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)     

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)     

The cDNA Sequence of NS3-Glycoprotein from Brassica campestris and its Homology to Related Proteins

A cDNA clone encoding NS₃-glycoprotein was isolated from stigmasof Brassica campestris. NS-glycoproteins correspond to the SLRI-glycoproteinsof B. oleracea and are highly conserved within the species.These data suggest that the NS-glycoproteins may play a rolein discrimination between species in the fertilization systemof Brassica. (Received September 4, 1992; Accepted November 9, 1992)     

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     

Control of self-incompatibility by CO2 gas in Brassica

Pollen tubes penetrated stigma papilla cells in flowers thatwere illegitimately (self) pollinated, after CO₂ treatment ofthese flowers. This shows that the self-incompatible reactionin Brassica can be removed by CO₂ gas. Ethylene gas was noteffective. (Received May 16, 1969; )     

Biosynthesis of glycoproteins involved in the pollen-stigma interaction of incompatibility in developing flowers of Brassica oleracea L.

De-novo synthesis of the S-allele-specific glycoproteins of Brassica oleracea is demonstrated in stigmas at different developmental stages. Excised stigmas incorporate ¹⁴C-labeled amino acids into their S-glycoproteins early in development and before the self-incompatibility response is acquired, but the rate of synthesis accelerates prior to anthesis, resulting in the accumulation of high levels of the S-glycoproteins in the stigma and coinciding with the acquisition of the pollen-stigma incompatibility response. Since the self-compatible and self-incompatible zones of developing inflorescences are very sharply delineated, a threshold quantity of S-glycoproteins appears to be critical for the onset of self-incompatibility. Incorporation experiments in which [^35Smethionine was applied to intact stigma surfaces indicate that the papillae are the main sites of synthesis of the S-specific glycoproteins.Abbreviations IEF isoelectric focusing - SC self-compatibility - SDS sodium dodecyl sulfate - SI self-incompatibility     

A Comparison of the Growth, Photosynthesis, Stomatal Conductance and Water Use Efficiency of Moricandia and Brassica Species

When grown in pots and well-watered, the relative growth ratesof the above ground parts of two species of Moricandia (M. arvensis,an intermediate C₃–C₄ species, and M. moricandioides,a C₃ species) were inferior to those of two cultivated Brassicaspecies (B. campestris and B. napus). The Moricandia specieshad thicker leaves (greater d.wt per unit leaf area) with morechlorophyll than the Brassica species and had slightly greaterrates of photosynthesis per unit leaf area at an irradiance(400–700 nm) of 2000 µmol quanta m^–2 s ^–1.Leaves of M. arvensis, known to have a CO₂ compensation pointbetween that of C₃ and C₄ species, had a lower ratio of theintercellular to atmospheric partial pressure of CO₂ (C₁/C_a)and a greater instantaneous water use efficiency (WUE) thanthose of M. moricandioides and the Brassica species. Carbon isotope discrimination (     

Con A — Peroxidase method: an improved procedure for staining S-glycoproteins in cellulose-acetate electrofocusing in crucifers

Summary In the analysis of stigma glycoproteins by cellulose acetate electrofocusing in self-incompatible crucifers, the staining method of the glycoproteins, described in the earlier report, has been improved by using Con A — peroxidase reactions to obtain a permanent profile of band patterns which are visible under day-light conditions. Identifying S alleles by the corresponding S-glycoproteins can be facilitated by the present S-glycoprotein analysis.     

An effective time for CO2 gas treatment in overcoming self-incompatibility in Brassica

An investigation was made to determine the effective time forCO₂ treatment in overcoming self-incompatibility in Brassica.CO₂ was effective when supplied to a self-pollinated flowerwhile hundreds of pollen grains were germinating on the stigma.Since the effective time coincides with the attachment of pollentubes to papilla cells, it is thought that CO₂ produces a metabolicchange in these cells during attachement. ¹Part of a thesis submitted for the Dr. of Agr. degree by thesenior author at Tohoku University. ²Present address: Faculty of Agriculture, Kobe University, Nada-ku,Kobe, Japan. (Received December 7, 1972; )     

The escape behavior of marine copepods in response to a quantifiable fluid mechanical disturbance

The threshold shear values needed to elicit the escape reactionto a quantifiable fluid mechanical disturbance were comparedbetween five free-swimming oceanic copepod species. The resultsindicate a significant difference in the threshold for differentspecies of copepods and between different age groups withina single species. In general, animals captured from more energeticregimes required a higher threshold than those captured frommore pacific locations. Labidocera madurae required the highestshear values with 51.5 s^–1 for 50% of the animals testedto elicit an escape reaction (S₅₀). Acartia tonsa and Euchaetarimana, in contrast, were behaviorally the most sensitive requiringan S₅₀ of only 1.5 and 4.1 s^–1, respectively, to initiatean escape reaction. Pleuromamma xiphias and Oithona requiredintermediate shear values with an S₅₀ of 7.2 and 8.1 s^–1.When compared to literature values, the threshold needed toelicit an escape reaction was consistently higher than averageenvironmental shear values. Threshold shear values also variedsignificantly with developmental stage. Naupliar stages of A.tonsarequired greater than six times the S₅₀ value required by adultsof the same species. This suggests that the higher vulnerabilityto predation of naupliar stages of copepods may not only reflectinferior escape strength, but may also result from the higherthreshold needed to elicit an escape reaction. This study supportsthe hypothesis that selective feeding patterns exhibited bypredators of copepods may be the result of the differentialbehavioral sensitivities of different species and developmentalstages of copepods.     

Identification of self-incompatibility-related glycoproteins in styles of apple (Malus x domestica)

In this study, stylar proteins of apple (Malus x domestica) which correlate with known intervarietal incompatibility relationships and have similar characteristics to the S-glycoproteins of Japanese pear (Pyrus serotina) were surveyed by two-dimensional gel electrophoresis (2D-PAGE). Varietal differences were detected in a group of glycoproteins having Mrs and pIs similar to those of the S-glycoproteins of Japanese pear. 2D-PAGE profiles of these glycoproteins were correlated with intervarietal incompatibility relationships. These glycoproteins reacted with antiserum raised against the S ⁴-glycoprotein of Japanese pear, a result suggesting that they may be the products of S-alleles in styles of apple. On the basis of the profiles of the putative S-glycoproteins, S-genotypes were proposed for each of the apple cultivars examined.     

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)     

Effect of Peroxydisulfate on Vigna radiata Abscission

K₂S₂O₈, applied to the basal end of cuttings of Vigna radiatastimulated leaf abscission in the light or dark. Because inhibitionof leaf sbscission in the dark by IAA was completely abolishedby K₂S₂O₈, and IAA decreased stimulation of abscission by K₂S₂O₈,destruction of IAA in the cuttings by K₂S₂O₈ is indicated. K₂S₂O₈had no effect on leaf abscission when applied as a foliar sprayor when roots of undisturbed seedlings were treated. When appliedproximally or distally to leafless explants, K₂S₂O₈ inhibitedpetiole abscission, and neither IAA nor ethylene had an effecton the inhibition. Although K₂S₂O₈ destroyed IAA in vitro, ithad no effect on abscission inhibitors in macerates of Vignaleaves and corn roots, nor did it destroy the biological activityof IAA added to such macerates. Substances liberated by macerationmay interfere with the ability of K₂S₂O₈ to destroy IAA. (Received May 2, 1981; Accepted August 24, 1981)     

Metabolism of Gibberellins in Cell-Free Extracts of Anthers from Normal and Dwarf Rice

Cell-free extracts were prepared from anthers of normal anddwarf rice (Oryza sativa L.), and the metabolism of radioisotope-labeledgibberellins in the extracts was analyzed by HPLC and gas chromatography-massspectrometry (GC/MS). GA₁₂ was converted to GA₁₅ and GA₃₄ inthe extracts. GA₂₀ was converted to GA¹, GA₈ and GA₂₉, but GA₉was converted only to GA₃₄. The extracts of the dwarf cultivar,Waito-C (dy mutant), showed the same 3ß-hydroxylationactivity as did those of the normal cultivar, Nihonbare, indicatingthat the dy gene is not expressed in the anthers. These resultssuggest that the regulation of the biosynthesis of gibberellinsin rice is organ-specific. (Received November 9, 1989; Accepted January 10, 1990)     

Biological Activities of the Methyl Ester of Gibberellin A73, a Novel and Principal Antheridiogen in Lygodium japonicum

The synthetic methyl ester of GA₇₃ (GA₇₃-Me) and the naturalantheridiogen of Lygodium japonicum showed almost the same activityto induce the formation of antheridia in dark-grown protonemataof L. japonicum at concentrations of 10^-14 M and higher. Thus,it appears that the principal antheridiogen in L. japonicumis GA₇₃-Me. GA₇₃-Me inhibited formation of ar-chegonia in light-grownprothallia of L. japonicum at concentrations of 10^-11 M andhigher and induced germination of spores in the dark in thisspecies at the same range of concentrations. GA₇₃(free acidform) promoted growth of seedlings of dwarf rice and hypocotylsof cucumber seedlings at dosages of and above 1 and 100 ng/plant,respectively. Eight compounds related to GA₇₃-Me, includingantheridiogens of Anemia phyllitidis and Anemia mexicana, wereactive in inducing an-theridial formation in L. japonicum, althoughtheir activities were considerably lower than that of GA₇₃-Me. (Received August 24, 1988; Accepted November 28, 1988)     

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     

Photosynthetic Characteristics of C3-C4 Intermediate Flaveria Species II. Kinetic Properties of Phosphoenolpyruvate Carboxylase from C3, C4 and C3-C4 Intermediate Species

The kinetic properties of phosphoenolpyruvate (PEP) carboxylasehave been studied among several Flaveria species: the C₃ speciesF. cronquistii, the C₃–C₄ species F. pubescens and F.linearis, and the C₄ species F. trinervia. At either pH 7 or8, the maximum activities (in µmol.mg Chl^–1.h^–1)for F. pubescens and linearis (187–513) were intermediateto those of the C₃ species (12–19) and the C₄ species(2,182–2,627). The response curves of velocity versusPEP concentration were hyperbolic for the C₃ and C₃–C₄species at either pH 7 or 8 while they were sigmoidal for theC₄ species at pH 7 and hyperbolic at pH 8. The K_m values forPEP determined from reciprocal plots were lowest in the C₃ species,and of intermediate value in the C₃–C₄ species comparedto the K' values of the C₄ species determined from Hill plotsat either pH 7 or 8. Glucose-6-phosphate (G6P) decreased theK_m values for PEP at both pH 7 and 8 in the C₃ and C₃–C₄species. In the C₄ species, G6P decreased the K' values at pH8 but increased the K' values at pH 7. In all cases, G6P hadits effect by influencing the activity at limiting PEP concentrationswith little or no effect on the maximum activity. At pH 8 andlimiting concentrations of PEP the degree of stimulation ofthe activity by G6P was greatest in the C₄ species, intermediatein F. linearis, a C₃–C₄ species, and lowest in the C₃species. In several respects, the PEP carboxylases of the C₃–C₄Flaveria species have properties intermediate to those of theC₃ and C₄ species. (Received April 30, 1983; Accepted August 22, 1983)