The role of arbuscular mycorrhizal symbiosis in improving plant water status under drought.

Arbuscular mycorrhizal fungi (AMF) have been presumed to ameliorate crop tolerance to drought. Here, we review the role of AMF in maintaining water supply to plants from drying soils and the underlying biophysical mechanisms. We used a soil-plant hydraulic model to illustrate the impact of several AMF mechanisms on plant responses to edaphic drought. The AMF enhance the soil's capability to transport water and extend the effective root length, thereby attenuating the drop in matric potential at the root surface during soil drying. The synthesized evidence and the corresponding simulations demonstrate that symbiosis with AMF postpones the stress onset limit, which is defined as the disproportionality between transpiration rates and leaf water potentials, during soil drying. The symbiosis can thus help crops survive extended intervals of limited water availability. We also provide our perspective on future research needs and call for reconciling the dynamic changes in soil and root hydraulics in order to better understand the role of AMF in plant water relations in the face of climate changes.

An arbuscular mycorrhizal fungus alters soil water retention and hydraulic conductivity in a soil texture specific way.

Arbuscular mycorrhizal fungi (AMF) alter plant water relations and contribute to soil structure. Although soil hydraulic properties depend on soil structure and may limit plant water uptake, little is known about how AMF influence soil water retention (the relation between the soil water content and soil water potential) and hydraulic conductivity in different soils. Instead, these soil hydraulic properties often are considered to be independent of AMF presence in experiments. We asked if this assumption holds true for both sand and loam. We grew maize plants either inoculated with Rhizophagus irregularis or with autoclaved inoculum in pots filled with quartz sand or loam soil until extraradical spread of the fungus throughout the pots was achieved. Each pot contained a hyphal compartment made of a soil sampling core (250 cm3) covered with a 20-µm nylon mesh to encourage fungus ingrowth but to exclude root ingrowth. We measured soil water retention and unsaturated hydraulic conductivity in these undisturbed root-free soil volumes. We observed that in loam harboring the mycorrhizal fungus, the soil water retention decreased, while in sand, it increased without detectable changes in the soil bulk density. The effects of the fungus on the soil water potential were strongest at low soil water contents in both soils. As a consequence of the altered water potentials in soils with the mycorrhizal fungus, soil hydraulic conductivity increased in loam but decreased in sand after fungus ingrowth. We conclude that in our study, the mycorrhizal fungus acted as a soil conditioner even distant from roots, which encouraged drainage in loams prone to sogginess but enhanced water storage in sands prone to quick desiccation. We recommend considering soil hydraulic properties as being dynamic in future studies on water relations of mycorrhizal plants.

Facilitation of plant water uptake by an arbuscular mycorrhizal fungus: a Gordian knot of roots and hyphae.

Arbuscular mycorrhizal (AM) fungi play a positive role in plant water relations, and the AM symbiosis is often cited as beneficial for overcoming drought stress of host plants. Nevertheless, water uptake via mycorrhizal hyphal networks has been little addressed experimentally, especially so through isotope tracing. In a greenhouse study conducted in two-compartment rhizoboxes, Medicago truncatula was planted in the primary compartment (PC), either inoculated with Rhizophagus irregularis or left uninoculated. Plant roots were either allowed to enter the secondary compartment (SC) or were restricted to the PC by root-excluding mesh. Substrate moisture was manipulated in the PC such that the plants were grown either in high moisture (15% of gravimetric water content, GWC) or low moisture (8% GWC). Meanwhile, the SC was maintained at 15% GWC throughout and served as a water source accessible (or not) by roots and/or hyphae. Water in the SC was labeled with deuterium (D) to quantify water uptake by the plants from the SC. Significantly, increased D incorporation into plants indicated higher water uptake by mycorrhizal plants when roots had access to the D source, but this was mainly explained by generally larger mycorrhizal root systems in proximity to the D source. On the other hand, AM fungal hyphae with access to the D source increased D incorporation into plants more than twofold compared to non-mycorrhizal plants. Despite this strong effect, water transport via AM fungal hyphae was low compared to the transpiration demand of the plants.

Brassinosteroids and sucrose transport in mycorrhizal tomato plants.

Silencing of SlSUT2 expression in tomato plants leads to a dwarfed phenotype, reduced pollen vitality and reduces pollen germination rate. Male sterility of flowers, together with a dwarfed growth behavior is reminiscent to brassinosteroid defective mutant plants. Therefore we aimed to rescue the SlSUT2 silencing phenotype by local brassinosteroid application. The phenotypical effects of SlSUT2 down-regulation could partially be rescued by epi-brassinolide treatment suggesting that SlSUT2 interconnects sucrose partitioning with brassinosteroid signaling. We previously showed that SlSUT2 silenced plants show increased mycorrhization and, this effect was explained by a putative sucrose retrieval function of SlSUT2 at the periarbuscular membrane. More recently, we reported that the symbiotic interaction between Solanaceous hosts and AM fungi is directly affected by watering the roots with epi-brassinolide. Here we show that the SlSUT2 effects on mycorrhiza are not only based on the putative sucrose retrieval function of SlSUT2 at the periarbuscular membrane. Our analyses argue that brassinosteroids as well as SlSUT2 per se can impact the arbuscular morphology/architecture and thereby affect the efficiency of nutrient exchange between both symbionts and the mycorrhizal growth benefit for the plant.

Brassinosteroids Affect the Symbiosis Between the AM Fungus Rhizoglomus irregularis and Solanaceous Host Plants.

Together with several proteins involved in brassinosteroid (BR) signaling and synthesis, the membrane steroid binding protein 1 (MSBP1) was identified within the interactome of the sucrose transporter of tomato (SlSUT2). We asked whether MSBP1 is also involved in BR signaling as assumed for the AtMSBP1 protein from Arabidopsis and whether it impacts root colonization with arbuscular mycorrhizal (AM) fungi in a similar way as shown previously for SlSUT2. In addition, we asked whether brassinosteroids per se affect efficiency of root colonization by AM fungi. We carried out a set of experiments with transgenic tobacco plants with increased and decreased MSBP1 expression levels. We investigated the plant and the mycorrhizal phenotype of these transgenic plants and tested the involvement of MSBP1 in BR metabolism by application of epi-brassinolide and brassinazole, an inhibitor of BR biosynthesis. We show that the phenotype of the transgenic tobacco plants with increased or reduced MSBP1 expression is consistent with an inhibitory role of MSBP1 in BR signaling. MSBP1 overexpression could be mimicked by brassinazole treatment. Interestingly, manipulation of MSBP1 expression in transgenic tobacco plants not only affected plant growth and development, but also the host plant responses toward colonization with AM fungi, as well as arbuscular architecture. Moreover, we observed that brassinosteroids indeed have a direct impact on the nutrient exchange in AM symbiosis and on the biomass production of colonized host plants. Furthermore, arbuscular morphology is affected by changes in MSBP1 expression and brassinolide or brassinazole treatments. We conclude that host plant growth responses and nutrient exchange within the symbiosis with AM fungi is controlled by brassinosteroids and might be impeded by the MSBP1 protein.

Atmospheric drought and low light impede mycorrhizal effects on leaf photosynthesis-a glasshouse study on tomato under naturally fluctuating environmental conditions.

Arbuscular mycorrhiza fungi (AMF) consume plant carbon and impact photosynthesis, but effects of AMF on plant gas exchange are transient and hardly predictable. This is at least partially because plant-internal nutrient-, water-, and sink-related effects, which can be influenced AMF, and atmospheric conditions integrate at the photosynthesis level. In nature and in plant production, plants face periodical and random short-term switches of environmental conditions that limit photosynthesis, which may impede stimulatory effects of AMF on leaf photosynthetic capacities. We hypothesized that mycorrhizal effects on plant internal-photosynthetic potentials will only translate to actual photosynthetic rates, if atmospheric conditions do not superimpose limitations to the photosynthetic process. We aimed to cover wide ranges of within and between-day variations in light intensities and vapor pressure deficits with an untargeted approach. We grew tomato plants hydroponically for 8 weeks in open pots and irrigated beyond pot water capacity every morning. Plants were inoculated or not with Funneliformis mosseae and were fertilized with a low-strength nutrient solution, which guaranteed good AMF colonization and comparable sets of mycorrhizal and non-mycorrhizal plants regarding developmental stage and leaf age. Instantaneous leaf photosynthesis was monitored continuously with transparent chambers during 3 days under naturally fluctuating greenhouse conditions on the two uppermost fully expanded leaves. We fitted mechanistic gas exchange models and modeled continuous daytime dynamics of net photosynthetic rates and stomatal conductance for representative sunlit canopies of random populations of mycorrhizal and non-mycorrhizal plants. Depending on time, mycorrhizal plants showed enhanced or decreased stomatal conductance over wide ranges of light intensities. Higher or lower stomatal opening in mycorrhizal plants became ineffective for photosynthetic rates under low light. In contrast and in accordance with the effects on stomatal conductance, photosynthetic rates were comparatively increased or decreased in mycorrhizal plants under high light conditions. This required at least moderate vapor pressure deficits. Under high atmospheric drought, stomatal conductance strongly declined in all plants, which also capped maximum photosynthetic rates under high light. Leaf photosynthetic capacities were higher in mycorrhizal plants when leaves contained more proteins and/or the plant-internal moisture stress was lower than in non-mycorrhizal plants. However, this only resulted in enhanced photosynthetic rates as long as leaves were not exposed to low radiation or high atmospheric drought. We conclude that light and atmospheric moisture are decisive factors for potential carbon cost and gain scenarios of plants associated with AMF.

Arbuscular Mycorrhizas: A Promising Component of Plant Production Systems Provided Favorable Conditions for Their Growth.

Arbuscular mycorrhizal (AM) fungi have become an attractive target as biostimulants in agriculture due to their known contributions to plant nutrient uptake and abiotic stress tolerance. However, inoculation with AM fungi can result in depressed, unchanged, or stimulated plant growth, which limits security of application in crop production systems. Crop production comprises high diversity and variability in atmospheric conditions, substrates, plant species, and more. In this review, we emphasize that we need integrative approaches for studying mycorrhizal symbioses in order to increase the predictability of growth outcomes and security of implementation of AM fungi into crop production. We briefly review known mechanisms of AM on nutrient uptake and drought tolerance of plants, on soil structure and soil hydraulic properties. We carve out that an important factor for both nutrient availability and drought tolerance is yet not well understood; the AM effects on soil hydraulic properties. We gave special emphasis to circular references between atmospheric conditions, soil hydraulic properties and plant nutrient and water uptake. We stress that interdisciplinary approaches are needed that account for a variability of atmospheric conditions and, how this would match to mycorrhizal functions and demands in a way that increased plant nutrient and water uptake can be effectively used for physiological processes and ultimately growth. Only with integrated analyses under a wide range of growing conditions, we will be able to make profound decisions whether or not to use AM in particular crop production systems or can adjust culture conditions in ways that AM plants thrive.

Blumenols as shoot markers of root symbiosis with arbuscular mycorrhizal fungi.

High-through-put (HTP) screening for functional arbuscular mycorrhizal fungi (AMF)-associations is challenging because roots must be excavated and colonization evaluated by transcript analysis or microscopy. Here we show that specific leaf-metabolites provide broadly applicable accurate proxies of these associations, suitable for HTP-screens. With a combination of untargeted and targeted metabolomics, we show that shoot accumulations of hydroxy- and carboxyblumenol C-glucosides mirror root AMF-colonization in Nicotiana attenuata plants. Genetic/pharmacologic manipulations indicate that these AMF-indicative foliar blumenols are synthesized and transported from roots to shoots. These blumenol-derived foliar markers, found in many di- and monocotyledonous crop and model plants (Solanum lycopersicum, Solanum tuberosum, Hordeum vulgare, Triticum aestivum, Medicago truncatula and Brachypodium distachyon), are not restricted to particular plant-AMF interactions, and are shown to be applicable for field-based QTL mapping of AMF-related genes.

Arbuscular Mycorrhiza Improves Substrate Hydraulic Conductivity in the Plant Available Moisture Range Under Root Growth Exclusion.

Arbuscular mycorrhizal fungi (AMF) proliferate in soils and are known to affect soil structure. Although their contribution to structure is extensively investigated, the consequences of those processes for soil water extractability and transport has, so far, gained surprisingly little attention. Therefore we asked, whether AMF can affect water retention and unsaturated hydraulic conductivity under exclusion of root ingrowth, in order to minimize plant driven effects. We carried out experiments with tomato inoculated with Rhizoglomus irregulare in a soil substrate with sand and vermiculite that created variation in colonization by mixed pots with wild type (WT) plants and mycorrhiza resistant (RMC) mutants. Sampling cores were introduced and used to assess substrate moisture retention dynamics and modeling of substrate water retention and hydraulic conductivity. AMF reduced the saturated water content and total porosity, but maintained air filled porosity in soil spheres that excluded root ingrowth. The water content between field capacity and the permanent wilting point (6-1500 kPa) was only reduced in mycorrhizal substrates that contained at least one RMC mutant. Plant available water contents correlated positively with soil protein contents. Soil protein contents were highest in pots that possessed the strongest hyphal colonization, but not significantly affected. Substrate conductivity increased up to 50% in colonized substrates in the physiologically important water potential range between 6 and 10 kPa. The improvements in hydraulic conductivity are restricted to substrates where at least one WT plant was available for the fungus, indicating a necessity of a functional symbiosis for this effect. We conclude that functional mycorrhiza alleviates the resistance to water movement through the substrate in substrate areas outside of the root zone.

Arbuscular Mycorrhiza Alleviates Restrictions to Substrate Water Flow and Delays Transpiration Limitation to Stronger Drought in Tomato.

Arbuscular mycorrhizal fungi (AMF) proliferate in soil pores, on the surface of soil particles and affect soil structure. Although modifications in substrate moisture retention depend on structure and could influence plant water extraction, mycorrhizal impacts on water retention and hydraulic conductivity were rarely quantified. Hence, we asked whether inoculation with AMF affects substrate water retention, water transport properties and at which drought intensity those factors become limiting for plant transpiration. Solanum lycopersicum plants were set up in the glasshouse, inoculated or not with Funneliformis mosseae, and grown for 35 days under ample water supply. After mycorrhizal establishment, we harvested three sets of plants, one before (36 days after inoculation) and the second (day 42) and third (day 47) within a sequential drying episode. Sampling cores were introduced into pots before planting. After harvest, moisture retention and substrate conductivity properties were assessed and water retention and hydraulic conductivity models were fitted. A root water uptake model was adopted in order to identify the critical substrate moisture that induces soil derived transpiration limitation. Neither substrate porosity nor saturated water contents were affected by inoculation, but both declined after substrates dried. Drying also caused a decline in pot water capacity and hydraulic conductivity. Plant available water contents under wet (pF 1.8-4.2) and dry (pF 2.5-4.2) conditions increased in mycorrhizal substrates and were conserved after drying. Substrate hydraulic conductivity was higher in mycorrhizal pots before and during drought exposure. After withholding water from pots, higher substrate drying rates and lower substrate water potentials were found in mycorrhizal substrates. Mycorrhiza neither affected leaf area nor root weight or length. Consistently with higher substrate drying rates, AMF restored the plant hydraulic status, and increased plant transpiration when soil moisture declined. The water potential at the root surface and the resistance to water flow in the rhizosphere were restored in mycorrhizal pots although the bulk substrate dried more. Finally, substrates colonized by AMF can be more desiccated before substrate water flux quantitatively limits transpiration. This is most pronounced under high transpiration demands and complies with a difference of over 1,000 hPa in substrate water potential.

Can Adverse Effects of Acidity and Aluminum Toxicity Be Alleviated by Appropriate Rootstock Selection in Cucumber?

Low-pH and aluminum (Al) stresses are the major constraints that limit crop yield in acidic soils. Grafting vegetable elite cultivars onto appropriate rootstocks may represent an effective tool to improve crop tolerance to acidity and Al toxicity. Two greenhouse hydroponic experiments were performed to evaluate growth, yield, biomass production, chlorophyll index, electrolyte leakage, mineral composition, and assimilate partitioning in plant tissues of cucumber plants (Cucumis sativus L. "Ekron") either non-grafted or grafted onto "P360" (Cucurbita maxima Duchesne × Cucurbita moschata Duchesne; E/C) or figleaf gourd (Cucurbita ficifolia Bouché; E/F). Cucumber plants were cultured in pots and supplied with nutrient solutions having different pH and Al concentrations: pH 6, pH 3.5, pH 3.5 + 1.5 mM Al, and pH 3.5 + 3 mM Al (Experiment 1, 14 days) and pH 6, pH 3.5, and pH 3.5 + 0.75 mM Al (Experiment 2, 67 days). Significant depression in shoot and root biomass was observed in response to acidity and Al concentrations, with Al-stress being more phytotoxic than low pH treatment. Significant decrease in yield, shoot, and root biomass, leaf area, SPAD index, N, K, Ca, Mg, Mn, and B concentration in aerial parts (leaves and stems) in response to low pH with more detrimental effects at pH 3.5 + Al. Grafted E/C plants grown under low pH and Al had higher yield, shoot, and root biomass compared to E/F and non-grafted plants. This better crop performance of E/C plants in response to Al stress was related to (i) a reduced translocation of Al from roots to the shoot, (ii) a better shoot and root nutritional status in K, Ca, Mg, Mn, and Zn concentration, (iii) a higher chlorophyll synthesis, as well as (iv) the ability to maintain cell membrane stability and integrity (lower electrolyte leakage). Data provide insight into the role of grafting on Al stress tolerance in cucumber.

Interaction of brassinosteroid functions and sucrose transporter SlSUT2 regulate the formation of arbuscular mycorrhiza.

Transgenic tomato plants with reduced expression of the sucrose transporter SlSUT2 showed higher efficiency of mycorrhization suggesting a sucrose retrieval function of SlSUT2 from the peri-arbuscular space back into the cell cytoplasm plant cytoplasm thereby limiting mycorrhiza fungal development. Sucrose uptake in colonized root cells requires efficient plasma membrane-targeting of SlSUT2 which is often retained intracellularly in vacuolar vesicles. Protein-protein interaction studies suggested a link between SISUT2 function and components of brassinosteroid biosynthesis and signaling. Indeed, the tomato DWARF mutant d(x) defective in BR synthesis (1) showed significantly reduced mycorrhization parameters. (2) The question has been raised whether the impact of brassinosteroids on mycorrhization is a general phenomenon. Here, we include a rice mutant defective in DIM1/DWARF1 involved in BR biosynthesis to investigate the effects on mycorrhization. A model is presented where brassinolides are able to impact mycorrhization by activating SUT2 internalization and inhibiting its role in sucrose retrieval.

The sucrose transporter SlSUT2 from tomato interacts with brassinosteroid functioning and affects arbuscular mycorrhiza formation.

Mycorrhizal plants benefit from the fungal partners by getting better access to soil nutrients. In exchange, the plant supplies carbohydrates to the fungus. The additional carbohydrate demand in mycorrhizal plants was shown to be balanced partially by higher CO2 assimilation and increased C metabolism in shoots and roots. In order to test the role of sucrose transport for fungal development in arbuscular mycorrhizal (AM) tomato, transgenic plants with down-regulated expression of three sucrose transporter genes were analysed. Plants that carried an antisense construct of SlSUT2 (SlSUT2as) repeatedly exhibited increased mycorrhizal colonization and the positive effect of plants to mycorrhiza was abolished. Grafting experiments between transgenic and wild-type rootstocks and scions indicated that mainly the root-specific function of SlSUT2 has an impact on colonization of tomato roots with the AM fungus. Localization of SISUT2 to the periarbuscular membrane indicates a role in back transport of sucrose from the periarbuscular matrix into the plant cell thereby affecting hyphal development. Screening of an expression library for SlSUT2-interacting proteins revealed interactions with candidates involved in brassinosteroid (BR) signaling or biosynthesis. Interaction of these candidates with SlSUT2 was confirmed by bimolecular fluorescence complementation. Tomato mutants defective in BR biosynthesis were analysed with respect to mycorrhizal symbiosis and showed indeed decreased mycorrhization. This finding suggests that BRs affect mycorrhizal infection and colonization. If the inhibitory effect of SlSUT2 on mycorrhizal growth involves components of BR synthesis and of the BR signaling pathway is discussed.

Photochemical processes, carbon assimilation and RNA accumulation of sucrose transporter genes in tomato arbuscular mycorrhiza.

Arbuscular mycorrhizal fungi enhance CO2 assimilation of their hosts which ensure the demand for carbohydrates of these obligate biotrophic microorganisms. Photosynthetic parameters were measured in tomato colonised or not by the arbuscular mycorrhizal fungus Glomus mosseae. In addition, carbohydrate contents and mRNA accumulation of three sucrose transporter genes were analysed. Mycorrhizal plants showed increased opening of stomata and assimilated significant more CO2. A higher proportion of the absorbed light was used for photochemical processes, while non-photochemical quenching and the content of photoprotective pigments were lower. Analysis of sugar contents showed no significant differences in leaves but enhanced levels of sucrose and fructose in roots, while glucose amounts stayed constant. The three sucrose transporter encoding genes of tomato SlSUT1, SlSUT2 and SlSUT4 were up-regulated providing transport capacities to transfer sucrose into the roots. It is proposed that a significant proportion of sugars is used by the mycorrhizal fungus, because only amounts of fructose were increased, while levels of glucose, which is mainly transferred towards the fungus, were nearly constant.