Sabine Lüthje

Properties of Guaiacol Peroxidase Activities Isolated from Corn Root Plasma Membranes

Although several investigations have demonstrated a plasma membrane (PM)-bound peroxidase activity in plants, this study is the first, to our knowledge, to purify and characterize the enzymes responsible. Proteins were extracted from highly enriched and thoroughly washed PMs. Washing and solubilization procedures indicated that the enzymes were tightly bound to the membrane. At least two distinct peroxidase activities could be separated by cation exchange chromatography (pmPOX1 and pmPOX2). Prosthetic groups were identified in fractions with peroxidase activity by absorption spectra, and the corresponding protein bands were identified by heme staining. The activities of the peroxidase enzymes responded different to various substrates and effectors and had different thermal stabilities and pH and temperature optima. Because the enzymes were localized at the PM and were not effected by p-chloromercuribenzoate, they were probably class III peroxidases. Additional size exclusion chromatography of pmPOX1 revealed a single activity peak with a molecular mass of 70 kD for the native enzyme, whereas pmPOX2 had two activity peaks (155 and 40 kD). Further analysis of these fractions by a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with heme staining confirmed the estimated molecular masses of the size exclusion chromatography.

Wound-induced apoplastic peroxidase activities: their roles in the production and detoxification of reactive oxygen species.

Production of reactive oxygen species (ROS) is a widely reported response of plants to wounding. However, the nature of enzymes responsible for ROS production and metabolism in the apoplast is still an open question. We identified and characterized the proteins responsible for the wound-induced production and detoxification of ROS in the apoplast of wheat roots (Triticum aestivum L.). Compared to intact roots, excised roots and leachates derived from them produced twice the amount of superoxide (O2(*-)). Wounding also induced extracellular peroxidase (ECPOX) activity mainly caused by the release of soluble peroxidases with molecular masses of 37, 40 and 136 kD. Peptide mass analysis by electrospray ionization-quadrupole time-of-flight-tandem mass spectrometry (ESI-QTOF-MS/MS) following lectin affinity chromatography of leachates showed the presence of peroxidases in unbound (37 kD) and bound (40 kD) fractions. High sensitivity of O2(*-)-producing activity to peroxidase inhibitors and production of O2(*-) by purified peroxidases in vitro provided evidence for the involvement of ECPOXs in O2(*-) production in the apoplast. Our results present new insights into the rapid response of roots to wounding. An important component of this response is mediated by peroxidases that are released from the cell surface into the apoplast where they can display both oxidative and peroxidative activities.

Oxidoreductases in plant plasma membranes.

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.

Membrane-bound guaiacol peroxidases from maize (Zea mays L.) roots are regulated by methyl jasmonate, salicylic acid, and pathogen elicitors

Plant peroxidases are involved in numerous cellular processes in plant development and stress responses. Four plasma membrane-bound peroxidases have been identified and characterized in maize (Zea mays L.) roots. In the present study, maize seedlings were treated with different stresses and signal compounds, and a functional analysis of these membrane-bound class III peroxidases (pmPOX1, pmPOX2a, pmPOX2b, and pmPOX3) was carried out. Total guaiacol peroxidase activities from soluble and microsomal fractions of maize roots were compared and showed weak changes. By contrast, total plasma membrane and washed plasma membrane peroxidase activities, representing peripheral and integral membrane proteins, revealed strong changes after all of the stresses applied. A proteomic approach using 2D-PAGE analysis showed that pmPOX3 was the most abundant class III peroxidase at plasma membranes of control plants, followed by pmPOX2a >pmPOX2b >pmPOX1. The molecular mass (63 kDa) and the isoelectric point (9.5) of the pmPOX2a monomer were identified for the first time. The protein levels of all four enzymes changed in response to multiple stresses. While pmPOX2b was the only membrane peroxidase down-regulated by wounding, all four enzymes were differentially but strongly stimulated by methyl jasmonate, salicylic acid, and elicitors (Fusarium graminearum and Fusarium culmorum extracts, and chitosan) indicating their function in pathogen defence. Oxidative stress applied as H2O2 treatment up-regulated pmPOX2b >pmPOX2a, while pmPOX3 was down-regulated. Treatment with the phosphatase inhibitor chantharidin resulted in distinct responses.

Quinones in plant plasma membranes — a missing link?

SummaryThe occurrence of a vitamin-K-like substance (naphthoquinone group) and flavins (flavin mononucleotide and flavin adenine dinucleotide) is demonstrated in plasma membranes isolated from maize (Zea mays L.) roots, on the basis of high-pressure liquid chromotography and spectral analysis. At least three NAD(P)H dehydrogenases could be purified to homogeneity from this plant material. Two of these proteins (25 and 30 kDa) reduce hexacyanoferrate III and quinones, while the third (41 kDa) reduces oxalacetic acid but not hexacyanoferrate III in the presence of NADH. Low-temperature spectra demonstrate the occurrence of a b-type cytochrome in plasma membranes isolated from maize roots. The latter compound could be reduced by ascorbic acid (E0′ > +80 mV) and shows an α-band maximum at 559 nm (at −196 °C). NADH-dependent cytochromeb reduction could be observed only in the presence of detergent and increased after preincubation with vitamin K3 (menadione). On the basis of the presented data a possible function of naphthoquinones in plasma membrane electron transfer is discussed.

Molecular components and biochemistry of electron transport in plant plasma membranes (review).

It 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.

Membrane-bound class III peroxidases: Identification, biochemical properties and sequence analysis of isoenzymes purified from maize (Zea mays L.) roots

The occurrence of three plasma membrane-bound class III peroxidases has been demonstrated for maize (Zea mays L.) roots [Mika and Lüthje (2003) Plant Physiol. 132:1489-1498]. In the present work a novel PM-bound peroxidase (pmPOX3) was partially purified. The experimental molecular mass of the heme protein was 38 kDa after size exclusion, and 57 kDa in non-reducing SDS-PAGE stained with the peroxidase substrates tetramethylbenzidine and H(2)O(2). The glycosylation of pmPOX1, pmPOX2b and pmPOX3 was shown by different approaches. The full length sequences of pmPOX1, pmPOX2b and pmPOX3 were identified by ESI-MS/MS and MALDI-TOF MS analysis in combination with in silico and in vivo cloning. Thus, we report the first sequence analysis of membrane-bound class III peroxidases. A partial gene analysis revealed two or three introns. Experimental and theoretical isoelectric points and molecular masses were compared. Targeting signals, the putative protein structures and the localization of the active center of the enzymes on the outside of the plasma membrane were deduced of the amino acid sequences. In contrast to other class III peroxidases, pmPOX1 seems to have a dimeric structure. Predictions of hydrophobic domains in comparison with solubilization experiments suggest an N-terminal transmembrane domain for the isoenzymes.

Hexabromoiridate IV as an electron acceptor: comparison with hexachloroiridate IV and hexacyanoferrate III

Three artificial electron acceptors potassium hexacyanoferrate (K3Fe(CN)6) ‘ferricyanide’ (HCF III), potassium hexachloroiridate (K2 IrCl6) (HCl IV), and potassium hexabromoiridate (K2IrBr6) (HBI IV), have been compared with respect to their reduction by roots, membrane permeability, phytotoxicity and their absorption spectra. In this paper it will be shown that HBI IV as well as HCI IV are more effective electron acceptors than HCF III in regard to their lower charge, redox potential and toxicity. The toxicity of these compounds is negligible when used in micromolar concentrations. Both do not permeate the plasma membrane of root cells. Low concentrations of all three electron acceptors stimulates elongation growth of Lepidium sativum L. roots. The reduction of HBI IV by maize roots was characterized and the effect of artificial auxins on the reduction rate of HBI IV has been investigated.

Salicylic acid changes the properties of extracellular peroxidase activity secreted from wounded wheat (Triticum aestivum L.) roots.

Summary. Wheat (Triticum aestivum L.) roots released proteins showing peroxidase activity in the apoplastic solution in response to wound stress. Preincubation of excised roots with 1 mM salicylic acid at pH 7.0 enhanced the guaiacol peroxidase activity of the extracellular solution (so-called extracellular peroxidase). The soluble enzymes were partially purified by precipitation with ammonium sulfate followed by size exclusion and ion exchange chromatography. Despite an increase in the total activity of secreted peroxidase induced by pretreatment of excised roots with salicylic acid, the specific activity of the partially purified protein was significantly lower compared to that of the control. Purification of the corresponding proteins by ion exchange chromatography indicates that several isoforms of peroxidase occurred in both control and salicylic acid-treated samples. The activities of the extracellular peroxidases secreted by the salicylic acid-treated roots responded differently to calcium and lectins compared with those from untreated roots. Taken together, our data suggest that salicylic acid changes the isoforms of peroxidase secreted by wounded wheat roots.

b-type cytochromes in plasma membranes of Phaseolus vulgaris hypocotyls, Arabidopsis thaliana leaves, and Zea mays roots

SummaryThe plasma membrane of higher plants contains more than one kind ofb-type cytochromes. One of these has a high redox potential and can be fully reduced by ascorbate. This component, the cytochromeb561 (cytb561), has its characteristic α-band absorbance close to 561 nm wavelength at room temperature. Cytb561 was first isolated from etiolated bean hook plasma membranes by two consecutive anion exchange chromatography steps. During the first step performed at pH 8, cytb561 did not bind to the anion exchange column, but otherb-type cytochromes did. In the second step performed at pH 9.9, cytb561 was bound to the column and was eluted from the column at an ionic strength of about 100 mM KCl. However, when the same protocol was applied to the solubilized plasma membrane proteins fromArabidopsis thaliana leaves and maize roots, the ascorbate-reducible cytb561 bound already to the first anion exchange column at pH 8 and was eluted also at an ionic strength of about 100 mM KCl. Otherb-type cytochromes than the ascorbate-reducible cytb561 from the plasma membranes of Arabidopsis leaves and maize roots showed similar Chromatographic characteristics to that of bean hypocotyls. These results demonstrate particular differences in the Chromatographic behavior of cytb561 from different sources.