Phytophthora pluvialis maintenance, spore production and detached needle assays.

Phytophthora pluvialis is an oomycete that primarily infects Pinus radiata and Pseudotsuga menziesii causing the destructive foliar disease red needle cast (RNC). Recent observations show that P. pluvialis can also infect western hemlock inducing resinous cankers. High-throughput and reproducible infection assays are integral to find key information on tree health and oomycete pathogenicity. In this protocol, we describe the propagation and spore induction of P. pluvialis, followed by detached needle assays for verification and quantification of virulence of P. pluvialis in P. radiata needles. These needle assays can be employed for high-throughput screening of tree needles with diverse genetic backgrounds. In downstream analysis, Quantitative PCR (qPCR) was utilized to assess relative gene expression, as exemplified by candidate RxLR effector protein PpR01. Additional techniques like RNA sequencing, metabolomics, and proteomics can be combined with needle assays and can offer comprehensive insights into P. pluvialis infection mechanisms.

Bi-directional Dual-flow-RootChip for Physiological Analysis of Plant Primary Roots Under Asymmetric Perfusion of Stress Treatments.

Due to technical limitations, research to date has mainly focused on the role of abiotic and biotic stress-signalling molecules in the aerial organs of plants, including the whole shoot, stem, and leaves. Novel experimental platforms including the dual-flow-RootChip (dfRC), PlantChip, and RootArray have since expanded this to plant-root cell analysis. Based on microfluidic platforms for flow stream shaping and force sensing on tip-growing organisms, the dfRC has further been expanded into a bi-directional dual-flow-RootChip (bi-dfRC), incorporating a second adjacent pair of inlets/outlet, enabling bi-directional asymmetric perfusion of treatments towards plant roots (shoot-to-root or root-to-shoot). This protocol outlines, in detail, the design and use of the bi-dfRC platform. Plant culture on chip is combined with guided root growth and controlled exposure of the primary root to solute changes. The impact of surface treatment on root growth and defence signals can be tracked in response to abiotic and biotic stress or the combinatory effect of both. In particular, this protocol highlights the ability of the platform to culture a variety of plants, such as Arabidopsis thaliana, Nicotiana benthamiana, and Solanum lycopersicum, on chip. It demonstrates that by simply altering the dimensions of the bi-dfRC, a broad application basis to study desired plant species with varying primary root sizes under microfluidics is achieved. Key features Expansion of the method developed by Stanley et al. (2018a) to study the directionality of defence signals responding to localised treatments. Description of a microfluidic platform allowing culture of plants with primary roots up to 40 mm length, 550 µm width, and 500 µm height. Treatment with polyvinylpyrrolidone (PVP) to permanently retain the hydrophilicity of partially hydrophobic bi-dfRC microchannels, enabling use with surface-sensitive plant lines. Description of novel tubing array setup equipped with rotatable valves for switching treatment reagent and orientation, while live-imaging on the bi-dfRC. Graphical overview Graphical overview of bi-dfRC fabrication, plantlet culture, and setup for root physiological analysis.(a) Schematic diagram depicting photolithography and replica molding, to produce a PDMS device. (b) Schematic diagram depicting seed culture off chip, followed by sub-culture of 4-day-old plantlets on chip. (c) Schematic diagram depicting microscopy and imaging setup, equipped with a media delivery system for asymmetric treatment introduction into the bi-dfRC microchannel root physiological analysis under varying conditions.

How plants solubilise seed fats: revisiting oleosin structure and function to inform commercial applications.

Plants store triacylglycerides in organelles called oil bodies, which are important fuel sources for germination. Oil bodies consist of a lipid core surrounded by an interfacial single layer membrane of phospholipids and proteins. Oleosins are highly conserved plant proteins that are important for oil body formation, solubilising the triacylglycerides, stabilising oil bodies, and playing a role in mobilising the fuel during the germination process. The domain structure of oleosins is well established, with N- and C-terminal domains that are hydrophilic flanking a long hydrophobic domain that is proposed to protrude into the triacylglyceride core of the oil body. However, beyond this general understanding, little molecular level detail on the structure is available and what is known is disputed. This lack of knowledge limits our understanding of oleosin function and concomitantly our ability to engineer them. Here, we review the state of play in the literature regarding oleosin structure and function, and provide some examples of how oleosins can be used in commercial settings.

Encoding, transmission, decoding, and specificity of calcium signals in plants.

Calcium acts as a signal and transmits information in all eukaryotes. Encoding machinery consisting of calcium channels, stores, buffers, and pumps can generate a variety of calcium transients in response to external stimuli, thus shaping the calcium signature. Mechanisms for the transmission of calcium signals have been described, and a large repertoire of calcium binding proteins exist that can decode calcium signatures into specific responses. Whilst straightforward as a concept, mysteries remain as to exactly how such information processing is biochemically implemented. Novel developments in imaging technology and genetically encoded sensors (such as calcium indicators), in particular for multi-signal detection, are delivering exciting new insights into intra- and intercellular calcium signaling. Here, we review recent advances in characterizing the encoding, transmission, and decoding mechanisms, with a focus on long-distance calcium signaling. We present technological advances and computational frameworks for studying the specificity of calcium signaling, highlight current gaps in our understanding and propose techniques and approaches for unravelling the underlying mechanisms.

A dual-flow RootChip enables quantification of bi-directional calcium signaling in primary roots.

One sentence summary: Bi-directional-dual-flow-RootChip to track calcium signatures in Arabidopsis primary roots responding to osmotic stress. Plant growth and survival is fundamentally linked with the ability to detect and respond to abiotic and biotic factors. Cytosolic free calcium (Ca2+) is a key messenger in signal transduction pathways associated with a variety of stresses, including mechanical, osmotic stress and the plants' innate immune system. These stresses trigger an increase in cytosolic Ca2+ and thus initiate a signal transduction cascade, contributing to plant stress adaptation. Here we combine fluorescent G-CaMP3 Arabidopsis thaliana sensor lines to visualise Ca2+ signals in the primary root of 9-day old plants with an optimised dual-flow RootChip (dfRC). The enhanced polydimethylsiloxane (PDMS) bi-directional-dual-flow-RootChip (bi-dfRC) reported here adds two adjacent inlet channels at the base of the observation chamber, allowing independent or asymmetric chemical stimulation at either the root differentiation zone or tip. Observations confirm distinct early spatio-temporal patterns of salinity (sodium chloride, NaCl) and drought (polyethylene glycol, PEG)-induced Ca2+ signals throughout different cell types dependent on the first contact site. Furthermore, we show that the primary signal always dissociates away from initially stimulated cells. The observed early signaling events induced by NaCl and PEG are surprisingly complex and differ from long-term changes in cytosolic Ca2+ reported in roots. Bi-dfRC microfluidic devices will provide a novel approach to challenge plant roots with different conditions simultaneously, while observing bi-directionality of signals. Future applications include combining the bi-dfRC with H2O2 and redox sensor lines to test root systemic signaling responses to biotic and abiotic factors.

Exploiting Structural Modelling Tools to Explore Host-Translocated Effector Proteins.

Oomycete and fungal interactions with plants can be neutral, symbiotic or pathogenic with different impact on plant health and fitness. Both fungi and oomycetes can generate so-called effector proteins in order to successfully colonize the host plant. These proteins modify stress pathways, developmental processes and the innate immune system to the microbes' benefit, with a very different outcome for the plant. Investigating the biological and functional roles of effectors during plant-microbe interactions are accessible through bioinformatics and experimental approaches. The next generation protein modeling software RoseTTafold and AlphaFold2 have made significant progress in defining the 3D-structure of proteins by utilizing novel machine-learning algorithms using amino acid sequences as their only input. As these two methods rely on super computers, Google Colabfold alternatives have received significant attention, making the approaches more accessible to users. Here, we focus on current structural biology, sequence motif and domain knowledge of effector proteins from filamentous microbes and discuss the broader use of novel modelling strategies, namely AlphaFold2 and RoseTTafold, in the field of effector biology. Finally, we compare the original programs and their Colab versions to assess current strengths, ease of access, limitations and future applications.

Interior design: how plant pathogens optimize their living conditions.

Pathogens use effectors to suppress host defence mechanisms, promote the derivation of nutrients, and facilitate infection within the host plant. Much is now known about effectors that target biotic pathways, particularly those that interfere with plant innate immunity. By contrast, an understanding of how effectors manipulate nonimmunity pathways is only beginning to emerge. Here, we focus on exciting new insights into effectors that target abiotic stress adaptation pathways, tampering with key functions within the plant to promote colonization. We critically assess the role of various signalling agents in linking different pathways upon perturbation by pathogen effectors. Additionally, this review provides a summary of currently known bacterial, fungal, and oomycete pathogen effectors that induce biotic and abiotic stress responses in the plant, as a first step towards establishing a comprehensive picture for linking effector targets to pathogenic lifestyles.

Host interactors of effector proteins of the lettuce downy mildew Bremia lactucae obtained by yeast two-hybrid screening.

Plant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes of many agriculturally important plant pathogens have been sequenced and vast candidate effector repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped physical interactions between B. lactucae effectors and lettuce candidate target proteins. Using a lettuce cDNA library-based yeast-two-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten interactors based on the number of independent colonies identified in the Y2H and two interactors that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged lettuce proteins and their interacting effectors. Importantly, relocalization of effectors or their interactors to the nucleus was observed for four protein-pairs upon their co-expression, supporting their interaction in planta.

Multiple downy mildew effectors target the stress-related NAC transcription factor LsNAC069 in lettuce.

To cause disease in lettuce, the biotrophic oomycete Bremia lactucae secretes potential RxLR effector proteins. Here we report the discovery of an effector-target hub consisting of four B. lactucae effectors and one lettuce protein target by a yeast-two-hybrid (Y2H) screening. Interaction of the lettuce tail-anchored NAC transcription factor, LsNAC069, with B. lactucae effectors does not require the N-terminal NAC domain but depends on the C-terminal region including the transmembrane domain. Furthermore, in Y2H experiments, B. lactucae effectors interact with Arabidopsis and potato tail-anchored NACs, suggesting that they are conserved effector targets. Transient expression of RxLR effector proteins BLR05 and BLR09 and their target LsNAC069 in planta revealed a predominant localization to the endoplasmic reticulum. Phytophthora capsici culture filtrate and polyethylene glycol treatment induced relocalization to the nucleus of a stabilized LsNAC069 protein, lacking the NAC-domain (LsNAC069ΔNAC ). Relocalization was significantly reduced in the presence of the Ser/Cys-protease inhibitor TPCK indicating proteolytic cleavage of LsNAC069 allows for relocalization. Co-expression of effectors with LsNAC069ΔNAC reduced its nuclear accumulation. Surprisingly, LsNAC069 silenced lettuce lines had decreased LsNAC069 transcript levels but did not show significantly altered susceptibility to B. lactucae. In contrast, LsNAC069 silencing increased resistance to Pseudomonas cichorii bacteria and reduced wilting effects under moderate drought stress, indicating a broad role of LsNAC069 in abiotic and biotic stress responses.

Recognition of lettuce downy mildew effector BLR38 in Lactuca serriola LS102 requires two unlinked loci.

Plant-pathogenic oomycetes secrete effector proteins to suppress host immune responses. Resistance proteins may recognize effectors and activate immunity, which is often associated with a hypersensitive response (HR). Transient expression of effectors in plant germplasm and screening for HR has proven to be a powerful tool in the identification of new resistance genes. In this study, 14 effectors from the lettuce downy mildew Bremia lactucae race Bl:24 were screened for HR induction in over 150 lettuce accessions. Three effectors-BLN06, BLR38 and BLR40-were recognized in specific lettuce lines. The recognition of effector BLR38 in Lactuca serriola LS102 did not co-segregate with resistance against race Bl:24, but was linked to resistance against multiple other B. lactucae races. Two unlinked loci are both required for effector recognition and are located near known major resistance clusters. Gene dosage affects the intensity of the BLR38-triggered HR, but is of minor importance for disease resistance.

Proteomic Profiling of the Microsomal Root Fraction: Discrimination of Pisum sativum L. Cultivars and Identification of Putative Root Growth Markers.

Legumes are a large and economically important family, containing a variety of crop plants. Alongside different cereals, some fruits, and tropical roots, a number of leguminosae evolved for millennia as crops with human society. One of these legumes is Pisum sativum L., the common garden pea. In the past, breeding has been largely selective on improved above-ground organs. However, parameters, such as root-growth, which determines acquisition of nutrients and water, have largely been underestimated. Although the genome of P. sativum is still not fully sequenced, multiple proteomic studies have been published on a variety of physiological aspects in the last years. The presented work focused on the connection between root length and the influence of the microsomal root proteome of four different pea cultivars after five days of germination (cultivar Vroege, Girl from the Rhineland, Kelvedon Wonder, and Blauwschokker). In total, 60 proteins were identified to have significantly differential abundances in the four cultivars. Root growth of five-days old seedlings and their microsomal proteome revealed a similar separation pattern, suggesting that cultivar-specific root growth performance is explained by differential membrane and ribosomal protein levels. Hence, we reveal and discuss several putative root growth protein markers possibly playing a key role for improved primary root growth breeding strategies.

Long-term iron deficiency: Tracing changes in the proteome of different pea (Pisum sativum L.) cultivars.

UNLABELLED: Iron deficiency (-Fe) is one of the major problems in crop production. Dicots, like pea (Pisum sativum L.), are Strategy I plants, which induce a group of specific enzymes such as Fe(III)-chelate reductase (FRO), Fe responsive transporter (IRT) and H(+)-ATPase (HA) at the root plasma membrane under -Fe. Different species and cultivars have been shown to react diversely to -Fe. Furthermore, different kinds of experimental set-ups for -Fe have to be distinguished: i) short-term vs. long-term, ii) constant vs. acute alteration and iii) buffered vs. unbuffered systems. The presented work compares the effects of constant long-term -Fe in an unbuffered system on roots of four different pea cultivars in a timely manner (12, 19 and 25days). To differentiate the effects of -Fe and plant development, control plants (+Fe) were analyzed in comparison to -Fe plants. Besides physiological measurements, an integrative study was conducted using a comprehensive proteome analysis. Proteins, related to stress adaptation (e.g. HSP), reactive oxygen species related proteins and proteins of the mitochondrial electron transport were identified to be changed in their abundance. Regulations and possible functions of identified proteins are discussed. SIGNIFICANCE: Pea (Pisum sativum L.) belongs to the legume family (Fabaceae) and is an important crop plant due to high Fe, starch and protein contents. According to FAOSTAT data (September 2015), world production of the garden pea quadrupled from 1970 to 2012. Since the initial studies by Gregor Mendel, the garden pea became the most-characterized legume and has been used in numerous investigations in plant biochemistry and physiology, but is not well represented in the "omics"-related fields. A major limitation in pea production is the Fe availability from soils. Adaption mechanisms to Fe deficiency vary between species, and even cultivars have been shown to react diversely. A label-free proteomic approach, in combination with physiological measurements, was chosen to observe four different pea cultivars for 5 to 25days. Physiological and proteome data showed that cultivar Blauwschokker and Vroege were more susceptible to -Fe than cultivar Kelvedon (highly efficient) and GftR (semi-efficient). Proteomic data hint that the adaptation process to long-term -Fe takes place between days 19 and 25. Results show that adaptation processes of efficient cultivars are able to postpone secondary negative effects of long-term -Fe, possibly by stabilizing the protein metabolic processing and the mitochondrial electron transport components. This maintains the cellular energy proliferation, keeps ROS production low and postpones the mitochondrial cell death signal.

Pre-fractionation strategies to resolve pea (Pisum sativum) sub-proteomes.

Legumes are important crop plants and pea (Pisum sativum L.) has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula Gaertner) allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins). Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed.

Two-dimensional phos-tag zymograms for tracing phosphoproteins by activity in-gel staining.

Protein phosphorylation is one of the most common post-translational modifications regulating many cellular processes. The phos-tag technology was combined with two-dimensional zymograms, which consisted of non-reducing IEF PAGE or NEPHGE in the first dimension and high resolution clear native electrophoresis (hrCNE) in the second dimension. The combination of these electrophoresis methods was mild enough to accomplish in-gel activity staining for Fe(III)-reductases by NADH/Fe(III)-citrate/ferrozine, 3,3'-Diaminobenzidine/H2O2 or TMB/H2O2 in the second dimension. The phos-tag zymograms can be used to investigate phosphorylation-dependent changes in enzyme activity. Phos-tag zymograms can be combined with further downstream analysis like mass spectrometry. Non-reducing IEF will resolve proteins with a pI of 3-10, whereas non-reducing NEPHGE finds application for alkaline proteins with a pI higher than eight. Advantages and disadvantages of these new methods will be discussed in detail.

Isolation of cellular membranes from lignin-producing tissues of Norway spruce and analysis of redox enzymes.

There are no earlier reports with successful isolation of plasma membranes from lignin-forming tissues of conifers. A method to isolate cellular membranes from extracellular lignin-producing tissue-cultured cells and developing xylem of Norway spruce was optimized. Modifications to the homogenization buffer were needed to obtain membranes from these phenolics-rich tissues. Membranes were separated by aqueous polymer two-phase partitioning. Chlorophyll a determination, marker enzyme assays and western blot analyses using antibodies for each membrane type showed that mitochondrial, chloroplastic and to a certain extent also ER and Golgi membranes were efficiently diminished from the upper phase, but tonoplast and plasma membranes distributed evenly between the upper and lower phases. Redox enzymes present in the partially purified membrane fractions were assayed in order to reveal the origin of H(2)O(2) needed for lignification. The membranes of spruce contained enzymes able to generate superoxide in the presence of NAD(P)H. Besides members of the flavodoxin and flavodoxin-like family proteins, cytochrome b5, cytochrome P450 and several stress responsive proteins were identified by nitroblue tetrazolium staining of isoelectric focusing gels and by mass spectrometry. Naphthoquinones juglone and menadione increased superoxide production in activity-stained gels. Some juglone-activated enzymes were preferentially using NADH. With NADH, menadione activated only some of the enzymes that juglone did, whereas with NADPH the activation patterns were identical. Duroquinone, a benzoquinone, did not affect superoxide production. Superoxide dismutase, ascorbate peroxidase, catalase and an acidic class III peroxidase isoenzyme were detected in partially purified spruce membranes. The possible locations and functions of these enzymes are discussed.

Class III peroxidases.

Class III peroxidases are heme-containing proteins of the secretory pathway with an extremely high number of isoenzymes, indicating the tremendous and important functions of this protein family. This chapter describes fractionation of the cell in subproteomes, their separation by polyacrylamide gel electrophoresis (PAGE) and visualization of peroxidase isoenzymes by heme and specific in-gel staining procedures. Soluble and membrane-bound peroxidases were separated by differential centrifugation. Aqueous polymer two-phase partitioning and discontinuous sucrose density gradient were applied to resolve peroxidase profiles of plasma membranes and tonoplast. Peroxidase isoenzymes of subproteomes were further separated by PAGE techniques such as native isoelectric focussing (IEF), high resolution clear native electrophoresis (hrCNE), and modified sodium dodecyl sulfate (modSDS)-PAGE. These techniques were used as stand-alone method or in combination for two-dimensional PAGE.

Alterations in Soluble Class III Peroxidases of Maize Shoots by Flooding Stress.

Due to changing climate, flooding (waterlogged soils and submergence) becomes a major problem in agriculture and crop production. In the present study, the effect of waterlogging was investigated on peroxidases of maize (Zea mays L.) leaves. The plants showed typical adaptations to flooding stress, i.e., alterations in chlorophyll a/b ratios and increased basal shoot diameter. Seven peroxidase bands could be detected by first dimension modified SDS-PAGE and 10 bands by first dimension high resolution Clear Native Electrophoresis that altered in dependence on plant development and time of waterlogging. Native isoelectric focusing revealed three acidic to neutral and four alkaline guaiacol peroxidases that could be further separated by high resolution Clear Native Electrophorese in the second dimension. One neutral peroxidase (pI 7.0) appeared to be down-regulated within four hours after flooding, whereas alkaline peroxidases (pI 9.2, 8.0 and 7.8) were up-regulated after 28 or 52 h. Second dimensions revealed molecular masses of 133 kDa and 85 kDa for peroxidases at pI 8.0 and 7.8, respectively. Size exclusion chromatography revealed native molecular masses of 30-58 kDa for peroxidases identified as class III peroxidases and ascorbate peroxidases by mass spectrometry. Possible functions of these peroxidases in flooding stress will be discussed.

IPG-strips versus off-gel fractionation: advantages and limits of two-dimensional PAGE in separation of microsomal fractions of frequently used plant species and tissues.

The crucial cellular role of membrane proteins is generally known for all life forms. Depending on the species, tissue, compartment, function and physiological condition, membranes differ in their protein and lipid profiles. Additionally, occurrence of microdomains hampers quantitative protein solubilisation and therefore membrane proteomics remain a major challenge. In the present study sample preparation (TCA/acetone and methanol/chloroform precipitation with and without SDS pre-solubilisation) for two-dimensional PAGE were compared for microsomal fractions of leaves (Arabidopsis thaliana, Nicotiana tabaccum, Pisum sativum) and roots (P. sativum, Zea mays). Generally, pre-solubilisation with SDS impaired the resolution of the gels. All samples showed higher spot yields with TCA/acetone precipitation. Finally, we compared the results of conventional 2D-PAGE (IPG/SDS-PAGE) and the combination of off-gel fractionation in the first-dimension, 10% urea-SDS-PAGE in the second-dimension. Results showed that more spots are present in the alkaline pH range after off-gel fractionation then on conventional 2D-PAGE. For the first time, off-gel fractionation was combined with SDS/SDS-PAGE and BAC/SDS-PAGE to improve the resolution after off-gel fractionation. Transmembrane domains and GRAVY were calculated for all significantly identified spots resulting from the MALDI-TOF-TOF mass spectrometry showing that in the second dimension after off-gel fractionation 10.3% more transmembrane proteins were identified compared to IPG/SDS-PAGE.

Alteration of plasma membrane-bound redox systems of iron deficient pea roots by chitosan.

Iron is essential for all living organisms and plays a crucial role in pathogenicity. This study presents the first proteome analysis of plasma membranes isolated from pea roots. Protein profiles of four different samples (+Fe, +Fe/Chitosan, -Fe, and -Fe/Chitosan) were compared by native IEF-PAGE combined with in-gel activity stains and DIGE. Using DIGE, 89 proteins of interest were detected in plasma membrane fractions. Data revealed a differential abundance of several spots in all samples investigated. In comparison to the control and -FeCh the abundance of six protein spots increased whereas 56 spots decreased in +FeCh. Altered protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. Besides stress-related proteins, transport proteins and redox enzymes were identified. Activity stains after native PAGE and spectrophotometric measurements demonstrated induction of a ferric-chelate reductase (-Fe) and a putative respiratory burst oxidase homolog (-FeCh). However, the activity of the ferric-chelate reductase decreased in -Fe plants after elicitor treatment. The activity of plasma membrane-bound class III peroxidases increased after elicitor treatment and decreased under iron-deficiency, whereas activity of quinone reductases decreased mostly after elicitor treatment. Possible functions of proteins identified and reasons for a weakened pathogen response of iron-deficient plants were discussed.

Phylogeny, topology, structure and functions of membrane-bound class III peroxidases in vascular plants.

Peroxidases are key player in the detoxification of reactive oxygen species during cellular metabolism and oxidative stress. Membrane-bound isoenzymes have been described for peroxidase superfamilies in plants and animals. Recent studies demonstrated a location of peroxidases of the secretory pathway (class III peroxidases) at the tonoplast and the plasma membrane. Proteomic approaches using highly enriched plasma membrane preparations suggest organisation of these peroxidases in microdomains, a developmentally regulation and an induction of isoenzymes by oxidative stress. Phylogenetic relations, topology, putative structures, and physiological function of membrane-bound class III peroxidases will be discussed.