A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi

Many plants have the capacity to obtain phosphate via a symbiotic association with arbuscular mycorrhizal (AM) fungi. In AM associations, the fungi release phosphate from differentiated hyphae called arbuscules, that develop within the cortical cells, and the plant transports the phosphate across a symbiotic membrane, called the periarbuscular membrane, into the cortical cell. In Medicago truncatula, a model legume used widely for studies of root symbioses, it is apparent that the phosphate transporters known to operate at the root-soil interface do not participate in symbiotic phosphate transport. EST database searches with short sequence motifs shared by known phosphate transporters enabled the identification of a novel phosphate transporter from M. truncatula, MtPT4. MtPT4 is significantly different from the plant root phosphate transporters cloned to date. Complementation of yeast phosphate transport mutants indicated that MtPT4 functions as a phosphate transporter, and estimates of the K(m) suggest a relatively low affinity for phosphate. MtPT4 is expressed only in mycorrhizal roots, and the MtPT4 promoter directs expression exclusively in cells containing arbuscules. MtPT4 is located in the membrane fraction of mycorrhizal roots, and immunolocalization revealed that MtPT4 colocalizes with the arbuscules, consistent with a location on the periarbuscular membrane. The transport properties and spatial expression patterns of MtPT4 are consistent with a role in the acquisition of phosphate released by the fungus in the AM symbiosis.     

Localization of proton-ATPase genes expressed in arbuscular mycorrhizal tomato plants

In arbuscular mycorrhizal symbioses, solutes such as phosphate are transferred to the plant in return for photoassimilates. The uptake mechanism is probably facilitated by a proton gradient generated by proton H⁺-ATPases. We investigated expression of Lycopersicon esculentum Mill. H⁺-ATPases in mycorrhizal and non-mycorrhizal plants to determine if any are specifically regulated in response to colonization. Tissue expression and cellular localization of H⁺-ATPases were determined by RNA gel blot analysis and in situ hybridization of mycorrhizal and non-mycorrhizal roots. LHA1, LHA2, and LHA4 had high levels of expression in roots and were expressed predominantly in epidermal cells. LHA1 and LHA4 were also expressed in cortical cells containing arbuscules. The presence of arbuscules in root sections was correlated with lower levels of expression of these two isoforms in the epidermis. These results suggest that LHA1 and LHA4 expression is decreased in epidermal cells located in regions of the root that contain arbuscules. This provides evidence of differential regulation between molecular mechanisms involved in proton-coupled nutrient transfer either from the soil or fungus to the plant.     

Medicago truncatula Mtha1-2 mutants loose metabolic responses to mycorrhizal colonization

Bidirectional nutrient transfer is one of the key features of the arbuscular mycorrhizal symbiosis. Recently we were able to identify a Medicago truncatula mutant (mtha1-2) that is defective in the uptake of phosphate from the periarbuscular space due to a lack of the energy providing proton gradient provided by the symbiosis specific proton ATPase MtHA1¹ In order to further characterize the impact of fungal colonization on the plant metabolic status, without the beneficial aspect of improved mineral nutrition, we performed leaf ion analyses in mutant and wildtype plants with and without fungal colonization. Although frequency of fungal colonization was unaltered, the mutant did not show a positive growth response to mycorrhizal colonization. This indicates that nutrient transfer into the plant cell fails in the truncated arbuscules due to lacking expression of a functional MtHA1 protein. The leaves of wildtype plants showed clear metabolic responses to root mycorrhizal colonization, whereas no changes of leaf metabolite levels of mycorrhizal mtha1-2 plants were detected, even though they were colonized. These results show that MtHa1 is indispensable for a functional mycorrhizal symbiosis and, moreover, suggest that fungal root colonization per se does not depend on nutrient transfer to the plant host.     

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.     

Construction of a phosphate transporter gene expression vector and its usage for tobacco transformation

A plant expression vector was constructed by inserting the phosphate transporter gene, LePT1, which was cloned from the tomato genome, into pCAMBIA2300. An agrobacterium-mediated system was used to transform tobacco and acquire transgenic plants. Analyses of the transgenic plants by PCR and RT-PCR indicated that the exogenous gene was integrated into and expressed by transgenic plants. The growth characteristics of T1 generation transgenic plants were examined, and the phosphate content of transgenic plants in a low-phosphate environment was found to be significantly higher than in wild-type plants.     

Uptake and transfer of nutrients in ectomycorrhizal associations: interactions between photosynthesis and phosphate nutrition

Energy-dispersive X-ray microanalytical investigations and microautoradiographic studies were carried out to examine whether the uptake and transfer of phosphate (P) by an ectomycorrhizal fungus is affected by the carbohydrate supply of its host plant. For this purpose, non-mycorrhizal seedlings of Pinus sylvestris L. and plants inoculated with the ectomycorrhizal basidiomycete Suillus bovinus (L. ex Fr.) Kuntze were placed in the dark for 7 days in advance of a P supply. The subcellular element distribution and the uptake and distribution of (33)P was analyzed in non-mycorrhizal and mycorrhizal roots of these plants and compared with plants kept constantly under normal light conditions (control plants). The results show that placing non-mycorrhizal plants in the dark in advance of the nutrient supply led to (1) a reduction of the subcellular contents of P, S and K, but to an increase in the cytoplasmic Na content, and (2) an increase of (33)P absorption and translocation to the shoot. It can be assumed that this increased inflow of (33)P in non-mycorrhizal plants was due to P starvation after suppressed photosynthesis and reduced respiration of these plants. The suppression of photosynthesis by an ectomycorrhizal host plant and the resulting lower carbohydrate supply conditions for the ectomycorrhizal fungus led to (1) a decrease of P absorption by the mycobiont, (2) a change of the P allocation in the fungal cell compartments of an ectomycorrhizal root, and (3) a reduction of P transfer to the host plant. However, microautoradiographic studies revealed that, under these conditions, P was also absorbed by the mycorrhizal fungus and translocated via the Hartig net to the host plant. In mycorrhizal roots of plants placed in the dark in advance of the nutrient supply, the cytoplasmic P content of the Hartig net was reduced and, instead, a high number of polyphosphate granules could be detected within the hyphae. The results indicate that the exchange processes between the symbionts in a mycorrhiza are possibly linked and that P uptake and translocation by an ectomycorrhizal fungus is also regulated by the carbohydrate supply of its host plant.     

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     

Transport processes at the plant-fungus interface in mycorrhizal associations: physiological studies

The roots of most plants form symbiotic associations with mycorrhizal fungi. The net flux of nutrients, particularly phosphorus (P), from the soil into the plant is greater in mycorrhizal than in comparable non-mycorrhizal plants. However despite the widespread occurrence of mycorrhizal associations the processes controlling the transfer of solutes between the symbionts are poorly understood. To understand the mechanisms regulating the transfer of solutes information about conditions at the interface between plant and fungus is needed.Measurements of apoplastic and intracellular electrical potential difference in leek roots colonised by mycorrhizal fungi and estimates of cytosolic pH in fungal hyphae are presented. These and the implications for plant/fungal mineral nutrition in vesicular-arbuscular mycorrhizas are discussed.     

Mycorrhizal status and diversity of fungal endophytes in roots of common buckwheat (Fagopyrum esculentum) and tartary buckwheat (F. tataricum)

To determine the mycorrhizal status and to identify the fungi colonising the roots of the plants, common buckwheat (Fagopyrum esculentum) and tartary buckwheat (F. tataricum) were inoculated with an indigenous fungal mixture from a buckwheat field. Root colonisation was characterised by the hyphae and distinct microsclerotia of dark septate endophytes, with occasional arbuscules and vesicles of arbuscular mycorrhizal fungi. Sequences of arbuscular mycorrhizal fungi colonising tartary buckwheat clustered close to the Glomus species group A. Sequences with similarity to the Ceratobasidium/Rhizoctonia complex, a putative dark septate endophyte fungus, were amplified from the roots of both common and tartary buckwheat. To the best of our knowledge, this is the first report of arbuscular mycorrhizal colonisation in tartary buckwheat and the first molecular characterisation of these fungi that can colonise both of these economically important plant species.