Salinity, oxidative stress and antioxidant responses in shoot cultures of rice

When shoot cultures derived from salt-sensitive Oryza sativavar. Taipei 309 were grown at 25C in medium containing 0.35M NaCl, responses to possible oxidative stress in the earlystages of exposure were observed. Overall levels of Mn-superoxidedismutase activity, Cu, Zn-superoxide dismutase activity andH₂O₂ were significantly elevated. After 1 d there was a notabledecline in tissue concentrations of GSH and a correspondingincrease in GSSG. However, after a further day, concentrationsof GSH and GSSG returned to concentrations normally encounteredin control cultures. Activities of ascorbate peroxidase andcatalase were similar whether the shoots were grown in the presenceor absence of NaCl. In contrast, there was an early increasein glutathione reductase activity in NaCl-exposed cultures,and no indication of extensive increases in lipid peroxidation.Thus although some indications of oxidative stress accompanyexposure of this salt-sensitive rice variety to salinity, mechanismsappear to exist within its shoot tissue to permit the toleranceof such oxidative stress. Key words: Salinity stress, hydrogen peroxide, glutathione, antioxidant enzymes, Oryza sativa     

生长素和氯化钙对盐胁迫下沙冬青幼苗的缓解作用

沙冬青(Ammopiptanthus mongolicus)是亚洲中部荒漠地区唯一的常绿阔叶灌木,本课题组已将其在天津引种栽培成功。为进一步了解生长素和氯化钙对沙冬青在盐胁迫下的缓解作用,用水培方法培养沙冬青幼苗,测定其在1.3%NaCl胁迫下经不同浓度的生长素和氯化钙处理后抗氧化酶活性、PSII光化学效率以及其它与植物抗性有关的生理指标的变化。结果表明,一定浓度的IBA和CaCl2处理可以促进盐胁迫下沙冬青幼苗的生长、提高叶绿素的含量、增强保护酶的活性、降低丙二醛含量,缓解了盐胁迫。但是当IBA、CaCl2浓度分别达到5和20mmol.L-1时则对沙冬青幼苗产生伤害。综合各项生理指标说明,CaCl对沙冬青盐胁迫的缓减作用要优于IBA。     

Der Einflu{beta} verschiedener Lichtqualitaten auf Chlorophyllgehalt und Wachstum von Gewebekulturen aus Crepis capillaris (L.) WALLR.

The influence of different light qualities on chlorophyll contentand growth of tissue cultures from Crepis capillaris (L.) WALLR. Tissue cultures from Crepis capillaris growing on media (M₁; M₂ ; M₂-E) formed chlorophyll and intact chloroplasts onlyin the short wave length region of the visible spectrum (350–550nm). In red light (600–700 nm) as well as in darknessthey lost their chlorophyll after 8–10 weeks. The growth of Crepis-cultures was strongly influenced by lightand the nitrogen of the medium. The highest increase in freshweight (425–485% increase in 3 weeks) was attained inred light or in darkness on M₂ by cultures which had lost theirchlorophyll completely. M₂ contains nitrates, ammonium saltsand amino acids. In contrast, the increase in fresh weight ofgreen cultures growing on M₂ in blue or white light was considerablylower (155–180% increase in 3 weeks). Omission of amino acids, (M₂-E), resulted in the reduction ofthe growth (increase of fresh weight in 3 weeks: 120%) of thechlorophyll-free cells growing in the dark. Green cultures behaveddifferently on M₂-E. In white light they attained an increasein fresh weight of 245%. This suggests that the growth promotingeffect of the amino acids can be replaced by light. Results with cultures growing on M₁, which contains neitherammonium salts nor amino acids, point in the same direction.Green cultures in white or blue light grew better (90–100%increase in fresh weight in 3 weeks) on this "deficient" mediumthan chlorophyll-free tissues in red light or in darkness (20–30%increase in fresh weight in 3 weeks). Some aspects of thesefindings which concern the effect of light on growth are discussed. (Received November 28, 1969; )     

Influence of Abscisic Acid on Nitrate Accumulation and Nitrate Reductase Activity in Potato Tuber Slices

Abscisic acid (ABA) at concentrations of 1 to 10 µg.ml^–1suppressed development of nitrate reductase activity in freshtuber slices of Solanum tuberosum L. incubated in KNO₃. Suppressionof activity was evident after 3 hr and continued for 20 hr beforerecovery. This recovery may be due to inactivation of the hormone.Nitrate accumulation was enhanced by ABA. At exogenous NO₃ levelsof 0.1 to 5 mM, the hormone enhanced both NO₃ accumulation andnitrate reductase activity. When applied 24 hr after incubation in NO₃, ABA promoted a markeddecline in enzyme activity in the absence of exogenous NO₃,but was less effective in the presence of NO₃. Slices incubatedin NO₃ and ABA also exhibited increased loss of enzyme activityupon removal of NO₃. Preincubating slices in the hormone for24 hr in a NO₃- free medium resulted in stimulation of nitratereductase activity. Addition of NO₃ resulted in a marked stimulationof enzyme activity over a period of 8–10 hr. The ABA response is not related to tissue levels of free aminoacids and is not affected by different NO₃ sources. These resultssuggest the ABA effect on nitrate reductase activity is influencedby NO₃ status of the cells. Where external NO₃ levels are lowit stimulated NRA while it inhibited activity where NO₃ contentis high. (Received May 12, 1981; Accepted October 12, 1981)     

Density dependent regulation of growth in L cell suspension cultures. 3. Elevation of specific activity of acetylcholinesterase

High density L cell suspension cultures were previously shown to remain viable for indefinite periods of time and to exhibit marked inhibition of DNA synthesis and mitosis while the fraction of total protein synthesis represented by collagen is increased. The present study demonstrates that regulation in this system extends to the activity of acetylcholinesterase found to be approximately 100-fold greater in the high density populations than in low density exponentially growing cultures. Kinetic studies of the increase of the activity, its fluctuation over an extended period of time and its decrease upon resumption of exponential growth after dilution of the cultures were performed. The data obtained indicate that the enzyme does not accumulate in high density populations merely as a result of the absence of net protein synthesis and cell division but that changes of its rates of synthesis and possibly degradation are involved. The expression of regulated acetylcholinesterase activity in a cell line of connective tissue origin is considered in relation to phenotype reprogramming and to cell membrane associated growth control mechanisms.     

Effect of silver nitrate on sugarcane cell suspension growth, protoplast isolation, ethylene production and shoot regeneration from cell suspension cultures

Silver nitrate (AgNO₃), an inhibitor of the physiological actionof ethylene, reduced cell growth, promoted ethylene production,increased the yield of protoplasts and reduced shoot regenerationfrom sugarcane heterogeneous cell suspension cultures. The increasein the rate of protoplast isolation from cultures treated withAgNO₃ (0 to 59 µM) correlate with an increase in endogenousethylene production by the cells. The addition to the culturemedium of chemicals that either inhibited (aminoethoxyvinylglycine,AVG) or promoted (aminocyclopropane-1-carboxylic acid, ACC)ethylene biosynthesis did not alter the number of protoplastsisolated from these cultures. However, protoplasts were isolatedwith AVG in combination with AgNO₃ even though ethylene productionwas inhibited. These results suggested that AgNO₃ may be havinganother more direct effect on protoplast release. One such sitemay be the cell wall or on cell metabolism conditioning cellsto release protoplasts after enzyme treatment. Key words: Sugarcane, cell suspension, protoplast, silver nitrate, ethylene     

Growth Response of Young Birch Trees (Betula pendulaRoth.) After Four and a Half Years of CO2Exposure

A field experiment consisting of 18 birch trees grown in opentop chambers in ambient and elevated CO₂concentrations was setup with the aim of testing whether the positive growth responseobserved in many short-term studies is maintained after severalgrowing seasons. We present the results of growth and biomassafter 4.5 years of CO₂exposure, one of the longest studies sofar on deciduous tree species. We found that elevated CO₂ledto a 58% increase in biomass at the end of the experiment. However,estimation of stem mass during the growing season showed thatelevated CO₂did not affect relative growth rate during the fourthgrowing season, and therefore, that the large accumulation ofbiomass was the result of an early effect on relative growthrate in previous years. Trees grown in elevated CO₂investedmore carbon into fine roots and had relatively less leaf areathan trees grown in ambient CO₂. In contrast with previous studies,acceleration of growth did not involve a significant declinein nutrient concentrations of any plant tissue. It is likelythat increased fine root density assisted the trees in meetingtheir nutrient demands. Changes in the species composition ofthe ectomycorrhizal fungi associated with the trees grown inelevated CO₂in favour of late successional species supportsthe hypothesis of an acceleration of the ontogeny of the treesin elevated CO₂.Copyright 1997 Annals of Botany Company Betula pendula; silver birch; elevated CO₂; growth; biomass allocation; ectomycorrhizas; tissue composition; nutrients; leaf morphology; specific leaf area; stomatal density; shoot structure     

Retardation of fetal brain cell growth during maternal starvation: Circulating factors versus altered cellular response

Maternal starvation inhibits fetal brain development during late gestation in the rat. To determine whether intrinsic or extrinsic factors might be the principal contributor to altered growth, brain cells from 20 day fetuses were cultured in a 96 well plate with MEM and 10% adult rat serum. Tissue growth was monitored by spectrophotometric measurement of the mitochondrial reduction of a chromagen 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT). After 1, 4 or 6 days incubation, MTT activity in non confluent cultures was shown to be directly related to tissue mass. When fetal brain cell cultures were incubated with 1% and 10% concentrations of adult rat serum, an 11-fold increase in MTT activity paralleled a 15-fold increase in tritiated thymidine incorporation. The impact of maternal starvation on fetal brain cell growth was examined by measuring MTT activity in fetal brain cells from fed and starved mothers. When cultures were incubated for 6 days with graded concentrations of fed adult serum (1.25–10%), the MTT response was slightly but consistently lower in cells from starved when compared with cells from fed mothers. By contrast, a marked difference in MTT activity which was paralleled by a lower DNA content became apparent when fetal rat brain cells were incubated with starved adult serum. Fetal serum and adult male serum were found to support growth equally well, while incubation of fetal brain cells with maternal sera resulted in lower MIT values than with the corresponding fetal sera. When cells were incubated with fetal sera pooled from starved mothers, MTT activity was decreased by 42 to 45%. A relative decrease in MTT activity was also apparent when cells were exposed to sera from starved mothers. Graded concentrations of starved fetal serum (2.5–10%) produced an increase in MTT activity that was consistently lower than similar concentrations of fed fetal serum, a finding suggesting a decrease in growth factors. Mixing fasted with fed serum did not correct the diminished growth, and indicated that an inhibitor might also be functioning to restrict growth. These findings therefore suggest that the principal determinants of diminished fetal brain growth during maternal starvation are not only intrinsic to the cells but are importantly related to the altered extrinsic factors in the fetal circulation.     

Studies in the Physiology of Parasitism: XXII. The Production of Pectolytic Enzymes by Pythium de Baryanum Hesse

The incorporation of sodium chloride in a synthetic medium stimulatedthe pectolytic activity of cultures of Pythium de Baryanum.The Cl^– ion appeared to be mainly responsible for thiseffect; on the other hand, presence of the Ca⁺⁺ ion depressedenzymic activity. Glucose, fructose, and mannose were about equally suitable forgrowth and enzyme production. Sucrose, if used as sole carbohydratesource, gave good mycelial growth but poor enzyme production,but if a small proportion was replaced by glucose, enzyme productionwas as good as on glucose itself. Galactose gave very poor growthand negligible enzyme production. For optimum production of pectolytic enzyme, glucose (or fructoseor mannose) requires to be autoclaved in a somewhat alkalinemedium—very conveniently with the K₂HPO₄ or K₃PO₄ of thenutrient medium. A yellow to brown coloration (due to caramelization)is produced in the process, but the stimulating factor is notbound up with the colouring substance. The same stimulatingeffect on enzyme production was obtained by adding to the nutrientmedium a small quantity of glucose which had been dry heatedat 150° C. for 20 minutes. Chromatographic analysis suggestedthat the stimulating substance was probably glyceraldehyde,though it is not excluded that other breakdown products of sugarsmay also play a part.     

Some Characteristics and Inhibitors of Indoleacetic Acid Oxidase from Tissue 2

Extracts from tissue cultures of crown-gall from Parthenocissuscatalysed the destruction of indoleacetic acid in vitro withoptimum activity at pH 4·5. The presence of two co-factors,Mn⁺⁺ and 2,4-dichlorophenol, was necessary for this activity,which was found to be strictly aerobic. Chlorogenic acid, caffeic acid, scopoletin, ferulic acid, andgibberellic acid markedly inhibited IAA-destruction. Chlorogenicacid inhibition was reversed by the addition of H₂O₂. Chlorogenicacid was not oxidized by the IAA-destroying system and did notbehave as a competitive inhibitor. The IAA-oxidase extract manifested peroxidase and phenolaseactivity with catechol and pyrogallol as substrates. However,this activity was greater at pH 7·0 than at the optimumfor IAA-oxidase activity, pH 4·5. Further evidence ofthe existence of these two enzymes in the intact tissue wasdemonstrated by histochemical studies. In tissue slices, peroxidaseactivity was very high and widely distributed while phenolaseactivity was low and restricted to localized centres of thetissue.     

Mechanism for the induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in HgCl2-treated sweet potaso root tissue

No 3-hydroxy-3-methylglutaryl coenzyme A reductase activitywas detected in microsomal fractions prepared from healthy andwounded sweet potato root tissues. However, there was considerableenzyme activity in the tissue discs when Hgcl₂ was applied afterincubation at 30?C for 18 hr. This increase in the enzyme activitywas followed by furano-terpene accumulation. Application ofcycloheximide to discs immediately after preparation completelyinhibited the increase in the enzyme activity when HgCl₂ wasapplied after incubation. In contrast, the increase was delayedfor about 4 hr, then the activity was enhanced, when CHI wasapplied after preliminary incubation. CHI completely inhibitedprotein synthesis when applied to the discs after the preliminaryincubation, as judged by the inhibition of the incorporationof ¹⁴C-leucine into protein and the inhibition of the increasein peroxidase activity which is synthesized de novo. These resultssuggest that the inactive precursor of HMG-CoA reductase issynthesized during the preliminary incubation in response onlyto wounding then it is converted into the active form aftertreatment with HgCl₂. (Received January 11, 1979; )     

Antioxidant Efficiency during Callus Initiation from Mature Rice Embryo

The changes in the activities of active oxygen scavenging enzymesand lipid peroxidation during callus formation from germinatingmature rice embryo was investigated. Superoxide dismutase (SOD)and catalase (CAT) activities were much lower during callusformation than during seedling growth indicating the decliningcapacity of the callus tissue to scavenge O^.-₂ and H₂O₂ respectively.Other H₂O₂-utilizing enzymes such as guaiacol peroxidase (GPOX)and ascorbate peroxidase (APOX) had also much lower activitiesduring the initial period of callus formation than during normalgermination but soon these enzyme activities were rapidly enhancedduring callus growth but declined during seedling growth. SinceH₂O₂ level was quite high in the callus tissue, it is probablethat GPOX and APOX are not efficient in decomposing H₂O₂ inthis tissue. Water soluble non-protein -SH compounds of whichGSH is the major component increased more rapidly during seedlinggrowth than during callus formation. This was reflected by thehigher activity of glutathione reductase (GR) in the seedlingtissue than in the callus tissue. Although peroxide and malondialdehydedid not accumulate during the callus initiation period, thefast decrease in the SOD and CAT activities indicates that duringthis transition period the tissue has increasing tendency towardsan oxidative state because of the weakening of the scavengingmechanism. The cellular environment, thereafter, becomes moreoxidizing during callus growth when compared with the normalseedling development in the absence of 2,4-D. (Received September 8, 1994; Accepted February 16, 1995)     

Changes in the specific activities of the galactose enzymes in E. coli under different growth conditions

Summary An increase in the specific activity of kinase and transferase is observed when late growth phase is compared with early exponential phase. The increase is more pronounced in the presence of saturating inducer concentrations than in uninduced cultures. Epimerase does not increase in late growth phase in the presence of D-fucose, provided the other enzymes of the galactose pathway are present. It is believed that increases observed are due to an increased rate of synthesis of the galactose enzymes in late growth phase, and that the lack of coordination found for epimerase is due to an inactivation of the latter enzyme, which occurs in the presence of D-fucose and needs an intact galactose pathway.     

Conversion of (+)-Dihydroquercetin to 3,4-cis-Leucocyanidin by a Reductase Extracted from Cell Suspension Cultures of Cryptomeria japonica

A soluble, NADPH-dependent reductase catalyzing the reductionof (+)-dihydroquercetin to 3,4-cis-leucocyanidin (5,7,3',4'-tetrahydroxyflavan-3,4-cis-diol)was demonstrated in an enzyme preparation from a cell suspensionculture of Japanese cedar (Cryptomeria japonica D. Don). TheK_m value for (+)-dihydroquercetin was 48µM. The enzyme,which was purified 26.2-fold, could also catalyze the reductionof (+)-dihydrokaempferol to 3,4-cis-leucopelargonidin (5,7,4'-trihydroxyflavan-3,4-cis-diol). The enzyme had a pH optimumof 7 and a molecular weight of 133,000. It was inhibited byCu²⁺ and iodoacetate, but not by p-chloromercuribenzoate. Duringthe growth stages of the cell suspension cultures, an increasein reductase activity proceeds an increase in procyanidin content,as might be expected. (Received November 25, 1987; Accepted April 11, 1988)     

Characterization and Induction of the Activity of 1-Aminocyclopropane-1-Carboxylate Oxidase in the Wounded Mesocarp Tissue of Cucurbita maxima

1-Aminocyclopropane-1-carboxylate (ACC) oxidase (ethylene-formingenzyme) was isolated from wounded mesocarp tissue of Cucurbitamaxima (winter squash) fruit, and its enzymatic properties wereinvestigated. The enzyme required Fe²⁺ and ascorbate for itsactivity as well as ACC and O₂ as substrates. The in vitro enzymeactivity was enhanced by CO₂. The apparent K_m value for ACCwas 175 µM under atmospheric conditions. The enzyme activitywas inhibited by sulfhydryl inhibitors and divalent cationssuch as Co²⁺, Cu²⁺, and Zn²⁺. ACC oxidase activity was induced at a rapid rate by woundingin parallel with an increase in the rate of ethylene production.The exposure of excised discs of mesocarp to 2,5-norbornadiene(NBD),an inhibitor of ethylene action, strongly suppressed inductionof the enzyme, and the application of ethylene significantlyaccelerated the induction of the activity of ACC oxidase inthe wounded mesocarp tissue. These results suggests that endogenousethylene produced in response to wounding may function in promotingthe induction of ACC oxidase. (Received January 13, 1993; Accepted April 15, 1993)     

The Aeration of Catharanthus roseus L. G. Don Suspension Cultures in Airlift Bioreactors: The Inhibitory Effect at High Aeration Rates on Culture Growth

An investigation was carried out to examine the effect of aerationon the growth of Catharanthus roseus suspension cultures inairlift bioreactors. A high aeration rate (0·86 v.v.m.)was found to inhibit the growth of cultures. Venting culturesat a high rate with low oxygen content gas mixtures was equallyinhibitory to culture growth, showing that high aeration wasnot inhibitory as a result of oxygen toxicity. The dissolvedcarbon dioxide tension was found to be lower in cultures operatedat high aeration than those operated at low aeration. Supplyingexogenous CO₂ to cultures at high aeration restored the CO₂tension to values normally encountered at a low aeration rate,and was found to alleviate the inhibitory effects at high aeration.However, further increasing the CO₂ supply to cultures was foundto be severely inhibitory to growth. Therefore, the growth ofC. roseus cultures is very sensitive to dissolved CO₂ concentration,growth being inhibited at values either higher or lower thanan optimum. Key words: Aeration, carbon dioxide, Catharanthus roseus suspension culture     

外源NO对南方红豆杉幼苗光合色素及抗氧化酶的影响

以4年生南方红豆杉幼苗为实验材料,通过对南方红豆杉幼苗喷施不同浓度外源一氧化氮（NO）供体硝普钠溶液（0、0.01、0.1、0.5和1 mmol·L^-1SNP）,测定光合色素含量、抗氧化酶活性、丙二醛（MDA）含量和过氧化氢（H₂O₂）含量等生理指标,以探讨不同浓度外源NO对南方红豆杉叶片光合色素和抗氧化酶的影响。结果表明：喷施低浓度（0.01、0.1 mmol·L^-1）SNP可显著提高南方红豆杉叶片的叶绿素a、叶绿素b、类胡萝卜素和总叶绿素含量,增加叶绿素a/b的比值,而喷施高浓度（0.5、1 mmol·L^-1）SNP降低了叶片的光合色素含量。随着外源NO供体浓度的增加,叶片过氧化氢酶(CAT)活性显著增加,过氧化物酶(POD)活性先增加后降低。此外,处理前期,低浓度SNP处理明显提高了抗坏血酸过氧化物酶(APX)活性,而高浓度SNP处理显著降低了APX活性,处理后期APX活性随SNP浓度的增加而显著下降。喷施低浓度SNP可有效提高超氧化物歧化酶(SOD)活性和增加可溶性蛋白含量,降低MDA和H₂O₂的含量,而喷施高浓度SNP显著增加了MDA和H₂O₂的含量。因此,低浓度的SNP（<0.5 mmol·L^-1）处理南方红豆杉幼苗,可增加其叶绿素含量,提高抗氧化酶活性,降低MDA和H₂O₂的含量,而高浓度的SNP（≥0.5 mmol·L^-1）处理会降低叶绿素含量,提高H₂O₂含量,增加细胞膜质过氧化程度,从而对南方红豆杉幼苗造成一定伤害。     

Regulation of Nitrate Reductase in Acetabularia mediterranea

Nitrate reductase (NR) activity has been characterized on cell-freeextracts of the marine unicellular green alga, Acetabulariamediterranea. Reduced methyl viologen and bromophenol blue aswell as NAD(P)H were effective electron donors for the enzyme. NADH-NR activity increased during cell development reachingits maximum level when the cells approached their maximum length.A substantial increase was also seen in enucleated cells. Theenzyme activity was only present in the green parts of the celland became diminished as the stalks bleached in ageing cells.Under a 12 h light: 12 h darkness photoperiod, NADH-NR activityexhibited pronounced daily fluctuations reaching its maximumon the first hour of illumination (this increase was impairedby cycloheximide) and a minimum in the middle of the dark period. In N-starved algal cultures, NR was constitutively present atappreciable levels and new synthesis took place in the presenceof either NO₃ or low concentrations of NH⁺₄ . However, in thepresence of this cation the enzyme remained mostly in its inactiveform, and could be reactivated in vitro with ferrycyanide. Key words: Nitrate reductase, Acetabularia, daily rhythms, nitrate, ammonium     

Inhibition of nitrate reductase from tomato leaves by adenosine-5'-diphosphate

ADP was found to inhibit the activity of nitrate reductase fromtomato leaves in vitro. No effects of ATP, AMP, adenosine andadenine could be detected. Orthophosphate promoted activityonly in the presence of high concentrations of NADH₂. It is suggested that nitrate reductase possesses characteristicsof a regulatory enzyme and that ADP brings about a transformationin the conformation of the enzyme, with a resulting decreasein its activity. (Received February 29, 1968; )