Roles of arbuscular mycorrhizas in plant phosphorus nutrition: interactions between pathways of phosphorus uptake in arbuscular mycorrhizal roots have important implications for understanding and manipulating plant phosphorus acquisition.

Does the enhanced tolerance of arbuscular mycorrhizal plants to water deficit involve modulation of drought‐induced plant genes?

In natural and agricultural ecosystems, arbuscular mycorrhizal (AM) fungi play a major role in plant nutrition. In AM symbiosis, the AM fungi extract mineral nutrients from the substrate and transfer them to the host plant. Inside the roots of the host plant, the intraradical hyphae form tree like structures (arbuscules) where the nutrients are released to the plant fungal interface. In return, the AM fungi receive carbohydrates from the plants. Specialized transport systems enable nutrient uptake from the substrate and translocation across membranes. As main components of organic molecules, phosphorus (P), nitrogen (N) and carbon (C) are of particular importance for symbiotic nutrient exchanges. This work is focused on a range of genes that encode proteins contributing to transport molecules (P, N and C nutrients) across cellular membranes in the plants Populus trichocarpa (poplar) and Sorghum bicolor (sorghum), and in the AM fungus Rhizophagus irregularis. In the AM fungus R. irregularis (formerly Glomus intraradices), we identified and characterized a novel functional ammonium transporter (AMT), GintAMT3. Quantification of transcript abundances in the extraradical mycelium (ERM) and the intraradical mycelium (IRM) during symbiosis with poplar and sorghum revealed that GintAMT3 was highly expressed in the IRM of AM roots. Phylogenetic analysis showed further, that the six glomeromycotan AMTs share high sequence similarity, but are distinct to AMTs of other fungal phyla. To functionaly analyze GintAMT3, we expressed GintAMT3 in a yeast deletion mutant devoid of all AMTs. The heterologous expression revealed that GintAMT3 is a low affinity transporter. Heterologous expression of GFP tagged GintAMT3 in yeast showed that GintAMT3 is localized in the plasma membrane and the vacuolar membrane. Further, we could show that expression of GinAMT3 is dependent on the N nutrition status and the fungal C status. Taken together, our data suggested that GintAMT3 is the main export carrier for ammonium at the arbuscular site. Using mRNA sequencing, we could show that low N availability significantly increased gene expression of the AM fungus, including genes involved in cell growth and membrane biogenesis as well as genes involved in signaling and metabolic processes. High abundances of genes related to N metabolism, including glutamine synthase, aminotransferase, AMTs as well as arginases, indicated a high turnover rate of N in the symbiotic root tissue. Depending on P availability, gene expression of AM phosphate transporters (PT) and AMT changed. Induction of PT and AMT under low-P availability indicated that the AM fungus transfers more nutrients to the host plant. Further, we identified amino acids transporters and H+/oligopeptide transporters specifically induced in mycorrhizal poplar roots, indicating that amino acids are transferred between the AM fungus and the plant. In poplar, we found that root colonization and low-N conditions resulted in the down-regulation of defense gene expression, suggesting that the plant stimulated symbiotic interactions with the AM fungus. We showed that root colonization specifically induced expression of known and newly identified PT and AMT in poplar and sorghum. Specific induction of nutrient transporters upon starvation strongly indicated that they are essential components of a functional symbiosis and suggested they are located in AM roots. Furthermore, root colonization suppressed the expression of genes involved in P starvation response, indicating that root colonization efficiently alleviated P stress of the plant. Moreover, we could show that the annual sorghum is more dependent on the AM fungus than the perennial poplar, but also that more P and possibly also more N is transferred from the AM fungus to the host plant. Non-mycorrhized sorghum accumulated similar quantities of P as AM sorghum under conditions, in which only the AM fungus had access to the P source. Poplar on the other hand accumulated less P in AM plants. In addition, we observed that a subset of poplar Pht1 transporters was regulated independently on the AM fungus, but depending on the P availability of the substrate. To deepen our understanding about symbiotic C exchange, we made transcriptome analysis and qRT-PCR to investigate the role of carbohydrate transporters in AM symbiosis between R. irregularis and, poplar and sorghum, respectively. In R. irregularis, the monosaccharide transporter GintMST2 was specifically induced in the IRM independently on the nutrient condition. Interestingly, we observed the down-regulation of many carbohydrate transporters in AM roots of poplar and sorghum. However, in poplar, we identified one carbohydrate transporter, which might be involved in symbiotic C transfer. In conclusion, our data on C transport suggested that carbohydrates are taken from the plant by the AM fungus instead of actively transferred to the fungus by the host plant. Taken together, the data summarized in my thesis add to our understanding of nutrient transport in AM symbiosis under different environmental conditions and help elucidating the underlying mechanisms. Regarding climate changes and resources shortening, a precise understanding of the efficiency of AM symbiosis may help to increase the efficiency of sustainable agriculture.

Recent research on arbuscular mycorrhizas has demonstrated that AM fungi play a significant role in plant phosphorus (P) uptake, regardless of whether the plant responds positively to colonization in terms of growth or P content. Here we focus particularly on implications of this finding for consideration of the balance between organic carbon (C) use by the fungi and P delivery (i.e. the C–P trade between the symbionts). Positive growth responses to arbuscular mycorrhizal (AM) colonization are attributed frequently to increased P uptake via the fungus, which results in relief of P deficiency and increased growth. Zero AM responses, compared with non-mycorrhizal (NM) plants, have conventionally been attributed to failure of the fungi to deliver P to the plants. Negative responses, combined with excessive C use, have been attributed to this failure. The fungi were viewed as parasites. Demonstration that the AM pathway of P uptake operates in such plants indicates that direct P uptake by the roots is reduced and that the fungi are not parasites but mutualists because they deliver P as well as using C. We suggest that poor plant growth is the result of P deficiency because AM fungi lower the amount of P taken up directly by roots but the AM uptake of P does compensate for the reduction. The implications of interplay between direct root uptake and AM fungal uptake of P also include increased tolerance of AM plants to toxins such as arsenate and increased success when competing with NM plants. Finally we discuss the new information on C–P trade in the context of control of the symbiosis by the fungus or the plant, including new information (from NM plants) on sugar transport and on the role of sucrose in the signaling network involved in responses of plants to P deprivation.

Differential effects of soil disturbance and plant residue retention on function of arbuscular mycorrhizal (AM) symbiosis are not reflected in colonization of roots or hyphal development in soil

Arbuscular mycorrhizal (AM) symbiosis can protect the host plants against the detrimental effects of the water deficit caused by osmotic stresses such as drought and salinity. Stomatal conductance (gs) and water use efficiency (WUE) are among the most studied water relations parameters in the mycorrhizal literature, since they are considered critical to the long-term performance of host plants in semiarid environments. Mycorrhizal effects on gs have been observed in about 50% of experiments involving AM and nonAM plants of similar size. In fact, gs rates usually are higher in AM than in nonAM plants, which implies that AM plants have a lower resistance to vapour transfer from inside the leaves to the atmosphere. AM and nonAM plants have also shown different critical points or thresholds of stomatal behaviour during drought episodes. The higher gs rates in AM plants have been associated with lower xylem-sap abscisic acid (ABA) and lower ABA fluxes to leaves in AM plants. On the other hand, it has been suggested that extraradical hyphae or increased root branching may allow mycorrhizal roots to better explore a particular soil volume, extending soil water depletion zones and giving a mycorrhizal root system more access to available water. In addition, it has been estimated that about half of the promotion of gs by AM fungi can be attributable to the soil colonization by AM fungi. Nevertheless, these results can vary when the host plant shows a water conservative strategy. Moreover, different AM fungal species have been shown to modulate also differently the physiological response, including gs, of host plant to drought. The AM influence on gs can also be modulated by environmental conditions such as irradiance, air temperature or leaf temperature. There are also several reports in the literature showing an increase of plant WUE by the AM symbiosis either under well watered or under osmotic stress conditions. The effects of AM symbiosis on WUE depend on the fungal species involved, without a correlation with the percentage of root infection. These effects have been rather related to higher net photosynthetic rate and optimal quantum yield of photosystem II in AM plants than in nonAM ones and with enhanced activities of carbon assimilatory enzymes such as Rubisco. In any case, specific studies dealing with the effect of AM symbiosis on leaf morphology are needed in order to ascertain how these parameters influence the WUE of the host plant.

Loquat (Eriobotrya japonica) is an evergreen tree with a shallow root system subjected to drought stress. We have found that AM fungi can alleviate drought stress by improving loquat nutrient uptake. However, the physiological mechanisms of improving drought tolerance have not been described so far in loquat mycorrhiza symbiosis. Funneliformis mosseae was used as arbuscular mycorrhizal fungus and loquat was selected as a model for an evergreen, woody plant. Thus, a pot experiment with four treatments was conducted. Growth, leaf water status, solute accumulation, oxidative damage to lipids, antioxidant activities, and phytohormones were evaluated by non-mycorrhizal (NM) and arbuscular mycorrhizal (AM) loquat plants growing under well-watered or drought-stressed conditions. Results showed that AM plants had higher dry-biomass production and leaf water potential than NM plants under drought-stressed conditions. The drought-stressed AM roots accumulated more proline than in NM roots, while not in leaves. Lipid peroxides of leaves and roots in drought-stressed AM plants were 26 and 61 % lower than in NM plants. The AM symbiosis may enhance osmotic adjustment in roots, contributing to maintaining a water potential gradient and water absorption from soil into the roots. The cumulative effects increased the AM plant tolerance to drought stress. The results are compared to findings reported hitherto mainly from short-lived, herbaceous AM plants in the literature.

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.

Arbuscular mycorrhizal (AM) symbioses are widespread in land plants but the extent to which they are functionally important in agriculture remains unclear, despite much previous research. We ask focused questions designed to give new perspectives on AM function, some based on recent research that is overturning past beliefs. We address factors that determine growth responses (from positive to negative) in AM plants, the extent to which AM plants that lack positive responses benefit in terms of nutrient (particularly phosphate: P) uptake, whether or not AM and nonmycorrhizal (NM) plants acquire different forms of soil P, and the cause(s) of AM ‘growth depressions’. We consider the relevance of laboratory work to the agricultural context, including effects of high (available) soil P on AM fungal colonisation and whether AM colonisation may be deleterious to crop production due to fungal ‘parasitism’. We emphasise the imperative for research that is aimed at increasing benefits of AM symbioses in the field at a time of increasing prices of P-fertiliser, and increasing demands on agriculture to feed the world. In other words, AM symbioses have key roles in providing ecosystem services that are receiving increasing attention worldwide.

We used medic (Medicago truncatula) to investigate effects of inoculation with two arbuscular mycorrhizal (AM) fungi and application of arsenate (AsV) and phosphate (Pi) on mechanisms underlying increased tolerance (in terms of growth) of AM plants to AsV. We tested the hypotheses that (1) inoculation with AM fungi results in down-regulation of MtPht1;1 and MtPht1;2 genes (encoding high-affinity Pi and AsV uptake systems in the direct root epidermal pathway) and up-regulation of the AM-induced MtPht1;4 (responsible for transfer of Pi from the arbuscular interface to cortical cells), and (2) these changes are involved in decreased As uptake relative to P uptake and hence increased As tolerance. We also measured expression of MtMT4, a Pi starvation-inducible gene, other genes encoding Pi uptake systems (MtPht 1;5 and MtPht1;6) and arsenate reductase (MtACR) and phytochelatin synthase (MtPCS), to gain insights into broader aspects of P transfers in AM plants and possible detoxification mechanisms. Medic responded slightly to AM colonization in terms of growth in the absence of As, but positively in terms of P uptake. Both growth and P responses in AM plants were positive when As was applied, indicating As tolerance relative to non-mycorrhizal (NM) plants. All AM plants showed high expression of MtPT4 and those inoculated with Glomus mosseae showed higher selectivity against As (shown by P/As molar ratios) and much lower expression of MtPht1;1 (and to some extent MtPht1;2) than Glomus intraradices-inoculated or NM plants. Results are consistent with increased P/As selectivity in AM plants (particularly those inoculated with G. mosseae) as a consequence of high P uptake but little or no As uptake via the AM pathway. However, the extent to which selectivity is dependent on down-regulation of direct Pi and AsV uptake through epidermal cells is still not clear. Marked up-regulation of a PCS gene and an ACR gene in AM plants may also be involved and requires further investigation.

Mycorrhizal symbiosis drives a carbon-dependent metabolic reprogramming in olive tree rhizosphere and leaves.

A glimpse into the past of land plants and of their mycorrhizal affairs: from fossils to evo‐devo

The antagonistic interplay between phosphorus (P) and zinc (Zn) in plants is well established. However, the molecular mechanisms mediating those interactions as influenced by arbuscular mycorrhizal (AM) symbiosis remain unclear. We investigated Zn concentrations, root AM symbiosis, and transcriptome profiles of maize roots grown under field conditions upon different P levels. We also validated genotype-dependent P-Zn uptake in selected genotypes from a MAGIC population and conducted mycorrhizal inoculation experiments using mycorrhizal-defective mutant pht1;6 to elucidate the significance of AM symbiosis in P-Zn antagonism. Finally, we assessed how P supply affects Zn transporters and Zn uptake in extraradical hyphae within a three-compartment system. Elevated P levels led to a significant reduction in maize Zn concentration across the population, correlating with a marked decline in AM symbiosis, thus elucidating the P-Zn antagonism. We also identified ZmPht1;6 is crucial for AM symbiosis and confirmed that P-Zn antagonistic uptake is dependent on AM symbiosis. Moreover, we found that high P suppressed the expression of the fungal RiZRT1 and RiZnT1 genes, potentially impacting hyphal Zn uptake. We conclude that high P exerts systemic regulation over root and AM hyphae-mediated Zn uptake in maize. These findings hold implications for breeding Zn deficiency-tolerant maize varieties.

Arbuscular mycorrhizal symbiosis and salicylic acid regulate aquaporins and root hydraulic properties in maize plants subjected to drought

Studies have suggested that increased root hydraulic conductivity in mycorrhizal roots could be the result of increased cell-to-cell water flux via aquaporins. This study aimed to elucidate if the key effect of the regulation of maize aquaporins by the arbuscular mycorrhizal (AM) symbiosis is the enhancement of root cell water transport capacity. Thus, water permeability coefficient (Pf ) and cell hydraulic conductivity (Lpc ) were measured in root protoplast and intact cortex cells of AM and non-AM plants subjected or not to water stress. Results showed that cells from droughted-AM roots maintained Pf and Lpc values of nonstressed plants, whereas in non-AM roots, these values declined drastically as a consequence of water deficit. Interestingly, the phosphorylation status of PIP2 aquaporins increased in AM plants subjected to water deficit, and Pf values higher than 12μms-1 were found only in protoplasts from AM roots, revealing the higher water permeability of AM root cells. In parallel, the AM symbiosis increased stomatal conductance, net photosynthesis, and related parameters, showing a higher photosynthetic capacity in these plants. This study demonstrates a better performance of AM root cells in water transport under water deficit, which is connected to the shoot physiological performance in terms of photosynthetic capacity.

In the arbuscular mycorrhizal (AM) symbiosis, plants satisfy part of their nitrogen (N) requirement through the AM pathway. In sorghum, the ammonium transporters (AMT) AMT3;1, and to a lesser extent AMT4, are induced in cells containing developing arbuscules. Here, we have characterized orthologs of AMT3;1 and AMT4 in four other grasses in addition to sorghum. AMT3;1 and AMT4 orthologous genes are induced in AM roots, suggesting that in the common ancestor of these five plant species, both AMT3;1 and AMT4 were already present and upregulated upon AM colonization. An artificial microRNA approach was successfully used to downregulate either AMT3;1 or AMT4 in rice. Mycorrhizal root colonization and hyphal length density of knockdown plants were not affected at that time, indicating that the manipulation did not modify the establishment of the AM symbiosis and the interaction between both partners. However, expression of the fungal phosphate transporter FmPT was significantly reduced in knockdown plants, indicating a reduction of the nutrient fluxes from the AM fungus to the plant. The AMT3;1 knockdown plants (but not the AMT4 knockdown plants) were significantly less stimulated in growth by AM fungal colonization, and uptake of both 15N and 33P from the AM fungal network was reduced. This confirms that N and phosphorus nutrition through the mycorrhizal pathway are closely linked. But most importantly, it indicates that AMT3;1 is the prime plant transporter involved in the mycorrhizal ammonium transfer and that its function during uptake of N cannot be performed by AMT4.