Localization of plasma membrane H+-ATPase in nodules of Phaseolus vulgaris L.

Campos, Francisco; Perez-Castiñeira, José R.; Villalba, José M.; Culiañez-Marciá, Francisco A.; Sánchez, Federico; Serrano, Ramón

doi:10.1007/bf00041388

Cited by 18 publications

(7 citation statements)

References 72 publications

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“…High enrichments of the V‐ATPase and P‐ATPase activities were detected in the tonoplast and PM fractions, respectively ( Table 1). This was confirmed by Western blot experiments using antibodies against PM H + ‐ATPase ( Campos et al . 1996 ), as well as antibodies against a tonoplast intrinsic protein (MIP‐F) ( Yamada et al .…”

Section: Resultsmentioning

confidence: 57%

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Two bean cell wall proteins more abundant during water deficit are high in proline and interact with a plasma membrane protein

et al. 2000

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SummaryTwo antigenically related glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell wall in response to water de®cit in Phaseolus vulgaris. In this report, we show that p33 and p36 are able to adhere to leaf protoplasts, and that they bind to plasma membrane (PM) vesicles in a divalent cationdependent manner. Data from the partial amino acid sequence of the p33 and p36 proteins indicate that they contain repeats of the decapeptide POVYKPOVEK; therefore, they are related to proline-rich proteins. Binding assays demonstrate that both proteins speci®cally bind to an 80 kDa PM protein. This binding is competed with a peptide that contains the RGD motif, as well as with ®bronectin, which also includes this sequence, suggesting that the 80 kDa PM protein has an integrin-like function whose natural ligands are p33 and p36. This is the ®rst case where a PM ligand for a higher plant cell wall protein has been identi®ed.

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Section: Resultsmentioning

confidence: 57%

“…(1999) . The purity of these fractions was verified by immunoblotting using antibodies raised against the Arabidopsis PM H + ‐ATPase ( Campos et al . 1996 ) and antibodies against the tonoplast MIP‐F protein from Mesembryanthemun crystallinum ( Yamada et al .…”

Section: Methodsmentioning

confidence: 99%

Two bean cell wall proteins more abundant during water deficit are high in proline and interact with a plasma membrane protein

et al. 2000

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“…A modi®ed version of the method of Towbin et al (1979) was employed for immunoblotting, using antibodies to P-type and V-type ATPases. The following were used: polyclonal antibodies against the small loop (Glu 118 to Gln241) of the plasma membrane H + -ATPase from Nicotiana plumbaginifolia (isoform PMA2: Morsomme et al 1996) expressed in Escherichia coli as a fusion protein with glutathione S-transferase; polyclonal antibodies against the central domain of Arabidopsis thaliana plasma membrane H + -ATPase (isoform AHA3; Campos et al 1996); and monoclonal antibodies to the 70-kDa and 60-kDa subunits of vacuolar H + -ATPase from oat roots (7A5 and 2E7, respectively; Ward et al 1992). Immunoreactive proteins were visualized using the BM Chemiluminescence system (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…In the only molecular study of PBM ATPase to date, Campos et al (1996) isolated a cDNA encoding a P-type ATPase from Phaseolus nodules, but immunolocalization and RNA hybridization suggested that this protein was localized in the uninfected cells of the nodule. These authors concluded that the PBM did not possess signi®cant P-type ATPase.…”

Section: Introductionmentioning

confidence: 98%

Localization of H + -ATPases in soybean root nodules

Fedorova

Thomson

Whitehead

et al. 1999

Planta

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The localization of H(+)-ATPases in soybean (Glycine max L. cv. Stevens) nodules was investigated using antibodies against both P-type and V-type enzymes. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies against tobacco and Arabidopsis H(+)-ATPases detected a single immunoreactive band at approximately 100 kDa. These antibodies recognized a protein of similar relative molecular mass in the crude microsomal fraction from soybean nodules and uninoculated roots. The amount of this protein was greater in PBM from mature nodules than in younger nodules. Immunolocalization of P-type ATPases using silver enhancement of colloidal-gold labelling at the light-microscopy level showed signal distributed around the periphery of non-infected cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infected and uninfected cells. Examination of nodule sections using confocal microscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as vesicles distributed throughout the infected cells of the central zone. Electron-microscopic examination of immunogold-labelled sections shows that P-type ATPase antigens were present on the PBM of both newly formed, single-bacteroid symbiosomes just released from infection threads, and on the PBM of mature symbiosomes containing two to four bacteroids. Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to detect this protein on symbiosome membranes. Only a very faint signal with this antibody was detected on Western blots of purified PBM. During nodule development, fusion of small symbiosomes to form larger ones containing multiple bacteroids was observed. Fusion was preceded by the formation of cone-like extensions of the PBM, allowing the membrane to make contact with the adjoining membrane of another symbiosome. We conclude that the major H(+)-ATPase on the PBM of soybean is a P-type enzyme with homology to other such enzymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids.

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“…Western blots were performed on selected fractions using antibodies specific to calreticulin, glutathione S-transferase (GST), mitochondrial aldehyde dehydrogenase (RF2), and plasma membrane proton-ATPase (PM H 1 -ATPase), which represented markers for the ER (Campos et al, 1996), cytosol (Marrs, 1996), mitochondria (Quitadamo et al, 2000), and plasma membrane (Liu et al, 2001), respectively. Calreticulin and PM H 1 -ATPase levels peaked in fractions with specific densities of 1.12 and 1.09 g mL 21 , respectively (Fig.…”

Section: Cyp80b1 Bbe and Cor Levels In Elicited Opium Poppy Cell Cumentioning

confidence: 99%

Sanguinarine Biosynthesis Is Associated with the Endoplasmic Reticulum in Cultured Opium Poppy Cells after Elicitor Treatment

et al. 2005

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Three key benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine-3#-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), were localized in cultured opium poppy (Papaver somniferum) cells by sucrose density gradient fractionation and immunogold labeling. CYP80B1 catalyzes the second to last step in the formation of (S)-reticuline, the last common intermediate in sanguinarine and morphine biosynthesis. BBE converts (S)-reticuline to (S)-scoulerine as the first committed step in sanguinarine biosynthesis, and COR catalyzes the penultimate step in the branch pathway leading to morphine. Sanguinarine is an antimicrobial alkaloid that accumulates in the vacuoles of cultured opium poppy cells in response to elicitor treatment, whereas the narcotic analgesic morphine, which is abundant in opium poppy plants, is not produced in cultured cells. CYP80B1 and BBE were rapidly induced to high levels in response to elicitor treatment. By contrast, COR levels were constitutive in the cell cultures, but remained low and were not induced by addition of the elicitor. Western blots performed on protein homogenates from elicitor-treated cells fractionated on a sucrose density gradient showed the cosedimentation of CYP80B1, BBE, and sanguinarine with calreticulin, and COR with glutathione S-transferase. Calreticulin and glutathione S-transferase are markers for the endoplasmic reticulum (ER) and the cytosol, respectively. In response to elicitor treatment, large dilated vesicles rapidly developed from the lamellar ER of control cells and fused with the central vacuole. Immunogold localization supported the association of CYP80B1 and BBE with ER vesicles, and COR with the cytosol in elicitor-treated cells. Our results show that benzylisoquinoline biosynthesis and transport to the vacuole are associated with the ER, which undergoes major ultrastructural modification in response to the elicitor treatment of cultured opium poppy cells.Alkaloids are a diverse group of approximately 12,000 low molecular weight, nitrogen-containing compounds found in about 20% of plant species. Morphine and sanguinarine are members of the large and diverse group of benzylisoquinoline alkaloids, of which more than 2,500 different structures have been identified in plants. Although morphine is a potent narcotic analgesic, it has been implicated in the stress-induced cross-linking of galacturonic-containing polysaccharides in the cell walls of opium poppy (Papaver somniferum) plants ( Morimoto et al., 2001). By contrast, sanguinarine, which shows specific toxic effects to herbivores and microbial pathogens, is proposed to function as an inducible defense compound (Schmeller et al., 1997). Benzylisoquinoline alkaloids share a common biosynthetic pathway, beginning with the condensation of two L-Tyr derivatives to produce the central precursor (S)-norcoclaurine ( Fig. 1; Samanani et al., 2004). Specific O-and N-methyltransferases convert (S)-norcoclaurine to (S)-N-methylcoclaurine (Facchini, 2001). The P450-depe...

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Localization of plasma membrane H+-ATPase in nodules of Phaseolus vulgaris L.

Cited by 18 publications

References 72 publications

Two bean cell wall proteins more abundant during water deficit are high in proline and interact with a plasma membrane protein

Two bean cell wall proteins more abundant during water deficit are high in proline and interact with a plasma membrane protein

Localization of H + -ATPases in soybean root nodules

Sanguinarine Biosynthesis Is Associated with the Endoplasmic Reticulum in Cultured Opium Poppy Cells after Elicitor Treatment

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