Characterization of two novel defense peptides from pea (Pisum sativum) seeds

A fraction that possesses antifungal activity against Aspergillus niger has been isolated from seeds of the pea (Pisum sativum) by ammonium sulfate fractionation followed by gel filtration on Sephadex G-75. On further purification by reverse-phase high performance liquid chromatography, two small cysteine-rich polypeptides were obtained (Psd1 and Psd2). They are localized primarily in vascular bundles and epidermis tissues of pea pods and exhibit high antifungal activity toward several fungi, displaying IC(50) values ranging from 0.04 to 22 microg/ml. This inhibitory activity decreases when A. niger growth medium is supplemented with cations such as Ca(2+), Mg(2+), Na(+), and K(+). Although the primary sequence of both Psd1 and Psd2 shows homology with other plant defensins, they cannot easily be assigned to any established group.     

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.     

The purification and characterization of a third storage protein (convicilin) from the seeds of pea (Pisum sativum L.).

A third storage protein, distinct from legumin and vicilin, has been purified from the seeds of pea (Pisum sativum L.). This protein has been named 'convicilin' and is present in protein bodies isolated from pea seeds. Convicilin has a subunit mol.wt. of 71 000 and a mol.wt. in its native form of 290 000. Convicilin is antigenically dissimilar to legumin, but gives a reaction of identity with vicilin when tested against antibodies raised against both proteins. However, convicilin contains no vicilin subunits and may be clearly separated from vicilin by non-dissociating techniques. Unlike vicilin, convicilin does not interact with concanavalin A, and contains insignificant amounts of carbohydrates. Limited heterogeneity, as shown by isoelectric focusing, N-terminal analysis, and CNBr cleavage, is present in convicilin isolated from a single pea variety; genetic variation of the protein between pea lines has also been observed.     

Abscisic acid levels in seeds of the gibberellin-deficient mutant lh-2 of pea (Pisum sativum)

The lh-2 mutation in garden pea ( Pisum sativum L.) blocks an early step in the gibberellin (GA) biosynthesis pathway, the three-step oxidation of ent -kaurene to ent -kaurenoic acid. As a result, only low levels of GAs, including the bioactive GA₁, are found in shoots and seeds of lh-2 plants. Mutant plants are dwarf in stature, and show increased seed abortion and decreased seed weight, compared with seeds of the tall wild-type (WT) progenitor (cv. Torsdag). The aberrant seed development of lh-2 plants is associated with reduced levels of GA₁ and GA₃, and with an accumulation of abscisic acid (ABA) in young seeds (pre-contact point). This ABA accumulation is typically 3- to 4-fold, and can be up to 6-fold, compared with control plants. To investigate whether the accumulation of ABA is partly responsible for causing the observed seed abortion in lh-2 plants, we constructed a double mutant between the lh-2 allele and wil . The wil mutation blocks ABA biosynthesis, and reduces ABA levels in young seeds by 10-fold. Introduction of the wil mutation reduces the endogenous ABA levels in young lh-2 seeds, but fails to rescue the seeds from abortion. This indicates that the effects of lh-2 on seed development are not mediated through increased ABA levels, and is consistent with previous evidence that GAs are the controlling factor underlying the lh-2 seed phenotype in pea.     

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.     

Respiratory potential and Se compounds in pea (Pisum sativum L.) plants grown from Se-enriched seeds

Selenium (Se) has been proved to be an essential element for humans and animals. However, less is known about its effects on plants. Pea plants were treated foliarly once (OT) and twice (TT) with Se solution during their flowering period. Seeds obtained from these plants contained 383 and 743 ng Se g(-1), respectively, and, together with control seeds from untreated plants (UT) containing 21 ng Se g(-1), were sown in soil in a greenhouse. Se content and its chemical form in young plants were studied, and its impact on plant respiratory potential, measured as terminal electron transport system (ETS) activity, determined. ETS activity was highest in young pea leaves with the highest Se content. Higher ETS activity possibly reflected increased glutathione peroxidase (GSH-Px) activity in mitochondria. The Se content of leaves and stems of plants grown from control seeds was similar to that in the seed, being around 40 ng Se g(-1). Se concentration in leaves of young plants grown from OT and TT seeds was 605%, and 1340% higher, respectively, than the control, and in their stems 355%, and 680% higher, respectively. The ratio of Se concentrations in OT and TT seeds was the same as in the leaves and stems in the young plants grown from them. SeMet was the major Se compound in Se-rich pea seeds and leaves, comprising 49% and 67% of the total Se content in OT and TT seeds, respectively, and 85% and 79% in the corresponding leaves.     

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.     

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.     

CO2 enrichment, drought stress and growth of Alaska pea plants (Pisum sativum)

The interaction of CO₂ enrichment and drought on water status and growth of pea plants was investigated. Pisum sativum L. (cv. Alaska) plants were grown from seeds in growth chambers using 350 and 675 μl I1 CO₂, a photon flux density of 600 μmol M-2 S-1, a 16 h photoperiod and a temperature regime of 20/14°C. The drought treatment was started at the beginning of branch initiation and lasted for 9 or 11 days. The water status of the plants was monitored daily by measuring total leaf water potential and stomatal conductance. The total leaf water potential of well-watered plants was not affected by the CO₂ level. Under draughting conditions total leaf water potential decreased, with a slower decrease under the high CO₂ regime, due, at least in part, to reduced stomatal conductance. Upon rewatering, total leaf water potential and stomatal conductance recovered within one day. High CO₂ counteracted the reduction in height and, to some extent, leaf area that developed in low CO₂ unwatered plants. Additional CO₂ had no effect on branch number and did not prevent the complete inhibition of branch development that resulted from drought stress. Removing the drought conditions resulted in a rapid recovery of the internal water status and also a rapid recovery of most, but not all, plant growth parameters.     

Design, expression, and crystallization of recombinant lectin from the garden pea (Pisum sativum)

The propeptide form of the lectin from the garden pea (Pisum sativum agglutinin) has been expressed in Escherichia coli by attaching its cDNA to an inducible promoter. By a number of criteria, including the ability to form dimers, hemagglutination titer, Western blot, and enzyme-linked immunosorbent assay, the resulting propeptide molecule is virtually indistinguishable from the mature proteolytically processed lectin isolated from peas. Preliminary crystallization experiments using the recombinant propeptide lectin yield crystals in space group P2(1)2(1)2(1) with a = 64.8 A, b = 73.8 A, and c = 109.0 A (1 A = 0.1 nm) that diffract to 2.8-A resolution. This unit cell size is quite similar to the unit cell determined for native pea lectin, suggesting that the overall structure of the recombinant prolectin is virtually identical.     

Purification and partial characterization of a glycoprotein from pea (Pisum sativum) with receptor activity for rhicadhesin,an attachment protein of Rhizobiaceae

Attachment of Rhizobium and Agrobacterium bacteria to cells of their host plants is a two-step process. The first step, direct attachment of bacteria to the plant cell wall, is mediated by the bacterial protein rhicadhesin. A putative plant receptor molecule for rhicadhesin was purified from cell walls of pea roots using a bioassay based on suppression of rhicadhesin activity. This molecule appeared to be sensitive to treatments with pronase or glycosidase. Its isoelectric point is 6.4, and its apparent molecular mass was estimated to be 32 kDa before and 29 kDa after glycosidase treatment, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ultrafiltration. The sequence of the first 29 N-terminal amino acids was determined: A-D-A-D-A-L-Q-D-L-C(?)-V-A-D-Y-A-S-V-I-L-V-N-G-F-A-S-K(Q)-(P/Q)-(L)-(I). No homology with known proteins was found. In the course of this research project the extracellular matrix protein vitronectin was reported to inhibit attachment of A. tumefaciens to carrot cells [29]. A variety of adhesive proteins, including vitronectin, contain a common cell attachment determinant with the sequence R-G-D. Since we could not detect other cell wall components able to suppress rhicadhesin activity, and since an R-G-D containing hexapeptide was also active as a receptor, we speculate that the plant receptor for rhicadhesin is a glycoprotein containing an R-G-D attachment site.     

Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.)

Polyribosomes which have template activity in the wheat germ system have been isolated from developing pea seeds. Some of the translation products have identical mobilities to the vicilin and legumin subunits by SDS-PAGE. Certain products were specifically immunoprecipitated with antisera prepared against purified vicilin and legumin fractions. Various RNA fractions including poly A-rich RNA have also been isolated from polyribosomes and shown to direct the synthesis of polyripeptides whose properties are similar to the storage protein subunits. The results are discussed in relationship to other investigations with seed storage protein biosynthesis in vitro.Abbreviations DTT dithiothreitol - SDS-PAGE SDS-polyacrylamide gel electrophoresis - TCA tricarboxylic acid     

Phylogenetic study of rhizobia nodulating pea (Pisum sativum) isolated from different geographic locations in Tunisia

Nodulated Pisum sativum plants showed the presence of native rhizobia in 16 out of 23 soil samples collected especially in northern and central Tunisia. A total of 130 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, dnaK and glnII) assigned 35 isolates to Rhizobium laguerreae, R. ruizarguesonis, Agrobacterium radiobacter, Ensifer meliloti and two putative genospecies. R. laguerreae was the most dominant species nodulating P. sativum with 63%. The isolates 21PS7 and 21PS15 were assigned to R. ruizarguesonis, and this is the first report of this species in Tunisia. Two putative new lineages were identified, since strains 25PS6, 10PS4 and 12PS15 clustered distinctly from known rhizobia species but within the R. leguminosarum complex (Rlc) with the most closely related species being R. indicum with 96.4% sequence identity. Similarly, strains 16PS2, 3PS9 and 3PS18 showed 97.4% and 97.6% similarity with R. sophorae and R. laguerreae, respectively. Based on 16S-23S intergenic spacer (IGS) fingerprinting, there was no clear association between the strains and their geographic locations. According to nodC and nodA phylogenies, strains of Rlc species and, interestingly, strain 8PS18 identified as E. meliloti, harbored the symbiotic genes of symbiovar viciae and clustered in two different clades showing heterogeneity within the symbiovar. All these strains nodulated and fixed nitrogen with pea plants. However, the strains belonging to A. radiobacter and the two remaining strains of E. meliloti were unable to nodulate P. sativum, suggesting that they were non-symbiotic strains. The results of this study further suggest that the Tunisian Rhizobium community is more diverse than previously reported.     

Cells of pea (Pisum sativum) that differentiate from G2 phase have extrachromosomal DNA.

Velocity sedimentation in an alkaline sucrose gradient of newly replicated chromosomal DNA revealed the presence of extrachromosomal DNA that was not replicated by differentiating cells in the elongation zone. The extrachromosomal DNA had a number average molecular weight of 12 X 10(6) to 15 X 10(6) and a weight average molecular weight of 25 X 10(6), corresponding to about 26 X 10(6) and 50 X 10(6) daltons, respectively, of double-stranded DNA. The molecules were stable, lasting at least 72 h after being formed. Concurrent measurements by velocity sedimentation, autoradiography, and cytophotometry of isolated nuclei indicated that the extrachromosomal molecules were associated with root-tip cells that stopped dividing and differentiated from G2 phase but not with those that stopped dividing and differentiated from G1 phase.     

Purification and characterization of N-acetylglutamate 5-phosphotransferase from pea (Pisum sativum) cotyledons.

N-Acetylglutamate 5-phosphotransferase (acetylglutamate kinase, EC 2.7.2.8) has been isolated from pea (Pisum sativum) cotyledons and purified 312-fold by using heat treatment, (NH4)2SO4 fractionation, affinity chromatography on ATP--Sepharose and ion-exchange chromatography on DEAE-cellulose. This preparation was shown on polyacrylamide-gel electrophoresis to yield one band staining with Coomassie Blue. The enzyme was shown by a variety of techniques to be composed of two different kinds of subunits, of mol.wts. 43000 and 53000 respectively. These subunits are arranged to give either a dimeric or tetrameric enzyme composed of equal numbers of each type of subunit. The dimeric and tetrameric enzyme forms are thought to be interconvertible, the equilibrium between these forms being influenced by the type of ligand bound to the subunits. Kinetic studies performed on the purified enzyme, indicated a random Bi Bi type of mechanism. The enzyme displayed apparent negative co-operativity with respect to one of its substrates, N-acetylglutamate; as a result, two Km values were found for this substrate, one at 1.9 X 10(-3) M and the other at 6.2 X 10(-3) M. A single Km value for ATP was found to be 1.7 X 10(-3) M. Allosteric regulation by arginine was also shown. A model, based on the Koshland, Némethy & Filmer [(1966) Biochemistry 5, 365-385] Sequential model, which adequately describes the kinetic and structural properties of N-acetylglutamate 5-phosphotransferase, is presented.     

Isolation and characterization of phytoferritin from pea (Pisum sativum) and Lentil (Lens esculenta).

Ferritin was isolated from the seeds of pea (Pisum sativum) and lentil (Lens esculenta). The homogeneity of the phytoferritins was established by polyacrylamide-gel electrophoresis. The subunit molecular weights were respectively 20 300 and 21 400 for hte pea and lentil proteins. A neutron low-angle scattering study established the molecular weight of the oligomer as 480 000 for pea apoferritin and 510 000 for lentil apoferritin. Although the quaternary structure of 24 polypeptide chains is preserved, the phytoferritins have a larger cavity in the interior than mammalian ferritins and can thus potentially store 1.2-1.4 times as much iron. The amino acid composition of the phytoferritins show some similarities to those of mammalian apoferritins; tryptic 'fingerprinting' reveals that there are many differences in the amino acid sequence of plant and mammalian apoferritins.     

The phenolic profile of pea (Pisum sativum): a phytochemical and pharmacological overview

Phytochemistry Reviews - Pisum sativum L., (Fabaceae), commonly known as dry, green or field pea, is one of the most popular and economically important legumes. It enjoys a worldwide culinary,...