Metabolic networks of Cucurbita maxima phloem

Metabolomic analysis aims at a comprehensive characterization of biological samples. Yet, biologically meaningful interpretations are often limited by the poor spatial and temporal resolution of the acquired data sets. One way to remedy this is to limit the complexity of the cell types being studied. Cucurbita maxima Duch. vascular exudates provide an excellent material for metabolomics in this regard. Using automated mass spectral deconvolution, over 400 components have been detected in these exudates, but only 90 of them were tentatively identified. Many amino compounds were found in vascular exudates from leaf petioles at concentrations several orders of magnitude higher than in tissue disks from the same leaves, whereas hexoses and sucrose were found in far lower amounts. In order to find the expected impact of assimilation rates on sugar levels, total phloem composition of eight leaves from four plants was followed over 4.5 days. Surprisingly, no diurnal rhythm was found for any of the phloem metabolites that was statistically valid for all eight leaves. Instead, each leaf had its own distinct vascular exudate profile similar to leaves from the same plant, but clearly different from leaves harvested from plants at the same developmental stage. Thirty to forty per cent of all metabolite levels of individual leaves were different from the average of all metabolite profiles. Using metabolic co-regulation analysis, similarities and differences between the exudate profiles were more accurately characterized through network computation, specifically with respect to nitrogen metabolism.     

Interaction of xylem and phloem during exudation and wound occlusion in Cucurbita maxima

Collection of cucurbit exudates from cut petioles has been a powerful tool for gaining knowledge on phloem sap composition without full notion of the complex exudation mechanism. Only few publications explicitly mentioned that exudates were collected from the basal side of the cut, which exudes more copiously than the apical side. This is surprising since only exudation from the apical side is supposedly driven by phloem pressure gradients. Composition of carbohydrates and pH values at both wounding sides are equal, whereas protein concentration is higher at the basal side. Apparently, exudation is far more complex than just the delivery of phloem sap. Xylem involvement is indicated by lower protein concentrations after elimination of root pressure. Moreover, dye was sucked into xylem vessels owing to relaxation of negative pressure after cutting. The lateral water efflux from the vessels increases turgor of surrounding cells including sieve elements. Simultaneously, detached parietal proteins (PP1/PP2) induce occlusion of sieve plates and cover wound surface. If root pressure is strong enough, pure xylem sap can be collected after removal of the occlusion plug at the wound surface. The present findings provide a mechanism of sap exudation in Cucurbita maxima, in which the contribution of xylem water is integrated.     

Synthesis of P-protein in mature phloem of Cucurbita maxima

Summary Cotyledons of Cucurbita maxima Duch. seedlings were provided with ¹⁴C-labeled amino acids for 12 h. Besides the bulk of labeled amino acids the sieve-tube exudate also carried labeled proteins. 80% of the incorporated radioactivity was found in the P-protein, 20% in a neutral protein, and traces were found in acidic proteins after fractionation on diethyl-aminoethyl cellulose columns. The radioactive elutes were characterized by autoradiographs of both disc- and sodium dodecyl sulfate-gelelectropherograms, and by isoelectric focusing. The P-protein fraction appeared with the void volume from the diethylaminoethyl-cellulose column. Obviously, this is the protein that gels when oxidized and that is reversibly precipitable giving rise to filaments when processed for electron microscopy. Its main component has a molecular weight of 115,000 Dalton. By isoelectric focusing this fraction separated into 3 proteins with isoelectric points of 9.8, 9.4, and 9.2. The isoelectric point 9.2-protein probably is identical with an oligomer of a 30,000 Dalton protein with neutral isoelectric point, which keeps 20% of the incorporated label. Microautoradiographs suggest that the labeled proteins were synthesized in companion cells. The results indicate that P-protein of Cucurbita maxima is synthesized continuously in mature phloem. It can be assumed that P-protein has a relatively high turn-over rate. Therefore it seems unlikely that P-protein is a structural protein.Abbreviations DEAE diethylaminoethyl - SDS sodium dodecyl sulfate - pI isoelectric point Supported by Deutsche Forschungsgemeinschaft.     

Subunit structure and interactions of the phloem proteins of Cucurbita maxima (pumpkin)

The two major proteins from the phloem exudate of Cucurbita maxima (pumpkin), PP1 and PP2, were stable in the absence of reducing agents after modification of their accessible cysteine residues with iodoacetamide. This permitted their purification without precautions to prevent oxidation. PP2, a lectin specific for oligomers of N-acetyl-D-glucosamine, was shown by sedimentation-equilibrium ultracentrifugation to be a dimer of Mr of 48000. Neither dithiothreitol nor tri-(N-acetyl-D-glucosamine) altered this value. The constituent polypeptides were linked by two buried disulphide bridges. PP2 behaved aberrantly on gel-filtration on both Sephadex and Bio-Gel unless tri-(N-acetyl-D-glucosamine) was added to the elution buffer; the Mr was then measured as 46000. Other proteins which bind oligomers of N-acetyl-D-glucosamine are also retarded on gel-filtration. Soluble phloem filaments were prepared by collection of exudate into deaerated buffer containing iodoacetamide but no reducing agent. Oxidative gellation of the filaments was prevented by rapid modification of their many accessible cysteine residues, and is assumed to have maintained the degree of polymerisation found in vivo. Those disulphide bridges which were present allowed the incorporation of approximately 60% of the PP1 and 80% of the PP2 into polymeric material. It is concluded that PP1 and PP2 are both structural proteins present in the filaments observable in vivo. PP2 had an elongated binding-site for oligomers of N-acetyl-D-glucosamine. It is suggested that this lectin immobilises bacteria and fungi to the cross-linked filaments which seal wounded phloem sieve-tubes, and thus maintains sterility.     

Characterization and sequencing of cDNA clone encoding the phloem protein PP2 of Cucurbita pepo

Direct N-terminal amino acid sequencing of the phloem protein 2 (PP2) from 3-month old Cucurbita pepo L. (pumpkin), purified by SDS-PAGE and blotted onto PVDF membrane, showed that the protein had a blocked N-terminus. However, after in situ cleavage of the polypeptide in a gel slice by cyanogen bromide, 75 residues of sequence on two cyanogen bromide fragments were determined. An oligonucle-otide probe based on this amino acid sequence was used to screen a cDNA library, constructed from mRNA of 3–5-day old seedling hypocotyls, in ZAP II. A cDNA clone (p11A) predicted an amino acid sequence of 218 residues, in full agreement with the sequences determined for two CNBr fragments of PP2, and suggests that the N-terminus of the protein is a blocked methionine residue which is cleaved off by CNBr. Two additional cDNA clones were sequenced but no heterogeneity in the PP2 sequence was found. The deduced amino acid sequence of C. pepo differs in nine residues from the recently published sequence of Cucurbita maxima (Bostwick et al., Plant Cell 4 (1992) 1539–1548). Southern blot showed that PP2 is encoded by a gene family with a relatively large number of members (estimated as 7–15 per haploid genome).     

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.Abbreviation SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Filament formation in vitro of a sieve tube protein from Cucurbita maxima and Cucurbita pepo

Summary Phloem proteins of the sieve tube exudate from Cucurbita maxima Duch. and Cucurbita pepo L. were investigated as to their filament forming ability in vitro. From the two main proteins (116000 dalton, 30000 dalton) only the 116000 dalton protein was found to form reversibly distinct filaments of 6–7 nm diameter upon removal of SH-protecting agents from the buffer, whereas the 30000 dalton protein was precipitated as amorphous material under these conditions. The protein filaments were similar to the filaments ocurring within the sieve tube cells in vivo.Abbreviations SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Diversity of the superfamily of phloem lectins (phloem protein 2) in angiosperms

Phloem protein 2 (PP2) is one of the most abundant and enigmatic proteins in the phloem sap. Although thought to be associated with structural P-protein, PP2 is translocated in the assimilate stream where its lectin activity or RNA-binding properties can exert effects over long distances. Analyzing the diversity of these proteins in vascular plants led to the identification of PP2-like genes in species from 17 angiosperm and gymnosperm genera. This wide distribution of PP2 genes in the plant kingdom indicates that they are ancient and common in vascular plants. Their presence in cereals and gymnosperms, both of which lack structural P-protein, also supports a wider role for these proteins. Within this superfamily, PP2 proteins have considerable size polymorphism. This is attributable to variability in the length of the amino terminus that extends from a highly conserved domain. The conserved PP2 domain was identified in the proteins encoded by six genes from several cucurbits, celery (Apium graveolens), and Arabidopsis that are specifically expressed in the sieve element-companion cell complex. The acquisition of additional modular domains in the amino-terminal extensions of other PP2-like proteins could reflect divergence from its phloem function.     

Molecular characterization of a phloem-specific gene encoding the filament protein, Phloem Protein 1 (PP1), from Cucurbita maxima

Sieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P-protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lectin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N-terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P-protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lectin, further suggesting an interaction between these phloem-specific proteins.     

Lipid Labeling in Normal and Nitrogen-Deficient Squash (Cucurbita maxima) Leaves

Squash (Cucurbita maxima Duchesne) plants were grown on normal and on nitrogen-deficient nutrients. The degrees of label incorporation into chloroplast lipids as well as non-chloroplast lipids were determined. Nitrogen-deficient tissues contain less chlorophyll, have a decreased chlorophyll a/b ratio, incorporate more label into phosphatidyl choline and phosphatidyl ethanolamine than into the chloroplast lipids such as mono- and digalactosyl diglycerides, have a reduced capacity to incorporate the hexose moieties into the glycolipids but normal capacity to incorporate bases into the phospholipids of non-chloroplast constituents, and have a normal level of total fatty acids even though the level of linolenate is decreased. All of this would suggest that the most evident changes in membrane lipid constituents during nitrogen-deficiency occur as changes in the chloroplast lipid constituents as opposed to the non-chloroplast lipid constituents.     

A new protein inhibitor of trypsin and activated Hageman factor from pumpkin (Cucurbita maxima) seeds

A protein inhibitor (CMTI-V; Mr 7106) of trypsin and activated Hageman factor (Factor XIIa), a serine protease involved in blood coagulation, has been isolated for the first time from pumpkin (Cucurbita maxima) seeds by means of trypsin-affinity chromatography and reverse phase high performance liquid chromatography (HPLC). The dissociation constants of the inhibitor complexes with trypsin and Factor XIIa have been determined to be 1.6 x 10(-8) and 4.1 x 10(-8) M, respectively. The primary structure of CMTI-V is reported. The protein has 68 amino acid residues and one disulfide bridge and shows a high level of sequence homology to the Potato I inhibitor family. Furthermore, its amino terminus consists of an N-acetylates Ser. The reactive site has been established to be the peptide bond between Lys44-Asp45. The modified inhibitor which has the reactive site peptide bond hydrolyzed inhibits trypsin but not the Hageman factor.     

Microbial population of the laimosphere of squash (Cucurbita maxima)

Summary The laimosphere, a term analogous to rhizosphere, describes the zone of influence of below-ground portions of shoots on soil microbial populations. Squash hypocotyls influenced microbial populations in soils 0–3 mm from hypocotyl surfaces. In this region, the laimosphere/soil ratio was 7, 36, and 7, respectively, for general bacteria, fluorescent pseudomonads, and actinomycetes.     

Different AT-rich satellite DNAs in Cucurbita pepo and Cucurbita maxima

Summary The AT-rich highly repeated satellite DNA of Cucurbita pepo (zucchini) and Cucurbita maxima (pumpkin) were cloned and their DNA structure was investigated. DNA sequencing revealed that the repeat length of satellite DNA in Cucurbita pepo is 349–352 base pairs. The percentage of AT-base pairs is about 61%. This satellite is highly conserved in restriction enzyme pattern and DNA sequence; sequence heterogeneity is about 10%. In contrast, the satellite DNA of Cucurbita maxima has a repeat length of 168–169 base pairs. This satellite is also rich in AT-base pairs (64%), existing in at least three different variants as revealed by restriction enzyme analysis and DNA sequencing. The sequence heterogeneity between these variants is about 15%. The two satellite DNAs showed no cross-hybridization to each other and sequence homology is only limited. Nevertheless, we found in the C. pepo genome a high amount of sequences resembling the satellite of C. maxima. In contrast, the satellite repeat of C. pepo is found in the C. maxima DNA only in a few copies. These observations were discussed with respect to satellite DNA evolution and compared to the data received from monocotyledonous species.     

Plastid differentiation during Cucurbita pepo (Cucurbitaceae) pollen grain development

The development of plastids in the pollen of Cucurbita pepo was followed from meiosis to pollen maturation by quantitative light and electron microscopy. Plastids are initially undifferentiated, then divide, and at late microspore stage differentiate into amyloplasts containing starch. Later the amyloplasts form lobes and divide. Amyloplasts containing a single starch grain are present from the early bicellular stage. Plastid development is considered in relation to such cytoembryological features as the pollen does not dehydrate at anthesis and germination begins 3 min after pollination.     

Adenosine triphosphatase in the phloem of Cucurbita

Summary The distribution of adenosine triphosphatase (ATPase) activity in the phloem of petioles and minor veins of Cucurbita maxima has been studied using a lead phosphate precipitation procedure. ATPase activity was localized in sieve elements, companion cells and parenchyma cells. Activity was found at the cell surfaces, associated with the dispersed P-protein of mature sieve elements, in mitochondria, sieve-element reticulum, and at specific regions of the cell walls. It is suggested that the ATPase at the phloem cell surfaces may function in intercellular transport of assimilates or ions, and that the ATPase activity associated with the P-protein may function in the translocation process or in callose deposition.     

Structure and interactions of the phloem lectin (phloem protein 2) Cus17 from Cucumis sativus

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Organization and characterization of Cucurbita phloem lectin genes

The phloem of pumpkin and squash contains a dimeric chitin-binding lectin called PP2 (phloem protein 2). We have isolated three genomic clones from pumpkin (Cucurbita maxima Duch.) that encoded PP2. One clone, gPC13-1, contained two PP2 genes that were 99.8% identical over a region of 3055 nucleotides. This conserved region included 1922 bp of 5 non-coding sequence, 844 bp of protein coding sequence (including two introns), and 289 bp of 3 non-coding sequence. To examine the conservation of the phloem lectin within the genus Cucurbita, we analyzed nine different species for PP2, its mRNA, and the genes that encode PP2. DNA blot analysis indicated that each species contained genes that encoded PP2, however, there was considerable restriction fragment length polymorphism (RFLP) among the species. PP2 gene copy number reconstructions indicated that PP2 is encoded by a small gene family (two to eight genes). Although a high level of PP2 DNA polymorphism existed among species, a single mRNA (ca. 1 kb) was detected in each species. PP2, affinity-purified from the vascular exudate of each species, reacted with PP2-specific antibodies; five species contained a single PP2 polypeptide while four species contained two PP2 polypeptides.     

The making of giant pumpkins: how selective breeding changed the phloem of Cucurbita maxima from source to sink

Despite the success of breeding programmes focused on increasing fruit size, relatively little is known about the anatomical and physiological changes required to increase reproductive allocation. To address this gap in knowledge, we compared fruit/ovary anatomy, vascular structure and phloem transport of two varieties of giant pumpkins, and their smaller fruited progenitor under controlled environmental conditions. We also modelled carbon transport into the fruit of competitively grown plants using data collected in the field. There was no evidence that changes in leaf area or photosynthetic capacity impacted fruit size. Instead, giant varieties differed in their ovary morphology and contained more phloem on a cross‐sectional area basis in their petioles and pedicels than the ancestral variety. These results suggest that sink activity is important in determining fruit size and that giant pumpkins have an enhanced capacity to transport carbon. The strong connection observed between carbon fixation, phloem structure and fruit growth in field‐grown plants indicates that breeding for large fruit has led to changes throughout the carbon transport system that could have important implications for how we think about phloem transport velocity and carbon allocation.     

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 g/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.