Structure of N-glycans on the S3- and S6-stylar self-incompatibility ribonucleases of Nicotiana alata

Self-incompatibility is a mechanism developed by many plantsto prevent inbreeding. The products of the selfincompatibility(S)-locus in the styles of solanaceous plants are a series ofglycoproteins with ribonuclease activity. In this study, wereport on the N-glycans from the stylar selfincompatibilityS₃- and S₆-ribonucleases of Nicotiana alata, which were enzymicallyreleased and fractionated by high-pH anion-exchange HPLC. Atotal of 14 N-glycans were identified and characterized by acombination of electrospray-ionization mass-spectrometry, ¹H-NMRspectroscopy, chemical degradation, and methylation analyses.This pattern of N-glycosylation is much more complex than thatpreviously found on the N.alata S₁- and S₂-RNases each of whichcontained only four N-glycans. N-glycan Nicotiana alata ribonuclease selfincompatibility     

Variations in and Inheritance of NS-Glycoprotein in Self-Incompatible Brassica campestris L.

The stigma of Brassica species contain NS-glycoproteins thatexhibit a high degree of structural homology to the S-glycoproteinsof self-incompatibility. Inheritance of and variations in theNS-glycoprotein were studied with reference to self-incompatibility.The detection of NS-glycoproteins was performed by cross-reactionwith an antiserum raised against a purified NS-glycoprotein.In B. campestris, four isoforms of the NS-glycoprotein weredifferentiated by their pI values, but their molecular weightswere identical to one another. The genes for these isoformsof NS-glycoprotein were controlled by alleles at a single locus,tentatively named the NS allele, which was independent of Salleles at both the protein and the DNA level. Segregation ofF₂ plants with respect to the self-incompatibility behaviorof pollen tubes can be explained by the S allele model, butit appears not to be affected by the NS alleles. NS-glycoproteinswere found in all 21 species of Brassica and its allies examinedto date. The pI values of these glycoproteins varied among differentspecies. In addition to the isoforms of the NS alleles, maturestigmas contained other groups of proteins that reacted weaklywith the antiserum against the NS-glycoprotein. (Received July 30, 1991; Accepted February 21, 1992)     

Identification of Stylar RNases Associated with Gametophytic Self-Incompatibility in Almond (Prunus dulcis)

Stylar proteins of 13 almond (Prunus dulcis) cultivars withknown S-genotypes were surveyed by IEF and 2D-PAGE combinedwith immunoblot and N-terminal amino acid sequence analysesto identify S-RNases associated with gametophytic self-incompatibility(SI) in this plant species. RNase activities corresponding toS_a and S_b, two of the four S-alleles tested, were identifiedby IEF and RNase activity staining. The S_a-RNase band reactedwith the anti-S₄serum prepared from Japanese pear (Pyrus serotina);no reaction with the antiserum was observed with the s_bRNaseband. When the s_a-RNase band was excised from an IEF gel stainedfor RNase activity, subjected to SDS-PAGE, and detected by immunoblotting,it appeared that this band consisted of a single protein thatreacted with the anti-s₄serum with M_r of about 28 kDa. With2D-PAGE and silver staining of the stylar extracts, all fourS-proteins could be successfully distinguished from each otherin the highly basic zone of the gel. Although S_b-, S_c-, andS_dproteins had roughly the same M_r of about 30 kDa, the S_c-proteinseemed to be slightly smaller than the S_b-protein and slightlylarger than the S_aprotein. In 2D-PAGE profiles as well, theS_a-protein had M_r of about 28 kDa, apparently smaller than theother three proteins. A bud sport, in which one of the two S-allelesof the original cultivar is impaired, was visualized as a lossof S_cprotein, which is consistent with the previous pollinationstudy. All four S-proteins reacted with the anti-S₄serum, probablybecause of the differing conformations of these S-proteins inthe IEF and 2D-PAGE gels. The S_a-protein in 2D-PAGE appearedto be identical to S_a-RNase in IEF; both bad the same M_r andwere reactive with the anti-S₄-serum. N-terminal amino acidsequence analysis of the four 5-proteins revealed that theywere highly homologous to each other and similar to the 5-RNasesof Malus, Pyrus, Scrophulariaceae, and Solanaceae. Taken together,RNases in the style are strongly suggested to be associatedwith the gametophytic SI of al- mond. This is the first reportidentofiying and characterizing S-RNase in almond. (Received July 11, 1996; Accepted December 26, 1996)     

Pollen-stigma Interaction in the Leguminosae: Constituents of the Stylar Fluid and Stigma Secretion of Trifolium pratense L.

The style of T. pratense is hollow, and the canal contains awatery secretion which forms the medium through which the pollentubes grow after penetrating the stigma head. In self-incompatiblegenotypes, incompatible pollen germinates freely and the tubespenetrate the stigma, but they are arrested in the canal afterpassing an inflated zone (entasis) proximal to the stigma head.The stylar fluid contains sucrose, glucose and traces of galactoseand arabinose, as well as a range of proteins. Comparison ofthe proteins in the stigma eluate and stylar fluid by microgradientgel electrophoresis shows that the spectra are broadly similar;but in addition to various minor differences, two major glycoproteinsare present in the stigma secretion which are absent from thestyle, while one in the stylar fluid is not represented in thestigma. Six esterase isoenzymes are present in the stylar fluid,and three of these also in the stigma eluate; there are alsodifferences in acid phosphatase isoenzymes. Leguminosae, Trifolium pratense L., pollen-stigma interaction, self-incompatibility, stigma eluate secretion, stylar secretion     

Microheterogeneity of N-glycosylation on a stylar self-incompatibility glycoprotein of Nicotiana alata

Gametophytic self-incompatibility, a mechanism that preventsinbreeding in some families of flowering plants, is mediatedby the products of a single genetic locus, the S-locus. Theproducts of the S-gene in the female sexual tissues of Nicotianaalata are an allelic series of glycoproteins with RNase activity.In this study, we report on the microheterogeneity of N-linkedglycosylation at the four potential N-glycosylation sites ofthe S²-glycoprotein. The S-glycoproteins from N.alata containfrom one to five potential N-glycosylation sites based on theconsensus sequence Asn-Xaa-Ser/Thr. The S²-glycoprotein containsfour potential N-glycosylation sites at Asn²⁷, Asn³⁷, Asn¹³⁸and Asn¹⁵⁰, designated sites I, n, IV and V, respectively. SiteIII is absent from the S₂-glycoprotein. Analysis of glycopeptidesgenerated from the S₂-glycoprotein by trypsin and chymotrypsindigestions revealed the types of glycans and the degree of microheterogeneitypresent at each site. Sites I (Asn²⁷) and IV (Asn¹³⁸) displaymicroheterogeneity, site II (Asn³⁷) contains only a single typeof N-glycan, and site V (Asn¹⁵⁰) is not glycosylated. The microheterogeneityobserved at site I on the S₂-glycoprotein is the same as thatobserved at the only site, site I, on the Srglycoprotein (Woodwardet al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylationconsensus sequence at site I is conserved in all S-glycoproteinsfrom other species of self-incompatible solanaceous plants,glycosylation at this site may be important to their function.No other post-translational modifications (e.g. O-glycosylation,phosphorylation) were detected on the S₂-glycoprotein. fertilization microheterogeneity N-glycans plants RNase     

The Anatomy, Histochemistry and Ultrastructure of Stigmas and Styles in Commelinaceae

The anatomy and ultrastructure of stigmas in 37 species of 13genera of Commelinaceae are described. The stigmas are papillate,papillae forming a dense fringe of cells around the mouth ofthe stylar canal in most species. The papillar cell wall iscovered by an unstructured cuticle of variable thickness andis of variable thickness because of small wall ingrowths. Thecuticle and the external surface of the papillar cell wall arevariably disrupted, particularly in the mid and basal regionsof the cell. This was not found in species of the genus Aploleiaor Callisia. The cell cytoplasm possesses all major organellesexcept chloroplasts and each cell is vacuolate. In all species except Aploleia mulitiflora the style comprisesan epidermis, a cortex and a hollow, tripartite canal whichis continuous into the ovary cavity. The three vascular strandsare positioned at the apex of each canal lobe. The canal cellsare elongate and tabular and the wall abutting the canal hasingrowths. The style in Aploleia is solid and the transmittingtissue comprises cells whose walls are electron opaque. Thecytoplasms of both types of cell are similar in content althoughthere is a single, large vacuole in canal cells and many smallvacuoles in transmitting tissue. The morphology, position and histochemistry of stigmatic andstylar exudate was similar in all ‘wet’ stigmas.Most of the exudate originates from the stylar canal althoughsignificant contributions are made by the papillae in stigmasof Coleotrype, Dichorisandra and Thyrsanthemum. There is no apparent relationship between stigma structure andthe presence of self-incompatibility. Stigma papillae, stylar canal, transmitting tissue, Commelinaceae     

Endotoxin-induced skeletal muscle contractile dysfunction: contribution of nitric oxide synthases

The aims of thisstudy were to assess the role of nitric oxide (NO) and the contributionof different NO synthase (NOS) isoforms in skeletal muscle contractiledysfunction in septic shock. Four groups of conscious rats wereexamined. Group 1 served as control; groups 2, 3, and4 were injected withEscherichia coli endotoxin [lipopolysaccharide (LPS), 20 mg/kg ip] and killed after 6, 12, and 24 h, respectively. Protein expression was assessed byimmunoblotting and immunostaining. LPS injection elicited a transientexpression of the inducible NOS isoform, which peaked 12 h after LPSinjection and disappeared within 24 h. This expression coincided with a significant increase in nitrotyrosine formation (peroxynitrite footprint). Muscle expression of the endothelial and neuronal NOSisoforms, by comparison, rose significantly and remained higher thancontrol levels 24 h after LPS injection. In vitro measurement of musclecontractility 24 h after LPS injection showed that incubation with NOSinhibitor (S-methyliosothiourea)restored the decline in submaximal force generation, whereas maximalmuscle force remained unaffected. We conclude that NO plays asignificant role in muscle contractile dysfunction in septic animalsand that increased NO production is due to induction of the inducibleNOS isoform and upregulation of constitutive NOS isoforms.

     

Pollination and Pollen-pistil Interaction in Oil Palm, Elaeis guineensis

Structural and cytochemical aspects of the pistil and detailsof pollination and pollen-pistil interaction were investigatedin the African oil palm (Elaeis guineensis Jacq.), an importantperennial oil crop. The stigma is trilobed, wet and papillate.The branched papillae are confined to a narrow linear zone oneach stigmatic lobe. Each stigmatic lobe harbours a deep stigmaticgroove, which runs adaxially along the surface. The stigmaticgroove is bordered by a well-defined layer of glandular cells,each of which has a pectinaceous cap on the inner tangentialwall. The style is hollow. The canal cells show thickeningson the inner tangential wall. The stigmatic groove and stylarcanal contain an extracellular matrix secreted by the canalcells which is rich in proteins, acidic polysaccharides andpectins. The canal cells at the base of the style are papillateand loosely fill the stylar canal. The stigma becomes receptivewhen the stigmatic lobes separate, and remains so for 24 h.Pollination is mediated by weevils as well as by the wind. Undernatural conditions the pollination efficiency was 100%. Pollinationinduces additional secretion in the stigmatic groove and stylarcanal. During post-pollination secretion, the pectinaceous capsof the cells lining the stigmatic groove are degraded. Pollengrains germinate on the stigmatic papillae and tubes grow onthe surface of the papillae, entering the stigmatic groove andadvancing along it into the stylar canal to eventually gainaccess to the locules. Pollen tubes are seen in the ovules 18–20h after pollination. Copyright 2001 Annals of Botany Company Arecaceae, Elaeis guineensis, African oil palm, pollination, stigmatic grove, stylar canal, Tenera hybrid, weevil     

The Structure and Cytochemistry of the Pistil of Sternbergia lutea (Amaryllidaceae)

Studies were carried out on structural and cytochemical aspectsof the pistil of Sternbergia lutea (L.) KerGawl. The stigmais of the wet papillate type; the papillae are unicellular andare arranged densely around the rim of a funnel-shaped stigma.The stigma exudate is limited and is confined to the bases ofthe papillae and the inner lining of the stigma. The papillaeare smooth in the distal part and are covered with intact cuticle-pelliclelining. The cuticle is disrupted at places towards the baseof the papillae releasing the exudate. The exudate is rich inpectins and other polysaccharides but poor in proteins and lipids.The papillae show dense cytoplasmic profiles with extensiveendoplasmic reticulum (ER), abundant mitochondria, polyribosomesand active dictyosomes. The style is hollow. The stylar cavityis surrounded by two to four layers of glandular cells. In theyoung pistil the canal is lined with a continuous cuticle, butin the mature pistil the cuticle becomes disrupted and the canalis filled with the secretion produced by the cells of the surroundingglandular tissue. Ultrastructurally, the cells of the glandulartissue are very similar to the stigmatic papillae. The innertangential wall of the cells bordering the canal is uniformlythicker than other walls. The secretion in the stylar canal,as well as the intercellular spaces of the glandular tissue,stain intensely for pectins and polysaccharides but poorly forproteins and lipids. Pollen tubes grow through the stylar canal.Structural and cytochemical details of the pistil of Sternbergiaare compared with other hollow-styled systems. Pistil, Sternbergia lutea (L.) Ker-Gawl., stigma and style, structure and cytochemistry     

S-Locus-Specific Glycoproteins Associated with Self-Incompatibility in Brassica campestris

Specific S-glycoproteins were isolated from three Brassica campestriscultivars homozygous with respect to the S-alleles S₈, S₉ andS₁₂. Amino acid sequences of various peptide fragments of theS-proteins were determined using a gas-phase protein sequencer,and a nearly complete amino acid sequence of the S₈-glycoproteinwas determined on the basis of the revised cDNA sequence ofthe B. oleracea S-specific glycoprotein. The lysyl endopeptidasefragments of S₉ and S₁₂-glycoproteins were aligned in comparisonwith the sequence of the S₈-glycoprotein. Although extensivesequence homology was evident among the three S-glycoproteins,the sequences of the middle part were relatively different fromeach other. The numbers and positions of N-glycosylation alsodiffered among the S-glycoproteins of Brassica species. (Received April 20, 1987; Accepted July 29, 1987)     

Crossability Barriers in Neotropical Heliconia

Artificial hybridization among species of neotropical Heliconiawas studied at two sites in Costa Rica, centralAmerica. At LasCruces Tropical Botanical Garden individuals in cultivationwere used as parents in crosses primarily between species withpendent inflorescences that normally are distributed allopatrically.At Finca La Selva normally sympatric species with either pendentor erect inflorescences were crossed in their natural habitats.Observation of pollen tube growth by means of fluorescence microscopyand seed set were used to determine the extent of crossability.Crossability barriers between the majority of species are strongand foreign pollen tubes are inhibited at the stigmatic surface,within the stylar tissue or within the ovary. The site of inhibitionis consistent for each pair of species, and is dependent onthe parentage and the direction of the cross. Although additionalisolating mechanisms, such as pollinator specificity and phenologicalseparation, are present in Heliconia, pre-fertilization crossabilitybarriers act as the ultimate mechanism to prevent hybridization.The type of barrier (stigmatic, stylar or ovarian) that existsbetween two species is not dependent upon the geographical distributionof the parental species or the specific types of pollinatorsthat visit them, but in some cases may indicate taxonomic relationships. Heliconia spp, isolating mechanisms, crossability barriers, progamic phase, hybridization, Costa Rica, hummingbirds, taxonomy, pollinator sharing     

Existence of a Common Glycoprotein Homologous to S-Glycoproteins in Two Self-incompatible Homozygotes of Brassica campestris

S-Glycoproteins (S-locus-specific glycoproteins) in Brassicaspecies are present only in stigmas and thought to play an importantrole in self-incompatibility system. The stigma extract containsalso several other glycoproteins reacting with the antiserumto S-glycoproteins, among which some glycoproteins from S₈S₈-and S₉S₉-homozygotes have the same pI value. Both of the glycoproteinswhich were tentatively termed NS₈- and NS₈S₉-glycoproteins,respectively, were isolated and analyzed. Those were revealedto be identical. Its amino acid sequence was homologous withthe S-glycoproteins in Brassica species. The NS-glycoproteinswere expressed at the same time and only in stigma as S-glycoproteins. (Received July 19, 1988; Accepted September 7, 1988)     

The Transmitting Tissue in Brugmansia suaveolens L.: Ultrastructure of the Stylar Transmitting Tissue

The stylar transmitting tissue of the angel's trumpet, Brugmansia(Datura) suaveolens, was studied at two developmental stages:about 6 d before anthesis and after anthesis. Histochemicallocalization of polysaccharides was carried out with PATAg andimmunohistochemistry with gold-conjugated antibodies recognizingpectins. Before anthesis the transmitting tissue forms a centralcore of polyhedral meristematic, still dividing, cells withnarrow intercellular spaces. Epitopes for unesterified pectinsare present in the walls and the spaces between the cells, whilemethylesterified pectins are confined to the middle lamellaand intercellular spaces. PATAg positive material and the antibodyagainst unesterified pectin was found in plasmalemma invaginationsand multivesicular bodies. Dictyosome cisternae and vesiclescontained epitopes for both kinds of pectins. Plastids are poorlydifferentiated and lack starch. Nutrients are stored as lipidbodies, which are digested by small vacuoles. After anthesisthe transmitting tract cells form cylindrical files separatedby voluminous spaces filled with a mucous secretion reactingwith PATAg and with the antibody against unesterified pectins.Dictyosome vesicles contain epitopes for the same kind of pectins.The cells are vacuolized and have leucoplasts. This study showspectin synthesis by different parts of the endomembrane systemand changes in pectin esterification during stylar development.Copyright1993, 1999 Academic Press Brugmansia suaveolens, immunocytochemistry, pectin, secretion, style, transmitting tissue     

Immunological Reactions of Single Pollen Grains, Electrophoresis and Enzymology of Pollen Protein Exudates

Single intact pollen grains of Oenothera organensis, when placedupon a thin layer of agar containing pollen antiserum, producecircular areas of precipitate. Pollen grains from an S₂S₂ plantdo not produce precipitate in S₆ antiserum. Pollen grains froman S₆S₆ plant and an S₂'S₄' self-compatible plant produce precipitatesin S₆ antiserum. Fifty per cent of the pollen grains from anS₂S₆ plant produce precipitate in S₆ antiserum. Protein diffusesinto buffer solutions from intact pollen grains within 212 min.As much as 40 per cent of the total protein diffuses out inan hour. Amylase and invertase were detected in the diffusatefrom pollen grains. Alkaline and acid phosphatases were confinedto the pollen grains and did not diffuse out. The serologicalprecipitates are specific to the incompatibility system.     

Metabolism of S-methylcysteine and pectin esterification in Chinese cabbage, Brassica pekinensis Rupr.

S-Methyl-L-cysteine was actively metabolized in Chinese cabbageand carbon from its methyl group was distributed into both thesoluble and insoluble fractions. The high incorporation of ¹⁴Cfrom the methyl group into the insoluble fraction after administeringof S-methyl-L-cysteine-¹⁴CH₃, and our previous results thatS-methyl-L-cysteine is demethylated to give cysteine, suggestthat S-methyl-L-cysteine might act as a methyl donor in Chinesecabbage. To obtain evidence for this possibility, incorporationof the methyl-¹⁴C of S-methyl-L-cysteine into methyl estersof pectic substances was investigated. Most of the ¹⁴C incorporatedinto pectic substances was liberated by treatment with dilutealkali and pectin esterase. The results show that S-methyl-L-cysteineacts as a methyl donor to form pectin ester. (Received October 12, 1971; )     

Characterization and Translation of Poly(A)+RNA from Wounded and Crown Gall Tissues of Potato Tubers

Poly(A)⁺ and poly(A)^–RNA from wounded potato tuber tissuesand crown gall tumors were separated from total RNA by oligodeoxythymidylicacid-cellulose affinity chromatography. The poly(A)⁺RNA wascharacterized by sucrose density gradient centrifugation, hybridizationwith ³(H)polyuridylic acid [Poly(U)] and in vitro translationin a rabbit reticulocyte lysate system. The tumor poly(A)⁺RNAwas a heterodisperse mixture from 3.5S to 35S. Upon poly(U)hybridization of the gradient fractions two major hybridizationpeaks at 7S and 21S and two peaks at 11S and 16S appeared. Inan in vitro translation system the poly(A)⁺RNA programmed thesynthesis of 23 different polypeptides of 9,000 to 79,800 daltonsmolecular weight as determined by SDS-polyacrylamide gel electrophoresis.The 21S poly(A)+RNA was about 5 times more active in in vitroprotein synthesis than the 7S poly(A)⁺RNA. The poly(A)⁺RNA from wounded tissues was also heterodisperse(from 4.5S to 31S) with a modal peak at 18S. This RNA codedfor at least 28 polypeptides, which were different from thoseof crown gall tumor tissues. On a per unit poly(A)⁺RNA basis the tumor RNA was slightly moreactive in translation than that from wounded tissues. The translationof tumor poly(A)⁺RNA was completely blocked by 0.5 mM 7-methylguanosine5'-phosphate, but not by 7-methylguanosine, suggesting the presenceof a 5'-cap structure. (Received May 15, 1982; Accepted June 30, 1982)     

The Site of the Self-incompatibility Mechanism in Tradescantia pallida

Cytochemical and ultrastructural investigations were carriedout on unpollinated, self-pollinated and cross-pollinated pistilsof Tradescantia pallida. A protein secretion was found to occuron the unicellular papillac of the stigma in increasing amountstowards the base of the cells. The active cytoplasm of the cellsseemed to be responsible for this secretion. Compatible cross-pollinationresulted in pollen tube growth through the secretion and downthe stylar canal, whereas self-pollination resulted in pollentube growth being halted at the base of the stigma. It is suggestedthat the secretion may play an important role in the self-incompatibilityresponse. Tradescantia pallida, self-incompatibility, self-pollination, cross-pollination     

Expression of nitric oxide synthase isoforms in normal ventilatory and limb muscles

Hussain, Sabah N. A., Qasim El-Dwairi, Mohammed N. Abdul-Hussain, and Dalia Sakkal. Expression of nitric oxidesynthase isoforms in normal ventilatory and limb muscles.J. Appl. Physiol. 83(2): 348-353, 1997.Nitric oxide (NO), an important messenger molecule withwidespread actions, is synthesized by NO synthases (NOS). In thisstudy, we investigated the correlation between fiber type and NOSactivity among ventilatory and limb muscles of various species. We alsoassessed the presence of the three NOS isoforms in normal skeletalmuscles and how various NOS inhibitors influence muscle NOS activity.NOS activity was detected in various muscles; however, NOS activity inrabbits and rats varied significantly among different muscles.Immunoblotting of muscle samples indicated the presence of both theneuronal NOS and the endothelial NOS isoforms but not thecytokine-inducible NOS isoform. However, these isoforms were expressedto different degrees in various muscles. Although the neuronal NOSisoform was detectable in the canine diaphragm, very weak expressionwas detected in rabbit, rat, and mouse diaphragms. The endothelial NOSisoform was detected in the rat and mouse diaphragms but not in thecanine and rabbit diaphragms. We also found thatN^G-nitro-L-arginine methyl ester,7-nitroindazole, andS-methylisothiourea werestronger inhibitors of muscle NOS activity than was aminoguanidine. These results indicate the presence of different degrees ofconstitutive NOS expression in normal ventilatory and limb muscles ofvarious species. Our data also indicate that muscle NOS activity is not determined by fiber type distribution but by other not yet identified factors. The functional significance of this expression remains to beassessed.

     

Structural and molecular analysis of self-incompatibility in almond (<Emphasis Type="Italic">Prunus dulcis</Emphasis>)

. A multi-approach was used to study different aspects of self-incompatibility (SI) in almond (Prunus dulcis). First, a population of almond cultivars was characterised as to their individual S-allele combination using separation of stylar protein extracts (non-equilibrium pH gradient electrofocusing) followed by staining for RNase activity, which led to the identification of one putative new allele and several new S-allele combinations. Second, a field pollination scheme was designed to study pollen tube progression and to obtain a spatial and temporal characterisation of this reproductive stage in both incompatible and compatible crosses. In addition, an anti-serum was raised against a synthetic peptide designed from an almond S-protein (S₈) and used for immunological in situ detection in pistil cryosections. S-RNases were found to accumulate intercellularly in the stylar transmitting tissue as previously reported for other rosaceous species. The results are discussed in view of the evolution of the gametophytic SI system and the models proposed for its mechanism. Gametophyte selection is also proposed as an important intraspecific barrier to fertilisation in this species.