Plant Oxygenases, Peroxidases
and Oxidases
Bioenergetics Group Colloquium Organized and Edited by J. Bowyer (Royal Holloway and Bedford N e w
College). 64 I s t Meeting held at Royal Holloway and Bedford N e w College, 17-20 December I99 I
Plant peroxidase gene expression and function
P. E. Kolattukudy, Royce Mohan," M. Aslam Bajar and Bruce A. Sherf
Ohio State Biotechnology Center and "Biochemistry Program, The Ohio State University, Columbus, Ohio 432 10,
U.S.A.
Flg. I
Peroxidases constitute an enzyme family that
displays tremendous diversity in form, function and
distribution throughout the plant kingdom [ 1 I.
Kecent studies on certain cell wall-associated
anionic peroxidases strongly suggest that they are
involved in cell wall fortification for defence
purposes [Z, 31. In this paper we will confine our
discussion to these recent findings.
I3C-n.m.r. (CP/MAS) spectra of untreated and
LiAIH,-reduced suberin from the wound-periderm of
potato tubers
Exhaustive reductive depolymerization could only remove the
aliphatic components (lower spectrum) leaving the aromatics
still attached to the wall carbohydrates
CH,O, CHOC,
CHOH. CH-CH
Cell wall-associated phenolics
One of the important functions of anionic peroxidases is to catalyse cross-linking and polymerization of cell wall-associated phenolic polymers [4].
1,ignin deposited in the walls and concentrated in
the middle lamella, is a well-known polymer composed of aromatic monomers IS]. Deposition of
phenolics on cell walls occur in many other contexts regulated by developmental and environmental factors [6]. For example, under conditions
that require reinforcement of cell walls, phenolics
somewhat similar to lignin are deposited on the
plasma membrane side of the cell wall. To this
phenolic matrix a polyester domain composed of
w-hydroxy fatty acids, the corresponding dicarboxylic acids, and some epoxy, di-, and trihydroxy acids, are attached to generate the
hydrophobic polymer, suberin [7]. Soluble waxes
are deposited with suberin to make the cell wall an
effective barrier to diffusion. Solid-state n.m.r. helps
us to understand the nature of this extremely complex cell wall-attached polymer [XI, which cannot
be separated from the wall (Fig. 1). Recent n.m.r.
studies showed that 50% of the suberized potato
periderm was composed of carbohydrates [O], confirming similar conclusions previously reached by
Abbreviations used: AHA. abscisic acid; P-GUS,
glucuronidase; nos. nopaline synthase gene.
p-
333
-
Untreated
r
240 220 200 180 160 140 120 100 80
%
60
40
20
tedious chemical analysis [ 61. The chemical nature
of phenolic-containing materials deposited on plant
cell walls under different conditions. such as when
attacked by fungi, is not understood well enough to
allow classification as lignin or suberin. Recently,
combined g.l.c./m.s. analysis of depolymerization
products from the polymeric material deposited in
tomato vascular tissues as a result of Verticillium
albo-atrum attack revealed that fungal attack
resulted in suberization [ 101. The phenolic
materials deposited on the walls may not always fit
I992
Biochemical Society Transactions
the description of lignin or suberin. IIowever, cell
wall-associated peroxidases may be involved in
their deposition.
334
Lignin-forming peroxidases
An anionic peroxidase gene from tobacco implicated in lignification was cloned [ 31, engineered for
overexpression and subsequently reintroduced into
tobacco 1111. A selected transgenic plant was
demonstrated to express the anionic peroxidase at
levels 1 0-fold over the non-transformed controls,
resulting in a concomitant twofold increase in lignin
content in stem and leaf tissues and a perplexing
phenotype of flowering-induced plant wilting. On
the other hand, lignin level was not affected in transgenic tobacco plants with a suppressed level of the
peroxidase.
Anionic peroxidases for suberization
We have postulated that the process of suberization
is regulated, in part, by the action of a cell wallassociated anionic peroxidase [ 121. An anionic peroxidase (PI 3.1 5) was purified from suberizing
potato tuber tissues [ 13 I and a cDNA encoding this
protein was cloned [ 14J. Using the potato cDNA as
a probe, two highly homologous tomato anionic
peroxidase genes (tapl and tap2) were subsequently cloned and characterized [ 151. Numerous
lines of evidence implicate a direct role for this
anionic peroxidase in the process of localized suberization of plant tissues responding to wounding or
pathogen assault. Appearance of anionic peroxidase
activity in wound-healing potato tuber tissues was
correlated with suberization I 161. Thorough washing of potato tuber discs inhibited suberization,
implicating a wound-generated molecular signal
that triggers suberization [ 171. The inhibition of
induction of anionic peroxidase activity and suberization could be reversed by application of exogenous abscisic acid (AHA) to the washed tissues.
Suberization and anionic peroxidase activity can
also be induced in potato callus cultures by AHA
[ 161. Suberization and anionic peroxidase gene activation were both severely reduced when potato
tuber discs were incubated in an atmosphere of
10% CO1 (R. Mohan, 1’. E. Kolattukudy, R. A. Boyd
& C. G. 1,aties. unpublished work). Fez+ deficiency
in bean plants caused a dramatic decrease in
anionic peroxidase activity and a concomitant
decrease in suberization of the roots [ 181. Addition
of Fe’+ to these plants restored the level of the
anionic peroxidase and suberization. Anionic peroxidase has been immunocytochemically localized
t o the walls of suberizing potato tissues 1191. Addi-
Volume 20
tionally, anionic peroxidase transcripts were
detected only during suberization of wound-healing
tissues of potato tubers and young tomato fruits
[15], and localized by in situ hybridization tech,niques using -”S-labelled anti-sense KNA, to the
wound-healing cells of suberizing potato tissues (R.
Mohan & P. E. Kolattukudy, unpublished work).
Regulation of anionic peroxidase gene
expression
Despite the existence of highly anionic peroxidase
isoenzymes in tobacco [ 3, 201, their low sequence
identity to the tomato anionic peroxidases precludes
their cross-hybridization during northern and
Southern analyses. Therefore, tobacco presents
itself as an ideal host for studying the regulation
patterns of tapl and tap2. The entire TAP-1 gene
was introduced into tobacco via the Agrobacteriummediated transformation method. Analysis of KNAs
from control and wound-healing tissues of transgenic tobacco plants verified that tapl expression is
equally responsive to wound induction in tobacco
as in tomato (M. A. Hajar & 1’. E. Kolattukudy,
unpublished work).
These results encouraged us to investigate the
individual expression patterns of tap1 and tap2 in
tobacco. The 5’-flanking region including the promoter and putative leader peptide sequences of tupf
(Fig. 2) was fused to the /?-glucuronidase (/?-GUS)
reporter gene and similarly a 1.56 kb fragment
derived from the 5’ end of tap2 was fused to the
@-GUS gene, and individually introduced into
tobacco. Tobacco plants transformed with the t a p / /
@-GUS construct activated the expression, upon
wounding, of the /?-GUS gene in cells immediately
surrounding the wound site (K. Mohan & 1’. E.
Kolattukudy, unpublished work). A temporal study
of /?-GUS gene activation in the wound-healing
transgenic leaf tissues showed a biphasic induction
profile. The early phase of induction could possibly
Structure of the toplOGUS construct introduced into
tobacco
The tapl sequences containing the promoter region (462 bp)
and coding sequences for a 74 amino acid putative leader
sequence and 9 amino acids of the mature protein were translationally fused t o the coding sequences of the /$GUS gene and
introdciced into tobacco.
putative 74 aa leader
Plant Oxygenases, Peroxidases and Oxidases
reflect cross-linking of pre-existing phenolics and
wall-phenolic deposition produced soon after
wounding, while the later phase might represent the
final stages of suberization. tapd//?-GUS transgenic
plants also responded to wounding by activating
P-GUS expression (K.Mohan & P. E. Kolattukudy,
unpublished work), indicating that both tapl and
tap2 are responsive to wound stress in vzvo.
Defence role for anionic peroxidase
gene expression
Comparison of the responses of two near-isogenic
tomato lines that differ in their resistance to V. alboatrum infection showed that upon petiole inoculation the resistant line deposited greater amounts of
vascular suberin. The resistance reaction was associated with the timely appearance of a high level of
the anionic peroxidase transcripts, whereas in the
near-isogenic susceptible line, fungal inoculation
activated the anionic peroxidase gene later and to a
much lower extent I 101. Such differential responses
between the resistant and susceptible tomato lines
were also evident when fruits of these plants were
wounded 1221. The differential anionic peroxidase
response could also be demonstrated in cell
cultures derived from the near-isogenic tomato lines
upon incubation with fungal wall preparations
(elicitors) of V. albo-atrum 1221. Cell suspension
cultures derived from the resistant line responded
to low amounts (ng of glucose equivalents/nd) of
fungal elicitor by inducing the anionic peroxidase
transcripts, whereas the susceptible line failed to
respond a t such levels, although the response could
be elicited with high levels (pg/niI) of the fungal
elicitor.
To test whether tobacco plants transformed
with the tapl /P-GUS construct could also respond
to infection by phytopathogenic fungi, transgenic
tobacco leaves were incubated with zoospores of
I’hytophthora parasitica. /?-GUS activity was induced
in the infected tissues, and this activity was localized
to the primary infection sites by staining with
X-Gluc. As the fungal infection spread P-GUS
expression was detected in the advancing lesion
areas (R. Mohan & P. E. Kolattukudy, unpublished
work). These observations are consistent with the
hypothesis that the physiological responses affecting host resistance occur within the cell populations
immediately preceeding infection zones [ 231.
The timely induction of anionic peroxidase for
cross-linking of phenolics could reinforce the plant
cell walls [6], thereby making them recalcitrant to
degradation by fungal hydrolytic enzymes known to
be produced during the pathogen ingress [24]. If
the anionic peroxidase catalyses such cross-linking,
plants expressing antisense transcripts for the
anionic peroxidase might be unable to mount the
defence barrier when wounded or attacked by
pathogens. T o ensure that both tap genes are suppressed, a part of the tap2 5‘-sequence fused tail-tohead to a part of DNA from the 5’-end of tapf was
cloned in an antisense orientation after a two CaMV
35s transcriptional enhancer (Fig. 3). Th‘IS construct
with a nopaline synthase (nos) terminator, was
introduced into tomato and potato plants via Agrobacterium-mediated transformation.
Northern analysis of the putative transformants identified several tomato and potato plants
expressing very high levels of the anti( taplItap2)
transcript. Extended northern and Southern
analyses have been performed on ;I single transgenic tomato plant selected for its high expression
of the antisense transcript and homozygous genotype for second-generation self-fertilized (S?)
progeny. Analysis of total KNAs extracted from
various tissues has confirmed a high, but somewhat
variable, expression level for the antisense transcript
in root, stem, leaf and green and red tomato fruits.
Southern analysis revealed insertion of the introduced anti( taplItap2) gene at two, unlinked, loca-
Structure of pSIN:aTI/TZ construct
Sequences coriesponding to a portion of the 5’-domain of exon-l from both tap1 and
tap2 were fused and cloned as a chimeric antisense gene behind tandem CaMV 355
enhancer elements The Agrobocterrum-to-plantshuttle vector employed i s pBlN I 9 [25]
-
I992
335
Biochemical Society Transactions
tions within the genome and, in contrast with
tomato cv. Castlemart [ 151, the genome of cv. Hetter
Hoy contains only one anionic peroxidase gene
ability to directly test the proposed function of
tomato and potato anionic peroxidases in mediating
the process of induced suberization.
(tap).
336
Several transgenic plants selected from each
group have subsequently been screened for their
ability to effectively block induced expression of
their respective native anionic peroxidase genes.
When these transgenic plants were treated with
either AHA or 1/. albo-utrum conidia, the anionic
peroxidase transcripts could not be detected by
northern blot analysis, whereas the transcripts were
readily detected in similarly treated untransformed
plants. The anionic proteins extracted from the
transgenic anti( tupl/tup2) tomato and control plants
were separated on 0% (w/v) acrylamide acid-gels
(pl I 5.0). Peroxidase-positive proteins were visualized as blue bands following staining for activity
with 4-chloro- 1-naphthol/I IIOL.Activity staining of
protein extracts from conidia- and AHA-treated
petioles and wounded fruits demonstrated that the
anionic peroxidase rvas absent in selected anti(tap//
tap2)-expressing plants, whereas the untransformed
tomato plants showed a single intensely stained
protein corresponding to anionic peroxidase.
Anionic peroxidases most probably play a
defensive role in other systems. Treatment of
soybean cotyledons by wounding or application of
fungal elicitor has recently demonstrated increased
expression of anionic peroxidase and a rapid deposition of wall-bound phenolic polymers [26]. A
pathogen-induced putative anionic peroxidase (PI
ca. 5.7) cDNA from wheat leaves infected with
Erysiphe graminis was recently cloned and
sequenced [27]. The presence of a characteristic
eukaryotic signal peptide suggests that this putative
peroxidase is secreted. An anionic peroxidase (PI
4.1) from the seed coat of soybean, with a postulated role in providing a barrier to protect the
enclosed embryo, was recently purified and found
to be induced 20 days post anthesis [2X]. Two
defence-related anionic peroxidase isoenzymes (PI
3.5 and 3.7) from tobacco associated with the cell
wall have been found to be induced systemically
upon inoculation of the plant with either tobacco
mosaic virus or Peronosporu tubucina [20].
The role of tomato and potato anionic peroxidases in defence is well founded ([ 10, 22, 291; R.
Mohan & P. E. Kolattukudy, unpublished work).
The unequivocal function of these enzymes, however, has yet to be determined. W e are now in a
position to critically examine the participatory role
of anionic peroxidases in the overall mechanism of
induced host-defence responses, as well as the
Volume 20
This work was supported by the NSP grant 1)CH
xx1900x.
1. Gaspar, T., I’enel. C. I,.. Thorpc. T‘. Ct (;reppin> I I.
(10x2) I’eroxidases: A Survey of Their 13iochemical
and I’hysiological Koles in Higher I’lants, I Iniversity
of Geneva Press, Geneva
2. Kolattukudy. 1’. E. (1980) Science 208, 000- 1000
3. Lagrimini, I,. M., Hurkhart, W., Moyer, M. &
Kothstein. S. (10x7) I’roc. Natl. Acad. Sci. I I.S.A. 84.
7542-7546
4. van lluystee. K. 13. (10x7) Annu. Rev. Plant I’hysiol.
38,205-2 17
5. Grisehach. f 1. (10x1) in The Hiochemistry of I’lants
(Stumpf, 1’. K. & Conn, E. I<., eds.), vol. 7, pp.
457-478, Academic Press, New York
6. Kolattukudy, 1’. 13. (10x1) Annu. Rev. I’lant I’hysiol.
32, 530-567
7. Kolattukudy, 1’. E. (10x4) Cali.J. I h t . 62, 201X-20.33
X. Kolattukudy. 1’. R. & Espelie, K. I:. (10XO) in Natural
Products of Woody I’lants (Kowe. J., cd.). vol. 1, pp.
304-307, Springer-Verlag I’ublishers, I leidelberg
0. Garhow, J. K., Ferrantello, I,. M. & Stark, R. 1.: (10x0)
Plant I’hysiol. 90. 783-787
10. Kohh, J., Lee, S. W., Mohan, K.& Kolattukudy, 1’. 13.
(1001) Plant I’hysiol. 97. 528-5.30
11. Lagrimini, I,. M., Ihdford, S. 81 Kothstein, S.(1000)
Plant Cell 2. 7- 1X
12. Kolattukudy, 1’. 1;. (1087) in The Ih-heniistry of
I’lants (Stumpf, 1’. K. & Conn, 13. R.. eds.), vol. (Iq pp.
2(J 1-3 14, Academic I’rcss, New York
13. Rspelie. K. E. & Kolattukudy, 1’. E. (1085) Arch.
Biochem. Hiophys. 240,539-545
14. Roberts, E., Kutchan. T. & Kolattukudy, 1’. E. (IOXX)
Want Mol. Hiol. 11. 15-26
1s. Koherts, E. & Kolattukudy, 1’. E. (10XO) Mol. <;en.
Genet. 217,223-231
If). Cottle, W. & Kolattukudy, 1’. E. (1982) Plant I’hysiol.
70,775-780
17. Soliday. C. I,., 1)ean. 1% €3. & Kolattukudy. 1’. I:.
(197X) Plant I’hysiol. 61, 170-174
18. Sijmons. 1’. C.. Kolattukudy. 1’. E. & Kenfait, 11. 1:.
(1985) Plant Physiol. 78, 115-120
10. Espelie, K. E., Franceschi, V. K.& Kolattukudy. 1’. E.
(1986) Plant I’hysiol. 81, 487-402
20. Ye. X. S., Pan, S. Q. & Kui.. J. (1000) I’hysiol.
Hiochem. 80. 1295-1299
2 1. Reference deleted
22. Mohan, R. & Kolattukudy, P. E. (1990) Plant I’hysiol.
92.27h-280
23. Graham, M. Y. & Graham. T. I,. (1001) Mol.
Plant-Microbiol. Interact. 4, 41 5-422
24. Keon. J. 1’. K., Hyrde, K. J. W. & Cooper, K. M. (1987)
in Fungal Infection of Plants (I’egg. G. F. & Ayres,
Plant Oxygenases, Peroxidases and Oxidases
1’. (;., cds.). pp. 1.33-157. Cambridge llniversity
I’rcss. Cambridge
25. Ikvan, hl. (1084) Nucleic Acids lies. 12. 871 1-8721
20. (;rahani. R I . Y.K: Graham. T. I,. (1901) I’lant I’hysiol.
97,1445-145s
27. lirbmann. (;., llcrtig. C.. Hull, J.. Xlauch. F.81 1)udler.
I<. (I001) I’lant Rlol. Ijiol. 16, 320-33 1
28. (;illikiii. J. W. K: Graham. J. S. (1001) I’lant I’hysiiol.
96. 2 14-220
20. Kolattukudy. 1’. E., I’odila. G. K., Shcrf, 1%. A., lhjar,
M. A. 81 Mohan. K.(1001) in Advances in Molecular
Genetics of I’lant-Microbe Interactions (1 ienneckc,
H. 81 Verma, I). 1’. S., eds.). pp. 242-240. Kluwcr
Academic I’ublishers. lklgium
licccivcd 18 I )ecembcr 100 1
~
Structure of plant and fungal peroxidases
Karen G. Welinder,’ J. Matthew MauroZand Leif N0rskov-Lauritsen3
‘Institute of Biochemical Genetics, University of Copenhagen, Bster Farimagsgade 2A, DK- I353 Copenhagen K,
Denmark, 2Center for Advanced Research in Biotechnology,9600 Gudelsky Drive, Rockville MD 20850, U.S.A. and
jNovo Nordisk NS, Novo Alle, 2880 Bagsvzrd. Denmark
Introduction
In the last few years R great number of new
sequences for haem-containing peroxidases (EC
1. I 1.1 .) have ;ippeared. Analyses of these sequences
have shown that peroxidases from plants, fungi, and
bacteria are structurally related and belong to the
plant peroxidase superfamily I 1-51, Ilaeni-containing peroxidases of ;inimal origin such as eosinophil,
thyro-, lacto-, and niyelo-peroxidases constitute a
separate superfamily (animal peroxidase superfamily) unrelated in structure to the plant peroxidases. The animal peroxidases appear to contain
;i
haem-binding domain similar to residues
200-5 10 of prostaglandin endoperoxide synthetase
121.
Several aspects of plant peroxidases M ere
recently reviewed 10, 71, including the kinetics of
horseradish peroxidase [ 81, the properties of yeast
cytochrome c peroxidase (CCP) [ 91, and the structure-function relationships of plant peroxidases [ 51.
‘I’he studies are most advanced for CCP for which a
high-resolution three-dimensional structure has
been available since 1084 I 101. Furthermore,
detailed structural and functional information has
been derived for the reaction intermediate Compound I. for complexes with CN, I;, NO and CO,
and for ;i large number of CCI’ active site mutants
(references in 15, 01). Similar information on other
plant-type peroxidases is lagging behind.
Itecently, however, Morita et al. [ 1 I ] presented ;i crystallographic model of a highly basic
horseradish peroxidase isoenzyme (I IRP ES).This
structure verifies that CCI’ is a valid prototype for
AI>[,r<*vi;ltioIis Llsed: C(-p, cytochrome c peroxidase;
I IHI’. horseradish peroxidase
the plant peroxidase superfamily. ‘I‘he haemin is
embedded in an apoprotein framework containing
10 dominating helices (A to J). At the closed proximal site a histidine residue (175 from helix 1; of
CCP) is ligated to the iron atom and hydrogen
bonded to an aspartate residue (2.35 of helix I I in
CCP), and at the accessible distal site a histidine
and arginine residue (52 and 48, respectively, from
helix I{ of CCI’) catalyse the reduction of hydrogen
peroxide. (The active sites and mechanisms of the
redox enzymes CCP. cytochrome /’-450, and catalase are reviewed and compared in 1121.) CCP
oxidizes cytochrome c and cannot serve as a model
for the reaction of plant peroxidases and their aromatic hydrogen donors. Information on these
aspects has been derived from recent experiments
on horseradish isoperoxidases using n.m.r. spectroscopy I 1.3, 14 ]* chemical modifications of peroxidases through ‘suicide’ reactions [ 15 and
correlations of the electronic properties of a series
of substrates and kinetic parameters [ 161 (reviewed
in 151).
Sequences of peroxidases and roles of
invariant residues
Figure 1 summarizes the characteristics of the
structures of plant, fungal, and bacterial peroxidases
based on presently available sequences and the
crystallographic model of CCP. Nine residues are
invariant. I;ive of these, ArglX, f Iis52, AsnX2,
IIis175 and Asp2.35, appear to assist the haem
group in peroxidase-spec.ifc catalysis (see I;ig. 2).
Three residues, Asp 100, Gly 120 and Argl.30, form
a buried salt bridge and a compact structural motif
as illustrated in Fig. 3. This salt bridge is invariant,
in contrast with the disulphide bridges, and links
I992
337