The peroxidase gene family of <i>Arabidopsis thaliana</i>

Proceedings Chapter

Reference

The peroxidase gene family of Arabidopsis thaliana

SIMON, Patrice, et al.

SIMON, Patrice, et al . The peroxidase gene family of Arabidopsis thaliana . In: Obinger, Christian ; Burner, Ursula ; Ebermann, Robert ; Penel, Claude & Greppin, Hubert. Plant peroxidases: biochemistry and physiology: IV International Symposium 1996:

proceedings . Genève : Université de Genève, Laboratoire de biochimie et physiologie végétales, 1996. p. 179-183

Available at:

http://archive-ouverte.unige.ch/unige:123665

Disclaimer: layout of this document may differ from the published version.

1 / 1

P.

SIMON ET AL

^t79

Plant Peroxidascs: B iochcm istry an d Ph1'siologv, C. Obingcr, U. Burncr,

R

Eberm:rnn, C. Pencl, H. Greppin, cds.

Univcrsity of Gcncva, 1996

TIIE PEROXIDASE GENE

FA1VIILY OF

A RA I}

I

D OPSIS T HAI, IA NA

P. Simon,

N.

Capelli, J. Flach, S.Overney,

M.Tognolli,

C. Penel and

H.

Greppin Laboratorv of Plant Biochemistry and Physiologv. Univcrsit_v of Geneva,

place dc I'Université 3. CH-l2l

I

Genèr'e 4. Stvitzerland

INTRODUCTION

The

classical

guaiacol

peroxidase

in

plants

is

represented

by

numerous

true or

^pseudo-

isozymes, the expression

of which

obeys spatio-temporal

regulation.

There has been interest

tbr a long time to purify

each specific

isotbrm in order to

evâluate

the intrinsic

biochemical

properlies that would

allor,v

a better

assessrnent

of the

putative physiological

rolc of

^thcsc

diverse and omnipresent enzymes.

Efforts to purify

peroxidases are

limited by the

available amounts and can also be bafiled by severe loss occurring during purification. These

eflorts

have therefore often been concentrated on the highly expressed and also purification docile isoforms.

Facing

this kind of difiiculties we

have developped

a

strategy

a few years ago to

obtain

sufficient amount of refractory and minoritary

peroxidases.

The

strategy

we follow is

the isolation

of

^molecular

cDNA

clones and the heterologous expression

in

an appropriate system, such as previously the Xenoptr,s oocytes and now the baculovirus/insect cells. These eukaryotic expression systems are reputated

to

perform effrcient expression and adequate posttranslational modifications.

We were

indeed able

to produce catalytically active and properly

maturated spinach

isoforms in

Xertoptts oocytes

(1,2). We

have

recently

switched

to the

baculovirus system, which

worked

efiiciently in the expression of the synthetic gene of HRP C (3). We have also chosen

the model

plant Arabitlop.si.ç

lhaliana in

the frame

of

a

joint EU-project

and we

have committed ourselves to the

systematic screening

for Arabidopsis cDNAs

encoding isoperoxidases,

which will be used in the next step for

heterologous expression

in

the baculovirus systems. The results obtained from this screening are presented here.

IVTATERIAL AND NIETHODS

In a first step we have

screened

a cDNA library

available

at the ABRC

(Arabidopsis Biological Resource Center at the Ohio State University), the

cDNA

library

of

T. Newman. The

library

was screened

with the

same probe derived

from the

conserved catalytical site

that

we uscd succcssfully

with

spinach (unpublishcd rcsults).

With thc

suddcn explosion

of EST

clones (from the Expressed Sequence Tag sequencing projects) and their availahility at the

ABRC

and also

from

individual French Labs,

we

also searched

for

peroxidase

EST

clones

with

available computer program facilities at the Center server

(4). A virtual

screening

of

the

EST

databases

with

the conserved catalytical site probe yielded partially the same results. The candidates were sequenced manually

with

Sequenase

(Amersham).

_Sequence

data were

analyzed

with

the programs of the Wisconsin software package (5).

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Figure l'

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I

new peroxidases and

of

the previously known peroxidases in Arabidopsis. Expfanations in the text.

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P.

SIMON ET AL. l8l

RESULTS AND DISCUSSION Arabidopsis

sequences

Partial screening of the

cDNA library

yielded about 50 positive clones. Around

thirty

clones were clearly identified as peroxidases by partial sequencing at the 5' end and could be àssigned

to five

different peroxidases. One

of

them revealed

to

be a cloning artefact and another one

to

an already ktrowtt Arubiùupsrs gene. Three representative clones

of the

remaining types have been completely sequenced. More than 150 clones were found in thc databank non exhaustive

Atprxrl

ÀLprxr5 À[prxca

Atprxcb

ÀLprxr 3

At preca

^LprxrS Atprxrl I

AE prxr2

Àtprxr?

At.prxr 9

AEprxrl0

Acprxr ⁴

At.prxr 5

Figure

2. Level

of

similarity between the Arabidopsis peroxidases.

screening, from

which it

was possible

to

select

l4

new peroxidases.

Eight of

these clones have been sequenced.

In

summary,

l9

different peroxidase clones, clearly

distinct from

each other and representing not only allelic variants, were retrieved from this cloning abundancy, and

l l of

them have been now

fully

sequenced. The alignment

of the 1l

encoded àminoacid iequences is shown in

figure

1 together

with

the three already known Arabidopsis sequences

(

6). Region

of similarity are boxed. The

naming

of the

sequences

is arbitrary, r

stands

for RNA

and the numbering reflects

the

chronological

order of

determination.

A

peroxidaser

expert

eye

will

easily recognize the typically conserved residues, i.e. the 8 Cys involved

in

disulfidà bridges, the

proximal

and

distal His

residues

of the catalytical

site, and

the Arg and Asp residuis

also involved in catalysis, all marked

with

a triangle (7). The preclicted signal pcptide cleavage site is indicated by a

dot. Two

of the eleven peroxidases have a C-terminal extension ancl

theil.orrog.

site

has been estimated

by

analogy

to

experimentally determined _cases (see

7) and is alio

punctuated

by a dot. The

degree

of

homology between

the

various Arabidopsis peroxidases ranges

from

3 loÂ

to

89

%

identity (52

to

92 ^o similarity), excluding the close relation between

Atprxca and Atpxcb

peroxidases.

The level of similarity is illustrated in Figure 2.

This relatively

low

degree

of

similarity between various peroxidases from the same plant has already been observed in spinach

(l)

and can be related

to

random cloning and analysisin opposition to

182 GENETICS

s

16s

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oprxr2 Hs

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rxca cb Àt

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LetaDl Stoxà

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0

c ^{atl E}

lNsanper

Brtp 1a

Lu f lxp

rl

23 14

Figure

3. Evolutionary _tree

of

plant peroxidases. The names are the entry names at the

EMBL

databank,

with

the exception

of

Arhrpa2 and Arhrpe5

which

are not

yet

available

in

databanks

and BrtpT which

corresponds

to ^Per_brara in Swissprot. Arabidopsis

peroxidases are designated by a triangle.

P. SIMON

ET AL

¹⁸³

selective cloning, that uses a probe that preferentially detects closely related sequences (8). The calculated

pls are all basic, and reach a value of ^{10.7 for} Atprxl0. One to 7

potential glycosylation sites are found.

Phylogeny

The

evolutionary relationships

with

available peroxidases

from other

plants

is

shown

in

a

phylogenic tree (Figure

3).

The

tree is only

approximate since

not all

sequences are complete and thcrcforc biascd

thc

calculations. Nine sequences were nevertheless omitted because

of

the excessive

size of the missing fragments. It is apparent from this tree that

Arabidopsis peroxidases, besides a Brassicaceae cluster, are disseminated among the various branches

with other plant

peroxidases, independently

of the taxonomic relation. This is best

illustrated by

Atpxrl

^which shows a clear cut high degree

of

similarity

to

a cotton peroxidase, isolated in the

top of the

tree.

This

situation

with

plant peroxidases was already depicted

in

previous works (1,9). The grouping

of

various peroxidases irrespective

of

the taxonomic relations derives most

likely from

some

functional

specificities rather

than

random molecular

variation on a

theme.

More

refined phylogenic analysis is needed

to

strengthen this hypothesis.

CONCLUSIONS AND PERSPECTIVES

Molecular analysis of the Arabidopsis

cDNA

clones has shown a great variety olperoxidases isozymes

in this model plant. It is

questionable r,vhether each

single

isozynrc

is linksd to

^a

specific peroxidative ftrnction. We rather think that the situation we obselve is lhe result of both intertwined redondant diversification and functional speciation.

With

all the available clones, we

are now

headed

towards the

heterologous expression

of the

recombinant

proteins,

the biochemical analysis

of

the active enzymes and

the

spatio-temporal expression analysis

of

the genes.

A

better understanding

of

peroxidase diversity should soon emanate from this work.

Acknnou,legrnents. Wc arc thankful to thc ABRC at the Ohio SLate Universitr.and to Dr T. Ncu'man at the MSU for providing the Est cloucs. This rcsearch is part of a joint EU-proiect (AfR2-CT9:l-1661) and is supportcd by the Sl iss Federal Ofhce of Science and Education (grânt OFES 93.0090).

îrtote addcd in ^prooJ, The

Atprxrl

to Atprxrl

I

sequcnccs are deposited

in

the EMBL database under the accession numbcrs X983l3 to X98323. respectir.ely.

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