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

Summary Proteins 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.     

Autotoxic potential of cucurbit crops

Soil sickness is often observed in cucurbit crops such as Citrullus lanatus, Cucumis melo and Cucumis sativus, but not in cucurbit crops such as Cucurbita moschata, Lagenaria leucantha and Luffa cylindrica. Results showed that root aqueous extracts of Citrullus lanatus, Cucumis melo and Cucumis sativus were autotoxic, but those of Cucurbita moschata, Momordica charantia and Luffa cylindrica were less autotoxic to the radicle elongation of respective species. Plant growth of Citrullus lanatus, Cucumis melo and Cucumis sativus were greatly inhibited by autotoxic substances released from powered root tissue at a rate of 1 g per seedling. Root exudates of Citrullus lanatus, Cucumis melo and Cucumis sativus were autotoxic to radicle elongation and seedling growth of respective species. However, root exudates of Citrullus lanatus did not inhibit radicle elongation of Cucurbita ficifolia, which is commonly used as rootstock for the grafting of Citrullus lanatus, Cucumis melo and Cucumis sativus to decrease soil-borne diseases in commercial production. It seems possible to overcome autotoxicity in cucurbit crops by grafting on Cucurbita ficifolia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development of novel chloroplast microsatellite markers for Cucumis from sequence database

The development of chloroplast microsatellite (cpSSR) markers in Cucumis species and analysis of their polymorphism and transferability were reported. Fifteen microsatellite markers, represented by mononucleotide repeats, were developed from the complete sequence of Cucumis sativus chloroplast genome. Intraspecific variation was successfully detected in C. sativus and C. melo and revealed mean 1.6 and 1.9 alleles per cpSSR locus, respectively. With the exception of two exon region-located cpSSR markers being monomorphic, each of the others amplified polymorphic fragments in C. sativus or C. melo. A total of 34 polymorphic loci were detected with these cpSSR markers in the two species. Transferability of the newly developed cpSSR markers was checked on an additional set of 41 Cucurbitaceae accessions (belonging to 12 different species), and except for two markers with no amplification in Cucurbita maxima, the others could be transferable to all the accessions tested. Of the 15 cpSSR markers, 14 markers generated fragments with expected band sizes and 13 markers detected interspecific polymorphism among the accessions. Intraspecific polymorphism was also observed within four Cucurbitaceae species excluding C. sativus and C. melo.     

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.     

Evaluation of restriction fragment length polymorphism in Cucumis melo

Summary The objectives of this study were to assess the degree of restriction fragment length polymorphism (RFLP) in Cucumis melo and to determine interrelationships among cultivated varieties. Initial screening of a genomic PstI library revealed that approximately 40% of the clones were repetitive. A total of 162 unique and low-copy sequence clones were hybridized to seven diverse accesions of C. melo and a C. sativus cultivar Pacer to evaluate RFLP variation. Of these, 130 probes (80%) detected a polymorphism between C. melo accessions and C. sativus, and the majority were polymorphic with more than one enzyme digest. In contrast, only 53 probes (33%) were useful in differentiating at least one of the seven accessions. Of those, only 9% were informative with more than one enzyme digest. This indicates that within C. melo, the differences among accessions are due to infrequent base substitutions, whereas between the two species, differences are mainly due to genome rearrangements such as insertions and deletions or numerous base substitutions. Of the informative probes, 34 were used in analyzing 44 C. melo lines to establish a data base of RFLP hybridization patterns. Percent similarity based on RFLP profiles was computed among lines and analyzed by principal component analysis, to visualize relationships among lines. There were clear demarcations among, but not within, muskmelon and honeydew groups.     

Phylogenetic relationships among Cucumis species based on the ribosomal internal transcribed spacer sequence and microsatellite markers

The phylogenetic relationships within the genus Cucumis (a total of 25 accessions belonging to 17 species) were studied using the nuclear ribosomal DNA internal transcribed spacer (ITS) region. The analysis included commercially important species such as melon (C. melo L.) and cucumber (C. sativus). Two additional cucurbit species, watermelon and zucchini, were also included as outgroups. The data obtained reflected the clustering of Cucumis species in four main groups, comprising accessions from cucumber, melon, C. metuliferus and the wild African species. Some of the species clustered in different positions from those reported in classifications previously described by other authors. The data obtained clearly identify a division between the 2n=2x = 14 species (C. sativus) and the 2n = 2x = 24 ones (C. melo and wild species). Within the wild species we identified a subgroup that included C. sagittatus and C. globosus. Oreosyce africana, also classified as Cucumis membranifolius, was shown to be nested within Cucumis. Three accessions previously classified as independent species were shown to be genotypes of Cucumis melo. A set of melon and cucumber SSRs were also used to analyse the Cucumis species and the results were compared with the ITS data. The differential amplification of the SSRs among the accessions made it possible to distinguish three main groups: melon, cucumber and the wild species, though with less detail than applying ITS. Some SSRs were shown to be specific for melon, but other SSRs were useful for producing PCR fragments in all species of the genus.We are grateful to NCRPIS, IPK in Gatersleben, Semillas Fitó S.A., Michel Pitrat and Fernando Nuez for providing seeds. We would also like to thank Vanessa Alfaro, Trinidad Martínez and Núria Galofré for their excellent technical assistance. This work was financed by project AGL2000-0360 of Spains Ministerio de Ciencia y Tecnología (MCYT). AJMs work was supported by a postdoctoral contract from Spains MCYT.     

Comparison of the ribosomal RNA genes in four closely relatedCucurbitaceae

Cucurbitaceae are characterized by a high copy number for nuclear ribosomal RNA genes. We have investigated the genomic ribosomal DNA (rDNA) of four closely related species of this family with respect to structure, length heterogeneity, and evolution. InCucumis melo (melon) there are two main length variants of rDNA repeats with 10.7 and 10.55kb.Cucumis sativus (cucumber) shows at least three repeat types with 11.5, 10.5, and 10.2kb.Cucurbita pepo (zucchini) has two different repeat types with 10.0 and 9.3kb. There are also two different repeat types inCucurbita maxima (pumpkin) of about 11.2 and 10.5kb. Restriction enzyme mapping of the genomic rDNA of these four plants and of cloned repeats ofC. sativus shows further heterogeneities which are due to methylation or point mutations. By comparison of the restriction enzyme maps it was possible to trace some evolutionary events in the family ofCucurbitaceae. Some aspects of regulation and function of the middle repetitive rRNA genes (here between 2000 and 10000 copies) are discussed.     

Evidence for graft transmission of structural phloem proteins or their precursors in heterografts of Cucurbitaceae

Interspecific and intergeneric grafts of Cucurbitaceae were used to study the mobility of structural P-proteins in the phloem. When Cucumis sativus L. scions were grafted onto Cucurbita rootstocks, at least nine additional proteins appeared on sodium dodecyl sulfate-polyacrylamide electrophoresis gels of scion exudate, 9–11 d after grafting. These proteins corresponded exactly to those of the respective Cucurbita sp. rootstock, including the filament-forming phloem protein PP1 and the phloem lectin PP2, as shown by the apparent molecular weights and peptide maps. According to probing at three sites, the additional proteins were evenly distributed within the scion. The appearance of additional proteins was correlated with the establishment of phloem bridges across the graft union. The developmental coincidence establishes that the structural proteins or their precursors are translocated in the phloem. This translocation was a universal phenomenon in Cucurbitaceae as shown by a comparative screening for additional proteins in eleven graft combinations, using Benincasa hispida (Thunb.) Cogn., Citrullus colocynthis (L.) Schrad., Cucumis melo L, C. sativus, Cucurbita ficifolia Bouché, Cucurbita maxima Duchesne ex Lam., and Trichosanthes cucumerina var. lobata Roxb. According to this screening, the direction of transmission of additional proteins depended upon the combination tested. While some graft partners failed to show exchange, some behaved as “donor” for additional proteins and still others could be both “donor” or “acceptor”. However, whether used as scion or stock, C. sativus was consistently identified as an acceptor. The occurrence of additional proteins in heterografts is discussed with regard to the transport mechanism of structural P-proteins in the phloem and its relationship to assimilate transport. Received: 18 February 1998 / Accepted: 12 May 1998     

Development of regeneration protocols for selected cucurbit cultivars

A shoot regeneration protocol was developed for five cultivars of theCucurbitaceae. The influence of the seed coat, an ethanol pre-treatment,different seed sources, NaOCl concentrations and treatment times ondecontamination were investigated. The effects of combinations of BA, kinetin,iP and TDZ with IAA in the culture medium on shoot regeneration of cotyledonaryexplants were tested. The cultivars Cucurbita maxima cv.A-line, C. maxima cv. Chicago Warted and C.pepo cv. Rolet, did not form shoots on any of the treatments.However, somatic embryos formed on C. maxima cv. ChicagoWarted explants. Cucumis melo cv. Hales Best 36 was highlyregenerable and shoots formed on all the treatments containing cytokinin. Theshoot regeneration response was significantly higher on BA-containing media thanwith the other cytokinins tested. Although Cucumis sativuscv. Ashley responded poorly, shoot development was observed on media containingBA or iP.     

Isolation and identification of the gibberellins of Cucumis sativus and Cucumis melo

Summary Thin-layer chromatography, gas-liquid chromatography, and mass spectrometry were used to identify gibberellins isolated from mature seeds of both Cucumis sativus (cucumber) and Cucumis melo (muskmelon). The gibberellins were extracted and purified by organic solvent fractionation, paper and thin-layer chromatography, and crystallization. Seeds of C. sativus were found to contain gibberellins A₁, A₃, A₄, and A₇ with A₁ the predominant species. Seeds of C. melo contained gibberellins A₁ and A₃ and a trace of A₅. Direct probe mass spectrometry of the gibberellins proved successful for identification purposes. Distinctive molecular ions and fragmentation patterns were obtained for each gibberellin.Journal Article No. 5664 from the Michigan Agricultural Experiment Station. This work was supported in part by a grant from the Herman Frasch Foundation.Portions were taken from a thesis submitted in partial fulfillment of the requirements for the Ph.D. degree, Michigan State University, 1971     

Analgesic and anti-inflammatory effects and safety profile of Cucurbita maxima and Cucumis sativus seeds

The premise of the pharmacology of natural product is to explore benefits of natural resources for the mankind. Medicines extracted from natural resources are considered as primary source for drug discovery. Thus, the current study was designed to evaluate the safety profile and explore the analgesic and anti-inflammatory activity of ethanol extract of Cucurbita maxima (C. maxima) and Cucumis sativus (C. sativus) seeds. These seeds are edible, good in taste and have been used for several therapeutic purposes. Acute toxicity of the seeds was evaluated by Lorke’s method while Eddy’s hot plate and tail immersion methods were used to assess analgesic activity in mice. Anti-inflammatory activity was evaluated by rat hind paw edema method. The seed extracts of C. maxima and C. sativus were found to be safe and showed significant analgesic and anti-inflammatory activity in comparison with the control group. The therapeutic effects of these extracts were almost comparable to aspirin and brufen. Therefore, the seeds can be used as effective analgesic and anti-inflammatory agents.     

Development and characterization of microsatellite markers in Cucumis

This study provides a set of useful SSR markers and describes their development, characterization and application for diversity studies.Sixty one Cucumis SSR markers were developed, most of them (46) from melon (Cucumis melo L.) genomic libraries. Forty of the markers (30 melon and 10 cucumber SSRs) were evaluated for length polymorphism in a sample of 13 melon genotypes and 11 cucumber (Cucumis sativus L.) genotypes. PCR-amplification revealed up to six size alleles among the melon genotypes and up to five alleles among the cucumber genotypes, with mean gene-diversity values of 0.52 and 0.28 for melon and cucumber, respectively. These differences are in accordance with the known narrower genetic background of the cucumber. SSR data were applied to phylogenetic analysis among the melon and cucumber genotypes. A clear distinction between the ’exotic’ groups and the sweet cultivated groups was demonstrated in melon. In cucumber, separation between the two sub-species, C.sativus var. sativus and C.sativus var. hardwickii,was obtained. Conservation of SSR loci between melon and cucumber was proven by sequence comparisons. Received: 17 April 2000 / Accepted: 16 May 2000     

Comparative mapping of ZYMV resistances in cucumber (Cucumis sativus L.) and melon (Cucumis melo L.)

Zucchini yellow mosaic virus (ZYMV) routinely causes significant losses in cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). ZYMV resistances from the cucumber population TMG1 and the melon plant introduction (PI) 414723 show different modes of inheritance and their genetic relationships are unknown. We used molecular markers tightly linked to ZYMV resistances from cucumber and melon for comparative mapping. A 5-kb genomic region (YCZ-5) cosegregating with the zym locus of cucumber was cloned and sequenced to reveal single nucleotide polymorphisms and indels distinguishing alleles from ZYMV-resistant (TMG1) and susceptible (Straight 8) cucumbers. A low-copy region of the YCZ-5 clone was hybridized to bacterial artificial chromosome (BAC) clones of melon and a 180-kb contig assembled. One end of this melon contig was mapped in cucumber and cosegregated with ZYMV resistance, demonstrating that physically linked regions in melon show genetic linkage in cucumber. However the YCZ-5 region segregated independently of ZYMV resistance loci in two melon families. These results establish that these sources of ZYMV resistances from cucumber TMG1 and melon PI414723 are likely non-syntenic.     

Xylem sap protein composition is conserved among different plant species

Xylem sap from broccoli (Brassica oleracea L. cv. Calabrais), rape (Brassica napus L. cv. Drakkar), pumpkin (Cucurbita maxima Duch. cv. gelber Zentner) and cucumber (Cucumis sativus L. cv. Hoffmanns Giganta) was collected by root pressure exudation from the surface of cut stems of healthy, adult plants. Total protein concentrations were in the range of 100 g ml^–1. One-dimensional gel electrophoresis (SDS–PAGE) resulted in 10–20 visible protein bands in a molecular mass range from 10 to 100 kDa. The main bands were cut out, digested with trypsin, and analysed using tandem mass spectrometry. Fifty bands resulted in amino acid sequence information that was used to perform database similarity searches. Sequences from 30 bands showed high homology to proteins present in databases. Among them, we found mostly peroxidases, but could also identify the lectin-like xylem protein XSP30, a glycine-rich protein, serine proteases, an aspartyl protease family protein, chitinases, and a lipid transfer protein-like polypeptide. Sequence analysis predicted apoplastic secretion signals for all database entries similar to the partial xylem protein sequences. This and the lack of cross-reactivity with phloem protein-specific antibodies suggest that the proteins really originate from the xylem and do not result from phloem contamination. Most of the highly similar proteins probably function in repair and defence reactions. Some of the most abundant proteins (peroxidases, chitinases, serine proteases) were present in xylem exudate of all species analysed, often in more than one band. This indicates an important basic role of these proteins in maintaining xylem function.Abbreviations CHT Chitinase - 1D One-dimensional - GRP Glycine-rich protein - SP Serine protease - SSP Subtilisin-like serine protease - POX Peroxidase     

Induction of ELF transmembrane potentials in relation to power-frequency electric field bioeffects in a plant root model system

Summary Seminal roots ofCucumis sativus andCucurbita maxima were exposed to 60 Hz electric fields of 100–500 Vm^–1 in a conducting aqueous inorganic growth medium. Root growth rates were measured to produce a dose-response relationship for each species. The species were selected for study because of their familial relationship, reported sensitivity to 60 Hz, 360 Vm^–1 electric fields, and differing average root cell sizes. The latter characteristic influences the magnitude of ELF membrane potentials induced by constant-strength applied electric fields, but does not affect the magnitude of the electric field strength tangent to the cell surface. The difference in average root cell size betweenC. sativus (smaller cells) andC. maxima (larger cells) was used to evaluate two alternate hypotheses that the observed effect on root growth is stimulated by [1] the electric field tangent to the cell surface, or (2) a field-induced perturbation in the normal transmembrane potential of the cells.The results of the dose-response relationship studies are qualitatively consistent with the hypothesis that the effect is elicited by induced transmembrane potentials. The smaller-celled roots showed a substantially higher response threshold [C. sativus; E ₀ ^TH 330 Vm^–1] than did the larger-celled species [C. maxima; E ₀ ^TH 200 Vm^–1]. At field strengths above the response thresholds in both species, the growth rate ofC. sativus roots was less affected than that ofC. maxima roots exposed to the same field strength.     

The effects of innovative silicon applications on growth and powdery mildew control in soilless-grown cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) and zucchini (<Emphasis Type="Italic">Cucurbita pepo</Emphasis> L.)

Silicon (Si) is one of the most beneficial microelements for several plants, in mediating the growth regulation in horticultural species. This research evaluated the effects of innovative Si-applications on. soilless-grown Cucumis sativus L. and Cucurbita pepo L. Crop growth, powdery mildew incidence and abiotic stress resistance were evaluated. Two experiments were carried out in a nonheated glasshouse on benches. Two new Si treatments (Si–Nanosponge complex, and one experimental fertilizer) were compared with the traditional K₂SiO₃. Topas^® EC 10 was used as control fungicide treatment. Biometric parameters, and incidence and severity of powdery mildew were measured. Cucumis sativus plants showed a severe powdery mildew infection, and no significant effect of the Si treatments was found. Cucurbita pepo plants were initially grown under lower disease pressure conditions, and the positive effect of Si treatments was found. The innovative use of Si–Nanosponge complex and the new experimental fertilizer can be considered a good alternative to traditional compounds for plant growth stimulation.     

Adaptation of cucurbit species to changes in substrate temperature: Root growth,antioxidants, and peroxidation

To investigate their response to changes in substrate temperatures, the roots from six species of cucurbit plants were exposed to 14°C, 24°C, or 34°C, while their aerial portions were maintained at natural ambient temperatures (23°C to 33°C). These species could be classified into three groups based on their stress response: Group A,Cucurbita ficifolia and C.maxima, heatsensitive but cold-tolerant; Group B,Cucumis sativus and C.melo, heat- and cold-sensitive; and Group C,Luffa cylindrica andBenincasa hispida, heat-tolerant but cold-sensitive. The highest growth rates and lowest contents of malondialdehyde (MDA) for plants in Groups A, B, and C were achieved at temperatures of 14°C, 24°C, and 24°C to 34°C, respectively. Superoxide dismutase (SOD) activity was lowest in the roots exposed to optimal growth temperatures while activities of catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (G-POD) operated coordinately in a complicated manner to prevent the accumulation of reactive oxygen species (ROS) in the root cells. Moreover, all plants, regardless of species, responded to unfavorable temperatures by increasing their synthesis of ascorbate and glutathione as well as by reducing the redox ratio of those two important antioxidants.     

The Cucumis plastome: physical map,intrageneric variation and phylogenetic relationships

Summary A restriction map of the Cucumis melo L. (melon) plastome was constructed by using several mapping approaches: single and double digestions of the chloroplast DNA (chlDNA) with endonucleases (XhoI, SmaI, SacI and PvuII) and hybridization to heterologous chlDNA probes and to isolated melon chlDNA fragments. Four plastome-coded genes were located using heterologous probes. The overall organization and gene position of the melon plastome was found to be similar to that of tobacco and other angiosperm species. Restriction patterns based on digestion of the chlDNA with nine endonucleases were obtained in over 20 wild species and cultivated varieties of Cucumis. These led to mutational analysis of the restiction sites yielding the most parsimonious phylogenetic tree of the Cucumis plastome. Most African species from a compact group (Anguria group) which is distant from the melon, the cucumber and a few other species (C. sagittatus, C. metuliferus and C. humifructus). All of these are also far apart from each other. The distribution of polymorphic restriction sites along the Cucumis plastome is described and conservative regions as well as hot spots are suggested.