis (RppsbA, RppsbD, RprbcL, and RprbcS) and genes coding for aquaporins or membrane transport proteins involved in K+ and/or Na+ uptake, translocation, or compartmentalization homeostasis (RpSOS1, RpHKT1, RpNHX1, and RpSKOR) in black locust. The results showed that salinity reduced the net photosynthetic rate, stomatal conductance, and relative water content in both non-mycorrhizal (NM) and AM plants; the reductions of these three parameters were less in AM plants compared with NM plants. Under saline conditions, AM fungi significantly improved the net photosynthetic rate, quantum efficiency of photosystem II photochemistry, and K+ content in plants, but evidently reduced the Na+ content. AM plants also displayed a significant increase in the relative water content and an evident decrease in the shoot/root ratio of Na+ in the presence of 200 mM NaCl compared with NM plants. Additionally, mycorrhizal colonization upregulated the expression of three chloroplast genes (RppsbA, RppsbD, and RprbcL) in leaves, and three genes (RpSOS1, RpHKT1, and RpSKOR) encoding membrane transport proteins involved in K+/Na+ homeostasis in roots. Expression of several aquaporin genes was regulated by AM symbiosis in both leaves and roots depending on soil salinity. This study suggests that the beneficial effects of AM symbiosis on the photosynthetic capacity, water status, and K+/Na+ homeostasis lead to the improved growth performance and salt tolerance of black locust exposed to salt stress.

The majority of vascular flowering plants can establish arbuscular mycorrhizal (AM) symbiosis with AM fungi. These associations contribute to plant health and plant growth against various environmental stresses. In the mutualistic endosymbiosis, the AM fungi deliver phosphate (Pi) to the host root through highly branched hyphae called arbuscules. The molecular mechanisms of Pi transfer from AM fungi to the plant have been determined, which are dominated by AM-specific Pi transporters belonging to the PHOSPHATE TRANSPORTER 1 (Pht1) family within the subfamily I. However, it is unknown whether Pht1 family proteins are involved in other regulations in AM symbiosis. Here, we report that the expression of EgPT8 is specifically activated by AM fungus Rhizophagus irregularis and is localized in root cortical cells containing arbuscules. Interestingly, knockdown of EgPT8 function does not affect the Eucalyptus grandis growth, total phosphorous (P) concentration, and arbuscule formation; however, the size of mature arbuscules was significantly suppressed in the RNAi-EgPT8 lines. Heterogeneous expression of EgPT4, EgPT5, and EgPT8 in the Medicago truncatula mutant mtpt4-2 indicates that EgPT4 and EgPT5 can fully complement the defects of mutant mtpt4-2 in mycorrhizal Pi uptake and arbuscule formation, while EgPT8 cannot complement the defective AM phenotype of the mtpt4-2 mutant. Based on our results, we propose that the AM fungi-specific subfamily I transporter EgPT8 has novel functions and is essential to arbuscule elongation. IMPORTANCE Arbuscular mycorrhizal (AM) formation in host root cortical cells is initiated by exchanges of diffusible molecules, among which Pi uptake is known as the important feature of AM fungi on symbiosis functioning. Over the last two decades, it has been repeatedly proven that most vascular plants harbor two or more AM-specific Pht1 proteins; however, there is no direct evidence regarding the potential link among these Pi transporters at the symbiotic interface. This work revealed a novel function of a structurally conserved protein involved in lateral arbuscule development. In total, we confirmed that three AM-specific Pht1 family proteins are nonredundant in Eucalyptus grandis and that EgPT8 is responsible for fungal arbuscule elongation of Rhizophagus irregularis.

To identify why tree growth differs by afforestation type is a matter of prime concern in forestry. A study was conducted to determine why oriental arborvitae (Platycladus orientalis) grows better in the presence of black locust (Robinia pseudoacacia) than in monoculture. Different types of stands (i.e., monocultures and mixture of black locust and oriental arborvitae, and native grassland as a control) were selected in the Loess Plateau, China. The height and diameter at breast height of each tree species were measured, and soil, shoot, and root samples were sampled. The arbuscular mycorrhizal (AM) attributes, shoot and root nutrient status, height and diameter of black locust were not influenced by the presence of oriental arborvitae. For oriental arborvitae, however, growing in mixture increased height and diameter and reduced shoot Mn, Ca, and Mg contents, AM fungal spore density, and colonization rate. Major changes in soil properties also occurred, primarily in soil water, NO 3-N, and available K levels and in soil enzyme activity. The increase in soil water, N, and K availability in the presence of black locust stimulated oriental arborvitae growth, and black locust in the mixed stand seems to suppress the development of AM symbiosis in oriental arborvitae roots, especially the production of AM fungal spores and vesicles, through improving soil water and N levels, thus freeing up carbon to fuel plant growth. Overall, the presence of black locust favored oriental arborvitae growth directly by improving soil water and fertility and indirectly by repressing AM symbiosis in oriental arborvitae roots.

Arbuscular mycorrhizal (AM) fungi can improve the lead (Pb) tolerance of host plants and accumulate intensive Pb in mycorrhizal roots. However, the detailed contribution of AM fungal extraradical hyphae to the plants' Pb uptake remains unknown. In this study, mulberry (Morus alba) colonized by the AM fungus (Rhizophagus irregularis) with light treatments were linked by fungal extraradical hyphae using a three-compartment system (pot test), and their differences in responding to Pb application were compared. Shading inhibited mulberry photosynthesis and the growth of mulberry. In this study, Pb application did not affect the colonization of R. irregularis when symbiosis had already formed as the root was not exposed to Pb during the colonization and formation of the AM fungal hyphae network. The R. irregularis preferred to transfer more Pb to the unshaded mulberry than to the shaded mulberry, a condition capable of providing more C supply for fungal survival than to low-light mulberry. The Pb transferred through the mycorrhizal pathway to mulberry had low mobility and might be compartmented in the root by R. irregularis until exceeding a threshold. The relatively high expressions of MaABCG16 with high Pb concentrations in plants suggest that MaABCG16 might play an important role in Pb translocation.

Arbuscular mycorrhizal (AM) fungi establish symbiosis and improve the lead (Pb) tolerance of host plants. The AM plants accumulate more Pb in roots than their non-mycorrhizal counterparts. However, the direct and long-term impact of AM fungi on plant Pb uptake has been rarely reported. In this study, AM fungus (Rhizophagus irregularis) colonized and non-colonized roots of Medicago truncatula were separated by a split-root system, and their differences in responding to Pb application were compared. The shoot biomass accumulation and transpiration were increased after R. irregularis inoculation, whereas the biomass of both colonized and non-colonized roots was decreased. Lead application in the non-colonized root compartment increased the R. irregularis colonization rate and up-regulated the relative expressions of MtPT4 and MtBCP1 in the colonized root compartments. Rhizophagus irregularis inoculation increased Pb uptake in both colonized and non-colonized roots, and R. irregularis transferred Pb to the colonized root segment. The Pb transferred through the colonized root segment had low mobility and might be sequestrated and compartmented in the root by R. irregularis. The Pb uptake of roots might follow water flow, which is facilitated by MtPIP2. The quantification of Pb transfer via the mycorrhizal pathway and the involvement of MtPIP2 deserve further study.

Eucalyptus grandis (E. grandis) has been reported to form a symbiosis with arbuscular mycorrhizal fungi (AMF), which plays an important role in improving plant tolerance of heavy metal. However, the mechanism of how AMF intercept and transport cadmium (Cd) at the subcellular level in E. grandis still remains to be researched. In this study, a pot experiment was conducted to investigate the growth performance of E. grandis under Cd stress and Cd absorption resistance of AMF and explored the Cd localization in the root by using transmission electron microscopy and energy dispersive X-ray spectroscopy. The results showed that AMF colonization could enhance plant growth and photosynthetic efficiency of E. grandis and reduce the translocation factor of Cd under Cd stress. After being treated with 50, 150, 300, and 500 μM Cd, the translocation factor of Cd in E. grandis with AMF colonization decreased by 56.41%, 62.89%, 66.67%, and 42.79%, respectively. However, the mycorrhizal efficiency was significant only at low Cd concentrations (50, 150, and 300 μM). Under 500 μM Cd concentration condition, the colonization of AMF in roots decreased, and the alleviating effect of AMF was not significant. Ultrastructural observations showed that Cd is abundant in regular lumps and strips in the cross-section of E. grandis root cell. AMF protected plant cells by retaining Cd in the fungal structure. Our results suggested that AMF alleviated Cd toxicity by regulating plant physiology and altering the distribution of Cd in different cell sites.

Arbuscular mycorrhizal (AM) fungi can form beneficial associations with the most terrestrial vascular plant species. AM fungi not only facilitate plant nutrient acquisition but also enhance plant tolerance to various environmental stresses such as drought stress. However, the molecular mechanisms by which AM fungal mitogen-activated protein kinase (MAPK) cascades mediate the host adaptation to drought stimulus remains to be investigated. Recently, many studies have shown that virus-induced gene silencing (VIGS) and host-induced gene silencing (HIGS) strategies are used for functional studies of AM fungi. Here, we identify the three HOG1 (High Osmolarity Glycerol 1)-MAPK cascade genes RiSte11, RiPbs2 and RiHog1 from Rhizophagus irregularis. The expression levels of the three HOG1-MAPK genes are significantly increased in mycorrhizal roots of the plant Astragalus sinicus under severe drought stress. RiHog1 protein was predominantly localized in the nucleus of yeast in response to 1 M sorbitol treatment, and RiPbs2 interacts with RiSte11 or RiHog1 directly by pull-down assay. Importantly, VIGS or HIGS of RiSte11, RiPbs2 or RiHog1 hampers arbuscule development and decreases relative water content in plants during AM symbiosis. Moreover, silencing of HOG1-MAPK cascade genes led to the decreased expression of drought-resistant genes (RiAQPs, RiTPSs, RiNTH1 and Ri14-3-3) in the AM fungal symbiont in response to drought stress. Taken together, this study demonstrates that VIGS or HIGS of AM fungal HOG1-MAPK cascade inhibits arbuscule development and expression of AM fungal drought-resistant genes under drought stress.

It is possible that arbuscular mycorrhizal fungi play a pivotal role in root development and Pb phytostabilization in plants grown in Pb-contaminated soil. In this study, a pot experiment was conducted over 4 months to evaluate the effects of Funneliformis mosseae strain BGCXJ01A on root characteristics of black locust (Robinia pseudoacacia L.) seedlings in Pb-contaminated soil. Four Pb treatments (0, 90, 900, and 3,000 mg kg-1) were applied to soil in the presence and absence of F. mosseae. Inoculation with F. mosseae prominently improved root length, surface area, volume, and tip number in the plants across all Pb treatments. The F. mosseae inoculation also increased root diameter and fork number, especially under high Pb treatments. The presence of F. mosseae significantly increased the root activity and root tolerance index. However, there was little difference in specific root length between inoculated and non-inoculated plants. The biomass of roots, stems, and leaves all increased following inoculation with F. mosseae. Inoculated plants had greater accumulation and translocation capacities for Pb in the roots and stems, but lower capacities were found in the leaves when compared with those in non-inoculated plants. These results highlight that F. mosseae can alleviate the toxic effects of Pb on root development and can immobilize Pb in the roots and stems of R. pseudoacacia grown in Pb-contaminated soil. This study provides a model system for phytoremediation of Pb-contaminated soil via reciprocal symbiosis between arbuscular mycorrhizal fungi and woody legumes.

Potassium in plants accounts for up to 10% dry weight, and participates in different physiological processes. Under drought stress, plant requires more potassium but potassium availability in soil solutes is lowered by decreased soil water content. Forming symbiosis with arbuscular mycorrhizal (AM) fungi not only enlarges exploration range of plant for mineral nutrients and water in soil, but also improves plant drought tolerance. However, the regulation of AM fungi on plant root potassium uptake and translocation from root to shoot was less reported. In current study, the effect of an AM fungus (Rhizophagus irregularis), potassium application (0, 2, and 8 mM), and drought stress (30% field capacity) on Lycium barbarum growth and potassium status was analyzed. Ten weeks after inoculation, R. irregularis colonized more than 58% roots of L. barbarum seedlings, and increased plant growth as well as potassium content. Potassium application increased colonization rate of R. irregularis, plant growth, potassium content, and decreased root/shoot ratio. Drought stress increased colonization rate of R. irregularis and potassium content. Expression of two putative potassium channel genes in root, LbKT1 and LbSKOR, was positively correlated with potassium content in root and leaves, as well as the colonization rate of R. irregularis. The increased L. barbarum growth, potassium content and genes expression, especially under drought stress, suggested that R. irregularis could improve potassium uptake of L. barbarum root and translocation from root to shoot. Whether AM fungi could form a specific mycorrhizal pathway for plant potassium uptake deserves further studies.

Glomalin-related soil protein (GRSP), a widespread glycoprotein produced by arbuscular mycorrhizal fungi (AMF), is crucial for ecosystem functioning and ecological restoration. In the present study, an investigation was conducted to comprehensively analyze the effects of heavy metal (HM) contamination on AMF status, soil properties, aggregate distribution and stability, and their correlations at different soil depths (0-10, 10-20, 20-30, 30-40 cm). Our results showed that the mycorrhizal colonization (MC), hyphal length density (HLD), GRSP, soil organic matter (SOM) and soil organic carbon (SOC) were significantly inhibited by Pb compared to Zn at 0-20 cm soil depth, indicating that HM had significant inhibitory effects on AMF growth and soil properties, and that Pb exhibited greater toxicity than Zn at shallow layer of soil. Both the proportion of soil large macroaggregates (>2000 μm) and mean weight diameter (MWD) were positively correlated with GRSP, SOM and SOC at 0-20 cm soil depth (P < 0.05), proving the important contributions of GRSP, SOM and SOC for binding soil particles together into large macroaggregates and improving aggregate stability. Furthermore, MC and HLD had significantly positive correlation with GRSP, SOM and SOC, suggesting that AMF played an essential role in GRSP, SOM and SOC accumulation and subsequently influencing aggregate formation and particle-size distribution in HM polluted soils. Our study highlighted that the introduction of indigenous plant associated with AMF might be a successful biotechnological tool to assist the recovery of HM polluted soils, and that proper management practices should be developed to guarantee maximum benefits from plant-AMF symbiosis during ecological restoration.

The sex-specific physical and biochemical responses in dioecious plants to abiotic stresses could result in gender imbalance, and how to ease the current situation by microorganisms is still unclear. Using native soil where poplars were grown, growth parameters, soil physicochemical properties in the rhizosphere soil of different sexes of Populus cathayana exposed to salt stress and exogenous arbuscular mycorrhizal (AM) inoculation were tested. Besides, the sex-specific microbial community structures in the rhizosphere soil of different sexes of Populus cathayana were compared under salt stress. To identify the sex-specific microbial community characteristics related to salinity and AM symbiosis, a combined qPCR and DGGE method was used to monitor microbial community diversity. Seedlings suffered severe pressure by salt stress, reflected in limited growth, biomass, and nutrient element accumulation, especially on females. Exogenous AM inoculation treatment alleviated these negative effects, especially under salt treatment of 75 mM. Compared with salt effect, exogenous AM inoculation treatment showed a greater effect on soil physical-chemical properties of both sexes. Based on DGGE results, salt stress negatively affected fungal richness but positively affected fungal Simpson diversity index, while exogenous AM inoculation treatment showed the opposite effect. Structural equation modeling (SEM) was performed to show the causal relationships between salt and exogenous AM inoculation treatments with biomass accumulation and microbial community: salt and exogenous AM inoculation treatment showed complicated effects on elementary concentrations, soil properties, which resulted in different relationship with biomass accumulation and microbial community. Salt stress had a negative effect on soil properties and microbial community structure in the rhizosphere soil of P. cathayana, whereas exogenous AM inoculation showed positive impacts on most of the soil physical-chemical properties and microbial community status.