Investigation of the Presence of Phenolic Compounds in Monocotyledonous Cell Walls, using UV Fluorescence Microscopy

Harris and Hartley (1976, 1980) demonstrated the presence offerulic acid in cell walls of certain monocotyledons using UVfluorescence microscopy (fluorescing green after treatment withammonium hydroxide solution). The presence or absence of thistype of fluorescence is apparently critical in higher levelsystematics of monocotyledons. In order to evaluate the significanceof this character, cell wall fluorescence was investigated ina range of monocotyledon species, particularly the AustralianXanthorrhoeaceae sensu lato (Bedford et al., 1986), which werenot investigated in earlier studies. This family is widely regardedas polyphyletic and was divided into several families by Dahlgren,Clifford and Yeo (1985). Some of its constituent genera, suchas Dasypogon, Kingia and Calectasia, have been linked with bothcommelinoid and non-commelinoid monocotyledons, and are of obscureaffinity. Some genera of Xanthorrhoeaceae sensu lato (Baxteria,Calectasia, Dasypogon and Kingia) show this type of green cellwall fluorescence and may therefore be more closely linked withthe commelinoid monocotyledons, rather than the Lilianae-Asparagales,as previously placed (Dahlgren et al., 1985).Copyright 1994,1999 Academic Press Asparagales, Dasypogonaceae, fluorescence, Hanguana, monocotyledons, systematics, Xanthorrhoeaceae     

Purification and characterization of mannose/glucose-specific lectin from seeds of <Emphasis Type="Italic">Trigonella foenumgraecum</Emphasis>

A lectin present in seeds of Trigonella foenumgraecum was isolated and purified by acid precipitation, salt fractionation, and affinity chromatography on mannan cross-linked agarose. SDS-PAGE revealed a single band corresponding to a molecular weight of 27,350 daltons. The lectin agglutinated trypsin-treated rat erythrocytes. Sugar specificity as determined by hemagglutination inhibition assay indicated that the lectin belongs to a glucose/mannose-specific group. The reaction of the lectin with glycoprotein was affected by pH changes. The carbohydrate binding specificity of the lectin was investigated by turbidity and activity measurements. As the lectin belongs to the Leguminoceae family, the specificity of the lectin for glucose/mannose renders it a valuable tool for Rhizobium-legume symbiosis. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 1, pp. 52–57.     

Isolation of a mannose-specific lectin from Escherichia coli and its role in the adherence of the bacteria to epithelial cells.

A high molecular weight protein aggregate, which agglutinates yeast cells, human epithelial cells and mouse lymphocytes, was isolated from extracts of Escherichia coli by differential centrifugation and gel filtration. The agglutination is specifically inhibited by d-mannose and its derivatives, the best inhibitor being p-nitrophenyl α-d-mannoside. Sodium dodecyl sulfate gel electrophoresis showed that the lectin consists of protein subunits with identical M_r of ～36500. The amino acid composition of the purified lectin is different from that reported for the type I pili protein, the K99 antigen and the major outer membrane protein Ia of E. coli. The protein appears to be located on the bacterial surface, and is probably involved in the mannose-specific adherence of E. coli to eukaryotic cells.     

Isolation and Characterization of Mitochondrial Nucleoids from the Yeast Pichia jadinii

Mitochondrial (mt) nucleoids were isolated with a high degreeof purity from the yeast Pichia jadinii, in which the mitochondrialDNA (mtDNA) is linear. Field-inversion gel electrophoresis (FIGE)revealed that significant amounts of mtDNA could be isolatedintact, as linear molecules of 41 kbp, from the isolated mt-nucleoids.Fifteen different proteins were detected in the mt-nucleoidfraction and, eight of these proteins bound to DNA. The patternsof mt-nucleoid proteins and of the DNA-binding proteins aftergel electrophoresis in the presence of SDS were somewhat differentfrom those of such proteins from Saccharomyces cerevisiae. Thecorresponding proteins isolated from the mt-nucleoids of fourother species of yeast in the genera Pichia and Williopsis alsodiffered from one another in terms of electrophoretic mobilityin the presence of SDS. In immunoblotting experiments, antibodiesthat had been raised against the 67-kDa protein of mt-nucleoidsfrom S. cerevisiae and the YMN-1 monoclonal antibody that isspecific for a 48-kDa protein in the mt-nucleoids from S. cerevisiaedid not recognize any proteins in the mt-nucleoids from Pichiajadinii and four other species of yeast. The results suggestthe considerable diversity of the proteins in the mt-nucleoidsof yeasts. (Received March 28, 1996; Accepted June 19, 1996)     

Purification and Partial Characterization of a Lectin from the Bark of Japanese Elderberry (Sambucus sieboldiana)

A lectin [Sambucus sieboldiana agglutinin (SSA)] was purifiedfrom the twigs of Sambucus sieboldiana by repeated affinitychromatography on fetuin-Sepharose. SSA had a molecular weight(Mr) of approximately 160 K on gel filtration and consistedof two types of subunit of which the molecular weights rangedfrom 31 to 37 K. SSA agglutinated human erythrocytes irrespectiveof their blood type and the hemagglutination was inhibited bya very low concentration of Neu5Ac(2-6)lactose, suggesting thatSSA has a carbohydrate-binding specificity similar to that ofthe lectin previously isolated from the bark of S. nigra (SNA).However, the Ouchterlony double-diffusion analysis of theselectins with an antibody raised against SSA showed that SSAwas immunologjcally related but not identical to SNA. (Received January 17, 1989; Accepted June 19, 1989)     

The Level of mRNA Transcribed from psaL, Which Encodes a Subunit of Photosystem I, Is Increased by Cytokinin in Darkness in Etiolated Cotyledons of Cucumber

A cDNA clone for an mRNA whose level increased within 2 h ofthe start of treatment with N⁶-benzyladenine in etiolated cotyledonsof cucumber was isolated by differential hybridization. ThecDNA was homologous to psaL, which encodes subunit XI (PSI-L)of photosystem I. The accumulation of psaL mRNA was specificallyinduced by cytokinins or light. (Received April 10, 1996; Accepted July 31, 1996)     

The Bark Lectin of Robinia pseudoacacia: Purification and Partial Characterization

A lectin was purified from the bark of Robinia pseudoacaciaby sequential ion-exchange chromatography on DEAE-Sepharoseand CM-Toyopearl. The purified lectin was estimated to havea molecular weight of 106 kDa and to be a homotetramer of subunitswith a molecular weight of 29 kDa. Antibodies raised againstthe bark lectin cross-reacted with a 29-kDa polypeptide duringWestern blot analysis, showing that the antibodies are specificfor the bark lectin. The antibodies against the lectin fromRobinia bark cross-reacted with polypeptides in extracts ofthe seeds and bark of Sophora japonica, indicating that thelectin from Robinia bark is immunologically related to the lectinsof Sophora. However, the antibodies did not cross-react withproteins from Robinia seeds and leaves. The first twenty aminoacid residues from the N-terminus of the lectin from Robiniabark were determined and compared with those of the Sophoralectins. (Received July 13, 1991; Accepted December 12, 1991)     

Evidence for a sugar receptor (lectin) in the peritrophic membrane of the blowfly larva, Calliphora erythrocephala Mg. (Diptera)

For the first time a sugar receptor (lectin) has been localized by electron microscopy in an invertebrate. The peritrophic membrane of the blowfly larva, Calliphora erythrocephala, is shown here to express lectins with high specificity for mannose. The lectin is restricted to the lumen side of the peritrophic membrane. The surface of the midgut epithelium is devoid of mannose-specific lectins. It is suggested that the midgut epithelium has lost these lectins during the course of evolution in favour of the peritrophic membrane which is secreted by specialized cells only at the beginning of the midgut.Peritrophic membranes and the midgut epithelium lack lectins specific for galactose. The lumen side of the peritrophic membrane of the larvae has mannose and/or glucose residues, and it is densely packed with two species of bacteria, Proteus vulgaris and P. morganii. These also have mannose-specific lectins as well as mannose residues on their pili. The existence of mannose-specific receptors and mannose residues on both, peritrophic membranes and bacteria, leads to the assumption of mutual adherence between the two surfaces.     

Isolation, Properties and a Possible Function of a Water-Soluble Chlorophyll a/b-Protein from Brussels Sprouts

A water-soluble Chl a/b-protein (CP673) was isolated and purifiedfrom Brussels sprouts (Brassica oleracea L. var. gemmifera DC).The protein had a molecular mass of 78 kDa and an isoelectricpoint of 4.7, consisted of three or four subunits of 22 kDaand was extremely heat-stable. Although CP673 contained aboutone Chl a per protein, the blue and red absorption bands ofChl a that consisted of three or four Chl a forms with differentabsorption maxima suggested that there are several differentmodes or sites of binding for Chl a. Chl a/b ratio of largerthan 10 also indicated that Chl b is present only in a smallfraction of CP673. The heterogeneity of CP673 in terms of compositionand binding of Chl suggests that Chl is not an intrinsic componentof the Chl-protein. Homology search showed that the N-terminalamino acid sequence of CP673 is highly homologous with thatof a 22 kDa protein that accumulates in water-stressed leavesof two Brassicaceae plants, rapeseed and radish, but not withthose of the light-harvesting Chl a/b-proteins of photosynthesis.A possible function of the water-soluble Chl-protein was discussed. (Received September 17, 1996; Accepted November 18, 1996)     

Biomechanical Responses of Chamaedorea and Spathiphyllum Petioles to Tissue Dehydration

Data are presented for the mechanical responses to dehydrationof petioles from two monocotyledons (Chamaedorea erumpens andSpathiphyllum Clevelandii). These data were used to test thehypothesis that the mechanical properties (elastic modulus Eand flexural stiffness EI) of petioles from C. erumpens arealtered significantly less by dehydration than those of petiolesfrom Spathiphyllum. Dehydration, resulting from either dryingat room temperature or from submergence in various concentrationsof mannitol solutions, produced significant increases in E anddecreases in EI (due to geometric distortions) in both youngand mature Spathiphyllum petioles. Similar trends were observedfor young petioles of C. erumpens, but significantly less sofor mature petioles of this species. Regardless of petiolarage, E increased allometrically as a linear function of tissuedensity, which in turn correlated with the volume fraction oflignified tissues in petioles; however, the proportional increaseof E as a function of tissue density was significantly greaterfor C. erumpens petioles than for Spathiphyllum. Anatomicalanalyses of petiolar transections indicated that Chamaedoreapetioles had larger volume fractions of lignified tissues thanthose of Spathiphyllum and that these tissues were located tomaximize stiffness. These data (and previously reported allometricrelationships between EI and petiolar length) shed light onthe difficulties in evaluating the ‘costs’ of committingtissues to mechanical support. Petioles, biomechanics, leaf anatomy, monocotyledons     

Mannose-specific isolectins with different hemagglutinating potencies isolated from Chinese daffodil (Narcissus tazetta var. chinensis) leaves

Three mannose-specific lectins exhibiting considerable similarities in NH₂-terminal amino acid sequence were isolated from leaves of the Chinese daffodil Narcissus tazetta (Family Amaryllidaceae). The purification protocol involved extraction with an aqueous buffer, anion exchange chromatography on DEAE-cellulose using stepwise elution with increasing salt concentrations, affinity chromatography on mannose-agarose, and FPLC-gel filtration on Superose 12. From the peak unadsorbed on DEAE-cellulose, and two peaks adsorbed on the ion exchanger and eluted respectively with 0.2 M Tris-HCl buffer and 0.5 M NaCl, were prepared fractions which yielded isolectins 1, 2, and 3 after adsorption on mannose-agarose and FPLC-gel filtraton. All three isolectins were homodimers with a molecular weight of 26 kDa. The lectin unadsorbed on DEAE-cellulose had the lowest, while the most strongly adsorbed lectin had the highest hemagglutinating activity.     

Purification of Cajanus cajan root lectin and its interaction with rhizobial lipopolysaccharide as studied by different spectroscopic techniques.

A lectin present in roots of Cajanus cajan seedlings was isolated and purified by affinity chromatography. Sugar specificity assayed by hemagglutination-inhibition activity indicated that lectin belongs to glucose/mannose-specific group. The root lectin was found to be mannose-specific from the second day onwards as it was reconfirmed by specific elution of different days' sample from mannose agarose matrix. The maximum interaction of lectin with goat IgM was obtained in 10-day-old sample, indicating the highest crude lectin content. Lectin (total amount of eluted protein) from different days soil sample showed a maximum amount in 10-day-old sample. For further studies, the lectin has been isolated from the roots of 10-day C. cajan seedlings and purified on mannose-CL agarose column by affinity chromatography. Lectin was found to be a dimer of 18.5-kDa subunit as revealed by SDS-PAGE. Tryptophan quenching fluorescence was studied for C. cajan root lectin. Secondary structure of C. cajan root lectin as studied by circular dichroism was found to be a typical beta-pleated sheet structure. The interaction of purified root lectin with C. cajan-specific rhizobial lipopolysaccharide and its inhibition by specific and nonspecific sugars was demonstrated by fluorescence and circular dichroism. Results discussed in this paper were studied for the first time by different spectroscopic methods, suggesting that C. cajan root lectin-lipopolysaccharide interaction is specific.     

Novel mannose-specific lectins found in torafugu, Takifugu rubripes: A review

In earlier work, we identified a novel mannose-specific lectin, termed pufflectin-s, from the skin mucus of torafugu, Takifugu rubripes. We here make a brief review on the lectin. The amino acid sequence of pufflectin-s shares sequence homology with mannose-binding lectins of monocotyledonous plants, and has conserved two of three carbohydrate recognition domains, QDNVY motifs, of these plant lectins. By site-directed mutagenesis, we verified that the QDNVY motif in the N-terminal region was critical to the mannose-binding function of pufflectin-s. RT-PCR and Northern blot analyses indicated that the pufflectin-s gene is expressed in the gill, oral cavity wall, esophagus, and skin. In addition, an isoform, pufflectin-i, which shares 91.4% amino acid identity with pufflectin-s, was isolated from the intestine. Using immunohistochemistry, pufflectin-s could be detected exclusively in the epithelial cells of the skin, gill, oral cavity wall and esophagus, whereas pufflectin-i was observed in both mucous and epithelial cells in the intestine. Nevertheless, mRNAs for both pufflectins were detected only in epithelial cells of these tissues with in situ hybridization. Pufflectin-s agglutinated some bacteria isolated from rearing water and from fish skin. This lectin also bound to a parasite, Heterobothrium okamotoi, suggesting that it may play an important role in the self-defense system of fugu.     

Purification of Lectin from Micropropagated Roots Derived from Aseptic Seedling of <Emphasis Type="Italic">Canavalia ensiformis</Emphasis> L.

A well-organized protocol has been developed for high frequency root germination from the seed of Canavalia ensiformis on Murashige and Skoog (MS) medium. Surprisingly, the seeds that were grown on the MS medium having no growth hormone showed the best response. Roots of 30 days old aseptic seedling were homogenized and a lectin from them was purified on Sephadex G-50 affinity column. The finding that final product is a pure lectin was confirmed by specific hemagglutinating property. The final root lectin yield was 0.6% and eluted as a single peak. Root lectin specific activity was 50 times more than the seed lectin. Sugar specificity activity by hemagglutination-inhibition assay indicated that lectin belongs to glucose/mannose-specific group. Interestingly, the lectin was found to be 25 kDa, similar to molecular mass of Concanavalin A purified from seed of C. ensiformis, as revealed by SDS–PAGE. Thus, Concanavalin A from either source can be used for development of transgenic crops that are capable of expressing lectin gene and hence can efficiently perform biological nitrogen fixation by giving rise to nodules in their root. The advantage of this method is that purification of Concanavalin A in tissue culture conditions is easier, handy and is less time consuming.     

Cloning and expression of a Xenopus laevis oocyte lectin and characterization of its mRNA levels during early development

cDNA clones encoding a soluble, calcium-dependent, melibiose-bindinglectin from Xenopus laevis oacytes have been isolated, characterized,and expressed in bacteria This lectin has been shown by othersto be localized in oocyte cortical granules where it ultimatelyis released and participates in the formation of the fertilizationenvelope. A lectin with similar specificity has been purifiedby others from blastula and immunolocalized to specific locationsin developing embryos, which suggests it may also function afterfertilization in regulating cell adhesion and migration. Wehave used melibiose affinity chromatography to isolate the oocytelectin (monomer molecular masses of about 45 and 43 kDa) andshown that after exhaustive treatment with N-glycanase, onlyone major protein band at 35 kDa was observed, suggesting thata single polypeptide with variable N-Linked glycosylation isexpressed in the oocyte. After obtaining internal peptide sequences,a PCR-based cloning approach allowed the isolation of full lengthcDNAs from an ovary     

Molecular Cloning and Characterization of a cDNA Clone That Encodes a Cdc2 Homolog from Nicotiana tabacum

We have isolated a cDNA clone (cdc2Nt1) that encodes a homologof p34^cdc2/CDC28 kinase from tobacco (Nicotiana tabacum). Thecdc2Ntl protein showed extensive similarity to other homologsof Cdc2 from plants. Complementation studies showed that thecdc2Ntl gene was able to overcome cell cycle arrest at boththe G1/S and the G2/M transitions of cdc28^ts mutants of buddingyeast, demonstrating that the cdc2Ntl protein was able to replacethe Cdc28 kinase at both the G1/S and the G2/M transitions.Analysis of gene expression demonstrated that the cdc2Ntl genewas transcribed constitutively throughout the cell cycle butthat it was preferentially expressed in actively dividing tobaccoBY-2 cells. (Received July 13, 1995; Accepted February 15, 1996)     

Isolation and characterization of a lectin gene from seeds of chickpea (Cicer arietinum L.).

A cDNA library was constructed in lambda TriplEx2 vector using poly (A(+)) RNA from immature seeds of Cicer arietinum. The lectin gene was isolated from seeds of chickpea through library screening and RACE-PCR. The full-length cDNA of Chichpea seed lectin(CpGL)is 972 bp and contains a 807 bp open reading frame encoding a 268 amino acid protein. Analysis shows that CpSL gene has strong homology with other legume lectin genes. Phylogenetic analysis showed the existence of two main clusters and clearly indicated that CpSL belonged to mannose-specific family of lectins. RT-PCR revealed that CAA gene expressed constitutively in various plant tissues including flower, leaf, root and stem. When chickpea lectin mRNA level was checked in developing seeds, it was higher in 10 DAF seeds and decreased throughout seed development.     

Occurrence of Form PIIcf Sieve-element Plastids in Xyris Species (Xyridaceae)

Mature leaf fragments from eight species ofXyris from the Serrado Cipó, State of Minas Gerais, Brazil, were preparedby the usual methods for electron microscopy. Ultrastructuralanalysis of phloem shows the occurrence of nacreous walls witha polylamellate structure in the sieve-elements ofX. tortilisand plastids similar to P-plastids, form PIIcf in the sieve-elementsof all investigated species. Sieve-element plastids; monocotyledons; Xyris