Distribution of electric potential and ion transport in the hypocotyl of Vigna sesquipedalis VI. The dual structure of radial electrogenic activity

The electrophysiological structure in bean hypocotyl was investigatedby the intracellular electrode method in combination with surfaceelectric potential (V_s) measurement and respiratory inhibitionby anoxia, with special reference to the membrane transportof ions and the formation of an absorption centre in the elongating(E) zone. The radial potential difference (V_sx: electric potentialdifference between the organ surface and a xylem vessel), onwhich axial distribution of V_a was dependent, comprised twocomponents; V_ax=V_px–V_ps. |V_px| (the potential differencebetween the inside of a parenchyma symplast and a xylem vessel)was at a maximum in the E-zone, while |V_ps| (the intracellularelectric potential with respect to the organ surface) was largestin the G-zone (mature zone), resulting in the characteristicdistribution pattern of V_s with a minimum in the E-zone. Therewere two independent electromotive forces which were both partiallydependent on respiration; one corresponding to V_ps located atthe surface of the parenchyma symplast (P) and the other toV_px located between P and the xylem (X). The electrogenic componentof Vpx was relatively small both in the hook (H) zone and theG-zone, but maximal in the E-zone of the hypocotyl. This resultwas consistent with the emergence of a maximum pH differencebetween P and X in the E-zone, where accumulation of K⁺ andwater were at a maximum, suggesting maximum activity of an H⁺-pumpextruding protons from P into X in exchange for K⁺. (Received July 17, 1978; )     

Structure and function of the elongation sink in the stems of higher plants I. Effects of anoxia and IAA on the growth rate and the spatially separate electrogenic ion pumps

Two spatially separate membrane potentials of the parenchymasymplast (7, 8) and the rate of elongation growth were measuredsimultaneously in bean hypocotyl segments. Both the membrane potentials, V_ps (electric potential differencebetween parenchyma symplast and organ surface) and V_px (electricpotential difference between parenchyma symplast and xylem),were rapidly depolarized by anoxia, and were repolarized withre-aeration. Anoxia reduced the growth rate by about 85%, andre-aeration restored it. Changes in the membrane potentialspreceded those in the growth rate by 30–50 sec. About 8 min after the application of aerosol generated from10^–3 M indole acetic acid (IAA) solution to the segments,V_ps began to hyperpolarize, and the growth rate increased witha delay of several minutes after the potential change and subsequentlybecame ten times the control rate. This hyperpolarization ofV_ps was due to the increase in the activity of the respiration-dependentelectrogenic ion pump at the outer surface membrane of the parenchymasymplast. A clear correlation was observed between the growthrate and pump activity (V_ps) with the change in IAA concentration. (Received December 6, 1979; )     

The Role of Membrane Potential for the Control of Elongation Growth of Vigna Hypocotyl: Response of a Hollow Cylinder to Osmotic and Ionic Stress

We have devised an experimental system of perfusion througha hollow cylinder of a Vigna hypocotyl to examine the controlmechanism of plant stem elongation. When the cylinder was subjectedto osmotic stress, it began to shrink and then spontaneouslyresumed elongation. Not only the membrane potential differencebetween the parenchyma symplast and the central bore (V_px),but also that between the parenchyma symplast and the organsurface (V_ps), showed hyperpolarization a few minutes afterthe cylinder began to shrink. Removal of the stress caused animmediate increase in elongation rate followed by depolarizationof both membrane potentials a few minutes later. When the cylinderwas subjected to KCl stress, V_px showed transient depolarizationand recovery, while V_ps showed only immediate hyperpolarization.Increasing the KCl concentration caused V_px to depolarize, andthe cylinder simultaneously to cease to elongate for about 5min,even when the osmotic concentration of the perfusion solutionwas kept almost constant. An inverse reaction was observed whenthe KCl concentration was decreased. These two reversible responses suggest that control of V_px mayregulate the elongation of hollow cylinders, and that the xylempump plays an important role in the regulation of intact stemelongation. (Received January 7, 1987; Accepted April 30, 1987)     

Effects of anoxia and high CO2 concentration on the electrogenic activity of leaf cell membrane in the dark

Resting membrane potential of a single cell in Antirrhinum leafdepolarized rapidly by about 40% under anoxia in the dark andrecovered to the normal level with reaeration. V_m in the dark comprised two fractions; one was passive diffusionpotential and the other was intimately dependent on aerobicmetabolism. The rapidity of the depolarization due to anoxiasuggested the possible functioning of an electrogenic mechanism,although the level of V_m was not much higher than the equilibriumpotential of K⁺ across membrane. 50% CO₂ in air brought about slightly more complex responses:a slight transient hyperpolarization and a subsequent depolarization.Sometimes one more transient depolarization took place as a"CO₂-off" effect. The total depolarization span was equal tothat caused by anoxia. 50% CO₂ under anoxia did not evoke furtherdepolarization but only hyperpolarization. (Received July 7, 1976; )     

Homeostatic Regulation of Membrane Potential by an Electrogenic Ion Pump against Change in the K+ Concentration of the Extra- and Intra-Organ Perfusion Solutions

The dependence of membrane potentials on changes in the extra-cellularK⁺ concentration [K⁺]_e was investigated in potato tuber sliceswith dripping perfusion, and in growing Vigna hypocotyl segmentswith pressurized intra-organ perfusion methods. Only under anoxiawere the membrane potential of potato tuber slices and the electricpotential difference between the parenchyma symplast and xylem(V_px) of Vigna hypocotyl segments depolarized markedly (46 mVand 42 mV/log[K⁺]_e unit, respectively) with increasing [K⁺]_eabove the critical values. The electric potential differencebetween the parenchyma symplast and organ surface (V_ps of thehypocotyl segments remained nearly unchanged up to 30 mEq [K⁺]_e.Under highly aerobic conditions the membrane potentials wererelatively independent of [K⁺]_e except at very high K⁺ concentrations.V_ps showed even hyperpolarization with the increasing KCl concentrationin the perfusion solution that is not in direct contact withthe surface membrane of the parenchyma symplast. The respiration-dependentelectrogenic components of the membrane potentials regularlyincreased with the increasing [K⁺]_e. A voltage-dependent homeostaticcontrol of membrane potential is discussed. (Received August 13, 1984; Accepted December 21, 1984)     

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)     

Effects of carbon dioxide on the spatially separate electrogenic ion pumps and the growth rate in the hypocotyl ofVigna sesquipedalis

Carbon dioxide, introduced into the gas phase of the experimental chamber, has distinct effects on two spatially separate membrane potentials and the rate of elongation growth in hypocotyl segments ofVigna sesquipedalis Wight. Both membrane potentials (V _ps andV _px=the electric potential difference between the parenchyma symplast and the surface of the hypocotyl, and that between the parenchyma symplast and the xylem, respectively) hyperpolarized rapidly but transiently at the introduction of CO₂. Prolonged exposure of the hypocotyl to high concentrations of CO₂ (above 10%) caused depolarization of membrane potentials above the level before CO₂ introduction. When CO₂ was replaced with air, the membrane potentials exhibited a distinct depolarization response of transient nature. The growth rate of the hypocotyl segments exhibited similar responses to CO₂ as did the membrane potentials (the increase and the decrease of the growth rate were corresponded to the hyperpolarization and the depolarization, respectively), but these responses always followed the changes of the membrane potentials. The CO₂-induced maximum hyperpolarization ofV _ps and the maximum increase of the growth rate were closely correlated. All these responses were strictly dependent on aerobic metabolism. These results indicate that CO₂ may regulate elongation growth in two ways: by affecting the activity of the electrogenic ion pump via intracellular acidification, and also by acting via apoplastic acidification as a wall-loosening acid.Symbols and abbreviations V _sx electric potential difference between the surface (S) and the xylem (X) of the hypocotyl - V _px electric potential difference between the inside of a parenchyma cell (P) andX - V _ps electric potential difference betweenP andS - V _ps (CO₂, max) the maximum value of CO₂-induced hyperpolarization ofV _ps - GR(CO₂, max) the maximum value of CO₂-induced increase of the growth rate - IAA indole-3-acetic acid     

Distribution of electric potential and ion transport in the hypocotyl of Vigna sesquipedalis IV. The effects of carbon monoxide and cyanide on the axial potential difference of hypocotyl segments

The effects of carbon monoxide and cyanide on the electric potentialdifference between both cut ends of a hypocotyl segment excisedfrom a bean embryo were investigated. Carbon monoxide definitely diminished the PD. This inhibitionwas photoreversible. The inhibition ratio was quantitativelyrelated to the [CO]/[O₂] ratio of the gas mixture. Prolongedexposure to a CO gas mixture induced spontaneous recovery ofthe PD, and this CO-resistant electrogenic activity was sensitiveto CN and also disappeared under anoxia. Cyanide also diminished the PD and often induced oscillationof the potential. The type of oscillation and the inhibitionratio varied with the concentration of CN. Sometimes CN pretreatmentcaused changes in the anoxia response of the potential. The time course of the spontaneous change in PD after cuttingof the hypocotylsegment also was investigated. (Received August 8, 1977; )     

Distribution of electric potential and ion transport in the hypocotyl of Vigna sesquipedalis II. Axial potential difference

Vital staining with pH indicator dyes made it possible to identifythe xylem with the specific channel A, reported previously,through which hydrogen ions flow generating a resting potentialdifference along the germ axis as their diffusion potential.The distribution of K+ concentration within this channel showedno similarity to electric potential distribution, in contrastto the distribution of H+. The axial P.D. between both ends of a segment cut from a hypocotylresponded reversibly to the change in O₂ tension of the surroundinggas phase. After air had been quickly replaced by N₂, a lagperiod appeared before the sudden potential drop took place.The lag period () was largely dependent on temperature. Apparentactivation energy of the process characterized by 1/ was 18Kcal/mole between 14–30?C, approximately equal to thatof the O₂-uptake within the same temperature range. The relationbetween O₂ concentration and the maximum rate of recovery ofP.D. from anoxia was of the Michaelis-Menten type; the apparentKm was calculated as 2.1 ? 10^–5M O₂ being of the sameorder as that of cytochrome oxidase in higher plants. The O₂-uptakerate "per unit of hypocotyl length" showed a distinct maximumin the elongating region where the axial distribution of bothelectric potential and pH within channel A had their minimums. (Received July 21, 1972; )     

Effects of Osmotic Stress, Salt Stress and IAA on the Regeneration of the Resting Membrane Potential after Excision of a Segment from an Intact Plant

Effects of osmotic stress, salt stress and IAA on the regenerationprocess of the transmembrane potential across the xylem/symplastinterface (V_px) of newly excised hypocotyl segments of Vignaseedlings were examined by means of the xylem perfusion method.It took about 8 h under ordinary conditions for an excised segmentto regenerate a membrane potential comparable with that of anintact seedling. Osmotic stress imposed by perfusion of 100-200mM sorbitol solution seemed to accelerate this process. Thiseffect diminished with the removal of sorbitol from the perfusionsolution. The increase in the negativity of V_px in this regenerationprocess resulted from the increases in both passive (V_px inN₂) and respiration-dependent components (V_px). NaCl (50–100mM) did not accelerate the regeneration of the total membranepotential, but significantly promoted an increase in V_px, i.e.electrogenic xylem pump activity. Perfusion of KC1 (50–100mM) or IAA (10^–4M) shortened the regeneration phase upto 2–3 h. The increase in total V_px under salt stressor IAA mainly resulted from the increase of V_px. The effectof those agents on the V_px is discussed, as is the questionof whether the increase in PD after excision should be interpretedas a recovery or regeneration from injury by excision, or asa so-called release from hormonal control. (Received September 3, 1987; Accepted March 14, 1988)     

Characterization of Sulfate Transport in the Green Alga Chlorella ellipsoidea

A rapid induction of sulfate transport was observed in the greenalga Chlorella ellipsoidea during sulfur-limited growth. Bothaffinity and V_max increased about five-fold within 6 h of transferringcells from Bold's basal medium with 350 µM MgSO₄ to sulfur-deficientBold's medium. High affinity sulfate transport was induced within15 min and reached maximum rate within 3 h of transferring cellsto sulfur-deficient condition, indicating that a new, high-affinity-sulfatetransport system is induced by sulfur starvation in C. ellipsoidea.Eadie-Hofstee plots of initial rates of sulfate uptake indicatedthat the K of sulfur-starved cells was about 17 µM. Bothsulfur-starved and unstarved cells grown in air had a V_max of1.5 times higher than that of high-CO₂ grown cells. Sulfatetransport was completely inhibited by 30 µM CCCP or 800µMKCN both in the light and the dark but transport in the lightwas not inhibited by 20 µM DCMU. Treatment with 50 µMor 500 µM vanadate caused 50% inhibition of uptake. Therate of sulfate uptake in the dark was twice that in the lightand was stimulated by low pH. These results suggest that thesulfate transport system in C. ellipsoidea is operated by protonsymport across the plasmamembrane which is partially mediatedby P-type ATPase and that these systems depend exclusively onenergy derived from oxidative phosphorylation in the mitochondria. (Received June 28, 1995; Accepted August 8, 1995)     

Effect of darkening the cotyledons on the growth and curvature of the sunflower hypocotyl: evidence of hydraulic signalling

When both cotyledons of light-grown sunflower seedlings (Helianthusannuus L.) were darkened by covering them with aluminium foil,the resulting increase in the rate of elongation of the hypocotylwas closely proportional to the associated reduction in seedlingtranspiration and to the increase in xylem water potential (_PX.Covering only one cotyledon induced curvature away from thatside. This response was associated with a higher _PX in the vascularbundles connected directly with the covered cotyledon than inthose connected with the illuminated cotyledon. The water contentof peripheral tissues of the hypocotyl below the covered cotyledonwas also higher than that of similar samples from below theilluminated cotyledon. These results are consistent with thehypothesis that the effect of darkening the cotyledons on thegrowth and curvature of the hypocotyl is mediated by hydraulicsignalling, characterized by transmission to the hypocotyl ofan increase in _PX resulting from the reduction in cotyledontranspiration. Key words: Cotyledons, Helianthus annuus, hydraulic signalling, hypocotyl, transpiration, water potential     

Hormonal Regulation of Hypocotyl Elongation in Lactuca sativa L.: I. EVIDENCE AGAINST THE INVOLVEMENT OF GIBBERELLINS IN THE ELONGATION OF HYPOCOTYL IN SEEDLINGS GROWN IN THE DARK

The kinetics of extension induced by GA₃¹ in the hypocotyl ofintact seedlings of Lactuca sativa are similar in the dark andin the light, and differs fundamentally from the kinetics ofelongation in the dark without GA₃. Both in continuous lightand in the dark, GA₃-induced promotion starts 24 h after incubation.In the dark, even low concentrations of GA₃, which do not affectthe length measured after 6 d when the extension of hypocotylalmost ceases, remove the lag period of 48 h which precedesextension, and prolong the high rate of elongation. FollowingGA₃ supply the hypocotyl length in the dark and in the lightdoes not differ until 48 h; thereafter the rate of elongationin the light is less, so that the final length of the hypocotylis 40 per cent shorter than that of the dark-grown seedlingswithout GA₃. IAA supplied apically to light-grown seedlings induces a weakpromotion at a concentration of 1 mg l^–1 only. With anincreasing concentration of GA₃ supplied simultaneously, theconcentration of IAA inducing a significant promotion decreases.A combined supply of both these regulators, however, does notrestore the light-mediated inhibition of hypocotyl elongationcompletely. The maximum decrease in hypocotyl length induced by the growthretardants AMO-1618, CCC, and B-9 supplied from the beginningin the dark does not exceed 70 per cent. Saturating doses ofGA₃ supplied in combination with any one of the retardants compensateonly a fraction of the decrease. The results have been interpreted to show that native GAs arenot involved in extension growth in the dark.     

Distribution of electric potential and ion transport in the hypocotyl of Vigna sesquipedalis III. Significance of the radial potential difference

The distribution pattern of the trans-root surface potentialalong the germ axis of Vigna sesquipedalis intimately dependedon the existence of oxygen as well as the potential difference(PD) between both cut ends of a cylindrical segment of the hypocotyl.In the grown-up region (basal region) of the hypocotyl, a largechange of the trans-root surface potential occurred with anoxia,but not in the elongating zone except in the transient phaseafter reaeration. The distribution pattern of the trans-root stump potential alongthe germ axis, measured just after die hypocotyl had been cutat various positions, was nearly flat. However, PD between diecut stump and a point on the hypocotyl surface at sufficientdistance from the stump had a large value. The results are not explainable based on the axial electromotiveforces alone. Consideration of equivalent circuits of the hypocotylhas led to die idea that radial electromotive forces largelycontribute to generation of die distribution pattern of thesurface electric potential. (Received June 30, 1977; )     

Requirement of sodium chloride for the action of gibberellic acid in stimulating hypocotyl elongation of a halophyte, Salicornia herbacea L.

NaCl stimulated hypocotyl elongation of the halophyte Salicorniaherbacea L. grown either in light or dark. Its optimal concentrationwas around 0.1–0.2 M and its promoting effect was muchmore prominent in the dark. Gibberellic acid at 10^–5 Mstimulated hypocotyl elongation in light but not in the dark.Indole-3-acetic acid and kinetin were ineffective in promotinghypocotyl elongation. In light, gibberellic acid and NaCl synergisticallyenhanced hypocotyl elongation when both were given simultaneously.The action of NaCl could be replaced by KCl, but not by mannitol.Osmotic pressure of the epidermis of the Salicornia hypocotylincreased in response to gibberellic acid and/or NaCl treatment.Na⁺ content in the hypocotyl increased with NaCl application.Gibberellic acid and NaCl when given alone increased the extensibilityof the hypocotyl cell wall. Synergistic interaction in increasingthe extensibility was observed between gibberellic acid andNaCl. Stress-relaxation analysis of mechanical properties ofthe hypocotyl wall revealed that gibberellic acid and NaCl actedsynergistically in decreasing minimum relaxation time. Basedon these results, a possible mechanism by which gibberellicacid and NaCl regulate hypocotyl elongation of Salicornia herbaceaL., a typical halophilic plant, is discussed. ¹ Present address: Laboratory of Biology, Tezukayama College,Gakuen Minami, Nara 631, Japan. (Received June 13, 1978; )     

Water Transport during Acid-Induced Growth of Excised Hypocotyl Sections of Vigna unguiculata under Xylem Perfusion

Elongation growth of abraded hypocotyl sections of Vigna unguiculataunder xylem perfusion was markedly promoted a few minutes afterthe application of an acid aerosol generated from a solutionof HCl. At the beginning of the acid-induced growth, intracellularpressure (Pⁱ) began to decrease and the membrane potential betweenthe symplast and the xylem apoplast (V^px) began to depolarize.Subsequently, Pⁱ and V^px remained at a reduced level and a depolarizedlevel, respectively, while the promotion of elongation growthcontinued for more than 4 hours. The electrogenic componentof the xylem membrane potential (V^px_act) gradually increasedto about twice that before acid treatment. There was a closecorrelation between the enhanced growth and the decrease inintracellular pressure within 30 min after application of acidbut little correltion after 60 min. By contrast, there was littlecorrelation between the promotion of growth and the activityof the xylem pump after 30 min while a close correlation wasobserved after 60 min. It is inferred that the acid-induced activation of water uptakeconsists of two major processes, in series, that are drivenby different forces: the rapid uptake of water for more than30 min, driven by hydrostatic force generated by loosening ofcell walls; and a long-lasting enhancement of water uptake forat least 4 h, which is driven by osmotic force that is generatedby the canal system within the xylem. (Received October 17, 1994; Accepted January 23, 1995)     

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     

Distribution of electric potential and ion transport in the hypocotyl of Vigna sesquipedalis I. Distribution of overall ion concentration and the role of hydrogen ion in generation of potential difference

Overall concentration of free inorganic ions distributes inthe hypocotyl of a bean seedling {Vigna sesquipedalis) at aconstant level (H⁺) or decreases monotonously from the cotyledonarynode towards the base (K⁺, Na⁺, Ca⁺⁺ and Mg⁺⁺, phosphate, NO₃^–).According to our theory, this is inconsistent with the distributionof electric potential having a definite minimum in the elongatingregion. The discrepancy can not be explained by regional variancein radial potential difference or histological differentiationin passive ionic permeability of the cell membrane. Short circuitcurrent observed through a hypocotyl segment corresponded toa net flux in ions of 10–24 pEq/cm².sec. It is questionable,however, whether this is due to active ion transport, whichcan be the source of electric potential difference, or is apassive flow due to histological heterogeneity in ion concentration. In order to investigate the latter possibility, pH of sap exudingfrom stumps made at various intervals along the hypocotyl axiswas measured, since H⁺ is the ion electro-osmotically most effective.pH Values of acropetal exudates distributed along the axis closelycorresponding to the distribution of electric potential. Thissuggests that potential distribution is determined by a passiveflow of H⁺ through a specific channel in the vascular system.The fact that H⁺ production and the uptake of ions and waterare most active at the elongating zone of hypocotyl is discussedfrom a physiological point of view. (Received December 3, 1969; )     

Participation of Peroxidase in the Metabolism of 3,4-Dihydroxyphenylalanine and Hydrogen Peroxide in Vacuoles of Vicia faba L. Mesophyll Cells

When leaves of Vicia faba were treated with H₂O₂ or visiblelight in the presence of methyl viologen (MV), the orange-redcompound dopachrome was formed transiently and melanin was accumulated.With the darkening of leaves, the level of 3,4-dihydroxyphenylalanine(DOPA) decreased and then recovered to the original level uponaddition of 1 mM H₂O₂. However, if leaves were incubated inthe presence of 10 mM H₂O₂, the level of DOPA decreased againafter the increase. The time course of the changes in levelsof DOPA observed during the accumulation of melanin as a resultof illumination in the presence of MV was very similar to thatobserved after the addition of 10 mM H₂O₂. Illumination of leavesin the absence of MV did not result in any accumulation of melanin,but the level of DOPA changed slightly. When isolated mesophyllcells were incubated in the dark, the level of DOPA decreased.Illumination of the cells stimulated this decrease. Tropolone,an inhibitor of phenol oxidase, did not inhibit and actuallystimulated the H₂O₂- and light-induced oxidation of DOPA andaccumulation of melanin in leaves. Tropolone also stimulatedthe decrease in the levels of DOPA both in the dark and in thelight in isolated mesophyll cells. These data suggest that aperoxidase-H₂O₂ system, and not phenol oxidase, participatesin the oxidation of DOPA. When DOPA was oxidized by a basicperoxidase isolated from V.faba leaves, an intermediate, whichwas perhaps dopaquinone and which was reducible by ascorbate,was formed. Based on the data, a discussion is presented ofthe physiological significance of the oxidation of DOPA by peroxidasein vacuoles. (Received March 4, 1991; Accepted May 21, 1991)     

Enzymic Activities Associated with the Ability of Aerial and Submerged Forms of Littorella uniflora (L.) Aschers to perform CAM

ABSRACT: Groenhof, A. C, Smirnoff, N. and Bryant, J. A. 1988. Enzymicactivities associated with the ability of aerial and submergedforms of Littorella uniflora (L.) Aschers to perform CAM.—J.exp. Bot. 39: 353-361. The submerged form of Littorella uniflora shows a full CAM modeof photosynthesis as shown by diel acid fluctuations and elevatedactivities (in comparison to non-submerged leaves) of the enzymesphosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme.Non-submerged plants exhibit no diel fluctuations of acidityand no changes in activity of NADP-malic enzyme or PEPC. PEPCactivity is low and NADP-malic enzyme is not detectable. Furthercharacterization of PEPC extracted from submerged plants duringthe light and dark periods of a diel cycle shows that the enzymeextracted in the dark is more active. In addition, the enzymeshows a decrease in K_m (PEP) and an increase in V_max in thepresence of glucose-6-phosphate, whilst in the presence of malateK_m (PEP) is increased and V_max decreased; this response to malateis only observed in the light and at pH 7.2. Molecular weightdeterminations using a Sephacryl S-300 column show that theenzyme extracted from plants during the dark period has an apparentmol. wt. of 375 KDa and the enzyme extracted from plants duringthe light period has an apparent mol. wt. of 307 KDa. Key words: Littorella uniflora (shoreweed), Crassulacean acid metabolism, PEP carboxylase, malic enzyme