Dinkar D. Sabnis

The isolation and some properties of a lectin (Haemagglutinin) from Cucurbita phloem exudate

The occurrence of high haemagglutinating (lectin) activity in phloem exudate from three cucurbit species is reported. The protein responsible for this lectin activity in Cucurbita maxima Duch. has been isolated by cation exchange chromatography on Sepharose and identified by gel electrophoresis. The lectin showed agglutinating activity at concentrations as low as 0.1 μg/ml. No sugar, including those transported in the phloem of these species, interacted with agglutination. The lectin could not be extracted from cucurbit seed, but appeared in 5-day old seedlings. The possible role of a lectin in the sieve element is discussed.

Immunocytochemical localisation of phloem lectin from Cucurbita maxima using peroxidase and colloidal-gold labels

Antibodies were raised against lectin purified from the sieve-tube exudate of Cucurbita maxima. Immunocytochemistry, using peroxidase-labelled antibodies and Protein A-colloidal gold, was employed to determine the location of the lectin within the tissues and cells of C. maxima and other cucurbit species. The anti-lectin antibodies bound to P-protein aggregates in sieve elements and companion cells, predominantly in the extrafascicular phloem of C. maxima. This may reflect the low rate of translocation in these cells. Under the electron microscope, the lectin was shown to be a component of P-protein filaments and was also found in association with the sieve-tube reticulum which lines the plasmalemma. The anti-lectin antibodies reacted with sieve-tube proteins from other species of the genus Cucurbita but showed only limited reaction with other genera. We suggest that the lectin serves to anchor P-protein filaments and associated proteins to the parietal layer of sieve elements.

Heterogeneity in phloem protein complements from different species : Consequences of hypotheses concerned with P-protein function.

Protein subunits present in phloem exudate from 17 cultivars, 5 species and 3 genera of the Cucurbitaceae have been fractionated by SDS-gel electrophoresis. The degree of difference in the phloem protein patterns appears to reflect the taxonomic relationships of the plants: there were major differences among genera, significant differences and similarities among species, and relatively few differences among cultivars of a single species. A representative of another family (Acer pseudoplatanus; Aceraceae) provided a completely different electrophoretic pattern. Each plant displayed a consistent protein pattern, irrespective of the organ from which exudate was obtained or the age and physiological status of the plant. Neither complete etiolation nor transition to the flowering phase effected any change in phloem proteins. The differences in phloem proteins between plants and the unusual properties of major subunits from different protein complements, render improbable many of the present ideas concerning the origin and function of P-protein.

Calcium-binding protein in sieve tube exudate.

A calcium-binding macromolecule, with an estimated molecular weight greater than 100,000, was detected in phloem exudate from Cucurbita maxima and related species. The macromolecule was a component of sieve tube sap, rather than a contaminant leached from cell walls or cut parenchyma cells during exudate collection. The protein nature of this macromolecule was deduced from its size, lability, susceptibility to proteolytic digestion, and by the dependence of calcium-binding activity on thiol-protecting agents. This protein is distinct from the major proteins of exudate and does not appear to be related to calmodulin.

A comparative analysis of phloem exudate proteins from Cucumis melo, Cucumis sativus and Cucurbita maxima by polyacrylamide gel electrophoresis and isoelectric focusing.

SummaryProteins in sieve tube exudate from Cucumis melo L., Cucumis sativus L. and Cucurbita maxima Duch. were analysed by gel electrophoresis and isoelectric focusing. Estimated molecular weights and isoelectric points for the major and minor proteins from each plant species are presented. Electrophoresis revealed striking differences between the protein complements of exudatc from the two genera investigated. Similarly, although a few exudate proteins from the two species of Cucumis possessed identical molecular weights, several major proteins were peculiar to each species. Isoelectric focusing of proteins in exudate samples from the three plants confirmed the marked differences in their protein complements. Furthermore, focusing also revealed differences between cultivars of Cucumis sativus. Both Cucumis sativus and Cucurbita maxima possessed relatively large amounts of basic proteins; these were absent in exudate from Cucumis melo. The implications of these results are discussed in relation to present concepts regarding the interrelationships and possible functional roles of P-proteins.

The heterogeneity of phloem exudate proteins from different plants: A comparative survey of ten plants using polyacrylamide gel electrophoresis.

SummaryProteins in sieve tube exudate from Ricinus communis L., Acer pseudoplatanus L., Aesculus hippocastanum L., Cucumis melo L., and two cultivars each of Cucumis sativus L., Cucurbita pepo L. and Cucurbita maxima Duchesne were fractionated and compared using polyacrylamide gel electrophoresis. Striking differences in major exudate proteins were displayed among the genera and species examined. Even cultivars within a single species, although showing general similarities, differed in some prominent proteins. Estimated molecular weights of the major exudate proteins from each plant are presented. The effects of reducing and chaotropic agents on the aggregation and subunit composition of exudate proteins from Cucumis sativus have been investigated. The problems involved in relating structure, function and biochemistry of P-protein are discussed.

Colchicine-binding protein from phloem and xylem of a higher plant.

SummaryColchicine has been demonstrated by ion-exchange and by gelfiltration assay to bind to a protein fraction derived from the higher plant Heracleum mantegazzianum. Colchicine-binding protein from a plant source was much more unstable than tubulin from animal preparations. The tissues of Heracleum vary in their content of colchicine-binding activity. No activity was obtained from non-vascular tissue. Phloem has at most, twice as much activity as xylem. The significance of these results is discussed in relation to a proposed degree of homology between P protein of phloem and microtubule protein.

Studies on the possible occurrence of actomyosin-like proteins in phloem

SummaryExtracts of large quantities of petioles and of isolated vascular tissues of Heracleum mantegazzianum have been analysed for actomyosin-like contractile proteins. Concentrated preparations, subjected to a standard isolation and purification procedure for actomyosin, failed to demonstrate either superprecipitation or viscosity changes in response to the addition of ATP and divalent cations. In addition, ATPase activities in phloem and xylem extracts have been fractionated by gel electrophoresis and characterised with regard to their substrate specificity, pH optima and ion requirements. Phloem extracts provide two phosphatases: one is non-specific in its substrate requirements; the other is a nucleoside triphosphatase but is stimulated only by monovalent cations and is also present in xylem extracts. All the enzymes are strongly inhibited by divalent cations and do not possess any of the characteristics of ATPases associated with contractile systems. The results are discussed in relation to the postulated involvement of contractile proteins in the translocation of sugars in phloem.

A phloem-specific, lectin-like protein is located in pine sieve-element plastids by immunocytochemistry

Two co-purifying phloem polypeptides of 24 and 25 kilodaltons (kDa) were isolated from homogenates of Pinus sabiniana Dougl. phloem by differential centrifugation, selective solubilization and electrophoresis, and rabbit antibodies raised against them. The antisera were found to be specific for doublet bands between 23 and 25 kDa in Western blots of whole phloem extracts of Pinus species; no xylem polypeptides were labelled, nor did labelling occur in blots of phloem extracts from other genera in the Pinaceae. Solubilized phloem polypeptides bind strongly to chitin (oligomeric N-acetylglucosamine) columns and are sensitive to thiol reagents, both characteristics which relate them to phloemspecific lectins isolated from angiosperm species (C. Allen, 1979, Biochem. J. 183, 133–137; A.K. Gietl et al., 1979, Planta 144, 367–371). Fluorescence microscopy and immuno-gold electron microscopic cytochemistry demonstrated antigenic sites specifically associated with protein crystals peculiar to the sieve-element plastids of the Pinaceae.

P-Protein in sieve elements : I. Ultrastructure after treatment with vinblastine and colchicine.

SummaryThe ultrastructure of P1 and P2 proteins in the sieve elements of Heracleum mantegazzianum is described. P1-protein tubules are closely associated with stacks of membranes, are often linked by short cross-bridges, and occasionally display a hexagonal packing. Incubation with the alkaloids vinblastine and colchicine had no discernible effects on the ultrastructure of the sieve elements at various stages during differentiation. Evidence for and against any similarities between P1-protein tubules and cytoplasmic microtubules is discussed.