Determination of pea (Pisum sativum L.) root lectin using an enzyme-linked immunoassay

Root lectins are believed to participate in the recognition between Rhizobium and its leguminous host plant. Among other factors, testing this hypothesis is difficult because of the very low amounts in which root lectins are produced. A double-antibody-sandwich enzyme-linked immunoassay, was used to determine nanogram quantities of pea lectin in root slime and salt extracts of root cell-wall material when pea seedlings were 4 and 7 d old. In addition, a critical NO ₃ ^- concentration (20 mM) which inhibited nodulation was found, and the lectin present in root slime and salt extracts of root cell walls of 4- and 7-d-old peas supplied with 20 mM NO ₃ ^- was comparatively determined. With the enzyme-linked immunoassay, lectin quantities ranging between 20 and 100 nanograms could be determined. The assay is not affected by monomeric mannose and glucose (pealectin haptens). The slime of the 4-d-old roots contained more lectin than the slime of the 7-d-old roots. Salt-extractable, cell-wall-associated lectin accumulated in the older roots. Nitrate affected slime and cell-wall production, and the extractability of cell-wall material in both age groups. The presence of NO ₃ ^- increased lectin in the slime, most notably in the younger roots; the relative amount of lectin in the slime was almost doubled. The cell-wall-associated, salt-extractable lectin decreased two- to threefold compared with the control group.Abbreviations ELISA enzyme-linked immunoassay - PTN 0.01 M phosphate buffer (pH 7.4), containing 0.15 M NaCl, 0.05% Tween-20 and 0.02% NaN₃ Dedicated to Professor A. Quispel on the occasion of his retirement     

Distribution of glucose/mannose-specific isolectins in pea (Pisum sativum L.) seedlings

We report on the distribution and initial characterization of glucose/mannose-specific isolectins of 4- and 7-d-old pea (Pisum sativum L.) seedlings grown with or without nitrate supply. Particular attention was payed to root lectin, which probably functions as a determinant of host-plant specificity during the infection of pea roots by Rhizobium leguminosarum bv. viciae. A pair of seedling cotyledons yielded 545±49 g of affinity-purified lectin, approx. 25% more lectin than did dry seeds. Shoots and roots of 4-d-old seedlings contained 100-fold less lectin than cotyledons, whereas only traces of lectin could be found in shoots and roots from 7-d-old seedlings. Polypeptides with a subunit structure similar to the precursor of the pea seed lectin could be demonstrated in cotyledons, shoots and roots. Chromatofocusing and isoelectric focusing showed that seed and non-seed isolectin differ in composition. An isolectin with an isoelectric point at pH 7.2 appeared to be a typical pea seed isolectin, whereas an isolectin focusing at pH 6.1 was the major non-seed lectin. The latter isolectin was also found in root cell-wall extracts, detached root hairs and root-surface washings. All non-seed isolectins were cross-reactive with rabbit antiserum raised against the seed isolectin with an isolectric point at pH 6.1. A protein similar to this acidic glucose/mannose-specific seed isolectin possibly represents the major lectin to be encountered by Rhizobium leguminosarum bv. viciae in the pea rhizosphere and at the root surface. Growth of pea seedlings in a nitrate-rich medium neither affected the distribution of isolectins nor their hemagglutination activity; however, the yield of affinity-purified root lectin was significantly reduced whereas shoot lectin yield slightly increased. Agglutination-inhibition tests demonstrated an overall similar sugar-binding specificity for pea seed and non-seed lectin. However root lectin from seedlings grown with or without nitrate supplement, and shoot lectin from nitrate-supplied seedlings showed a slightly different spectrum of sugar binding. The absorption spectra obtained by circular dichroism of seed and root lectin in the presence of a hapten also differed. These data indicate that nutritional conditions may affect the sugar-binding activity of non-seed isolectin, and that despite their similarities, seed and non-seed isolectins have different properties that may reflect tissue-specialization.Abbreviations IEF isoelectric focusing - MW molecular weight - pI isoelectric point - Psl1, Psl2 and Psl3 pea isolectins - SDSPAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis The authors wish to thank Professors L. Kanarek and M. van Poucke for helpful discussions.     

Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots

The lectin on the surface of 4- and 5-dold pea roots was located by the use of indirect immunofluorescence. Specific antibodies raised in rabbits against pea seed isolectin 2, which crossreact with root lectins, were used as primary immunoglobulins and were visualized with fluorescein- or tetramethylrhodamine-isothiocyanate-labeled goat antirabbit immunoglobulin G. Lectin was observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. On both types of cells, lectin was concentrated in dense small patches rather than uniformly distributed. Lectin-positive young hairs were grouped opposite the (proto)xylematic poles. Older but still-elongating root hairs presented only traces of lectin or none at all. A similar pattern of distribution was found in different pea cultivars, as well as in a supernodulating and a non-nodulating pea mutant. Growth in a nitrate concentration which inhibits nodulation did not affect lectin distribution on the surface of pea roots of this age. We tested whether or not the root zones where lectin was observed were susceptible to infection by Rhizobium leguminosarum. When low inoculum doses (consisting of less than 10⁶ bacteria·ml^-1) were placed next to lectin-positive epidermal cells and on newly formed root hairs, nodules on the primary roots were formed in 73% and 90% of the plants, respectively. Only a few plants showed primary root nodulation when the inoculum was placed on the root zone where lectin was scarce or absent. These results show that lectin is present at those sites on the pea root that are susceptible to infection by the bacterial symbiont.Abbreviations FITC fluorescein isothiocyanate - TRIC tetramethylrhodamine isothiocyanate     

Glycolytic enzymes in non-photosynthetic plastids of pea (Pisum sativum L.) roots

The presence of the glycolytic enzymes from hexokinase to pyruvate kinase in plastids of seedling pea (Pisum sativum L.) roots was investigated. The recoveries, latencies and specific activities of each enzyme in different fractions was compared with those of organelle marker enzymes. Tryptic-digestion experiments were performed on each enzyme to determine whether activities were bound within membranes. The results indicate that hexokinase (EC 2.7.1.2) and phosphoglyceromutase (EC 5.4.2.1) are absent from pea root plastids. The possible function of the remaining enzymes is considered.Abbreviations GADPH glyceraldehyde 3-phosphate dehydrogenase - PFK phosphofructokinase - PFP pyrophosphate: fructose 6-phosphate 1-phosphotransferase Bronwen A. Trimming gratefully acknowledges the award of a studentship from the Science and Engineering Research Council     

The immuno-histochemical localization of lectin in pea seeds (Pisum sativum L.)

The lectin from the garden pea (Pisum sativum L.) has been localized at the ultrastructural level by the unlabeled peroxidase-antiperoxidase procedure of L.A. Sternberger et al. (1970, J. Histochem. Cytochem 18, 315–333) in 24 h imbibed seeds. Upon examination by light microscopy and transmission electron microscopy, the lectin was only found in the protein bodies of cotyledons and embryo axis. Cell walls as well as membraneous fractions were completely devoid of lectin. These results are discussed in relation to the possible physiological function of seed lectins.Abbreviations PBS phosphate-buffered saline - TBS Tris-buffered saline - PAP-complex horseradish peroxidase-antihorseradish peroxidase soluble complex - NGS normal goat serum - TBS^* Tris-buffered saline containing 0.5 M NaCl, pH 7.6     

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     

Plasmatubules in transfer cells of pea (Pisum sativum L.)

Plasmatubules are tubular evaginations of the plasmalemma. They have previously been found at sites where high solute flux between apoplast and symplast occurs for a short period and where wall proliferations of the transfer cell type have not been developed (Harris et al. 1982, Planta 156, 461–465). In this paper we describe the distribution of plasmatubules in transfer cells of the leaf minor veins of Pisum sativum L. Transfer cells are found in these veins associated both with phloem sieve elements and with xylem vessels. Plasmatubules were found, in both types of transfer cell and it is suggested that the specific distribution of the plasmatubules may reflect further membrane amplification within the transfer cell for uptake of solute from apoplast into symplast.     

Capacity for fermentation in roots and Rhizobium nodules of Pisum sativum L.

The aim of this work was to compare the capacities for fermentation and synthesis of malate from phosphoenolpyruvate in roots and Rhizobium nodules of Pisum sativum. The nodules and the cortices and apices of roots had similar activities of glycolytic enzymes and enzymes of ethanolic and lactic fermentation when expressed on a protein basis. The activity of phosphoenolpyruvate carboxylase was similar in nodules and apices, and three to four fold lower in cortices. All three tissues had very high activities of malate dehydrogenase, significant activity of NADP-malic enzyme, and no detectable activity of phosphoenolpyruvate carboxykinase. These results do not support the belief that nodules have a substantially greater capacity to convert phosphoenolpyruvate to malate than roots, or that there are major qualitative differences in the pathways of fermentation of nodules and roots.Abbreviation PEP phosphoenolpyruvate     

Changes in mitochondrial DNA levels during development of pea (Pisum sativum L.)

The percentage of mitochondrial DNA (mtDNA) present in total DNA isolated from pea tissues was determined using labeled mtDNA in reassociation kinetics reactions. Embryos contained the highest level of mtDNA, equal to 1.5% of total DNA. This value decreased in light- and dark-grown shoots and leaves, and roots. The lowest value found was in dark-grown shoots; their total DNA contained only 0.3% mtDNA. This may be a reflection of increased nuclear ploidy levels without concomitant mtDNA synthesis. It was possible to compare the mtDNA values directly with previous estimates of the amount of chloroplast DNA (ctDNA) per cell because the same preparations of total DNA were used for both analyses. The embryo contained 1.5% of both mtDNA and ctDNA; this equals 410 copies of mtDNA and 1200 copies of ctDNA per diploid cell. Whereas mtDNA levels decreased to 260 copies in leaf cells of pea, the number of copies of ctDNA increased to 10300. In addition, the levels of ctDNA in first leaves of dark-grown and light-transferred pea were determined, and it was found that leaves of plants maintained in the dark had the same percentage of ctDNA as those transferred to the light.Abbreviations ctDNA chloroplast DNA - mtDNA mitochondrial DNA     

Interaction of gibberellic and indole-3-acetic acid on root formation in pea (Pisum sativum L.) epicotyl cuttings

Indole-3-acetic acid (IAA) strongly enhanced rooting of etiolated pea epicotyl cuttings while gibberellic acid (GA₃) enhanced rooting only slightly. The promoting effects of the hormones appeared not until 14 d after the onset of treatment. When GA₃ and IAA were applied together, the initiation of rooting started already after 6 d after onset of treatment. It is suggested that gibberellin plays an important role, in combination with auxin, in the initiation of root formation in Pisum cuttings.Abbreviations IAA Indole-3-acetic acid - GA₃ Gibberellic acid     

Purification and properties of nitrite reductase from roots of pea (Pisum sativum cv. Meteor)

Nitrite reductase (EC 1.6.6.4) prepared from pea roots was found to be immunologically indistinguishable from pea leaf nitrite reductase. Comparisons of the pea root enzyme with nitrite reductase from leaf sources showed a close similarity in inhibition properties, light absorption spectrum, and electron paramagnetic resonance signals. The resemblances indicate that the root nitrite reductase is a sirohaem enzyme and that it functions in the same manner as the leaf enzyme in spite of the difference in reductant supply implicit in its location in a non-photosynthetic tissue.Abbreviations DEAE diethylaminoethyl - EPR electron paramagnetic resonance - NIR nitrite reductase - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

The supply of reducing power for nitrite reduction in plastids of seedling pea roots (Pisum sativum L.)

Plastids were separated from extracts of pea (Pisum sativum L.) roots by sucrose-density-gradient centrifugation. The incubation of roots of intact pea seedlings in solutions containing 10 mM KNO₃ resulted in increased plastid activity of nitrite reductase and to a lesser extent glutamine synthetase. There were also substantial increases in the activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenases. No other plastid-located enzymes of nitrate assimilation or carbohydrate oxidation showed evidence of increased activity in response to the induction of nitrate assimilation. Studies with [1-¹⁴C]-and [6-¹⁴C]glucose indicated that there was an increased flow of carbon through the plastid-located pentose-phosphate pathway concurrent with the induction of nitrate assimilation. It is suggested that there is a close interaction through the supply and demand for reductant between the pathway of nitrite assimilation and the pentose-phosphate pathway located in the plastid.     

Accumulation of 14C from exogenous labelled auxin in lateral root primordia of intact pea seedlings (Pisum sativum L.)

When [¹⁴C]indol-3yl-acetic acid was applied to the apical bud of 5-day old dwarf pea seedlings which possessed unbranched primary roots, a small amount of ¹⁴C was transported into the root system at a velocity of 11–14 mm h^-1. Most of the ¹⁴C which entered the primary root accumulated in the young lateral root primordia, including the smallest detectable (20–30 mm from the primary root tip). In older (8-d old) seedlings in which the primary root bore well-developed lateral roots, ¹⁴C also accumulated in the tertiary root primordia. In contrast, little ¹⁴C was detected in the apical region of the primary root or, in older plants, in the apices of the lateral roots.Abbreviations IAA indol-3yl-acetic acid     

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.     

Soluble and microsomal glutatione S-transferase activities in pea seedlings (Pisum sativum L.)

Epicotyl and primary leaves of pea seedlings (Pisum sativum L., var. Alaska) were found to contain soluble and microsomal enzymes catalyzing the addition of glutathione to the olefinic double bond of cinnamic acid. Glutathione S-cinnamoyl transfer was also obtained with enzyme preparations from potato slices and cell suspension cultures of parsley and soybean.The pea transferases had pH-optima between pH 7.4 and 7.8 K_m-values were 0.1–0.4 mM and 1–4 mM for cinnamic acid and glutathione, respectively. V-values were between 2–15 nmol mg^-1 protein x min.Chromatography on Sephacryl S-200 indicated that the soluble pea glutathione S-cinnamoyl transferase activity existed in molecular weight forms of 37,000, 75,000, and 150,000. The glutathione-dependent cleavage of the herbicide fluorodifen was catalyzed by a different soluble enzyme activity which eluted in molecular weight positions of 47,000 and/or 82,000.The microsomal fraction from pea primary leaves also catalyzed the conjugation of the carcinogen benzo[]pyrene with glutathione.Abbreviations GSH glutathione - DDE 1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethylene - DDMU l-Chloro-2,2-bis-(4-chlorophenyl)-ethylene     

Transfer of exogenous auxin from the phloem to the polar auxin transport pathway in pea (Pisum sativum L.)

When [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA) was applied to the upper surface of a mature foliage leaf of garden pea (Pisum sativum L. cv. Alderman), ¹⁴C effluxed basipetally but not acropetally from 30-mm-long internode segments excised 4 h after the application of [1-¹⁴C]IAA. This basipetal efflux was strongly inhibited by the inclusion of 3.10^–6 mol· dm³ N-1-naphthylphthalamic acid (NPA) in the efflux buffer. In contrast, when [¹⁴C] sucrose was applied to the leaf, the efflux of label from stem segments excised subsequently was neither polar nor sensitive to NPA. The [1-¹⁴C]IAA was initially exported from mature leaves in the phloem — transport was rapid and apolar; label was recovered from aphids feeding on the stem; and label was recovered in exudates collected from severed petioles in 20 mM ethylenediaminetetraacetic acid. No ¹⁴C was detected in aphids feeding on the stems of plants to which [1-¹⁴C]IAA had been applied apically, even though the internode on which they were feeding transported considerable quantities of label. Localised applications of NPA to the stem strongly inhibited the basipetal transport of apically applied [1-¹⁴C]IAA, but did not affect transport of [1-¹⁴C]IAA in the phloem. These results demonstrate for the first time that IAA exported from leaves in the phloem can be transferred into the extravascular polar auxin transport pathway but that reciprocal transfer probably does not occur. In intact plants, transfer of foliar-applied [1-¹⁴C]IAA from the phloem to the polar auxin transport pathway was confined to immature tissues at the shoot apex. In plants in which all tissues above the fed leaf were removed before labelling, a limited transfer of IAA occurred in more mature regions of the stem.Abbreviations IAA indol-3yl-acetic acid - EDTA ethylenediaminetetraacetic acid - NPA N-1-naphthylphthalamic acid We are grateful to the Nuffield Foundation for supporting this research under the NUF-URB95 scheme and for the provision of a bursary to A.J.C. We thank Professor Dennis A. Baker for constructive comments on a draft of this paper and Mrs. Rosemary Bell for her able technical assistance.