Evaluating the Potential Value of Natural Product Cuminic Acid against Plant Pathogenic Fungi in Cucumber.

Fusarium wilt and anthracnose are two major diseases which limit the yield and quality of cucumber worldwide. Cuminic acid was extracted from the seed of Cuminum cyminum L. The mean EC50 values of cuminic acid for inhibiting mycelial growth and zoospore germination of five Fusarium oxysporum f. sp. cucumerinum strains were 25.66 ± 3.02 µg/mL and 15.99 ± 2.19 µg/mL, and of four Colletotrichum lagenarium (Pass.) Ellis and Halsted strains were 29.53 ± 3.18 µg/mL and 18.41 ± 2.78 µg/mL, respectively. In greenhouse experiments, cuminic acid at 2000 µg/mL exhibited 70.77% protective and 62.63% curative efficacies against F. oxysporum, and 65.43% protective and 55.46% curative efficacies against C. lagenarium. Moreover, the translocation behavior of cuminic acid, determined by high performance liquid chromatography (HPLC), showed that it could be readily absorbed and transported upward and downward in cucumber. Importantly, superoxide dismutase (SOD) and pyphenol oxidase (PPO) activities of cucumber leaves treated with cuminic acid increased significantly. All results indicated that cuminic acid showed antifungal activity, and could be used as a botanical fungicide in disease management. This study encourages further investigation on the mechanism of action of cuminic acid and the development of alternative antifungal drugs.

SELDI-TOF MS-based discovery of a biomarker in Cucumis sativus seeds exposed to CuO nanoparticles.

Metal oxide nanoparticles (NPs) can inhibit plant seed germination and root elongation via the release of metal ions. In the present study, two acute phytotoxicity tests, seed germination and root elongation tests, were conducted on cucumber seeds (Cucumis sativus) treated with bulk copper oxide (CuO) and CuO NPs. Two concentrations of bulk CuO and CuO NPs, 200 and 600ppm, were used to test the inhibition rate of root germination; both concentrations of bulk CuO weakly inhibited seed germination, whereas CuO NPs significantly inhibited germination, showing a low germination rate of 23.3% at 600ppm. Root elongation tests demonstrated that CuO NPs were much stronger inhibitors than bulk CuO. SELDI-TOF MS analysis showed that 34 proteins were differentially expressed in cucumber seeds after exposure to CuO NPs, with the expression patterns of at least 9 proteins highly differing from those in seeds treated with bulk CuO and in control plants. Therefore, these 9 proteins were used to identify CuO NP-specific biomarkers in cucumber plants exposed to CuO NPs. A 5977-m/z protein was the most distinguishable biomarker for determining phytotoxicity by CuO NPs. Principal component analysis (PCA) of the SELDI-TOF MS results showed variability in the modes of inhibitory action on cucumber seeds and roots. To our knowledge, this is the first study to demonstrate that the phytotoxic effect of metal oxide NPs on plants is not caused by the same mode of action as other toxins.

[Role of phytochrome in organ formation processes in Cucumis sativus L].

The role of phytochrome B in the organogenesis process in the apical and axillary shoot meristems during early ontogenesis stages in cucumber Cucumis sativus L. at photoperiods (day/night) 10/14, 16/8 h, and continuous light in comparison with wild type plants and phytochrome B-deficient mutant (lh-mutant) was investigated. In mutant phytochrome B, deficiency caused inhibition of initiation of leaves both in the leading shoot and off-shoots and increased the number of flower buds (IV stage of organogenesis). With continuous light, the number of off-shoots and flowers during stage IV of organogenesis in wild-type plants increased twofold in comparison with the mutant. Short-term temperature drops did not induce floral ontogenesis in mutants but increased the number of off-shoots in both experimental variants during a long photoperiod and continuous light situations. We propose that phytochrome B, by increasing the compactness of chromatin, may facilitate coordination of ontogenesis processes with changing environmental conditions.

Characterization of CsSEF1 gene encoding putative CCCH-type zinc finger protein expressed during cucumber somatic embryogenesis.

Somatic embryos obtained in vitro are a form of vegetative reproduction that can be used in artificial seed technology, as well as a model to study the principles of plant development. In order to isolate the genes involved in somatic embryogenesis of the cucumber (Cucumis sativus L.), we utilized the suppression subtractive hybridization (SSH). One of the obtained sequences was the CsSEF1 clone (Cucumis sativus Somatic Embryogenesis Zinc Finger 1), with a level of expression that sharply increased with the induction of embryogenesis. The full length cDNA of CsSEF1 encodes the putative 307 amino acid long protein containing three zinc finger motifs, two with CCCH and one with the atypical CHCH pattern. The CsSEF1 protein shows significant similarity to other proteins from plants, in which the zinc fingers arrangement and patterns are very similar. Transcripts of CsSEF1 were localized in the apical part of somatic embryos, starting as early as the polarity was visible and in later developmental stages marking the cotyledon primordia and procambium tissues. As a result of transferring an antisense fragment of CsSEF1 into Arabidopsis thaliana abnormalities in zygotic embryos and also in cotyledons and root development were observed.

[Daily temperature gradients and processes of organogenesis in apical meristem of Cucumis sativus L].

We studied the influence of daily temperature gradients on organogenesis in apical and axil shoot meristems at different developmental stages in Cucumis sativus L. The level of organogenic activity of meristems was determined according to the number of leaf primordia on the main and lateral shoots, number of 2nd order shoots, and rudiments of flowers of different levels of development. At the studied ontogenetic stages (mesotrophic seedling or juvenile state), plants were grown under the controlled conditions: photoperiod 12 h, light intensity 100 Wt/m2, range of mean daily temperatures 20 ... 30 degrees C, and daily temperature gradients -20 ... +20 degrees C. After the temperature treatment, some plants were returned to the optimal, for growth and development, conditions for two weeks (aftereffect). Three types of organogenic activity of meristems in response to the influence of variable daily temperatures were described: stimulation, inhibition, or absence of effect. The phenomenon of stimulation includes two subtypes: optimization, when a maximum effect, observed at other constant temperatures, was attained under the influence of variable temperatures and maximization, when maximum values markedly exceeded those at constant temperatures. The patterns described are preserved on the whole in the aftereffect of daily temperatures.

Cucumber (Cucumis sativus L.).

We describe two novel Agrobacterium tumefaciens-based methods of cucumber transformation. The first involves direct regeneration from leaf microexplants selected on kanamycin-containing medium. The second involves regeneration from a long-term established embryogenic suspension culture emitting green autofluorescence (GAF) and selection on medium containing hygromycin. In the latter method, GAF was used as a reporter, thereby allowing a simple and reliable identification of transgenic cells with a high regeneration capacity. (No false positives were observed.) The transformation efficiency in the leaf microexplants fluctuated from 0.8 to 6.5% of the primary explants, whereas in the embryogenic suspension-cultured cells it varied from 6.4 to 17.9% of the aggregates. In the GAF method, the step involving the elimination of the Agrobacterium cells by antibiotics could be omitted; however, this reduced the transformation efficiency to about 3%. The time required from inoculation to regenerated plant in the greenhouse was the same for both methods, but the GAF method required more preinoculation time than the leaf microexplant method.

The perisperm-endosperm envelope in Cucumis: structure, proton diffusion and cell wall hydrolysing activity.

BACKGROUND: and Aims The envelope surrounding the embryo in cucurbit seed, which consists of a single layer of live endosperm cells covered by lipid- and callose-rich layers, is reported to show semi-permeability and also to act as the primary barrier to radicle emergence. Structure, development and permeability of the envelope and activity of cell wall hydrolases during germination of cucumber and muskmelon seeds were investigated. METHODS: Sections of seeds were stained with aniline blue and Sudan III. Proton diffusion and endo-beta-mannanase activity were detected by tissue printing. A gel-diffusion assay was performed to quantify endo-beta-mannanase activity, while the activity of beta-glucanase was determined with laminarin as the substrate and glucose formation measured using the GOD-POD method. KEY RESULTS: The lipid layer differentiated during seed development in cucumber in the epidermis of a multilayered nucellus, whereas the callose layer appeared to develop outside the endosperm cell layer. Accordingly, the envelope has been called the perisperm-endosperm (PE) envelope. Chloroform treatment of seeds, which resulted in a substantial reduction in Sudan staining of the lipid layer, also enhanced the permeability of the PE envelope to 2,3,5-triphenyltetrazolium chloride. Proton diffusion occurred when the PE envelopes from seeds had their inner surface in contact with bromocresol purple-containing agarose gels, but not when their outer surface was in contact. Substantial endo-beta-mannanase activity was present in the caps of the PE envelopes, whereas a marked increase in beta-glucanase activity was observed in radicles prior to germination. CONCLUSIONS: The lipid layer seems to contribute to the semi-permeability of the PE envelope. The diffusion of protons might create an acidic environment conducive to the activity of cell wall hydrolases, namely endo-beta-mannanase (EC 3.2.1.78) and beta-glucanase [beta(1-->3)glucanohydrolase; EC 3.2.1.6], which, in turn, may play a role in the weakening of the PE envelope necessary for the protrusion of the radicle in cucumber and muskmelon seeds.

The modulating effect of the perisperm-endosperm envelope on ABA-inhibition of seed germination in cucumber.

Abscisic acid (ABA) markedly reduced the germination of developing seeds at much lower concentrations (ABA50=0.1 mM) compared with that of mature seeds (ABA50=1.6 mM) in cucumber (Cucumis sativus L. cv. Green long). The perisperm-endosperm (PE) envelope in developing seeds showed partly differentiated lipid and callose layers, considerable ABA biosynthetic activity in endosperm cells, and appreciable permeability to applied ABA. The decrease in the sensitivity of seeds to applied ABA was coincident with the complete development of lipid and callose layers, diminished ABA biosynthetic activity in endosperm cells in imbibed mature seeds, and moderate permeability of the PE envelope to applied ABA. Decoated seeds pretreated with chloroform showed decreased germination (ABA50=0.4 mM) in response to applied ABA and increased ABA permeation through the PE envelope. ABA thus allowed to permeate into embryonic tissues substantially reduced the pregerminative activity of beta-glucanase in the radicles. The structure and biophysical/biochemical properties of the PE envelope seem to modulate the effect of ABA on the germination of developing and mature cucumber seeds.

The cloning of sequences differentially transcribed during the induction of somatic embryogenesis in cucumber (Cucumis sativus L.).

Somatic embryogenesis in cucumber cell suspension culture is a convenient tool to study differential gene expression, particularly during the early stages of this process. In this study, we used the cucumber somatic embryogenesis system to detect genes that were differentially transcribed during the induction of embryo development. We identified and cloned 120 candidate cDNA fragments from differential display gels. The selected cDNAs were confirmed by reverse northern, and 83 were sequenced. The obtained sequences represent 64 independent transcripts. The search for similarities in the databases gave a significant result in 16 cases. The potential involvement of these sequences in somatic embryogenesis is discussed.

Developing a scale-up system for the micropropagation of cucumber (Cucumis sativus L.): the effect of growth retardants, liquid culture and vessel size.

We investigated the effect of the growth retardant flurprimidol, the phase of the culture medium (solid versus liquid) and the size of the liquid culture vessel (250-ml flask versus 2.5-l airlift bioreactor) on the micropropagation of cucumber (Cucumis sativus L.) from nodal explants. Flurprimidol at concentrations of 0.1-2 mg l(-1) caused considerable growth retardation but increased, albeit slightly, the number of branches and buds and stimulated (solid medium) or reduced (liquid medium) the accumulation of NO(3)-and PO(4)(3-). Flurprimidol had varying effects on the accumulation of soluble sugars and antioxidant compounds. Bioreactor-derived plants showed an increased fresh weight and size but a decreased content of macronutrients, solid sugars, ascorbic acid and free antioxidant phenolics. The majority (95%) of the plants were successfully acclimatized after being graft on squash. The perspective for an efficient, commercial-level use of bioreactors in combination with growth retardants of this commercially important vegetable species is discussed.

Effect of chilling on the gravitropic response of 'Poinsett 76' cucumber (Cucumis sativus L.) radicles.

Germinating 'Poinsett 76' cucumber (Cucumis sativus) seeds are chilling sensitive, and subsequent radicle elongation is inhibited by exposure to nonfreezing temperatures below 10 degrees C. Reorienting germinated seeds with 5-mm-long radicles from a vertical to a horizontal position induced gravitropic curvature within 2 hours at 10 to 25 degrees C without significantly affecting the rate of radicle elongation. However, neither elongation nor curvature occurred in similar seeds held at 2.5 or 5 degrees C. Chilling seeds with 5-mm-long radicles at 2.5 degrees C for 18 hours significantly reduced the subsequent rate of radicle elongation at 25 degrees C by 47% compared with nonchilled control, while gravitropic curvature was reduced by only 34%. After 36 hours of recovery at 25 degrees C, the difference was even more pronounced; elongation was reduced by 26% while curvature was reduced by only 6%. Clearly, gravitropic curvature was less chilling sensitive than radicle elongation, despite the fact that differential elongation of cells in the radicle are needed to produce curvature. Exposing seeds with 5-mm-long radicles to a heat shock of 45 degrees C for 4 to 10 minutes significantly diminished the chilling-induced reduction in radicle elongation and gravitropic curvature.

Fatty acid beta-oxidation in glyoxysomes. Characterization of a new tetrafunctional protein (MFP III).

We describe a new form of a multifunctional protein possessing the enzyme activities of delta 3, delta 2-enoyl-CoA isomerase, 3-hydroxyacyl-CoA epimerase, L-3-hydroxyacyl-CoA dehydrogenase and L-3-hydroxyacyl-CoA forming 2-trans-enoyl-CoA hydratase. This isoform, characterized by a molecular mass of 81 kDa and an isoelectric point above pH 9, was designated MFP III. Along with the tetrafunctional 76.5 kDa MFP II and the trifunctional 74 kDa MFP I, MFP III participates in degradation of fatty acid in glyoxysomes during mobilization of fat reserves. In combination with thiolase, MFP III encompasses all activities to degrade 3-cis-enoyl-CoAs to acetyl-CoA.