Studies on the Isolation,Identification and Bioactivities of Daucosterol in the Roots ot Paeonia lactiflora

A white crystalline powder substance (m.p. 290–292℃; [a]23D=-48.3˚) was iso- lated from the roots of Paeonia lactiflora. It was identified by chemical and spectral analysis as β-sitosterol-D-glucoside, i.e. daucosterol. It amounts to 4×10-5 of the dry material Bioassays showed that daucosterol increased the fresh weight of cucumber cotyledons, promoted the elongation of wheat coleoptiles, and delayed the destruction of chlorophyll. Daucosterol also induced the initiation of root and leaf of isolated cucumber cotyledons. These phenomena showed that daucosterol was an active phytosetroid and exhibited both auxin-like and kinetin-like activities.     

Oxidative DNA damage in cucumber cotyledons irradiated with ultraviolet light

DNA was isolated from the cotyledons of cucumber seedlings irradiated with ultraviolet (UV)-C (254 nm) or UV-B+UV-A (280–360 nm; maximum energy at 312 nm) at various fluence rates and durations. Following enzymatic hydrolysis of DNA, the content of 8-hydroxy-2-deoxyguanosine [(8-OHdG), 8-oxo-7,8-dihydro-2-deoxyguanosine], a well-established biomarker closely identified with carcinogenesis and aging in animal cells, was determined using a high-performance liquid chromatograph equipped with an electrochemical detector. The levels of 8-OHdG increased with UV-C and UV-B irradiation in a fluence-dependent manner. This increase was also observed in etiolated cotyledons that had been excised from dark-grown cucumber seedlings and then cultured in vitro under UV light: monochromatic UV light at 270 nm or 290 nm increased the 8-OHdG level considerably, while UV at wavelengths above 310 nm had only small effects. In situ detection of H₂O₂ and quantification of H₂O₂ in plant extracts revealed that H₂O₂ accumulated in cotyledons irradiated with UV light. These results suggest that UV irradiation induces oxidative DNA damage in plant cells.     

Phytochrome-mediated control of respiratory activities of mitochondria in cotyledons of dark-grown cucumber seedlings

Mitochondria isolated from cotyledons of dark-grown cucumber ( Cucumber sativus L., cv. Shimotsuki-Aonaga) seedlings after illumination with continuous far-red light showed an increased capacity for oxidation of malate or α-ketoglutarate, as compared with those from cotyledons of non-illuminated seedlings. This increase is supposed to be caused by phytochrome action (high irradiance response). Exogenous NAD⁺ had no effect on the rate of the oxidation of α-ketoglutarate or malate by mitochondria isolated from far-red light-treated cotyledons, but it enhanced the oxidation rate of mitochondria from control cotyledons to the level of mitochondria from light-treated ones. The NAD (NAD⁺+ NADH) content was higher in mitochondria isolated from continuously far-red light-treated cotyledons than in mitochondria from controls. The NAD content was also increased by the treatment with a red light pulse and this response was reversed by a subsequent far-red light pulse. It is proposed that phytochrome controls respiratory activities of cucumber mitochondria by changing the size of the NAD pool in the mitochondria.     

离体培养黄瓜子叶诱导雌花的研究

采用添加Spd和IAA的MS培养基培养离体黄瓜子叶,研究了Spd和IAA对雌花诱导的协同作用,及昼夜温差、培养基中N素和pH值对雌花诱导的影响。结果表明,分别添加Spd、IAA时的雌花诱导率和单株雌花数偏低或为0,12 mg·L^-1 Spd与0.01mg·L^-1 IAA 配合时的诱导效果明显高于单独处理的,而对照组未见雌花,说明Spd和IAA对雌花诱导的协同作用显著。在0、2、6、10℃昼夜温差,60、70、80、90 mmol·L^-1的N素含量和pH 5.4、5.8、6.2、6.6的培养条件下,70 mmol·L^-1 N、6℃温差和pH 6.2时的雌花诱导效果较好,表明适当提高昼夜温差、培养基中N素和pH值有利于黄瓜子叶的雌花诱导。     

Regulation by ABA of beta-Conglycinin Expression in Cultured Developing Soybean Cotyledons

The regulation of cotyledon-specific gene expression by exogenously applied abscisic acid (ABA) was studied in developing cultured cotyledons of soybean (Glycine max L. Merr. cv Provar). When immature cotyledons were cultured in modified Thompson's medium, the addition of ABA resulted in an increased concentration of the β-subunit of β-conglycinin, one of the major storage proteins of soybean seeds. The amount of the α′-and α-subunits of β-conglycinin was relatively unaffected by the ABA treatment. When fluridone, an inhibitor of carotenoid biosynthesis that has been shown to decrease ABA levels in plant tissues, was added to the medium the level of ABA and the β-subunit decreased in the cotyledons. Increasing the concentration of sucrose in the culture medium caused an increase in the concentration of ABA and β-subunit in the cotyledons. When in vitro translation products from RNA isolated from cotyledons cultured with ABA were immunoprecipitated with antiserum against β-conglycinin, there was an increased amount of pre-β-subunit polypetide compared to the translation products from RNA isolated from control cotyledons. The pre-β-subunit polypeptide was not detected in translation products from RNA isolated from fluridone-treated cotyledons. Nucleic acid hybridization reactions showed that the level of β-subunit mRNA was higher in ABA-treated cotyledons compared to the control, and was lower in the fluridone-treated cotyledons. We have shown that exogenous ABA is able to modulate the accumulation of the β-subunit of β-conglycinin in developing cultured soybean cotyledons.     

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.     

Water stress in cucumber cotyledons infected withXanthomonas campestris pv.cucurbitae

Xanthomonas compestris pv.cucurbitae strain 62 was isolated from diseased cucumber seedlings grown in an open field in Egypt and identified in this study. Inoculation of cucumber seedlings with this strain resulted in reduction of relative water content (RWC) and increase in free proline content of infected cotyledons. RWC was found to be decreased by the increase of disease severity. The reverse was true for free proline content which was found to be increased by the increase of disease severity. These results indicate that water stress increased in direct proportion to the disease severity. Highly significant increase in free proline content was found to occur in organs of cucumber seedlings inoculated with strain 62 and showing disease symptoms on cotyledons only. This implied that water stress was not limited to symptomatic cotyledons but extended to the whole seedling.     

表油菜素内酯对黄瓜子叶过氧化物酶活性和可溶性蛋白含量的影响

０．５ｍｇ／Ｌ的表油菜素内酯（ｅｐｉ－ＢＲ）能显著地促进黄瓜子叶叶绿素ａ、叶绿素ｂ的含量和叶绿素ａ／叶绿素ｂ比值的下降,表明ｅｎｉ－ＢＲ能促进子叶的衰老。从过氧化物酶（ＰＯＤ）的活性测定及同工酶谱发现ｅｐｉ－ＢＲ可提高其活性,暗示它可能通过提高子叶ＰＯＤ的活性而加速子叶叶绿素的降解。另一方面,ｅｐｉ－ＢＲ促进子时可溶性蛋白含量的下降,其中主要是ＲｕＢＰ羧化酶含量的下降,同时,ｅｐｉ－ＢＲ引起子叶游离氨基酸的累积。     

Thiolase mRNA translated in vitro yields a peptide with a putative N-terminal presequence

Thiolase is part of the fatty acid oxidation machinery which in plants is located within glyoxysomes or peroxisomes. In cucumber cotyledons, proteolytic modification of thiolase takes place during the transfer of the cytosolic precursor into glyoxysomes prior to the intraorganellar assembly of the mature enzyme. This was shown by size comparison of the in vitro synthesized precursor and the 45 kDa subunit of the homodimeric glyoxysomal form. We isolated a full-length cDNA clone encoding the 48 539 Da precursor of thiolase. This plant protein displayed 40% and 47% identity with the precursor of fungal peroxisomal thiolase and human peroxisomal thiolase, respectively. Compared to bacterial thiolases, the precursor of the plant enzyme was distinguished by an N-terminal extension of 34 amino acid residues. This putative targeting sequence of cucumber thiolase shows similarities with the cleavable presequences of rat peroxisomal thiolase and plant peroxisomal malate dehydrogenase.     

Lepidimoide Promotes Light-Induced Chlorophyll Accumulation in Cotyledons of Sunflower Seedlings

The effect of disaccharide lepidimoide on light-induced chlorophyll accumulation was studied in cotyledons of sunflower (Helianthus annuus L.) seedlings and detached cucumber (Cucumis sativus L.) cotyledons. From studies on the structure-activity relationships of lepidimoide, its analogs, and sucrose with respect to light-induced chlorophyll accumulation in the cotyledons of sunflower seedlings, both lepidimoide and the free carboxylic acid of lepidimoide (lepidimoic acid) showed the highest promoting activity, whereas the hydrogenated lepidimoide, which lacks a double bond in the C4, 5 position in uronic acid, showed lower activity than lepidimoide; however, sucrose exhibited very weak activity. These results suggest that lepidimoide acts as a new type of plant growth regulator, not simply as a carbon source providing energy. Lepidimoide promoted not only light-induced chlorophyll accumulation in sunflower cotyledons but also light-induced 5-aminolevulinic acid content, which is considered to be a rate-limiting step in chlorophyll biosynthesis. Lepidimoide with cytokinin stimulated the accumulation of chlorophyll and 5-aminolevulinic acid additively. In detached cucumber cotyledons, lepidimoide also promoted light-induced chlorophyll accumulation. These results indicate that lepidimoide, in cooperation with cytokinin, causes light-induced chlorophyll accumulation in the cotyledons of several dicot plant species by affecting the level of 5-aminolevulinic acid. Received April 4, 1997; accepted September 28, 1998     

Pyruvate metabolism in mitochondria from cucumber cotyledons during early seedling development

Mitochondria were isolated from cucumber cotyledons during earlyseedling growth, and their capacity for pyruvate metabolisminvestigated. The rate of pyruvate oxidation was low. Evidenceis presented that suggests that this is due to low activitiesof the pyruvate transporter. Key words: Cotyledon, cucumber, germination, pyruvate oxidation     

Synthesis of Chloroplast Proteins during Germination and Early Development of Cucumber

Cell-free protein synthesizing systems have been used to study the developmental changes in the synthesis of chloroplast proteins in the cotyledons of cucumber seedlings grown in the light or in the dark. Escherichia coli and wheat germ in vitro protein synthesizing systems have been used to assay the changes in the levels of the mRNA's coding for ribulose 1,5-bisphosphate carboxylase (RuBPCase). The large subunit of cucumber RuBPCase has been identified among the translation products of the E. coli system. The wheat germ system translates the cucumber mRNA coding for the small subunit of RuBPCase to produce a 25,000 molecular weight precursor polypeptide. Plastids isolated from light-grown cotyledons were used to study developmental changes in their capacity to synthesize protein. The data obtained indicate that in the light there is an initial 48-hour period of accumulation of the mRNA's coding for the large and small subunits of RuBPCase, coupled with an increase in the capacity of the isolated plastids to synthesize protein. This is followed by a decline. This decline is not reflected in the accumulation of RuBPCase in the cotyledons which remains constant over the period of study.     

Insect feeding on cucumber mediated by rhizobacteria-induced plant resistance

Select strains of plant growth-promoting rhizobacteria (PGPR) were evaluated in greenhouse experiments with cucumber for induction of resistance against cucumber beetle (Diabrotica undecimpunctata howardi Barber) feeding and the beetle-transmitted cucurbit wilt disease. When beetles were given a choice between PGPR-treated and nontreated cucumber, their feeding on stems and cotyledons and the severity of wilt symptoms were significantly lower on PGPR-treated plants. HPLC analysis demonstrated that cotyledons from PGPR-treated plants contained significantly lower concentrations of the cucumber beetle feeding stimulant cucurbitacin than nontreated plants. These results suggest that a mechanism for PGPR-induced resistance against cucumber beetle feeding may involve a change in the metabolic pathway for cucurbitacin synthesis.     

A cDNA encoding a putative SPF1-type DNA-binding protein from cucumber

A cDNA clone encoding a polypeptide with homology to the novel SPF1 DNA-binding protein of sweet potato has been isolated from a cDNA library from RNA of senescing cucumber (Cucumis sativus, L.) cotyledons. Comparison of the two sequences reveals similar features which may be important in the evolution and function of this protein, including a duplicated region of about 56 amino acids (aa). The first half of the duplicated region is enriched in basic aa and is very highly conserved, both within and between each polypeptide. In contrast, the second half of the duplicated region is poorly conserved within each polypeptide, but highly conserved when cucumber and sweet potato sequences are compared. Southern blot analysis with cucumber DNA shows a simple hybridisation pattern indicating one or very few genes. Northern blot analysis shows that the expression of the cucumber gene increases in cotyledons as they expand and become photosynthetic and remains high in senescence. The possibility that the cucumber SPF1-type protein may be involved in carbohydrate regulation of gene expression is discussed.     

In vitro assembly of microtubules from tubulins of several higher plants

Tubulins were isolated by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4B) and ion exchange (DEAE-Sephacel) chromatography from several higher plants (mung bean, pea, whole pod bean, zucchini, cucumber seedlings and carrot suspension cultured cells). All these higher plant tubulins readily polymerized to microtubules in a polymerization medium containing GTP, Mg2+, EGTA, leupeptin and DMSO. Tubulins from mung bean, pea and whole pod bean showed identical behaviour on polyacrylamide gel electrophoresis but differed from carrot zucchini and cucumber tubulin. Consequently, tubulin of higher plants seems to have different molecular properties in different plant species.     

Nonspecific intercellular protein trafficking probed by green-fluorescent protein in plants

Summary Plasmodesmata mediate intercellular transport of proteins, nucleic acids, and small molecules in plants. We show that transiently produced green-fluorescent protein (GFP) trafficked intercellularly in the epidermis of sink leaves, but not of source leaves, in tobacco and cucumber. In contrast, the protein did not traffic in either sink or source leaves of tomato. On the other hand, the protein spread extensively from cell to cell in the epidermis of all leaves and stems ofArabidopsis thaliana as well as in young hypocotyls and cotyledons of tomato and cucumber. GFP could traffic from epidermis to ground tissues in hypocotyls but not in cotyledons of cucumber. GFP fused to a number of mutant forms of the cucumber mosaic virus 3a movement protein (CMV 3a MP) failed to traffic from cell to cell, suggesting that GFP does not have a specific motif for plasmodesmal trafficking. Our data, together with previous findings, indicate that plasmodesmata can mediate both specific and nonspecific intercellular trafficking of proteins. Furthermore, our data suggest that nonspecific protein trafficking is controlled by species-, development-, organ-, and tissue-specific factors. Since GFP can readily traffic from cell to cell, it raises the questions of how metabolites are compartmentalized intercellularly in a plant and of whether some endogenous plant proteins traffic nonspecifically from cell to cell to perform physiological functions yet to be elucidated.Abbreviations CMV cucumber mosaic virus - GFP green-fluorescent protein - MP movement protein - SEL size exclusion limit     

Action of ultraviolet radiation (UV-B) upon cuticular waxes in some crop plants

The surface structure and composition of surface lipids were examined in leaves of barley, bean, and cucumber seedlings grown in a growth chamber under white light and low levels of ultraviolet (UV-B; 280–320 nm) radiation. The cuticular wax of cucumber cotyledons and bean leaves appeared as a thin homogeneous layer, whereas on barley leaves crystal-like structures could be observed under these irradiation conditions. Principally, the amount of cuticular wax found in barley leaves was five times greater than in bean or cucumber leaves. The prediominant wax components were primary alcohols in barley, primary alcohols and monoesters in bean, and alkanes in cucumber cotyledons. Irradiation with enhanced UV-B levels caused an increase of total wax by about 25% in all plant species investigated. Aldehydes, detected as a minor constituent of cucumber and barley wax, increased twofold. Distribution patterns of the homologs within some wax classes were different at low and enhanced UV-B levels. In general, the distribution of the homologs was shifted to shorter acyl chain lengths in wax of leaves exposed to enhanced UV-B levels. This was most apparent in cucumber wax, less in bean or barley wax. The UV-B-caused effects upon cucumber wax were mainly due to a response by the adaxial surface of the leaf.Abbreviation UV-B Ultraviolet radiation (280–320 nm)