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Summary Polypeptides from barley ( Hordeum vulgare ) mesophyll vacuoles were characterized by immunoblotting after SDS gel electrophoresis. A polyspecific rabbit antiserum raised against barley vacuolar proteins enriched in tonoplast membranes was used. Approximately 17 vacuolar polypeptides of different electrophoretic mobilities reacted with the anti-vacuole serum. Vacuolar polypeptides could be readily immunostained in lanes loaded with vacuoplasts. In vacuoplasts derived from young, 7 day old primary leaves, the major immunoreactive tonoplast polypeptide had an apparent molecular mass of approximately 26 kDa. This protein was developmentally regulated. Within 8 days, it disappeared gradually from the vacuoplasts and could barely be detected in 15 day old leaves.

Transport of malate and chloride across the tonoplast of barley mesophyll vacuoles has been compared. Whereas malate inhibited chloride uptake, 1,2,3-benzenetricarboxylic acid, a potent inhibitor of malate uptake which is assumed not to cross the tonoplast, was ineffective. In contrast to chloride uptake, malate uptake was inactivated by pyridoxal phosphate and phenylglyoxal. The malate transport system could be protected against pyridoxal phosphate when a substrate such as 1,2,3-benzenetricarboxylic acid was present in the preincubation medium. It is concluded that the transfer of malate and chloride across the tonoplast is mediated by different systems.

The vacuoles of mesophyll cells are capable of storing amino acids which are mobilized on demand. An amino-acid carrier was characterized by studying the efflux of vacuolar amino acids from isolated mesophyll vacuoles using the silicon oil layer centrifugation technique. Efflux was slow in the absence of ATP. It was stimulated by a factor of more than 5 by the addition of ATP. A similar stimulation was brought about by adenylyl imidodiphosphate ( Ado PP [NH] P ), suggesting that efflux activation by ATP did not involve ATP hydrolysis. GTP, CTP, UTP and ADP were not effective, or were much less effective than ATP, in activating the amino-acid transport system. Neutral amino acids inhibited ATP-stimulated amino-acid efflux by an allosteric mechanism. External leucine, valine, phenylalanine and methionine were more effective in inhibiting efflux than alanine, arginine, lysine or glutamic acid when present outside the vacuole. Protein-modifying agents such as N -ethylmaleimide and p -chloromercuriphenylsulfonic acid activated amino-acid efflux from the vacuoles, whereas proteinase treatment led to an inhibition. Stimulation by ATP of amino-acid export from the vacuole and inhibition by specific amino acids may be part of a mechanism to stabilize cytoplasmic amino-acid levels during protein synthesis.

Vacuoles were isolated from primary leaves of barley (Hordeum vulgare L.) by mechanical breakage of protoplasts, and their polypeptide composition analyzed by two-dimensional gel electrophoresis. Vacuoplasts which consist of the vacuole, a portion of the plasmalemma and of the cytoplasma were prepared from protoplasts by ultracentrifugation. By comparing the vacuolar polypeptide pattern with polypeptide patterns of isolated chloroplasts and of vacuoplasts, vacuolar polypeptides could clearly be distinguished from polypeptides derived from cross-contaminating cell compartments. At least 14 polypeptides of apparent molecular mass between 12 and 76 kilodaltons and an isoelectric point between 4.5 and 7.6 could be attributed to the tonoplast fraction of the vacuole, and 35 polypeptides to the soluble fraction of the vacuole. Several lectins with different specificity were employed to characterize the degree and nature of glycosylation of vacuolar polypeptides. Concanavalin A bound to a large number of polypeptides. Three out of the 14 tonoplast polypeptides exhibited detectable carbohydrate moieties and almost two-thirds of the surveyed soluble polypeptides were glycosylated.

Chloroplasts prepared from lysed protoplasts of barley mesophyll contain 2–8% of the total acid proteinase activity. This residual activity is not associated with intact chloroplasts isolated by means of density gradient centrifugation. Vacuoles isolated from lysed protoplasts contain 80–85% of the total acid proteinase activity, indicating that the enzyme(s) which is presumably responsible for the degradation of chloroplastic proteins is located largely in the central vacuoles of mesophyll cells.

Intact chloroplasts and vacuoles were isolated from mesophyll protoplasts of barley. The chloroplasts occupied about 15% of the cellular volume and contained 75% of the protein, whereas the vacuoles occupied about 80% of the volume and contained less than 4% of total cellular protein. Contamination of the vacuolar fraction by foreign protein is included in these values. Chlorophyll was absent from the vacuolar fraction, but less than 1% of several extra-vacuolar marker proteins were still present. The vacuoles contained hydrolytic enzymes. Several of them (α-mannosidase, α-galactosidase, N-acetylglucosaminidase) were soluble, whereas part of the activity of others semimented with the tonoplasts during centrifugation. Attached proteins could be released from the membranes during freezing in the presence of NaCl. One-dimensional gel electrophoretic separation of soluble vacuolar proteins under non-denaturing conditions yielded more than 10 protein bands. A comparative analysis was performed of thylakoids and vacuoles which were subfractionated into tonoplasts and soluble vacuolar constituents. Sodium dodecyl sulfate gel electrophoresis separated about 15 polypeptides of the soluble fraction which reacted with silver reagent. The tonoplast fraction yielded about 20 bands. A similar number of bands was observed when vacuoles incubated with the (14)C-labelled SH-reagent N-ethylmaleimide were analysed for radioactive polypeptides. Silverstaining of the polypeptides and their SH-content did not correlate. Several polypeptides of the vacuolar fraction had molecular weights very similar to the molecular weights of known chloroplast proteins. However, with the exception of the two subunits of ribulose-1,5-bisphosphate carboxylase, contamination of the vacuolar fraction by chloroplast proteins could be ruled out as a possible cause of the close correspondence. The lipophilic carboxylic-group reagent N,N'-dicyclohexylcarbodiimide ([(14)C]DCCD) reacted with several polypeptides of thylakoids and tonoplasts. However, the labelling patterns were different. The most heavily labelled polypeptide of thylakoids was the 8-kDa polypeptide of the basal part of the coupling factor CF0. Tonoplast polypeptides heavily labelled with [(14)C]DCCD had molecular weights of 24, 28, and 56 kDa. The vacuolar 8-kDa polypeptide remained unlabelled.

In primary leaves of barley allowed to senesce under natural conditions, carotenoids, like chlorophylls, disappear gradually. Commercial horse-radish peroxidase catalizes the oxidation of lutein to unknown colorless products. This reaction depends on the presence of 2,4 dichlorophenol. It is independent of peroxide but is nullified in the presence of catalase. Preparations of thylakoids from barley chloroplasts show an activity with features comparable to those of horse-radish peroxidase.

Null-mutations of the Arabidopsis FKBP-like immunophilin TWISTED DWARF1 (TWD1) gene cause a pleiotropic phenotype characterized by reduction of cell elongation and disorientated growth of all plant organs. Heterologously expressed TWD1 does not exhibit cis-trans-peptidylprolyl isomerase (PPIase) activity and does not complement yeast FKBP12 mutants, suggesting that TWD1 acts indirectly via protein-protein interaction. Yeast two-hybrid protein interaction screens with TWD1 identified cDNA sequences that encode the C-terminal domain of Arabidopsis multidrugresistance-like ABC transporter AtPGP1. This interaction was verified in vitro. Mapping of protein interaction domains shows that AtPGP1 surprisingly binds to the N-terminus of TWD1 harboring the cis-trans peptidyl-prolyl isomerase-like domain and not to the tetratrico-peptide repeat domain, which has been shown to mediate protein-protein interaction. Unlike all other FKBPs, TWD1 is shown to be an integral membrane protein that colocalizes with its interacting partner AtPGP1 on the plasma membrane. TWD1 also interacts with AtPGP19 (AtMDR1), the closest homologue of AtPGP1. The single gene mutation twd1-1 and double atpgp1-1/atpgp19-1 (atmdr1-1) mutants exhibit similar phenotypes including epinastic growth, reduced inflorescence size, and reduced polar auxin transport, suggesting that a functional TWD1-AtPGP1/AtPGP19 complex is required for proper plant development.

International audience; Metal immobilization away from metabolically active sites within the cell represents the last step in both the homeostasis of metals and the detoxification of metal in excess. Assessment of the importance of this step requires having access to the in vivo speciation of metals. Evolving techniques have made it possible to acquire more reliable in situ profiling of: (i) spatio-temporal accumulation of metal, (ii) characterization of the metal-ligands complexes and determination of the structure of the different bio-ligands involved. The chapter ”metal immobilization: where and how?” presents the role of different metal-chelators in plants, based on examples from works using non-invasive techniques and genetic approaches at both the whole plant, cellular and subcellular levels. The aim of the chapter is to give a survey of the key molecules and processes involved in metal immobilization in plants, on the basis of direct and robust evidences of the in vivo speciation of metals.