Plasma membrane ATPase and H+ transport activities in microsomal membranes from mycorrhizal tomato roots

ATPase activity, ATP-dependent H⁺ transport and the amount of antigenic tomato plasma membrane H⁺-APTase have been analysed in membrane vesicles isolated from Glomus mosseae- or Glomus intraradices-colonized roots and from non-mycorrhizal tomato roots. Microsomal protein content was higher in mycorrhizal than in control roots. The specific activity of the plasma membrane H⁺-ATPase was not affected by mycorrhizal colonization, although this activity increased in membranes isolated from mycorrhizal roots when expressed on a fresh weight basis. Western blot analysis of microsomal proteins using antibodies raised against the Arabidopsis thaliana plasma membrane H⁺ - ATPase showed that mycorrhizal colonization did not change the relative amount of tomato plasma membrane ATPase in the microsomes. However, on a fresh weight basis, there was a greater amount of this protein in roots of mycorrhizal plants. In addition, mycorrhizal membranes showed a higher specific activity of the vanadate-sensitive ATP-dependant H⁺ transport than membranes isolated from control roots. These results suggest that mycorrhiza might regulate the plasma membrane ATPase by increasing the coupling efficiency between H⁺ transport and ATP hydrolysis. The observed effects of mycorrhizal colonization on plasma membrane H⁺-ATPase were independent of the AM fungal species colonizing the root system.     

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco

In arbuscular mycorrhizas, H⁺-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526–S532). The proton-pumping H⁺-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H⁺-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H⁺-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of β-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H⁺-ATPase activity in the periarbuscular membrane results from selective induction of two H⁺-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface. Received: 23 October 1999 / Accepted: 7 February 2000     

A H+-ATPase That Energizes Nutrient Uptake during Mycorrhizal Symbioses in Rice and Medicago truncatula

Most plant species form symbioses with arbuscular mycorrhizal (AM) fungi, which facilitate the uptake of mineral nutrients such as phosphate from the soil. Several transporters, particularly proton-coupled phosphate transporters, have been identified on both the plant and fungal membranes and contribute to delivering phosphate from fungi to plants. The mechanism of nutrient exchange has been studied in plants during mycorrhizal colonization, but the source of the electrochemical proton gradient that drives nutrient exchange is not known. Here, we show that plasma membrane H⁺-ATPases that are specifically induced in arbuscule-containing cells are required for enhanced proton pumping activity in membrane vesicles from AM-colonized roots of rice (Oryza sativa) and Medicago truncatula. Mutation of the H⁺-ATPases reduced arbuscule size and impaired nutrient uptake by the host plant through the mycorrhizal symbiosis. Overexpression of the H⁺-ATPase Os-HA1 increased both phosphate uptake and the plasma membrane potential, suggesting that this H⁺-ATPase plays a key role in energizing the periarbuscular membrane, thereby facilitating nutrient exchange in arbusculated plant cells.     

The auxin-inducible phosphate transporter AsPT5 mediates phosphate transport and is indispensable for arbuscule formation in Chinese milk vetch at moderately high phosphate supply

Phosphorus is a macronutrient that is essential for plant survival. Most land plants have evolved the ability to form a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, which enhances phosphate (Pi) acquisition. Modulation of Pi transporter systems is the master strategy used by mycorrhizal plants to adapt to ambient Pi concentrations. However, the specific functions of PHOSPHATE TRANSPORTER 1 (PHT1) genes, which are Pi transporters that are responsive to high Pi availability, are largely unknown. Here, we report that AsPT5, an Astragalus sinicus (Chinese milk vetch) member of the PHT1 gene family, is conserved across dicotyledons and is constitutively expressed in a broad range of tissues independently of Pi supply, but is remarkably induced by indole-3-acetic acid (auxin) treatment under moderately high Pi conditions. Subcellular localization experiments indicated that AsPT5 localizes to the plasma membrane of plant cells. Using reverse genetics, we showed that AsPT5 not only mediates Pi transport and remodels root system architecture but is also essential for arbuscule formation in A. sinicus under moderately high Pi concentrations. Overall, our study provides insight into the function of AsPT5 in Pi transport, AM development and the cross-talk between Pi nutrition and auxin signalling in mycorrhizal plants.     

Linking lipid transfer with reduced arbuscule formation in tomato roots colonized by arbuscular mycorrhizal fungus under low pH stress

Arbuscules are the core structures of arbuscular mycorrhizae (AM), and arbuscule development is regulated by environmental stress, e.g., low pH. Recent studies indicate that lipid transfer from plants is essential for AM fungal colonization; however, the role of lipid transfer in arbuscule formation and the dynamics of lipid accumulation in arbuscules under low pH stress are far from well understood. In the symbiosis of tomato and Rhizophagus intraradices under contrasting pH conditions (pH 4.5 vs. pH 6.5), we investigated arbuscule formation, nutrient uptake, alkaline phosphatase activity and lipid accumulation; examined the gene expression involved in phosphate transport, lipid biosynthesis and transfer and sugar metabolism; and visualized the lipid dynamics in arbuscules. Low pH greatly inhibited arbuscule formation, in parallel with reduced phospholipid fatty acids accumulation in AM fungus and decreased P uptake. This reduction was supported by the decreased expression of plant genes encoding lipid biosynthesis and transfer. More degenerating arbuscules were observed under low pH conditions, and neutral lipid fatty acids accumulated only in degenerating arbuscules. These data reveal that, under low pH stress, reduced lipid transfer from hosts to AM fungi is responsible for the inhibited arbuscule formation.     

Molecular approaches to study plasma membrane H+-ATPases in arbuscular mycorrhizas

The activity of H⁺-ATPases of plant and fungi generates an electrochemical gradient of H⁺ across the cell plasma membrane that drives a number of secondary transport systems, including those responsible for the translocation of cations, anions, amino acids and sugars. During the last years, several studies have been aimed at elucidating the role of plasma membrane H⁺-ATPases in the nutrient exchange processes taking place between the plant and the fungus in arbuscular mycorrhizal (AM) symbiosis. This paper reviews present knowledge about plasma membrane H⁺-ATPases and experimental evidence supporting the involvement of H⁺-ATPases of both organisms in the bidirectional transport of nutrients between partners. Molecular strategies that will provide further information on the function and regulation of plasma membrane H⁺-ATPases in AM symbiosis are presented and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis

Plants acquire essential mineral nutrients such as phosphorus (P) and nitrogen (N) directly from the soil, but the majority of the vascular plants also gain access to these mineral nutrients through endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. In AM symbiosis, the fungi deliver P and N to the root through branched hyphae called arbuscules. Previously we identified MtPT4, a Medicago truncatula phosphate transporter located in the periarbuscular membrane that is essential for symbiotic phosphate transport and for maintenance of the symbiosis. In mtpt4 mutants arbuscule degeneration occurs prematurely and symbiosis fails. Here, we show that premature arbuscule degeneration occurs in mtpt4 mutants even when the fungus has access to carbon from a nurse plant. Thus, carbon limitation is unlikely to be the primary cause of fungal death. Surprisingly, premature arbuscule degeneration is suppressed if mtpt4 mutants are deprived of nitrogen. In mtpt4 mutants with a low N status, arbuscule lifespan does not differ from that of the wild type, colonization of the mtpt4 root system occurs as in the wild type and the fungus completes its life cycle. Sulphur is another essential macronutrient delivered to the plant by the AM fungus; however, suppression of premature arbuscule degeneration does not occur in sulphur-deprived mtpt4 plants. The mtpt4 arbuscule phenotype is strongly correlated with shoot N levels. Analyses of an mtpt4-2 sunn-1 double mutant indicates that SUNN, required for N-mediated autoregulation of nodulation, is not involved. Together, the data reveal an unexpected role for N in the regulation of arbuscule lifespan in AM symbiosis.     

Inorganic phosphate uptake in intact vacuoles isolated from suspension-cultured cells of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don under varying Pi status

Inorganic phosphate (Pi) uptake across the vacuolar membrane of intact vacuoles isolated from Catharanthus roseus suspension-cultured cells was measured. Under low Pi status, Pi uptake into the vacuole was strongly activated compared to high Pi status. Since Pi uptake across the vacuolar membrane is correlated with H⁺ pumping, we examined the dependency of H⁺ pumping on plant Pi status. Both H⁺ pumping and the activities of the vacuolar H⁺-pumps, the V-type H⁺-ATPase and the H⁺-PPase were enhanced under low Pi status. Despite this increase in H⁺ pumping, Western blot analysis showed no distinct increase in the amount of proton pump proteins. Possible mechanisms for the activation of Pi uptake into the vacuole under low Pi status are discussed. Miwa Ohnishi and Tetsuro Mimura contributed equally to this work.     

Mycorrhizal Networks Interacting with Litter Improves Nutrients and Growth for One Plant through the Vary of N/P Ratio under Karst Soil

Arbuscular mycorrhizae (AM) fungi affect nutrient uptake for host plants, while it is unclear how AM fungi interacting with soil litter affect plant growth and nutrient utilization through mycorrhizal networks in karst soil of deficient nutrients beyond the rhizosphere. An experiment was conducted in a microcosm composed of a planting compartment for Cinnamomum camphora seedlings with or without Glomus mosseae fungus (M⁺ vs. M⁻ ) and an adjacent litter compartment containing or not containing additional litter material of Arthraxon hispidus (L⁺ vs. L⁻ ), where the compartments are connected either by nylon mesh of 20 μm or 0.45 μm which either allow available mycorrhizal networks within the litter compartment or prevent mycelium entering into the litter compartment (N⁺ vs. N⁻ ). Plant biomass and nutrients were measured. The results showed that the addition of litter changed the symbiotic process in mycorrhizal colonization, spore, and hyphal density, which when in association with the host plant then affected the biomass, and accumulations of N (nitrogen) and P (phosphorus) in the individual plant as well as root, stem, and leaf respectively. AM fungi increased N and P accumulations and N/P ratio in individual plants and plant tissues. A decrease of the N/P ratio of the individual plant was observed when AM fungus interacted significantly with litter through mycorrhizal networks in the litter compartment. The results indicate that the C. camphora seedlings benefited from litter in nutrient utilization of N and P through the vary of N/P ratio when accessing mycorrhizal networks. These findings suggest that mycorrhizal networks interacting with litter improve growth and nutrients of N and P for plants through the vary of N/P ratio in order to alleviate nutrient limitation under karst soil.     

A purple acid phosphatase,GmPAP33, participates in arbuscule degeneration during arbuscular mycorrhizal symbiosis in soybean

Arbuscules are the central structures of the symbiotic association between terrestrial plants and arbuscular mycorrhizal (AM) fungi. However, arbuscules are also ephemeral structures, and following development, these structures are soon digested and ultimately disappear. Currently, little is known regarding the mechanism underlying the digestion of senescent arbuscules. Here, biochemical and functional analyses were integrated to test the hypothesis that a purple acid phosphatase, GmPAP33, controls the hydrolysis of phospholipids during arbuscule degeneration. The expression of GmPAP33 was enhanced by AM fungal inoculation independent of the P conditions in soybean roots. Promoter‐β‐glucuronidase (GUS) reporter assays revealed that the expression of GmPAP33 was mainly localized to arbuscule‐containing cells during symbiosis. The recombinant GmPAP33 exhibited high hydrolytic activity towards phospholipids, phosphatidylcholine, and phosphatidic acid. Furthermore, soybean plants overexpressing GmPAP33 exhibited increased percentages of large arbuscules and improved yield and P content compared with wild‐type plants when inoculated with AM fungi. Mycorrhizal RNAi plants had high phospholipid levels and a large percentage of small arbuscules. These results in combination with the subcellular localization of GmPAP33 at the plasma membrane indicate that GmPAP33 participates in arbuscule degeneration during AM symbiosis via involvement in phospholipid hydrolysis.     

Growth responses and N and P use efficiency of three Alnus species as affected by arbuscular-mycorrhizal colonisation

In this study it was determined how different species ofAlnus (A. cordata, A. incana and A. glutinosa) responded tocolonisation by arbuscular mycorrhizal (AM) fungi (Glomusmosseae or Glomus intraradices) with regard togrowth and their ability to acquire and utilise nitrogen and phosphorus.Non-mycorrhizal plants but with phosphorus added, were used as control. InA. glutinosa the application of 75 ppm P hadsimilar effect on growth and P acquisition as did AM. Nevertheless,A.cordata and A. incana grew poorly when suppliedwith 75 ppm of P and required AM symbiosis for optimum growth andNand P uptake. The percentage increases in shoot dry biomass in AM colonised ascompared with P-fertilised plants were 441 (A. cordata)and644 (A. incana) whilst AM-colonised A.glutinosa matched P-fertilised plants. Plant shoot N/P ratioincreased in response to AM-colonisation indicating that mycorrhizal effects onN uptake are greater than on P uptake. Information concerning the directinfluence of AM on N acquisition and nutrient use efficiency byAlnus species is important. AM-colonisation provides anexcellent biological mechanism by which Alnus plantsbecamemore efficient P-users. That Alnus sp. are highlymycorrhizal-dependent plants was apparent because AM-colonisation was criticalfor growth of A. incana and A.cordata. In this respect, for maximizing the efficient uptake anduseof N and P, under the growth conditions provided, Alnusplants need to be mycorrhizal. AM symbiosis seems decisive for the successfulestablishment of Alnus sp. in revegetation strategies. Thelow N and P availability in soils where Alnus species areuseful candidates in any recolonisation and reclamation process emphasises theneed to investigate systems by which N and P uptake byAlnus plants can be enhanced.     

Plant–fungus competition for nitrogen erases mycorrhizal growth benefits of Andropogon gerardii under limited nitrogen supply

Considered to play an important role in plant mineral nutrition, arbuscular mycorrhizal (AM) symbiosis is a common relationship between the roots of a great majority of plant species and glomeromycotan fungi. Its effects on the plant host are highly context dependent, with the greatest benefits often observed in phosphorus (P)‐limited environments. Mycorrhizal contribution to plant nitrogen (N) nutrition is probably less important under most conditions. Moreover, inasmuch as both plant and fungi require substantial quantities of N for their growth, competition for N could potentially reduce net mycorrhizal benefits to the plant under conditions of limited N supply. Further compounded by increased belowground carbon (C) drain, the mycorrhizal costs could outweigh the benefits under severe N limitation. Using a field AM fungal community or a laboratory culture of Rhizophagus irregularis as mycorrhizal inoculants, we tested the contribution of mycorrhizal symbiosis to the growth, C allocation, and mineral nutrition of Andropogon gerardii growing in a nutrient‐poor substrate under variable N and P supplies. The plants unambiguously competed with the fungi for N when its supply was low, resulting in no or negative mycorrhizal growth and N‐uptake responses under such conditions. The field AM fungal communities manifested their potential to improve plant P nutrition only upon N fertilization, whereas the R. irregularis slightly yet significantly increased P uptake of its plant host (but not the host's growth) even without N supply. Coincident with increasing levels of root colonization by the AM fungal structures, both inoculants invariably increased nutritional and growth benefits to the host with increasing N supply. This, in turn, resulted in relieving plant P deficiency, which was persistent in non‐mycorrhizal plants across the entire range of nutrient supplies.     

Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles

In response to the colonization by arbuscular mycorrhizal (AM) fungi, plants reprioritize their phosphate (Pi)-uptake strategies to take advantage of nutrient transfer via the fungus. The mechanisms underlying Pi transport are beginning to be understood, and recently, details of the regulation of plant and fungal Pi transporters in the AM symbiosis have been revealed. This review summarizes recent advances in this area and explores current data and hypotheses of how the plant Pi status affects the symbiosis. Finally, suggestions of an interrelationship of Pi and nitrogen (N) in the AM symbiosis are discussed.     

The H+-ATPase HA1 of Medicago truncatula Is Essential for Phosphate Transport and Plant Growth during Arbuscular Mycorrhizal Symbiosis

A key feature of arbuscular mycorrhizal symbiosis is improved phosphorus nutrition of the host plant via the mycorrhizal pathway, i.e., the fungal uptake of Pi from the soil and its release from arbuscules within root cells. Efficient transport of Pi from the fungus to plant cells is thought to require a proton gradient across the periarbuscular membrane (PAM) that separates fungal arbuscules from the host cell cytoplasm. Previous studies showed that the H⁺-ATPase gene HA1 is expressed specifically in arbuscule-containing root cells of Medicago truncatula. We isolated a ha1-2 mutant of M. truncatula and found it to be impaired in the development of arbuscules but not in root colonization by Rhizophagus irregularis hyphae. Artificial microRNA silencing of HA1 recapitulated this phenotype, resulting in small and truncated arbuscules. Unlike the wild type, the ha1-2 mutant failed to show a positive growth response to mycorrhizal colonization under Pi-limiting conditions. Uptake experiments confirmed that ha1-2 mutants are unable to take up phosphate via the mycorrhizal pathway. Increased pH in the apoplast of abnormal arbuscule-containing cells of the ha1-2 mutant compared with the wild type suggests that HA1 is crucial for building a proton gradient across the PAM and therefore is indispensible for the transfer of Pi from the fungus to the plant.