Oxygen-mediated cold-acclimation in cucumber (Cucumis sativus) seedlings

Cold acclimation of etiolated cucumber seedlings, consisting of cooling at 12°C for 48 h followed by a warming period at 25°C, led to tolerance to subsequent chilling at 2°C. Tolerance, as evidenced by freedom from chilling injury and continued growth, developed during the warming period in a time-course manner for 12 h but decreased with prolonged warming. A similar increase and subsequent decrease was also observed in the content of palmitic, linoleic and linolenic acids in total lipid fraction from cucumber hypocotyl tissue. During the warming period supra-ambient oxygen stimulated, whereas subambient oxygen inhibited, the increase in fatty acid content as well as development of chilling tolerance. A strong correlation between oxygen-mediated changes in fatty acid content and associated development of cold tolerance suggests that both these processes are interrelated. Cold acclimation, but not cold stress, led to an increase followed by a decrease in CO₂ evolution suggesting that a respiratory upsurge is yet another feature of cold acclimation in cucumbers.     

Induction of chilling tolerance in cucumber (Cucumis sativus) seedlings by endogenous and applied ethanol

Five-day-old etiolated cucumber ( Cucumis sativus L.) seedlings cv. Marketmore held at 2°C for 72 h developed chilling injury, resulting in desiccation and collapse of the hypocotyl tissue and eventual plant death. Hypoxia-induced accumulation of ethanol and acetaldehyde led to tolerance of subsequent chilling, as evidenced by continued hypocotyl growth and freedom from injury. Attenuated accumulation of volatiles by applied bisulfite reduced the development of hypoxia-induced chilling tolerance in seedlings. In seedlings held in normoxia cold tolerance was induced by applied ethanol vapors, whereas acetaldehyde had a marginal effect, suggesting that hypoxia-induced cold tolerance may arise from the accumulation and activity of ethanol. Cold tolerance was also induced by exposure of seedlings to volatile anesthetics including n -propanol, n -butanol, chloroform and halothane, suggesting that ethanol activity may result from fluidization of membrane lipids. This view is consistent with results which showed that ethanol activity was not associated with lipid metabolism. However, development of cold tolerance in ethanol-enriched tissues was time dependent, indicating that ethanol activity probably also entails biosynthetic event(s).     

Endogenous salicylic acid accumulation is required for chilling tolerance in cucumber (Cucumis sativus L.) seedlings

Salicylic acid (SA) is an important plant hormone, and its exogenous application can induce tolerance to multiple environmental stresses in plants. In this study, we examine the potential involvement of endogenous SA in response to chilling in cucumber (Cucumis sativus L.) seedlings. A low temperature of 8 °C induces a moderate increase in endogenous SA levels. Chilling stimulates the enzymatic activities and the expression of genes for phenylalanine ammonia-lyase (PAL) and benzoic acid-2-hydroxylase rather than isochorismate synthase. This indicates that the PAL enzymatic pathway contributes to chilling-induced SA production. Cucumber seedlings pretreated with SA biosynthesis inhibitors accumulate less endogenous SA and suffer more from chilling damage. The expression of cold-responsive genes is also repressed by SA inhibitors. The reduction in stress tolerance and in gene expression can be restored by the exogenous application of SA, confirming the critical roles of SA in chilling responses in cucumber seedlings. Furthermore, the inhibition of SA biosynthesis under chilling stress results in a prolonged and enhanced hydrogen peroxide (H₂O₂) accumulation. The application of exogenous SA and the chemical scavenger of H₂O₂ reduces the excess H₂O₂ and alleviates chilling injury. In contrast, the protective effects of SA are negated by foliar spraying with high concentrations of H₂O₂ and an inhibitor of the antioxidant enzyme. These results suggest that endogenous SA is required in response to chilling stress in cucumber seedlings, by modulating the expression of cold-responsive genes and the precise induction of cellular H₂O₂ levels.     

Oxidative stress in cucumber (Cucumis sativus L) seedlings treated with acifluorfen

Treatment of diphenyl ether herbicide acifluorfen-Na (AF-Na) to intact cucumber (Cucumis sativus L cv Poinsette) seedlings induced overaccumulation of protoporphyrin IX in light (75 mumole m-2 s-1). The extra-plastidic protoporphyrin IX accumulated during the light exposure disappeared within two hours of transfer of acifluorofen-treated seedlings to darkness. The dark disappearance was due to re-entry of migrated protoporphyrin IX into the plastid and its subsequent conversion to protochlorophyllide. In light, protoporphyrin IX acted as a photosensitizer and caused generation of active oxygen species. The latter caused damage to the cellular membranes by peroxidation of membrane lipids that resulted in production of malondialdehyde. Damage to the plastidic membranes resulted in damage to photosystem I and photosystem II reactions. Dark-incubation of herbicide-sprayed plants before their exposure to light enhanced photodynamic damage due to diffusion of the herbicide to the site of action. Compared to control, in treated samples the cation-induced increases in variable fluorescence/maximum fluorescence ratio and increase in photosystem II activity was lower due to reduced grana stacking in herbicide-treated and light-exposed plants.     

Auxin,biosynthesis of ethylene and sex expression in cucumber (Cucumis sativus)

Ethylene production, level of 1-aminocyclopropane-1-carboxylic acid (ACC) and activity of the ethylene forming enzyme (EFE) were higher in apices of gynoecious cucumber (Cucumis sativus cv. Alma) as compared to monoecious cucumber (C. sativus cv. Elem). Application of indole-3-acetic acid (IAA) enhanced ethylene and ACC production in both cultivars. The stimulatory effect of IAA was more pronounced in gynoecious apices. Induction of ethylene production and accumulation of ACC resulting from treatment with IAA were effectively blocked by aminoethoxyvinylglycine (AVG). Content of endogenous IAA, measured by an enzyme immunoassay, was lower in gynoecious cucumber as compared to monoecious one. Treatment of gynoecious plants with the antiauxins -(p-chlorophenoxy)isobutyric acid (PCIB) and -naphthaleneacetic acid (-NAA) did not inhibit female sex expression.It appears that although exogenous IAA enhances ACC and ethylene production, endogenous IAA might not have a major role in the control of sex expression in cucumber of the Beit-Alfa type.Prof. Rudich passed away in May 1986.     

Purification and characterization of arginine decarboxylase from cucumber (Cucumis sativus) seedlings

A simple, reproducible and rapid protocol for the purification of arginine decarboxylase fromCucumis sativus seedlings has been standardised. The purification steps involved ion-exchange chromatography on diethylaminoethyl-cellulose followed by gel filtration on Sephadex G-l 50. The purified enzyme preparation migrated as a single stainable band on Polyacrylamide gels at both basic and acidic pH, but under denaturing and reducing conditions on sodium dodecyl sulphate-polyacrylamide gels resolved into polypeptides of molecular weight 48,000,44,000 and 15,000. However, in the absence of 2-mercaptoethanol on electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, the enzyme moved as single band with a molecular weight of 150,000. Evidence was obtained to indicate that these three polypeptides were probably derived from a single larger molecular weight enzyme. On storage of the purified protein, the 48,000 species was preferentially degraded to smaller polypeptides. The preliminary data suggested that the 48,000 and 44,000 species shared many common tryptic peptides as revealed by finger printing of the [¹²⁵I ]-labelled protein. The purified enzyme was a glycoprotein and had aK _m of 0.5 mM for arginine. Its activity was stimulated by dithiothrietol and pyridoxal phosphate. EDTA did not inhibit the enzyme activity. Mn²⁺ at 1 mM stimulated arginine decarboxylase activity but was inhibitory at higher concentration     

Acclimation of chlorophyll biosynthetic reactions to temperature stress in cucumber (Cucumis sativus L.)

The adaptive responses of the greening process of plants to temperature stress were studied in cucumber (Cucumis sativus L. cv. Poinsette) seedlings grown at ambient (25 °C), low (7 °C) and high (42 °C) temperatures. Plastids isolated from these seedlings were incubated at different temperatures and the net syntheses of various tetrapyrroles were monitored. In plastids isolated from control seedlings grown at 25 °C, the optimum temperature for synthesis of Mg-protoporphyrin IX monoester or protochlorophyllide was 35 °C. Temperature maxima for Mg-protoporphyrin IX monoester and protochlorophyllide syntheses were shifted to 30 °C in chill-stressed seedlings. The net synthesis of total tetrapyrroles was severely reduced in heat-stressed seedlings and the optimum temperature for Mg-protoporphyrin IX monoester or protochlorophyllide synthesis shifted slightly towards higher temperatures, i.e. a broader peak was observed. To further study the temperature acclimation of seedlings with respect to the greening process, tetrapyrrole biosynthesis was monitored at 25 °C after pre-heating the plastids (28–70 °C) isolated from control, chill- and heat-stressed seedlings. In comparison to 28 °C-pre-heated plastids the percent inhibition of protochlorophyllide synthesis in 40 °C-pre-heated plastids was higher than for the control (25 °C-grown) in chill-stressed seedlings and lower than for the control in heat-stressed seedlings. Maximum synthesis of total tetrapyrroles and protoporphyrin IX was observed when chloroplasts were heated at 50 °C, which was probably due to heat-induced activation of the enzymes involved in protoporphyrin IX synthesis. Prominent shoulders towards lower or higher temperatures were seen in chill-stressed or heat-stressed seedlings, respectively. The shift in optimum temperature for tetrapyrrole biosynthesis in chill- and heat-stressed seedlings was probably due to acclimation of membranes possibly undergoing desaturation or saturation of membrane lipids. Proteins synthesized in response to temperature-stress may also play an important role in conferring stress-tolerance in plants. Received: 8 October 1998 / Accepted: 19 November 1998     

Effect of seed pretreatment by magnetic field on the sensitivity of cucumber (Cucumis sativus) seedlings to ultraviolet-B radiation

The effects of seed pretreatment by magnetic field (MF) on the impacts of ultraviolet-B (UV-B) radiation were tested using cucumber (Cucumis sativus) seedlings in phytotron. Soaked cucumber seeds were placed in MF of various strengths (0, 0.2 and 0.45 T). After germination the seeds were sowed in homogeneous garden soil and grown, then cucumber seedlings were exposed to 0 (as control) and 3.5 kJ m⁻² UV-B irradiation, respectively. Some effects of UV-B radiation and MF-pretreatment as well as their combination were investigated. MF-pretreatment increased seed germination rate, seedling growth and development, although also increased lipid oxidation and ascorbic acid contents. On the other hand, our results provided evidence that seed MF-pretreatment increased the sensitivity of cucumber seedlings to UV-B radiation. The seedling growth and development were significantly decreased by the combination of UV-B irradiation and MF-pretreatment. This combination also increased oxidative pressure and decreased actual quantum yield of PS II. Leaf UV-B absorbing compound was increased by MF-pretreatment or UV-B irradiation, whereas their combination significantly decreased it. These results suggested that the harmful effects of combination were partially due to the inhibition of secondary metabolism.     

A cotyledon screen for resistance to scab (Cladosporium cucumerinum) in cucumber (Cucumis sativus) seedlings

An efficient protocol for differentiating cucumber (Cucumis sativus) seedlings resistant and susceptible to scab (Cladosporium cucumerinum) has been developed by examining the interacting factors of spore density and cotyledon growth stage. The procedure permitted both resistant and susceptible plants to be recovered. Seedlings were grown at 20°C and inoculated 1 day after emergence by placing a 5 μl inoculum droplet (2 × 10⁶ spores/ml) on the newly expanded cotyledons. Seedlings were incubated in a darkened dew chamber for 48 h at 20°C and 100% r.h., and then grown for 6 days at 20°C. Plants could be rated as resistant or susceptible 8 days after inoculation.     

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings

A purified preparation of arginine decarboxylase fromCucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine andPi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase,viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3–4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine andvice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.     

Development of glutamate:glyoxylate aminotransferase in the cotyledons of cucumber (Cucumis sativus) seedlings.

Glutamate:glyoxylate aminotransferase had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration (Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. Glutamate:glyoxylate aminotransferase activity was already present in the etiolated cotyledons of cucumber (Cucumis sativus) seedlings, and increased during greening. The enzyme was present only in the cytosol of the etiolated cotyledons and appeared in the peroxisomes during greening. The enzyme was purified to homogeneity from the cytosol of the etiolated cotyledons and from the peroxisomes of the green cotyledons of cucumber seedlings. The two enzyme preparations had nearly identical enzymic and physical properties. On the basis of these findings, roles of glutamate:glyoxylate aminotransferase in the glycollate pathway in photorespiration, and the mechanism of its appearance in the peroxisomes during greening, are discussed.     

Boron toxicity is alleviated by hydrogen sulfide in cucumber (Cucumis sativus L.) seedlings

Boron (B) is an essential micronutrient for plants, which when occurs in excess in the growth medium, becomes toxic to plants. Rapid inhibition of root elongation is one of the most distinct symptoms of B toxicity. Hydrogen sulfide (H₂S) is emerging as a potential messenger molecule involved in modulation of physiological processes in plants. In the present study, we investigated the role of H₂S in B toxicity in cucumber (Cucumis sativus) seedlings. Root elongation was significantly inhibited by exposure of cucumber seedlings to solutions containing 5 mM B. The inhibitory effect of B on root elongation was substantially alleviated by treatment with H₂S donor sodium hydrosulfide (NaHS). There was an increase in the activity of pectin methylesterase (PME) and up-regulated expression of genes encoding PME (CsPME) and expansin (CsExp) on exposure to high B concentration. The increase in PME activity and up-regulation of expression of CsPME and CsExp induced by high B concentration were markedly reduced in the presence of H₂S donor. There was a rapid increase in soluble B concentrations in roots on exposure to high concentration B solutions. Treatment with H₂S donor led to a transient reduction in soluble B concentration in roots such that no differences in soluble B concentrations in roots in the absence and presence of NaHS were found after 8 h exposure to the high concentration B solutions. These findings suggest that increases in activities of PME and expansin may underlie the inhibition of root elongation by toxic B, and that H₂S plays an ameliorative role in protection of plants from B toxicity by counteracting B-induced up-regulation of cell wall-associated proteins of PME and expansins.     

Effect of chilling, heat shock, and vigor on the growth of cucumber (Cucumis sativus) radicles

Chilling at 2.5°C reduced the subsequent growth of cucumber ( Cucumis sativus L.) radicles at 25°C. The reduction in radicle growth was linear for 1–3 days of chilling at ≈10% per day of treatment, but then it increased in a non-linear pattern until subsequent radicle growth was all but eliminated by 6 days of chilling. A heat shock of 40°C for 4–12 min increased chilling tolerance such that 4 days of chilling caused only a 36% decrease in radicle growth, compared to 66% for seedlings not heat shocked. Heat shocks were only able to protect that part of radicle growth that was in excess of the linear decrease in radicle growth projected from 0–3 days. There appear to be two effects of chilling on radicle growth. The first inhibition of subsequent growth was linear and was not affected by heat shocks. The second inhibition was much more severe; it appeared after 3 days of chilling and could be prevented by heat shock. Seeds classified with different levels of vigor (i.e., different initial rates of growth) did not respond significantly different to chilling stresses following heat-shock treatments.     

A novel role for cyanide in the control of cucumber (Cucumis sativus L.) seedlings response to environmental stress

The effects of potassium cyanide (KCN) pretreatment on the response of cucumber (Cucumis sativus L.) plants to salt, polyethylene glycol (PEG) and cold stress were investigated in the present study. Here, we found that KCN pretreatment improved cucumber seedlings tolerance to stress conditions with maximum efficiency at a concentration of 20 µM. The results showed that pretreatment with 20 µM KCN alleviated stress‐induced oxidative damage in plant cells and clearly induced the activity of alternative oxidase (AOX) and the ethylene production. Furthermore, the structures of thylakoids and mitochondria in the KCN‐pretreated seedlings were less damaged by the stress conditions, which maintained higher total chlorophyll content, photosynthetic rate and photosystem II (PSII) proteins levels than the control. Importantly, the addition of the AOX inhibitor salicylhydroxamic acid (1 mm ; SHAM) decreased plant resistance to environmental stress and even compromised the cyanide (CN)‐enhanced stress tolerance. Therefore, our findings provide a novel role of CN in plant against environmental stress and indicate that the CN‐enhanced AOX might contribute to the reactive oxygen species (ROS) scavenging and the protection of photosystem by maintaining energy charge homoeostasis from chloroplast to mitochondria.     

Intraspecific differences in Cucumis sativus sensitivity to ultraviolet-B radiation

Cucumber ( Cucumis sativus L.) cultivars Marketmore, Lama, XPH 1187, XPH 1484 and Sprint 440 (N) were grown in a greenhouse under two levels of biologically effective ultraviolet-B ( UV -B) radiation (daily dose: 0 and 11.6 kJ m⁻² UV-B_BE) for 31 days. Significant intraspecific differences were observed in plant height, number of leaves, leaf area and total dry weight. Based upon total biomass accumulation, Marketmore was found to be the most tolerant, and XPH 1484 the most sensitive to UV-B radiation. The dose response of accumulation of UV absorbing compounds (measured as absorbance of methanolic extracts) in leaf tissues showed an increase in UV absorbing compounds with UV-B dose in Marketmore, Sprint 440 (N) and XPH 1187. In Lama and XPH 1484, however, doses below 8.7 kJ m⁻² UV-B_BE produced no change in UV absorbing compounds. This study suggests that intraspecific differences in UV-B radiation sensitivity in cucumber may be related to inherent differences in the accumulation of UV absorbing compounds in leaves.     

Effects of metal elements on acid phosphatase activity in cucumber (Cucumis sativus L.) seedlings

Acid phosphatases (E.C.3.1.3.2) are a group of enzymes widely distributed in nature, which nonspecifically catalyze the hydrolysis of a variety of phosphate esters in pH ranges from 4 to 6 and play a major role in the supply and metabolism of phosphate in plants. The objective of the present study was to investigate the in vitro effects of some metals on the activity of acid phosphatase in cucumber seedlings (Cucumis sativus L.) and to determine their kinetic parameters. The enzyme was assayed with Hg, Cd, Mn, Pb, Zn, K and Na at the 0.001–1 mM range using ATP, PPi and β-glycerol phosphate as substrates. Mn, Na and Cd did not significantly alter the enzyme activity. K caused a broad activation at low concentrations and an inhibition at high concentrations (10 mM) and lead caused no inhibition. Acid phosphatase was inhibited by Hg and Zn and the inhibition type and IC₅₀ values were determined for these metals. Hg presented a mixed inhibition type with PPi and ATP as substrates and uncompetitive inhibition with β-glycerol phosphate as substrate. Zn presented competitive inhibition for ATP as substrate, and a mixed inhibition type with PPi and β-glycerol phosphate as substrate. IC₅₀ values were 0.02, 0.3 and 0.15 mM for Hg, and 0.056, 0.035 and 0.24 mM for Zn with ATP, PPi and β-glycerol phosphate as substrates, respectively. Analysis of these results indicates that Zn is a more potent inhibitor of acid phosphatase from cucumbers than Hg.     

Complex structure of the ribosomal DNA spacer of Cucumis sativus (cucumber)

Summary The nuclear 18 S, 5.8 S and 25 S ribosomal RNA genes (rDNA) of Cucumis sativus (cucumber) occur in at least four different repeat types of 10.2, 10.5, 11.5, and 12.5 kb in length. The intergenic spacer of these repeats has been cloned and characterized with respect to sequence organization. The spacer structure is very unusual compared to those of other eukaryotes. Duplicated regions of 197 bp and 311 bp containing part of the 3 end of the 25 S rRNA coding region and approximately 470 bp of 25 S rRNA flanking sequences occur in the intergenic spacer. The data from sequence analysis suggest that these duplications originate from recombination events in which DNA sequences of the original rDNA spacer were paired with sequences of the 25 S rRNA coding region. The duplicated 3ends of the 25 S rRNA are separated from each other mostly by a tandemly repeated 30 bp element showing a high GC-content of 87.5%. In addition, another tandemly repeated sequence of 90 bp was found downstream of the 3flanking sequences of the 25 S rRNA coding region. These results suggest that rRNA coding sequences can be involved in the generation of rDNA spacer sequences by unequal crossing over.     

Aspartyl proteinase from cucumber (Cucumis sativus) seeds. Preparation and characteristics

Aspartyl proteinase (EC 3.4.23) from cucumber seeds was purified by ammonium sulphate fractionation, chromatography on immobilized pepstatin and gel filtration on Sephacryl S-200. The preparation obtained, homogeneous on polyacrylamide-gel electrophoresis in acidic and alkaline media, has a molecular mass of 42,000, pI of 5.2, and shows the highest activity with denatured haemoglobin at pH 3.2. The proteinase is stable in slightly alkaline medium, whereas it is inactivated in acidic medium, especially in the presence of NaCl. The enzyme activity is affected neither by the inhibitors of serine proteinases, sulfhydryl-proteinases and metalloproteinases, nor by divalent metal ions, whereas the enzyme is inactivated by the inhibitors of aspartyl proteinases: 1,2,3-epoxy(p-nitrophenoxy)propane, diazoacetyl-DL-norleucine and pepstatin.