Peroxisomal xanthine oxidoreductase: characterization of the enzyme from pea (Pisum sativum L.) leaves

The presence and properties of the enzyme xanthine oxidoreductase (XOR) in peroxisomes from pea (Pisum sativum L.) leaves were studied using biochemical and immunological methods. The activity analysis showed that, in leaf peroxisomes, the superoxide-generating XOR form, xanthine oxidase (XOD), was predominant over the xanthine dehydrogenase form (XDH), with a XDH/XOD ratio of 0.5. However, in crude extracts of pea leaves, the XDH form was more abundant, with a XDH/XOD ratio of 1.6. The native molecular mass of the peroxisomal XOR determined by polyacrylamide gel electrophoresis was 290kDa. Using western blot assays, we identified an immunoreactive band of 59kDa that was not affected by the reducing reagent DTT or endogenous proteases. The analysis of pea leaves by electron microscopy immunogold labeling with affinity-purified antibodies against rat XOD confirmed that this enzyme was localized in the matrix of peroxisomes, as well as in chloroplasts and cytosol. In pea plants subjected to abiotic stress by cadmium, the activity of the peroxisomal XOR was reduced, whereas its protein level expression increased. The results confirmed that leaf peroxisomes contain XOR, and suggest that this peroxisomal metalloflavoprotein enzyme is involved in the mechanism of response of pea plants to abiotic stress by heavy metals.     

Glutathione reductase from pea leaves: response to abiotic stress and characterization of the peroxisomal isozyme

The glutathione reductase (GR; EC 1.6.4.2) isozyme present in peroxisomes has been purified for the first time, and its unequivocal localization in these organelles, by immunogold electron microscopy, is reported. The enzyme was purified c. 21-fold with a specific activity of 9523 units mg(-1) protein, and a yield of 44 microg protein kg(-1) leaves was obtained. The subunit size of the peroxisomal GR was 56 kDa and the isoelectric point was 5.4. The enzyme was recognized by a polyclonal antibody raised against total GR from pea (Pisum sativum) leaves. The localization of GR in peroxisomes adds to chloroplasts and mitochondria where GR isozymes are also present, and suggests a multiple targeting of this enzyme to distinct cell compartments depending on the metabolism of each organelle under the plant growth conditions. The expression level of GR in several organs of pea plants and under different stress conditions was investigated. The possible role of peroxisomal GR under abiotic stress conditions, such as cadmium toxicity, high light, darkness, high temperature, wounding and low temperature, is discussed.     

Localization of nitric-oxide synthase in plant peroxisomes

The presence of nitric-oxide synthase (NOS) in peroxisomes from leaves of pea plants (Pisum sativum L.) was studied. Plant organelles were purified by differential and sucrose density gradient centrifugation. In purified intact peroxisomes a Ca(2+)-dependent NOS activity of 5.61 nmol of L-[(3)H]citrulline mg(-1) protein min(-1) was measured while no activity was detected in mitochondria. The peroxisomal NOS activity was clearly inhibited (60-90%) by different well characterized inhibitors of mammalian NO synthases. The immunoblot analysis of peroxisomes with a polyclonal antibody against the C terminus region of murine iNOS revealed an immunoreactive protein of 130 kDa. Electron microscopy immunogold-labeling confirmed the subcellular localization of NOS in the matrix of peroxisomes as well as in chloroplasts. The presence of NOS in peroxisomes suggests that these oxidative organelles are a cellular source of nitric oxide (NO) and implies new roles for peroxisomes in the cellular signal transduction mechanisms.     

Antioxidative enzymes from chloroplasts, mitochondria, and peroxisomes during leaf senescence of nodulated pea plants

In this work the influence of the nodulation of pea (Pisum sativum L.) plants on the oxidative metabolism of different leaf organelles from young and senescent plants was studied. Chloroplasts, mitochondria, and peroxisomes were purified from leaves of nitrate-fed and Rhizobium leguminosarum-nodulated pea plants at two developmental stages (young and senescent plants). In these cell organelles, the activity of the ascorbate-glutathione cycle enzymes ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), and the ascorbate and glutathione contents were determined. In addition, the total superoxide dismutase (SOD) activity, the pattern of mitochondrial and peroxisomal NADPH-generating dehydrogenases, some of the peroxisomal photorespiratory enzymes, the glyoxylate cycle and oxidative metabolism enzymes were also analysed in these organelles. Results obtained on the metabolism of cell organelles indicate that nodulation with Rhizobium accelerates senescence in pea leaves. A considerable decrease of the ascorbate content of chloroplasts, mitochondria, and peroxisomes was found, and in these conditions a metabolic conversion of leaf peroxisomes into glyoxysomes, characteristic of leaf senescence, took place.     

Oligomerization and chaperone activity of a plant 2-Cys peroxiredoxin in response to oxidative stress

Plant 2-Cys peroxiredoxins (2-Cys Prxs) have been reported to localize to chloroplasts and perform antioxidative roles during plant development and photosynthesis. In this study, we identified that, in addition to the well-known function of thioredoxin (Trx)-dependent peroxidase, the plant 2-Cys Prx in Chinese cabbage 2-Cys Prx1, designated C2C-Prx1, also behaves as a molecular chaperone under oxidative stress conditions, like the yeast and mammalian 2-Cys Prxs. By the chaperone function of C2C-Prx1, the protein efficiently prevented the denaturation of citrate synthase and insulin from heat shock and dithiothreitol (DTT)-induced chemical stresses. Also, the protein structure of C2C-Prx1 was shown to have discretely sized multiple structures, whose molecular sizes were in the diverse ranges of low molecular weight (LMW) proteins to high molecular weight (HMW) protein complexes. The dual functions of C2C-Prx1 acting as a peroxidase and as a molecular chaperone are alternatively switched by heat shock and oxidative stresses, accompanying with its structural changes. The peroxidase function predominates in the lower MW forms, but the chaperone function predominates in the higher MW complexes. The precise regulation of C2C-Prx1 structures and functions may play a pivotal role in the protection of plant chloroplasts from photo-oxidative stress.     

Increased levels of peroxisomal active oxygen-related enzymes in copper-tolerant pea plants

The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes containing Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties of Pisum sativum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme Cu,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxisomal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The higher activities of these peroxisomal active oxygen-related enzymes in Cu-tolerant plants suggest the involvement of reactive oxygen intermediates (O₂⁻, OH) in the mechanism of Cu lethality, and also imply a function for peroxisomal Mn-SOD in the molecular mechanisms of plant tolerance to Cu in Pisum sativum L.     

The dual-targeted plant sulfiredoxin retroreduces the sulfinic form of atypical mitochondrial peroxiredoxin

Sulfiredoxin (Srx) couples the energy of ATP hydrolysis to the energetically unfavorable process of reducing the inactive sulfinic form of 2-cysteine peroxiredoxins (Prxs) to regenerate its active form. In plants, Srx as well as typical 2-cysteine Prx have been considered as enzymes with exclusive chloroplast localization. This work explores the subcellular localization of Srx in pea (Pisum sativum) and Arabidopsis (Arabidopsis thaliana). Immunocytochemistry, analysis of protein extracts from isolated intact organelles, and cell-free posttranslational import assays demonstrated that plant Srx also localizes to the mitochondrion in addition to plastids. The dual localization was in line with the prediction of a signal peptide for dual targeting. Activity tests and microcalorimetric data proved the interaction between Srx and its mitochondrial targets Prx IIF and thioredoxin. Srx catalyzed the retroreduction of the inactive sulfinic form of atypical Prx IIF using thioredoxin as reducing agent. Arabidopsis Srx also reduced overoxidized human Prx V. These results suggest that plant Srx could play a crucial role in the regulation of Prx IIF activity by controlling the regeneration of its overoxidized form in mitochondria, which are sites of efficient reactive oxygen species production in plants.     

Origin and evolution of the peroxisomal proteome

Background  

Peroxisomes are ubiquitous eukaryotic organelles involved in various oxidative reactions. Their enzymatic content varies between species, but the presence of common protein import and organelle biogenesis systems support a single evolutionary origin. The precise scenario for this origin remains however to be established. The ability of peroxisomes to divide and import proteins post-translationally, just like mitochondria and chloroplasts, supports an endosymbiotic origin. However, this view has been challenged by recent discoveries that mutant, peroxisome-less cells restore peroxisomes upon introduction of the wild-type gene, and that peroxisomes are formed from the Endoplasmic Reticulum. The lack of a peroxisomal genome precludes the use of classical analyses, as those performed with mitochondria or chloroplasts, to settle the debate. We therefore conducted large-scale phylogenetic analyses of the yeast and rat peroxisomal proteomes.     

Expression of the Cymbidium ringspot virus 33-kilodalton protein in Saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal

Open reading frame 1 in the viral genome of Cymbidium ringspot virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.     

Plant peroxisomes,reactive oxygen metabolism and nitric oxide

In plant cells, as in most eukaryotic organisms, peroxisomes are probably the major sites of intracellular H2O2 production, as a result of their essentially oxidative type of metabolism. Like mitochondria and chloroplasts, peroxisomes also produce superoxide radicals (O2*-) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29, and 32 kDa have been shown to generate O2*- radicals. Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the four enzymes of the ascorbate-glutathione cycle plus ascorbate and glutathione, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of plant peroxisomes. The presence of the enzyme nitric oxide synthase (NOS) and its reaction product, nitric oxide (NO*), has been recently demonstrated in plant peroxisomes. Different experimental evidence has suggested that peroxisomes have a ROS-mediated cellular function in leaf senescence and in stress situations induced by xenobiotics and heavy metals. Peroxisomes could also have a role in plant cells as a source of signal molecules like NO*, O2*- radicals, H2O2, and possibly S-nitrosoglutathione (GSNO). It seems reasonable to think that a signal molecule-producing function similar to that postulated for plant peroxisomes could also be performed by human, animal and yeast peroxisomes, where research on oxy radicals, antioxidants and nitric oxide is less advanced than in plant peroxisomes.     

Evidence for the Presence of the Ascorbate-Glutathione Cycle in Mitochondria and Peroxisomes of Pea Leaves

The presence of the enzymes of the ascorbate-glutathione cycle was investigated in mitochondria and peroxisomes purified from pea (Pisum sativum L.) leaves. All four enzymes, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), were present in mitochondria and peroxisomes, as well as in the antioxidants ascorbate and glutathione. The activity of the ascorbate-glutathione cycle enzymes was higher in mitochondria than in peroxisomes, except for APX, which was more active in peroxisomes than in mitochondria. Intact mitochondria and peroxisomes had no latent APX activity, and this remained in the membrane fraction after solubilization assays with 0.2 M KCl. Monodehydroascorbate reductase was highly latent in intact mitochondria and peroxisomes and was membrane-bound, suggesting that the electron acceptor and donor sites of this redox protein are not on the external side of the mitochondrial and peroxisomal membranes. Dehydroascorbate reductase was found mainly in the soluble peroxisomal and mitochondrial fractions. Glutathione reductase had a high latency in mitochondria and peroxisomes and was present in the soluble fractions of both organelles. In intact peroxisomes and mitochondria, the presence of reduced ascorbate and glutathione and the oxidized forms of ascorbate and glutathione were demonstrated by high-performance liquid chromatography analysis. The ascorbate-glutathione cycle of mitochondria and peroxisomes could represent an important antioxidant protection system against H2O2 generated in both plant organelles.     

Peroxisomal manganese superoxide dismutase: Purification and properties of the isozyme from pea leaves

The peroxisomal manganese superoxide dismutase (perMn‐SOD; EC 1.15.1.1) was purified to homogeneity for the first time from peroxisomes of pea ( Pisum sativum L.) leaves. Peroxisomes were isolated from pea leaves by sucrose density‐gradient centrifugation, and then perMn‐SOD was purified from these organelles by two purification steps involving anion‐exchange and gel‐filtration fast protein liquid chromatography. Pure peroxisomal Mn‐SOD had a specific activity of 2 880 units per mg protein and was purified 3 000‐fold, with a yield of about 7 µg enzyme per kg pea leaves. The relative molecular mass determined for perMn‐SOD was 92 000, and it was composed of four equal subunits of 27 kDa. Ultraviolet and visible absorption spectra of the enzyme showed two absorption maxima at 278 and 483 nm, respectively, and two shoulders at 290 and 542 nm. By isoelectric focusing (pH 5‐7), an isoelectric point of 5.53 was determined for perMn‐SOD. In immunoblot assays, purified Mn‐SOD was recognized by a polyclonal antibody against mitochondrial Mn‐SOD (mitMn‐SOD) from pea leaves. The amino acid sequence of the N‐terminal region of the purified peroxisomal enzyme was determined. A 100% identity was found with the mitMn‐SOD from pea leaves, and high identities were also found with Mn‐SODs from other plant species.     

Purification of heart and liver mitochondrial carnitine acetvltransferase

Heart and liver mitochondrial, as well as liver peroxisomal, carnitine acetyltransferase was purified to apparent homogeneity and some properties, primarily of heart mitochondrial carnitine acetyltransferase, were determined. Hill coefficients for propionyl-CoA are 1.0 for each of the enzymes. The molecular weight of heart mitochondrial carnitine acetyltransferase, determined by SDS-PAGE, is 62,000. It is monomeric in the presence of catalytic amounts of substrate. Polyclonal antibodies against purified rat liver peroxisomal carnitine acetyltransferase precipitate liver and heart mitochondrial and liver peroxisomal carnitine acetyltransferase, but not liver peroxisomal carnitine octanoyltransferase. Liver peroxisomes, mitochondria, and microsomes and heart mitochondria all give multiple bands on Western blotting with the antibody against carnitine acetyltransferase. Major protein bands occur at the molecular weight of carnitine acetyltransferase and at 33 to 35 kDa.     

Structural and functional regulation of eukaryotic 2-Cys peroxiredoxins including the plant ones in cellular defense-signaling mechanisms against oxidative stress

The ubiquitously distributed peroxiredoxins (Prxs) have been shown to have diverse functions in cellular defense‐signaling pathways. They have been largely classified into three Prx classes, 2‐Cys Prx, atypical 2‐Cys Prx and 1‐Cys Prx, which can be distinguished by how many Cys residues they possess and by their catalytic mechanisms. Proteins belonging to the typical 2‐Cys Prx group containing the N‐terminal peroxidatic Cys residue undergo a cycle of peroxide‐dependent oxidation to sulfenic acid and thiol‐dependent reduction during H₂O₂ catalysis. However, in the presence of high concentrations of H₂O₂ and catalytic components, including thioredoxin (Trx), Trx reductase and NADPH, the sulfenic acid can be hyperoxidized to cysteine sulfinic acid. The overoxidized 2‐Cys Prxs are slowly reduced by the action of the adenosine 5′‐triphosphate‐dependent enzyme, sulfiredoxin. Upon exposure of cells to strong oxidative or heat‐shock stress conditions, 2‐Cys Prxs change their protein structures from low‐molecular weight to high‐molecular weight complexes, which trigger their functional switching from peroxidases to molecular chaperones. The C‐terminal region of 2‐Cys Prx also plays an essential role in this structural conversion. Thus, proteins with truncated C‐termini are resistant to overoxidation and cannot regulate their structures or functions. These reactions are primarily guided by the active site peroxidatic Cys residue, which serves as an ‘H₂O₂‐sensor’ in cells. The reversible structural and functional switching of 2‐Cys Prxs provides cells with a means to adapt to external stresses by presumably activating intracellular defense‐signaling systems. In particular, plant 2‐Cys Prxs localized in chloroplasts have dynamic protein structures that undergo major conformational changes during catalysis, forming super‐complexes and reversibly attaching to thylakoid membranes in a redox‐dependent manner.     

Copper–zinc superoxide dismutase is a constituent enzyme of the matrix of peroxisomes in the cotyledons of oilseed plants

The number and type of isoforms of superoxide dismutase (SOD) and their activities were compared in mitochondria and peroxisomes isolated from cotyledons of three different oilseed seedlings. Mitochondrial and peroxisomal isoforms of SOD could be distinguished in nondenaturing polyacrylamide gels by their differential sensitivities to KCN and/or H₂O₂. The type of SOD was not the same for each organelle in each of the three oilseed species. For example, a single Mn–SOD was found in cotton and cucumber mitochondria, whereas four CuZn–SODs were present in mitochondria from sunflower. At least one CuZn–SOD isoform was found in the peroxisomes of all three species. Cucumber peroxisomes contained both a CuZn–SOD and a Mn–SOD, cotton peroxisomes contained a single CuZn–SOD, whilst four separate CuZn–SODs, but no Mn–SOD were found in sunflower peroxisomes. Using antibodies against CuZn–SOD from watermelon peroxisomes or from chloroplasts of Equisetum , a single polypeptide of c . 16·5 kDa was detected on immunoblots of peroxisomal fractions from the three species. Post-embedment, electron-microscopic double immunogold-labelling showed that CuZn–SOD, with malate synthase used as marker enzyme of peroxisomes, was localized in the matrix of these organelles of all three species. These results suggest that CuZn–SOD is a characteristic matrix enzyme of peroxisomes in oilseed cotyledons.     

Immunocytochemical evidence for a peroxisomal localization of manganese superoxide dismutase in leaf protoplasts from a higher plant

The controversial question of the intracellular location of manganese-containing superoxide dismutase in higher plants was examined under a new experimental approach by applying the more rigorous and specific methods of immunocytochemistry to protoplasts isolated fromPisum sativum L. leaves. Manganese superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from 15 kg of leaves ofPisum sativum L. Rabbits were immunized with the mangano enzyme and the antibody specific for pea manganese superoxide dismutase was purified and found not to contain antigenic sites in common with (i) human manganese superoxide dismutase, (ii) iron superoxide dismutase from eitherEscherichia coli or higher plants, or (iii) plant or animal cuprozinc-superoxide dismutase.Pisum sativum L. manganese superoxide dismutase only appears to have antigenic determinants similar to other manganese superoxide dismutases from higher land plants. The antibody to pea Mn-superoxide dismutase was used to locate the enzyme in protoplasts isolated from young pea leaves by indirect immunofluorescence, and by electron microscopy using the unlabelled antibody peroxidase-antiperoxidase method. Results from immunofluorescence showed that chloroplasts were devoid of specific fluorescence which appeared scattered over the cytosolic spaces among chloroplasts, and demonstrate the absence of manganese superoxide dismutase inside chloroplasts. The metalloenzyme was found to be localized only in peroxisomes, whereas mitochondria, the traditionally accepted site for this enzyme in many eukaryotic organisms, did not show any specific staining. The possible subcellular roles of manganese superoxide dismutase inPisum sativum L. leaves are discussed in the light of its peroxisomal location.     

Integral membrane polypeptides of rat liver peroxisomes: topology and response to different metabolic states

Rats were treated with clofibrate, a hypolipidemic drug, and with thyroxine. Both drugs which are known to cause peroxisome proliferation, and a concomitant increase in peroxisomal fatty acid beta-oxidation activity in liver increased one of the major integral peroxisomal membrane polypeptides (PMPs), with apparent molecular mass of 69-kDa, six- and twofold, respectively. On the other hand hypothyroidism caused a decrease in peroxisomal fatty acid beta-oxidation activity and considerably lowered the concentration of PMP 69 in the peroxisomal membrane. Two other PMPs with apparent molecular masses of 36 and 22 kDa were not influenced by these treatments. The PMPs with apparent molecular masses of 42, 28, and 26 kDa were shown to be derived from the 69-kDa polypeptide by the activity of a yet uncharacterized endogenous protease during isolation of peroxisomes. Limited proteolysis of intact peroxisomes using proteinase K and subtilisin further substantiated that some portion of the 69-kDa polypeptide extends into the cytoplasm. The 36- and the 22-kDa polypeptides were accessible to proteolytic attack to a much lower extent and, therefore, are supposed to be rather deeply embedded within the peroxisomal membrane. It is demonstrated that peroxisomal acyl-CoA synthetase, an integral PMP extending partially into the cytoplasm, and PMP 69 are not identical polypeptides. Comparison of the peroxisomal membrane with that of mitochondria and microsomes revealed that the 69- and 22-kDa polypeptides as well as the bifunctional protein of the peroxisomal fatty acid beta-oxidation pathway were specifically located only in peroxisomes. Considerable amounts of a polypeptide cross-reacting with the antiserum against the 36-kDa polypeptide were found in mitochondria.     

Rotenone-insensitive NAD(P)H dehydrogenases in plants: Immunodetection and distribution of native proteins in mitochondria

Antisera produced against peptides deduced from potato nda1 and ndb1, homologues of yeast genes for mitochondrial rotenone-insensitive NADH dehydrogenases, recognise respective proteins upon expression in Escherichia coli. In western blots of potato (Solanum tuberosum L.) mitochondrial proteins, the NDB and NDA antibodies specifically detect polypeptides of 61 and 48 kDa, respectively. The proteins are found in mitochondria of flowers, leaves and tubers. Different signal intensities are seen relative to other respiratory chain components when organs are compared, indicating variations in relative abundance of dehydrogenases within the plant. The antibodies detect single polypeptides, of similar size as in potato, in mitochondria from several plant species. No specific cross-reaction was found in chloroplasts, but a weak NDA signal of 50 kDa was found in microsomes, possibly associated with peroxisomes. Two-dimensional native/SDS-PAGE analyses indicate that both NDA and NDB proteins reside as higher molecular mass forms, possibly oligomeric. The NDB immunoreactive protein is released by sonication of mitochondria, but is resistant to extraction by digitonin and partially to Triton X-100. In comparison, the NDA protein remains bound to the inner membrane at sonication or digitonin treatment, but can be solubilised with Triton. Investigation of a beetroot (Beta vulgaris L.) induction system for external NADH dehydrogenase indicates that the NDB antibody does not recognise the induced external NADH dehydrogenase in this species, but possibly an external NADPH dehydrogenase.