Mycorrhizal Fungal Effects on Plant Growth, Osmolytes, and CsHsp70s and CsPIPs Expression in Leaves of Cucumber under a Short-Term Heat Stress.

Arbuscular mycorrhizal (AM) fungi enhance plant stress tolerance, but it is unclear whether AM fungi affect heat tolerance in cucumbers. This study aimed to analyze how an AM fungus, Diversispora versiformis, affected growth, chlorophyll, five osmolytes, and plasma membrane intrinsic proteins (PIPs) and heat shock protein 70 (Hsp70) gene expression in cucumber leaves after a short-term (80 h) heat stress. Heat treatment significantly reduced root AM fungal colonization rate (0.26 folds). Heat treatment also distinctly suppressed plant height, stem diameter, and biomass, whereas AM fungal inoculation improved these growth variables as well as the chlorophyll index, with the benefit being more obvious under heat than under no-heat stress conditions. Heat treatment triggered differential changes in osmolytes (sucrose, fructose, and betaine) of inoculated and uninoculated cucumbers, whereas inoculation with AM fungus significantly raised leaf sucrose, fructose, glucose, betaine, and proline levels when compared to non-AM fungal inoculation. Heat treatment increased the expression of two (CsPIP1;6 and CsPIP2;1) of eight CsPIPs in inoculated and uninoculated plants, whereas AM fungal inoculation up-regulated the expression of CsPIP1;6, CsPIP2;1, and CsPIP2;6 under heat stress conditions. Hsp70s expressed differently in inoculated and uninoculated plants under heat versus no-heat stress, with 6 of 11 CsHsp70s down-regulated in inoculated plants. Under heat stress conditions, AM fungus only up-regulated CsHsp70-8 expression in 11 Hsp70s, while another eight CsHsp70s were down-regulated. Heat treatment and AM fungal inoculation both increased the expression of CsHsp70-8 and CsPIP1;6. It was concluded that AM fungus-inoculated cucumbers have high levels of growth, chlorophyll, and osmolytes under heat stress and do not require high CsPIPs and CsHsp70s expression to tolerate a short-term heat treatment.

Two different strategies of Diversispora spurca-inoculated walnut seedlings to improve leaf P acquisition at low and moderate P levels.

Walnut (Juglans regia) is an important nut tree species in the world, whereas walnut trees often face inadequate phosphorus (P) levels of soil, negatively limiting its growth and yield. Arbuscular mycorrhizal fungi (AMF) can colonize walnut roots, but whether and how AMF promotes walnut growth, physiological activities, and P acquisition is unclear. The present study aimed to evaluate the effects of Diversispora spurca on plant growth, chlorophyll component concentrations, leaf gas exchange, sugar and P concentrations, and expression of purple acid phosphatase (PAP) and phosphate transporter (PT) genes in leaves of J. regia var. Liaohe 1 seedling under moderate (100 µmol/L P) and low P (1 µmol/L P) levels conditions. Three months after inoculation, the root mycorrhizal colonization rate and soil hyphal length were 45.6-53.2% and 18.7-39.9 cm/g soil, respectively, and low P treatment significantly increased both root mycorrhizal colonization rate and soil hyphal length. Low P levels inhibited plant growth (height, stem diameter, and total biomass) and leaf gas exchange (photosynthetic rate, transpiration rate and stomatal conductance), while AMF colonization significantly increased these variables at moderate and low P levels. Low P treatment limited the level of chlorophyll a, but AMF colonization did not significantly affect the level of chlorophyll components, independent on soil P levels. AMF colonization also increased leaf glucose at appropriate P levels and leaf fructose at low P levels than non-AMF treatment. AMF colonization significantly increased leaf P concentration by 21.0-26.2% than non-AMF colonization at low and moderate P levels. Low P treatment reduced the expression of leaf JrPAP10, JrPAP12, and JrPT3;2 in the inoculated plants, whereas AMF colonization up-regulated the expression of leaf JrPAP10, JrPAP12, and JrPT3;2 at moderate P levels, although AMF did not significantly alter the expression of JrPAPs and JrPTs at low P levels. It is concluded that AMF improved plant growth, leaf gas exchange, and P acquisition of walnut seedlings at different P levels, where mycorrhizal promotion of P acquisition was dominated by direct mycorrhizal involvement in P uptake at low P levels, while up-regulation of host PAPs and PTs expressions at moderate P levels.

Metabolomics reveals arbuscular mycorrhizal fungi-mediated tolerance of walnut to soil drought.

BACKGROUND: Arbuscular mycorrhizal fungi (AMF) have a positive effect on drought tolerance of plants after establishing reciprocal resymbiosis with roots, while the underlying mechanism is not deciphered. Metabolomics can explain the mechanism of plant response to environmental stress by analyzing the changes of all small molecular weight metabolites. The purpose of this study was to use Ultra High Performance Liquid Chromatography Q Exactive Mass Spectrometer to analyze changes in root metabolites of walnut (Juglans regia) after inoculation with an arbuscular mycorrhizal fungus Diversispora spurca under well-watered (WW) and drought stress (DS). RESULTS: Sixty days of soil drought significantly inhibited root mycorrhizal colonization rate, shoot and root biomass production, and leaf water potential in walnut, while AMF inoculation significantly increased biomass production and leaf water potential, accompanied by a higher increase magnitude under DS versus under WW. A total of 3278 metabolites were identified. Under WW, AMF inoculation up-regulated 172 metabolites and down-regulated 61 metabolites, along with no changes in 1104 metabolites. However, under DS, AMF inoculation up-regulated 49 metabolites and down-regulated 116 metabolites, coupled with no changes in 1172 metabolites. Among them, juglone (a quinone found in walnuts) as the first ranked differential metabolite was up-regulated by AMF under WW but not under DS; 2,3,5-trihydroxy-5-7-dimethoxyflavanone as the first ranked differential metabolite was increased by AMF under DS but not under WW. The KEGG annotation showed a large number of metabolic pathways triggered by AMF, accompanied by different metabolic pathways under WW and DS. Among them, oxidative phosphorylation and phenylalanine metabolism and biosynthesis were triggered by AMF in response to WW and DS, where N-acetyl-L-phenylalanine was induced by AMF to increase under DS, while decreasing under WW. CONCLUSION: This study provides new insights into the metabolic mechanisms of mycorrhiza-enhanced drought tolerance in walnuts.

Extraradical Mycorrhizal Hyphae Promote Soil Carbon Sequestration through Difficultly Extractable Glomalin-Related Soil Protein in Response to Soil Water Stress.

Soil water stress (WS) affects the decomposition of soil organic carbon (SOC) and carbon (C) emissions. Glomalin, released by arbuscular mycorrhizal fungi into soil that has been defined as glomalin-related soil protein (GRSP), is an important pool of SOC, with hydrophobic characteristics. We hypothesized that mycorrhizal fungi have a positive effect on SOC pools under soil WS for C sequestration in GRSP secreted by extraradical mycorrhizal hyphae. A microsystem was used to establish a root chamber (co-existence of roots and extraradical mycorrhizal hyphae) and a hyphal chamber (the presence of extraradical mycorrhizal hyphae) to study changes in plant growth, leaf water potential, soil aggregate stability, SOC, GRSP, C concentrations in GRSP (CGRSP), and the contribution of CGRSP to SOC after inoculating Rhizophagus intraradices with trifoliate orange (Poncirus trifoliata) in the root chamber under adequate water (AW) and WS. Inoculation with R. intraradices alleviated negative effects on leaf water potential and plant growth after 7 weeks of WS. Soil WS decreased SOC and mean weight diameter (MWD), while AMF inoculation led to an increase in SOC and MWD in both chambers, with the most prominent increase in the hyphal chamber under WS. The C concentration in easily extractable GRSP (EE-GRSP) and difficultly extractable GRSP (DE-GRSP) was 7.32 - 12.57 and 24.90 - 32.60 mg C/g GRSP, respectively. WS reduced CGRSP, while AMF mitigated the reduction. Extraradical mycorrhizal hyphae increased GRSP production and CGRSP, along with a more prominent increase in DE-GRSP under WS than under AW. Extraradical mycorrhizal hyphae increased the contribution of CDE-GRSP to SOC only under WS. CEE-GRSP and CDE-GRSP were significantly positively correlated with SOC and MWD. It is concluded that extraradical mycorrhizal hyphae prominently promoted C sequestration of recalcitrant DE-GRSP under soil WS, thus contributing more organic C accumulation and preservation in aggregates and soil C pool.

An endophytic fungus, Piriformospora indica, enhances drought tolerance of trifoliate orange by modulating the antioxidant defense system and composition of fatty acids.

A cultivable endophytic fungus, Piriformospora indica, improves growth and enhances stress tolerance of host plants, but the underlying mechanisms remain unknown. We hypothesized that P. indica enhanced the drought tolerance of the host by regulating the antioxidant defense system and composition of fatty acids. Trifoliate orange (Poncirus trifoliata) seedlings were inoculated with P. indica under ample water and drought stress to analyze the change in plant growth, reactive oxygen species (ROS) levels, antioxidant enzyme activities, non-enzymatic antioxidant concentrations, fatty acid compositions, and expressions of both antioxidant enzyme genes and fatty acid desaturase (FAD) genes. The 9-week soil water deficit significantly increased the colonization of P. indica to roots, and P. indica promoted the increase of shoot biomass under drought. Soil drought triggered an elevation of hydrogen peroxide in roots, while the inoculated plants had lower levels of ROS (hydrogen peroxide and superoxide anion radicals) and lower degree of membrane lipid peroxidation (based on malondialdehyde levels) under drought. Drought treatment also elevated ascorbic acid and glutathione concentrations, and the elevation was further amplified after P. indica inoculation. Inoculated plants under drought also recorded significantly higher iron-superoxide dismutase (Fe-SOD), manganese-superoxide dismutase (Mn-SOD), peroxidases, catalase, glutathione reductase and ascorbate peroxidase activities, accompanied by up-regulation of PtFe-SOD and PtCu/Zn-SOD expressions. Inoculation with P. indica significantly increased total saturated fatty acids (e.g., C6:0, C15:0, C16:0, C23:0 and C24:0) concentration and reduced total unsaturated fatty acids (e.g., C18:1N9C, C18:2N6, C18:3N3, C18:1N12 and C19:1N9T) concentrations, leading to a decrease in the unsaturation index of fatty acids, which may be associated with the up-regulation of PtFAD2 and PtFAD6 and down-regulation of PtΔ9. It was concluded that the colonization of P. indica can activate enzyme and non-enzyme defense systems and regulate the composition of fatty acids under drought, thus alleviating the oxidative damage to the host caused by drought.

Arbuscular mycorrhiza induces low oxidative burst in drought-stressed walnut through activating antioxidant defense systems and heat shock transcription factor expression.

Arbuscular mycorrhizal fungi (AMF) have important roles in enhancing drought tolerance of host plants, but it is not clear whether and how AMF increase drought tolerance in walnut (Juglans regia). We hypothesized that AMF could activate antioxidant defense systems and heat shock transcription factors (Hsfs) transcription levels to alleviate oxidative damage caused by drought. The walnut variety 'Liaohe No. 1' was inoculated with Diversispora spurca and exposed to well-watered (WW, 75% of the maximum soil water capacity) and drought stress (DS, 50% of the maximum soil water capacity) for 6 weeks. Plant growth, antioxidant defense systems, and expressions of five JrHsfs in leaves were studied. Such drought treatment inhibited root mycorrhizal colonization, while plant growth performance was still improved by AMF inoculation. Mycorrhizal fungal inoculation triggered the increase in soluble protein, glutathione (GSH), ascorbic acid (ASC), and total ASC contents and ascorbic peroxidase and glutathione reductase activities, along with lower hydrogen peroxide (H2O2), superoxide anion radical (O2 •-), and malondialdehyde (MDA) levels, compared with non-inoculation under drought. Mycorrhizal plants also recorded higher peroxidase, catalase, and superoxide dismutase activities than non-mycorrhizal plants under drought. The expression of JrHsf03, JrHsf05, JrHsf20, JrHsf22, and JrHsf24 was up-regulated under WW by AMF, while the expression of JrHsf03, JrHsf22, and JrHsf24 were up-regulated only under drought by AMF. It is concluded that D. spurca induced low oxidative burst in drought-stressed walnut through activating antioxidant defense systems and part Hsfs expressions.

Cloning of a CHS gene of Poncirus trifoliata and its expression in response to soil water deficit and arbuscular mycorrhizal fungi.

Flavonoids are secondary metabolites widely found in plants with antioxidants, of which chalcone synthase (CHS) is a key enzyme required in flavonoid synthesis pathways. The objective of this study was to clone a CHS gene from trifoliate orange (Poncirus trifoliata) and analyze its biological information and partial functions. A PtCHS gene (NCBI accession: MZ350874) was cloned from the genome-wide of trifoliate orange, which has 1156 bp in length, encoding 391 amino acids, with a predicted protein relative molecular mass of 42640.19, a theoretical isoelectric point of 6.28, and a lipid coefficient of 89.82. The protein is stable, hydrophilic, and high sequence conservation (92.49% sequence homology with CHS gene of other species). PtCHS was highly expressed in stems, leaves and flowers, but very low expression in roots and seeds. Soil water deficit could up-regulate expressions of PtCHS in leaves. An arbuscular mycorrhizal fungus, Funneliformis mosseae, significantly increased plant biomass production, CHS activity, expressions of PtCHS, and total flavonoid content in leaves and roots, independent of soil water status. Total flavonoids were significantly positively correlated with PtCHS expression in leaves only and also positively with root mycorrhizal colonization. Such results provide insight into the important functions of PtCHS in trifoliate orange.

Mycorrhizal fungi regulate daily rhythm of circadian clock in trifoliate orange under drought stress.

The circadian rhythm of plants is associated with stress responses; however, it is not clear whether increased host plant drought tolerance by arbuscular mycorrhizal fungi (AMF) is associated with changes in the circadian clock. The present study aimed to analyze the effect of Funneliformis mosseae (Nicol. & Gerd.) Schüßler & Walker on the circadian clock gene expression patterns in trifoliate orange (Poncirus trifoliata L. Raf.) along with gas exchange, abscisic acid (ABA) levels and antioxidant enzyme gene expression under well-watered (WW) and drought stress (DS) conditions. Plant growth, net photosynthetic rate, stomatal conductance and ABA levels were significantly higher in AMF- than in non-AMF-inoculated plants regardless of soil water regimes. Six circadian clock genes, including PtPRR7, PtLHY, PtCCA1, PtGI, PtPIF3 and PtSRR1, were identified and showed rhythmic expression patterns over the course of the day. The AMF inoculation reduced the expression of most circadian clock genes in different time periods. However, AMF treatment significantly increased PtPRR7 and PtGI expression at 5:00 p.m. under WW and DS conditions, PtLHY expression at 1:00 a.m. and PtSRR1 expression at 9:00 p.m. At 1:00 a.m., AMF inoculation up-regulated the expression of the circadian clock genes PtPRR7, PtCCA1, PtLHY and PtPIF3 and the antioxidant enzyme genes PtFe-SOD, PtMn-SOD, PtCu/Zn-SOD, PtPOD and PtCAT1. Correlation analysis revealed that these changes in circadian clock gene expression were associated with antioxidant enzyme gene expression, root ABA and gas exchange. We concluded that mycorrhizal fungi have the ability to regulate the daily rhythm of the circadian clock in trifoliate orange plants in response to drought.

Metabolomics Analysis Reveals Drought Responses of Trifoliate Orange by Arbuscular Mycorrhizal Fungi With a Focus on Terpenoid Profile.

Soil water deficit seriously affects crop production, and soil arbuscular mycorrhizal fungi (AMF) enhance drought tolerance in crops by unclear mechanisms. Our study aimed to analyze changes in non-targeted metabolomics in roots of trifoliate orange (Poncirus trifoliata) seedlings under well-watered and soil drought after inoculation with Rhizophagus intraradices, with a focus on terpenoid profile. Root mycorrhizal fungal colonization varied from 70% under soil drought to 85% under soil well-watered, and shoot and root biomass was increased by AMF inoculation, independent of soil water regimes. A total of 643 secondary metabolites in roots were examined, and 210 and 105 differential metabolites were regulated by mycorrhizal fungi under normal water and drought stress, along with 88 and 17 metabolites being up-and down-regulated under drought conditions, respectively. KEGG annotation analysis of differential metabolites showed 38 and 36 metabolic pathways by mycorrhizal inoculation under normal water and drought stress conditions, respectively. Among them, 33 metabolic pathways for mycorrhization under drought stress included purine metabolism, pyrimidine metabolism, alanine, aspartate and glutamate metabolism, etc. We also identified 10 terpenoid substances, namely albiflorin, artemisinin (-)-camphor, capsanthin, ß-caryophyllene, limonin, phytol, roseoside, sweroside, and α-terpineol. AMF colonization triggered the decline of almost all differential terpenoids, except for ß-caryophyllene, which was up-regulated by mycorrhizas under drought, suggesting potential increase in volatile organic compounds to initiate plant defense responses. This study provided an overview of AMF-induced metabolites and metabolic pathways in plants under drought, focusing on the terpenoid profile.

Differential Effects of Exogenous Glomalin-Related Soil Proteins on Plant Growth of Trifoliate Orange Through Regulating Auxin Changes.

Multiple functions of glomalin released by arbuscular mycorrhizal fungi are well-recognized, whereas the role of exogenous glomalins including easily extractable glomalin-related soil protein (EE-GRSP) and difficultly extractable glomalin-related soil protein (DE-GRSP) is unexplored for plant responses. Our study was carried out to assess the effects of exogenous EE-GRSP and DE-GRSP at varying strengths on plant growth and chlorophyll concentration of trifoliate orange (Poncirus trifoliata) seedlings, along with changes in root nutrient acquisition, auxin content, auxin-related enzyme and transporter protein gene expression, and element contents of purified GRSP. Sixteen weeks later, exogenous GRSP displayed differential effects on plant growth (height, stem diameter, leaf number, and biomass production): the increase by EE-GRSP and the decrease by DE-GRSP. The best positive effect on plant growth occurred at exogenous EE-GRSP at ½ strength. Similarly, the GRSP application also differently affected total chlorophyll content, root morphology (total length, surface area, and volume), and root N, P, and K content: positive effect by EE-GRSP and negative effect by DE-GRSP. Exogenous EE-GRSP accumulated more indoleacetic acid (IAA) in roots, which was associated with the upregulated expression of root auxin synthetic enzyme genes (PtTAA1, PtYUC3, and PtYUC4) and auxin influx transporter protein genes (PtLAX1, PtLAX2, and PtLAX3). On the other hand, exogenous DE-GRSP inhibited root IAA and indolebutyric acid (IBA) content, associated with the downregulated expression of root PtTAA1, PtLAX1, and PtLAX3. Root IAA positively correlated with root PtTAA1, PtYUC3, PtYUC4, PtLAX1, and PtLAX3 expression. Purified EE-GRSP and DE-GRSP showed similar element composition but varied in part element (C, O, P, Ca, Cu, Mn, Zn, Fe, and Mo) concentration. It concluded that exogenous GRSP triggered differential effects on growth response, and the effect was associated with the element content of pure GRSP and the change in auxins and root morphology. EE-GRSP displays a promise as a plant growth biostimulant in citriculture.

The Change in Fatty Acids and Sugars Reveals the Association between Trifoliate Orange and Endophytic Fungi.

Endophytes have the ability to improve plant nutrition alongside their agronomic performance, among which arbuscular mycorrhizal fungi provide the most benefits to their host. Previously, we reported for the first time that an arbuscular mycorrhizal-like fungus Piriformospora indica had the ability to colonize roots of trifoliate orange (Poncirus trifoliata) and conferred positive effects on nutrient acquisition. Present study showed the changes in fatty acids and sugars to unravel the physiological and symbiotic association of trifoliate orange with P. indica and an arbuscular mycorrhizal fungus, Funneliformis mosseae singly or in combination. All the endophytic fungi collectively increased fructose, glucose, and sucrose content in leaves and roots, along with a relatively higher increase with P. indica inoculation than with F. mosseae alone or dual inoculation. Treatment with P. indica increased the concentration of part unsaturated fatty acids such as C18:3N6, C20:2, C20:3N6, C20:4N6, C20:3N3, C20:5N3, C22:1N9, and C24:1. Additionally, P. indica induced the increase in the concentration of part saturated fatty acids such as C6:0, C8:0, C13:0, C14:0, and C24:0. F. mosseae hardly changed the content of fatty acids, except for increase in C14:0 and C20:5N3. Double inoculation only reduced the C21:0, C10:0, C12:0, C18:3N3, and C18:1 content and increased the C20:5N3 content. These endophytic fungi up-regulated the root PtFAD2, PtFAD6, PtΔ9, and PtΔ15 gene expression level, coupled with a higher expression of PtFAD2 and PtΔ9 by P. indica than by F. mosseae. It was concluded that P. indica exhibited a stronger response, for sugars and fatty acids, than F. mosseae on trifoliate orange. Such results also reveal the Pi (an in vitro culturable fungus) as a bio-stimulator applying to citriculture.

Arbuscular Mycorrhizal Fungi Alleviate Drought Stress in Trifoliate Orange by Regulating H+-ATPase Activity and Gene Expression.

A feature of arbuscular mycorrhiza is enhanced drought tolerance of host plants, although it is unclear whether host H+-ATPase activity and gene expression are involved in the physiological process. The present study aimed to investigate the effects of an arbuscular mycorrhizal fungus (AMF), Funneliformis mosseae, on H+-ATPase activity, and gene expression of trifoliate orange (Poncirus trifoliata) seedlings subjected to well-watered (WW) and drought stress (DS), together with the changes in leaf gas exchange, root morphology, soil pH value, and ammonium content. Soil drought treatment dramatically increased H+-ATPase activity of leaf and root, and AMF inoculation further strengthened the increased effect. A plasma membrane (PM) H+-ATPase gene of trifoliate orange, PtAHA2 (MW239123), was cloned. The PtAHA2 expression was induced by mycorrhization in leaves and roots and also up-regulated by drought treatment in leaves of AMF-inoculated seedlings and in roots of AMF- and non-AMF-inoculated seedlings. And, the induced expression of PtAHA2 under mycorrhization was more prominent under DS than under WW. Mycorrhizal plants also showed greater photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate and better root volume and diameter than non-mycorrhizal plants under DS. AMF inoculation significantly increased leaf and root ammonium content, especially under DS, whereas it dramatically reduced soil pH value. In addition, H+-ATPase activity was significantly positively correlated with ammonium contents in leaves and roots, and root H+-ATPase activity was significantly negatively correlated with soil pH value. Our results concluded that AMF stimulated H+-ATPase activity and PtAHA2 gene expression in response to DS, which resulted in great nutrient (e.g., ammonium) uptake and root growth, as well as low soil pH microenvironment.

Mycorrhizal response strategies of trifoliate orange under well-watered, salt stress, and waterlogging stress by regulating leaf aquaporin expression.

Aquaporins (AQPs) involved in water and small molecule transport respond to environmental stress, while it is not clear how arbuscular mycorrhizal fungi (AMF) regulate AQP expression. Here, we investigated the change in leaf water potential and expression level of four tonoplast intrinsic proteins (TIPs), six plasma membrane intrinsic proteins (PIPs), and four nodin-26 like intrinsic proteins (NIPs) genes in trifoliate orange (Poncirus trifoliata) inoculated with Funneliformis mosseae under well-watered (WW), salt stress (SS), and waterlogging stress (WS). Root AMF colonization and soil hyphal length collectively were reduced by SS and WS. Under WW, inoculation with AMF gave diverse responses of AQPs: six AQPs up-regulated, three AQPs down-regulated, and five AQPs did not change. Such up-regulation of more AQPs under mycorrhization and WW partly accelerated water absorption, thereby, maintaining higher leaf water potential. However, under SS, all the fourteen AQPs were dramatically induced by AMF inoculation, which improved water permeability of membranes and stimulated water transport of the host. Under WS, AMF colonization almost did not induce or even down-regulated these AQPs expressions with three exceptions (PtTIP2;2, PtPIP1;1, and PtNIP1;2), thus, no change in leaf water potential. As a result, mycorrhizal plants under flooding may have an escape mechanism to reduce water absorption. It is concluded that AMF had different strategies in response to environmental stresses (e.g. SS and WS) by regulating leaf AQP expression in the host (e.g. trifoliate orange).

Elucidating the Mechanisms Underlying Enhanced Drought Tolerance in Plants Mediated by Arbuscular Mycorrhizal Fungi.

Plants are often subjected to various environmental stresses during their life cycle, among which drought stress is perhaps the most significant abiotic stress limiting plant growth and development. Arbuscular mycorrhizal (AM) fungi, a group of beneficial soil fungi, can enhance the adaptability and tolerance of their host plants to drought stress after infecting plant roots and establishing a symbiotic association with their host plant. Therefore, AM fungi represent an eco-friendly strategy in sustainable agricultural systems. There is still a need, however, to better understand the complex mechanisms underlying AM fungi-mediated enhancement of plant drought tolerance to ensure their effective use. AM fungi establish well-developed, extraradical hyphae on root surfaces, and function in water absorption and the uptake and transfer of nutrients into host cells. Thus, they participate in the physiology of host plants through the function of specific genes encoded in their genome. AM fungi also modulate morphological adaptations and various physiological processes in host plants, that help to mitigate drought-induced injury and enhance drought tolerance. Several AM-specific host genes have been identified and reported to be responsible for conferring enhanced drought tolerance. This review provides an overview of the effect of drought stress on the diversity and activity of AM fungi, the symbiotic relationship that exists between AM fungi and host plants under drought stress conditions, elucidates the morphological, physiological, and molecular mechanisms underlying AM fungi-mediated enhanced drought tolerance in plants, and provides an outlook for future research.

Arbuscular Mycorrhizal Fungi Regulate Polyamine Homeostasis in Roots of Trifoliate Orange for Improved Adaptation to Soil Moisture Deficit Stress.

Soil arbuscular mycorrhizal fungi (AMF) enhance the tolerance of plants against soil moisture deficit stress (SMDS), but the underlying mechanisms are still not fully understood. Polyamines (PAs) as low-molecular-weight, aliphatic polycations have strong roles in abiotic stress tolerance of plants. We aimed to investigate the effect of AMF (Funneliformis mosseae) inoculation on PAs, PA precursors, activities of PA synthases and degrading enzymes, and concentration of reactive oxygen species in the roots of trifoliate orange (Poncirus trifoliata) subjected to 15 days of SMDS. Leaf water potential and total chlorophyll levels were comparatively higher in AMF-inoculated than in non-AMF-treated plants exposed to SMDS. Mycorrhizal plants recorded a significantly higher concentration of precursors of PA synthesis such as L-ornithine, agmatine, and S-adenosyl methionine, besides higher putrescine and cadaverine and lower spermidine during the 15 days of SMDS. AMF colonization raised the PA synthase (arginine decarboxylase, ornithine decarboxylase, spermidine synthase, and spermine synthase) activities and PA-degrading enzymes (copper-containing diamine oxidase and FAD-containing polyamine oxidase) in response to SMDS. However, mycorrhizal plants showed a relatively lower degree of membrane lipid peroxidation, superoxide anion free radical, and hydrogen peroxide than non-mycorrhizal plants, whereas the difference between them increased linearly up to 15 days of SMDS. Our study concluded that AMF regulated PA homeostasis in roots of trifoliate orange to tolerate SMDS.

Mycorrhizal symbiosis down-regulates or does not change root aquaporin expression in trifoliate orange under drought stress.

Arbuscular mycorrhizas absorb water from soil to host plants, while the relationship between mycorrhizas and aquaporins (AQPs, membrane water channel proteins, which function in water transport) in mycorrhizal plants is unclear. In this study, Funneliformis mosseae-colonized trifoliate orange (Poncirus trifoliata) seedlings were grown in pots fitted with 37-µm nylon meshes at the bottom of each pot to allow mycorrhizal hyphae absorb water from an outer beaker. The expression of seven plasma membrane intrinsic proteins (PIPs) genes, six tonoplast intrinsic proteins (TIPs) genes, and four nodulin-26 like intrinsic proteins (NIPs) genes were analyzed in roots of both well-watered (WW) and drought stressed (DS) plants. The six-week DS plants dramatically increased hyphal water absorption rate by 1.4 times, as compared with WW plants. Mycorrhizal plants exhibited greater plant growth performance, leaf water status (water potential and relative water content), and gas exchange under both WW and DS conditions. Mycorrhizal inoculation induced diverse expression patterns in these AQPs under WW: up-regulation of PtNIP1;1, PtPIP2;1, and PtPIP2;5, down-regulation of PtNIP1;2, PtNIP6;1, PtPIP1;2, PtPIP1;5, PtPIP2;8, PtTIP1;1, PtTIP1;2, PtTIP1;3, and PtTIP5;1, and no changes in other AQPs. However, the expression of PtPIPs and PtNIPs was down-regulated by mycorrhizal inoculation under DS, and PtTIPs was not induced by mycorrhizal colonization under DS. The expression pattern of AQPs in response to mycorrhizas under DS is a way of mycorrhizal plants to minimize water loss.

Mycorrhizas enhance drought tolerance of citrus by altering root fatty acid compositions and their saturation levels.

Arbuscular mycorrhizas (AMs) have the ability to enhance drought tolerance of citrus, but the underlying mechanisms have not been clearly elucidated. Considering the strong association of cell membrane fatty acid (FA) unsaturation with plant drought tolerance, the present study hypothesized that AM fungi (AMF) modulated the composition and unsaturation of FAs to enhance drought tolerance of host plants. Drought-sensitive citrus rootstocks, trifoliate orange (Poncirus trifoliata) seedlings, were inoculated with AMF (Funneliformis mosseae) for 3 months and were subsequently exposed to drought stress (DS) for 8 weeks. Mycorrhizal seedlings exhibited better plant growth performance, higher leaf water potential and lower root abscisic acid concentrations under both well-watered (WW) and DS conditions. Arbuscular mycorrhiza fungus inoculation considerably increased root methyl oleate (C18:1), methyl linoleate (C18:2) and methyl linolenate (C18:3N3) concentrations under both WW and DS conditions, and root methyl palmitoleate (C16:1) concentrations under WW, while it decreased root methyl stearate (C18:0) levels under both WW and DS. These changes in the composition of FAs of mycorrhized roots resulted in higher unsaturation index of root FAs, which later aided in reducing the oxidative damage on account of lower concentration of malondialdehyde and superoxide radicals. The changes of these FAs were a result of AMF-up-regulating root FA desaturase 2 (PtFAD2), FA desaturase 6 (PtFAD6) and Δ9 FA desaturase (PtΔ9) genes under WW and PtFAD2, PtFAD6 and Δ15 FA desaturase (PtΔ15) genes under DS conditions. Our results confirmed that mycorrhization brought significant changes in root FA compositions, in addition to regulation of gene expression responsible for increasing the unsaturation level of FAs, a predisposing physiological event for better drought tolerance of citrus.

Common mycorrhizal networks activate salicylic acid defense responses of trifoliate orange (Poncirus trifoliata).

Citrus canker, caused by Xanthomonas axonopodis pv. citri ('Xac'), is an important quarantine disease in citrus crops. Arbuscular mycorrhizal fungi (AMF) form symbiotic interactions with host plants and further affect their disease resistance, possibly by modulating the activity of salicylic acid (SA), a key phytohormone in disease resistance. Common mycorrhizal networks (CMNs) can interconnect plants, but it is not yet clear whether CMNs promote resistance to citrus canker and, if so, whether SA signaling is involved in this process. To test this possibility, we used a two-chambered rootbox to establish CMNs between trifoliate orange (Poncirus trifoliata) seedlings in chambers inoculated (treated) or not (neighboring) with the AMF, Paraglomus occultum. A subset of the AMF-inoculated seedlings were also inoculated with Xac (+AMF+Xac). At 2 d post-inoculation (dpi), compared with the +AMF-Xac treatment, neighboring seedlings in +AMF+Xac treatment had lower expression levels of the SA biosynthetic genes, PtPAL, PtEPS1, and PtPBS3, but higher SA levels, which attributed to the upregulation of PtPAL and PtPBS3 in treated seedlings and the transfer of SA, via CMNs, to the neighboring seedlings. At 4 dpi, the pathogenesis-related (PR) protein genes, PtPR1, PtPR4, and PtPR5, and the transcriptional regulatory factor gene, PtNPR1, were activated in neighboring seedlings of +AMF+Xac treatment. At 9 dpi, root phenylalanine ammonia-lyase activity and total soluble phenol and lignin concentrations increased in neighboring seedlings of +AMF+Xac treatment, likely due to the linkage and signal transfer, via CMNs. These findings support the hypothesis that CMNs transfer the SA signal from infected to neighboring healthy seedlings, to activate defense responses and affording protection to neighboring plants against citrus canker infection.

Mycorrhiza stimulates root-hair growth and IAA synthesis and transport in trifoliate orange under drought stress.

Root-hair growth and development regulated by soil microbes is associated with auxin. In this background, we hypothesized that mycorrhizal fungal inoculation induces greater root-hair growth through stimulated auxin synthesis and transport under water stress conditions. Trifoliate orange (Poncirus trifoliata) was inoculated with an arbuscular mycorrhizal (AM) fungus (Funneliformis mosseae) under well-watered (WW) and drought stress (DS) for 9 weeks. Compared with non-AM seedlings, AM seedlings displayed significantly higher density, length, and diameter of root hairs and root indoleacetic acid (IAA) level, whereas lower total root IAA efflux, regardless of soil moisture status. Root PtYUC3 and PtYUC8 involved in IAA biosynthesis were up-regulated by mycorrhization under WW and DS, whereas AM-modulated expression in PtTAA1, PtTAR2, PtYUC4, and PtYUC6 depended on status of soil moisture. Mycorrhizal inoculation down-regulated the transcript level of root auxin efflux carriers like PtPIN1 and PtPIN3, whereas significantly up-regulated the expression of root auxin-species influx carriers like PtABCB19 and PtLAX2 under DS. These results indicated that AMF-stimulated greater root-hair growth of trifoliate orange under DS that is independent on AMF species is related with mycorrhiza-modulated auxin synthesis and transport, which benefits the host plant to enhance drought tolerance.

Alleviation of drought stress by mycorrhizas is related to increased root H2O2 efflux in trifoliate orange.

The Non-invasive Micro-test Technique (NMT) is used to measure dynamic changes of specific ions/molecules non-invasively, but information about hydrogen peroxide (H2O2) fluxes in different classes of roots by mycorrhiza is scarce in terms of NMT. Effects of Funneliformis mosseae on plant growth, H2O2, superoxide radical (O2·-), malondialdehyde (MDA) concentrations, and H2O2 fluxes in the taproot (TR) and lateral roots (LRs) of trifoliate orange seedlings under well-watered (WW) and drought stress (DS) conditions were studied. DS strongly inhibited mycorrhizal colonization in the TR and LRs, whereas mycorrhizal inoculation significantly promoted plant growth and biomass production. H2O2, O2·-, and MDA concentrations in leaves and roots were dramatically lower in mycorrhizal seedlings than in non-mycorrhizal seedlings under DS. Compared with non-mycorrhizal seedlings, mycorrhizal seedlings had relatively higher net root H2O2 effluxes in the TR and LRs especially under WW, as well as significantly higher total root H2O2 effluxes in the TR and LRs under WW and DS. Total root H2O2 effluxes were significantly positively correlated with root colonization but negatively with root H2O2 and MDA concentrations. It suggested that mycorrhizas induces more H2O2 effluxes of the TR and LRs, thus, alleviating oxidative damage of DS in the host plant.