Immunochemical studies on the specificity of soybean agglutinin

The specificity of the purified soybean agglutinin has been studied immunochemically by quantitative precipitin and quantitative precipitin inhibition assays. The lectin is precipitated by human A and Le^a blood-group substance, by the products of the second, third, fourth, and fifth stages of periodate oxidation of a human H blood-group substance (JS), and by precursor blood-group substances, as well as by a pig-submaxillary mucin having blood-group A activity, by partially hydrolyzed blood-group B substances (Pl fraction), and by group C streptococcal polysaccharide. The activity is attributable to terminal α-linked 2-acetamido-2-deoxy-d-galactopyranosyl or to α- or β-d-galactopyranosyl residues. The lectin did not precipitate with human blood-group H substances, with the product of the first stage of periodate oxidation (JS), with streptococcal group A polysaccharide, or with pig-submaxillary mucin devoid of blood-group A activity, and is poorly precipitated by blood-group B substances. Inhibition of precipitation with various monosaccharides indicated that the lectin is strongly specific for 2-acetamido-2-deoxy-d-galactose and for its oligosaccharides, and to a lesser extent for d-galactose and its oligosaccharides; the α-glycosides of both sugars were slightly more reactive than the β-glycosides of 2-acetamido-2-deoxy-d-galactose, and both α- and β-glycosides were more active than the free monosaccharides. Aromatic α- and β-glycosides of 2-acetamido-2-deoxy-d-galactose and d-galactose were better inhibitors than the corresponding methyl or ethyl compounds. The blood-group A trisaccharide α-d-GalNAcp-(1→3)-β-d-Galp-(1→3)-d-GlcNAc was more active than the disaccharide lectins by the use of precipitation with polysaccharides, as well as inhibition reactions, is essential to the understanding of their reactivity with cell-surface receptors.     

Role of carbohydrate moieties in peanut (Arachis hypogaea) peroxidases.

The activities of a cationic (C.PRX) and an anionic peroxidase isolated from peanut (Arachis hypogaea)-cell suspension culture were drastically reduced when they were deglycosylated with glycopeptidase F or oxidized by 10 mM-periodate. In contrast with the controls, the deglycosylated or the oxidized peroxidases were much more susceptible to proteolytic degradation. In radiolabelling experiments with [35S]methionine, the non-glycosylated C.PRX was synthesized in the tunicamycin-treated cultures and secreted into the medium. Examination of the C.PRX polypeptides by SDS/polyacrylamide-gel electrophoresis followed by fluorography showed that the non-glycosylated form had an Mr of approx. 31,000, which is about 78% of that of the glycosylated form. Our results suggest that carbohydrates may not be essential for peroxidase secretion, but that stabilization of the peroxidase molecules and acquisition by these isoenzymes of a catalytically active conformation is linked directly or indirectly to glycosylation.     

Histochemical specificity of beta-galactose binding lectins from Arachis hypogaea (peanut) and Ricinus communis (castor bean)

Previous histochemical studies have demonstrated disparities in the binding of two lectins with a nominal specificity for terminal beta-D-galactose. Biochemical studies have shown that the most complementary structure for binding peanut agglutinin (PNA) is the terminal disaccharide Gal-(beta 1----3)-GalNAc, whereas the most complementary structure for binding Ricinus communis agglutinin I (RCA I) is the terminal disaccharide Gal-(beta 1----4)-GlcNAc. However, it is not known if only these differences in affinity account for the different histochemical staining reactions observed on tissue sections. In the present study we compared the staining patterns of PNA and RCA I by inhibiting in situ the binding of each lectin conjugated to horseradish peroxidase (HRP) with increasing concentrations of unlabeled PNA or RCA I. The PNA-HRP conjugate did not stain most tissue sites suspected of containing an abundance of glycoconjugates with terminal Gal-(beta 1----4)-GlcNAc. Moreover, unlabeled PNA failed to significantly inhibit strong RCA I-HRP staining in these sites. In loci thought to contain variable amounts of glycoconjugates with terminal Gal-(beta 1----3)-GalNAc, unlabeled RCA I decreased PNA-HRP reactivity only slightly or not at all, whereas weak to strong RCA I-HRP staining was diminished or abolished by unlabeled PNA. The results suggest that PNA staining is restricted to glycoconjugates with terminal Gal-(beta 1----3)-GalNAc. RCA I apparently reacts most strongly with glycoconjugates having the terminal disaccharide Gal-(beta 1----4)-GlcNAc, but also stains sites containing a moderate to abundant amount of glycoconjugates with the terminal Gal-(beta 1----3)-GalNAc sequence.     

The surface glycoproteins of human skin fibroblasts detected after electrophoresis by the binding of peanut (Arachis hypogaea) agglutinin and Ricinus communis (castor-bean) agglutinin I.

A new methodology was developed to study the cell-surface glycoproteins of cultured human skin fibroblasts. This was based on the binding of a variety of biotinyl-lectins to nitrocellulose electrophoretic transfers of total fibroblast lysates after separation in sodium dodecyl sulphate/polyacrylamide gels, followed by reaction with avidin-biotinyl-peroxidase complexes and detection with 3,3'-diaminobenzidine. The technique proved to be very sensitive and a large number of glycoproteins were detected by binding of concanavalin A and wheat-germ agglutinin. Binding of peanut agglutinin and to a lesser extent of Ricinus communis agglutinin I were found to be dependent on prior removal of sialic acid residues from the glycoproteins. Since by treatment of intact viable cells with neuraminidase only external sialic acid residues were removed, peanut agglutinin and Ricinus communis agglutinin I could thus be utilized for selective detection of cell-surface glycoproteins. Also, because peanut agglutinin was known to bind preferentially to oligosaccharides of the O-glycosidic type, and Ricinus communis agglutinin I to those of the N-glycosidic type, the two lectins were complementary in displaying the surface glycoproteins and in providing information about their oligosaccharide composition.     

Trypanosoma cruzi I and Trypanosoma cruzi II: recognition of sugar structures by Arachis hypogaea (peanut agglutinin) lectin

Epimastigote culture forms of different isolates of Trypanosoma cruzi from different mammal hosts, humans, and vectors were tested with FITC-conjugated peanut agglutinin lectin (PNA-FITC). The parasites maintained in axenic medium, liver infusion tryptose. were evaluated by flow cytometric analyses; whereas T. cruzi I (Tcl), which is associated with the sylvatic transmission cycle, was labeled in high percentages with PNA (88-99.2%), T. cruzi II (TcII) (parasites associated with domiciliar cycle) and T. cruzi, zymodeme 3 (Tc/Z3) (also associated with the sylvatic cycle) were labeled in low percentages (TcII, 0-26% and Tc/Z3, 0-12.6%). It was demonstrated that it is possible to differentiate the 2 main T. cruzi subpopulations, TcI and TcII, using Arachis hypogaea. These results also showed a higher variability in TcII in terms of PNA binding.     

Pterocarpenes elicited by Aspergillus caelatus in peanut (Arachis hypogaea) seeds

The substituted pterocarpenes named aracarpene-1 (1) and aracarpene-2 (2) were isolated from wounded peanut seeds challenged by a strain of Aspergillus caelatus. The structures of these putative phytoalexins were determined by interpretation of NMR and MS data. The aracarpenes were investigated for their antifungal and antibacterial activities as well as antioxidant, anti-inflammatory, and cytotoxic activities in mammalian cells. Aracarpene-2 demonstrated high antibacterial properties against tested gram-positive and gram-negative bacteria, whereas aracarpene-1 displayed low antibacterial properties against the same bacteria. Both compounds had no antifungal activity against Aspergillus flavus. Together with peanut stilbenoids that are also produced in the challenged seeds, these compounds may represent a class of low-molecular weight peanut metabolites with a defensive role(s) against pathogenic microorganisms.     

Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.)

A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/–) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F₂ progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F₂ progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut.     

Effects of aluminium on the peanut (Arachis hypogaea L.)/ Bradyrhizobium symbiosis

The effects of low activities of the monomeric Al species, Al³⁺, Al(OH)₂ ⁺ and Al(OH)²⁺, on the peanut/Bradyrhizobium symbiosis were examined in solution culture. In flowing solution culture, growth of the host plant was depressed at activities ≥5 μM. Neither shoot dry weight, root dry weight nor root length were inhibited by 3 μM Al, an activity which reduced nodule number by 70%. Low nodule number was compensated for, at this activity, by an increase in weight per nodule. In non-flowing solution culture of similar composition, survival of a streptomycin resistant mutant of Bradyrhizobium spp. NC92 in the bulk solution or in the rhizosphere of peanut roots was unaffected by 20 μM Al. The site of infection by Bradyrhizobium was examined by scanning electron microscopy. Lateral root axils of plants exposed to ≥2 μM Al did not display the rosette of multicellular root hairs which is characteristic in normal plants. The detrimental effects of Al on nodulation appear to be related to structural changes at the site of infection which are observed at Al activities too low to cause any depression in growth of the host plant, including root length, and at activities of Al which do not affect survival of the free-living Bradyrhizobium.     

Diniconazole's effect on peanut (Arachis hypogaea L.) growth and development

Greenhouse nutrient solution studies demonstrated that diniconazole will decrease peanut (Arachis hypogaea L.) shoot growth when either root or shoot applied. Root growth and development were decreased by root and, to a lesser extent, by shoot uptake of diniconazole. Diniconazole is apparently xylem translocated, but not phloem translocated. Concentrations of 200 ppb ES isomer of diniconazole in nutrient solution (root uptake) increased specific leaf weight and starch deposits in the leaf. Field applications of 193 g ES isomer ha^–1 of diniconazole reduced main stem height by 33%, leaf area index by 16%, and total vegetative dry weight by 19%, but had no effect on average leaf size. Decreased germination of seeds from plants treated with 1435 g ha^–1 diaminozide was associated with increased seed dormancy. Seed dormancy was counteracted by either ethylene gas or storage for 150 days after harvest. Soil applications of diniconazole were more effective than foliar appliations in reducing vine growth. Diniconazole's ER isomer is a broad spectrum fungicide that reduced damage (when compared to the control) bySclerotium rolfsii andRhizoctonia solani. The reduced damage by these diseases was thought to be the primary reason for the significant pod yield increase (when compared to the control) observed with the diniconazole treatments. In drought-stressed plots, populations of the two-spotted spider mite (Tetranychus urticae) were increased by diniconazole.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee by the University of Georgia or the U.S. Department of Agriculture and does not imply UGA or USDA approval to the exclusion of other products or vendors that also may be suitable.     

Thidiazuron promotes high frequency regeneration of peanut (Arachis hypogaea) plants in vitro

Multiple shoots were induced on Valenciatype peanut (Arachis hypogaea L.) explants cultured in vitro on a nutrient medium supplemented with thidiazuron. Zygotic embryos excised from mature seeds were germinated on Murashige-Skoog nutrient medium, and the resulting plantlets (8 days-old) were used as a source of explants. When cultured on a nutrient medium with increasing levels of thidiazuron (0.5 to 30 mg/l), expiants from various parts of the peanut plant (except the root) produced multiple shoot primordia which subsequently developed into individual shoots. Hypocotyl and cotyledon explants produced shoots in higher numbers than other explants (20 shoots per hypocotyl explant at all thidiazuron concentrations and 15 shoots per cotyledon explant at 30 mg/l). Shoots rooted normally on a basal Murashige-Skoog medium containing charcoal and developed into healthy and fertile plants when planted in soil.Abbreviations TDZ thidiazuron - MSO Murashige and Skoog (1962) basal medium - BA 6-benzylaminopurine     

Mass spectrometric characterization of isoform variants of peanut (Arachis hypogaea) stem lectin (SL-I)

Matrix assisted laser desorption/ionization–time-of-flight (MALDI–TOF) mass spectrometric (MS) analysis of purified Arachis hypogaea stem lectin (SL-I) and its tryptic digests suggested it to be an isoformic glucose/mannose binding lectin. Two-dimensional gel electrophoresis of SL-I indicated six isoforms (A1–A6), which were confirmed by Western blotting and MALDI–TOF MS analysis. Comparative analysis of peptide mass spectra of the isoforms matched with A. hypogaea lectins with three different accession numbers (Q43376_ARAHY, Q43377_ARAHY, Q70DJ5_ARAHY). Tandem mass spectrometric (MS/MS) analysis of tryptic peptides revealed these to be isoformic variants with altered amino acid sequences. Among the peptides, the peptide T12 showed major variation. The ¹⁹⁹Val–Ser–Tyr–Asn²⁰² sequence in peptide T12 of A1 and A2 was replaced by ¹⁹⁹Leu–Ser–His–Glu²⁰² in A3 and A4 (T12′) while in A5 and A6 this sequence was ¹⁹⁹Val–Ser–Tyr–Val²⁰² (T12″). Peptide T1 showed the presence of ¹⁰Asn in the isoforms A1–A5 while in A6 this amino acid was replaced by ¹⁰Lys (T1′). Overall amino acid sequence as identified by MS/MS showed a high degree of similarity between A1, A2 and among A3, A4, A5. Carbohydrate binding domain and adenine binding site seem to be conserved.     

Somatic embryogenesis from the axillary meristems of peanut (Arachis hypogaea L.)

Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed from the caulogenic meristems in the axils. Exposure of 2 weeks in primary medium with 90.5 μM 2,4-D suppressed the shoot tip differentiation temporarily which then regained the ability to form the shoot on withdrawal of 2,4-D. Exposure of 4 weeks in primary medium with 90.5 μM 2,4-D suppressed the shoot tip differentiation irreversibly. No shoot formation was noted from the tips in any of the cultures which were in secondary medium with 13.6 μM 2,4-D. Development of somatic embryos directly from axillary meristems was confirmed histologically. Conversion frequency of these embryos was 11%. Thus, in this report, we describe a method to obtain somatic embryos from the determined organogenic buds of the axillary meristem, by culturing the nodal explant vertically on embryo induction medium. It also displays the possibility of obtaining both embryogenic and organogenic potential in two parts of the same explant simultaneously. The possibility of extending this approach for genetic transformation in in vivo system through direct DNA delivery or Agrobacterium injection in meristems can also be explored. Using Agrobacterium rhizogenes, we have demonstrated the possibility of gene transfer in the axillary meristems of seed-derived plumule explant.     

Proteomics reveals elevated levels of PR 10 proteins in saline-tolerant peanut (Arachis hypogaea) calli.

The proteome of a salinity-tolerant Arachis hypogaea L. callus cell line was compared with its sensitive counterpart. Several low molecular weight proteins were detected by two-dimensional electrophoresis as being unique or significantly elevated in the tolerant line. The identities of several of these proteins were established as PR 10 proteins using tandem Mass Spectrometry and are shown to be phosphorylated on the basis of staining with the phosphorylation-specific stain, Pro-Q Diamond. Our results suggest that these differentially phosphorylated PR 10 proteins may play an important role in mediating salinity stress responses.     

花生镉污染研究进展

花生既是世界主要的油料作物,又是重要的植物蛋白来源和食品加工原料．随着花生直接食用和食品加工的不断增加,国际上对花生籽粒Cd含量问题越来越关注．我国是世界上重要的花生生产国和出口国．近年来,花生Cd含量偏高已经成为制约我国出口贸易的重要因素．本文从花生籽粒Cd富集能力、花生Cd含量的种内差异、籽粒中Cd的分布规律、影响花生籽粒Cd积累的机制和降低花生籽粒Cd含量技术等方面,对花生Cd污染研究的现状与问题进行了论述．指出在花生cd污染控制方面有2种策略可以考虑,一是降低花生对土壤Cd的吸收;二是控制Cd向籽粒的迁移富集．为此需要从3个方面加强对花生籽粒Cd积累机制的研究,即花生根系活性特征参数及其与籽粒Cd积累的关系;花生果荚Cd吸收机制及其对籽粒Cd含量的贡献;花生植株体内Cd迁移机制及其与籽粒Cd含量的关系．     

Gene transfer into peanut (Arachis hypogaea L.) by Agrobacterium tumefaciens

Introduction of foreign genes into plant tissues via Agrobacterium tumefaciens based vectors requires specific knowledge of Agrobacterium-host compatibility. Therefore, to develop a transformation protocol for peanut (Arachis hypogaea L.), five Brazilian cultivars were screened with four wild-type A.tumefaciens strains. Successful transformation was dependent on specific bacterial strain-plant cultivar interactions and strain A281 was the most effective for tumor induction. Tumors displayed hormone autonomous growth, were opine positive and contained DNA that was homologous to the T-DNA of the inciting strain. Tumors induced on seed and seedling explants by A281 (pTD02) also expressed the reporter genes gus and npt-II contained in the binary vector. These results show that peanut is a permissive host for the acceptance of genes from specific A.tumefaciens gene vectors.Abbreviations GUS ß-glucuronidase (EC 3.2.1.31) - NPT-II neomycin phosphotransferase II (EC 2.7.1.95) - EDTA ethylene-diamine-tetracetic acid     

Plant regeneration through somatic embryogenesis in peanut (Arachis hypogaea L.)

Somatic embryos were induced from immature cotyledons and immature embryonal axis ofArachis hypogaea L. on L-6 basal medium supplemented with NAA, picloram or 2,4-D at 5–50 mg 1^-1. Immature embryonal axis produced a higher number of somatic embryos in comparison with immature cotyledons. The highest number of responding cultures was produced on medium supplemented with NAA (50 mg 1^-1), while the highest average number of somatic embryos per culture was produced on medium with 2,4-D (10 or 20 mg 1^-1) and picloram (30 mg 1^-1) from cotyledons. The somatic embryos developed into plants on basal medium supplemented with activated charcoal and about 100 plants were successfully transferred to the field. Acknowledgement: The authors wish to thank Nuclear Agriculture Division, BARC for supplyingA. hypogaea seeds and Mr. R.M. Mudliar for photography.     

Agrobacterium tumefaciens mediated gene transfer in peanut (Arachis hypogaea L.)

Transgenic peanut plants were produced using Agrobacterium mediated gene transfer. Primary leaf explants of peanut were co-cultivated with Agrobacterium tumefaciens LBA 4404 harbouring the binary plasmid pBI 121 (conferring -glucuronidase activity and resistance to kanamycin) and cultured on regeneration medium supplemented with kanamycin to select putatively transformed shoots. They were rooted and plants were transferred to soil. Stable integration and expression of the transgenes were confirmed by NPT II assay, Southern blot hybridization and GUS assay.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - SDS Lauryl sulfate     

土壤类型与作物基因型对花生籽实镉积累的影响

以花生主产区的棕壤和潮土为供试土壤,以种植面积最大的白沙1016、花育22和湛油27基因型花生(Arachis hypogaea)为供试作物,采用不添加Cd(对照)和添加Cd(1.5 mg· kg-1)处理进行盆栽试验,研究土壤类型和作物基因型对花生籽实Cd积累的影响.结果表明:两种土壤对照处理的3种基因型花生籽实Cd含量均低于国家食品安全标准,而Cd处理下均高于食品安全标准.同种土壤Cd处理3种基因型花生籽实Cd含量显著高于对照,不同基因型间表现为湛油27＞白沙1016＞花育22,棕壤花生籽实Cd含量及总量均高于潮土.对照处理3种基因型花生籽实Cd生物富集系数均大于1.0,Cd处理下多小于1.0,表明花生籽实对土壤中Cd的累积能力较强,土壤Cd含量进一步增加时,其生物富集能力降低.