Multiple forms of ADP-glucose pyrophosphorylase from tomato fruit.

ADP-glucose pyrophosphorylase (AGP) was purified from tomato (Lycopersicon esculentum Mill.) fruit to apparent homogeneity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme migrated as two close bands with molecular weights of 50,000 and 51,000. Two-dimensional polyacrylamide gel electrophoresis analysis of the purified enzyme, however, revealed at least five major protein spots that could be distinguished by their slight differences in net charge and molecular weight. Whereas all of the spots were recognized by the antiserum raised against tomato fruit AGP holoenzyme, only three of them reacted strongly with antiserum raised against the potato tuber AGP large subunit, and the other two spots (with lower molecular weights) reacted specifically with antisera raised against spinach leaf AGP holoenzyme and the potato tuber AGP small subunit. The results suggest the existence of at least three isoforms of the AGP large subunit and two isoforms of the small subunit in tomato fruit in vivo. The native molecular mass of the enzyme determined by gel filtration was 220 +/- 10 kD, indicating a tetrameric structure for AGP from tomato fruit. The purified enzyme is very sensitive to 3-phosphoglycerate/inorganic phosphate regulation.     

Organ‐specific localization and molecular properties of three soluble invertases from Lilium longiflorum flower buds

Three soluble invertase (EC 3.2.1.26) isoforms from Easter lily ( Lilium longiflorum Thunb. cv. Nellie White) flower buds were purified to apparent homogeneity. Non‐denaturing PAGE showed one band for all three invertases that corresponded to the invertase activity. SDS‐PAGE of purified invertase I gave a single band at 78 kDa, whereas invertases II and III gave three bands at 54, 52 and 24 kDa. Antibodies against tomato fruit acid invertase and Urtica dioica leaf acid invertase recognized all three invertase isoforms, whereas antibodies against wheat coleoptile acid invertase recognized only 56‐ and 54‐kDa bands of invertases II and III. Antibodies against wheat coleoptile invertase recognized the 54‐ and 52‐kDa proteins from crude extracts of all flower organs, and a 72‐kDa protein in both leaf and bulb scale extracts. All three invertases bound to Con‐A peroxidase. Deglycosylation of invertase I with glycopeptidase F was complete and resulted in a peptide of 75 kDa. Invertases II and III were deglycosylated partially by glycopeptidase F and resulted in proteins of 53, 51, 50 and 22 kDa. Invertase I was localized only in anther and filament, whereas the other two isoforms were present in all flower organs.     

Identification and immunolocalization of superoxide dismutase isoenzymes of olive pollen

Gametophytic tissues of plants are an area largely neglected in the broad literature on free radical processes in plants. In order to study the mechanisms of protection against oxidative stress in pollen, the presence of the key antioxidative enzyme superoxide dismutase (SOD; EC 1.15.1.1) was investigated. Crude extracts of olive tree ( Olea europaea L.) pollen were subjected to native PAGE in 10% polyacrylamide gels. The SOD activity staining of gels showed the presence of four isoenzymes. All the SODS were completely inhibited by 2 m M KCN and 5 m M H₂O₂, and therefore belong to the family of CuZn‐SODS. Isoelectric focusing (pH 3.5‐7) of crude extracts and further detection of SOD activity allowed determination of isoelectric points for the four isoforms, namely 4.60, 4.78, 5.08 and 5.22. The cross‐reactivity of pollen extracts with a polyclonal antibody to cytosolic CuZn‐SOD from spinach leaves was assayed by western blotting. After SDS‐PAGE and immunoblotting, a major polypeptide band of about 16.5 kDa was detected, which is characteristic of the subunit of most CuZn‐SODS. Immunocytochemical studies at TEM level using the same antiserum showed that CuZn‐SOD was localized in the cytoplasm of both vegetative and generative cells, and also in material adhered to the pollen wall. The olive pollen CuZn‐SODS could function in the protection against oxidative stress during pollen development.     

Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) is one of the major enzymes involved in starch biosynthesis in higher plants. We report here the molecular cloning of two cDNAs encoding so far uncharacterized isoforms (AGP S2 and AGP S3) of the potato enzyme. Sequence analysis shows that the two polypeptides are more homologous to previously identified large subunit polypeptides from potato and other plant species than to small subunit isoforms. This observation suggests that AGP S2 and AGP S3 represent novel large subunit polypeptides. agpS2 is expressed in several tissues of the potato plant, including leaves and tubers. Expression was stronger in sink leaves than in source leaves, indicating developmental regulation. In leaves, agpS2 expression was induced 2- to 3-fold by exogenous sucrose; therefore, agpS2 represents a new sucrose-responsive gene of starch metabolism. Expression of agpS3 was restricted to tubers: no agpS3 expression could be seen in leaves of different developmental stages, or when leaves were incubated in sucrose. Therefore, agpS3 represents the only AGPase gene so far characterized from potato, which is not expressed in leaves. Conversely, all four AGPase isoforms known from potato are expressed in tubers.     

Induction of phenylpropanoid metabolism by Pseudomonas fluorescens against tomato spotted wilt virus in tomato

Pseudomonas fluorescens (two native strains, one collection strain and their strain mixtures in all possible combinations) when applied through seed, seedling dip, soil and on leaf significantly reduced the tomato spotted wilt virus (TSWV) disease. InP. fluorescens-treated plants, the peroxidase and phenylalamine ammonia-lyase activity increased. Accumulation of phenolic compounds and lignin were shown to be increased in theP. fluorescens-treated plants. Isoperoxidase native PAGE indicated that the peroxidase isoforms in tomato plants induced by fluorescent pseudomonads were different from the control plants; this suggests that the general phenylpropanoid pathway is probably stimulated in tomato plants treated which in turn led to significant reduction in TSWV.     

Purification and characterization of strongly chitin‐binding chitinases from salicylic acid‐treated leek (Allium porrum)

Six chitinases (EC 3.2.1.14) were purified from salicylate‐treated leek ( Allium porrum L.). They all strongly bind to chitin and can roughly be divided into two groups. One group has blocked N‐termini, is completely inhibited by 1 m M AgNO₃, has a relatively narrow pH optimum, a temperature optimum of 40°C and cannot degrade the tetramer of chitin. The other group has unblocked N‐termini showing homology to the chitin‐binding lectin WGA and is therefore considered as class I chitinases. This group is only moderately inhibited by 1 m M AgNO₃ (30%), has a relatively broad pH optimum, has a higher temperature optimum (50 to 60°C) and can degrade the tetramer of chitin to dimers. Furthermore, all isoforms have molecular masses around 34 kDa as estimated by SDS‐PAGE. They have isoelectric points ranging from 4 to 8 and no detectable lysozyme activity. Two isoforms investigated in more detail differ in their antifungal potential.     

Mapping of the ADP-glucose pyrophosphorylase genes in barley

cDNA probes encoding the barley endosperm ADP-glucose pyrophosphorylase (AGP) small subunit (bepsF2), large subunit (bepl10), and leaf AGP large subunit (blpl) were hybridized with barley genomic DNA blots to determine copy number and polymorphism. Probes showing polymorphism were mapped on a barley RFLP map. Probes that were not polymorphic were assigned to chromosome arms using wheat-barley telosomic addition lines. The data suggested the presence of a single-copy gene corresponding to each of the cDNA probes. In addition to the major bands, several weaker cross-hybridizing bands indicated the presence of other, related sequences. The weaker bands were specific to each probe and were not due to cross-hybridization with the other probes examined here. The endosperm AGP small subunit (bepsF2) majorband locus was associated with chromosome 1P and designated Aga1. The endosperm AGP large subunit (bepl10) major-band locus was mapped to chromosome 5M and designated Aga7. The endosperm AGP large-subunit minor bands were not mapped. The leaf AGP large-subunit major band was associated with chromosome 7M and designated Aga5. One of the leaf AGP large-subunit minor bands was mapped to chromosome 5P and designated Aga6. A clone for the wheat endosperm AGP large-subunit (pAga7) hybridized to the same barley genomic DNA bands as the corresponding barley probe indicating a high degree of identity between the two probes.     

ADP-Glucose Pyrophosphorylase Is Localized to Both the Cytoplasm and Plastids in Developing Pericarp of Tomato Fruit

The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.     

Virus induced gene silencing of three putative prolyl 4-hydroxylases enhances plant growth in tomato (Solanum lycopersicum)

Proline hydroxylation is a major posttranslational modification of hydroxyproline-rich glycoproteins (HRGPs) that is catalyzed by prolyl 4-hydroxylases (P4Hs). HRGPs such as arabinogalactan proteins (AGPs) and extensios play significant roles on cell wall structure and function and their implication in cell division and expansion has been reported. We used tobacco rattle virus (TRV)-based virus induced gene silencing to investigate the role of three tomato P4Hs, out of ten present in the tomato genome, in growth and development. Eight-days old tomato seedlings were infected with the appropriate TRV vectors and plants were allowed to grow under standard conditions for 6 weeks. Lower P4H mRNA levels were associated with lower hydroxyproline content in root and shoot tissues indicating successful gene silencing. P4H-silenced plants had longer roots and shoots and larger leaves. The increased leaf area can be attributed to increased cell division as indicated by the higher leaf epidermal cell number in SlP4H1- and SlP4H9-silenced plants. In contrast, SlP4H7-silenced plants had larger leaves due to enhanced cell expansion. Western blot analysis revealed that silencing of SlP4H7 and SlP4H9 was associated with reduced levels of JIM8-bound AGP and JIM11-bound extensin epitopes, while silencing of SlP4H1 reduced only the levels of AGP proteins. Collectively these results show that P4Hs have significant and distinct roles in cell division and expansion of tomato leaves.     

Ceruloplasmin fragmentation is implicated in 'free' copper deregulation of Alzheimer disease

A dysfunction in copper homeostasis seems to occur in Alzheimer’s disease (AD). We recently demonstrated that an excess of non-ceruloplasmin-copper (i.e. ‘free’ copper) correlates with the main functional and anatomical deficits as well as the cerebrospinal markers of the disease, thus suggesting that copper contributes to AD neurodegeneration. Aim of this study was to investigate the profile of serum ceruloplasmin isoforms immunoreactive protein in relation to copper dysfunction in AD. Twenty-five AD patients and 25 controls were included in the study. All subjects underwent individual measurements of serum ceruloplasmin and copper concentrations, and the amount of ‘free’ copper was computed for each copper and ceruloplasmin pair. Serum samples were also pooled and analyzed by two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and western blot analysis. The mean concentration of ’free’ copper resulted higher in AD patients than in controls. Ceruloplasmin 2-D PAGE western blot analysis of pooled sera showed in the AD samples low-molecular-weight spots in the <50 kDa range that were not detected in controls’ pooled sera (p < 0.029). Our data indicate a ceruloplasmin fragmentation in the serum of AD patients, possibly related to ‘free’ copper deregulation in this disease.     

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.     

Analysis of the Functional Interaction of Arabidopsis Starch Synthase and Branching Enzyme Isoforms Reveals that the Cooperative Action of SSI and BEs Results in Glucans with Polymodal Chain Length Distribution Similar to Amylopectin

Starch synthase (SS) and branching enzyme (BE) establish the two glycosidic linkages existing in starch. Both enzymes exist as several isoforms. Enzymes derived from several species were studied extensively both in vivo and in vitro over the last years, however, analyses of a functional interaction of SS and BE isoforms are missing so far. Here, we present data from in vitro studies including both interaction of leaf derived and heterologously expressed SS and BE isoforms. We found that SSI activity in native PAGE without addition of glucans was dependent on at least one of the two BE isoforms active in Arabidopsis leaves. This interaction is most likely not based on a physical association of the enzymes, as demonstrated by immunodetection and native PAGE mobility analysis of SSI, BE2, and BE3. The glucans formed by the action of SSI/BEs were analysed using leaf protein extracts from wild type and be single mutants (Atbe2 and Atbe3 mutant lines) and by different combinations of recombinant proteins. Chain length distribution (CLD) patterns of the formed glucans were irrespective of SSI and BE isoforms origin and still independent of assay conditions. Furthermore, we show that all SS isoforms (SSI-SSIV) were able to interact with BEs and form branched glucans. However, only SSI/BEs generated a polymodal distribution of glucans which was similar to CLD pattern detected in amylopectin of Arabidopsis leaf starch. We discuss the impact of the SSI/BEs interplay for the CLD pattern of amylopectin.     

Isolation, characterization and significance of papaya β‐galactanases to cell wall modification and fruit softening during ripening

β‐Galactosidases (EC 3.2.1.23) from ripe papaya ( Carica papaya L. cv. Eksotika) fruits having galactanase activities were fractionated by a combination of cation exchange and gel‐filtration chromatography into three isoforms, viz., β‐galactosidase I, II and III. The native proteins of the respective isoforms have apparent molecular masses of 67, 67 and 55 kDa, each showing one predominant polypeptide upon SDS‐PAGE of about 31 and 33 kDa for β‐galactosidases I and III, respectively, and of 67 kDa for β‐galactosidase II. The β‐galactosidase I protein, which was undetectable in immature fruits, appeared to be specifically accumulated during ripening. The β‐galactosidase II protein was present in developing fruits, but its level seemed to decrease with ripening. β‐Galactosidase I seemed to be an important softening enzyme; its activity increased dramatically (4‐ to 8‐fold) to a peak early during ripening and correlated closely with differential softening as related to position in the fruit tissue. The inner mesocarp tissue was softer, and its wall pectins were modified earlier and firmness decreased more rapidly during ripening compared to the outer mesocarp tissue. β‐Galactosidase II also may contribute significantly to softening because of its ability to catalyse increased solubility and depolymerization of pectins as well as through its ability to modify the alkali‐soluble hemicellulose fraction of the cell wall. The physiological significance of both β‐galactosidase isoforms may partly be attributed to their functional capacity as β‐(1,4)‐galactanases.     

Molecular characterization of four chitinase cDNAs obtained fromCladosporium fulvum-infected tomato

Complementary DNA clones encoding acidic and basic isoforms of tomato chitinases were isolated fromCladosporium fulvum-infected leaves. The clones were sequenced and found to encode the 30 kDa basic intracellular and the 26 and 27 kDa acidic extracellular tomato chitinases previously purified (M.H.A.J. Joostenet al., in preparation). A fourth truncated cDNA which appears to encode an extracellular chitinase with 82% amino acid similarity to the 30 kDa intracellular chitinase was also isolated. Characterization of the clones revealed that the 30 kDa basic intracellular protein is a class I chitinase and that the 26 and 27 kDa acidic extracellular proteins which have 85% peptide sequence similarity are class II chitinases. The characterized cDNA clones represent four from a family of at least six tomato chitinases. Southern blot analysis indicated that, with the exception of the 30 kDa basic intracellular chitinase, the tomato chitinases are encoded by one or two genes. Northern blot analysis showed that the mRNA encoding the 26 kDa acidic extracellular chitinase is induced more rapidly during an incompatibleC. fulvum-tomato interaction than during a compatible interaction. This difference in timing of mRNA induction was not observed for the 30 kDa basic intracellular chitinase.