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。     

CYTOLOGICAL AND BIOCHEMICAL ASPECTS OF CYTOKININ-ENHANCED GROWTH OF RADISH (RAPHANUS SATIVUS) COTYLEDONS

Light and electron microscopy revealed no stimulation of oleosome disappearance or chloroplast development associated with zeatin-enhanced growth of excised radish cotyledons. Accelerated protein body breakdown was the only detectable effect of zeatin treatment. Isocitrate lyase activity, disappearance of total lipids, and appearance of galactolipids, phospholipids, and chlorophyll were unaffected by zeatin. Results indicate that cytokinins do not accelerate conversion of radish cotyledons from storage to photosynthetic organs.     

Chloroplast development in 4-thiouridine-cultured radish seedlings IV. Properties of cellular organelles

The effect of 4-thiouridine (4SU) on metabolic activities oforganelles other than plastids was investigated by examininggrowth parameters, cellular enzyme activities and metabolicbehaviors of greening radish seedlings germinated and grownwith 4SU. Seedling growth was not severely affected by 4SU culture.Mitochondrial, peroxisomal and microsomal marker enzymes andcytoplasmic enzyme activities were not generally changed inthe 4SU-cultured radish cotyledons. The respiration rate ofmitochondria isolated from 4SU-cultured cotyledons was alsonormal. Some soluble enzymes of the isolated plastids had normalor even higher activities. However, chloroplastic pigments andribulose bisphosphate carboxylase activity decreased with increasingconcentration of 4SU in the culture medium. We concluded thatthe inhibitory effect of 4SU was limited to chloroplast development. (Received December 4, 1979; )     

Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings XI. Inhibition of RNA Synthesis by 4-Thiouridine Nucleotides during Germination of Radish Seeds

The effects of 4-thiouridine and its metabolites on RNA synthesisin radish (Raphanus sativus) cotyledons were investigated. The4-thiouridine nucleotides, 4-thioUTP and 4-thioUDP, were foundto inhibit DNA-dependent RNA polymerase activities of isolatednuclei and of chloroplasts from radish cotyledons. These inhibitorsappeared to compete with UTP for its binding to RNA polymerase.Neither 4-thiouridine nor 4-thioUMP inhibited RNA polymeraseactivities. Reduced RNA accumulation accompanied by the inhibition of protochlorophyllideaccumulation was observed in cotyledons of dark-grown radishseedlings germinated and grown with 4-thiouridine. On the otherhand, 4-thiouridine had no effect on chloroplast developmentin the control seedlings germinated and grown without 4-thiouridine.These results suggest that the inhibition of chloroplast developmentby 4-thiouridine may in part be due to the inhibition of RNAsynthesis by 4-thiouridine nucleotide during germination, resultingin inhibition of etioplast development. (Received December 9, 1985; Accepted June 26, 1986)     

ffects of Some Inhibitors on Potassium-and IAA-Induced Adventitious Root Formation of Excised Cucumber Cotyledon

The effects of some inhibitors on potassium- and IAA-induced rooting were studied adopting the root-formation bioassay in the excised cucumber ( Cucumis sativus L. ) cotyledon. 5-fluomuracil at 7 Ï 10-4 – 7 Ï10-1 mmol/L and cycloheximide at 3.5 Ï 10-4 – 1.05 Ï10-2 mmol/L significantly inhibited potassium- and IAA-induced adventitious root formation of the excised cucumber cotyledons, respectively. Na3VO4 at 0.1 – 1.0 mmol/L obviously inhibited potassium and IAA-induced adventitious rooting of the excised cucumber cotyledons, and similar inhibitory effect was found with 2,3,5-triiodobenzoic acid (TIBA) at 2 Ï 10-4 – 2 Ï 10-l mmol/L.There is a close relationship between potassium and IAA-induced adventitious rooting and the promotive effect of potassium on rooting is possibly brought about via affecting the endogenous level of IAA.     

Formation of Tyrosine Phenol-lyase by Bacteria

Formation of protochlorophyllide and ribulose-1,5-diphosphate carboxylase, both of which were constituents proper to chloroplast, was inhibited in the radish cotyledons grown with 4-thiouridine (0.5 mm), though the growth of the seedlings, activities of a cytoplasmic enzyme glucose-6-phosphate dehydrogenase and of a mitochondrial enzyme glutamate dehydrogenase in the cotyledones were not affected. Photo-induced formation of chlorophyll pigments and of chloroplastic ribosomal RNAs in the 4-thiouridine treated cotyledones were depressed as compared with controls. Interestingly, the effect of 4-thiouridine, however, disappeared with continuous illumination. Based upon these results, specific development of the chloroplast is discussed.     

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.     

The effect of the embryo axis and exogenous sucrose on lipolysis and glyoxysomal enzyme development in Ricinus communis (castor bean) endosperm and cotyledons

Post-germinative growth in castor bean ( Ricinus communis L. cv. Hale) seedlings was investigated to determine whether lipolytic enzyme synthesis and lipid breakdown was a function of the embryo axis or simply based on a source-sink mechanism connected with sucrose produced during mobilization of storage lipid. Endosperm and cotyledons were excised from the embryo axis at 24 h intervals and were then incubated in Petri dishes containing water or 0.1 M sucrose for 24 h. Excised endosperm showed similar or higher malate synthase (MS, EC 4.1.3.2) and isocitrate lyase (ICL, EC 4.1.3.1) activities and increased lipolysis when compared with endosperm obtained from similarly intact seedlings of the same age. In contrast, cotyledonary ICL and MS activity was up to 50% lower and lipolysis was only slightly affected in excised material when compared with cotyledons obtained from intact seedlings. Incubating endosperm in sucrose had no effect on the development of the above enzyme activities or lipid content, when compared with material incubated in water only. In contrast, cotyledonary MS and ICL activities were up to 70% lower in sucrose and lipolysis substantially inhibited. Lipid breakdown and the development of lipolytic enzyme activity in cotyledons seem to be dependent on the presence of the endosperm. It is concluded that enzyme regulation in castor bean seedlings cannot entirely be explained by axis control or source-sink relationships.     

邻啡罗啉-Cu诱发小麦离体叶片的氧化损伤

邻啡罗啉-Cu处理小麦叶片,加快O2^-的产生,提高SOD和POD活性,加速叶绿体色素和蛋白质降解,因而MDA积累增加。暗示邻啡罗啉-Cu有诱导植物组织产生活性氧、促进过氧化的作用。     

Studies on {partial}-amino levulinic acid deydratase in radish cotyledons during chloroplast development

The subcellular localization and biosynthetic site of 8-aminolevulinic acid dehydratase [EC 4.2.1.24 [EC] , ALAD] were investigatedin relation to chloroplast development in radish cotyledons. ALAD was mainly located in the chloroplasts and cytoplasm. Mostof the ALAD in the chloroplasts was readily released by hypotonicshock. The enzyme was also found in the proplastids of etiolatedcotyledons. The normal increase in the activity of ALAD in the chloroplastsas well as the cytoplasm was inhibited by cycloheximide butunaffected by D-threo chloramphenicol and kanamycin during thegreening of radish cotyledons. We concluded that the ALAD inboth the cytoplasm and chloroplasts was synthesized on the cytoplasmic80S-ribosomes. This suggests that the ALAD formed on the 80S-ribosomesmight be incorporated into chloroplasts during their development. When etiolated radish seedlings were illuminated, ALAD in boththe cytoplasm and chloroplasts increased up to the point ofthe full development of the chloroplasts, and thereafter itdecreased. (Received August 20, 1975; )     

Lipid-body lipoxygenase is expressed in cotyledons during germination prior to other lipoxygenase forms

Lipid bodies are degraded during germination. Whereas some proteins, e.g. oleosins, are synthesized during the formation of lipid bodies of maturating seeds, a new set of proteins, including a specific form of lipoxygenase (LOX; EC 1.13.11.12), is detectable in lipid bodies during the stage of fat degradation in seed germination. In cotyledons of cucumber (Cucumis sativus L.) seedlings at day 4 of germination, the most conspicuous staining with anti-LOX antibodies was observed in the cytosol. At very early stages of germination, however, the LOX form present in large amounts and synthesized preferentially was the lipid-body LOX. This was demonstrated by immunocytochemical staining of cotyledons from 1-h and 24-h-old seedlings: the immunodecoration of sections of 24-h-old seedlings with anti-LOX antiserum showed label exclusively correlated with lipid bodies of around 3 m in diameter. In accordance, the profile of LOX protein isolated from lipid bodies during various stages of germination showed a maximum at day 1. By measuring biosynthesis of the protein in vivo we demonstrated that the highest rates of synthesis of lipid-body LOX occurred at day 1 of germination. The early and selective appearance of a LOX form associated with lipid bodies at this stage of development is discussed.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DIC differential interference contrast - FITC fluorescein-5-isothiocyanate - LOX lipoxygenase This work was financially supported by the Deutsche Forschungsgemeinschaft, SFB 286 (I.F., A.N., H.K.) and SFB 363 (B.H., C.W.).     

Electron microscope studies on the morphogenesis of plastids

Streptomycin sulphate (2 mg/ml) did not affect the formation of proplastids or the elaboration of prolamellar bodies. The plastids of the streptomycin (SM)-treated cotyledons contained both crystalline prolamellar bodies and ribosomes, and were undistinguishable from the plastids of the water-grown cotyledon. However, plastids from dark-grown SM-treated cotyledons were no longer able to differentiate to more advanced stages of development, even after exposure to light. The plastids of light and dark-grown SM-treated cotyledons often contained prolamellar bodies and abnormal giant grana. Variegation developed in the cotyledons germinated in Hoagland's solution plus SM. The plastids in pale green tissue contained stroma-lamellae and one or two giant grana, whereas in those of pale yellow tissue, many osmiophilic globules, large vacuoles and crystal bodies were observed. It is suggested that the formation of prolamellar bodies may depend on cytoplasmic protein synthesis whereas functional stroma- and grana-lamellae may depend on protein synthesis within the plastids. The inhibitory effects of SM on protein synthesis were used as a tool to test this hypothesis. This work was carried out in the Department of Botany, University of California, Davis, by Grant-GB-11906 from National Science Foundation of U.S.A.     

Effect of root formation on 14C-glycine uptake and incorporation into protein by attached and excised cotyledons of Helianthus annuus

Root formation was found to extend the life-span of excised cotyledons of Helianthus annuus L. markedly. Excised cotyledons of 12-day-old plants attained longer life-span, higher root number and total root length than cotyledons excised before or after 12 days. Protein content of attached cotyledons reached a maximum level 12 days after the commencement of germination followed by a decrease. Cotyledons excised 8 days after sowing showed maximum level of protein content 44 days after excision followed by a decrease. The increase was correlated with the full development of roots. The incorporation of ¹⁴C-glycine into protein followed a pattern similar to the protein content, both in attached and detached cotyledons.     

Altered lignin composition in phenylalanine ammonia-lyase-inhibited radish seedlings: implications for seed-derived sinapoyl esters as lignin precursors

We earlier reported that when phenylalanine ammonia-lyase (PAL) activity in radish seedlings was inhibited by the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP), soluble sinapoyl esters carried over from the seed were converted to wall-bound esters in young cotyledons. We now report that these soluble sinapoyl esters may also be converted into lignin in the cotyledons. When radish seedlings were grown in the presence of 100 microM AIP, lignin formation (determined as lignothioglycolic acid) was inhibited ca. 74% in the cotyledons and ca. 80% in hypocotyls plus roots. The syringyl to guaiacyl (S/G) ratio in the lignin of AIP-grown plants, as determined by alkaline cupric oxidation and from Fourier-transform infrared (FT-IR) spectra, was higher in cotyledons, but lower in hypocotyls plus roots, as compared to plants grown on distilled water. These results support the view that soluble sinapoyl esters preformed in seeds may contribute to the syringyl moiety of lignin in cotyledons during early seedling development and that there is no appreciable transport of soluble sinapoyl esters from cotyledons to the hypocotyls and roots.     

Evidence that Ethylene is not Involved in Red-light-induced Stimulation of Chlorophyll Formation in Etiolated Cucumber Cotyledons

The involvement of ethylene in red-light-induced stimulationof chlorophyll (Chl) formation was studied because one of thered-light effects on Chl formation (the lateappearing effect)interacts with the ethylene effect in 3-day-old excised etiolatedcotyledons of cucumber (Cucumis sativus L. cv. Aonagajibai).Ethylene production by etiolated cotyledons of intact seedlingsin the dark is enhanced by a red-light pulse, but the effectdoes not occur in excised cotyledons. Application of ethylenein the dark to 3-day-old intact seedlings has little effecton Chl formation in the cotyledon during subsequent continuousillumination, although ethylene pretreatment of 5-day-old seedlingssignificantly stimulates Chl formation. Removal of endogenousethylene by mercuric perchlorate [Hg(ClO₄)₂] does not specificallysuppress the red-light action on Chl formation in both attachedand excised cotyledons. Inhibition of ethylene synthesis byaminoethoxyvinylglycine does not affect the red-light effecton Chl formation in excised cotyledons. These facts indicatethat ethylene does not operate as a mediator of red light instimulating Chi formation in either attached or excised cotyledons. (Received December 13, 1981; Accepted March 30, 1981)     

Ontogeny of microbodies (glyoxysomes) in cotyledons of dark-grown watermelon (Citrullus vulgaris Schrad.) seedlings

The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.Abbreviations CAT catalase - DAB 3,3 diaminobenzidine tetrahydrochloride - ER endoplasmic reticulum - GOX glycolate oxidase - HPR hydroxypyruvate reductase - HVEM high-voltage electron microscopy - ICL isocitrate lyase - MS malate synthase - RER rough endoplasmic reticulum In the figures bars represent 0.1 m (if not stated otherwise)     

Cytokinin Activity of Benzoylaminodeazapurines, Pentanoylaminodeazapurines and their Corresponding Purine Analogs in Five Bioassays

Cytokinin activities of 6-benzoylamino-1, 6-benzoylamino-3-, 6-pentanoylamino-1- and 6-pentanoylamino-3-deazapurines and their corresponding purine analogs, 6-benzoylaminopurine and 6-pentanoylaminopurine, were examined using five bioassay systems, tobacco callus growth, bud formation on tobacco callus, lettuce seed germination, fresh weight increase of radish cotyledons and retardation of chlorophyll degradation in radish cotyledons. 6-Benzoylamino- and 6-pentanoylamino-1-deazapurines showed stronger cytokinin activity than their corresponding purine analogs in all bioassays used. In tobacco callus growth, 6-benzoylamino-1-deazapurine was nearly as active as zeatin, one of the most active adenylate cytokinins. On the other hand, 6-benzoylamino- and 6-pentanoylamino-3-deazapurines were as active as or less active than corresponding purine analogs.     

Metabolic profiling of oxylipins in germinating cucumber seedlings--lipoxygenase-dependent degradation of triacylglycerols and biosynthesis of volatile aldehydes

A particular isoform of lipoxygenase (LOX) localized on lipid bodies was shown by earlier investigations to play a role in initiating the mobilization of triacylglycerols during seed germination. Here, further physiological functions of LOXs within whole cotyledons of cucumber (Cucumis sativus L.) were analyzed by measuring the endogenous amounts of LOX-derived products. The lipid-body LOX-derived esterified (13 S)-hydroperoxy linoleic acid was the dominant metabolite of the LOX pathway in this tissue. It accumulated to about 14 micromol/g fresh weight, which represented about 6% of the total amount of linoleic acid in cotyledons. This LOX product was not only reduced to its hydroxy derivative, leading to degradation by beta-oxidation, but alternatively it was metabolized by fatty acid hydroperoxide lyase leading to formation of hexanal as well. Furthermore, the activities of LOX forms metabolizing linolenic acid were detected by measuring the accumulation of volatile aldehydes and the allene oxide synthase-derived metabolite jasmonic acid. The first evidence is presented for an involvement of a lipid-body LOX form in the production of volatile aldehydes.     

Chloroplast development in 4-thiouridine-cultured radish seedlings III. Photochemical activities in isolated plastids

Accumulation of chlorophyll, development of photosystem I andII activities and contents of chloroplastic components wereinvestigated in greening radish seedlings germinated and grownwith 4-thiouridine (4SU). The development of photosystem I activityprior to that of photosystem II was observed also in the 4SU-culturedgreening radish cotyledons in which chlorophyll accumulationwas inhibited up to 60–80% of that of the control. Photochemicalactivities expressed on a plastid protein basis decreased withthe increase of 4SU in the culture medium. In contrast to ferredoxinand ferredoxin-NADP reductase, which were present in significantamounts in the treated cotyledons, chloroplastic cytochromes(f, b₅₅₉ and b₆ decreased in the plastids from 4SU-culturedcotyledons. These results suggest that 4SU interferes in partwith protein synthesis in plastids and thereby with chloroplastdevelopment. (Received December 4, 1979; )