油菜甾醇内酯类化合物结构与活性的相关性

比较了19种油菜甾醇内酯类似物和有关甾体化合物在水稻叶片倾斜及萝卜幼苗生长试验中的生物活性。表油菜甾醇内酯(24—Epi—BR)在两个系统中都具有很强的生物活性。C_2位失去羟基(香蒲甾醇)仅在水稻试验中有高活性,改变C_22位侧链结构(2α,3α双羟基—6—酮—23,24—双失碳—β—高-5α—胆烷酸甲酯)在萝卜试验中仍有活性。     

Regulators of cell division in plant tissues. XXX. Cytokinin metabolism in relation to radish cotyledon expansion and senescence

Kinetic studies of formation of glucosides of 6-benzylaminopurine (BAP) in excised radish cotyledons indicated that the 3-, 7-, and 9-glucosides (N-glucosides) were each formed directly from BAP. The 7- and 9-glucosides of BAP and the 7-glucoside of zeatin exhibited great stability in the cotyledons, but the 3-glucoside was converted to free BAP and to the 7- and 9-glucosides of BAP. When³H-labeled zeatin was supplied to developed cotyledons, at high concentrations (100 μM), 7-glucosylzeatin was the principal metabolite, but an appreciable proportion of the extracted³H was due to O-glucosylzeatin. In immature cotyledons, as used in the radish cotyledon cytokinin bioassay, this O-glucoside was shown to be converted into zeatin 7-glucoside probably via free zeatin. Metabolism of BAP and zeatin in radish cotyledons was studied in relation to cytokinin-induced cotyledon expansion. Cytokinin N-glucosides were not metabolites responsible for the observed cytokinin-induced expansion, and were not detoxification products, or deactivation products formation of which was coupled with cytokinin action. However, the free base, its riboside, and nucleotide were possible active forms of BAP associated with cotyledon expansion. The possible significance of cytokinin N-glucosides is discussed. Senescent and nonsenescent cotyledons differed in their metabolism of BAP, zeatin, and zeatin riboside. Senescence was associated principally with a reduction in ability to form 7-glucosylzeatin, enhanced metabolism to adenine derivatives, and an inability to form appreciable amounts of 3-glucosyl-BAP. A two-dimensional thin layer chromatography (TLC) system, based on adjoining layers of cellulose and silica gel, for separating zeatin metabolites is described. This does not completely separate zeatin and zeatin riboside from the corresponding dihydro-compounds. A reversed phase TLC method for achieving these separations is also reported.     

An improved disk bioassay for determining activities of plant growth regulators

An improved filter paper disk bioassay for determining activities of plant growth regulators was developed and evaluated. Indole-3-acetic acid (IAA), (±)-abscisic acid (ABA), and 6-furfurylamino-purine (kinetin) were dissolved in 95% ethanol at fixed dilutions. Specific concentrations of each growth regulator were then evenly dropped onto individual 6-cm paper disks and the solvent evaporated. The activities of the above three water-insoluble plant growth regulators in both solution and the disk assay were compared using the excised cucumber (Cucumis sativus L.) cotyledon root formation bioassay (IAA), wheat (Triticum aestivum L.) coleoptile straight growth bioassay (ABA), and cucumber cotyledon expansion bioassay (kinetin), and similar results were obtained at each concentration. The possible principle of this method has been studied using the cucumber cotyledon expansion bioassay. The results suggested that the disks, as carriers, had highly dispersed kinetin molecules on them and greatly accelerated the dissolution and diffusion of kinetin from disks to water.     

Synthesis and Comparative Cytokinin Activities of N-(Purin-6-yl)-d- and -l-amino Acid Methyl Esters

Six pairs of enantiomeric N-(purin-6-yl)amino acid methyl esters were synthesized and tested for their cytokinin activities by three bioassay systems, the growth of tobacco callus, the seed germination of lettuce and the fresh weight increase of excised radish cotyledons.l-(—)-Antipodes were as a whole more active than the corresponding d-(+)-isomers in the tobacco callus and seed germination tests, whereas an uniform tendency was not observed in the radish cotyledon expansion. The discussions were focused on the effects on the biological response of configurational arrangements around the asymmetric center at α-position to the adenine ring and on species difference of cytokinin receptor molecules.     

Regulators of cell division in plant tissues. XXX. Cytokinin metabolism in relation to radish cotyledon expansion and senescence

Kinetic studies of formation of glucosides of 6-benzylaminopurine (BAP) in excised radish cotyledons indicated that the 3-, 7-, and 9-glucosides (N-glucosides) were each formed directly from BAP. The 7- and 9-glucosides of BAP and the 7-glucoside of zeatin exhibited great stability in the cotyledons, but the 3-glucoside was converted to free BAP and to the 7- and 9-glucosides of BAP. When³H-labeled zeatin was supplied to developed cotyledons, at high concentrations (100 M), 7-glucosylzeatin was the principal metabolite, but an appreciable proportion of the extracted³H was due to O-glucosylzeatin. In immature cotyledons, as used in the radish cotyledon cytokinin bioassay, this O-glucoside was shown to be converted into zeatin 7-glucoside probably via free zeatin.Metabolism of BAP and zeatin in radish cotyledons was studied in relation to cytokinin-induced cotyledon expansion. Cytokinin N-glucosides were not metabolites responsible for the observed cytokinin-induced expansion, and were not detoxification products, or deactivation products formation of which was coupled with cytokinin action. However, the free base, its riboside, and nucleotide were possible active forms of BAP associated with cotyledon expansion. The possible significance of cytokinin N-glucosides is discussed.Senescent and nonsenescent cotyledons differed in their metabolism of BAP, zeatin, and zeatin riboside. Senescence was associated principally with a reduction in ability to form 7-glucosylzeatin, enhanced metabolism to adenine derivatives, and an inability to form appreciable amounts of 3-glucosyl-BAP.A two-dimensional thin layer chromatography (TLC) system, based on adjoining layers of cellulose and silica gel, for separating zeatin metabolites is described. This does not completely separate zeatin and zeatin riboside from the corresponding dihydro-compounds. A reversed phase TLC method for achieving these separations is also reported.     

Cytokinin-like effects of caffeine in bioassays

Cytokinin-like effects of pure caffeine were tested in bioassays specific for this hormonal activity [radish cotyledon growth and chlorophyll (Chl) biosynthesis in cucumber cotyledon and tobacco cell suspension] and in cell elongation bioassays [elongation of segments from soybean internode and internode elongation in dwarf cultivars of guandu (Cajanus cajan) and mucuna (Mucuna deeringiana)]. 6-Benzyl-aminopurine and kinetin (KIN) were used for comparison with caffeine. Although weaker than those given by cytokinins, positive responses were observed in all specific bioassays and in elongation of soybean internodes. A remarkable synergistic effect between caffeine and KIN was observed for the synthesis of Chl in the tobacco cell suspension bioassay, in which different concentrations of the alkaloid were combined with a single concentration of KIN. The hormone-like effect of caffeine might be related to the resemblance between caffeine and adenine derivatives. This revised version was published online in August 2006 with corrections to the Cover Date.     

Adsorption of peroxidase on Celite 545 directly from ammonium sulfate fractionated white radish (Raphanus sativus) proteins

This paper demonstrates the direct immobilization of peroxidase from ammonium sulfate fractionated white radish proteins on an inorganic support, Celite 545. The adsorbed peroxidase was crosslinked by using glutaraldehyde. The activity yield for white radish peroxidase was adsorbed on Celite 545 was 70% and this activity was decreased and remained 60% of the initial activity after crosslinking by glutaraldehyde. The pH and temperature-optima for both soluble and immobilized peroxidase was at pH 5.5 and 40°C. Immobilized peroxidase retained higher stability against heat and water-miscible organic solvents. In the presence of 5.0 mM mercuric chloride, immobilized white radish peroxidase retained 41% of its initial activity while the free enzyme lost 93% activity. Soluble enzyme lost 61% of its initial activity while immobilized peroxidase retained 86% of the original activity when exposed to 0.02 mM sodium azide for 1 h. The K_m values were 0.056 and 0.07 mM for free and immobilized enzyme, respectively. Immobilized white radish peroxidase exhibited lower V_max as compared to the soluble enzyme. Immobilized peroxidase preparation showed better storage stability as compared to its soluble counterpart.     

Purification and characterization of dipeptidase hydrolyzing L-cysteinylglycine from radish cotyledon

Dipeptidase activity was detected in the soluble fraction of radish (Raphanus sativus L.) cotyledon, and the purified enzyme had a specific activity of 7.32 nkat/mg protein for hydrolyzing L-cysteinylglycine. The dipeptidase was found to be a hexameric metalloenzyme, composed of homological 55 kDa-subunits. This is the first glutathione catabolism-related dipeptidase isolated from higher plants.     

Regulators of cell division in plant tissues XXIX. The activities of cytokinin glucosides and alanine conjugates in cytokinin bioassays

In a number of cytokinin bioassays, the activities of the following compounds were compared: 3-, 7-, and 9-glucosides of 6-benzylaminopurine (BAP); 7- and 9-glucosides of zeatin; O-glucosides of zeatin, dihydrozeatin, and their ribosides; 9-alanine conjugates of zeatin, and BAP. The bioassays included the radish cotyledon, theAmaranthus betacyanin, the oat leaf senescence, and the tobacco pith callus. Cytokinin activity was markedly reduced by 7- and 9-glucosylation in nearly all bioassays, but 3-glucosylation of BAP and O-glucosylation of the zeatin sidechain usually had little effect on activity. However, there were two notable exceptions to this generalization: the activity of O-glucosylzeatin markedly exceeded that of zeatin in the oat leaf senescence assay; 9-glucosyl-BAP and free BAP were similarly active in retarding the senescence of radish leaf discs. The 9-alanine conjugate of zeatin (lupinic acid) and of BAP were markedly less active than zeatin and BAP, respectively, in all bioassays, but the responses evoked by these conjugates at high concentrations in theAmaranthus bioassay approached those caused by the corresponding base. The activities of several new compounds related to the alanine conjugate of BAP were also assessed. To serve as a guide in the selection of the most suitable bioassay for detection of the above-mentioned cytokinin conjugates, the lowest detectable amounts in selected bioassays have been compared.     

Purification, properties and alternate substrate specificities of arginase from two different sources: Vigna catjang cotyledon and buffalo liver

Arginase was purified from Vigna catjang cotyledons and buffalo liver by chromatographic separations using Bio-Gel P-150, DEAE-cellulose and arginine AH Sepharose 4B affinity columns. The native molecular weight of an enzyme estimated on Bio-Gel P-300 column for Vigna catjang was 210 kDa and 120 kDa of buffalo liver, while SDS-PAGE showed a single band of molecular weight 52 kDa for cotyledon and 43 kDa for buffalo liver arginase. The kinetic properties determined for the purified cotyledon and liver arginase showed an optimum pH of 10.0 and pH 9.2 respectively. Optimal cofactor Mn++ ion concentration was found to be 0.6 mM for cotyledon and 2 mM for liver arginase. The Michaelis-Menten constant for cotyledon arginase and hepatic arginase were found to be 42 mM and 2 mM respectively. The activity of guanidino compounds as alternate substrates for Vigna catjang cotyledon and buffalo liver arginase is critically dependent on the length of the amino acid side chain and the number of carbon atoms. In addition to L-arginine cotyledon arginase showed substrate specificity towards agmatine and L-canavanine, whereas the liver arginase showed substrate specificity towards only L-canavanine.     

The effect of cytokinins on in vitro cultured inbred lines of Raphanus sativus var. radicula Pers. with genetically determined tumorigenesis

The higher plant tumors are convenient models for studying the genetic control mechanism of plant cell division. There are two types of tumors: induced by the pathogenic factor and genetically determined. The development of both tumor types was related to the changes in cytokinin metabolism and/or signal transduction. In this work, the effect of synthetic cytokinins on the in vitro morphogenesis of cotyledon explants and isolated apices of radish seedlings was studied in several inbred radish lines (Raphanus sativus var. radicula Pers.) that differed in their in vivo tumorigenic properties. It was noted that root formation was stronger affected by kinetin while the treatment with thidiazuron tended to induce active callus formation in cotyledon explants of all inbred lines, except IIa. Growing with benzyladenine produced an intermediate effect as regards all morphogenetic responses. Cytokinin treatment of tumorigenic lines enhanced necrotic development in cotyledon explants. Culturing isolated apices of regenerated plants produced tumors anatomically and morphologically similar to those developing in vivo. Some of the lines nontumorigenic in vivo with enhanced formation of calli on cotyledon explants also developed tumors on apical explants in vitro when treated with cytokinins. These data suggest that different mechanisms for tumor formation operate in various radish lines. The radish lines are classified into three types: (1) necrotic lines with tumor formation putatively related to endogenous cytokinin level, (2) callus-forming lines with cell division enhanced in response to cytokinins, and (3) necrosis-and callus-forming lines with both mechanisms of tumor formation involved.     

Changes in some enzymes of microbodies and plastid development in excised radish cotyledons: effect of narciclasine

Narciclasine (NCS), isolated from mucilage of Narcissus bulb, showed inhibitory effects on growth and plastid development of excised radish cotyledons. NCS (0.1 mumol/L) started to show inhibitory effects on isocitrate lyase and hydroxypyruvate reductase activities after 24 h incubation in light. When NCS concentration was increased to 10 mumol/L, the activities of both enzymes are completely inhibited. From ultrastructural studies, NCS markedly prevented the degradation of protein bodies and lipid bodies, as well as chloroplast formation of excised radish cotyledons. There was only little degradation of protein and lipid bodies, and almost no chloroplast formation in the excised radish cotyledon treated with 1 mumol/L NCS. Therefore, our results provide clear evidence that NCS inhibited the transition of glyoxysomes and peroxisomes, and chloroplast development.     

萝卜离体子叶衰老与膜脂过氧化的关系

萝卜离体子叶在光下或暗中衰老及激素调节衰老过程中,作为叶片衰老指标的叶绿素和蛋白质含量的降低,发生在MDA含量增高之前,更早于SOD活性的下降。表明由SOD活性降低所导致的膜脂过氧化的增强,并非衰老的原初反应,而是叶片衰老到一定程度的生理变化。因此,至少在萝卜离体子叶上,不能将其衰老的启动归因于受SOD控制的膜脂过氧化作用导致的膜累积性质变。     

Purification and Characterization of Dipeptidase Hydrolyzing L-Cysteinylglycine from Radish Cotyledon

Dipeptidase activity was detected in the soluble fraction of radish (Raphanus sativus L.) cotyledon, and the purified enzyme had a specific activity of 7.32 nkat/mg protein for hydrolyzing L-cysteinylglycine. The dipeptidase was found to be a hexameric metalloenzyme, composed of homological 55 kDa-subunits. This is the first glutathione catabolism-related dipeptidase isolated from higher plants.     

Improved germination bioassays for allelopathy research

A new method for germination bioassays in allelopathy was evaluated. Inspired by pharmacology, allelochemical quantity to test on different target seeds was calculated according to seeds biometry. The first step consisted in measuring volume, mass, surface and shape of Lactuca sativa and Raphanus sativus seeds. The radish seed mass, surface, volume and contact area biometric parameters were respectively 10, 2, 6 and 1.7 that of lettuce. Two germination bioassay sets were compared: (i) a conventional one, testing the same concentration of allelochemical (2-benzoxazolinone termed as “BOA”) on the two species and, (ii) a biometrics enhanced seed test (“BEST” method), employing quantities of the chemical that were calculated in proportion to seed biometry parameters. The conventional method indicated that 1 mM BOA slowed and decreased germination rate (radish 50 %, lettuce 10 %) whereas 10 and 0.1 μM did not induce any effect. The BEST method offered more differentiated results: applied BOA according to seed volume induced significant inhibition of radish germination (both quantities), while lettuce was only affected by the highest dose. The same occurred when considering grain surface and contact area. Thus, the BEST assay showed more clearly that radish was more sensitive to BOA than lettuce. Compared to a setup with identical test compound concentrations for all tested species, the BEST method provides more differentiated results contributing to a more realistic comparative susceptibility assessment.     

Cytokinin and anticytokinin activity of some 4-substituted 1H-pyrazoles and 8-aza analogues of adenine

Using specific bioassays i.e. radish cotyledon expansion, betacyanin synthesis in Amaranthus caudatus, and senescence retardation of isolated leaf explants, six 4-substituted 1-H pyrazoles and five 8-aza adenine analogues were tested for their cytokinin- and anticytokinin activity. Most of the pyrazole derivatives showed some cytokinin-like activity and enhanced the effect of 10^–5 M BA. 8-Aza substituted adenines were found to be cytokinin antagonists; in the bioassays used they were inactive when applied alone but blocked the action of 10^–5 M BA when applied simultaneously.     

NCS对离体萝卜子叶微体中酶活性及细胞超微结构的影响

研究了天然生物活性物质narciclasine(NCS)对离体萝卜子叶光下生长发育期间叶绿素含量变化、异柠檬酸裂解酶及羟基丙酮酸还原酶活性的影响;并对萝卜子叶细胞的超微结构变化进行了观察。结果表明,NCS明显抑制光下培养的离体萝卜子叶叶绿素含量及鲜重增加,对异柠檬酸裂解酶及羟基丙酮酸还原酶活性也显示出明显的抑制作用。电镜观察显示,NCS对蛋白体及脂质体的降解、叶绿体的发育也表现出强烈的抑制效应。NCS的各种抑制作用均随其浓度的增加而增加,而且高浓度的NCS(10^-5mol／L)基本上完全阻止了离体萝卜子叶的光下生长及其转绿。     

A New Root-Formation Test

This paper describes a new and convenientt rootformation test. It is excised cucumber cotyledon root-formation test. The experimental results showed that the mininum level of IAA detectable in this bioassay was 0.3–100 ppm and the number of roots formed in excised cucumber cotyledon was proportional to the logarithm of the IAA concentration in the range of 3.0–30 ppm. This bioassay is specific for natural and synthetic auxins. It had less error and better repeatability than others.     

Cytokinin-like activities of nucleocyclitols

Although the 9-substituted adenines are commonly inactive as cytokinins, the nucleocyclitol 3-(-adenin-9-yl)-3-deoxy-1,5,6-tri-0-(methylsulfonyl)-muco-inositol (NI) proved to be active in the following bioassays: cell proliferation in soybean cotyledon callus tissue, cell expansion in excised radish cotyledons, and delay of senescence in detached leaves. In these assays, the effect of the compound, applied at the same molar concentration as benzyl adenine, was lower or less uniform than BA. NI completely failed to promote germination of lettuce seeds in conditions of secondary dormancy or thermodormancy, whre BA is effective. NI can substitute for BA in some though not all of the numerous responses evoked by cytokinins.     

Mechanisms of constraints: the contributions of selection and genetic variance to the maintenance of cotyledon number in wild radish

The evolutionary mechanisms underlying the maintenance of invariant traits are poorly understood, partly because the lack of variance makes these mechanisms difficult to study. Although the number of cotyledons that plant species produce is highly canalized, populations of plants frequently contain individuals with abnormal cotyledon numbers. In a garden study with 1857 wild radish plants from 75 paternal half-sibling families, 89 (almost 5%) had cotyledon numbers less or greater than two. We found evidence for direct selection on cotyledon number, but no evidence for additive genetic variation for cotyledon number. In spite of the very large sample size, our power to detect variation and selection was hampered by the small number of individuals (10) producing more than two cotyledons. Thus, our results provide support for both a lack of genetic variation and selection as reasons for the current lack of variation in wild radish cotyledon number.