Tài liệu Báo cáo Y học: Structural diversity and transcription of class III peroxidases from

Structural diversity and transcription of class III peroxidases from

Arabidopsis thaliana

Karen G. Welinder1,2, Annemarie F. Justesen1

, Inger V. H. Kjærsga˚rd1

, Rikke B. Jensen1

,

Søren K. Rasmussen3

, Hans M. Jespersen1 and Laurent Duroux2

1

Department of Protein Chemistry, University of Copenhagen, Denmark; 2

Department of Biotechnology, Aalborg University,

Denmark; 3

Plant Genetics, Risø National Laboratory, Denmark

Understanding peroxidase function in plants is complicated

by the lack of substrate specificity, the high number of genes,

their diversity in structure and our limited knowledge of

peroxidase gene transcription and translation. In the present

study we sequenced expressed sequence tags (ESTs) enco￾ding novel heme-containing class III peroxidases from

Arabidopsis thaliana and annotated 73 full-length genes

identified in the genome. In total, transcripts of 58 of these

genes have now been observed. The expression of individual

peroxidase genes was assessed in organ-specific EST libraries

and compared to the expression of 33 peroxidase genes

which we analyzed in whole plants 3, 6, 15, 35 and 59 days

after sowing. Expression was assessed in root, rosette leaf,

stem, cauline leaf, flower bud and cell culture tissues using

the gene-specific and highly sensitive reverse transcriptase￾polymerase chain reaction (RT-PCR).We predicted that 71

genes could yield stable proteins folded similarly to horse￾radish peroxidase (HRP). The putative mature peroxidases

derived from these genes showed 28–94% amino acid

sequence identity and were all targeted to the endoplasmic

reticulum by N-terminal signal peptides. In 20 peroxidases

these signal peptides were followed by various N-terminal

extensions of unknown function which are not present in

HRP. Ten peroxidases showed a C-terminal extension

indicating vacuolar targeting. We found that the majority of

peroxidase genes were expressed in root. In total, class III

peroxidases accounted for an impressive 2.2% of root ESTs.

Rather few peroxidases showed organ specificity. Most

importantly, genes expressed constitutively in all organs and

genes with a preference for root represented structurally

diverse peroxidases (< 70% sequence identity). Further￾more, genes appearing in tandem showed distinct express￾ion profiles. The alignment of 73 Arabidopsis peroxidase

sequences provides an easy access to the identification of

orthologous peroxidases in other plant species and will

provide a common platform for combining knowledge of

peroxidase structure and function relationships obtained in

various species.

Keywords: EST; expression analysis by RT-PCR; peroxi￾dase gene annotation; peroxidase structure; propeptides.

Peroxidase enzymes have challenged chemists and biologists

for more than 70 years and have been used in a great

number of analytical applications [1]. The majority of

peroxidases contain an extractable heme (Fe3+ protopor￾phyrin IX) center, whereas others contain a cytochrome c

type heme, a selenium center or a vanadium center.

Peroxidases react first with a peroxide to yield highly

oxidizing intermediates with redox potentials up to

1000 mV and thereafter with a variety of organic or

inorganic reducing substrates, which are often oxidized to

form radicals. Peroxidase activity was detected early in

horseradish roots (reviewed in [1]), which is still the major

source of commercial heme peroxidases. In addition,

peroxidases have been isolated from a variety of plant,

animal, fungal and bacterial sources. The bacterium

Escherichia coli expresses a single intracellular heme peroxi￾dase with dual catalase–peroxidase activities [2], a finding

confirmed by its genome sequence [3]. Mitochondrial yeast

cytochrome c peroxidase, chloroplast and cytosol plant

ascorbate peroxidases are rather similar in amino acid

sequence to the bacterial enzymes, and they are collectively

referred to as class I peroxidases [4]. These intracellular

peroxidases appear to function as protective peroxide

scavengers and they constitute in plants a small family of

7–10 genes, encoding both soluble and membrane bound

enzymes [5]. White-rot fungi like Phanerochaete chrysospo￾rium and Trametes versicolor contain a small gene family

encoding approximately 10 different lignin-degrading or

Mn-dependent heme peroxidases. In contrast, the ink cap

fungus Coprinus cinereus contains only a single peroxidase

gene [6,7]. The extracellular fungal peroxidases (class II) can

participate in secondary metabolism under conditions of

limited nutritional supply [8]. The classical plant peroxidases

(class III) are targeted via the endoplasmic reticulum (ER)

to the outside of the plant cell or to the vacuole. They are

Correspondence to K. G. Welinder, Department of Biotechnology,

Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg,

Denmark. Fax: + 45 98141808, Tel.: + 45 96358467,

E-mail: [email protected]

Abbreviations: AtP, transcribed A. thaliana (class III) peroxidase;

BP, barley peroxidase; dbEST, database of ESTs; ef-1a, elongation

factor-1a; EST, expressed sequence tag; HRP, horseradish

peroxidase; SBP, soybean peroxidase; TC, tentative consensus.

Notes: Equal contributions were made to this work by A. F. J., L. D.

and H. M. J. The GenBank accession numbers for the nucleotide

sequence data produced are listed in Table 1.

(Received 19 August 2002, revised 8 October 2002,

accepted 15 October 2002)

Eur. J. Biochem. 269, 6063–6081 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03311.x