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In the present study nitrate uptake by maize (Zea mays L.) roots was investigated in the presence or absence of ferricyanide (hexacyanoferrate III) or dicumarol. Nitrate uptake caused an alkalization of the medium. Nitrate uptake of intact maize seedlings was inhibited by ferricyanide while the effect of dicumarol was not very pronounced. Nitrite was not detected in the incubation medium, neither with dicumarol-treated nor with control plants after application of 100 microM nitrate to the incubation solution. In a second set of experiments interactions between nitrate and ferricyanide were investigated in vivo and in vitro. Nitrate (1 or 3 mM) did neither influence ferricyanide reductase activity of intact maize roots nor NADH-ferricyanide oxidoreductase activity of isolated plasma membranes. Nitrate reductase activity of plasma-membrane-enriched fractions was slightly stimulated by 25 microM dicumarol but was not altered by 100 microM dicumarol, while NADH-ferricyanide oxidoreductase activity was inhibited in the presence of dicumarol. These data suggest that plasma-membrane-bound standard-ferricyanide reductase and nitrate reductase activities of maize roots may be different. A possible regulation of nitrate uptake by plasmalemma redox activity, as proposed by other groups, is discussed.

Intact plants can reduce external oxidants by an appearingly trans-membrane electron transport. In vivo an increase in net medium acidification accompanies the reduction of the apoplastic substrate. Up to now, several NAD(P)H dehydrogenases, b-type cytochromes, and a phylloquinone have been identified and partially purified from plant plasma membranes. The occurrence of a quinone in the plasma membrane of maize roots supports the hypothetical model of a proton-transferring redox system, i.e., an electron transport chain with a quinone as mobile electron and proton carrier. In the present study the trans-membrane electron transport system of intact maize (Zea mays L.) roots was investigated. Flow-through and ionostat systems have been used to estimate the electron and proton transport activity of this material. Application of 4,4,4-trifluoro-1-(2-thienyl)-butane-1,3-dione (thenoyltrifluoroacetone) inhibited the reduction of ferricyanide in the incubation solution of intact maize roots up to 70%. This inhibition could not be washed off by rinsing the roots with fresh incubation medium. The acidification of the medium induced after ferricyanide application was inhibited to about 62%. The effects of thenoyltrifluoroacetone on proton fluxes in the absence of ferricyanide have been characterized in a pH-stat system. The net medium acidification by maize roots was inhibited up to 75% by thenoyltrifluoroacetone in the absence of ferricyanide, while dicumarol inhibited net acidification completely. The inhibition of H(+)-ATPase activity was estimated with plasma membrane vesicles isolated by phase partitioning and treated with 0.05% (w/v) Brij 58. ATP-dependent proton gradients and Pi release were measured after preincubation with the effectors. The proton pumping activity by those plasma membrane vesicles was inhibited by dicumarol (53.6%) and thenoyltrifluoroacctone (77.8%), while the release of Pi was unaffected by both inhibitors.

Electron transporting oxidoreductases at biological membranes mediate several physiological processes. While such activities are well known and widely accepted as physiologically significant for other biological membranes, oxidoreductase activities found at the plasma membrane of plants are still being neglected. The ubiquity of the oxidoreductases in the plasma membrane suggests that the activity observed is of major importance in fact up to now no plant without redox activity at the plasmalemma is known. Involvement in proton pumping, membrane energization, ion channel regulation, iron reduction, nutrient uptake, signal transduction, and growth regulation has been proposed. However, positive proof for one of the numerous theories about the physiological function of the system is still missing. Evidence for an involvement in signalling and regulation of growth and transport activities at the plasma membrane is strong, but the high activity of the system displayed in some experiments also suggests function in defense against pathogens.

SummaryIt is worthwhile emphasizing the importance of electron transport across lipid membranes. Mitochondrial and electron transport in chloroplasts were elucidated in great detail many years ago. Plasma membrane-bound electron transfer may be involved in several processes such as membrane energization, signalling, regulation of transport and/or growth, and generation or scavenging of free radicals. We here give an overview of plasma membrane-bound electron transfer, of possible compounds of the electron transporting systems isolated from plasma membranes, and of their biochemical characteristics. Both the progress made in purification of redox enzymes and compounds and data from biochemical characterization of the activities found, support the discussion concerning models of the molecular structure of the electron transport systems of plant plasma membranes.

We investigated the change of root net proton excretion of seedlings of Triticum aestivum L. and Zea mays L. with daily variation of illumination using a multi-channel pH-stat system. We found an increase of net proton excretion during darkness and a drop after the beginning of illumination. Inhibition of carotenoid biosynthesis by norflurazone and photooxidation of chlorophylls did not change the periodicity or its induction. The induction of diurnal periodicity was possible with blue, green and red light. After induction the oscillation of net proton excretion continued for at least two cycles under constant light. We conclude that net H+ excretion of wheat and maize roots may be regulated by an endogenous clock or by a signal from the leaves. The nature of such a hypothetical signal remains unknown.

Iron tolerance of rice (Oryza sativa L.) was investigated using an oxygen depleted hydroculture system. Treatment with high concentrations of Fe2+ induced yellowing and bronzing symptoms as well as iron coatings at the root surface. Root and shoot growth were inhibited by increasing iron concentration in the medium. All symptoms were more pronounced in an iron sensitive cultivar (IR 64) compared to an iron tolerant one (IR 9764-45-2). Superoxide dismutase and peroxidase activity of root extracts of IR 97 were about twice that of IR 64 in untreated control plants. No significant increase of peroxidase activity was detected with increasing iron concentration in the medium. Catalase activity of IR 64 was slightly higher than that of IR 97, independent of iron concentration.

We investigated changes of thiols (GSH, GSSG, and cysteine) induced by transplasma membrane electron transport after addition of artificial electron acceptors and the influence of the thiol level on redox activity. GSH, GSSG, and cysteine content of maize (Zea mays L. cv. Golden Bantam) roots and coleoptile segments was determined by high performance liquid chromatography with a fluorescence detector. GSSG increased after treatment with 0.8 mM diamide, an SH-group oxidizer. GSH level of roots increased after treatment with diamide, while GSH levels of coleoptiles decreased. Incubation of roots with the GSH biosynthesis inhibitor buthionine-D,L-sulfoximine for 6 days lowered the glutathione level up to 80%. However, the GSH/GSSG ratio of maize roots remained constant after treatment with both effectors. The GSH/GSSG ratio and the glutathione level were changed by addition of artificial electron acceptors like hexacyanoferrate (III) or hexabromoiridate (IV), which do not permeate the plasma membrane. Hexacyanoferrate (III) reduction was inhibited up to 25% after the cellular glutathione level was lowered by treatment with diamide or buthionine-D,L-sulfoximine. Proton secretion induced by reduction of the electron acceptors was not affected by both modulators. The change in glutathione level is different for roots and coleoptiles. Our data are discussed with regard to the role of GSH in electron donation for a plasma membrane bound electron transport system.

Proton-induced x-ray emission was used to investigate the penetration of compounds of the membrane-impermeant electron acceptors hexabromoiridate IV, hexachloroiridate IV, and hexacyanoferrate III into corn (Zea mays L.) roots. Maps of the heavy element distribution in cross-sections of fixed, epoxy-embedded roots showed for hexabromoiridate IV small amounts of Br in samples treated for 24 h with concentrations normally used in physiological experiments (0.02 mM). After treatment with high concentrations (0.8 mM) of these complexes, Fe and Ir as well as Br were found in root cross-sections. In samples taken at a distance of 5 mm behind the root tip, we found an even distribution of Fe, Ir, and Br over the whole cross-section. In samples taken 15 mm behind the root tip, about 99% of both Br and Ir was confined to the rhizodermal cell layer. The distribution did not change with the complex used. These data are consistent with the view that apoplastic diffusion of the electron acceptors was blocked by the hypodermal Casparian band.

Right-side-out plasma membrane vesicles of soybean (Glycine max (L.) Merr.) hypocotyl were loaded with NADH by electroporation. After washing it was possible to measure a decrease of NADH within the vesicles. Addition of the membrane impermeant electron acceptor hexacyanoferrate III to the experimental solution caused an increase of NADH oxidation, while the acceptor was reduced. NADH oxidation and reduction of the external acceptor were influenced by calcium and by calmoudin inhibitors. Ca2+ up to 100 μM stimulates the NADH:hexacyanoferrate III oxidoreductase activity, while millimolar concentrations inhibit. The stimulation is Ca2+ specific and sensitive to calmodulin inhibitors. The membrane impermeant inhibitor, calmidazolium, is by far the strongest one, while the permeant compounds fluphenazine, trifluoperazine and W7 are less active. Compound W5 turned out to be practically inactive.

The correlation of the effects of vitamin K3 and dicumarol (an anti-vitamin K in pharmaceutical applications) on the trans plasma membrane electrical potential difference of maize roots with the reduction of the artificial electron acceptors hexacyanoferrate (III) or hexabromoiridate (IV) and the concomitant enhancement of acidification of the incubation medium was investigated. Vitamin K3 depolarized the plasma membrane of Zea mays L. roots, while dicumarol had no significant effect on the membrane potential. Plants treated with vitamin K3 for 30 min followed by intense rinsing showed higher reduction of hexabromoiridate (IV) than hexacyanoferrate (III), as well as a stimulated acidification of the incubation medium. Depolarization of the plasma membrane by hexacyanoferrate (III) or hexabromoiridate (IV) decreased after an incubation with vitamin K3. Pretreatment with dicumarol caused an inhibition of hexacyanoferrate (III) reduction and medium acidification as well as depolarization by K3. The reduction of hexabromoiridate (IV) was not affected by dicumarol pretreatment. The proton secretion associated with the reduction was slightly lowered. According to our results, it seems possible that vitamin K3 acts as an electron acceptor for the plasmalemma electron transport system of maize roots whereas dicumarol appears to inhibit electron and proton transport.

In this work we demonstrate the effect of different ascorbate redox states on the plasma membrane potential and on proton secretion of onion root cells. Ascorbate and dehydroascorbate induced plasmalemma hyperpolarization and stimulated acidification of the incubation medium. Both effects were transient and a repolarization of the membrane to the resting potential was reached within 10–15 min. The free radical of ascorbate had a permanent stimulatory effect on both parameters: a concentration dependent hyperpolarization of about 25 mV, partially inhibited by vanadate, could be observed immediately after addition. Medium acidification was increased by a factor of 20 and sustained for hours in the presence of the free radical. Additionally, ascorbate may increase the potassium permeability of the membrane, thus hyperpolarizing at low external potassium concentrations. Possible interactions of the free radical with the energization of the plasma membrane via an NAD(P)H-ascorbate free radical reductase (EC 1.6.5.4) will be discussed.

Three artificial electron acceptors of different E'0 and charge, hexacyanoferrate (III) (K3Fe(CN)6), hexachloroiridate (IV) (K2IrCl6), and hexabromoiridate (IV) (K2IrBr6), were compared with respect to their rate of reduction by roots of Zea mays L., the concomitant proton secretion, and to the effect on plasmalemma depolarization. It has been shown that these plasma membrane impermeable electron acceptors were reduced by a plasmalemma reductase activity. At low concentrations proton secretion was slightly inhibited, at higher concentrations, however, the rate of proton secretion was stimulated. The root cell plasmalemma showed a transient depolarization after addition of all three electron acceptors. The depolarization was concentration-dependent for the iridate complexes but not for hexacyanoferrate (III). For both iridate complexes maximum depolarization was reached at 50 ??mol dm-3. A hypothetical model as an explanation of the redox dependent proton secretion will be given.

Extracellular matrix (ECM) organization is a complex process that requires the coordinated efforts of many molecules. For the regulation of collagen fiber diameter, the proteoglycan decorin appears to be of major relevance. To investigate the role of decorin in the process of (photo-)aging in more detail, full-thickness punch biopsies were isolated from human buttock skin. Single exposure with two minimal erythemal doses of solar simulated irradiation caused down-regulation of decorin mRNA in young (n = 5) and old subjects (n = 5) after 24 h. Interestingly, decorin mRNA was elevated with age. To test the hypothesis that a decreased collagen-to-decorin-ratio impairs collagen structure we also investigated collagens I and III gene expression. Both were down-regulated with increasing age and after single UV-irradiation. As determined by laser capture microdissection-quantitative real time-Polymerase chain reaction (n = 11), decorin is mostly present in the reticular dermis while being absent from the papillary dermis. Minor expression was also observed in the epidermis. However, in contrast to full-thickness skin biopsies age-dependent changes in collagens I, III, and decorin expression could not be observed with this methodology indicating technical limitations. Together with our finding that collagens I and III mRNA are similarly expressed in the reticular and papillary dermis and are down-regulated by UV, our studies support the idea of a major role of decorin in ECM organization. Altered expression of decorin mRNA in the different dermal strata and a decrease in the collagen-to-decorin ratio inflicted by both age and ultraviolet irradiation possibly affect collagen bundle diameter and subsequently the mechanical properties of human skin.

The plant plasma membrane contains redox proteins able to mediate a trans-membrane electron flow. This electron flow might be responsible for the generation of the active oxygen species observed as a reaction to pathogen attack or stress. Vitamin K1 could be identified as a possible lipid soluble electron carrier in plant plasma membrane preparations. Such a function would be analogous to coenzyme Q in animal plasma membranes. What we are going to outline in this contribution is a concept of how the electron transport system of the plant plasma membrane could interact with quinones, thus contributing to the metabolism of free radicals in plants.

Are plants stacked neutrophils? Curious question, one might think. However, after reading some up-to-date reviews on the topic of plant pathogen defense the idea becomes inescapable. In literature, on journal covers [e.g., Plant & Cell Physiol 38 (10), 1997], in congress presentations and, we fear, also in the minds of scientists, the NADPH oxidase complex known from neutrophil phagolysosomes has been transplanted, complete with all its regulatory components, to the plasma membrane (PM) of plants. In other words, the plant defense system is proposed to be a clone of the mammalian’s oxidative burst.

At the beginning of the 1980s scientists working on plasma membrane (PM)-bound oxidoreductase (redox) activities had to go through thorough discussion in order to persuade their colleagues that electron transport at the PM of plants was no mere fiction but reality. Nowadays, the need to prove the sheer existence of the system(s) no longer exists; however, because the PM redox system of intact plants has, up till now, refused detection without electron acceptors that were added to the experimental solution, the most difficult of the old arguments of doubt still lingers on: What might such a system be good for? Today there are some answers to this question (Bienfait and Luttge 1988; Crane et al. 1991a,b; Navas 1991; Rubinstein 1992; see also the complete issue of Protoplasma 184 (1–4), 1995); yet it appears that there are too many answers and no plain way to decide which one is right. In experiments with PM-redox activity an impermeant electron acceptor, e.g. ferricyanide, is almost always added to the experimental solution, and it is therefore not clear what the system would do, if anything, without such a treatment.

The action of the 4-hydroxycoumarins dicumarol and warfarin, antagonists of probable vitamin K type components of the plasma membrane electron-transport system, on plasma membrane redox activity of intact maize roots was compared. Both effectors inhibited electron transfer to extracellular hexacyanoferrate III. While the effect of the strongly lipophilic dicumarol on the electron-transport system was irreversible by rinsing, the inhibition caused by the hydrophilic warfarin could be reverted completely by exchange of the incubation medium. We take these results as possible evidence for the integration of dicumarol into the plasma membrane. The action of warfarin may be confined to enzymic sites freely accessible from the aqueous apoplasmic solution.

Modulation of plasma membrane-bound NADH:hexacyanoferrate III oxidoreductase activities by dicumarol and warfarin was investigated with plasma membrane vesicles of Zea mays L. (cv. Sil Anjou 18) roots, prepared by aqueous two phase partitioning. Vesicles were about 65% right-side out orientated as demonstrated by enzyme latency of vanadate sensitive ATPase activity. Dicumarol or warfarin, respectively, inhibited NADH:hexacyanoferrate III oxidoreductase activity in a concentration-dependent manner and inhibition could be reversed partially by addition of quinones