Resource allocation to growth or luxury consumption drives mycorrhizal responses.

Highly variable phenotypic responses in mycorrhizal plants challenge our functional understanding of plant-fungal mutualisms. Using non-invasive high-throughput phenotyping, we observed that arbuscular mycorrhizal (AM) fungi relieved phosphorus (P) limitation and enhanced growth of Brachypodium distachyon under P-limited conditions, while photosynthetic limitation under low nitrogen (N) was exacerbated by the fungus. However, these responses were strongly dependent on host genotype: only the faster growing genotype (Bd3-1) utilised P transferred from the fungus to achieve improved growth under P-limited conditions. Under low N, the slower growing genotype (Bd21) had a carbon and N surplus that was linked to a less negative growth response compared with the faster growing genotype. These responses were linked to the regulation of N : P stoichiometry, couples resource allocation to growth or luxury consumption in diverse plant lineages. Our results attest strongly to a mechanism in plants by which plant genotype-specific resource economics drive phenotypic outcomes during AM symbioses.

Costs of acquiring phosphorus by vascular land plants: patterns and implications for plant coexistence.

Content Summary 1420 I. Introduction 1421 II. Root adaptations that influence P acquisition 1422 III. Costs of P acquisition: general 1423 IV. Costs of P acquisition that are independent of soil P concentrations 1423 V. Costs of P acquisition that increase as soil P concentrations decline 1424 VI. Discussion and conclusions 1424 Acknowledgements 1425 References 1425 SUMMARY: We compare carbon (and hence energy) costs of the different modes of phosphorus (P) acquisition by vascular land plants. Phosphorus-acquisition modes are considered to be mechanisms of plants together with their root symbionts and structures such as cluster roots involved in mobilising or absorbing P. Phosphorus sources considered are soluble and insoluble inorganic and organic pools. Costs include operating the P-acquisition mechanisms, and resource requirements to construct and maintain them. For most modes, costs increase as the relevant soil P concentration declines. Costs can thus be divided into a component incurred irrespective of soil P concentration, and a component describing how quickly costs increase as the soil P concentration declines. Differences in sensitivity of costs to soil P concentration arise mainly from how economically mycorrhizal fungal hyphae or roots that explore the soil volume are constructed, and from costs of exudates that hydrolyse or mobilise insoluble P forms. In general, modes of acquisition requiring least carbon at high soil P concentrations experience a steeper increase in costs as soil P concentrations decline. The relationships between costs and concentrations suggest some reasons why different modes coexist, and why the mixture of acquisition modes differs between sites.

Plant growth responses to elevated atmospheric CO2 are increased by phosphorus sufficiency but not by arbuscular mycorrhizas.

Capturing the full growth potential in crops under future elevated CO2 (eCO2) concentrations would be facilitated by improved understanding of eCO2 effects on uptake and use of mineral nutrients. This study investigates interactions of eCO2, soil phosphorus (P), and arbuscular mycorrhizal (AM) symbiosis in Medicago truncatula and Brachypodium distachyon grown under the same conditions. The focus was on eCO2 effects on vegetative growth, efficiency in acquisition and use of P, and expression of phosphate transporter (PT) genes. Growth responses to eCO2 were positive at P sufficiency, but under low-P conditions they ranged from non-significant in M. truncatula to highly significant in B. distachyon Growth of M. truncatula was increased by AM at low P conditions at both CO2 levels and eCO2×AM interactions were sparse. Elevated CO2 had small effects on P acquisition, but enhanced conversion of tissue P into biomass. Expression of PT genes was influenced by eCO2, but effects were inconsistent across genes and species. The ability of eCO2 to partly mitigate P limitation-induced growth reductions in B. distachyon was associated with enhanced P use efficiency, and requirements for P fertilizers may not increase in such species in future CO2-rich climates.

Soybean SAT1 (Symbiotic Ammonium Transporter 1) encodes a bHLH transcription factor involved in nodule growth and NH4+ transport.

Glycine max symbiotic ammonium transporter 1 was first documented as a putative ammonium (NH4(+)) channel localized to the symbiosome membrane of soybean root nodules. We show that Glycine max symbiotic ammonium transporter 1 is actually a membrane-localized basic helix-loop-helix (bHLH) DNA-binding transcription factor now renamed Glycine max bHLH membrane 1 (GmbHLHm1). In yeast, GmbHLHm1 enters the nucleus and transcriptionally activates a unique plasma membrane NH4(+) channel Saccharomyces cerevisiae ammonium facilitator 1. Ammonium facilitator 1 homologs are present in soybean and other plant species, where they often share chromosomal microsynteny with bHLHm1 loci. GmbHLHm1 is important to the soybean rhizobium symbiosis because loss of activity results in a reduction of nodule fitness and growth. Transcriptional changes in nodules highlight downstream signaling pathways involving circadian clock regulation, nutrient transport, hormone signaling, and cell wall modification. Collectively, these results show that GmbHLHm1 influences nodule development and activity and is linked to a novel mechanism for NH4(+) transport common to both yeast and plants.

Mycorrhizal responses in wheat: shading decreases growth but does not lower the contribution of the fungal phosphate uptake pathway.

Effects have been investigated of reduced C supply (induced by shade) on arbuscular mycorrhizal (AM) colonisation, mycorrhizal growth responses (MGRs) and on AM-mediated and direct uptake of phosphate (Pi) (using (32)P) in wheat, a plant that does not usually respond positively to AM colonisation. Shading markedly reduced growth and shoot/root dry weight ratios of both AM and non-mycorrhizal wheat, indicating decreased photosynthetic C supply. However, shading had very little effect on percent root length colonised by Rhizophagus irregularis or Gigaspora margarita or on MGRs, which remained slightly positive or zero, regardless of shade; there were no growth depressions under shade. By 6 weeks, when the contributions of the AM pathway were measured with (32)P supplied in small hyphal compartments, R. irregularis had supplied 23 to 28% of shoot P with no significant effect of shading. Data show that reduced C availability did not reduce the contribution of the AM pathway to plant P, so the fungi were not acting physiologically as parasites. These results support our previous hypothesis that lack of positive MGR is not necessarily the outcome of excessive C use by the fungi or failure to deliver P via the AM pathway.

Nutrient requirements differ in two Pedicularis species in the absence of a host plant: implication for driving forces in the evolution of host preference of root hemiparasitic plants.

BACKGROUND AND AIMS: Facultative root hemiparasitic plants generally have a wide host range, but in most cases show an obvious host preference. The reasons for the marked difference in growth performance of hemiparasites when attached to different hosts are not fully understood. In this study, the hypothesis was tested that hemiparasites showing a preference for different hosts have different nutrient requirements. METHODS: Two facultative root hemiparasitic Pedicularis species (P. rex and P. tricolor) with a different host dependency and preference were used to test their responses to inorganic solutes. The effects of nitrogen, phosphorus and potassium on growth of the hemiparasitic plants not attached to a host were determined, using an orthogonal design in pot cultivation under greenhouse conditions. Variables including biomass, shoot nutrient concentration, root:shoot (R:S) ratios and the number of haustoria were measured. KEY RESULTS: As in autotrophic plants, nutrient deficiency reduced dry weight (DW) and nutrient concentrations in the root hemiparasites. Nitrogen and phosphorus significantly influenced growth of both Pedicularis species, while potassium availability influenced only shoot DW of P. rex. Nitrogen had far more effect on growth of P. rex than on P. tricolor, while phosphorus deficiency caused more marked growth depression in P. tricolor than in P. rex. Pedicularis rex grew faster than P. tricolor in a range of nutrient supplies. Different patterns of biomass allocation between the two Pedicularis species were observed. While P. rex invested more into roots (particularly fine rootlets) than P. tricolor, the number of haustoria produced by P. rex was relatively much lower than that produced by P. tricolor, which had a much smaller root system. CONCLUSIONS: The two Pedicularis species differ in nutrient requirements and biomass allocation. Distinct interspecific traits in growth and nutrient requirements can be driving forces for the differential interactions between hemiparasites and their hosts.

Direct and indirect influences of arbuscular mycorrhizal fungi on phosphorus uptake by two root hemiparasitic Pedicularis species: do the fungal partners matter at low colonization levels?

BACKGROUND AND AIMS: Because most parasitic plants do not form mycorrhizal associations, the nutritional roles of arbuscular mycorrhizal (AM) fungi in them have hardly been tested. Some facultative root hemiparasitic Pedicularis species form AM associations and hence are ideal for testing both direct and indirect effects of AM fungi on their nutrient acquisition. The aim of this study was to test the influence of AM inoculation on phosphorus (P) uptake by Pedicularis rex and P. tricolor. METHODS: (32)P labelling was used in compartmented pots to assess the contribution of the AM pathway and the influence of AM inoculation on P uptake from a host plant into the root hemiparasites. Laboratory isolates of fungal species (Glomus mosseae and G. intraradices) and the host species (Hordeum vulgare 'Fleet') to which the two Pedicularis species showed obvious responses in haustorium formation and growth in previous studies were used. KEY RESULTS: The AM colonization of both Pedicularis spp. was low (<15 % root length) and only a very small proportion of total plant P (<1 %) was delivered from the soil via the AM fungus. In a separate experiment, inoculation with AM fungi strongly interfered with P acquisition by both Pedicularis species from their host barley, almost certainly because the numbers of haustoria formed by the parasite were significantly reduced in AM plants. CONCLUSIONS: Roles of AM fungi in nutrient acquisition by root parasitic plants were quantitatively demonstrated for the first time. Evidence was obtained for a novel mechanism of preventing root parasitic plants from overexploiting host resources through AM fungal-induced suppression of the absorptive structures in the parasites.

The reduced mycorrhizal colonisation (rmc) mutation of tomato disrupts five gene sequences including the CYCLOPS/IPD3 homologue.

Arbuscular mycorrhizal (AM) symbiosis in vascular plant roots is an ancient mutualistic interaction that evolved with land plants. More recently evolved root mutualisms have recruited components of the AM signalling pathway as identified with molecular approaches in model legume research. Earlier we reported that the reduced mycorrhizal colonisation (rmc) mutation of tomato mapped to chromosome 8. Here we report additional functional characterisation of the rmc mutation using genotype grafts and proteomic and transcriptomic analyses. Our results led to identification of the precise genome location of the Rmc locus from which we identified the mutation by sequencing. The rmc phenotype results from a deletion that disrupts five predicted gene sequences, one of which has close sequence match to the CYCLOPS/IPD3 gene identified in legumes as an essential intracellular regulator of both AM and rhizobial symbioses. Identification of two other genes not located at the rmc locus but with altered expression in the rmc genotype is also described. Possible roles of the other four disrupted genes in the deleted region are discussed. Our results support the identification of CYCLOPS/IPD3 in legumes and rice as a key gene required for AM symbiosis. The extensive characterisation of rmc in comparison with its 'parent' 76R, which has a normal mycorrhizal phenotype, has validated these lines as an important comparative model for glasshouse and field studies of AM and non-mycorrhizal plants with respect to plant competition and microbial interactions with vascular plant roots.

Unraveling the Influence of Arbuscular Mycorrhizal Colonization on Arsenic Tolerance in Medicago: Glomus mosseae is More Effective than G. intraradices, Associated with Lower Expression of Root Epidermal Pi Transporter Genes.

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.

Inoculation with arbuscular mycorrhizal fungi suppresses initiation of haustoria in the root hemiparasite Pedicularis tricolor.

BACKGROUND AND AIMS: Plant parasitism and arbuscular mycorrhizal (AM) associations have many parallels and share a number of regulatory pathways. Despite a rapid increase in investigations addressing the roles of AM fungi in regulating interactions between parasitic plants and their hosts, few studies have tested the effect of AM fungi on the initiation and differentiation of haustoria, the parasite-specific structures exclusively responsible for host attachment and nutrient transfer. In this study, we tested the influence of AM fungi on haustorium formation in a root hemiparasitic plant. METHODS: Using a facultative root hemiparasitic species (Pedicularis tricolor) with the potential to form AM associations, the effects of inoculation were tested with two AM fungal species, Glomus mosseae and Glomus intraradices, on haustorium initiation in P. tricolor grown alone or with Hordeum vulgare 'Fleet' (barley) as the host plant. This study consisted of two greenhouse pot experiments. KEY RESULTS: Both AM fungal species dramatically suppressed intraspecific haustorium initiation in P. tricolor at a very low colonization level. The suppression over-rode inductive effects of the parasite's host plant on haustoria production and caused significant growth depression of P. tricolor. CONCLUSIONS: AM fungi had strong and direct suppressive effects on haustorium formation in the root hemiparasite. The significant role of AM fungi in haustorium initiation of parasitic plants was demonstrated for the first time. This study provides new clues for the regulation of haustorium formation and a route to development of new biocontrol strategies in management of parasitic weeds.

Fresh perspectives on the roles of arbuscular mycorrhizal fungi in plant nutrition and growth.

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.

Two sympatric root hemiparasitic Pedicularis species differ in host dependency and selectivity under phosphorus limitation.

Parasitic biology of Pedicularis L. (Orobanchaceae) has been underinvestigated despite its wide distribution and potential ecological significance. To better understand the parasitic aspects of the root hemiparasites, host-parasite interactions were investigated with two sympatric Pedicularis species, Pedicularis rex C. B. Clarke and Pedicularis tricolor Hand.-Mazz., at two developmental stages. Plant DW, shoot phosphorus (P) content, root:shoot ratio and number of haustoria were measured in Pedicularis grown with either a host plant or a plant of its own species in pot experiments. In addition, effects of parasitism and intraspecific competition on growth and biomass allocation in four host species belonging to three major functional groups (grasses, legumes and forbs) were investigated. The two Pedicularis species showed obvious host preference, but preferred different host species. Interactions between Pedicularis and their hosts depended on both species identity and developmental stages of the partners. Overall, P. rex showed much weaker host dependency and less damage to hosts than P. tricolor. Interspecific variations were observed among different host species in their responses to intraspecific competition and parasitism. We concluded that different Pedicularis-host pairs showed different interaction patterns. Sympatric Pedicularis may have differential influence on plant community structure and productivity.

Roles of arbuscular mycorrhizas in plant nutrition and growth: new paradigms from cellular to ecosystem scales.

Root systems of most land plants form arbuscular mycorrhizal (AM) symbioses in the field, and these contribute to nutrient uptake. AM roots have two pathways for nutrient absorption, directly through the root epidermis and root hairs and via AM fungal hyphae into root cortical cells, where arbuscules or hyphal coils provide symbiotic interfaces. New physiological and molecular evidence shows that for phosphorus the mycorrhizal pathway (MP) is operational regardless of plant growth responses (positive or negative). Amounts delivered cannot be determined from plant nutrient contents because when responses are negative the contribution of the direct pathway (DP) is reduced. Nitrogen (N) is also delivered to roots via an MP, but the contribution to total N requirement and the costs to the plant are not clear. The functional interplay between activities of the DP and MP has important implications for consideration of AM symbioses in ecological, agronomic, and evolutionary contexts.

Underground friends or enemies: model plants help to unravel direct and indirect effects of arbuscular mycorrhizal fungi on plant competition.

*We studied the effects of two arbuscular mycorrhizal (AM) fungi, singly or together, on the outcome of competition between a host (tomato cultivar, wild-type (WT)) and a surrogate nonhost (rmc, a mycorrhiza-defective mutant of WT) as influenced by the contributions of the direct and AM phosphorus (P) uptake pathways to plant P. *We grew plants singly or in pairs of the same or different genotypes (inoculated or not) in pots containing a small compartment with (32)P-labelled soil accessible to AM fungal hyphae and determined expression of orthophosphate (P(i)) transporter genes involved in both AM and direct P uptake. *Gigaspora margarita increased WT competitive effects on rmc. WT and rmc inoculated with Glomus intraradices both showed growth depressions, which were mitigated when G. margarita was present. Orthophosphate transporter gene expression and (32)P transfer showed that the AM pathway operated in single inoculated WT, but not in rmc. *Effects of AM fungi on plant competition depended on the relative contributions of AM and direct pathways of P uptake. Glomus intraradices reduced the efficiency of direct uptake in both WT and rmc. The two-fungus combination showed that interactions between fungi are important in determining outcomes of plant competition.

The fate and efficacy of benomyl applied to field soils to suppress activity of arbuscular mycorrhizal fungi.

A systematic application of the fungicide benomyl was used to follow up the suppression of arbuscular mycorrhizal (AM) colonization and to determine its fungitoxic activity and persistence at different depths. Repeated applications of benomyl reduced AM colonization mainly in the upper 0-4 cm layer of the treated soils. Furthermore, AM colonization decreased with soil depth. The activity and persistence of this fungicide was reduced over small changes in depth in the first 10 cm of the soil profile beneath a semiarid herbland at Brookfield Conservation Park (South Australia). Repeated applications of the fungicide only slightly increased the levels of toxicity in the soils, probably because of biodegradation of the fungicide in soils with a recent history of exposure to the fungicide. The decline in fungicide activity at depth was correlated with a decline in the suppressive effect of the fungicide on the activity of AM fungi.

More than a carbon economy: nutrient trade and ecological sustainability in facultative arbuscular mycorrhizal symbioses.

Symbiosis is well recognized as a major force in plant ecology and evolution. However, there is considerable uncertainty about the functional, ecological and evolutionary benefits of the very widespread facultative arbuscular mycorrhizal (AM) associations, in which the plants can grow and reproduce whether or not they are colonized by AM fungi. Here we address the significance of new research findings that are overturning conventional views that facultative AM associations can be likened to parasitic fungus-plant associations. Specifically, we address the occurrence and importance of phosphate uptake via AM fungi that does not result in increases in total phosphorus (P) uptake or in plant growth, and possible signalling between AM fungi and plants that can result in plant growth depressions even when fungal colonization remains very low. We conclude that, depending on the individual AM fungi that are present, the role of facultative AM associations in the field, especially in relation to plant competition, may be much more subtle than has been previously envisaged.

Different arbuscular mycorrhizal fungi induce differences in cellular responses and fungal activity in a mycorrhiza-defective mutant of tomato (rmc).

The reduced mycorrhizal colonisation (rmc) mutant of tomato forms different phenotypes with different arbuscular mycorrhizal (AM) fungi. Our aim was to characterise microscopically the cellular responses in plant and fungus in order to reveal how these varied when colonisation was blocked at different stages. Synchronised colonisation coupled with vital staining, autofluorescence and laser scanning confocal microscopy (LSCM) were used to determine how long the AM fungi stay alive during the interactions with rmc, whether nuclear repositioning occurred in the same way as in wild-type interactions and whether there was evidence for deployment of defence responses. The results showed that (1) all the AM fungi tested were attracted to roots of rmc, on which they developed active external mycelium and appressoria, the latter sometimes in higher numbers than on the wild type; (2) plant cellular responses, such as nuclear movement, occurred only when the AM fungus was able to penetrate the epidermal cells of rmc; and (3) plant defence responses such as autofluorescence were observed only transiently and callose deposition was not involved in blocking AM fungi in rmc. The results demonstrate that multi-step AM colonisation is not only an outcome of cellular processes influenced by both plant and fungus, but is also modified by the capacity of different AM fungi to respond to the plant phenotype induced by the rmc mutation.

Plant growth depressions in arbuscular mycorrhizal symbioses: not just caused by carbon drain?

* This study investigated effects of plant density and arbuscular mycorrhizal (AM) colonization on growth and phosphorus (P) nutrition of a cultivar of wheat (Triticum aestivum) that often shows early AM-induced growth depressions. * Two experiments were conducted. Expt 1 had three plant densities and one soil P concentration. Expt 2 had two plant densities and two P concentrations. Plants were grown in calcareous P-fixing soil, inoculated with Glomus intraradices or Gigaspora margarita, or noninoculated (nonmycorrhizal (NM)). Glomus intraradices colonized well and caused a growth depression only in Expt 1. Gigaspora margarita caused large growth depressions in both experiments even though it colonized poorly. * The results showed that growth depressions were mitigated by changes in relative competition for soil P by NM and AM plants, and probably by decreasing carbon costs of the fungi. * The different effects of the two fungi appear to be attributable to differences in the balance between P uptake by the fungal pathway and direct uptake via the roots. These differences may be important in other AM symbioses that result in growth depressions. The results show that mycorrhizal growth responses of plants grown singly may not apply at the population or community level.