SUMMARYCytochemical methods were used to localize ATPase activity in different phases of development of the vesicular-arbuscular mycorrhizal association between Allium cepa L. and Glomus intraradices Schenck & Smith. In the absence of inhibitors the pattern of lead deposition (indicative of ATPase activity) at the arbuscular interface was similar to that previously published, whether material was prefixed in glutaraldehyde or formaldehyde. Considerable activity was detected along both plant and fungal plasma membranes and in the interfacial matrix associated with young arbuscules; this decreased with senescence of arbuscules. The fungal plasma membranes of external and intercellular hyphae also showed ATPase activity, but the peripheral plant membranes interfacing with these stages of fungal development showed little or none. The only precipitates observed in the absence of ATP were in the fungal vacuoles.Inhibitors of plasma membrane H'^-ATPase (DES, SW26 and vanadate) greatly reduced the lead precipitate due to ATPase activity on the fungal and plant membranes and completely inhibited its formation in the interfacial matrix. Vanadate has been reported to inhibit ATPases other than the H"^-ATPase, and the other inhibitors may also be less specific than previously believed. Molybdate, which inhibits non-specific phosphatase activity but not the H+-ATPase, completely inhibited the interfacial activity but consistently left a fine precipitate along the plant membrane surrounding arbuscular hyphae and on the plasma membrane of intercellular and arbuscular trunk hyphae. Fine arbuscular branches often completely lacked activity after treatment with molybdate. EDTA, which chelates Mg^+ necessary for Mg^^-ATPase activity, and low temperature prevented the formation of membraneassociated lead deposition in the presence of ATP, although scattered precipitates were formed over the plant and fungal cytoplasm.Incubation with /?-glycerophosphate of material prefixed with either glutaraldehyde or formaldehyde gave a picture different from that with ATP, Precipitates were heavy along the peripheral host plasma membrane and tonoplast but no or only light enzyme reactions were detected in the interfacial matrix and along the adjacent plant membrane surrounding young arbuscules, increasing in cells where arbuscular senescence was occurring. These enzyme activities were completely inhibited by molybdate but not by DES, The present observations strongly suggest that plasma membrane H-'-ATPase is a component of the ATPhydrolysing activities in host-fungus interfaces of VA mycorrhiza. Interpretation of the iocaiization of ATPase activities using cytochemical methods is discussed, together with possible changes in the distribution of the enzymes following mycorrhizal infection and their role in nutrient transfer between the symbionts.

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 beta-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.

SUMMARYThe ultrastructural distribution of acid and alkaline phosphatase in 6-week-old onion roots infected by Glomus mosseae has been investigated cytochennically. Significant acid phosphatase activity was only observed in the little vacuolated, immature terminal arbuscule branches of the mycorrhizal fungus whilst strong alkaline a-naphthyl phosphatase and /?-glycerophosphatase activities were localized within the vacuoles of the mature arbuscular and intercellular hyphae. In the host cells neither acid nor alkaline phosphatase distribution was modified with vesiculararbuscular mycorrhiza formation. These results are discussed in relation to previously reported mycorrhiza-specific alkaline phosphatase Gianinazzi, 1976, 1978) and the metabolism of phosphorus in vesicular-arbuscular mycorrhizal systems.

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