Hexachloroiridate IV as an Electron Acceptor for a ... - NCBI

Plant Physiol. (1988) 86, 1044-1047 0032-0889/88/86/1044/04/$01 .00/0

Hexachloroiridate IV as an Electron Acceptor for a Plasmalemma Redox System in Maize Roots' Received for publication July 8, 1987 and in revised form December 2, 1987

HARTWIG LUTHEN AND MICHAEL BOTTGER* Institut fur Allgemeine Botanik der Universitat Hamburg, Ohnhorststrasse 18, D-2000 Hamburg 52, West Germany meets the criteria with the exception of (c) (during its slow decay, it releases small amounts of cyanide). Searching for other candidates, complexes were again the first choice, especially those that are substitutionally inert during reduction. HCF III shares this important property with HCI IV. The brownish substance (absorption maxima at 488 and 415 nm-see Fig. 1) is reduced to colorless hexachloroiridate (III) (10): [IrCl6] [IrCl6]3 Eo = 0.87 V eHexachloroiridate IV Hexachloroiridate III The aim of our investigation was to determine whether HCI IV meets the criteria listed above and if the physiological properties of HCI reduction (especially control by divalent cations and hormones) can be compared to experiments with HCF III reduction, and to investigate a possible coupling to proton secretion.

ABSTRACT Hexachloroiridate IV, a new artificial electron acceptor for the constitutive plant plasma membrane redox system has been investigated. It appeared not to permeate through biological membranes. Due to its higher redox potential, it is a more powerful electron acceptor than hexacyanoferrate III (ferricyanide) and even micromolar concentrations are rapidly reduced. Hexachloroiridate IV increased H+ efflux over a concentration range of 0.05 to 0.1 millimolar. Lower concentrations slightly inhibited proton extrusion. Calcium stimulated both proton and electron transfer rates. Like hexacyanoferrate III-reduction, irridate reduction was inhibited by auxin.

Following the pioneering work of Craig and Crane (6, 7), plant plasma membrane redox systems have been investigated in vivo (2-9, 13) and in vitro (11, 12) by several groups. Two types of redox activity have been distinguished (2, 13). One is induced by iron deficiency and is present in dicots and monocots but not in grasses ('Turbo System'). The second one, the constitutive system ('Standard System') is generally present in all plant materials investigated so far. The inducible mechanism reduces ferric chelates. Other electron acceptors have been reported (13). The investigation of the constitutive system has been restricted to the use of HCF III as reduced substrate. This appeared to us to be a serious limitation in our eyes, since it could be argued that evidence for the postulation of a general electron transfer system (4, 5, 8) based on the reduction of a single substance might be too weak. The main reason for this unsatisfactory situation seemed to be the absence of an electron accepting compound suitable for physiological studies which satisfies the following criteria: (a) the substance has to have a sufficiently high redox potential to be reduced by the plants; (b) the reduction has to be accompanied by a change in absorption to allow photometric determinations. The extinction coefficient has to be high enough; (c) both reduced and oxidized form have to be sufficiently stable. Products of degradation should not be toxic; (d) both forms should not permeate the plasma membrane; and (e) if proton secretion is to be measured simultaneously, the reduction has to take place without release of protons. HCF 1112 seems to be a lucky accident of chemistry, since it

MATERALS AND METHODS

Plant Material. Seeds of Zea mays L. cv Goldprinz obtained from C. Sperling, Luneburg, F.R.G., were soaked in running tap water for 12 h and then sterilized in 10% H,O0 for 30 min. The seedlings were grown on moist paper in the dark at 26°C. Twelve intact seedlings were selected and the seeds mounted on plastic trays to allow roots to dip into a 16 ml plastic tube containing nutrient solution containing 10 mm KCl and 1 mm CaCl2. The plants were adapted to hydroculture and constant pH in the nutrient medium for 12 h at an irradiance of 150 ,umol m-2 s-- '. Measurement of Net e- and H+ Efflux. Proton fluxes were measured by means of a computer-controlled pH stat consisting of dilutors (Microlab M. Hamilton, Bonaduz, Switzerland), AD-converters (type pH 530 D WTW, Weilheim, FRG) and a processor (Apple II Europlus, 48K or 64K). The experiments were performed at pH 5.5. In a similar way, the concentration of HCI IV was kept constant by continuous titration with a 10 mM HCI IV solution. The amount of titrator needed was automatically calculated, added and registered after measurement of E(488 7(X) nm) in a flow-through cuvette by a photometer (LKB Ultrospec). Reduction rates could thus be monitored permanently. Further details of the redoxstat will be published in Bottger and Hilgendorf (5). The incubation medium contained 10 mM KCl and 1 mm CaCI, unless otherwise specified. Proton and electron release of 12 roots (approximately 1 g root fresh weight) was monitored; the roots were weighed after the experiment. Estimation of HCI Uptake by the Roots. In order to estimate the amount of HCI taken up by the plants the following technique ' Funding from the Deutsche Forschungsgemeinschaft is gratefully ac- was applied: The plants were treated with HCI IV. Immediately after application, an aliquot was taken as a control and stabilized knowledged. 2 Abbreviations: HCF III, hexacyanoferrate III; HCI IV, hexachloro- with oxone. After some hours, when iridate was completely reiridate IV; HCI III, hexachloroiridate III (HCI and HCF are abbrevi- duced by the plants, a sample was taken and oxidized with oxone ations of the correct IUPAC nomenclature of complex compounds); E,, for 5 h. Simultaneously, solutions of HCI III of known concentrations were oxidized with oxone to be used as standards. The redox potential; NAA, naphthylacetic acid. 1044

HEXACHLOROIRIDATE REDUCTION BY MAIZE ROOTS extinction of the formed HCI IV was measured at 488 nm, original concentrations of HCI III could thus be determined.

1045

Table I. Results of HCI III Reoxidation Experiments Plants were treated with HCI IV at the indicated concentration. After they had nearly completely reduced the compound, the formed HCI III was reoxidized to HCI IV using oxone. Concentrations of HCI IV were determined photometrically before and after the experiment. The high recovery rate indicates that the loss of extinction during the incubation with HCI IV is not due to HCI-uptake. HCI IV after HCI IV before Incubation Incubation and Reoxidation

RESULTS Principal Redox Reactions of HCI. With a redox potential of 0.87 V (10), HCI has a much higher oxidizing power than HCF III (En = 0.36 V). This could enhance interfering side-reactions. For instance, the redox potential of the water-oxygen couple is 0.79 V, so that HCI IV might be also reduced by water: 2 [IrC16P2- + H210 -- 2 [IrCl6]3- + 2 H+ + 1/2 02 % LM The resulting very slow decoloration of aqueous solutions has to 1 51.5 49.4 96 be carefully monitored if reduction of HCI IV by plants is in2 44.0 42.7 97 vestigated. Therefore, control experiments without plants were 3 50.0 48.0 96 performed to compensate for this background effect. In any case, 4 43.6 45.3 104 the spontaneous reaction with water was about one order of magnitude slower than the rapid reduction of iridate by the plants. Spectrophotometric Determination of HCI IV. Figure 1 shows a spectrum of a 0.06 mm HCI IV solution. There is a broad peak at about 420 nm, and a sharper and more intense one at 488 nm. The difference spectrum of HCI IV versus HCI III (not shown) indicates that measurement at 700 nm is best to compensate for changes in turbidity as both components have the same very low extinction at this wavelength. With 3680 mol- ' cm- 1, the extinction coefficient of HCI IV is high enough to allow determination of HCI IV at micromolar concentrations. Uptake by the Plants. A decline of extinction might be caused E by a rapid HCI uptake by the plants. This could mimic a reduction and both processes could not be easily distinguished. The reoxidation experiments were complicated by the fact that the commercially available HCI III used for the standards was only of 93% purity and contains 2 mol H20 per mol (Alfa Chemical Corp., personal communication). Correcting for the impurity, the recovery was between 96 and 104%. Table I shows some typical results of individual experiments. Thus it seems reasonable to state that a possible iridate uptake was too small to be 50 responsible for the loss of extinction. The low permeability of [HCI IV) (,M) biological membranes to HCI has been recently confirmed by FIG. 2. Dependence of HCI IV reduction and proton secretion on experiments with isolated spinach chloroplast. Oxygen formation was only increased by HCI IV if the chloroplasts were osmotically HCI IV concentration. The rate of spontaneous iridate reduction was shocked (M Frentzen, personal communication, data not shown). subtracted from the rate of reduction. The bars represent standard errors l-c

in the reduction rate of single, typical experiments. At least five measurements of proton and electron flux were done at each concentration indicated.

0.0. 0.2-

0.1

400

600

800 nm

WAVELENGTH FIG. 1. Absorption-spectrum of a 0.06 mm solution of HCI IV. The spectrum shows peaks at 420 and 488 nm. Note the low extinction at 700 nm.

Dependence of HCI Reduction on Iridate Concentration. The dependence of iridate reduction and proton secretion on photometrically determined HCI IV concentration is shown in Figure 2. Iridate reduction was measurable even at very low concentration, and it was significantly higher than the spontaneous reduction occurring without plants. At very low levels (1-5 ,uM), HCI IV inhibited proton secretion. This was also observed with HCF III (5). At higher concentrations, the proton secretion rate increased, but the absolute increase of proton secretion was lower than the increase of electron transfer. It might be that both HCI IV and HCF III reduce the activity of one proton pump (ATPase?) but stimulate another one (redox driven proton pump?). If the plants were taken out after the experiment, acidification and iridate reduction immediately dropped to the level of the control experiment without plants (data not shown). It can be excluded that the plants excreted a substance that catalyzed the spontaneous reduction of HCI IV. In general, the concentration dependence of HCI IV reduction was similar to the already known HCF III reduction (5), but the concentrations needed to obtain equivalent effects are lower than for HCF III by a factor of 100. Wall-bound peroxidases could mediate redox processes without

LOTHEN AND BOTTGER

1046

the involvement of transmembrane steps. However, their participation in HCI IV reduction is improbable because they would require NAD(P)H. In contrast to some previous investigations we strictly avoided the application of external NADH or NADPH in our experiments. Dependence of HCI IV Reduction on Calcium Concentration. High calcium concentrations greatly enhance proton extrusion and transmembrane electron transfer in the presence of HCF III (1, 5). It has been argued that the divalent cation screens the charge on the membrane surface and facilitates the access of the acceptor to the membrane. It was, therefore, interesting to investigate the possible effects of calcium on HCI IV reduction. At medium iridate concentrations (20 .M), 1 mM Ca2 + increased both acidification and electron transfer (Fig. 3). Dependence of HCI Reduction on Oxygen Concentration. It has been recently found that HCF III reduction by maize roots was dependent on 02 concentration (5). A reversible rise in electron transfer was observed if 02 partial pressure was reduced to below 2 kPa. With iridate, these results could not be reproduced. In some experiments, there was a drop in reduction rate at very Iow 02 concentrations (0.2 kPa). The lack of the typical dependence on oxygen partial pressure is probably due to the higher redox potential (10) which is even slightly higher than the H20/02 couple. It seems that, due to its high redox potential, HCI IV competes much more efficiently with oxygen than HCF III does. Hormone Effects. Auxin effects on HCF III reduction have been reported in carrot cells (7) and maize roots (5). IAA could not be used in our experiments, since it is oxidized by HCI IV. The synthetic auxin analogs 2,4-D and ax-NAA significantly decreased electron transfer and proton efflux (Fig. 4). The inactive isomer ,3-NAA had no effect on both (data not shown). As with HCF III (5), the effect required a low electron acceptor concentration, since otherwise the electron flow seems to be too high to be regulated by the hormone. Fusicoccin greatly enhanced proton secretion but does not act on electron transfer on HCI IV. After pretreatment with NAA, the expected inhibition (5) of transmembrane electron transfer could not be clearly detected (data not shown). Generally, the hormone effects are not as dramatic as with HCF III, which might be because of the high ,

,

+

C%Ja C%Ja Cs 0

C= E;E

+

C-Ja C-Ja (_a.I s C-3a2CZ C.)

E

E C E.)

C.

E

1.0

HCI IV Reduction Hv Efflux FIG. 3. Effect of calcium on proton secretion and HCI IV reduction in presence of HCI IV. Similar stimulations have been reported from HCF III reduction. The bars represent standard errors in the reduction rate of single, typical experiments. At least five measurements of proton and electron flux were done at each concentration indicated.

Plant Physiol. Vol. 86, 1988

AUXIN 14

C=

AUXIN

0.4-

E~~~~~~~ C2

_,

0.2-

1

3 2 time (hours)

L

FIG. 4. Kinetics of the effects of the auxins NAA (2 ,UM, 0, El) and 2,4-D (10 ,M, *, U) on proton secretion (EC, *) and HCI IV reduction (0, *). The assay was performed at pH 5.5, the iridate concentration was 0.01 mM, the medium contained 10 mm KCI and no Ca2 . Results of individual, typical experiments are shown.

oxidizing power of HCI IV. This produced a high electron flow even at low iridate concentrations.

DISCUSSION A new electron acceptor for the constitutive electron transfer system in maize roots has been described. Although the redox potential of the acceptor is slightly higher than that of the H20/02 couple, this side reaction does not contribute to the reduction of HCI IV and to the acidification of the medium to a disturbing extent. The physiological properties of HCI IV and HCF III reduction were directly comparable and they resembled each other markedly: both shared similar concentration dependencies. Both were influenced in the same way by auxin and calcium. For the iron deficiency-induced electron transfer, a broader range of acceptors has been previously described. This system has been demonstrated to be present in many monocots and dicots but not in grasses. It is therefore absent in our maize roots. The constitutive system, however, seemed up to now to be restricted to HCF III as electron acceptor. Since HCF III is an iron containing molecule, it could not be excluded that the only physiological significance of this kind of reduction is reduction of Fe3+ before iron uptake. HCI IV is a non-iron electron acceptor, and its reduction seems to be controlled by the same factors as electron transfer to HCF III. This might indicate that transmembrane electron transfer is a much more general and therefore important function of cells and tissues. It is found in many species and it is not restricted to plants (8). Redox activity seems to be tightly coupled to the generation of a proton gradient and evidence exists that proton pumping is an important task of this redox system even if no artificial electron acceptor is offered in the

HEXACHLOROIRIDATE REDUCTION BY MAIZE ROOTS bulk phase of the outer medium (1, 4, 5). HCI IV has a very high redox potential. It might impair the function of other plasmalemma proteins than the plasmalemma redox function, for example channels, co-transporters, pumps, and proteins containing SH-groups. This might influence proton and electron transfer. On the other hand, we could demonstrate previously that the actions of SH-blockers and HCF III on proton transport are additive and independent effects (4). There is still a need for a broad variety of useful electron acceptors covering the whole range of redox potentials between 0 and 870 mV. Acknowledgments-We are grateful to M. Frentzen for contributing data from studies with isolated chloroplasts and T. Lambertsen for stimulating and helpful discussion.

LITERATURE CITED RANJEVA, G

MARIGO 1986 Cations stimulate proton

pumping

1.

BELKOURA

2.

in Catharanthus roseus cells: implication of a redox system. Plant Cell Environ 9: 653-656 BIENFAIT HF 1985 Regulated redox processes at the plasmalemma of plant root cells

M,

R

and their function in iron

uptake. Bioenerg

Biomembr 17: 73-83

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3. BOTTGER M 1986 Proton translocation systems at the plasmalemma and their possible regulation by auxin. Acta Hortic 179: 83-93 4. BOTTGER M, H LUTHEN 1986 Possible linkage between NADH-oxidation and proton secretion in Zea mays L. roots. J Exp Bot 37: 666-675 5. BOTTGER M, F HILGENDORF 1988 Hormone action on transmembrane electron and H+ transport. Plant Physiol 86: 1038-1043 6. CRAIG TA. FL CRANE 1981 Evidence for a transplasma-membrane electron transport system in plant cells. Proc Indiana Acad Sci 90: 150-155 7. CRAIG TA. FL CRANE 1982 Hormonal control of a transplasmamembrane electron system in plant cells. Proc Indiana Acad Sci 91: 150-154 8. CRANE FL. IL SUN. MG CLARK. C GREBING. H Low 1985 Transplasmamembrane redox systems in growth and development. Biochim Biophys Acta 811: 233-264 9. FEDERICO R. CE GIARTOSIO 1983 A transplasma-membrane electron transfer system in maize roots. Plant Physiol 73: 182-184 10. GEORGE P, GIH HANIA 1957 A potentiometric study of the chloroiridatechloroiridite couple. J Chem Soc 1957: 3048-3052 11. MACRI F. A VIANELLO 1986 Independence of transplasmamembrane proton gradient from NAD(P)H-ferricyanide oxidoreduction in maize roots microsomes. Plant Sci 43: 25-30 12. SANDELIUs AS, R BARR. FL CRANE. JD MORRE 1986 Redox reactions of plasma membranes isolated from soybean hypocotyls by phase partition. Plant Sci 48: 1-10 13. SIJMONS PC, HF BIENFAIT 1983 Source of electrons for extracellular Fe (III) reduction in iron deficient bean roots. Physiol Plant 59: 409-415