Tissue browning of in vitro cultures of Scots pine: Role of peroxidase and polyphenol oxidase

Callus cultures from shoot tips of mature Scots pine ( Pinus sylvestris L.) were characterized by rapid browning and an inability to regenerate. The peroxidase (POD) and polyphenol oxidase (PPO) activities and relationship to browning in such cultures were compared with embryogenic and non-embryogenic cultures of Scots pine, started from immature embryos of three different pine clones. The browning in callus cultures derived from pine buds was visible approximately after 2 weeks of culture, and continued thereafter until the callus was dark brown and poorly growing. The non-embryogenic cultures induced from immature embryos showed either light yellow coloring or browning, whereas the embryogenic cultures showed browning. POD activity increased during the first 4 weeks in callus tissue initiated from pine buds, and was significantly higher than in pine buds or cultures derived from immature embryos. The ability of cultures initiated from pine buds to oxidize catechol was notably high compared with cultures initiated from immature embryos, regardless of the time of measurement. Addition of catalase revealed that both POD and PPO were able to use catechol as substrate. An antibody raised against broad bean ( Vicia faba ) chloroplast PPO was used to recognize PPO. One polypeptide with a molecular mass of 50 kDa was detected in all pine samples on SDS-PAGE and non-denaturing PAGE. Another polypeptide with a molecular mass of 70 kDa was shown exclusively in the light-yellow non-embryogenic cultures. The results suggest that especially the high POD activities in callus tissues started from mature trees cause rapid and early browning and possibly subsequent cell death.     

Compartmentation of solutes and water in developing sugarcane stalk tissue

Previous studies have suggested that the apoplast solution of sugarcane stalk tissue contains high concentrations of sucrose, but the accuracy of these reports has been questioned because sucrose leakage from damaged cells may have influenced the results. In this study, the solute potential of the apoplast and symplast of the second (immature), tenth, twentieth, thirtieth, and fortieth internodes of field-grown sugarcane (Saccharum spp. hybrid) stalk tissue was determined by two independent methods. Solute potential of the apoplast was measured either directly by osmometry from solution collected by centrifugation, or inferred from the initial water potential of fully hydrated tissue determined by thermocouple psychrometry before the tissue was progressively dehydrated for generation of water potential isotherms. Both methods produced nearly identical values ranging from −0.6 to −1.8 megapascals for immature and mature tissue, respectively. The solute potential of the symplast determined by either method ranged from −1.0 to approximately −2.2 megapascals for immature and mature internodes, respectively. Solute quantitation by HPLC agreed with concentrations inferred from osmometry. Washing thirtieth internode tissue in deionized water increased pressure potential from 0.29 to 1.96 megapascals. The apoplast of mature sugarcane stalk tissue is a significant storage compartment for sucrose containing as much as 25% of the total tissue water volume and as much as 21% of the stored sucrose.     

Cloning,characterization and expression of two new polyphenol oxidase cDNAs from <Emphasis Type="Italic">Agaricus bisporus</Emphasis>

Two new polyphenol oxidase (PPO) cDNAs (PPO3 and PPO4 cDNAs, accession numbers GQ354801 and GQ354802, respectively) were obtained by RACE-PCR from Agaricus bisporus. PPO3 cDNA was 1844 bp in length with an open reading frame of 1731 bp, while PPO4 cDNA was 2042 bp with an open reading frame of 1836 bp. PPO3 and PPO4 cDNAs, with 52% identity at the nucleic acid level, encoded a 576-amino acid protein of 66.3 kDa and 611-amino acid protein of 68.3 kDa, respectively. Mature forms of PPO3 and PPO4 were characterized after removing the specific C-terminal region and expressed in Escherichia coli BL21 (DE3) RIPL using pGEX-4T-1 vector. The expressed proteins were probed by the anti-A. bisporus PPO antibody but without PPO activity. This indicated that the recombinant mature PPO3 and mature PPO4 could not form an active center in prokaryotic expression system.     

Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem: a potential role for the ShSUT1 sucrose transporter

A transporter with homology to the SUT/SUC family of plant sucrose transporters was isolated from a sugarcane (Saccharum hybrid) stem cDNA library. The gene, designated ShSUT1, encodes a protein of 517 amino acids, including 12 predicted membrane-spanning domains and a large central cytoplasmic loop. ShSUT1 was demonstrated to be a functional sucrose transporter by expression in yeast. The estimated K_m for sucrose of the ShSUT1 transporter was 2 mM at pH 5.5. ShSUT1 was expressed predominantly in mature leaves of sugarcane that were exporting sucrose and in stem internodes that were actively accumulating sucrose. Immunolocalization with a ShSUT1-specific antiserum identified the protein in cells at the periphery of the vascular bundles in the stem. These cells became lignified and suberized as stem development proceeded, forming a barrier to apoplasmic solute movement. However, the movement of the tracer dye, carboxyfluorescein from phloem to storage parenchyma cells suggested that symplasmic connections are present. ShSUT1 may have a role in partitioning of sucrose between the vascular tissue and sites of storage in the parenchyma cells of sugarcane stem internodes.     

Sugar transport: Occurrence of trehalase activity in sugar cane

Summary Trehalase activity was detected in extracts of roots, leaves, and stalk tissue from sugar cane. The enzyme was not bound to cell particulates, and had a pH optimum of 6.2 and a Michaelis constant for trehalose of 1×10^-4 M. The level of enzyme detected in mature stalk tissue was too low to account for glucose transport into tissue slices. The enzyme level was high in immature stalk tissue in which the vacuolar sugar pool turns over rapidly. Trehalose synthesis and breakdown may be part of a system for transport of hexose out from the vacuole.     

Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase

Three cDNA clones were isolated which code for the ubiquitous chloroplast enzyme, polyphenol oxidase (PPO), from Vicia faba. Analysis of the cloned DNA reveals that PPO is synthesized with an N-terminal extension of 92 amino acid residues, presumed to be a transit peptide. The mature protein is predicted to have a molecular mass of 58 kDa which is in close agreement to the molecular mass estimated for the in vivo protein upon SDS-PAGE. Differences in the DNA sequence of two full-length and one partial cDNA clones indicate that PPO is encoded by a gene family. Analysis of the deduced amino acid sequence shows that the chloroplast PPO shares homology with the 59 kDa PPOs in glandular trichomes of solanaceous species. A high degree of sequence conservation was found with the copper-binding domains of the 59 kDa tomato PPO as well as hemocyanins and tyrosinases from a wide diversity of taxa.     

Integrin-like protein at the invaginated plasma membrane of epidermal cells in mature leaves of the marine angiosperm<Emphasis Type="Italic"> Zostera marina</Emphasis> L.

By immunoblotting with anti-human integrin polyclonal antibodies (1, 3 or 5), a single distinct band of about 60 kDa was detected in total protein extracts from mature leaves of the seagrass Zostera marina L., but no band was detected in total protein extracts from immature seagrass leaves, freshwater grass leaves or Arabidopsis thaliana (L.) Heynh. leaves. This integrin-like protein was detected by indirect immunofluorescence microscopy on the surface of non-spherical protoplasts of epidermal cells isolated from mature seagrass leaves using an anti-integrin 3 polyclonal antibody. Electron-microscopic analyses with the same antibody indicated that this integrin-like protein was localized specifically in the invaginated plasma membrane of epidermal cells in mature seagrass leaves. Therefore, this integrin-like protein of about 60 kDa may be involved in the developmentally regulated invagination of the plasma membrane in epidermal cells of the seagrass Z. marina.     

Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity was successfully down-regulated in sugarcane using constitutively expressed antisense and untranslatable forms of the sugarcane PFP-β gene. In young internodal tissue activity was reduced by up to 70% while no residual activity could be detected in mature tissues. The transgenic plants showed no visible phenotype or significant differences in growth and development under greenhouse and field conditions. Sucrose concentrations were significantly increased in the immature internodes of the transgenic plants but not in the mature internodes. This contributed to an increase in the purity of the immature tissues, resembling an early ripening phenotype. Both the immature and mature internodes of the transgenic plants had significantly higher fibre contents. These findings suggest that PFP influences the ability of young, biosynthetically active sugarcane culm tissue to accumulate sucrose but that the equilibrium of the glycolytic intermediates, including the stored sucrose, is restored when ATP-dependent phosphofructokinase and the residual PFP activity is sufficient to sustain the required glycolytic flux as the tissue matures. Moreover, it suggests a role for PFP in glycolytic carbon flow, which could be rate limiting under conditions of high metabolic activity.     

Developmental Changes in the Anatomy of the Sugarcane Stem in Relation to Phloem Unloading and Sucrose Storage

Transverse sections of immature and mature sugarcane internodes were investigated anatomically with white and fluorescence light microscopy. The pattern of lignification and suberization was tested histo-chemically. Lignification began in the xylem of vascular bundles and progressed through the sclerenchymatic bundle sheath into the storage parenchyma. Suberization began in parenchyma cells adjacent to vascular bundle sheaths and spread to the storage parenchyma and outer sheath cells. In mature internodes most of the storage parenchyma was lignified and suberized to a significant degree, except in portions of walls of isolated cells. The pattern of increasing lignification and suberization in maturing internodes more or less paralleled an increase of sucrose in stem tissue. In mature internodes having a high sucrose concentration, the vascular tissue was surrounded by thick-walled, lignified and suberized sclerenchyma cells. The apoplastic tracer dyes triso-dium 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS) and amido black 10 B, fed into cut ends of the stalk, wereconfined to the vascular bundles in all internodes above the one that was cut — with no dye apparently in storage parenchyma tissue. Thus both structural and experimental evidence is consistent with vascular tissue being increasingly isolated from the storage parenchyma as maturation of the tissue proceeds. We conclude that in mature internodes the pathway for sugars from the phloem to the storage parenchyma is symplastic. The data suggest that an increasingly greater role for a symplastic pathway of sugar transfer occurs as the tissue undergoes lignification/suberization.     

The prophenoloxidase from the wax moth Galleria mellonella: purification and characterization of the proenzyme

A prophenoloxidase (PPO) was purified from the hemolymph of the larvae of Galleria mellonella. A 135-fold purification of the proenzyme with 25% yield was achieved by a combination of different chromatographic methods. An alternative micropreparation of pure PPO by a novel method for native electrophoresis in polyacrylamide gel is also described. The molecular mass of the native PPO was estimated to be 300 kDa by the pore-limit gradient electrophoresis in polyacrylamide gel. In the presence of sodium dodecyl sulphate, two closely migrating subunits of 80 and 83 kDa were detected under non-reducing conditions. The PPO was shown to be a glycoprotein and its isoelectric point was 6.2. The amino-acid composition of the purified protein was similar to the PPO from Bombyx mori. The monospecific antibody raised against the purified PPO crossreacted with the (pro)phenoloxidase in hemolymph of Manduca sexta. The activation of the PPO with chymotrypsin was investigated and two proteins of 67 and 50 kDa were found to be products of the proteolytic cleavage. The N-terminus of the G. mellonella PPO was blocked, but eleven partial internal sequences were determined after fragmentation of the purified PPO with trypsin. Three of these peptides exhibited significant homology with highly conserved sequences found in arthopod hemocyanins and insect storage proteins, which indicates that the PPO belongs to this family.     

Detection of Colletotrichum falcatum causing red rot of sugarcane by enzyme-linked immunosorbent assay

Red rot disease of sugarcane caused by Colletotrichum falcatum Went is one of the most destructive diseases of sugarcane (Saccharum officinarum L.) worldwide. The pathogen spreads primarily through infected sugarcane setts and hence the use of disease-free setts is essential to prevent the disease. In order to develop immunological method for detection of C. falcatum, two proteins with molecular weights of 27 kDa and 45 kDa were purified from the mycelium of C. falcatum race Cf 05 and used as antigen source to raise polyclonal antibodies in NewZealand white rabbit. The developed polyclonal antibodies were tested for detection of C. falcatum by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The polyclonal antibodies specifically detected C. falcatum in extracts from infected plants, both in immunoblot and ELISA. The ELISA results showed that the developed polyclonal antibodies were highly specific to C.falcatum. The developed antibodies were very sensitive and could detect C.falcatum proteins even at a dilution of 1:50,000. Higher ELISA absorbance values were recorded even at an antigen dilution of 1:500. In western blot analysis, protein bands with molecular weights of 27 kDa and 45 kDa reacting to antisera raised against 27 kDa and 45 kDa mycelial proteins of C. falcatum, respectively, were detected in protein samples from red rot infected canes. The high specific reactivity and sensitivity of the antisera indicate its potential suitability for ELISA-based detection of C. falcatum.     

Isolation and Characterization of Goldfish cdc2, a Catalytic Component of Maturation-Promoting Factor

We have isolated a cdc2 cDNA from a library constructed from immature goldfish oocytes. The isolated clone has a PSTAVR sequence, instead of the PSTAIR sequence common to cdc2 in other species. Its product was characterized by monoclonal antibodies against its C-terminal amino acid sequence. The antibodies recognized an anti-PSTAIR-reactive 35 kDa protein in immature oocyte extracts, which was not recognized by anti-goldfish cdk2 antibody. In addition to the 35 kDa cdc2, mature oocytes contained a 34 kDa cdc2, which was a component of MPF purified from carp eggs. Upon gel filtration column, the 35 kDa cdc2 migrated at monomeric position, while the 34 kDa cdc2 migrated at around 100 kDa, where cyclin B also comigrated. These results strongly suggest that the 35 kDa protein is monomeric inactive cdc2, while the 34 kDa protein is cyclin B-bound active cdc2. The finding that the 35 kDa inactive cdc2 does not form a complex with any other proteins in immature oocytes is in contrast to the situation in Xenopus and starfish, in which cdc2-cyclin B complex exists already as pre-MPF in immature oocytes.     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Purification and characterization of a motility initiating protein from caprine epididymal plasma

Numerous reports have appeared on the occurrence of undefined protein factors in male reproductive fluids that promote motility of mature sperm and initiate forward motility in the immature (immotile) caput‐epididymal sperm. This study reports for the first time purification to apparent homogeneity of a motility initiating protein (MIP) from epididymal plasma and its characterization using the caprine sperm model. It is a 125 kDa (approximately) dimeric protein made up of two subunits: 70 and 54 kDa. MIP is an acidic protein with an isoelectric point of 4.75. The motility protein at 30 µg/ml (240 nM) level showed nearly maximal motility‐promoting activity. MIP is heat stable and it is maximally active at pH 8. It is a glycoprotein that binds with high affinity to concanavalin A and it contains mannose, galactose, and N‐acetyl glucosamine approximately in the ratios of 6:1:6. It is sensitive to the actions of α‐mannosidase and β‐N‐acetylglucoseaminidase thereby demonstrating that the sugar side chain of the glycoprotein is essential for its biological activity. Epididymal plasma is its richest source. It is also capable of enhancing forward motility of mature cauda‐sperm. Its antibody markedly inhibits sperm motility. MIP antibody is highly immunospecific and it recognizes both the subunits. MIP causes significant increase of the intrasperm level of cyclic AMP. MIP: the physiological motility‐activating protein has potential for use as a contraceptive vaccine and for solving some of the problems of human infertility and animal breeding. J. Cell. Physiol. 222:254–263, 2010. © 2009 Wiley‐Liss, Inc.     

The production and characterization of a new monoclonal antibody to the trefoil peptide human spasmolytic polypeptide

Summary Human spasmolytic polypeptide (hSP) is a member of the growing family of trefoil peptides which are expressed in discrete regions of the body, most notably the gastrointestinal tract. Much of the research into the localization of the spasmolytic polypeptide has relied on hybridization in situ to detect its mRNA, due to the absence of a suitable antibody. The aim of the present study was to develop and characterize a monoclonal antibody against the human spasmolytic polypeptide, using a combination of immunohistochemistry and hybridization in situ.After immunoblotting, the antibody detected a 14 kDa protein in gastrointestinal tissue extracts from the stomach and small intestine only. Using immunohistochemistry, human spasmolytic polypeptide showed a distinctive staining pattern in the duodenum which co-localized with its mRNA. The co-localization of the immunoreactive peptide with its mRNA provides good evidence that the antibody truly recognized human spasmolytic polypeptide.     

Lateral movement of water and sugar across xylem in sugarcane stalks

Laterally connected vascular bundles in the nodes of sugarcane (Saccharum species cv. Pindar) stalks allow a rapid redistribution of water across the stalk should the vascular continuity be partly disrupted. Tritiated water supplied to the roots exchanged rapidly between the xylem and storage tissue so that net movement up the stalk was slow. The half-time for exchange in a labeled stalk was about 4 hours so that the entire water content of a sugarcane stalk can turn over at least once in a single day. No rapid flux of sugar between xylem and phloem or xylem and storage tissue was detected. Functional xylem contained only low sugar concentrations: less than 0.3% w/v in the stalk and less than 0.02% w/v in the leaf. Previous reports of high sugar levels (9% w/v) in sugarcane stalk xylem reflect some degree of xylem blockage followed by a slow equilibration with free space sugars in the storage tissue.