Isolation of pisumin,a novel antifungal protein from legumes of the sugar snap pea Pisum sativum var macrocarpon

An antifungal protein with a novel N-terminal sequence GVGAAYGCFG and a molecular mass of 31 kDa was isolated from the legumes of the sugar snap pea Pisum sativum var. macrocarpon. The protein, designated pisumin, exhibited antifungal activity against Coprinus comatus and Pleurotus ostreatus and much weaker activity against Fusarium oxysporum and Rhizoctonia solani. Pisumin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) of 6 microM. Pisumin was similar to other leguminous antifungal proteins in that it was adsorbed on Affi-gel blue gel and CM-Sepharose.     

Sativin: a novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon

An antifungal protein designated sativin was isolated from the legumes of the sugar snap (also known as honey pea) Pisum sativum var. macrocarpon. The procedure entailed extraction, affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The protein exhibited a molecular weight of 38 kDa in SDS-polyacrylamide gel electrophoresis. It possessed an N-terminal amino acid sequence which showed similarity to those of miraculin (a sweet protein) and pisavin (a ribosome-inactivating protein from Pisum sativum var arvense Poir manifesting similarity to miraculin). Unlike pisavin, however, sativin demonstrated negligible ribonuclease activity and inhibited translation in a rabbit reticulocyte lysate system with a very low potency (IC50= 14 microM). Sativin exerted antifungal activity against Fusarium oxysporum, Coprinus comatus and Pleurotus ostreatus but not against Rhizoctonia solani.     

The major albumin protein from pea (Pisum sativum L.)

The major albumin protein in storage parenchyma tissue of developing peas has been localised at an ultrastructural level by immunocytochemistry. Tissue was fixed in buffered aldehyde and embedded in LR White resin which was polymerised by addition of catalyst. Sections were labelled by the indirect method of absorption of Protein A-gold to specifically bound antibodies. This method gives high levels of specific labelling on sections which retain good ultrastructural preservation and have high contrast after conventional staining. The albumin is located throughout the cytoplasm although no labelling was found associated with the endoplasmic reticulum, Golgi apparatus, vacuoles-protein bodies or other organelles.Abbreviation PMA pea major albumin protein     

The lectin-binding protein from the pea (Pisum sativum); properties and interactions

The lectin-binding protein (lectin binder) from the garden pea (Pisum sativum) was studied. It is a glycoprotein composed of four subunits of about 50 000 Da. Its amino-acid composition and molecular mass differ from those of lectin and of storage proteins. The interaction between lectin and lectin binder is demonstrated and quantified by several different methods and is shown to be specifically sugar-dependent. A biological function of lectin binders and lectins is discussed.     

Characterization of a lectin-binding storage protein from pea (Pisum sativum)

From the storage proteins of the pea (Pisum sativum), the fraction which interacts with the pea lectin by the sugar-binding site was studied. By electrophoretical subunit patterns and other criteria, this fraction resembles the group of the 7S storage proteins (vicilins). The fraction was resolved into subunits by micropreparative SDS PAGE. The N-terminal sequences of the individual subunits were determined. Most of these are identical with published vivilin subunit sequences; therefore this lectin-binding fraction belongs to the vicilins. Selected subunits and tryptic fragments were analysed for amino-acid compositions. Though unequivocal assignments to vicilin segments were possible, significant differences could be recognized, in particular in the tryptic fragments.     

Embryogenesis of the pea (Pisum sativum)

Summary The most striking internal feature of the suspensor cells inPisum is the abundant occurrence of a plastid containing spherical bodies consisting of intertwined bundles of tubules. These tubular complexes are not typical prolamellar bodies and they are not converted into grana. Cytochemical reactions indicate that they are proteinaneous. The participation of this plastid in the possible nutritional function of the suspensor is discussed but it is pointed out that critical experimental evidence is needed before the role of the suspensor and its contents in embryogenesis can be understood.Supported by a grant from the Australian Research Grants Committee.     

The solubilization of a glucuronyltransferase involved in pea (Pisum sativum var. Alaska) glucuronoxylan synthesis.

A glucuronyltransferase involved in glucuronoxylan biosynthesis was obtained from the epicotyls of 1-week-old etiolated pea (Pisum sativum var. Alaska) seedlings and was solubilized in Triton X-100, a non-ionic detergent. The enzyme was inactivated by SDS and inhibited by Derriphat 160 and cholic acid. The enzyme was active in the presence of NN-dimethyldodecylanium-N-oxide, but was not solubilized by it. The stimulatory effect of UDP-D-xylose on the particulate and solubilized enzymes was the same, but the optimum Mn2+ concentration was lower for the solubilized enzyme, and the product formed by the solubilized enzyme has altered structure and solubility properties. Gel filtration of the solubilized enzyme on Sepharose CL-6B permitted partial separation of the stimulatory effect of UDP-D-xylose from the activity in the absence of UDP-D-xylose. The solubilized enzyme was more stable than the particulate enzyme and could be stored for 2 weeks at -20 degrees C without loss of activity.     

Transient protein expression in three Pisum sativum (green pea) varieties

The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 ( 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.     

Hollow heart of pea (Pisum sativum)

Book reviewed in this article: The Way Ahead in Plant Breeding. Proceedings of the Sixth Congress of Eucarpia. Edited by F. G. H. Lupton , G. Jenkins and R. Johnson . Basic Electron Microscope Techniques. By M. A. Hayat . The Logit Transformation (with special reference to its uses in bioassay). By W. D. Ashton . Families of Frequency Distributions. By J. K. Ord .     

Verticillium wilt of pea (Pisum sativum)

A wilt disease of garden pea (Pisum sativum) caused by Verticillium dahliae is described and the range of pathogenicity of the isolate investigated. It is pathogenic to potato, sweet pea, antirrhinum and broad bean and isolates of V. dahliae from potato, lucerne and sweet pea and V. albo-atrum from lucerne are pathogenic to pea. Since the most common disease symptoms, acropetal progression of chlorosis and necrosis of the leaves followed by premature defoliation are indistinguishable from natural senescence, it is probable that disease and senescence symptoms are confused in the field. The premature defoliation results in marked reduction in green leaf area, leaf dry weight and pod yield.     

Regeneration of shoots from pea (Pisum sativum) hypocotyl explants

A sequential indole-3-acetic acid (IAA)-zeatin treatment was applied to Pisum sativum hypocotyl explants, resulting in shoot formation from 50% of the explants. Shoots were easily rooted and transplantable plants could be obtained in 3 months. The method has been applicable to the 5 cultivars tested. Histological examination of explants suggests the shoots to be of de novo origin, which would make the system suitable for transformation experiments.     

Lipid biosynthesis by isolated plastids from greening pea, Pisum sativum

Isolated etioplasts from 8-day-old dark-grown pea seedlings incorporated [1-(14)C]acetate into lipid at a relatively low rate. Plastids from seedlings that had been illuminated for at least 2 hr showed an enhanced incorporation provided the plastids were illuminated during incubation with the labeled acetate. Dark incubation or the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased the acetate-incorporating activity of the developing chloroplasts to the level observed with etioplasts. Light had a marked effect on the type of fatty acid into which acetate was incorporated by the developing chloroplasts. Unsaturated fatty acids (mostly oleic acid) accounted for 60-80% of the incorporated label if the plastids were illuminated, but in the dark or in the presence of DCMU the unsaturated acids accounted for only 0-15% of the label incorporated into lipid. The effect of ATP on incorporation was dependent on the maturity of the chloroplasts; mature pea chloroplasts were inhibited by ATP, whereas in developing plastids there was a slight stimulation by ATP. Inhibition of acetate incorporation into lipid by DCMU appears to be due to inhibition of noncyclic phosphorylation. Incorporation was restored by reduced 2,3,5,6-tetramethylphenylenediamine, which restored phosphorylation, but not by reduced N,N,N',N'-tetramethylphenylenediamine.     

Induction of somatic embryos in pea,Pisum sativum L.

Using low concentrations of picloram (0.06 mg/l), embryoids were formed on the surface of leaf-derived callus of pea, Pisum sativum L. (c.v. Dippes Gelbe Victoria) upon transfer to liquid medium. After some days in culture, embryoids spontaneously separated from the calli, and developed into torpedo-shaped embryos, which were transferred to solid medium. In a second series of experiments, embryos were also formed by mutant 489C and a genetic line of Pisum arvense, which additionally exhibited embryogenesis also from epicotyl-derived callus. Some of the embryos showed root formation, but no shoot morphogenesis occurred. In a limited number of cases, an additional root was formed in the apparent shoot apical region after 2–5 days.     

Identification of ultraviolet-B responsive genes in the pea, Pisum sativum L.

In the present work, UV-B-repressible and UV-B-inducible genes were identified in the pea, Pisum sativum L., by rapid amplification of 3' cDNA ends through use of the polymerase chain reaction. Of the UV-B-repressible clones, psUVRub and psUVDeh represent genes encoding Rubisco activase and dehydrin, respectively. A third clone, psUVZinc, did not correspond closely in overall nucleotide sequence to any gene registered in GenBank; however, a short deduced peptide shared similarity with the photosystem-II reaction center X protein of the chlorophyll a+c-containing alga, Odontella sinensis. The UV-B-inducible clones, psUVGluc, psUVAux and psUVRib, were related to genes encoding #-1, 3-glucanase, auxin-repressed protein, and a 40S ribosomal protein, respectively. The modulation of these pea genes indicates how UV-B, through its actions as a physical stressor, affects several important physiological processes in plants.     

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.