Comparison of the developmental patterns of mitochondrial malate dehydrogenase between mung bean and cucumber cotyledons during and following germination

The development of mitochondrial NAD⁺-malate dehydrogenase (EC 1.1.1.37) in mung bean and cucumber cotyledons was followed. using the antibody raised against it, during and following germination. The developmental patterns were quite different between the two. In cucumber, the content of mitochondrial malate dehydrogenase continued to increase through 3–4 days after the beginning of imbibition. This was, at least in part, due to active synthesis of the enzyme protein, and the synthesis seemed to be regulated by the availability of the translatable mRNA for the enzyme. In mung bean, on the other hand, the enzyme was present in dry cotyledons at a rather high concentration, and remained at a constant level between day 1 and day 3 after the reduction of the content to one-half its initial level during the first day. De novo synthesis of the enzyme could not be detected in mung bean cotyledons by pulse-labeling experiment.     

Modulation of Chlorophyll, Carotene and Xanthophyll Formation by Penicillin, Benzyladenine and Embryonic Axis in Mung Bean (Phaseolus aureus L.) Cotyledons

Penicillin stimulated the synthesis of pigments in the cotyledonsof intact embryos and excised cotyledons of mung bean (Phaseolusaureus L.) and enhanced benzyladenine-induced accumulation ofchloroplast pigments. The presence of the embryonic axis duringlight exposure proved to be beneficial for chlorophyll synthesisby the cotyledons whereas its presence in dark germination producedan adverse effect. The possible involvement of nucleic acidand protein synthesis in light-regulated chlorophyll formationis suggested. The stimulating effect on pigment synthesis providedby penicillin in this system seems to involve a maintenanceof nucleic acid and protein synthesis. Phaseolus aureus L., mung bean, pigment synthesis, cotyledons     

Cytokinin Induces a Rapid Decrease in the Levels of mRNAs for Catalase, 3-Hydroxy-3-methylglutaryl CoA reductase, Lectin and Other Unidentified Proteins in Etiolated Cotyledons of Cucumber

Large-scale differential hybridization was performed to examinerapid changes in gene expression caused by a phytohormone, cytokinin,in etiolated cotyledons of cucumber (Cucumis sativus L.). Weisolated 86 cDNA clones for mRNAs whose levels decreased within2 h of the start of treatment with N⁶-benzyladenine (BA). Partialnucleotide sequences showed that some of the cDNAs were homologousto those for catalase, 3-hydroxy-3-methylglutaryl CoA reductase(HMGR) and a lectin. This is the first report that the levelsof the mRNAs for those proteins are regulated by a cytokininin darkness. Together with previous results [Teramoto et al.(1993) Physiol. Plant. 87: 584, (1994) Planta 193: 573, (1995)Planta 196: 387], the present study suggests that the cytokininact to lower the levels of mRNAs transcribed from various genesin etiolated cotyledons. (Received May 18, 1995; Accepted August 17, 1995)     

萌发绿豆子叶衰老期间的呼吸作用与线粒体功能的改变

暗中培养的绿豆幼苗子叶在萌发后3—4天时,外观出现衰老征状,6天后子叶凋落。随子叶日龄的增加,子叶的呼吸强度一直下降,呼吸商始终小于1。当外加L—苹果酸、a—酮戊二酸、琥珀酸和NADH为底物测定离体线粒体氧化活性时,衰老子叶的线粒体对上述四种底物的氧化活性有不同程度的增加;抗氰呼吸也有所升高。子叶衰老时,线粒体的ADP／O和呼吸控制(RC值均降低);线粒体ATPase水解ATP的活性升高。衰老绿豆子叶线粒体氧化磷酸化偶联效率的降低和ATPase水解活性的增强是与线粒体结构改变相联系的一种功能变化,它导致能量亏缺,并进一步加速了衰老的恶化进程。     

Effects of spermidine on the synthesis of α-amylase in cotyledons of mung bean seedlings

When cotyledons of mung bean [ Vigna radiata (L.) Wilczek] were treated with spermidine (3 m M ) during the first 6 h of imbibition, the development of α-amylase activity in cotyledons during the following 3 days was severely inhibited (75%) This inhibition was due to a slower accumulation of α-amylase protein, which in turn resulted from an inhibition of α-amylase synthesis. The rise in the level of α-amylase mRNA in cotyledons was also inhibited by spermidine treatment. However, the degree of inhibition of mRNA accumulation (40%) was not so marked as that of the activity of α-amylase synthesis (80%). These results are discussed in relation to the mode of action of spermidine on α-amylase expression.     

Casein kinase activity in etiolated Cucumis sativus cotyledons

Two calcium- and light-dependent protein kinases have been reported in etiolated Cucumis sativus cotyledons (Vidal et al. 2007). In the present work, we studied casein kinase (CK) activity in etiolated cucumber cotyledons of in-gel and in vitro kinase assays, using specific CK inhibitors, and ATP and GTP as phosphate donors. Two proteins with CK activity were detected in both casein gels and autophosphorylation assays. One of them, with a molecular mass of approximately 36 kDa, showed biochemical CK1 characteristics: it was inhibited by specific CK1 inhibitors and only used ATP as phosphate donor. The second, with a molecular mass of approximately 38 kDa, had CK2 characteristics; it used both ATP and GTP as phosphate donors, was inhibited by all specific CK2 inhibitors, and was recognized by a polyclonal antibody directed against the α catalytic subunit of a CK2 from tobacco. The kinase activity of the CK2 detected in etiolated cucumber cotyledons showed circadian rhythmicity in both in vitro and in-gel casein phosphorylation and in autophosphorylation assays. Thus, our results suggest that the CK2 of approximately 38 kDa could be related to the circadian oscillator of C. sativus cotyledons.     

Peroxidase and Glucose-6-phosphate Dehydrogenase Levels in Cotyledons of Cucumis sativus (L.)

Peroxidase and glucose-6-phosphate dehydrogenase become increasinglyactive in cucumber cotyledons excised from the plant althoughthere is a fall in total protein content. The increases areinhibited by dinitrophenol, D, and L-threo-chloramphenicol,cycloheximide, puromycinj and actinomycin D, and it is concludedthat the enzymes are synthesized de novo. Disc electrophoresisof cotyledon extracts revealed three bands of peroxidase, oneof which becomes much enhanced in excised cotyledons. Citrullineaccumulates markedly in excised cotyledons. If the shoot ofcucumber plants is cut away above the cotyledons, very largeincreases in peroxidase and glucose-6-phosphate dehydrogenaseare observed although yellowing and protein loss are delayed.     

Cytokinins in Root Nodules of Phaseolus mungo

Four cytokinins have been separated from extracts of root nodulesof Phaseolus mungo by thin-layer chromatography. Their activitywas determined on the basis of their ability to induce betacyaninsynthesis in cotyledons of Amaranthus caudatus. Zeatin and itsriboside showed greater activity than N⁶ (²-(isopentenyl)) aminopurine and its riboside in the bioassay. Phaseolus mungo, mung bean, cytokinins, isopentenyl, amino-purine, zeatin, betacyanin synthesis, Amaranthus caudatus     

Formation of Chlorophyll-Protein Complexes in Greening Cucumber Cotyledons in Light and then in Darkness

The changes in chlorophyll-protein complexes (CPs) in cucumbercotyledons during illumination and subsequent dark incubationwere studied by SDS-polyacrylamide gel electrophoresis. Whenetiolated cucumber seedlings were illuminated, chlorophyll wassynthesized and CPs were formed. In the early phase of greening(6 h of illumination), light-harvesting chlorophyll a/b-proteincomplex (LHCP) was the main GP. As the greening proceeded, P700chlorophyll a-protein complex (CP1) accumulated. When 6-h illuminatedseedlings were transferred to darkness, CP1 accumulated concomitantlywith a decrease in LHCP without new chlorophyll synthesis. Thechanges in the amounts of CPs in the dark became smaller withthe progress of greening and were not observed after 72 h ofillumination. These changes were confirmed by examining thechlorophyll/P700 ratio and the low temperature absorption spectrumof cotyledons. These results suggest that in the early phaseof greening, CPs were unstable and their chlorophyll moleculeseasily exchanged with those of other kinds of CPs. (Received October 14, 1982; Accepted December 1, 1982)     

Exposure to alcohol vapours reduces chilling-induced injury of excised cucumber cotyledons, but not of seedlings or excised hypocotyl segments

Cucumber (Cucumis sativus L. cv. Poinsett 76) seedlings withfully expanded cotyledons, and excised cotyledons, first trueleaves, hypocotyl segments and fruit mesocarp discs were exposedto vapours from a series of aqueous alcohol solutions of 0 to320 mM methanol, ethanol, n-propanol, n-butanol, and n-pentanolduring chilling at 2.5C for 5 d. Certain concentrations ofeach alcohol reduced subsequent chilling-induced ion leakagefrom the cotyledons and leaves. Exposure of cotyledons to certainmethanol or ethanol solutions also reduced chilling-inducedethylene production, but not carbon dioxide production. In contrast,exposing cucumber seedlings with fully expanded cotyledons tothe same series of alcohol concentrations that resulted in reducedchilling-induced ion leakage and ethylene production of excisedcotyledons actually increased chilling injury of the seedlings.The hypocotyl region directly below the cotyledons was the siteof chilling-induced injury and contained the most chilling-sensitivehypocotyl tissue. Exposing hypocotyl segments excised from thissensitive region to alcohol solutions did not significantlyreduce chilling-inducedion leakage. Exposing excised cucumbercotyledons or hypocotyl segments to equivalent osmotic nonvolatilesolutions of mannitol and glycerol at 2.5C or to alcohol solutionsat 12.5C had no significant effect on the rate of ion leakage.For the series of alcohols used, the relationship between thelog of the alcohol concentration that minimized chilling-inducedion leakage from cucumber cotyledon discs held at 2.5C for5 d and the log of the partition coefficient of the alcoholinto olive oil or the log of the molecular weight of the alcoholswas highly significant. The same concentrations of alcoholsthat reduced chilling-inducedion leakage also reduced stomatalaperture as measured as decreased porosity of excised cotyledons.The correlation between reduced chilling injury and stomatalconductance of cotyledons exposed to a series of ethanol solutionswas highly significant. It appears that alcohols may reducechilling injury of cucumber cotyledons by inducing stomata closure.Sufficient endogenously synthesized ethanol accumulated in discsheld in N2 at 10C for 1 d to confer tolerance to chilling at2.5C for 5 d. Key words: Anaerobic, Cucumis sativus, ethanol, ion leakage, stomatal conductance     

The relationship between transpiration and chlorophyll synthesis in etiolated squash cotyledons

Etiolated cotyledons of squash (Cucurbita moschata Duch. var.melonaeformis Makino cv. Tokyo) treated with kinetin, KCl orfusicoccin in the dark increased both transpiration in the darkand chlorophyll synthesis after exposure to light. The minimumperiod of kinetin treatment to stimulate transpiration was similarto that to accelerate chlorophyll synthesis. On the other hand,treatment of cotyledons with vaseline or abscisic acid markedlyinhibited both transpiration and chlorophyll synthesis, suggestingthat cytokinin-induced stimulation of chlorophyll synthesiscould be mediated through the effect on the stomatal opening. (Received December 7, 1977; )     

Photosynthetic Electron Transport in Plastids in Detached Cucumber Cotyledons Treated with 6-Benzylaminopurine and Potassium

Etiolated cotyledons of cucumber (Cucumis sativus L.) were excisedand incubated for 16 days on agar in the absence or in the presenceof 6-benzylaminopurine (BA) (3 µM) or KCl (10 mM) undercontinuous light. Treatment with BA or KCl increased growthand greening of cotyledons, KCl being particularly active duringthe last few days of the incubation. The rate of photosynthetic electron transport reached a maximumafter 3 days, as did the chlorophyll content, and then it declinedby approximately 95%. An uncoupling from phosphorylation wasobserved in older cotyledons. BA and KCl increased the rateof electron flow per cotyledon, and KCl delayed the onset ofthe decline in this rate. PS I and PS II activities per chlorophyllunit were lower in BA-treated cotyledons, but KCl-treated cotyledonsbehaved like the controls. Fluorescence analysis indicated that the level of active chlorophyllsinvolved in the transfer of energy in PS II decreased between3 and 7 days under all conditions tested. In BA-treated cotyledons,the proportion of active chlorophylls was consistently lowerthan that in the control. Thus, it appears that, in the absenceof exogenous K⁺, cytokinin has only a low antisenescence effecton photosynthetic activity, and that K⁺ may enhance the photosyntheticelectron flow between plastoquinone and plastocyanin via thecytochrome b₆-f complex. (Received August 2, 1989; Accepted January 23, 1990)     

Induction and Repair of Damage to DNA in Cucumber Cotyledons Irradiated with UV-B

Photoinduced lesions in DNA, namely, cyclobutane pyrimidinedimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts[(6-4)photoproducts], in cucumber cotyledons that had been irradiatedwith naturally occurring levels of UV-B (290–320 nm) werequantitated by enzyme-linked immunosorbent assays with monoclonalantibodies specific to each type of photolesion. Induction ofthese photolesions was dependent on temperature and their extentwas reduced by simultaneous irradiation with white light. Thedark repair of both types of photolesion was undetectable. Light-dependentremoval of (6-4)photoproducts was very slow, with 50% removalin 4 h. By contrast, 50% of initial CPDs were removed within15 min. Both photorepair processes were dependent on the intensityof white light and were sensitive to temperature. These resultsindicate that high photolyase activity is present in cucumbercotyledons and that repair activities in cucumber cotyledonsare different from those reported in Arabidopsis, in which (6-4)photoproductsare photorepaired more rapidly than CPDs. (Received October 13, 1995; Accepted December 28, 1995)     

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)     

A re-examination of 5-aminolevulinic acid synthesis by isolated intact,developing chloroplasts: The O2 requirement in the light

Synthesis of 5-aminolevulinic acid (ALA) in organello was re-examined with developing chloroplasts isolated from greening cucumber (Cucumis sativus L. var. Beit Alpha) cotyledons. In the dark, ALA accumulated in the presence of ATP, reducing power (NADPH and glucose-6-phosphate), glutamate and levulinic acid (or 4,6-dioxoheptanoic acid).Under continuous illumination there was no requirement for added ATP and reducing power, unless DCMU was added or O₂ was removed, indicating that ATP and reducing power could be supplied endogenously by photosynthesis in the presence of O₂. No mitochondrial involvement could be demonstrated in this system. Under anaerobic conditions in the light oxaloacetic acid (OAA) could replace O₂ and permit a high accumulation of ALA. The fact that OAA could replace O₂ suggests that an acceptor of non-cyclic electron flow may be required to provide ATP or some other cofactor of ALA synthesis. The phosphorylation uncoupler, 2,4-dinitrophenol, inhibited ALA synthesis. Light-dependent ALA in air was strongly inhibited by methylene blue (MB) and NaN₃, but only very slightly by KCN.     

Inter-organ Control of Greening in Etiolated Cucumber Cotyledons

Inter-organ control of greening in etiolated cucumber (Cucumis sativus L. cv. Aonagajibae) cotyledons was investigated. Four- or six-day-old excised or intact etiolated cucumber cotyledons were illuminated under aerobic conditions. Excised cotyledons without hypocotyl hooks produced chlorophyll without a prolonged lag phase and the rate of chlorophyll formation was not depressed if they were illuminated immediately after excision. If the excised cotyledons were incubated in the dark before illumination, chlorophyll accumulation at the end of 6 h of continuous illumination was remarkably lowered as the dark period lengthened, especially in 6-day-old cotyledons. The rapid loss of chlorophyll-forming capacity of excised cotyledons during dark preincubation suggests a stimulatory effect of hypocotyls on the greening in cotyledons. The treatment of excised cotyledons with bleeding sap in the dark for 18 h resulted in the promotion of chlorophyll formation during subsequent continuous illumination. Partial fractionation of bleeding sap with organic solvents and paper chromatography indicates that the active substances showed the same behavior as cytokinins. These facts add weight to the hypothesis that cytokinins from roots flow into cotyledons and stimulate greening.     

Synthesis and Turnover of {alpha}-Amylase in Cotyledons of Germinating Vigna mungo Seeds: Effects of Exogenously Applied End-Products and Plant Hormones

Pulse-chase experiments indicated that the higher levels ofa-amylase in detached and incubated cotyledons of Vigna mungothan those in cotyledons attached to the embryonic axis weredue to both faster synthesis and slower degradation of the enzymein the detached cotyledons than in the attached cotyledons.Levels of a-amylase in the cotyledons were examined in termsof possible effects of end-products and the effects of exogenouslyapplied plant hormones and growth regulators. Levels of a-amylaseactivity and content were reduced by high concentrations ofglucose and sucrose, and it is suggested that this effect wascaused mostly by osmotic stress and partly by end-product repression.The level of a-amylase was nearly twice that in controls after1 to 10µM GA₃ had been applied to the cotyledons. In addition,0.1 mM kinetin, 0.1 mM 2,4-D and 0.1 to 0.S mM naphthaleneaceticacid also increased the level by 34% to 66% as compared to thecontrol. ABA and uniconazole both prevented the synthesis ofa-amylase. (Received July 4, 1994; Accepted November 14, 1994)     

Effect of Sink-Source Manipulations on the Photosynthetic Rate and Carbohydrate Content of Cucumber Cotyledons

Mayoral, M. L., Plaut, Z. and Reinhold, L. 1985. Effect of sink-sourcemanipulations on the photosynthetic rate and carbohydrate contentof cucumber cotyledons.-J. exp. Bot. 36 1551–1558. The photosynthetic rate of cucumber cotyledons (Cucumis sativuscv. Dahla) reached a maximum value of 12 mg dm^–2 h^–1,10 d after emergence. In 12-d-old seedlings removal of one cotyledondoubled the CO₂ fixation rate of the other, as observed 3 dafter treatment. When the primary leaf was removed, the photosyntheticrate of the cotyledons was decreased by 33%. At this stage ofgrowth elimination of the roots as a sink for assimilates bygirdling the hypocotyl affected neither the photosynthetic ratenor the carbohydrate content of the cotyledons. By contrast,in 18-d-old seedlings removal of the first leaf brought abouta 42% increase in the photosynthetic rate of the cotyledons.The simultaneous removal of the first leaf and one cotyledondoubled the rate of CO₂ fixation of the remaining cotyledon.Girdling the hypocotyl lowered the photosynthetic rate of thecotyledons by 73%. In both 12- and 18-d-old seedlings a decreaseor increase in the sink-source ratio was correlated with anincrease or a decrease respectively in the carbohydrate contentof the cotyledons. The stomatal resistance of the cotyledonswas not affected by any of the treatments. The effect of sink-sourcemanipulations on photosynthesis and on the level of carbohydratespresent in the cotyledons was more evident in those seedlingsgrowing under high light intensity (580 µE m^–2 s^–1),than in those exposed to 300 µE m^–2 s^–1 Key words: Sink-source relationship, cotyledons, photosynthesis     

In vitro Culture of Immature Cotyledons of Soya Bean (Glycine max L. Merr.)

An in vitro procedure promoting the rapid growth and proteinincrease of soya bean cotyledons has been developed. The amountof protein synthesized varied greatly depending on the nitrogen(N) source provided. Glutamine was the most effective N source,while inorganic forms of N were ineffective. Growth and proteinsynthesis were both more rapid in vitro than in vivo. Underthe best conditions, soya bean cotyledons increased 8-fold bothin dry weight and in protein in 6 days. The formation of the7S and 11S storage proteins in vitro was similar to that invivo. Hence, this in vitro culture method is appropriate forstudying legume seed storage protein synthesis under controlledconditions.