Regulation of lettuce hypocotyl elongation by gibberellic acid. Interaction of gibberellic acid and gibberellin synergists - dihydroconiferyl alcohol, pestalotin and a triazinone compound

The effect of three gibberellin synergists on lettuce hypocotylelongation was studied. Dihydroconiferyl alcohol isolated fromlettuce plants and (–)-(6S, 1'S)-pestalotin isolated fromPestalotia cryptomeriaecola enhanced the promoting effect ofgibberellic acid on hypocotyl elongation of lettuce seedlingswith and without the cotyledons. On the other hand, TA, a triazinonecompound, did not enhance the gibberellin effect. The actionof (–)-(6S, 1'S)-pestalotin was strongly inhibited bycompetitive inhibitors of dihydroconiferyl alcohol such as caffeic,ferulic and trans-cinnamic acids. Of the two stereoisomers ofpestalotin, (+)-(6R, 1'R)-pestalotin enhanced the gibberellineffect but (+)-(6R, 1'S)-epipestalotin did not. (+)-(6R, 1'S)-Epipestalotinstrongly inhibited the action of (–)-(6S, 1'S)-pestalotinand dihydroconiferyl alcohol. TA did not affect the action ofdihydroconiferyl alcohol. Stress-relaxation analysis of the mechanical properties of thelettuce hypocotyl cell wall demonstrated that gibberellic acidcaused cell wall loosening and dihydroconiferyl alcohol andpestalotin did not influence this gibberellin effect. The action mechanism of gibberellin synergists is discussedbased on these results. (Received December 22, 1978; )     

Catecholamine stimulation of the gibberellin action that induces lettuce hypocotyl elongation

Effects of catecholamines and their derivatives on gibberellicacid (GA)-induced lettuce hypocotyl elongation was studied,because catecholamines have a chemical structure similar tothe dihydroconiferyl alcohol that has been isolated from lettucecotyledons as a GA synergist. Epinephrine, norepinephrine, dopamineand 3,4-dihydroxymandelic acid synergistically enhanced thepromoting effect of GA on hypocotyl elongation. In contrast,metanephrine, normetanephrine, DOPA and 3-methoxy-4- hydroxymandelicacid did not enhance the GA effect. The action of catecholamineswas inhibited by trans-cinnamic acid which competitively inhibitedthe action of dihydroconiferyl alcohol; this suggests that thereceptor site for catecholamines is the same as that for dihydroconiferylalcohol. The basic ethyl acetate fraction from lettuce seedlingssynergistically enhanced the GA effect. TLC analyses of thisbasic ethyl acetate fraction revealed that the chromatographicarea corresponding to authentic catecholamines could enhancethe GA effect. From these results, a possible role for catecholamines in theregulation of lettuce hypocotyl elongation caused by GA wasposited, and is discussed here. (Received May 15, 1979; )     

Enhancement of Gibberellic Acid-stimulated Hypocotyl Elongation of Lettuce (Lactuca sativa L. cv. Grand Rapids) by Phlorizin and Light

The interaction of light, gibberellic acid (GA₃), and phlorizinin the growth of lettuce (Lactuca sativa L. cv. ‘GrandRapids’) hypocotyls was investigated. At all concentrationsof GA₃, phlorizin enhanced GA₃-induced growth at luminous intensitiesabove 50 ft-c (continuous light). Without GA₃, phlorizin hadno effect on hypocotyl growth in the light but it inhibitedgrowth in the dark. Both seedlings and hypocotyl sections respondedto phlorizin in the presence of GA₃. There was no iteractionbetween phlorizin and KCl. Water-growth was severly inhibitedby light. GA₃,-induced growth was slightly inhibited by light,and then only at luminous intensities above 50 ft-c. Thus, relativeto H₂O-growth, GA₃-induced growth increased with increasingluminous intensity up to 450 ft-c, where it reached saturation.It seems that a synergism may exist between light and GA₃ aswell as between phlorizin and GA₃. Lactuca sativa L, lettuce, hypocotyl elongation, gibberellic acid, phlorizin, light     

Enzyme system catalyzing formation of cis-3-hexenal and n-hexanal from linolenic and linoleic acids in Japanese silver (Farfugium iaponicum Kitamura) leaves

Leaf alcohol (cis-3-hexenol) and leaf aldehyde (trans-2-hexenal)are responsible for the green odor in leaves and fruits. cis-3-Hexenal,a precursor of cis-3-hexenol and trans-2-hexenal, was producedfrom linolenic acid by a homogenate of Farfugium japonicum (Japanesesilver) leaves. n-Hexanal was produced from linoleic acid bya homogenate of the leaves. The enzyme system catalyzing formationof C₆-aldehydes from linolenic and linoleic acids was localizedin chloroplast lamellae, and required oxygen for reaction. C₁₈-unsaturatedfatty acids such as linolenic acid, linoleic acid and -linolenicacid, which have carboxyl groups and cis-1, cis-4-pentadienesystems including a double bond at C-12, acted as substrates,and C₆-aldehydes (cis-3-hexenal or n-hexanal), but not C₉-aldehydes,were produced from them. The properties of the enzyme systemin chloroplasts were as follows: optimal pH 7.0; stable at pH5 to 7; thermolabile and no activity at 50?C. These propertieswere very similar to those of tea chloroplasts. The enzyme systemcould be solubilized from chloroplasts by 2% Triton X-100, butwas very unstable in solubilized form. (Received July 9, 1976; )     

Asparagusic acid, a new plant growth inhibitor in etiolated young asparagus shoots

Yellow prisms of asparagusic acid, with a molecular formulaof C₄H₆O₂S₂ were isolated from etiolated asparagus tissues (Asparagusofficinalis L.). This acid inhibits growth in lettuce and otherseedlings when applied in concentrations of 6.67x10^–7Mto 6.67xl0^–7M. The extent of activity was very similarto that of abscisic acid. ¹ A well known shift reagent in the NMR spectrum (1). (Received April 12, 1972; )     

Inhibition of Ethylene Production by Fatty Acids in Fruit and Vegetable Tissues

Fatty acids of chain length from C₄ to C₁₂ inhibited ethyleneproduction in wounded albedo tissue of Hassaku (Citrus hassakuHort. ex Tanaka) fruit. Of the fatty acids tested, caprylicacid (C₈) and capric acid (C₁₀) were the most effective. Lauricacid (C₁₂) was less effective, and caproic acid (C₆) and butyricacid (C₄) were the least effective. Caprylic acid at 5 mM markedlyinhibited ethylene production in not only wounded albedo tissueof citrus fruit but also apple (Malus sylvestris Mill.) cortex,tomato (Lycopersicon esculentum Mill.) pericarp, cucumber (Cucumissativus L.) cortex, banana (Musa AAA group Cavendish subgroup)pulp, broccoli (Brassica oleracea L.) floret, spinach (Spinaciaoleracea L.) leaf, lettuce (Lactuca sativa L.) leaf and mungbean (Vigna radiata [L.] Wilczek) hypocotyl. Caprylic acid inhibitedethylene production at the step of conversion of l-aminocyclopropane-l-carboxylicacid to ethylene. The inhibition could be partially relievedby transferring the tissue to caprylic acid-free medium. (Received June 15, 1982; Accepted August 13, 1982)     

Comparative Potency of Nine Gibberellins

Gibberellins A₁ to A₉ have been compared, each at several doselevels, in bioassays based on extension of stems of dwarf gardenpea (Pisum sativum), dwarf bean (Phaseolus vulgaris) and Lunariaannua, of hypocotyls of cucumber (Cucumis sativus) and lettuce(Lactuca sativa), and of leaf sheaths of three dwarf mutants(d–1, d–3, d–5) of maize (Zea mays). GibberellinsA₃ (gibberellic acid) and A₇ are of high potency in most bioassays.A₈ is of negligible potency in all and is probably not a functionalhormone. The other gibberellins show a more or less marked tendencyto specificity. The plants used as bioassay material also differin the specificity of their response. Some, for example, maizedwarfs d–3 and d–5 and lettuce, respond well tomost gibberellins; others, for example, cucumber, respond onlyto a few; extreme specificity is shown by Lunaria annua which,in the unvernalized condition, responds by stem elongation onlyto gibberellin A₇. Dose/response curves of the various gibberellinsare usually parallel, but certain exceptions to this have beenfound. Possible explanations of specificity are discussed inrelation to the results obtained, and it is concluded that insufficientevidence is available to make it possible to draw any validconclusions. Current definitions of gibberellins, whether basedon chemical structure or biological activity, are unsatisfactory.     

Effects of Repetitive Acid Immersion, Red Light, and Gibberellin A3 Treatments on Phytochrome-mediated Germination Control in Skotodormant Lettuce Seeds

Dry lettuce seeds (Lactuca sativa L. cv. Grand Rapids), whichreceived 5 min far-red light (FR) 0.5 h after the onset of waterimbibition, showed 17% and 50% germination without and withacid immersion treatment (pH 0.1) for 1 h and rinsing with water,respectively. The acid treatment caused only 6% germinationor less in FR-treated seeds held for 10 to 30 d in dark storage.The 10 to 30 d skotodormant seeds did not respond to red light(R) or gibberellin A₃ (GA₃) singly, but showed 84% or higherpercentage germination if 1 h acid immersion was given beforeR or GA₃. The 20 d skotodormant seeds, which received R treatmentat day 10 but remained dormant showed 89% germination with onlyacid treatment. Similar values were obtained with 30 d skotodormantseeds which received one or two R treatments at day 10 or 20,i.e. the only requirement for these R-treated dormant seedswas an acid immersion. This releases the skotodormancy and rendersthe seeds more sensitive to R or GA₃, but the skotodormancywas initiated again if no light or hormone treatments were givenimmediately. The repetitive R or GA₃ treatments, which did notcause skotodormant seeds to germinate, lessened the degree ofskotodormancy. The germination of these skotodormant seeds canonly be induced by the synergistic action of R and GA₃. In thisstudy, GA₃ caused higher germination percentages in R-treatedskotodormant seeds than R stimulated in GA3-treated seeds. Itis suggested that (i) repetitive R or Ga₃ treatments maintaina high endogenous level of the far-red-absorbing form of phytochrome(P_fr) and GA activity, respectively, (ii) the accumulated stableintermediates of phytochrome persist in fully-imbibed skotodormantseeds for up to 20 d, without phytochrome expressing its functionuntil the seeds are acidified and (iii) a model is formulatedto interpret the results of acidification, growth promotersand R effects on germination of light-sensitive lettuce seeds. Key words: Phytochrome, Latuca saliva, seed germination, dark reversion of phytochrome, gibberellin A₃, acidification, skotodormancy     

Control by Light of Hypocotyl Elongation and Levels of Endogenous Gibberellins in Seedlings of Lactuca sativa L.

Four 13-hydroxygibberellins, gibberellin A₁ (GA₁), 3-epi-GA₁,GA₁₉ and GA₂₀ were identified by full-scan GC/MS in extractsof lettuce seedlings (Lactuca sativa L. cv. Grand Rapids). Theresults suggest that the early-13-hydroxylation biosyntheticpathway to GA₁ functions in the lettuce seedlings. It was alsofound that GA₁ is active per se in the control of hypocotylelongation in lettuce seedlings. To investigate the relationshipbetween control by light of hypocotyl elongation and levelsof endogenous GAs in lettuce, endogenous levels of GAs werequantified by radioimmunoassay in seedlings that had been grownfor 5 days in the dark (5D) and in those that had been grownfor 4 days in the dark and then under white light for 1 day(4D1L). The endogenous level of GA₁ in the upper and lower partsof hypocotyls in 5D seedlings was about four times higher thanthat in 4D1L seedlings. The response of explants (hypocotylsegments with cotyledons) from dark-grown seedlings to GA₁ isknown to be similar in the dark and under white light when theexplants are treated with inhibitors of the biosynthesis ofGA. Therefore, the above information suggests that the highlevel of GA₁ in hypocotyls of dark-grown seedlings is responsiblefor the rapid elongation of hypocotyl, while irradiation bywhite light decreases the endogenous level of GA₁ in the hypocotylswith a resultant decrease in the rate of hypocotyl elongation. (Received March 13, 1992; Accepted May 21, 1992)     

Interaction of Moisture Stress and Three Phenolic Compounds on Lettuce Seed Germination

No interactions between water stress and three phenolic acids(p-coumaric, caffeic and ferulic acids) on lettuce (Lactucasativa L. var. Grand Rapids) seed germination were found. Probitanalysis indicated that mechanisms of action of water stressand the phenolic inhibitors were similar. The relative effectivenessof the compounds was p-coumaric > ferulic > caffeic. Nointeraction was found between p-coumaric and ferulic acid, whereasantagonism was found between caffeic acid and each of the othertwo phenolic acids. Lactuca sativa L., lettuce, germination, phenolic compounds, moisture stress, allelopathy, seed     

Ethylene Biosynthesis and Xylogenesis in Lactuca Pith Explants Cultured In Vitro in the Presence of Auxin and Cytokinin: The Effect of Ethylene Precursors and Inhibitors

The possible involvement of ethylene in the induction of xylemdifferentiation was studied in lettuce (Lactuca saliva L. cv.Romaine) pith parenchyma explants. The addition of the ethyleneprecursors L-methionine (0.25 µM), S-adenosylmethionine(25 µM) and 1-aminocyclopropane-l-carboxylic acid (0.01µM), or the ethylene-releasing agent 2-chloroethylphosphonicacid (1.0 µM), to a standard IAA-kinetin-containing mediumenhanced xylogenesis compared to control explants cultured inthe absence of these compounds. In the presence of the ethyleneinhibitors aminoethoxyvinylglycine, Co(NO₃)₂ and AgNO₃, xylogenesiswas inhibited. Inhibition of xylogenesis by aminoethoxyvinylglycine(75 µM), Co(NO₃)₂ (50 µM) and AgNO₃ (6.0 µM)was reversed by exogenous 1-aminocyclopropane-l-carboxylic acid(0.01 µM), 2-chloroethylphosphonic acid (5.0 µM)and L-methionine (0.25 µM), respectively. Ethylene productionby explants cultured on media containing L-methionine or 1-aminocyclopropane-l-carboxylicacid was greater than the biosynthesis of ethylene by explantscultured in the absence of these compounds. The incorporationof 2-chloroethylphosphonic acid into the culture medium resultedin higher rates of ethylene production compared to explantscultured on the IAA-kinetin medium. The presence of either aminoethoxyvinylglycineor Co(NO₃)₂ inhibited ethylene production by explants culturedon the IAA-kinetin medium. The data support the hypothesis thatethylene plays a positive role in the initiation of xylem differentiation. Key words: Xylogenesis, Differentiation, Ethylene, IAA, Kinetin, Lactuca sativa     

Seasonal Changes in Activities of Enzymes Responsible for the Formation of C6-aldehydes and C6-alcohols in Tea Leaves, and the Effects of Environmental Temperatures on the Enzyme Activities

When tea leaves were homogenized and incubated, the volatileC₆-compounds hexanal, cis-3-hexenal, cis-3-hexenol and trans-2-hexenalwere formed much more by summer leaves than by winter leavesof tea plants (Camellia sinensis). The enzymes lipolytic acylhydrolase (LAH), lipoxygenase, fatty acid hydroperoxide lyase(HPO lyase) and alcohol dehydrogenase (ADH) and an isomerizationfactor were responsible for the sequential reactions of C₆-compoundformation from linoleic and linolenic acids in tea leaf lipids,and there were seasonal changes in their activities. The tealeaf enzymes were of 3 types: LAH and lipoxygenase, which hadhigh activities in summer leaves and low activities in winterleaves; ADH, which had low activity in summer leaves and highactivity in winter ones; and HPO lyase and the isomerizationfactor, which did not seem to have any effect on the rate ofC₆-compound formation throughout the year. Changes in enzymeactivities were induced by shifts in the environmental air temperaturerather than by the age of the leaves. The combined activitiesof these enzymes determined the amounts and compositions ofthe volatile C₆-compounds formed, which are the factors thatcontrol the quality of the raw leaves processed for green tea. (Received October 6, 1983; Accepted December 20, 1983)     

Enhancement of Peroxidase-Dependent Oxidation of Sinapyl Alcohol by an Apoplastic Component, 4-Coumaric Acid Ester Isolated from Epicotyls of Vigna angularis L.

A water-soluble component that enhanced the peroxidase-dependent(POX-dependent) oxidation of sinapyl alcohol was isolated fromepicotyls of Vigna angularis. This compound was an ester of4-coumaric acid and a hexose, and it was found in both the apoplastand the symplast. The ester was oxidized by a basic POX isozyme(K_m, about 20 µM) and by an acidic POX isozyme (K_m, about40 µM) that had been partially purified from the apoplasticfraction of epicotyls of V. angularis. These POX isozymes oxidizedsinapyl alcohol at only a very low rate, but a 15-fold enhancementwas observed upon addition of the ester. The concentrationsof the ester required for the half-maximal enhancement weresimilar to the K_m values of the ester for its oxidation by therespective isozymes. The apoplastic concentration of the esterwas higher than 130 µM, suggesting that this ester mightact as a donor of electrons to the apoplastic POX isozymes insitu. Coniferyl alcohol also enhanced the POX-catalyzed oxidationof sinapyl alcohol. The concentrations of coniferyl alcoholrequired for half-maximal enhancement of the oxidation of sinapylalcohol were about 23 and 250 µM when reactions were catalyzedby the basic and acidic POXs, respectively. These values weresimilar to the K_m values of coniferyl alcohol for its oxidationby the respective isozymes. These results suggest that 4-coumaricacid ester and coniferyl alcohol, if it is present in the apoplast,can enhance the POX-dependent oxidation of sinapyl alcohol inthe apoplast of epicotyls of V. angularis. (Received July 1, 1996; Accepted February 5, 1997)     

Diffusion of Oxyleghaemoglobin

The diffusion of oxyleghaemoglobin, prepared from soybean rootnodules, was measured at 24°C in agar and agarose gels ofvarious strengths, or in 1% agarose containing 0-18% (w/v) bovineserum albumin, to simulate the protein content of the cytoplasmof root nodule cells. Values of D_p, the diffusion coefficient,were unaffected (D_p = 11·8 x 10^-11 m² s^-1; s.e.m. 0·3x 10^-11) until the protein concentration exceeded 6%, abovewhich D_p declined sharply. With 18% bovine serum albumin, theconcentration of total soluble protein calculated to be presentin the cytoplasm of infected cells, where most of the leghaemoglobinis located in vivo, D_p was 5·9 x 10^-11 m² s^-1. Theseresults are discussed in relation to leghaemoglobin-facilitateddelivery of O₂ to the respiring N₂-fixing bacteroids in rootnodule cells.Copyright 1993, 1999 Academic Press Bacteroids, diffusion, Glycine max, N₂ fixation, oxyleghaemoglobin, soybean, root nodules     

Ferric chelate reduction by sunflower (Helianthus annuus L.) leaves: influence of light, oxygen, iron-deficiency and leaf age

The presence of ferric chelate reducing activity in sunflower[Helianthus annuus L.) leaves has been studied by submergingleaf discs in a solution with Fe(III)-ethylenediaminetetra-acetate(FeEDTA), batho-phenanthroline disulphonate (BPDS) and vacuuminfiltration. The effect of different factors on the Fe(III)reduction rate was studied. Ferric reduction rate was about10-fold higher in the light than in darkness. The light effectwas greatly inhibited by 3-(3,4-dichloro-phenyl)-1,1-dimethylurea(DCMU), a photosystem II inhibitor. Several inhibitors of redoxsystems [cis-platinum (II) diamine dichloride (cis-platin),p-nitro-phenylacetate (p-NPA) and p-hydroxymercuribenzoic acid(pHMB)] decreased the FeEDTA reduction rate. The greatest inhibitionwas produced by the - SH group reagent pHMB (17% of control,in light). The FeEDTA reduction rate was much higher in theabsence of O₂ than with air or 100% O₂. Superoxide dismutase(SOD) decreased FeEDTA reduction with air in the light. Youngleaves reduced Fe(III)-chelate at a higher rate than did olderleaves. In iron-deficient plants, leaves did not exhibit enhancedferric chelate-reducing activity as was observed in roots. Itis suggested that at least two different redox systems or twostates of the same redox system work in the light and in darkness. Key words: Iron, leaves, plasma membrane-redox, light, oxygen level     

Voltage-dependent stimulation of the Na(+)-K(+) pump by insulin in rabbit cardiac myocytes

Insulin enhancesNa⁺-K⁺ pump activity in various noncardiactissues. We examined whether insulin exposure in vitro regulates Na⁺-K⁺ pump function in rabbit ventricularmyocytes. Pump current (I_p) was measured using thewhole-cell patch-clamp technique at test potentials(V_ms) from 100 to +60 mV. When theNa⁺ concentration in the patch pipette([Na]_pip) was 10 mM, insulin caused aV_m-dependent increase in I_p.The increase was ~70% when V_m was at nearphysiological diastolic potentials. This effect persisted afterelimination of extracellular voltage-dependent steps and whenK⁺ and K⁺-congeners were excluded from thepatch pipettes. When [Na]_pip was 80 mM, causingnear-maximal pump stimulation, insulin had no effect, suggesting thatit did not cause an increase in membrane pump density. Effects oftyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I inpipette solutions suggested that the insulin-induced increase inI_p involved activation of tyrosine kinase,phosphatidylinositol 3-kinase, and protein phosphatase 1, whereasprotein phosphatase 2A and protein kinase C were not involved.

     

Glandular Hairs and Secreted Material inSalvia blepharophyllaBrandegee ex Epling Grown in Italy

Morphological and histological investigations of cuticle andindumentum, as well as identification of the main componentsof the secreted material, were carried out forSalvia blepharophyllaBrandegeeex Epling. Besides non-glandular hairs, three types of glandulartrichomes (peltate and capitate) are described and comparedwith trichomes in other species. The histological findings andchemical analysis of the essential oil and leaf surface extractsrevealed a complex secretion product. GC-MS analysis of theessential oil showed that eugenol,cis-3-hexenyl benzoate,cis-jasmone,trans-nerolidol,benzyl alcohol and C₁₉-C₂₃n-alkanes were the main identifiablecomponents, whereas the flavonoids nuchensin and pedalitin,the neo-clerodane diterpenoid salvianduline D, and the triterpenoidsursolic acid and     

Regeneration of Ribulose 1,5-bisphosphate and Ribulose 1,5-bisphosphate carboxylase/oxygenase Activity Associated with Lack of Oxygen Inhibition of Photosynthesis at Low Temperature

The nature of the lack of oxygen inhibition of C₃-photosynthesisat low temperature was investigated in white clover (Trifoliumrepens L.). Detached leaves were brought to steady-state photosynthesisin air (34 Pa p(CO²), 21 kPa p(O₂), balance N₂) at temperaturesof 20°C and 8°C, respectively. Net photosynthesis, ribulose1,5-bisphosphate (RuBP) and ATP contents, and ribulose 1,5-bisphosphatecarboxylase/oxygenase (RuBPCO) activities were followed beforeand after changing to 2·0 kPa p(O₂). At 20°C, lowering p(O₂) increased net photosynthesis by37%. This increase corresponded closely with the increase expectedfrom the effect on the kinetic properties of RuBPCO. Conversely,at 8°C net photosynthesis rapidly decreased following adecrease in p(O₂) and then increased again reaching a steady-statelevel which was only 7% higher than at 21 kPa p(O₂). The steady-staterates of RuBP and associated ATP consumption were both estimatedto have decreased. ATP and RuBP contents decreased by 18% and33% respectively, immediately after the change in p(O₂) suggestingthat RuBP regeneration was reduced at low p(O₂) due to reducedphotophosphorylation. Subsequently, RuBP content increased again.Steady-state RuBP content at 2·0 kPa p(O₂) was 24% higherthan at 21 kPa p(O₂). RuBPCO activity decreased by 22%, indicatingcontrol of steady-state RuBP consumption by RuBPCO activity. It is suggested that lack of oxygen inhibition of photosynthesisat low temperature is due to decreased photophosphorylationat low temperature and low p(O₂). This may be due to assimilateaccumulation within the chloroplasts. Decreased photophosphorylationseems to decrease RuBP synthesis and RuBPCO activity, possiblydue to an acidification of the chloroplast stroma. Key words: Oxygen inhibition, photosynthesis, ribulose bisphosphate carboxylase/oxygenase     

Chlorophyll Metabolism in Higher Plants VI. Involvement of Peroxidase in Chlorophyll Degradation

The following phenolics were found to be essential for peroxidase-dependentchlorophyll bleaching: 2,4-dichlorophenol (DCP), p-coumaricacid (HCA), phenol, p-hydroxyphenylacetic acid, p-hydroxybenzoicacid, p-hydroxyacetophenone, resorcinol and umbelliferone. Mostof them are monophenols with electron-attracting groups at thep-position. The short-lived radicals generated by horseradishperoxidase (HRP)-phenolics-H₂O₂ reaction might be involved inthis reaction. Tobacco leaf enzyme preparation with peroxidaseactivity for guaiacol could also degrade chlorophyll with suchphenolics. In addition, tobacco leaf methanol extract couldsubstitute for chlorophyll bleaching as an electron donor inthe absence of phenolics. In place of free H₂O₂, the glycolate-glycolateoxidase (GOX) system could degrade chlorophyll in [peroxidase$phenolics]-dependentbleaching. This chlorophyll bleaching system was inhibited by peroxidaseinhibitors, radical scavengers, reducing reagents, and carotenoids.Ascorbate and glutathione stopped chlorophyll bleaching withGSSG reductase and NADPH. The role of ascorbate and glutathionein peroxidase activity for controlling the chlorophyll degradationrate is discussed. (Received January 28, 1985; Accepted July 23, 1985)     

Mechanisms of Na+-K+ pump regulation in cardiac myocytes during hyposmolar swelling

We have previously demonstrated that the sarcolemmalNa⁺-K⁺pump current(I_p) in cardiacmyocytes is stimulated by cell swelling induced by exposure tohyposmolar solutions. However, the underlying mechanism has not beenexamined. Because cell swelling activates stretch-sensitive ionchannels and intracellular messenger pathways, we examined their rolein mediating I_pstimulation during exposure of rabbit ventricular myocytes to ahyposmolar solution.I_p was measuredby the whole cell patch-clamp technique. Swelling-induced pumpstimulation altered the voltage dependence ofI_p. Pumpstimulation persisted in the absence of extracellularNa⁺ and under conditions designedto minimize changes in intracellular Ca²⁺, excluding an indirectinfluence on I_pmediated via fluxes through stretch-activated channels. Pumpstimulation was protein kinase C independent. The tyrosine kinaseinhibitor tyrphostin A25, the phosphatidylinositol 3-kinase inhibitorLY-294002, and the protein phosphatase-1 and -2A inhibitor okadaic acidabolished I_pstimulation. Our findings suggest that swelling-induced pumpstimulation involves the activation of tyrosine kinase,phosphatidylinositol 3-kinase, and a serine/threonine proteinphosphatase. Activation of this messenger cascade maycause activation by the dephosphorylation of pump units.