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

Phytochemistry 72 (2011) 1124–1135

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Phylogeny, topology, structure and functions of membrane-bound class III peroxidases in vascular plants Sabine Lüthje ⇑, Claudia-Nicole Meisrimler, David Hopff, Benjamin Möller University of Hamburg, Biocenter Klein Flottbek, Dept. Plant Physiology, Ohnhorststrasse 18, 22609 Hamburg, Germany

a r t i c l e

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Article history: Available online 4 January 2011 Keywords: Membrane-bound class III peroxidases Oxidative stress Plasma membrane Plant proteomics Tonoplast

a b s t r a c t 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. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Haem-containing plant peroxidases (EC 1.11.1.7) are divided into two classes (Duroux and Welinder, 2003). Class I peroxidases are intracellular isoenzymes related to bacterial peroxidases, whereas class III peroxidases are isoenzymes of the secretory pathway. Due to their peroxidative or hydroxylic reaction cycles, peroxidases are involved in a variety of processes. Besides cell wall-related reactions, peroxidases fulfil important functions in metabolic pathways and stress-related processes (Almagro et al., 2009; Cosio and Dunand, 2009; Díaz et al., 2004; Hiraga et al., 2001; Mika et al., 2004; Passardi et al., 2005; Sottomayor et al., 2008). They play a key role in the detoxification of reactive oxygen species (ROS) during cellular metabolism and oxidative stress (De Gara, 2004). In vascular plants the number of class III peroxidases is extremely high. In the Arabidopsis (Arabidopsis thaliana (L.) HEYNH) Abbreviations: 2D, two-dimensional; APx, ascorbate peroxidase; AtAPx, Arabidopsis thaliana-ascorbate-peroxidase; AtPrx, Arabidopsis thaliana-peroxidase; CarAPx, Cicer arietinum-peroxidase; CrPrx, Catharanthus roseus-peroxidase; DIGE, differential in-gel electrophoresis; DIM, detergent insoluble membranes; ER, endoplasmatic reticulum; GFP, green fluorescent protein; GPI, glycerophosphatidylinosityl; OsPrx, Oryza sativa-peroxidase; MtPrx, Medicago truncatula-peroxidase; Ndh, NAD(P)H dehydrogenase; pmPOX2a, plasma membrane peroxidase 2a; Prx, peroxidase; PsPrx, Pisum sativum-peroxidase; Rboh, respiratory burst oxidase homolog; ROS, reactive oxygen species; SbPrx, Sorghum bicolor-peroxidase; SOD, super oxid dismutase; TMD, transmembrane domain; TPO, thyroid peroxidase; TrPrx, Trifolium repens-peroxidase; ZmPrx, Zea mays-peroxidase. ⇑ Corresponding author. E-mail address: [email protected] (S. Lüthje). 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.11.023

genome 73 genes are coding for class III peroxidases (Duroux and Welinder, 2003). Monocotyledonous plants evolved some additional peroxidase clusters not found in dicotyledonous plants. The rice (Oryza sativa L.) genome is coding for 138 class III peroxidases (Passardi et al., 2004). A search of the protein sequences which were contained in the peroxibase (http://peroxibase.toulouse.inra.fr/), as at August 2010, revealed sequences for at least 143 distinct class III peroxidases in maize (Zea mays L.). Post-transcriptional and posttranslational modifications of peroxidase transcripts can generate several other isoenzymes (Tognolli et al., 2002; Welinder et al., 2002). Although many soluble intracellular and extracellular peroxidases have been characterised in great detail (Hiraga et al., 2001; Shigeoka et al., 2002), less is known on membrane-bound isoenzymes. Besides thylakoid-bound ascorbate peroxidases (AtAPx05, AtAPx06; Asada et al., 1996; Jespersen et al., 1997; Kieselbach et al., 2000; Newman et al., 1994; Yabuta et al., 2002), three types of membrane-bound class I peroxidases have been predicted with a high sequence similarity to cytosolic peroxidases (Jespersen et al., 1997). Type 1 is associated with microbodies (e.g. GhAPx01, AtAPx03, CkAPx03), whereas location of the other two types (SoAPx04 and McAPx06) is still unknown (Bunkelmann and Trelease, 1996; Jespersen et al., 1997; Ishikawa et al., 1998; Narendra et al., 2006; Nito et al., 2001; Yamaguchi et al., 1995). Both chloroplastic and cytosolic-like ascorbate peroxidases interact with the membrane via a C-terminal membrane-spanning segment. Class III peroxidases have been identified at the thylakoid, tonoplast and the plasma membrane (Costa et al., 2008; Laloue et al., 1997; Mika and Lüthje, 2003; Mika et al., 2008; Sottomayor and

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Ros Barceló, 2003; Sottomayor et al., 2008; Zapata et al., 1998). Analysis of all class III peroxidases predicted from the maize genome revealed an unexpected high number (47%) of membranebound isoenzymes. TMHMM predicted N-terminal transmembrane domains in these isoenzymes and PSORT suggested a location in the plasma membrane (n = 37), endoplasmic reticulum (ER) membrane (n = 29), Golgi (n = 2) or tonoplast (n = 1) (Möller et al., 2001; Sonnhammer et al., 1998). Finally, thyroid peroxidase (TPO) located in the plasma membrane of vertebrates belongs to the peroxidase superfamily 2 of humans and animals (McDonald and Pearce, 2009). TPO is the key enzyme in thyroid hormone production and a universal autoantigen in autoimmune thyroid diseases. This paper will focus on membrane-bound class III peroxidases, analyse their phylogenetic relations, topology and putative structures, and organisation in microdomains. Specific functions of membrane-bound peroxidases will be discussed. 2. Evidence for membrane-bound class III peroxidases Thirty years ago in vitro experiments presented first evidence for a plasma membrane-bound peroxidase activity in vascular plants. An increased oxygen consumption was observed by intact protoplasts in the presence of extracellular NAD(P)H (Lin, 1982; Pantoja and Willmer, 1988). Sealed and right-side out (apoplastic side out) vesicles of plasma membranes isolated from several plant species and tissues showed peroxidase-like NAD(P)H oxidase activities (Vianello and Macri, 1991). Due to these observations and estimation of enzyme latency peroxidase activity has been suggested to be located at the apoplastic surface of the plasma membrane. Guaiacol peroxidase activity, however, could be observed also with sealed and inside-out plasma membrane vesicles (Bischoff and Lüthje, unpublished). The NADH oxidation by plasma membranes isolated from cauliflower (Brassica oleracea) inflorescences increased in the presence of phenolic compounds and decreased after incubation with typical peroxidase inhibitors (Askerlund et al., 1987). In plasma membrane preparations of other Brassicaceae seedlings, oxidation of tryptophan was observed in the presence of H2O2 (Ludwig-Müller et al., 1990; Ludwig-Müller and Hilgenberg, 1992). Plasma membranes isolated from soybean (Glycine max L.) roots showed a peroxidase activity in the presence of substrates like o-dianisidine, guaiacol, and ascorbate (Vianello et al., 1997). The oxidation of ascorbate was strongly stimulated by phenolic acids, like ferulic and caffeic acid. Oxidation of guaiacol or o-dianisidine was stimulated by calcium and inhibited by cyanide or azide.

In addition to these experiments, antibodies specific for apoplastic peroxidases were used to detect plasma membrane-bound peroxidases by immunogold labelling and electron microscopy in situ (Crevecoeur et al., 1997; Hu et al., 1989; Penel and Castillo, 1991). However, contaminations by soluble proteins in plasma membrane preparations have been discussed (Bérczi and Asard, 2003). The presence of peroxidases in plasma membrane preparations appeared to depend largely on the final membrane washing procedure (Askerlund et al., 1987; Mika et al., 2010). Side-effects by attached soluble isoenzymes could not be excluded for some of the above studies. Meanwhile our group identified full length amino acid sequences of three class III peroxidases (ZmPrx01, ZmPrx66, ZmPrx70) purified from highly enriched plasma membranes of maize roots (Mika and Lüthje, 2003; Mika et al., 2008). Proteomic studies identified further class III peroxidases (Table 1), ascorbate peroxidases and glutathione peroxidases in plasma membrane preparations of different plant species and tissues (Table 2). In mesophyll cells of Madagascar Periwinkle (Catharanthus roseus (L.) G. Don) a basic class III peroxidase (CrPrx01) is located at the inner surface of the tonoplast as shown by GFP-fusion constructs (Costa et al., 2008; Sottomayor and Ros Barceló, 2003). CrPrx01 is involved in secondary metabolism, i.e. the oxidation of alkaloids. Finally, a thylakoid-bound class III peroxidase has been described for barley (Hordeum vulgare L.) that has a high affinity for hydroquinones (Casano et al., 2000; Martín et al., 2004). The sequence of the protein, however, has not been identified so far and interaction with the thylakoid membrane has to be further investigated. 3. Orthologs and phylogenic relations Phylogenetic analyses have been presented for class III peroxidases of Arabidopsis and rice (Duroux and Welinder, 2003; Passardi et al., 2004). Comparison of ZmPrx66 and ZmPrx70 with the amino acid sequences derived from all genes coding for secretory peroxidases in rice showed that the maize peroxidases shared the highest sequence identity with peroxidases of subgroup IV.3, while ZmPrx01 was arranged with peroxidases in subgroup I.4 (Mika et al., 2008). ZmPrx66 and two other peroxidases from maize (ZmPrx6 and ZmPrx42) were grouped with OsPrx110. The homology of these three peroxidase sequences to only one peroxidase from rice suggests that they are gene duplicates. Plasma membrane-bound peroxidases found in roots of older maize plants, i.e. ZmPrx24, ZmPrx58, and ZmPrx81 (Table 1) clus-

Table 1 Class III peroxidases identified in highly enriched plasma membranes isolated from different plant species. Class III peroxidases identified in proteome studies with highly enriched plasma membrane fractions or whole cell extracts are listed in the table. The accession numbers are given in accordance to NCBI or UniProt (⁄). The denoted protein name was found in the Peroxibase (http://peroxibase.toulouse.inra.fr/). BGP1, barley grain peroxidase 1; DIM, detergent-insoluble membranes; MW, theoretical molecular weight; pI, calculated isoelectric point; PM, plasma membrane; Prx, peroxidase. Acc. No. ⁄

Q40069 Q8L3W2⁄ Q6EUS1⁄ A5H8G4⁄ A5H452⁄ A5H454⁄ B4FH68⁄ B4FHG3⁄ B4FG39⁄ P24102⁄ AC123575_10.1 ABO77633.1 BAD97436.1 Q9ZP15⁄

Protein

Species

Tissue

Cell fraction

MW [kD]

pI

Refs.

Prx BGP 1 OsPrx95 OsPrx27 ZmPrx01 ZmPrx70 ZmPrx66 ZmPrx58 ZmPrx24 ZmPrx81 AtPrx22 AtPrx69 MtPrx16 PsPrx13 TrPrx02

H. vulgare O. sativa O. sativa Z. mays Z. mays Z. mays Z. mays Z. mays Z. mays A. thaliana M. truncatula M. truncatula P. sativum T. repens

Aleuroane Roots Cell culture Roots Roots Roots Roots Roots Roots Mature stems Roots Pea roots Roots Pea roots

PM PM PM PM PM PM PM PM PM Cell extract DIM-PM PM PM PM

32.0 37.6 33.8 38.3 33.4 33.4 37.5 38.7 36.6 38.1 36.0 38.3 38.3 36.5

7.57 5.63 8.45 6.81 8.39 8.94 6.22 5.66 8.08 5.66 9.47 7.5 7.5 9.11

Hynek et al. (2009) Cheng et al. (2009) Natera et al. (2008) Mika et al. (2008) Mika et al. (2008) Mika et al. (2008) Hopff et al. (2010) Hopff et al. (2010) Hopff et al. (2010) Minic et al. (2007) Lefebvre et al. (2007) Meisrimler et al. (2009) Meisrimler et al. (2009) Meisrimler et al. (2009)

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Table 2 Class I peroxidases and glutathinone peroxidases identified in plasma membrane preparations of different plant species and tissues. Ascorbate peroxidases (APx) and glutathione peroxidases (GPx) identified in proteomic studies with highly enriched plasma membrane fractions of different plant species. The accession number was obtained from NCBI or UniProt (⁄). The denoted protein name was found in the Peroxibase (http://peroxibase.toulouse.inra.fr/). Topology and predicted location of proteins were calculated by bioinformatics tools as indicated. AA, amino acids; MW, theoretical molecular weight in kD; pI, isoelectric point; TMD, transmembrane domain.

a b c d e

Acc. No.

Protein

Species

Tissue

AAa

MWa

pIa

TMHMMb

HMMTOPc

TargetPd

PSORTe

Refs.

AAQ88105 Q10N21 Q6ZJJ1 Q9FEV2 B6T5N2⁄ Q05431 Q9LYB4 O48646 Q9SXT2

OsAPx03 OsAPx01 OsAPx04 OsGPx02 ZmGPx04 AtAPx01 AtGPX05 AtGPx066 CarAPx01

O. sativa O. sativa O. sativa O. sativa Z. mays A. thaliana A. thaliana A. thaliana C. arietinum

Cell culture Cell culture Cell culture Cell culture Roots Cell culture, mature stems Cell culture, leaf Cell culture, leaf Leaf

291 250 291 169 170 250 173 232 177

32.3 64.1 31.8 19.5 19.3 27.6 19.3 25.6 19.3

8.26 4.95 7.74 8.8 7.59 5.72 9.28 9.38 4.6

1 TMD 0 1 TMD 0 0 0 0 0 0

1 TMD 0 0 0 0 0 0 1 TMD 0

– – Mito – – – – Chl Secretory

Mito Peroxi PM Cyt Cyt Cyt Chl Chl Peroxi

Natera et al. (2008) Natera et al. (2008) Natera et al. (2008) Natera et al. (2008) Hopff et al. (2010) Santoni et al. 1998 Marmagne et al. (2007) Marmagne et al. (2007) Katam et al. (2009)

AA/MW/pI (http://www.expasy.ch/tools/protparam.html). TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/). HMMTOP (http://www.enzim.hu/hmmtop/index.html). TargetP (http://www.cbs.dtu.dk/services/TargetP/). PSort (http://psort.hgc.jp/form.html).

tered together with subgroup II of the rice peroxidases. ZmPrx24 and ZmPrx58 shared highest similarity with OsPrx60, whereas ZmPrx81 clustered together with OsPrx61–64. The homology of ZmPrx24 and ZmPrx58 sequences to only one peroxidase from rice suggests that they are also gene duplicates. Maize peroxidases predicted with a location in endomembrane systems clustered together with subgroups I.2 and I.4 of the rice peroxidases. The putative ER-bound ZmPrx101 shared highest sequence similarity with OsPrx14 (subgroup I.4); the putative Golgi-bound ZmPrx08 clustered together with OsPrx105 and OsPrx106, and the putative tonoplast-bound ZmPrx02 with OsPrx45, both in subgroup I.2. A comparison of the maize peroxidases with class III peroxidases from Arabidopsis showed highest similarity for ZmPrx01 to AtPrx19a (score 264, identity 45%), for ZmPrx66 to a putative peroxidase (score 356, identity 58%), and for ZmPrx70 to AtPrx52 (score 416, identity 64%), respectively. The sequence difference between AtPrx19a and AtPrx39 are two amino acids (99% identity). The Arabidopsis peroxidases seem to be extracellular isoenzymes with a cleavable N-terminal signal peptide that do not contain any transmembrane domain. According to Duroux and Welinder (2003) peroxidases may be suggested as different with a sequence similarity <70%, whereas real orthologs should have a sequence similarity >90%. Although orthologs of the maize peroxidases (ZmPrx01, ZmPrx66, and ZmPrx70) appear not to exist in Arabidopsis, 26 class III peroxidases of Arabidopsis were predicted to have either a N-terminal transmembrane anchor or ß-barrel transmembrane segments or both (http://aramemnon.botanik.uni-koeln.de/). Strongest evidence for transmembrane domains was given for AtPrx25, AtPrx43, AtPrx48, AtPrx53, and AtPrx54. These peroxidases and isoenzymes with a sequence identity >90 % to the class III peroxidases identified in plant plasma membranes (Table 1) were used for in silico analysis. 4. Topology In silico analysis of ZmPrx01, ZmPrx66, and ZmPrx70 have been presented in the past (Mika et al., 2008). In accordance with the biochemical classification of these isoenzymes as class III peroxidases and corresponding to the location of the purified proteins at the plasma membrane (Mika and Lüthje, 2003), N-terminal ER signal peptides have been predicted for their amino acid sequences. PSORT even predicted a non-cleavable signal peptide in ZmPrx70 and a location of the enzyme at the plasma membrane

or ER membrane (Table 3). Besides this isoenzyme, five other class III peroxidases have been suggested with locations in the ER or plasma membrane by this prediction program. Most of the putative membrane-bound peroxidases were predicted as secretory proteins. Besides cleavable N-terminal signal peptides, transmembrane domains have been calculated by several prediction programs for all class III peroxidases shown in Table 4. These results further support a membrane location of the peroxidases. Among the proteins selected from the Aramemnon database, AtPrx25 and AtPrx48 were predicted with a location in the plasma membrane, whereas AtPrx43 was predicted with location in the vacuole (Table 3). Usually N-terminal signal peptides of soluble secretory proteins from eukaryotic organisms were cleaved off during the maturation of these proteins. The N-terminus is modified in the endoplasmic reticulum, the signal peptide is cleaved off and the resulting N-terminal position is blocked by pyroglutamate (Welinder and Larsen, 2004). In contrast, the signal peptide of membrane-bound proteins may remain and due to its hydrophobicity function as an N-terminal transmembrane anchor. So far the search algorithms used by bioinformatics tools are not able to make exact predictions of this possibility yet. ZmPrx01, AtPrx43, and CrPrx01 have a C-terminal extension predicted as a vacuolar signal peptide. GFP-fusion constructs of CrPrx01 and CrPrx02 were found to be associated with the central vacuole and cell wall associated structures (Kumar et al., 2007; Sottomayor et al., 2008), whereas location of ZmPrx01 and AtPrx43 have to be verified. Although the C-terminus of the amino acid sequence of ZmPrx01 was postulated to function as a propeptide resulting in the targeting of the protein to the vacuole, purification of the protein from highly enriched plasma membrane preparations does not suggest such a targeting (Mika et al., 2008). C-terminal prolongations of distinct lengths were found in the amino acid sequences of horseradish peroxidase, apoplastic and cell wall-associated peroxidases (Carpin et al., 1999; Blee et al., 2001; Welinder, 1979). All homologs to ZmPrx01 from monocotyledonous plants contained a C-terminal prolongation that was missing in the sequences from dicotyledonous plants (Mika et al., 2008). Thus, specific properties of the propeptides might be important for the location of the mature proteins in different species. Most of the bioinformatics tools, however, predicted an extracellular location of ZmPrx01 (Table 3). The only class III peroxidase from maize with a prediction for the tonoplast was ZmPrx02. Anchoring by a C-terminal a-helix to the membrane has been predicted for class I peroxidases and members of the peroxidase

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Table 3 Putative structure, predicted posttranslational modification and localisation of membrane-bound class III peroxidases. Membrane-bound peroxidases of Arabidopsis, as indicated in the Aramemnon database, and the proteins listed in table 1 were used for in silico analysis. The accession number was obtained from NCBI or UniProt (⁄). The denoted protein name is given in accordance with the Peroxibase (http://peroxibase.toulouse.inra.fr/). Templates of the RCSB Protein Data Bank (http://www.pdb.org/pdb/home/home.do) were used for structural modelling. The score indicates the identity of amino acids between target and template in percent. Structure and location were predicted by bioinformatics tools as indicated. AA, amino acid; Chl, chloroplast; ER, endoplasmic reticulum; extra, extra cellular; Locexper, location experimental, Mito, mitochondria; NGlyc, N-glycosylation, PM, plasma membrane, –S–S–, disulfide bonds, Vacu, vacuole.

a b c d e f

Acc. No.

Protein

AAa

Template

Score

b-sheets

a-helices

Cys

–S–S–

NGlycb

Locexper

PSORTc

BaCelLOd

MultiLoce

TargetPf

Q6EUS1⁄ Q8L3W2⁄ EF178277⁄ B4FHG3⁄ B4FH68⁄ EF059719⁄ EF059717⁄ B4FG39⁄ XP_002455565 C5X5K3⁄ P24102⁄ O80822⁄ Q9SZH2⁄ O81755⁄ Q42578⁄ Q9FG34⁄ AC123575_10.1 ABO77633.1 BAD97436.1 Q9ZP15⁄

OsPrx27 OsPrx95 ZmPrx01 ZmPrx24 ZmPrx58 ZmPrx66 ZmPrx70 ZmPrx81 SbPrx75 SbPrx144 AtPrx22 AtPrx25 AtPrx43 AtPrx48 AtPrx53 AtPrx54 AtPrx69 MtPrx16 PsPrx13 TrPrx02

321 361 367 364 355 320 321 344 371 318 349 328 326 404 335 358 168 354 357 329

1schB 3hdlA 3hdlA 1bgpA 1bgpA 1schA 1schB 1qgjA 3hdlA 1schB 1gwuA 3hdlA 1pa2A 1pa2A 1pa2A 1pa2A 1qgjA 1fhfB 1fhfB 3hdlA

59 42 56 51 51 65 65 38 57 64 68 45 41 36 100 85 51 68 69 45

10 11 2 7 7 2 4 6 9 10 9 11 9 9 7 7 2 8 8 10

16 17 13 21 21 15 15 17 19 17 18 17 18 20 19 19 6 17 17 18

8 8 11 9 10 9 9 9 11 9 10 9 8 9 9 9 5 10 10 9

4 4 4 2 2 4 4 3 4 4 4 4 4 4 4 4 1 4 4 4

1 5 4 2 1 4 3 3 5 3 5 1 0 2 4 7 2 2 3 1

PM PM PM PM PM PM PM PM – – extract – – – – – PM – PM –

Extra PM Extra Extra Extra Tylakoid ER/PM Extra Extra ER/PM Extra PM Extra PM Extra Extra ER Extra Extra Extra

Secretory Secretory Secretory Chl Chl Secretory Secretory Secretory Secretory Secretory Secretory Secretory Secretory Secretory nuc nuc Secretory Secretory Secretory Secretory

Extra Extra Chl Mito Chl Extra Chl Extra Chl Extra Extra Mito Vacu Golgi Extra Extra Extra Extra Extra Extra

Secretory Secretory Secretory Mito Secretory Secretory Secretory Secretory Secretory Secretory Secretory Secretory Secretory Secretory Secretory Secretory Secretory Secretory Secretory Secretory

ProtParam (http://www.expasy.ch/tools/protparam.html); Structural prediction by Swiss Modell (http://swissmodel.expasy.org/). Prediction of N-glycosylation by NetGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/). Prediction of cellular localisation was done by PSort (http://psort.hgc.jp/form.html). BaCelLO (http://gpcr2.biocomp.unibo.it/bacello/pred.htm). MultiLoc (http://www-bs.informatik.uni-tuebingen.de/Services/MultiLoc/index_html). TargetP (http://www.cbs.dtu.dk/services/TargetP/).

Table 4 Analysis of the topology of putative membrane-bound class III peroxidases in vascular plants. In silico analysis of various membrane-bound peroxidases in terms of occurrence of signal petides and transmembrane domains (TMD) using different prediction models. The accession number was obtained from NCBI or UniProt (⁄). AA, amino acid. Acc. No.

Q6EUS1⁄ Q8L3W2⁄ EF178277⁄ B4FHG3⁄ B4FH68⁄ EF059719⁄ EF059717⁄ B4FG39⁄ XP_002455565 C5X5K3⁄ P24102⁄ O80822⁄ Q9SZH2⁄ O81755⁄ Q42578⁄ Q9FG34⁄ AC123575_10.1 ABO77633.1 BAD97436.1 Q9ZP15⁄ a b c d e f

Protein

OsPrx27 OsPrx95 ZmPrx01 ZmPrx24 ZmPrx58 ZmPrx66 ZmPrx70 ZmPrx81 SbPrx75 SbPrx144 AtPrx22 AtPrx25 AtPrx43 AtPrx48 AtPrx53 AtPrx54 AtPrx69 MtPrx16 PsPrx13 TrPrx02

AA

361 321 367 364 355 320 321 344 371 318 349 328 326 404 335 358 168 354 357 329

Phobiosa

SignalPb

Signal

TM

1–27 1–28 1–33 1–32 1–29 1–25 1–29 1–29 1–35 1–21 1–23 – 1–32 – – – 1–23 1–27 1–24 1–26

– 1 – – – – – – – – – 1 1 1 1 1 – – – –

TMD

TMD TMD TMD TMD TMD

1–27 1–28 1–33 1–32 1–29 1–25 1–29 1–29 1–35 1–21 1–23 1–34 1–32 1–18 1–30 1–31 1–23 1–27 1–27 1–26

SoSui Signalc

TMHMMd

PRED-TMRe

HMMTOPf

1–21 3 TMD 1–33 1–38 1–35 1–29 1–29 1–29 1–33 1–27 1–23 1 TMD 3 TMD 1–28 1 TMD 1 TMD 1–23 1–25 1–25 1–27

0 3 1 1 1 1 0 1 1 1 1 1 2 1 1 1 1 1 0 1

1 2 1 1 1 1 1 1 1 1 1 1 3 2 1 1 1 2 2 1

1 3 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 2 1 1

TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD

TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD

TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD TMD

Phobius (http://phobius.sbc.su.se/). SignalP (http://www.cbs.dtu.dk/services/SignalP/). SoSuiSignal (http://bp.nuap.nagoya-u.ac.jp/sosui/sosuisignal/sosuisignal_submit.html). TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Pred-TMR (http://athina.biol.uoa.gr/PRED-TMR/input.html). HMMTOP (http://www.enzim.hu/hmmtop/index.html).

superfamily 2, e.g. thylakoid-bound ascorbate peroxidases and human TPO (Bianco et al., 2002; Jespersen et al., 1997). Due to the predicted cleavable N-terminal signal peptides, anchoring of class III peroxidases by N-terminal transmembrane domains has to be verified.

5. Structure In peroxidases functions of well-conserved amino-acid residues like haem, substrate, and calcium binding-sites have been studied in the past. Due to these data, the hypothetical structure of mem-

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brane-bound peroxidases could be predicted by comparison with known structures of peroxidases with adequate sequence similarity. Hypothetical three-dimensional (3D) models have been published for CrPrx01, ZmPrx01, ZmPrx66, and ZmPrx70 (Mika et al., 2008; Sottomayor et al., 2008). In silico analysis of the structures of membrane-bound class III peroxidases revealed 13–21 a-helices and between two and 11 b-sheets (Table 3). Conserved amino-acid residues were identified for haem, substrate, and calcium bindingsites. Disulfide-bonds and glycosylation-sites are indicated in Table 3. Although all peroxidases contain between eight and 11 cysteine residues with similar positions in the sequence, predicted models of ZmPrx24, ZmPrx58, and ZmPrx81 revealed less disulfide-bonds compared to the other peroxidases. The structure of these peroxidases may need further elucidation in the future. Membrane-bound maize peroxidases have two to seven b-sheets and 13–21 a-helices (Table 3). Haem, substrate, and calcium binding-sites could be identified in the amino acid sequences. Details of the structure of ZmPrx70 are presented in Fig. 1. Besides

the tertiary structure of the protein, the two substrate channels are shown by the model. The smaller one gives access to the hydrophilic side chains which are able to form hydrogen bonds with substrates. The bigger one presents largely hydrophobic faces due to the delocalised p orbitals with the charged iron at the centre (Gumiero et al., 2010). A modulation of the area around the haem group might hypothetically influence the substrate affinity. Hypothetical tertiary structures were derived for all class III peroxidases identified in plasma membranes of maize roots (Table 1) using the X-ray crystallography coordinates for the known structures of the homologous barley (Hordeum vulgare L.) seed peroxidase BP1 (Henriksen et al., 1997), horseradish peroxidase C (Carlsson et al., 2004; Henriksen et al., 1999), Arabidopsis peroxidase N (Mirza et al., 1999), Arabidopsis peroxidase A2 (Nielsen et al., 2001; Østergaard et al., 1999), soybean peroxidase (Henriksen et al., 2000), glycosylated peroxidase of royal palm tree (Watanabe et al., 2010), and peanut peroxidase (Schuller et al., 1996) using SWISS-MODEL and the Swiss-PDB Viewer (Guex and Peitsch, 1997), which are available at www.expasy.ch/spdbv/. As shown in Fig. 2, structures of the globular proteins are quite similar and substrate channels could be identified. In accordance with their acidic isoelectric points, ZmPrx01, ZmPrx24, and ZmPrx58 showed appreciable negative charges at their surface. In contrast the basic peroxidases ZmPrx66, ZmPrx70, and ZmPrx81 present considerable surface areas with positive charges. The area of positive charges increased with the pI of the isoenzymes. Fig. 3 presents hypothetical structural models for representatives of putative membrane-bound class III peroxidases that have been predicted with a location at the plasma membrane or endomembrane systems. Alignments of these four amino acid sequences confirmed the described analogy and revealed a high amino acid property conservation and alignment quality in homologous regions (Fig. 4). The known structures of glycosylated peroxidase of royal palm tree (Watanabe et al., 2010), Arabidopsis peroxidase N (Mirza et al., 1999), and peanut peroxidase (Schuller et al., 1996) were used as templates for modelling the tertiary structures by PyMOL (http://pymol.org/). All four derived models of ZmPrx02 (tonoplast), ZmPrx08 (Golgi), ZmPrx70 (plasma membrane) and ZmPrx101 (ER) showed high similarities and revealed 14 (ZmPrx08 and ZmPrx70) or 15 (ZmPrx02 and ZmPrx101) a-helices and two b-sheets at almost identical positions, as well as four disulfide-bonds, a typical and conserved element of class III peroxidases (Fig. 3). The overall structure, even though there are differences in the amino acid sequence, remains similar. Slightly changes in a-helix-orientation and more frequent changes of random coil regions are seen. The observed differences of these peroxidases in their tertiary structure may be due to an adaptation on different substrates. The haem group as prosthetic group stays in the centre of all observed 3D-structures accessible through two channels.

6. Protein–protein interaction and organisation in microdomains

Fig. 1. Structural model of ZmPrx70. The hypothetical model was prepared by PyMOL (http://pymol.org/) and presents the structural characteristics of class III peroxidases. The front view (A) shows the typical haem group coloured in light grey, two short b-strands in yellow, 14 a-helices in red and four conserved disulfides in blue. The haem group is connected to the outside by one singular huge and one smaller access channel (B). The putative huge channel facing one of the plan sides of the protoporphyrine ring system with its central iron.

Nowadays it became clear that most physiological processes are carried out by protein assemblies, rather than by single proteins (Alberts, 1998; Engelman, 2005). For example, participation of a soluble extracellular peroxidase in a putative and high molecular mass protein complex has been demonstrated in cowpea (Vigna unguiculata L.) (Fecht-Christoffers et al., 2003; Führs et al., 2009). Putative and high molecular mass protein complexes have been detected in plasma membrane preparations by blue native PAGE (Kjell et al., 2004; Katz et al., 2007). In plasma membranes of maize roots participation of class III peroxidases in protein assemblies was suggested by high resolution clear native PAGE and specific

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Fig. 2. Maize peroxidases 3-D surface renditions. The models were constructed using the X-ray crystallography coordinates of the most homologue peroxidase (http:// swissmodel.expasy.org/). Swiss-Pdb Viewer (Guex and Peitsch, 1997; http://spdbv.vital-it.ch/) was used to calculate the surface and the electrostatic potential. Colours represent surface charge: white, neutral; blue, positive; and red, negative. The channel leading to the centrally located haem can be observed on the left and was marked with an arrow. Front and back view are presented in the middle and on the right column. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

in-gel staining (Lüthje et al., 2009). Size exclusion chromatography and non-reducing SDS–PAGE suggest that ZmPrx01 may be organised as dimers (Mika et al., 2008), whereas pmPOX2a had an unexpected high relative molecular mass of 155 kD (Mika and Lüthje, 2003). A recent investigation suggests a relative molecular mass of 63 kD for the pmPOX2a monomer (Mika et al., 2010). The peroxidase corresponding to this protein band, however, has not been identified so far. The physiological functions of the putative protein complex and interaction partners are still unknown. Proteomic approaches demonstrated the occurrence of glycosylphosphatidylinositol-(GPI)-anchored and transmembrane proteins in detergent-insoluble membranes (DIM) of plants (Peskan et al., 2000; Borner et al., 2003, 2005; Morel et al., 2006). DIM are postulated to be specialised microdomains with specific lipid composition (so-called lipid rafts) that acts as molecular sorting machines capable of co-ordinating the spatiotemporal organisation of signal transduction pathways, transport proteins and others

within selected areas of the plasma membrane (Bhat and Panstrugah, 2005; Martin et al., 2005). Peroxidases identified in maize plasma membranes appeared tightly bound to the membrane. The proteins could not be washed off by different washing procedures and were only partially soluble by Triton X-114 (Mika and Lüthje, 2003). Fig. 5 shows the distribution of maize peroxidases in the supernatant and in the detergent resistant pellet after solubilisation of plasma membranes by nonionic detergents at 4 °C. Occurrence of class III peroxidases in the pellet supports location of these enzymes in microdomains (Lüthje, 2007). As shown in Table 1, a peroxidase precursor has been identified in Triton X-100 insoluble membranes of barrel clover (Medicago truncatula Gaertner) (Lefebvre et al., 2007). Organisation in microdomains has also been speculated for CrPrx01 (Sottomayor et al., 2008). Such microcompartmentation of membrane-bound peroxidases suggests a function in dependence on other specialised membrane proteins.

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Fig. 3. Amino acid sequence alignment of maize peroxidases with a location at the plasma membrane or in endomembrane systems. Amino acid alignment of ZmPrx02 (tonoplast), ZmPrx08 (Golgi), ZmPrx70 (plasma membrane) and ZmPrx101 (ER). The colouring of residues takes place according to their physiochemical properties. Also shown is the grade of amino acid property conservation, the alignment quality (BLOSUM62 score) and the resulting consensus sequence.

Fig. 4. 3D-Structures of maize peroxidases with a location at the plasma membrane or in endomembrane systems. Structural comparison of ZmPrx02 (First row a–c), ZmPrx08 (second row d–f), ZmPrx70 (third row g–i) and ZmPrx101 (fourth row j–l) in front view, side view and top view. All four peroxidases show a very similar secondary and tertiary structure with only small differences in their steric order.

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7M Ur ea

T X-100 * (1:3)

SB-1 2 ( 1:3) SDS (1 :3 )

DoMa* (1 :3)

(1:5) Digitonin *

Solubilisate

Chaps (1

:3)

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170 130 100 70 55 40 35 25 15

DIM

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Fig. 5. Evidence for membrane-bound class III peroxidases in DIM. Plasma membranes were prepared from 5-days old maize roots by aqueous two-phase partitioning (Lüthje et al., 1998). Proteins were solubilised by different detergents at the protein to detergent ratios as indicated in brackets. The solubilisate and the detergent-insoluble membranes (DIM) were separated by non-reducing SDS–PAGE (4–18% acrylamide gradient gels). Class III peroxidases were visualised by guaiacol staining in the presence of H2O2 in sodium acetate buffer, pH 5.5. Pre-stained markers are indicated in the left lane. Non-ionic detergents (⁄): Digitonin, Triton X100, DoMA, dodecylmaltoside, and ionic detergents: SDS, sodium dodecyl sulfate, SB-12, sulfobetanine 12, CHAPS, 3-[(3-cholamido-propyl)-dimethyl-ammonio]-1propanesulfonate.

source of ROS at plant plasma membranes (Simon-Plas et al., 2002). Both Rboh and peroxidases have been identified in DIM (Lefebvre et al., 2007; Morel et al., 2006). Microdomains of plant plasma membranes consist mainly of glycosphingolipids and free sterols (Borner et al., 2005; Laloi et al., 2007; Mongrand et al., 2004). Glycosylceramides contain polyunsaturated and very long chain fatty acids that could be a target for lipid peroxidation during oxidative stress. High amounts of these compounds were detected in the plasma membrane of maize roots (Bohn et al., 2001). As shown in the hypothetical model in Fig. 6, ROS produced by Rboh could be dismutated by apoplastic superoxide dismutase (SOD), whereas H2O2 may be detoxified by extracellular and membrane-bound class III peroxidases. Membrane-bound peroxidases could probably not only detoxify H2O2 directly at the site of origin to guarantee the optimal protection of the membrane, but could also, protect specific functional regions of the membrane (Lüthje, 2007). In contrast to soybean peroxidase (Vianello et al., 1997), ZmPrx01, ZmPrx66, and ZmPrx70 do not use ascorbate as a substrate (Mika and Lüthje, 2003). Although natural substrates have to be elucidated further, hydroquinones or flavonoids have been suggested as substrates for the maize peroxidases (Mika et al., 2004). On one hand, occurrence of NAD(P)H-dependent quinone reductases and a naphtoquinone has been demonstrated in plasma membranes of maize roots (Lüthje et al., 1998). On the other hand, flavonoids are a class of secondary plant phenolics with significant antioxidant and chelating properties, oxidised by plant peroxidases and induced by oxidative stress (Dixon and Paiva, 1995; Mika et al., 2004). Location of CrPrx01 in specific microdomains of the tonoplast together with putative alkaloid transporters has been discussed (Sottomayor et al., 2008). This would allow a coupled alkaloid transport through the tonoplast with oxidation by CrPrx01. The authors further proposed that the substrate H2O2 could be generated by a putative NADPH oxidase co-localised with CrPrx01 and alkaloid transporters in the membrane. Location of Rboh in the tonoplast, however, has not been demonstrated yet. Rboh, NADPH oxidases (NOX 1–5) and dual oxidases (Duox) belong to the flavocytochrome b family (Lambeth et al., 2000; Sumimoto, 2008). A recent study using co-immuno precipitation demonstrated that Duox and TPO locate closely together in plasma membranes of human thyrocytes (Song et al., 2010). This association was modulated by H2O2. Optimisation of working efficiency

POX

2 O2 2 OPOX

7. Function of membrane-bound class III peroxidases Most functions postulated for plasma membrane-bound peroxidases can also be fulfilled by soluble and cell wall-bound extracellular peroxidases (Hiraga et al., 2001; Kawano, 2003; Ros Barceló, 1997; Vianello and Macri, 1991). Thus, it can be speculated that there may be some functions specific for membrane-bound class III peroxidases, i.e. a membrane protective function (De Gara, 2004; Lüthje, 2007; Mika et al., 2004; Song et al., 2010). High amounts of H2O2 are produced intracellular, at the plasma membrane and in the apoplast due to cellular processes, as response to several stress factors, or external sources in plant–pathogen interactions (Bolwell et al., 2002; Jackson and Taylor, 1996; Schraudner et al., 1996; Schützendübel and Polle, 2002). Homologs of the respiratory burst oxidase (Rboh) were suggested as a major

Cu/Zn SOD

out Rboh

in

14-3-3

Rac5

Fig. 6. Hypothetical model of membrane-bound peroxidases interacting with microdomains and ROS producing and scavenging systems. Plasma membranes are organised in microdomains (so-called lipid rafts). Rboh, the major source of ROS, was found to be located in detergent-insoluble membranes and to be regulated by 14-3-3 proteins and GTPase (Rac5). Superoxide dismutase (SOD), apoplastic soluble peroxidases and membrane-bound peroxidases may regulate the level of AOS at the membrane.

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for Duox and minimisation of H2O2 concentration at the membrane by TPO have been suggested for this microcompartmentation. Microcompartmentation and membrane repair mechanisms have been investigated for peroxisomal and thylakoid-bound ascorbate peroxidases in detail. Enzymes responsible for the regeneration of ascorbate (Halliwell–Foyer–Asada cycle) are localised in several compartments including peroxisomes and chloroplasts (Foyer and Noctor, 2009). A location of membrane-bound ascorbate peroxidases outside peroxisomes or glyoxysomes has been postulated to allow scavenging of H2O2 that has leaked from the microbodies (Yamaguchi et al., 1995). The thylakoid-bound ascorbate peroxidase binds to the membrane next to photosystem I (Asada et al., 1996; Kieselbach et al., 2000; Yoshimura et al., 1998). Superoxide anion radicals produced at photosystem I were dismutated by SOD and ascorbate peroxidase scavenges the H2O2. In addition to this system a thylakoidassociated class III peroxidase with high affinity to hydroquinone, possibly plastoquinone, has been detected at the thylakoid membrane (Casano et al., 1999; Laloue et al., 1997; Zapata et al., 1998). Reconstitution experiments with the native enzyme support electron flow from the chloroplastic Ndh complex via plastoquinone to the class III peroxidase at the thylakoid (Casano et al., 2000; Martín et al., 2004). The Ndh complex regulates the redox level of cyclic electron transporters by providing electrons that are removed by the Mehler reaction and the coordinated action of SOD and peroxidase when transporters become over reduced (Zapata et al., 2005). Although substrates may be different for membrane-bound peroxidases, a general function of these enzymes appears to be membrane protection against oxidative damage, which occurred during plant development and biotic or abiotic stress. It may be further speculated, that all membrane systems contain comparable systems for this essential function.

8. Regulation of membrane-bound class III peroxidases The plasma membrane is the outer permeability barrier of the cell. Besides functions in transport processes and signal transduction, the plasma membrane is the first target for stress factors (heavy metals, pathogens, salinity etc.). The thylakoid membrane, however, is the first target in photorespiration. If the hypothesis of a function in oxidative stress for membrane-bound peroxidases is true, these isoenzymes are regulated during plant development and additionally by abiotic or biotic stress factors. Transcript levels or enzyme activities of membrane-bound ascorbate peroxidases of spinach (Spinacea oleracea L.) were not changed in response to high light intensity, drought, salinity, and treatment with methyl viologen or abscisic acid (Yoshimura et al., 2000). In contrast to these class I peroxidases, the activity of the thylakoid-associated class III peroxidase was developmentally regulated and increased under photo-oxidative stress (Casano et al., 1999). The enzyme appears to be involved in leaf senescence and programmed cell death (Zapata et al., 2005). As shown in Fig. 7, the peroxidase profile of plasma membranes isolated from roots of 5-days old maize seedlings is quite different compared to that of 18-days old plants. Instead of the three differentiated spots in the neutral pH range of the 5-days old plants, a single spot appears after 18 days. Plasma membranes of young maize roots showed three peroxidases with a molecular mass between 35 and 40 kDa, while plasma membranes of older plants showed more acidic isoforms with higher molecular masses. In addition neutral isoformes with high molecular masses were detected. This observation suggests a developmentally regulation of plasma membrane-bound class III peroxidases. The identification of

pH 10

5

170 130 100 70 55 40 35 25 15

5 days-old pH 10

5

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18 days-old

Fig. 7. Peroxidase profiles of plasma membranes in dependence on the state of development. Maize plants were grown for 5 days or for 18 days in hydroponic solution in the presence of 100 lM iron. Plasma membranes were prepared from roots by aqueous two-phase partitioning (Lüthje et al., 1998), solubilised by the non-ionic detergent digitonin and separated by 2D-PAGE (IEF: Ampholytes pH 2–11 mixed with pH 9–11 at a ratio of 1–3/non-reducing SDS–PAGE: 4–18% acrylamide gradient gels). Class III peroxidases were visualised as described in Fig. 5. The pH gradient is given on the top, pre-stained molecular mass markers are shown on the left.

ZmPrx01, ZmPrx66 and ZmPrx70 in 5-days old maize seedlings, and the detection of ZmPrx58 in the plasma membrane of 18-days old maize roots further support this hypothesis (Hopff et al., 2010; Mika et al., 2010). Proteomic approaches have been used to investigate the dynamics of the plasma membrane proteome under control and stress conditions. Proteome analysis of plasma membrane preparations of rice demonstrated that OsPrx95 is up-regulated in root tips of a salt sensitive rice cultivar after 4 h, but down-regulated after 8 h of salt stress (Cheng et al., 2009). Differential in-gel electrophoresis (DIGE) experiments demonstrated regulation of several proteins in the plasma membrane of pea (Pisum sativum) roots after elicitation (Meisrimler et al., 2009). Most of these proteins were down-regulated, whereas class III peroxidases were up-regulated by the elicitor. Proteins of iron-deficient plants were differentially regulated in comparison to the control and showed a lower abundance of membrane-bound class III peroxidases. Similar results have been observed for plasma membranes of maize roots by shot-gun proteomics (Hopff et al., 2010). In both species plasma membrane-bound class III peroxidases were up-regulated by chitosan, but the effect was significant lower in iron-deficient plants compared to the control (Meisrimler et al., 2009; Mika et al., 2010). This observation may be one explanation why iron-deficient plants are more susceptible to pathogen infection.

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amount of class III peroxidases appears to have a membrane location. This observation suggests a general function of peroxidases in membrane protection. In contrast to membrane-bound class I peroxidases, membrane-bound class III peroxidases are developmentally and stress regulated. Natural substrates, location and precise functions of plasma membrane-bound peroxidases have to be further elucidated. The biochemical characteristics of membrane-bound class III peroxidases predicted with a location in endomembrane systems have to be investigated in the future. The organisation of peroxidases in putative protein assemblies opens a new field in our understanding on the function of this protein family.

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Acknowledgements We thank François Clement Perrineau (University Hamburg) for reading the manuscript. Work carried out by the authors was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) and the University of Hamburg (Ph.D. student’s grant HmbNFG to C.M. and D.H.). References

130 100 70 55 40 35 25

Fe-toxicity Fig. 8. Peroxidase profiles of plasma membranes in dependence on iron supply. Maize plants were grown for 18 days either in the absence of iron or in the presence of excess iron (500 lM). Plasma membranes were prepared by aqueous two-phase partitioning, solubilised by the non-ionic detergent digitonin and separated by 2DPAGE (IEF: Ampholytes 2–11mixed with 9–11 at a ratio of 1–3/non-reducing SDS– PAGE: 4–18% acrylamide gradient gels). Class III peroxidases were visualised by ingel activity staining as described in Fig. 5. The pH gradient is given on the top, prestained molecular mass markers are shown on the left.

Fig. 8 shows the differences in the peroxidase profiles of plasma membrane preparations isolated from maize plants grown either under iron-deficiency or iron toxicity. In both samples peroxidases with similar properties were found in the acidic pH range. If the difference observed between the two samples are due to post-transcriptional modifications needs further investigation. The change of the molecular mass indicates a possible glycosylation. Other isoformes observed in Fe-deficient samples showed a neutral to acid pI and molecular masses higher than 55 kDa. In contrast neutral to alkaline isoformes with high molecular masses were found under iron toxicity. Shot-gun proteomics of plasma membranes isolated from maize roots suggest that ZmPrx58 and ZmPrx81 were induced or up-regulated under iron toxicity (Hopff et al., 2010). Short term treatment of 5-days old maize roots with several signalling compounds and effectors showed a differential regulation of ZmPrx01, ZmPrx66, ZmPrx70, and pmPOX2a (Mika et al., 2010). Although data on the regulation of these peroxidases at the transcriptional level are not available yet, the data at hand suggest an essential function of plasma membrane-bound class III peroxidases in oxidative stress. 9. Concluding remarks Membrane-bound peroxidases have been identified in all membrane systems investigated. Besides ascorbate peroxidases, a huge

Alberts, B., 1998. The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92, 291–294. Almagro, L., Gómez Ros, L.V., Belchi-Navarro, S., Bru, R., Ros Barceló, A., Pedreño, M.A., 2009. Class III peroxidases in plant defence reactions. J. Exp. Bot. 60, 377– 390. Asada, K., Miyake, C., Ogawa, K., Hossain, M.A., 1996. Microcompartmentation of ascorbate peroxidase and regeneration of ascorbate from ascorbate radical: its dual role in chloroplasts. In: Obinger, C., Burner, U., Ebermann, R., Penel, C., Greppin, H. (Eds.), Proceedings of the IV. International Symposium on Plant Peroxidases: Biochemistry and Physiology. University of Vienna, Austria and University of Geneva, Switzerland, pp. 163–167. Askerlund, P., Larsson, C., Widell, S., Møller, I.M., 1987. NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences. Physiol. Plant. 71, 9–19. Bérczi, A., Asard, H., 2003. Soluble proteins, an often overlooked contaminant in plasma membrane preparations. Trends Plant Sci. 8, 250–251. Bhat, R.A., Panstrugah, R., 2005. Lipid rafts in plants. Planta 223, 5–9. Bianco, A.C., Salvatore, D., Gereben, B., Berry, M.J., Larsen, P.R., 2002. Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr. Rev. 23, 38–89. Blee, K.A., Blee, S.C., Richard, G., Zimmerlin, A., Davies, D.R., Bolwell, G.P., 2001. Molecular identification and expression of the peroxidases responsible for the oxidative burst in French bean (Phaseolus vulgaris L.) and related members of the gene family. Plant Mol. Biol. 47, 607–620. Bohn, M., Heinz, E., Lüthje, S., 2001. Lipid composition and fluidity of plasma membranes isolated from corn (Zea mays L.) roots. Arch. Biochem. Biophys. 387, 35–40. Bolwell, G.P., Bindschedler, L.V., Blee, K.A., Butt, V.S., Davies, D.R., Gardner, S.L., Gerrish, C., Minbayeva, F., 2002. The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J. Exp. Bot. 53, 1367–1376. Borner, G.H.H., Lilley, K.S., Stevens, T.J., Dupree, P., 2003. Identification of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A proteomic and genomic analysis. Plant Physiol. 132, 568–577. Borner, G.H.H., Sherrier, D.J., Weimar, T., Michaelson, L.V., Hawkins, N.D., MacAskill, A., Napier, J.A., Beale, M.H., Lilley, K.S., Dupree, P., 2005. Analysis of detergentresistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts. Plant Physiol. 137, 104–116. Bunkelmann, J., Trelease, R.N., 1996. Ascorbate peroxidase. A prominent membrane protein in oilseed glyoxysomes. Plant Physiol. 110, 589–598. Carlsson, G.H., Nicholls, P., Svistunenko, D., Berglund, G.I., Hajdu, J., 2004. Complexes of horseradish peroxidase with formate, acetate, and carbon monoxide. Biochemistry 44, 635–642. Carpin, S., Crèvecoeur, M., Greppin, H., Penel, C., 1999. Molecular cloning and tissuespecific expression of an anionic peroxidase in zucchini. Plant Physiol. 120, 799–810. Casano, L.M., Martin, M., Zapata, J.M., Sabater, B., 1999. Leaf age- and paraquat dependent effects on the levels of enzymes protecting against photooxidative stress in barley. Plant Sci. 149, 13–22. Casano, L.M., Zapata, J.M., Martin, M., Sabater, B., 2000. Chlororespiration and poising of cyclic electron transport. Plastoquinone as electron transporter between thylakoid NADH dehydrogenase and peroxidase. J. Biol. Chem. 275, 942–948. Cheng, Y., Qi, Y., Zhu, Q., Chen, X., Wang, N., Zhao Chen, H., Cui, X., Xu, L., Zhang, W., 2009. New changes in the plasma-membrane-associated proteome of rice roots under salt stress. Proteomics 9, 3100–3114.

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S. Lüthje et al. / Phytochemistry 72 (2011) 1124–1135

Cosio, C., Dunand, C., 2009. Specific functions of individual class III peroxidase genes. J. Exp. Bot. 60, 391–408. Costa, M.M.R., Hilliou, F., Duarte, P., Pereira, L.G., Almeida, I., Leech, M., Memelink, J., Barcelo, A.R., Sottomayor, M., 2008. Molecular cloning and characterization of a vacuolar class III peroxidases involved in the metabolism of anticancer alkaloids in Catharanthus roseus. Plant Physiol. 146, 403–417. Crevecoeur, M., Pinedo, M., Greppin, H., Penel, C., 1997. Peroxidase activity in shoot apical meristem from spinach. Acta Histochem. 99, 177–186. De Gara, L., 2004. Class III peroxidases and ascorbate metabolism in plants. Phytochem. Rev. 3, 195–205. Díaz, J., Pomar, F., Bernal, Á., Merino, F., 2004. Peroxidases and the metabolism of capsaicin in Capsicum annuum L.. Phytochem. Rev. 3, 141–157. Dixon, R.A., Paiva, N., 1995. Stress-induced phenylpropanoid metabolism. Plant Cell 7, 1085–1097. Duroux, L., Welinder, K.G., 2003. The peroxidase gene family in plants: a phylogenetic overview. J. Mol. Evol. 57, 397–407. Engelman, D.M., 2005. Membranes are more mosaic than fluid. Nature 438, 578– 580. Fecht-Christoffers, M.M., Braun, H.P., Lemaitre-Guillier, C., Van Dorsselaer, A., Horst, W.J., 2003. Effect of manganese toxicity on the proteome of the leaf apoplast in Cowpea. Plant Physiol. 133, 1935–1946. Foyer, C.H., Noctor, G., 2009. Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications. Antioxidants Redox Signal. 11, 861–905. Führs, H., Götz, S., Specht, A., Erban, A., Gallien, S., Heintz, D., Van Dorsselaer, A., Kopka, J., Braun, H.P., Horst, W.J., 2009. Characterization of leaf apoplastic peroxidases and metabolites in Vigna unguiculata in response to toxic manganese supply and silicon. J. Exp. Bot. 60, 1663–1678. Guex, N., Peitsch, M.C., 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714– 2723. Gumiero, A., Murphy, E.J., Metcalfe, C.L., Moody, P.C., Raven, E.L., 2010. An analysis of substrate binding interactions in the heme peroxidase enzymes: a structural perspective. Arch. Biochem. Biophys. 500, 13–20. Henriksen, A., Mirza, O., Indiani, C., Teilum, K., Smulevich, G., Welinder, K.G., Gajhede, M., 2000. Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem–apoprotein interactions. Protein Sci. 10, 108–115. Henriksen, A., Smith, A.T., Gajhede, M., 1999. The structures of the horseradish peroxidase C-ferulic acid complex and the ternary complex with cyanide suggest how peroxidases oxidize small phenolic substrates. J. Biol. Chem. 274, 35005–35011. Henriksen, A., Welinder, K.G., Gajhede, M., 1997. Structure of barley grain peroxidase refined at 1.9-A resolution. A plant peroxidase reversibly inactivated at neutral pH. J. Biol. Chem. 273, 2241–2248. Hynek, R., Svensson, B., Nørregaard Jensen, O., Barkholt, V., Finnie, C., 2009. Enrichment and identification of integral membrane proteins from barley aleuron layers by reverse-phase chromatography, SDS-Page, and LC-MS/Ms. J. Proteome. Res. 5, 3105–3113. Hiraga, S., Sasaki, K., Ito, H., Ohashi, Y., Matsui, H., 2001. A large family of class III plant peroxidases. Plant Cell Physiol. 42, 462–468. Hopff, D., Wienkopf, S., Lüthje, S., 2010. Regulation of the plasma membrane proteome of maize roots due to iron stress as revealed by shotgun proteomics. Proteomlux 2010, October 18th to 20th, 2010, International Conference on Proteomics in Plants, Microorganisms and Environment, Luxembourg, p. 65. Hu, C., Smith, R., Van Huystee, R., 1989. Biosynthesis and localization of peanut peroxidases: a comparison of the cationic and the anionic isozymes. Plant Physiol. 135, 391–397. Ishikawa, T., Yoshimura, K., Sakai, K., Tamoi, M., Takeda, T., Shigeoka, S., 1998. Molecular characterization and physiological role of a glyoxysome-bound ascorbate peroxidase from spinach. Plant Cell Physiol. 39, 23–34. Jackson, A.O., Taylor, C.B., 1996. Plant–microbe interactions: life and death at the interface. Plant Cell 8, 1651–1668. Jespersen, H.M., Kjærsgard, V.H., Østergaard, L., Welinder, K.G., 1997. From sequence analysis of three novel ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase. Biochem. J. 326, 305–310. Katam, R., Basha, S.M., Suravajhala, P., Pechan, T., 2009. Analysis of peanut leaf proteome. J. Proteome Res. 9, 2236–2254. Katz, A., Waridel, P., Shevchenko, A., Pick, U., 2007. Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel electrophoresis and nano-LC-MS/Ms analysis. Mol. Cell Proteomics 7, 1459–1472. Kawano, T., 2003. Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction. Plant Cell Rep. 21, 829–837. Kieselbach, T., Bystedt, M., Hynds, P., Robinson, C., Schroder, W.P., 2000. A peroxidase homologue and novel plastocyanin located by proteomics to the Arabidopsis chloroplast thylakoid lumen. FEBS Lett. 480, 271–276. Kjell, J., Rasmusson, A.G., Larsson, H., Widell, S., 2004. Protein complexes of the plant plasma membrane resolved by Blue Native PAGE. Physiol. Plant. 212, 546–555. Kumar, S., Dutta, A., Sinha, A.K., Sen, J., 2007. Cloning, characterization and localization of a novel basic peroxidase gene from Catharanthus roseus. FEBS J. 274, 1290–1303. Laloi, M., Perret, A.M., Chatre, L., Meiser, S., Cantrel, C., Vaultier, M.N., Zachowski, A., Bathany, K., Schmitter, J.M., Vallet, M., Lessire, R., Hartmann, M.A., Moreau, P., 2007. Insights into the role of specific lipids in the formation and delivery of

lipid microdomains to the plasma membrane of plant cells. Plant Physiol. 143, 461–472. Laloue, H., Weber-Lofti, F., Lucau-Danila, A., Guillemaut, P., 1997. Identification of ascorbate and guaiacol peroxidase in needle chloroplasts of spruce trees. Plant Physiol. Biochem. 35, 341–346. Lambeth, J.D., Cheng, G., Arnold, R.S., Edens, W.A., 2000. Novel homologs of gp91phox. TIBS 25, 459–461. Lefebvre, B., Furt, F., Hartmann, M.A., Michaelson, L.V., Carde, J.P., Sargueil-Boiron, F., Rossignol, M., Napier, J.A., Cullimore, J., Bessoule, J.J., Mongrand, S., 2007. Characterization of lipid rafts from Medicago truncatula root plasma membranes: a proteomic study reveals the presence of a raft–associated redox system. Plant Physiol. 144, 408–418. Lin, W., 1982. Responses of corn root protoplasts to exogenous reduced nicotinamide adenine dinucleotide: oxygen consumption, ion uptake and membrane potential. Proc. Natl. Acad. Sci. USA 79, 3773–3776. Ludwig-Müller, J., Hilgenberg, W., 1992. Tryptophan oxidizing enzyme and basic peroxidase isoenzymes in Arabidopsis thaliana (L.) Heynh.: are they identical? Plant Cell Physiol. 33, 1115–1125. Ludwig-Müller, J., Rausch, T., Lang, S., Hilgenberg, W., 1990. Plasma membranebound high pI peroxidase isoenzymes convert tryptophan to indole-3acetaldoxime. Phytochemistry 29, 1397–1400. Lüthje, S., 2007. Plasma membrane redox systems: lipid rafts and protein assemblies. Prog. Bot. 69, 169–200. Lüthje, S., Hopff, D., Schmitt, A., Meisrimler, C.N., Menckhoff, L., 2009. Hunting for low abundant redox proteins in plant plasma membranes. J. Proteomics 72, 475–483. Lüthje, S., Van Gestelen, P., Córdoba-Pedregosa, M.C., González-Reyes, J.A., Asard, H., Villalba, J.M., Böttger, M., 1998. Quinones in plant plasma membrane – amissing link? Protoplasma 205, 43–51. Marmagne, A., Ferro, M., Meinnel, T., Bruley, C., Kuhn, L., Garin, J., Barbier-Brygoo, H., Ephritikhine, G., 2007. A high content in lipid-modified peripheral proteins and integral receptor kinases features in the Arabidopsis plasma membrane proteome. Mol. Cell Proteomics 6, 1980–1996. Martín, M., Casano, L.M., Zapata, J.M., Guéra, A., Del Campo, E.M., SchmitzLinneweber, C., Maier, R.M., Sabater, B., 2004. Role of thylakoid Ndh complex and peroxidase in the protection against photo-oxidative stress: fluorescence and enzyme activities in wild-type and ndhF-deficient tobacco. Physiol. Plant. 122, 443–452. Martin, S.W., Glover, B.J., Davies, J.M., 2005. Lipid microdomains–plant membranes get organized. TiPS 10, 263–265. McDonald, D.O., Pearce, S.H., 2009. Thyroid peroxidase forms thionamide-sensitive homodimers: relevance for immunomodulation of thyroid autoimmunity. J. Mol. Med. 87, 971–980. Meisrimler, C.N., Renaut, J., Sergeant, K., Lüthje, S., 2009. Regulation of the plasma membrane proteome of iron deficient pea roots after treatment with fungal elicitor. Proteomics. COST FA0603-Meeting, May 5th to 6th, 2009, Classical and Novel Approaches in Plant Proteomics, Viterbo, Italy, p. 10. Mika, A., Boenisch, M.J., Hopff, D., Lüthje, S., 2010. Membrane-bound guaiacol peroxidases are regulated by methyl jasmonate, salicylic acid, and pathogen elicitors. J. Exp. Bot. 61, 831–841. Mika, A., Buck, F., Lüthje, S., 2008. Membrane-bound class III peroxidases: identification, biochemical properties and sequence analysis of isoenzymes purified from maize (Zea mays L.) roots. J. Proteomics 71, 412–424. Mika, A., Lüthje, S., 2003. Properties of guaiacol peroxidase activities isolated from corn root plasma membranes. Plant Physiol. 132, 1489–1498. Mika, A., Minibayeva, F., Beckett, R., Lüthje, S., 2004. Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species. Phytochem. Rev. 3, 173–193. Minic, Z., Jamet, E., Négroni, L., der Garabedian, P.A., Zivy, M., Jouanin, L., 2007. A sub-proteome of Arabidopsis thaliana mature stems trapped on Concanavalin A is enriched in cell wall glycoside hydrolases. J. Exp. Bot. 58, 2503–2512. Mirza, O., Henriksen, A., Østergaard, L., Welinder, K.G., Gajhede, M., 1999. Arabidopsis thaliana peroxidase N: structure of a novel neutral peroxidase. Acta Crystallogr. D: Biol. Crystallogr. 56, 372–375. Möller, S., Croning, M.D.R., Apweiler, R., 2001. Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17, 646–653. Mongrand, S., Morel, J., Laroche, J., Claverol, S., Card, J.P., Hartmann, M.A., Bonneu, M., Simon-PLas, F., Lessire, R., Bessoule, J.-J., 2004. Lipid rafts in higher plant cells. J. Biol. Chem. 279, 36277–36286. Morel, J., Claverol, S., Mongrand, S., Furt, F., Fromentin, J., Bessoule, J.J., Blein, J.P., Simon-Plas, F., 2006. Proteomics of plant detergent-resistant membranes. Mol. Cell Proteomics 5, 1396–1411. Narendra, S., Venkataramani, S., Shen, G., Wang, J., Pasapula, V., Lin, Y., Kornyeyev, D., Holaday, A.S., Zhang, H., 2006. The Arabidopsis ascorbate peroxidase 3 is a peroxisomal membrane-bound antioxidant enzyme and is dispensable for Arabidopsis growth and development. J. Exp. Bot. 57, 3033–3042. Natera, S.H., Ford, K.L., Cassin, A.M., Patterson, J.H., Newbigin, E.J., Bacic, A., 2008. Analysis of the Oryza sativa plasma membrane proteome using combined protein and peptide fractionation approaches in conjunction with mass spectrometry. J. Proteome Res. 7, 1159–1187. Newman, T., De Bruijn, F.J., Green, P., Keegstra, K., Kende, H., McIntosh, L., Ohlrogge, J., Raikhel, N., Somerville, S., Thomashow, M., 1994. Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol. 106, 1241–1255. Nielsen, K.L., Indiani, C., Henriksen, A., Feis, A., Becucci, M., Gajhede, M., Smulevich, G., Welinder, K.G., 2001. Differential activity and structure of highly similar

S. Lüthje et al. / Phytochemistry 72 (2011) 1124–1135 peroxidases. Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase A2 and horseradish peroxidase A2. Biochemistry 40, 11013–11021. Nito, K., Yamaguchi, K., Kondo, M., Hayashi, M., Nishimura, M., 2001. Pumpkin peroxisomal ascorbate peroxidase is localized on peroxisomal membranes and unknown membranous structures. Plant Cell Physiol. 42, 20–27. Østergaard, L., Teilum, K., Mirza, O., Mattsson, O., Petersen, M., Welinder, K.G., Mundy, J., Gajhede, M., Henriksen, A., 1999. Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification. Plant Mol. Biol. 44, 231–243. Pantoja, O., Willmer, C.M., 1988. Redox activity and peroxidase activity associated with the plasma membrane of guard-cell protoplasts. Planta 174, 44–50. Passardi, F., Cosio, C., Penel, C., Dunand, C., 2005. Peroxidases have more functions than a swiss army knife. Plant Cell Rep. 24, 255–265. Passardi, F., Longet, D., Penel, C., Dunand, C., 2004. The class III peroxidase multigenic family in rice and its evolution in land plants. Phytochemistry 65, 1879–1893. Penel, C., Castillo, F.J., 1991. Peroxidases of plant plasma membranes, apoplastic ascorbate, and relation of redox activities to plant pathology. In: Crane, F.L., Morre, D.J., Loew, H. (Eds.), Oxidoreduction at the Plasma Membrane, vol. II. CRC Press, Boca Raton, FL, pp. 121–147. Peskan, T., Westermann, M., Oelmüller, R., 2000. Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants. Eur. J. Biochem. 267, 6989–6995. Ros Barceló, A., 1997. Lignification in plant cell walls. Int. Rev. Cytol. 176, 87–132. Santoni, V., Rouquie, D., Doumas, P., Mansion, M., Boutry, M., Degand, H., Dupree, P., Packman, L., Sherrier, J., Prime, T., Bauw, G., Posada, E., Rouzé, P., Dehais, P., Sahnoun, I., Barlier, I., Rossigniol, M., 1998. Use of a proteome strategy for tagging proteins present at the plasma membrane. Plant J. 16, 633–641. Schraudner, M., Langebartels, C., Sandermann Jr., H., 1996. Plant defence systems and ozone. Biochem. Soc. Trans. 24, 456–461. Schuller, D.J., Ban, N., Huystee, R.B., McPherson, A., Poulos, T.L., 1996. The crystal structure of peanut peroxidase. Structure 4, 311–321. Schützendübel, A., Polle, A., 2002. Plant responses to abiotic stresses: heavy metalinduced oxidative stress and protection by mycorrhization. J. Exp. Bot. 53, 1351–1365. Shigeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T., Yabuta, Y., Yoshimura, K., 2002. Regulation and function of ascorbate peroxidase isoenzymes. J. Exp. Bot. 53, 1305–1319. Simon-Plas, F., Elmayan, T., Blein, J.P., 2002. The plasma membrane oxidase NtrbohD is responsible for AOS production in elicited tobacco cells. Plant J. 31, 137–147. Song, Y., Ruf, J., Lothaire, P., Dequanter, D., Andry, G., Willemse, E., Dumont, J.E., Van Sande, J., De Deken, X., 2010. Association of duoxes with thyroid peroxidase and its regulation in thyrocytes. J. Clin. Endocrinol. Metab. 95, 375–382. Sonnhammer, E.L., von Heijne, G., Krogh, A., 1998. A hidden Markov model for predicting transmembrane helices in protein sequences. In: Proceedings of the Sixth International Conference on Intelligent Systems, Molecular Biology, pp. 175–182.

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Sottomayor, M., Duarte, P., Figueiredo, R., Ros Barceló, A., 2008. A vacuolar class III peroxidase and the metabolism of anticancer indole alkaloids in Catharanthus roseus. Plant Signall. Behav. 3, 899–901. Sottomayor, M., Ros Barceló, A., 2003. Peroxiase from Catharanthus roseus (L.) G. Don and the biosynthesis of a-30 -40 -anhydrovinlastine: a specific role for a multifunctional enzyme. Protoplasma 222, 97–105. Sumimoto, H., 2008. Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species. FEBS J. 275, 3249–3277. Tognolli, M., Penel, C., Greppin, H., Simon, P., 2002. Analysis and expression of the class III peroxidase large gene family in Arabidopsis thaliana. Gene 288, 129– 138. Vianello, A., Macri, F., 1991. Generation of superoxide anion and hydrogen peroxide at the surface of plant cells. J. Bioenerg. Biomembr. 23, 409–423. Vianello, A., Zancani, M., Nagy, G., Macri, F., 1997. Guaiacol peroxidase associated to soybean root plasma membranes oxidizes ascorbate. J. Plant Physiol. 150, 573– 577. Watanabe, L., de Moura, P.R., Bleicher, L., Nascimento, A.S., Zamorano, L.S., Calvete, J.J., Sanz, L., Perez, A., Bursakov, S., Roig, M.G., Shnyrov, V.L., Polikarpov, I., 2010. Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia). J. Struct. Biol. 169, 226–242. Welinder, K.G., 1979. Amino acid sequence studies of horseradish peroxidase. Amino and carboxyl termini, cyanogens bromide and tryptic fragments, the complete sequence, and some structural characteristics of horseradish peroxidase. Eur. J. Biochem. 96, 483–502. Welinder, K.G., Justesen, A.F., Kjaersgard, I.V., Jensen, R.B., Rasmussen, S.K., Jespersen, H.M., Duroux, L., 2002. Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana. Eur. J. Biochem. 269, 6063–6081. Welinder, K.G., Larsen, Y.B., 2004. Covalent structure of soybean seed coat peroxidase. Biochim. Biophys. Acta 1698, 121–126. Yabuta, Y., Motoki, T., Yoshimura, K., Takeda, T., Ishikawa, T., Shigeoka, S., 2002. Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress. Plant J. 32, 915–925. Yamaguchi, K., Mori, H., Nishimura, M., 1995. A novel isoenzyme of ascorbate peroxidase localized on glyoxysomal and leaf peroxisomal membranes in pumpkin. Plant Cell Physiol. 36, 1157–1162. Yoshimura, K., Ishikawa, T., Nakamura, Y., Tamoi, M., Takeda, T., Tada, T., Nishimura, K., Shigeoka, S., 1998. Comparative study on recombinant chloroplastic and cytosolic ascorbate peroxidase isoenzymes of spinach. Arch. Biochem. Biophys. 353, 55–63. Yoshimura, K., Yabuta, Y., Ishikawa, T., Shigeoka, S., 2000. Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stresses. Plant Physiol. 123, 223–233. Zapata, J.M., Guéra, A., Esteban-Carrasco, A., Martín, M., Sabater, B., 2005. Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhFdefective tobacco. Cell Death Different 12, 1277–1284. Zapata, J.M., Sabater, B., Martin, M., 1998. Identification of a thylakoid peroxidase of barley which oxidizes hydroquinone. Phytochemistry 48, 1119–1123.