Role of extensin peroxidase in tomato (Lycopersicon esculentum Mill.) seedling growth

 It is proposed that inhibition of extensin peroxidase activity leads to a less rigid cell wall and thus promotes cell expansion and plant growth. A low-molecular-weight inhibitor derived from the cell walls of suspension-cultured tomato cells was found to completely inhibit extensin peroxidase-mediated extensin cross-linking in vitro at a concentration of 260 μg/ml. The inhibitor had no effect upon guaiacol oxidation catalyzed by extensin peroxidase or horseradish peroxidase. We have demonstrated that the light-irradiated inhibition of plant growth may be partially offset by inhibition of endogenous extensin peroxidase activity. Overall plant growth was enhanced by up to 15% in the presence of inhibitor relative to control plants. Inhibitor-treated and illuminated tomato hypocotyls grew up to 15% taller than untreated controls. The inhibitor had no effect upon etiolated plants over a 15-d period, suggesting that only low levels of peroxidase-mediated cross-linking can be found in the cell walls of etiolated plants. SDS-PAGE/Western blots of ionically bound protein from both etiolated and illuminated hypocotyls identified a doublet at 57/58.5 kDa which is immuno-reactive with antibodies raised to tomato extensin peroxidase. Levels of the 58.5-kDa protein, determined by SDS-PAGE, were at least threefold higher in illuminated tomato hypocotyls than in etiolated hypocotyls. Three fold higher levels of extensin peroxidase, elevated in-vitro extensin cross-linking activity and 15% higher levels of cross-linked, non-extractable extensin were observed in illuminated tomato hypocotyls compared with etiolated tomato hypocotyls. This suggests that white-light inhibition of tomato hypocotyl growth appears to be mediated, at least partially, by deposition of cell wall extensin, a process regulated by M_r-58,500 extensin peroxidase. Our results indicate that the contribution of peroxidase-mediated extensin deposition to plant cell wall architecture may have an important role in plant growth. Received: 22 July 1999 / Accepted: 11 October 1999     

Tomato LeAGP-1 is a plasma membrane-bound, glycosylphosphatidylinositol-anchored arabinogalactan-protein

Arabinogalactan-proteins (AGPs) are a class of highly glycosylated, hydroxyproline-rich glycoproteins that function in plant growth and development. Tomato LeAGP-1 represents a major AGP expressed in cultured cells and plants. Based on cDNA and amino acid sequence analyses along with carbohydrate and other biochemical analyses, tomato LeAGP-1 is hypothesized to be a classical AGP localized to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Here, this hypothesis was tested and supported with the following experiments. First, tomato ( Lycopersicon esculentum , cv. UC82B) cotyledon protoplasts were isolated following cell wall digestion with cellulase and pectinase, and LeAGP-1 was immunolocalized to the plasma membrane with a LeAGP-1 antibody. Second, LeAGP-1 was shown to be a major AGP component in plasma membrane vesicles from tomato cv. Bonnie Best suspension-cultured cells by Western blot analysis with the LeAGP-1 antibody. Third, fluorescence microscopy of plasmolysed, transgenic tobacco ( Nicotiana tabacum BY-2) suspension-cultured cells expressing a green fluorescent protein (GFP)-LeAGP-1 fusion product demonstrated localization to the plasma membrane and Hechtian threads. Fourth, the GFP-LeAGP-1 fusion protein was present in plasma membrane preparations from these transgenic tobacco cells by Western blot analysis with a GFP antibody. Fifth, GFP-LeAGP-1 secreted into the culture media contained ethanolamine, presumably attached to the C-terminal amino acid residue, consistent with its processing and release from the plasma membrane. Thus, these data support the hypothesis that LeAGP-1 is localized to the plasma membrane via a GPI anchor and suggest possible roles for LeAGP-1 in cellular signalling and matrix remodelling.     

Isolation, characterization and immunolocalization of a novel, modular tomato arabinogalactan-protein corresponding to the LeAGP-1 gene

Arabinogalactan-proteins (AGPs) are a family of hydroxy-proline-rich glycoproteins implicated to function in plant growth and development. This report focuses on a novel, modular AGP found in tomato, LeAGP-1, which was predicted by DNA cloning and herein verified at the protein level as a major AGP component. LeAGP-1 was isolated from tomato suspension-cultured cells and verified to be an AGP by precipitation with (beta-D-galactosyl)3 Yariv phenylglycoside and by amino acid composition analysis. Furthermore, LeAGP-1 was determined to correspond to LeAGP-1 clones based on three criteria: (1) amino acid composition identity, (2) amino acid sequence identity, and (3) specific immunoreactivity of glycosylated and deglycosylated LeAGP-1 with an antibody developed against the highly basic subdomain predicted from LeAGP-1 clones. The antibody was also used to immunolocalize LeAGP-1 in tomato to the cell surface of suspension-cultured cells, maturing metaxylem elements in young internodes and petioles, and stylar transmitting tissue cells. At the subcellular level, LeAGP-1 immunolocalized to the cell walls of these particular cells as well as to intercellular spaces between stylar transmitting tissue cells. LeAGP-1 now emerges as one of the most comprehensively studied AGPs in terms of (1) characterization at the genomic DNA, cDNA and protein levels, (2) known organ-specific and developmentally regulated mRNA expression patterns, (3) development of an antibody against a unique, peptide subdomain which specifically recognizes LeAGP-1 in its glycosylated and deglycosylated states, and (4) immunolocalization of a single, well-defined AGP molecule at the tissue and subcellular levels.     

Phenylcoumaran benzylic ether reductase, a prominent poplar xylem protein, is strongly associated with phenylpropanoid biosynthesis in lignifying cells

 It has previously been shown (D.R. Gang et al., 1999, J Biol Chem 274: 7516–7527) that the most abundant protein in the secondary xylem of poplar (Populus trichocarpa cv. `Trichobel') is a phenylcoumaran benzylic ether reductase (PCBER), an enzyme involved in lignan synthesis. Here, the distribution and abundance of PCBER in poplar was studied at both the RNA and protein level. The cellular expression pattern was determined by immunolocalization of greenhouse-grown plants as well as of a field-grown poplar. Compared to other poplar tissues, PCBER is preferentially produced in the secondary xylem of stems and roots and is associated with the active growth period. The protein is present in all cells of the young differentiating xylem, corresponding to the zone of active phenylpropanoid metabolism and lignification. In addition, PCBER is located in young differentiating phloem fibers, in xylem ray parenchyma, and in xylem parenchyma cells at the growth-ring border. Essentially the same expression pattern was observed in poplars grown in greenhouses and in the field. The synthesis of PCBER in phenylpropanoid-synthesizing tissues was confirmed in a bending experiment. Induction of PCBER was observed in the pith of mechanically bent poplar stems, where phenylpropanoid metabolism is induced. These results indicate that the products of PCBER activity are synthesized mainly in lignifying tissues, suggesting a role in wood development. Received: 28 September 1999 / Accepted: 15 March 2000     

Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases

 A new system to study programmed cell death in plants is described. Tomato (Lycopersicon esculentum Mill.) suspension cells were induced to undergo programmed cell death by treatment with known inducers of apoptosis in mammalian cells. This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and DNA fragmentation. In search of processes involved in plant apoptotic cell death, specific enzyme inhibitors were tested for cell-death-inhibiting activity. Our results showed that proteolysis plays a crucial role in apoptosis in plants. Furthermore, caspase-specific peptide inhibitors were found to be potent inhibitors of the chemical-induced cell death in tomato cells, indicating that, as in animal systems, caspase-like proteases are involved in the apoptotic cell death pathway in plants. Received: 5 August 1999 / Accepted: 14 March 2000     

The Rhodococcus fascians-plant interaction: morphological traits and biotechnological applications

Rhodococcus fascians is a Gram-positive bacterium that infects dicotyledonous and monocotyledonous plants, leading to an alteration in the normal growth process of the host. The disease results from the modulation of the plant hormone balances, and cytokinins are thought to play an important role in the induction of symptoms. Generally, on the aerial parts of the plants, existing meristems were found to be most sensitive to the action of R. fascians, but, depending on the infection procedure, differentiated tissues as well gave rise to shoots. Similarly, in roots not only actively dividing cells, but also cells with a high competence to divide were strongly affected by R. fascians. The observed symptoms, together with the determined hormone levels in infected plant tissue, suggest that auxins and molecules of bacterial origin are also involved in leafy gall formation. The complexity of symptom development is furthermore illustrated by the necessary and continuous presence of the bacteria for symptom persistence. Indeed, elimination of the bacteria from a leafy gall results in the further development of the multiple embryonic buds of which it consists. This interesting characteristic offers novel biotechnological applications: a leafy gall can be used for germplasm storage and for plant propagation. The presented procedure proves to be routinely applicable to a very wide range of plants, encompassing several recalcitrant species. Received: 14 January 1999 / Accepted: 19 June 1999     

Expression of a Petunia inflata pectin methyl esterase in Solanum tuberosum L. enhances stem elongation and modifies cation distribution

Transgenic potato (Solanum tuberosum L.) plants were constructed with a Petunia inflata-derived cDNA encoding a pectin methyl esterase (PME; EC 3.1.1.11) in sense orientation under the control of the cauliflower mosaic virus 35S promoter. The PME activity was elevated in leaves and tubers of the transgenic lines but slightly reduced in apical segments of stems from mature plants. Stem segments from the base of juvenile PME-overexpressing plants did not differ in PME activity from the control, whereas in apical parts PME was less active than in the wild-type. During the early stages of development stems of these trangenic plants elongated more rapidly than those of the wild-type. Further evidence that overexpression of a plant-derived PME has an impact on plant development is based on modifications of tuber yield, which was reduced in the transgenic lines. Cell walls from transgenic tubers showed significant differences in their cation-binding properties in comparison with the wild-type. In particular, cell walls displayed increased affinity for sodium and calcium, while potassium binding was constant. Furthermore, the total ion content of transgenic potatoes was modified. Indications of PME-mediated differences in the distribution of ions in transgenic plants were also obtained by monitoring relaxations of the membrane potential of roots subsequent to changes in the ionic composition of the bathing solution. However, no effects on the chemical structure of pectin from tuber cell walls could be detected. Received: 24 March 1999 / Accepted: 20 August 1999     

NaAGP4 is an arabinogalactan protein whose expression is suppressed by wounding and fungal infection inNicotiana alata

Arabinogalactan proteins (AGPs) are proteoglycans secreted by plant cells that have been implicated in plant growth and development. Most AGPs cloned to date possess highly labile glycosylphosphatidylinositol (GPI) lipid anchors. These anchors transiently attach AGPs to the plasma membrane before they are released into the cell wall following GPI anchor hydrolysis. We have isolated and partially sequenced the protein core of an AGP purified from styles of Nicotiana alata. The protein sequence data were utilised to clone the AGP's gene, NaAGP4. This AGP shares about 78% sequence identity with the tomato AGP LeAGP-1. RNA gel blot analyses of different plant organs indicate that NaAGP4 is expressed in the same tissues and at similar levels as LeAGP-1. Furthermore, NaAGP4 like LeAGP-1 is rapidly suppressed by tissue wounding and by pathogen infection. We believe NaAGP4 and LeAGP-1 are the first described examples of orthologous AGPs from different plant species. In contrast, another AGP from N. alata, NaAGP1, is comparatively unaffected by wounding and pathogen infection, although this AGP is expressed in similar tissues and at similar levels as NaAGP4.     

Drought induces fructan synthesis and 1-SST (sucrose: sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.)

 Seeds of Cichorium intybus L. var. foliosum cv. Flash were sown in acid-washed vermiculite and grown in a controlled-environment growth chamber. After 1 month of growth, plantlets did not contain sucrose:sucrose 1-fructosyltransferase (1-SST), the key enzyme in fructan biosynthesis. No fructan could be observed. Some of the plants were submitted to drought for 2 weeks. Glucose, fructose and sucrose concentrations increased in roots and leaves of stressed plants and the fructan concentration in roots and leaves was ten times higher than in control plants. The onset of fructan synthesis coincided with the increase in 1-SST activity in roots. Expression of the 1-SST gene could be observed in roots and leaves of stressed plants. Received: 12 July 1999 / Accepted: 16 October 1999     

Promiscuous,non-catalytic,tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>) plants

We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth.     

Micromechanics and anatomical changes during early ontogeny of two lianescent Aristolochia species

 The mechanical properties of young stems of Aristolochia macrophylla Lam. and Aristolochia brasiliensis Mart. et Zucc. were studied during elongation growth and primary differentiation. Data for the modulus of elasticity, for the viscoelastic behaviour caused by longitudinal tension and for the shear modulus resulting from torsion around a longitudinal axis were related to the underlying structural changes by quantitative analysis of stem anatomy, tissue distribution, ultrastructure, and cell wall biochemistry. The orientation of cellulose microfibrils was determined by light microscopy and small-angle X-ray diffraction, and the lignin content was determined by thioglycolic acid derivatization and spectroscopic quantification. It was demonstrated that the increase in stability during early development is due to the complementary effects of increase in cell wall material, lignification, and cellulose microfibril alignment. A detailed micromechanical model, considering internal prestresses, is proposed to explain the characteristic biphasic stress-strain behaviour as well as the strain-hardening observed. Received: 22 March 1999 / Accepted 9 September 1999     

Overexpression of tomato LeAGP-1 arabinogalactan-protein promotes lateral branching and hampers reproductive development

LeAGP-1 is a glycosylphosphatidylinositol (GPI)-anchored arabinogalactan-protein (AGP) in tomato (Lycopersicon esculentum). Patterns of mRNA expression and protein localization for LeAGP-1 indicate that it likely functions in certain aspects of plant growth and development. To elucidate LeAGP-1 function(s), transgenic tomato plants expressing enhanced green fluorescent protein (GFP) fused to LeAGP-1 [GFP-LeAGP-1] or two LeAGP-1 variants, one lacking the C-terminal GPI-anchor domain [GFP-LeAGP-1DeltaC] and the other lacking the lysine-rich domain [GFP-LeAGP-1DeltaK], under the control of the CaMV35S promoter were produced using Agrobacterium-mediated transformation. Transgenic T0 and T1 lines with high levels of both GFP-LeAGP-1 mRNA and protein: (i) were significantly shorter; (ii) were highly branched; (iii) produced more flower buds, but most of these flowers did not mature, resulting in less fruit production; and (iv) produced seeds that were significantly smaller than normal seeds. Overexpression of LeAGP-1DeltaK had a similar or even more pronounced effect on plant vegetative and reproductive growth, while the effect of LeAGP-1DeltaC overexpression on plant reproduction was minimal. These results indicate that the GPI anchor is critical for LeAGP-1 function. As the phenotype of GFP-LeAGP-1 overexpressing transgenic plants is similar to that of cytokinin-overproducing plants, mRNA expression patterns of LeAGP-1 under different hormone treatments were examined. Cytokinins upregulated LeAGP-1 mRNA expression, while auxins and ABA inhibited LeAGP-1 mRNA expression. Based on these results, GPI-anchored LeAGP-1 most likely functions in plant growth and development in concert with auxin/cytokinin signaling.     

Growth, ageing and death of a photoautotrophic plant cell culture

 Batch cultures of photoautotrophic cell suspensions of Chenopodiumrubrum L., growing in an inorganic medium on CO₂ under a daily balanced light–dark regime of 16 : 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3–4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohomones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. Received: 30 April 1999 / Accepted: 21 August 1999     

Characterization of prosystemin expressed in the baculovirus/insect cell system reveals biological activity of the systemin precursor

 Tomato (Lycopersicon esculentum Mill.) prosystemin in fusion with a viral signal peptide was expressed in Sf21 insect cell cultures after infection with recombinant baculoviruses. Prosystemin was purified from culture supernatants and its identity was confirmed by N-terminal sequence and mass-spectral analyses. Recombinant prosystemin was found to be equally active as compared to systemin in inducing the expression of wound-response genes in tomato plants. In cultured cells of L. peruvianum, prosystemin elicited a rapid alkalinization of the growth medium. The timing and dose-dependence of the alkalinization response were found to be identical for prosystemin and systemin, respectively. Prosystemin-triggered defense responses were inhibited by a competitive antagonist of systemin activity, indicating that the systemin sequence within the primary structure of prosystemin determines its activity. Received: 30 August 1999 / Accepted: 6 December 1999     

A cytoskeletal basis for wood formation in angiosperm trees: the involvement of microfilaments

The cortical microfilament (MF) component of the cytoskeleton within axial elements of the secondary vascular system of the angiosperm tree, Aesculus hippocastanum L. (horse-chestnut) was studied using transmission electron microscopy of ultrathin sections and indirect immunofluorescence microscopy of actin in thick sections. As seen by electron microscopy, MF bundles have a net axial orientation within fusiform cambial cells and their secondary vascular derivatives (i.e. in the axial xylem and phloem parenchyma, xylem fibres, vessel and sieve elements, and companion cells). Immunofluorescence studies, however, reveal that this axial orientation can be more accurately described as a helix of extremely high pitch; it is a persistent feature of all axial secondary vascular elements during their development. Helical MF arrays are the only arrangement seen in secondary phloem cells. However, in addition to helices, other MF arrays are seen in secondary xylem cells. For example, fibres possess ellipses of MFs associated with simple-pit formation, and vessel elements possess circular arrays of MFs that associate with the developing inter-vessel bordered pits, ray–vessel contact pits, and with the perforation plate. Linear MF arrays are seen co-oriented with the developing tertiary wall-thickenings in vessel elements. The possible roles of MFs during the cytodifferentiation of secondary vascular cells is discussed, and compared with that of microtubules. Received: 7 June 1999 / Accepted: 23 December 1999     

AtAGP18 is localized at the plasma membrane and functions in plant growth and development

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins (HRGPs). AtAGP17, 18 and 19 comprise the lysine-rich classical AGP subfamily in Arabidopsis. Overexpression of GFP–AtAGP17/18/19 fusion proteins in Arabidopsis revealed localization of the fusion proteins on the plant cell surface of different organs. Subcellular localization of the fusion proteins at the plasma membrane was further determined by plasmolysis of leaf trichome cells. To elucidate AtAGP17/18/19 function(s), these AGPs were expressed without the green fluorescent protein (GFP) tag under the control of 35S cauliflower mosaic virus promoter. In contrast to AtAGP17/AtAGP19 overexpressors which showed phenotypes identical to wild-type plants, AtAGP18 overexpressors displayed several phenotypes distinct from wild-type plants. Specifically, these overexpressors had smaller rosettes and shorter stems and roots, produced more branches and had less viable seeds. Moreover, these AtAGP18 overexpressors exhibited similar phenotypes to tomato LeAGP-1 overexpressors, suggesting these two AGP genes may have similar function(s) in Arabidopsis and tomato.     

Minor vein structure and sugar transport in Arabidopsis thaliana

Leaf and minor vein structure were studied in Arabidopsis thaliana (L.) Heynh. to gain insight into the mechanism(s) of phloem loading. Vein density (length of veins per unit leaf area) is extremely low. Almost all veins are intimately associated with the mesophyll and are probably involved in loading. In transverse sections of veins there are, on average, two companion cells for each sieve element. Phloem parenchyma cells appear to be specialized for delivery of photoassimilate from the bundle sheath to sieve element-companion cell complexes: they make numerous contacts with the bundle sheath and with companion cells and they have transfer cell wall ingrowths where they are in contact with sieve elements. Plasmodesmatal frequencies are high at interfaces involving phloem parenchyma cells. The plasmodesmata between phloem parenchyma cells and companion cells are structurally distinct in that there are several branches on the phloem parenchyma cell side of the wall and only one branch on the companion cell side. Most of the translocated sugar in A. thaliana is sucrose, but raffinose is also transported. Based on structural evidence, the most likely route of sucrose transport is from bundle sheath to phloem parenchyma cells through plasmodesmata, followed by efflux into the apoplasm across wall ingrowths and carrier-mediated uptake into the sieve element-companion cell complex. Received: 5 October 1999 / Accepted: 20 November 1999     

Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components

The alga Chlamydomonas reinhardtii contains cytoplasmic vacuoles that are often filled with a dense granule that is released from the cell by exocytosis. Purified granules contained polyphosphate, complexed with calcium and magnesium, as the predominant inorganic components. Antiserum was raised against the major 70-kDa protein in granules purified from wall-deficient (cw15) mutants, which reacted on immunoblots with larger glycoprotein complexes in purified cell wall fractions from wild-type cells. Confocal fluorescence microscopy detected binding of these antibodies predominantly at the periphery of wall-containing C. reinhardtiiy1 cells but primarily to loci in the interior of cells of the cw15 strain. Immunoelectron microscopy demonstrated that the 70-kDa protein was localized in vacuolar granules and the trans-Golgi network in sections of cw15 cells but not in the cytosol or chloroplast. Treatment of cells with a dye, fluorescent in its protonated form, indicated that the pH within vacuoles was lower than that in the cytosol, which suggested that the vacuoles are similar to lysosomes. Thus, the vacuoles may serve a dual function to provide an environment for degradation within the cell and also serve as a vehicle for secretion of specific proteins. Received: 29 September 1999 / Accepted: 20 November 1999     

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