Báo cáo khoa học: The molecular basis of heme oxygenase deficiency in the pcd1 mutant of pea pot

The molecular basis of heme oxygenase deficiency
in the pcd1 mutant of pea
Philip J. Linley1,*, Martin Landsberger1,†, Takayuki Kohchi2, Jon B. Cooper1 and Matthew J. Terry1
1 School of Biological Sciences, University of Southampton, UK
2 Graduate School of Biostudies, Kyoto University, Sakyo, Japan

Keywords
biliverdin; photomorphogenesis;
phytochrome; plastid; structural modelling
Correspondence
M. J. Terry, School of Biological Sciences,
University of Southampton, Bassett
Crescent East, Southampton, SO16 7PX,
UK
Fax: +44 2380 594459
Tel: +44 2380 592030
E-mail:
/>Present address
*Graduate School of Biostudies, Kyoto
University, Sakyo, Kyoto 6068502, Japan
AG Molekulare Kardiologie, Klinik fur
ă
Innere Medizin B, Universitat Greifswald,
ă
17487 Greifswald, Germany
Database
The nucleotide sequences data for pea HO1
are available in the DDBJ ⁄ EMBL ⁄ GenBank
databases under the accession numbers
AF276228 (HO1 cDNA), AF276229 (HO1
genomic sequence from cultivar Solara),

AF276230 (HO1 genomic sequence from
cultivar Torsdag).

The pcd1 mutant of pea lacks heme oxygenase (HO) activity required for
the synthesis of the phytochrome chromophore and is consequently
severely deficient in all responses mediated by the phytochrome family of
plant photoreceptors. Here we describe the isolation of the gene encoding
pea heme oxygenase 1 (PsHO1) and confirm the presence of a mutation in
this gene in the pcd1 mutant. PsHO1 shows a high degree of sequence
homology to other higher plant HOs, in particular with those from other
legume species. Expression of PsHO1 increased in response to white light,
but did not respond strongly to narrow band light treatments. Analysis of
the biochemical activity of PsHO1 expressed in Escherichia coli demonstrated requirements for reduced ferredoxin, a secondary reductant such as
ascorbate and an iron chelator for maximum enzyme activity. Using the
crystal structure data from homologous animal and bacterial HOs we have
modelled the structure of PsHO1 and demonstrated a high degree of structural conservation despite limited primary sequence homology. However,
the catalytic site of PsHO1 is larger than that of animal HOs indicating
that it may accommodate an ascorbate molecule in close proximity to the
heme. This could provide an explanation for why plant HOs show a strong
and saturable dependence on this reductant.

(Received 23 January 2006, revised
17 March 2006, accepted 7 April 2006)
doi:10.1111/j.1742-4658.2006.05264.x

Light influences almost all aspects of plant growth and
development and the quantity, quality, direction and
duration of light in the environment is monitored by
plants using a variety of photoreceptors [1]. One
important class of photoreceptors are the phytochromes that mediate a broad range of responses to

red and far-red light including germination, growth,
development of the photosynthetic apparatus and
flowering [2]. In flowering plants, the phytochromes
are encoded by a small gene family and have both
unique and redundant roles in regulating these processes. The phytochromes are now known to be

Abbreviations
BV, biliverdin IXa; EST, expressed sequence tag; GST, glutathione S-transferase; HO, heme oxygenase; pcd1, phytochrome chromophoredeficient 1 mutant; PFB, phytochromobilin.

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P. J. Linley et al.

widespread in nature, and phytochrome-like proteins
have also been identified in most photosynthetic organisms and even many nonphotosynthetic bacteria [3].
The phytochromes are photoreversible chromoproteins that, in plants, utilize the linear tetrapyrrole
chromophore, phytochromobilin (PFB), which is covalently bound to an apoprotein of approximately
120 kDa [4,5]. The phytochrome chromophore is synthesized in two steps from heme. In the first step,
biliverdin IXa (BV) is produced from the oxidative
cleavage of heme by the enzyme heme oxygenase (HO;
EC 1.14.99.3). Although the substrates and products
of this enzyme are identical to those of animal and
bacterial HOs, there are a number of significant biochemical and functional differences between them [6–
8]. Plant HOs are soluble proteins that utilize reduced
ferredoxin as a reductant [9–11], while the animal
enzyme uses cytochrome P450 reductase and is membrane bound via a hydrophobic C-terminal extension

[7]. In plants the major product of the reaction, BV, is
then converted to 3Z-PFB by a ferredoxin-dependent
PFB synthase [12]. The precursor of the bound phytochrome chromophore is thought to be 3E-PFB, but
evidence for an isomerase that accomplishes this reaction is currently lacking.
Mutants that are unable to synthesize the phytochrome chromophore have proved to be important in
developing our understanding of the role of the phytochromes in light-regulated plant development [13]. As
all phytochromes appear to use the same chromophore,
this class of mutants lack responses mediated by all
phytochrome species. The most extensively studied
phytochrome chromophore mutants are the hy1 and hy2
mutants of Arabidopsis thaliana [14] and the aurea and
yellow-green-2 (yg-2) mutants of tomato [15]. Typically,
these mutants have elongated stems or hypocotyls,
reduced red and far-red responses during de-etiolation
and characteristic pale yellow-green pigmentation
resulting from reduced chlorophyll and anthocyanin
content. These mutants have now been cloned with HY1
and YG-2 shown to encode HOs [10,16,17] and HY2
and AUREA, PFB synthase [12,18].
Another important mutant in this class is the phytochrome chromophore-deficient 1 (pcd1) mutant of pea
[19]. This mutant was isolated from an EMS-mutagenesis screen and has pale yellow-green foliage and elongated internodes. Seedlings of pcd1 failed to de-etiolate
in far-red light, had severely reduced sensitivity to red
light and lacked spectrophotometrically detectable
phytochrome indicating that pcd1 contained less than
1% of wild-type phytochrome levels [19]. Moreover,
isolated etioplasts were unable to synthesize BV from
heme, but retained the ability to convert BV to PFB.

A heme oxygenase-deficient mutant of pea

This suggested that pcd1 was a HO-deficient mutant
[19]. The pcd1 mutant, like other chromophore
mutants, not only continues to be a useful tool for
understanding a variety of photomorphogenic
responses [20,21], but as the only known heme degradation mutant in a legume species may be an important resource in the study of nodulation and nitrogen
fixation. Root nodules contain exceedingly high concentrations of heme and thus heme metabolism is of
great interest in this tissue [22]. To better understand
the role of HOs in plants generally and more specifically in legumes, we have characterized the pcd1 mutant
at the molecular level and demonstrated that the
PCD1 gene corresponds to HO1.

Results
Isolation of heme oxygenase 1 from pea
Degenerate primers PS1.FOR and PS1.REV were
designed with reference to previously identified plant
HO1 genes and HO1-like sequences from plant
expressed sequence tag (EST) databases. An RT-PCR
reaction with these primers using RNA isolated from
light-grown pea (cultivar Solara) amplified a 403 bp partial cDNA sequence. The partial cDNA showed more
than 70% nucleotide sequence identity with the corresponding region of Arabidopsis thaliana HO1 (AtHO1)
and was used as the basis for the design of gene specific
primers. The 5¢- and 3¢-ends of the pea HO1 cDNA were
obtained by rapid amplification of cDNA ends (RACE).
Both the 5¢- and 3¢-RACE reactions used a universal primer in combination with gene specific primers PsGSP1
and PsGSP2, respectively (see Experimental procedures). The resulting full-length sequence of PsHO1
consisted of 849 bp, encoding a polypeptide of 283
amino acid residues with a predicted molecular mass of
32 794 Da (GenBank accession AF276228). A proposed
N-terminal chloroplast transit peptide of 59 amino acid
residues was identified by the ChloroP algorithm [23],

leaving a mature polypeptide with a predicted molecular mass of 25 937 Da (see Fig. 1A). The HO1 RNA
transcript was found to be approximately 1.5 kb including 5¢- and 3¢-untranslated regions (data not shown).
Complete sequences for plant HO1s from Arabidopsis, tomato and rice have been reported previously
[10,17,24]. A sequence alignment of the regions encoding the proposed mature protein regions is shown in
Fig. 1A. A total of 60% of residues are conserved
between all four sequences with almost all amino acids
conserved in the HO signature sequence identified by
comparison with animal HOs [10]. In PsHO1 this
signature sequence corresponds to Q194–I203 (Fig. 1A,

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Fig. 1. The pea HO1 gene. (A) Sequence alignment of HO1 proteins
from pea, Arabidopsis, rice and tomato. Fully conserved residues
are highlighted with a black background and functionally conserved
residues by a grey background. The region corresponding to the HO
signature sequence identified in animal HOs is underlined. The
N-terminal targeting sequences have been removed. (B) Diagrammatic alignment of the genomic DNA and cDNA sequences of
PsHO1 indicating the location of intron-exon boundaries. The position of the mutation in pcd1 causing premature chain termination is
indicated. Numbers refer to the first and last nucleotides of the
exons, respectively. (C) Phylogenetic tree of plant HO-like
sequences. The protein sequence for the mature region of PsHO1
was aligned with mature sequences of previously identified plant

HO proteins and HOs identified from expressed sequence tag (EST)
databases. Only EST sequences that were considered to encode
the entire mature HO sequence, including some reconstructed from
two or more separate ESTs with identical overlapping sequences,
were included in the analysis. Predicted N-terminal chloroplast transit peptide domains were removed based on predictions using ChloroP. Sequences were aligned by CLUSTALW and analysed using the
PHYLIP PHYLOGENY INFERENCE PACKAGE (Felsenstein, 1993, version 3.5c,
distributed by the author at Department of Genetics, University of
Washington, Seattle, USA) with Synechocystis ho-1 as the outgroup
sequence. Construction was by the parsimony method with 1000
bootstrap replicates and a consensus tree was generated. The
grouping of pea HO1 within the Leguminosae is shown.

black underline) and contains several residues that
contact the bound heme molecule in human HO-1 [25].
The genomic sequences of both pea cv. Solara and
cv. Torsdag HO1 were isolated by PCR amplification
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using primers PsHO1.FOR2 and PsHO1.REV. Pea
HO1 (PsHO1) genomic sequences for the two cultivars
were identical and spanned 2.5 kb including three
introns of 711 bp, 811 bp and 110 bp, respectively
(Fig. 1B). The positions of the three introns are conserved in comparison to HO1 genes from rice, tomato
and AtHO3 and AtHO4. In AtHO1 the first and second introns are also identical, but the third intron is
absent with the third and fourth exons encoded as a
single continuous exon. The genomic sequences of
both pea cv. Solara and cv. Torsdag HO1 have been
submitted to GenBank (AF276229 and AF276230,
respectively). The PsHO1 genomic sequence from pcd1
was amplified using the same primer combination. A

single base pair change was found at nucleotide 1199
(G fi A) resulting in a codon alteration from W163 to
a stop signal (Fig. 1B). The mutation site is upstream
of several highly conserved amino acid sequences
between higher plant HO1s including the HO signature
sequence (Fig. 1A).
To investigate whether pea contained additional
HO1-like sequences, we probed genomic DNA from
pea cv. Torsdag seedlings with the mature PsHO1 coding region. The pattern of bands hybridizing to the
HO1 probe was consistent with the presence of only
one HO1-like sequence in pea (data not shown). Several attempts were also made with new PCR primers
optimized to different conserved regions of plant HOs,

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P. J. Linley et al.

including HO2-specific sequences, to amplify additional HOs from pea. Despite the use of many primer
combinations with a range of cDNA templates we
were unable to isolate any additional HO sequences.
A phylogenetic tree of plant HOs was constructed
using the phylip algorithm (Fig. 1C). The tree was
constructed using only complete HO protein sequences
from a combination of published sources [10,16,
17,24,26] and EST databases. For the purposes of the
alignment the N-terminal chloroplast transit peptide
domain was removed from all sequences as this region
is highly variable and not subject to the same evolutionary constraints as the regions encoding the catalytic region of the polypeptide. Two main divisions of
plant HOs can be identified: namely the HO1-like and

HO2-like sequences. The HO identified in this study
clearly groups with the HO1-like sequences supporting
its designation as PsHO1. Plant HO1s group into a
number of families based upon established taxonomic
divisions. Consistent with this, PsHO1 clearly groups
with other sequences from the Leguminosae such as
Medicago truncatula and soybean (Fig. 1C). Interestingly, while only single examples of HO2-like
sequences have been found in each species, Arabidopsis, soybean, apple and maize all have two or more
HO1-like sequences. In each case the HO1-like
sequences show greater similarity to each other than to
HOs from other species. This pattern is most likely to
result from gene duplication of an ancestral copy of
HO1 following speciation and therefore pea does not
necessarily contain more than one HO1-like sequence.
PsHO1 expression in wild-type and pcd1 plants
We examined the expression of PsHO1 in wild-type
pea and the pcd1 mutant by RNA gel blotting. For
these experiments plants were grown in the dark for
5 days then transferred to continuous white light for
72 h (leaf and stem tissue) or kept in the dark for a
further 3 days (root tissue). Figure 2A shows that in
wild-type plants, PsHO1 expression was found at a
high level in all the tissues examined. In contrast, in
pcd1 plants PsHO1 expression was barely detectable
even though the mutation in pcd1 causes a premature
translation termination not a defect in transcription.
We also examined PsHO1 protein levels in dark-grown
wild-type and mutant pea seedlings. Figure 2B shows
that an antibody raised to AtHO1 (HY1) recognizes a
major band at approximately 29 kDa in wild-type

seedlings. This band was completely absent in pcd1
seedlings consistent with the presence of the premature
translation termination codon in the mutant gene. The
pcd2 mutant of pea is deficient in PFB synthase and

A heme oxygenase-deficient mutant of pea

Fig. 2. Expression of PsHO1. (A) RNA gel blot showing expression
of PsHO1 in wild-type and pcd1 pea leaf, stem and root tissue from
plants germinated in the dark and grown in constant white light for
72 h. (B) Western blot of total protein extracted from dark-grown
wild-type, pcd1 and pcd2 seedlings and probed with an antibody
raised against the Arabidopsis HY1 protein.

exhibits a typical phytochrome chromophore-deficient
phenotype [21,27]. Analysis of PsHO1 protein levels in
a pcd2 mutant background indicated no differences
from wild-type (Fig. 2B) indicating that the loss of
PsHO1 in pcd1 was not simply the consequence of
chromophore deficiency on seedling development and
that the absence of the next enzyme in the pathway
had no apparent effect on PsHO1 protein levels. In
addition, no band of smaller molecular mass was
detected in pcd1 suggesting that any translated PsHO1
protein is degraded in this mutant (Fig. 2B).
Since PsHO1 plays a key role in photomorphogenesis
[19] we examined the regulation of PsHO1 expression by
light during de-etiolation. Dark-grown seedlings were
transferred into white light and expression of PsHO1
was followed by RNA gel blotting over 3 days. Figure 3A shows that PsHO1 expression increased approximately two-fold after white light treatment in both stem

and leaf tissue. Maximum expression was observed after
48 h with no further increase seen at 72 h. Although the
expression profile was similar between stem and leaf tissue one significant difference was that PsHO1 showed a
sharp peak of expression at 4 h in stem tissue, but not in
leaf tissue. We further investigated the regulation of
PsHO1 expression by light in stem tissue using narrow
waveband light sources. As shown in Fig. 3B, under red
light there was again a strong (three-fold) transient

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Fig. 3. Light regulation of PsHO1 expression. Graphs showing densitometric quantification of relative band intensities of PsHO1 transcripts from RNA gel blots after correction for 18S rRNA levels. (A)
The effect of white light on PsHO1 expression in leaves and stems.
Total RNA was extracted from leaf and stem tissue of seedlings
grown in the dark for 5 days and transferred to continuous white
light for 0, 4, 8, 12, 24, 48 and 72 h. (B) The effect of red and farred light on PsHO1 expression in stems. Total RNA was extracted
from stem tissue of seedlings grown in the dark for 5 days and
transferred to continuous red or far-red light for 0, 4, 8, 12, 24, 48
and 72 h. Data shown are the mean and standard error of three
independent experiments.

induction of PsHO1 although the peak was somewhat
later than seen under white light (8 h vs. 4 h). A small

4 h peak in expression was also seen under far-red light
(Fig. 3B), but not under blue light (data not shown).
Thus it is likely that this acute induction response is
under phytochrome control. In general, the sustained,
almost two-fold induction of PsHO1 under white light
was not reproducibly seen under any of the narrow
waveband treatments either in stem or leaf tissue.
Biochemical activity of PsHO1
To confirm that PsHO1 encodes a HO and to further
characterize its properties we expressed mature PsHO1
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Fig. 4. Biochemical characterization of purified recombinant PsHO1.
(A) Coomassie Brilliant Blue R stained SDS ⁄ PAGE gel of protein
fractions from the overexpression of mature PsHO1 fused to glutathione S-transferase. (B) Absorption spectra following the conversion of heme to BV IXa by recombinant PsHO1 between 300 and
800 nm. Arrows indicate the direction of the major changes in
absorption over the course of the measurements. (C) Michaelis–
Menten plot of the PsHO1 reaction for heme concentrations of
1, 2, 5, 10 and 20 lM. Data shown are the mean and standard error
of 2–3 independent measurements. Inset: Lineweaver–Burk plot of
the same data.

(i.e. without the predicted transit peptide) as a fusion
protein with GST in Escherichia coli. As shown in
Fig. 4A, the purified GST-HO1 fusion protein was

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P. J. Linley et al.

A heme oxygenase-deficient mutant of pea

digested with thrombin and the mature PsHO1 protein further purified prior to use (see Experimental
procedures for details). The yield of PsHO1 protein
was routinely in the range 6.5–9 mgỈL)1 culture. We
measured HO activity of PsHO1 by following conversion of heme to BV IXa spectrophotometrically
(Fig. 4B). Absorbance was monitored between 300 and
800 nm with bound heme showing strong absorbance
at 398 nm and BV IXa at 376 nm and 665 nm. Over a
period of 20 min the bound heme peak decreases substantially with a concomitant rise in the BV IXa
absorbance maxima (Fig. 4B). Coupled oxidation of
heme also results in BV formation, but with a mixture
of four IX isomers as the macrocycle is cleaved nonspecifically. We therefore analysed the reaction products by HPLC and confirmed that PsHO1 exclusively
synthesized the IXa isomer of BV (data not shown).
The reaction rate for the formation of BV IXa was
determined by monitoring absorbance at 665 nm for
10 min at 2-s intervals for heme concentrations
between 1 and 20 lm (Fig. 4C). The reaction showed
normal Michaelis-Menten kinetics and the rate of BV
IXa formation with 10 lm heme in the presence of
reduced ferredoxin, ascorbate and an iron chelator
(desferroxamine) was 47.8 nmol BV IXa h)1Ỉmg protein)1 (Table 1). We characterized the contribution of
these assay components by omitting them individually.
In the absence of ferredoxin the reaction rate
decreased to 46.5% of the complete reaction while the
absence of ascorbate reduced the rate to 25.7%. The
largest reduction in rate was observed when desferroxamine was omitted with a rate of only 4.0 nmol BV
IXa h)1Ỉmg protein)1 or 8.4% of the complete reaction. Using the data shown in Fig. 4C, we determined
the kinetic constants for the HO reaction from a Lineweaver-Burk plot (Fig. 4C, insert). The Vmax value for

the complete reaction was estimated as 63.3 nmol BV
IXa h)1Ỉmg protein)1 with a Km for heme of 3.1 lm.
When the assays were performed in the absence of
Table 1. Effect of assay components on activity of PsHO1. Reactions were performed for 10 min using 10 lM heme as substrate
and other reaction components as described in Experimental procedures. The rate of BV IXa formation was determined by following
absorbance at 665 nm and the data shown is the mean and range
of two experiments.
Assay
components

Reaction rate
(nmol BV IXa h)1Ỉmg protein)1)

% complete

Complete
Ferredoxin
Ascorbate
Desferroxamine

47.8
22.2
12.3
4.0

–
46.4
25.7
8.4

±
±
±
±

3.7
6.6
0.2
1.6

ascorbate or desferroxamine a loss of true MichaelisMenten kinetics was observed and Lineweaver–Burk
plots from these data were not linear.
Structural predictions for PsHO1
To gain further insight into the function of PsHO1 we
have attempted to obtain structural information on
this enzyme using modelling algorithms and published
high resolution crystal structures. Crystal structures
have now been solved for HOs from human [25], rat
[28] pathogenic bacteria [29,30] and cyanobacteria
[31,32]. Despite the limited sequence identity between
members of the HO family, the tertiary structures are
remarkably conserved suggesting that modelling the
structure of pea HO1 would be likely to generate a
realistic model of the tertiary structure and its active
site interactions. This is supported by the observation
that many key residues, predominantly those associated with heme binding, are conserved across all
sequences. A predicted structure for the PsHO1 protein was generated using the programme modeller
based on an alignment of PsHO1 with human HO-1,
rat HO-1, Synechocystis ho-1 and Corynebacterium
diptheriae HmuO (see Fig. S1). Although PsHO1 is

only 13–21% identical to these HOs (Fig. S1), as
shown in Fig. 5A, the overall fold of the protein is
very similar with the relative position of the seven
major a-helices highly conserved with those of published structures [25,28,30]. The heme-binding pocket
is also broadly similar with the conserved His residue
that serves as the proximal heme ligand lying directly
below the predicted location of the bound heme
(Fig. 5B). However, there was one clear difference
between PsHO1 and other HOs in the heme binding
pocket. The predicted PsHO1 structure appeared to
contain a large space above the bound heme molecule
(Fig. 5B). Modelling AtHO1 by the same method identified a similar pocket with the same location and size
(data not shown), but this pocket was not present in
any of the animal or bacterial enzymes examined.
Since plant HOs have been shown to exhibit saturable
binding of ascorbate [11], which is likely to function
directly in the HO reaction as a cofactor, we hypothesized that the space adjacent to the bound heme might
accommodate an ascorbate molecule in a suitable position to participate in the HO reaction. We therefore
attempted to model ascorbate into our PsHO1 structure. As shown in Fig. 5C an ascorbate molecule was
readily accommodated in this space at a suitable dis˚
tance (predicted to be 3.3 A) to interact with the heme.
Six residues in the protein, Glu96, Phe120, His207,
Ile214, Tyr231 and Ser274 are also suitably placed,

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˚
within 2.4–3.8 A, to interact with the ascorbate (see
Table S1 for predicted atomic distances). The ability to
model an ascorbate molecule in close proximity to the
heme suggests a possible explanation for the strong
dependence of the plant HO reaction on ascorbate.

Discussion
The pcd1 mutant lacks a functional HO

Fig. 5. A structural model of PsHO1. A three-dimensional model of
the structure of pea HO was produced using the program MODELLER
and the structural co-ordinates for human HO-1, rat HO-1, Synechocystis ho-1 and Corynebacterium diptheriae HmuO (see Experimental procedures for details). (A) Overall fold of PsHO1 including
position of the bound heme molecule. (B) Close up of heme binding pocket. An asterisk indicates the space above the plane of the
heme molecule, that is present in plant, but not animal or bacterial,
proteins. (C) As for (B) with an ascorbate (Asc) molecule introduced
to the space adjacent to heme. Amino acid residues that could
interact with ascorbate are indicated.

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We have isolated a gene for the enzyme HO1 from pea
(PsHO1) encoding a polypeptide of 283 amino acid
residues. Consistent with the plastid localization of
PFB synthesis and with other known plant HOs [6],
PsHO1 contains a predicted N-terminal chloroplast
targeting sequence of 59 residues. The intron–exon

structure of the PsHO1 gene is conserved with relation
to other plant HOs. Sequencing of the genomic PsHO1
sequence from the pcd1 mutant revealed a point mutation resulting in the conversion of Trp163 (W163) to a
stop codon. This premature chain termination in pcd1
destabilized the PsHO1 mRNA resulting in a severe
reduction in mRNA levels in all tissues. Furthermore,
any truncated protein synthesized would lack many
key residues including the distal alpha helix of the
heme binding pocket and the HO signature sequence
(Q194-I203 in PsHO1). It is therefore likely that pcd1
is a null mutant for HO1.
The mutation in PsHO1 can fully account for the
observed phenotype of the pcd1 mutant, which lacks
holophytochrome and is consequently deficient in
responses mediated by all seedling phytochromes [19].
Interestingly, mature pcd1 plants, like chomophoredeficient mutants in other species, gradually recover
their ability to respond to phytochrome-mediated photomorphogenic signals. New internodes on 3 week-old
pcd1 plants respond normally to end-of-day far-red
(EOD-FR) treatments [19] indicating that phyB function in mature pcd1 plants is no longer compromised.
This suggests that a minimum level of PFB must accumulate in pcd1 plants to permit the formation of holophytochrome. Mutiple HO genes have been identified
in most plant species examined to date and an obvious
explanation for the recovery of phytochrome responses
in pcd1 is that additional HOs are functional at this
developmental stage. Indeed, additional HOs have
been shown to contribute to phytochrome responses in
Arabidopsis [17,33]. Multiple HO1-like genes have
been identified in a number of species including Arabidopsis, soybean, apple, maize and lettuce and in all
cases these gene families have greater sequence identity
within their family than with HOs from other species
(see Fig. 1C). Evolutionarily this suggests that each

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P. J. Linley et al.

family derived from gene duplication events postspeciation. Despite several attempts with numerous primer
combinations no further HO sequences could be isolated from pea. Southern blot results supported the presence of only PsHO1 as an HO1-like sequence. HO2
genes have been isolated from Arabidopsis, sorghum
tomato, M. truncatula and soybean. Attempts were
also made to isolate an HO2-like gene from pea but
these were unsuccessful. This was possibly due to the
relatively poor level of sequence identity between
HO2s in comparison with HO1s making primer design
more difficult. The low level of sequence conservation
between HO1s and HO2s would also account for the
failure of the PsHO1 probe to detect any HO2-like
sequences in Southern blot experiments. Whatever the
basis of the recovery of phytochrome responses in
older pcd1 plants, they still retain their pale phenotype
in maturity [19]. Since this is likely to be the result of
feedback inhibition within the tetrapyrrole pathway
[21], it suggests that any additional HOs are not able
to fully compensate for the loss of PsHO1 even in
mature plants. Clearly, more work needs to be undertaken to resolve this issue, but it is interesting to note
that there are precedents for this type of observation.
In Arabidopsis there are three genes encoding
NADPH : protochlorophyllide oxidoreductase, but
only a single gene has been identified in pea and
cucumber despite extensive attempts reported by the

authors to isolate additional genes [34].
Regulation of PsHO1 expression
The expression of PsHO1 is moderately induced by
light with an increase of approximately two-fold after
48 h of white light treatment. A moderate increase in
expression has also been noted for AtHO1 [16] and
indeed this level of response is seen for genes encoding
many tetrapyrrole synthesis enzymes [35]. We have
hypothesized that small white-light induced increases
in gene expression such as those seen for HO1 (and,
for example, GSA) may be the result of increased signals from the chloroplast to nucleus reflecting the promotion of chloroplast development and division under
prolonged white light [36]. This contrasts to the strong
photoreceptor-mediated induction of some key tetrapyrrole-related genes such as HEMA1 [35,37]. Since
the requirement for PFB is likely to be just as high
prior to illumination as afterwards, a major increase in
PsHO1 expression would not be expected. It is likely
that the observed increase is less driven by the need
for chromophore synthesis as it is for the increased
requirement for heme degradation in the developing
chloroplasts.

A heme oxygenase-deficient mutant of pea

One interesting feature of PsHO1 expression was the
apparent acute response evident in stem tissue in which
there was a transient induction peaking at about 4–8 h
after the start of the light treatment. This has not been
seen before as similar experiments with Arabidopsis
have necessarily used cotyledon ⁄ leaf tissue [35]. The
induction under red and (to some extent) far-red light,

but not under blue light suggests that this acute
response is phytochrome mediated. Further experiments on the kinetics of this response, and the use of
phytochrome-deficient mutants, will be needed to confirm this. Phytochrome plays a particularly important
role in regulating stem elongation, but is less stable in
the active Pfr form. It is possible that phytochrome
promotes chromophore synthesis to ensure a constant
supply of new holophytochrome during the crucial
early stages of de-etiolation. Since stems do not possess the surfeit of plastids present in leaf tissue, chromophore synthesis may be more limiting in stem
tissue.
Structure and function of PsHO1
Recombinant mature PsHO1 enzyme was shown to be
active in the conversion of heme to BV IXa. Maximal
activity required the presence of ferredoxin, ascorbate
and an iron chelator, in this case desferroxamine.
These requirements match those determined for maximum activity of AtHO1 and are consistent with a plastid-localized enzyme [11]. The kinetic parameters for
the PsHO1 reaction were also similar to those previously reported for a variety of HOs. PsHO1 had a Km
value for heme of 3.1 lm compared to 1.3 lm for
recombinant AtHO1 [11] and 3 lm for recombinant
human HO-1 [38]. The strongest dependence of PsHO1
activity was for the presence of an iron chelator to
accept the iron atom released from the cleaved heme
macrocycle. Free iron has a very low solubility level
in vivo (10)18 m) and is chelated by ferritin protein
complexes to maintain iron concentrations at approximately 10)7 m as required by the cell [39]. In Arabidopsis, four ferritin genes have been identified all of
which possess predicted chloroplast transit peptides
[40]. This is perhaps not surprising since it has been
reported that 90% of cellular iron is found in chloroplasts [41]. The iron chelator nicotianamine has been
suggested to play a role in controlling iron availability
for ferrochelatase [42] and endogenous iron chelators
may also be important regulators of HO activity

within the chloroplast.
We also observed a strong dependence of the HO
reaction on ascorbate. Ascorbate appears to be particularly important for the maximum activity of algal [9]

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P. J. Linley et al.

and plant [11] HOs, where it may function in the
reduction of verdoheme to Fe3+-BV [11]. As the
requirement for ascorbate was saturable, it was further
proposed that it functions as a cofactor in the HO
reaction [11]. To understand the structure of PsHO1,
and in particular the environment around the active
site, in more detail we have modelled the structure
based on published structural co-ordinates of HOs
from other species. Our results suggest that although
the basic structural folds of the enzyme are well conserved there was a significant difference within the active site, with considerably more space in the vicinity of
the bound heme. It is possible to model an ascorbate
molecule into this space suggesting a possible mechanism for the saturation kinetics of ascorbate in the HO
reaction. Six residues were identified as potentially
interacting with the bound ascorbate: Glu96, Phe120,
His207, Ile214, Tyr231 and Ser274 (Fig. 5C; Table S1).
A variety of enzymes that bind ascorbate have been
investigated previously. Soybean ascorbate peroxidase

[43], myrosinase from Sinapsis alba [44], and hyaluronate lyase from Streptococcus [45] all utilize an Arg
residue to bind to one of the oxygen atoms of ascorbate either via a salt bridge or, in the case of ascorbate
peroxidase, hydrogen bonding. One other reported
example, xylose isomerase from Streptomyces, utilizes
a His residue (Protein Data Bank, 1X1D [46]); and it
is possible that His207 fulfils the major role in ascorbate binding in PsHO1. Interestingly, this residue is
completely conserved in plant HOs, but is replaced by
an Asp in animal and bacterial HOs. Of the other
potential interacting residues Glu96, Phe120, Ile214,
Tyr231 and Ser274 are all conserved in plant HO1s
(with the exception of Glu96 and Ser274 in AtHO4),
but are not present in HO2s. They are also all absent
in animal and bacterial sequences with the exception
of Tyr231, which is conserved in bacteria. Instead
Glu96 is changed to a Met in animal sequences and a
Val in cyanobacteria, Phe120 becomes a Val or Leu in
mammalian and other animal ⁄ bacterial sequences,
respectively, Ile214 is a Leu in cyanobacterial and animal HOs, Tyr231 is a Phe in animal sequences and
Ser274 is always changed to Asn. Thus the potential
ascorbate interacting residues are highly conserved in
plant HOs, but not at all conserved in other HOs.
Instead the animal and cyanobacterial sequences contain a number of residues that prevent ascorbate binding. The human HO1 protein contains a Leu residue
(L147) in the equivalent position to Ile214 in the pea
HO1 sequence that restricts the space for ascorbate
binding as does Phe37 and Arg136. The Synechocystis
HO1 also contains this Arg (R127) and also a Phe residue (F203) that both prevent ascorbate binding. All of
2602

these residues are absent in all plant HO sequences
examined.

The modelling results provide a possible explanation
for the saturable stimulation of plant HO activity by
ascorbate, but clearly further information is required
to verify this hypothesis. We have initiated crystallization trials to obtain experimentally determined structural data on the active site environment. Why plant
HOs should show this ascorbate interaction when
other HOs do not is also unknown. Ascorbate has
been shown to stimulate cyanobacterial HOs to some
extent [47–49], although the cyanobacterial enzyme
shows greater activity with the alternative reductant,
Trolox [47,48]. This contrasts with the situation for
plant and algal HOs, for which ascorbate is far more
effective [9,11]. Chloroplasts contain very high concentrations of ascorbate [50] and perhaps plant HOs have
evolved to take full advantage of this.
In conclusion we have demonstrated that the pcd1
mutant of pea has a mutation in the HO1 gene and
have characterized pea HO1 at both the gene and protein level. Pea is an important system in which to study
nodulation and nitrogen fixation. Heme has a crucial
role to play in these processes as the cofactor of plant
hemoglobins that are present at very high concentrations in root nodules [22] and therefore heme synthesis
has been studied extensively in these tissues ([51] and
references therein). PsHO1 was very strongly expressed
in root tissue and the pcd1 mutant therefore represents
a useful genetic tool with which to investigate the role
of heme in this system. Recently it has also been suggested that HO has a role in antioxidant defence in
soybean nodules [52] and thus may have an additional
and crucial function in this important biological
process.

Experimental procedures
Plant material

The pcd1 mutant was originally isolated from pea (Pisum
sativum) cultivar Solara [19] and subsequently backcrossed
into the Torsdag cultivar. Seeds were kindly provided by
J. L. Weller (University of Tasmania, Australia). Wild-type
pea and the pcd1 mutant were grown on sifted damp
Vermiperl (William Sinclair Horticulture Ltd, Lincoln,
UK). Plant material for DNA or RNA extraction for
cDNA isolation was grown for 7–10 days in a controlled
environment growth chamber at 23 °C in 16 h white light
(250 lmolỈm)2Ỉs)1) photoperiods. Plants grown for analysis of PsHO1 expression were germinated in the dark
(22 °C) for 5 days and transferred to continuous light
treatments at 22 °C for the period indicated. Broad band

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P. J. Linley et al.

white light (400–700 nm) was provided by fluorescent
tubes at 320 lmolỈm)2Ỉs)1 in a controlled environment
cabinet (Percival Scientific Inc, Boone, IA, USA; model
I-36HILQ). Narrow band sources were provided by LED
displays in environmental control chambers (Percival Scientific Inc.; model E-30LED) as described previously [53].
Red light (R) had a fluence rate of 75 lmolỈm)2Ỉs)1; farred light (FR) was passed through two filters (#116 and
#172; Lee Filters, Andover, UK) to remove k < 700 nm
resulting in a final fluence rate of 10 lmolỈm)2Ỉs)1 and
blue light (B) was 9.2 lmolỈm-2Ỉs-1. Plants for the isolation of root material were germinated in closed sterile
pots on damp filter paper in the dark.

Cloning

A partial PsHO1 cDNA homologous to Arabidopsis HO1
was amplified from RT-PCR products of total RNA isolated from P. sativum cv. Solara using degenerate primers
PS1.FOR 5¢-GAG GAN ATG AGN TTN GTN GCN
ATG AGA-3¢ and PS1.REV 5¢-CCA CCA GCA NTA
TGN GNA AAG TAG AT-3¢. Amplification products were
ligated into pCR2.1 (TOPO TA cloning kit; Invitrogen Ltd,
Paisley, UK) and introduced into One ShotỊ TOP10F¢
competent cells (Invitrogen Ltd). The PsHO1 cDNA ends
were amplified by RACE (SMARTTM RACE kit, BD Biosciences Clontech, Palo Alto, CA, USA) using the Universal Primer (supplied) and gene specific primers PsGSP1
(5¢-RACE) 5¢-GCC TGG GGG TCG TTC TGA GAC
AAA TC-3¢ and PsGSP2 (3¢-RACE) 5¢-CGG AAG AGA
GAG CCG TGA CGA AGT G-3¢. The genomic
PsHO1 sequence was amplified from total genomic DNA
of P. sativum cv. Solara, cv. Torsdag and pcd1 using primers PsHO1.FOR 5¢-ACA CCC TCC GTG CAC TCA ACT
CT-3¢ and PsHO1.REV 5¢-AGA GTT TGG GCC AGA
GTA TCA GGA-3¢.

Northern analysis
Tissue samples (50–150 mg fresh weight) were collected and
RNA isolation was performed as described previously [53].
Denaturing RNA gels with 1.5% agarose were used to separate RNA samples denatured at 65 °C in the presence of
50% (v ⁄ v) formamide for 5 min [54]. Electrophoretically
separated RNA was transferred to Hybond-N membrane
(Amersham Biosciences, Amersham, Buckinghamshire,
UK) by capillary blotting. Probes were labelled with [a32P]dCTP using random hexanucleotide priming (Rediprime II
kit, Amersham Biosciences). Membranes were prehybridized and hybridized in the presence of 50% (v ⁄ v) formamide at 42 °C and, following hybridization, were washed to
a final stringency 0.2 · NaCl ⁄ Cit, 0.1% SDS at 42 °C. The
PsHO1 probe consisted of the coding region for the mature
protein isolated as a 690 bp fragment. Any variation in
sample loading was shown by reprobing the membranes

A heme oxygenase-deficient mutant of pea

with a flax 18S rRNA fragment. Blots were exposed to
X-ray film (Kodak Biomax MS, Amersham Biosciences)
and densitometry readings of the resulting images were performed with a digital imaging system (Alpha Innotech
Corp, San Leandro, CA, USA) using the Alphaease software package.

Immunoblotting
WT, pcd1 and pcd2 seedlings were grown in the dark for
10 days. For each genotype, five seedlings were harvested
and 1 cm segments (from the top) were weighed, ground in
liquid N2 and heated at 65 °C for 20 min in 400 lL
2 · SDS sample buffer (62.5 mm Tris ⁄ HCl pH 6.8 containing 10% (w ⁄ v) glycerol, 2% (w ⁄ v) SDS, 5% (v ⁄ v) 2-mercaptoethanol and 0.002% (w ⁄ v) bromophenol blue). Samples
were then centrifuged at top speed for 10 min at 4 °C in a
bench-top microcentrifuge, diluted four-fold in sample buffer and loaded directly onto a 15% (w ⁄ v) SDS ⁄ PAGE gel.
Proteins were then separated by electrophoresis and blotted
onto polyvinylidene difluoride membranes (Immobilon-P;
Sigma-Aldrich Company Ltd, Dorset, UK) using standard
protocols. HO was detected using a rabbit polyclonal antibody raised to AtHO1 [10] with a goat antirabbit IgGalkaline phosphatase conjugate as the secondary antibody
(Sigma-Aldrich Company Ltd).

Expression of recombinant PsHO1
The coding sequence for the mature PsHO1 (excluding the
coding region for the transit peptide) was amplified using
primers PSGEX.FOR (5¢-GTT ATT GGA TCC GCG
ACC ACG TC-3¢) and PSGEX.REV (5¢-CCA GGA ATT
CAG GAT AGT ATT AGA C-3¢), ligated into pGEX-2T
(Amersham Biosciences) and transformed into E. coli BL21
DE3 cells. HO1 was expressed a fusion protein with glutathione S-transferase (GST) from Schistosoma japonicum.

Cells were grown in Luria broth (LB) overnight at 30?C
and then diluted 100-fold into 500 mL LB broth with
100 lgỈmL)1 ampicillin. Expression of the fusion protein
was induced after 3 h by the addition of 0.1 mm isopropyl
b-d-1-thiogalactopyranoside (IPTG). Cells were harvested
after 3 h by centrifugation and lysed by sonication. The
soluble protein fraction was applied to a 5 mL GSTrap
column (Amersham Biosciences) and the fusion protein was
isolated according to the manufacturer’s protocol. The sample was concentrated using Centricon YM-10 centrifugal
filter units (Millipore UK Ltd, Watford, UK) and the
glutathione elution agent was removed by addition of phosphate buffered saline and further centrifugation. The GST
binding domain was cleaved from the fusion protein by
incubation with thrombin protease according to the GST
overexpression protocol and the mature PsHO1 protein
recovered using the GSTrap column. Centricon filters were
again used to concentrate the eluted protein.

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A heme oxygenase-deficient mutant of pea

P. J. Linley et al.

Heme oxygenase assay

References

The heme oxygenase assay was carried out essentially as
described by Muramoto et al. [11] using 75 lg recombinant
PsHO1 with the exception that an NADPH regenerating
system (100 lm NADP+, 1 mgỈmL-1 glucose-6-phosphate,
0.5 units yeast glucose-6-phosphate dehydrogenase (SigmaAldrich Company Ltd)) was used. The reaction was started
by the addition of the 10 lm hemin unless otherwise stated.
Spinach (Spinacia oleracea) ferredoxin and spinach
ferredoxin NADP+ reductase were obtained from the Sigma-Aldrich Company Ltd. The reaction was monitored by
taking absorption spectra between 300 and 800 nm for
20 min. Reaction rates for the formation of BV IXa were
determined by measuring absorbance at 665 nm (2-s intervals) for 10 min. Heme concentrations were varied from 1
to 20 lm with the sequential omission of ferredoxin, ascorbate and the iron chelator, desferroxamine. Values for Vmax
and Km were calculated from Lineweaver–Burk plots.

Modelling the structure of PsHO1
A three-dimensional model of the structure of pea HO was
produced on the basis of the alignment of its amino acid
sequence (minus the N-terminal transit sequence) with HOs
from human, rat and the bacteria, Corynebacterium diptheriae and Synechocystis. The C-terminal membrane anchor
domains were removed from the mammalian sequences.
This initial sequence alignment was produced using the program malign [55]. Modelling of the tertiary structure was
performed by the program modeller [56] using the following structural co-ordinates: human HO-1 (1 N45 [25]), rat
HO-1 (1DVE [28]), Corynebacterium diptheriae HmuO
(1IW0 [30]), and Synechocystis PCC 6803 ho-1 (1WE1 [31]).
The modeller program implements comparative protein
structure modelling by satisfaction of spatial restraints
derived from known structures with similar sequences to the
target molecule and also performs energy minimization of
the resulting model. The active site heme ligand in the model
of the pea HO was derived from that found in the human

enzyme. Visual inspection of the model was performed
using the molecular graphics program turbo–frodo
(Biographics, Marseille, France).

Acknowledgements
Thanks to J. L. Weller (University of Tasmania, Australia) for providing the seeds used in this study. This
work was supported by BBSRC grant 51 ⁄ P10948 and,
in the initial stages, by a Royal Society University
Research Fellowship to MJT and by a Grant-in-Aid
for Research for the Future Program 00L01605 and a
grant for Scientific Research (C) 16580076 from the
Japan Society for the Promotion of Science (JSPS) to
TK.
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Supplementary material
The following supplementary material is available
online:
Table S1. Predicted atomic distances between ascorbate, heme and pea HO1 protein residues. Distances
were determined from the structural model of PsHO1
shown in Fig. 5.
Fig. S1. Sequence alignment of HO proteins. The protein sequences used to produce the structural model of
pea HO1 shown in Fig. 5 were initially aligned for this
analysis using the malign program. The identical
alignment shown here was generated using ClustalW
and produced using boxshade 3.21. The pea HO1
sequence used is lacking the N-terminal 59 aa encoding
the chloroplast transit peptide while the C-terminal
membrane anchor domains were removed from the
mammalian sequences. The sequences used were
human HO-1 (human; 20% identical to pea HO1), rat
HO-1 (rat; 19%), Synechocystis PCC 6803 ho-1 (Syn;
13%) and Corynebacterium diptheriae HmuO (Dip;
21%). Black squares indicate identical residues and
grey squares similar residues. The six amino acids that
potentially interact with a bound ascorbateas listed in
Table S1 are shown by black arrows (note that the
residue numbers in the figure do not correspond
exactly to those in the table because of the absence of
the chloroplast transit peptide sequence).
This material is available as part of the online article
from

FEBS Journal 273 (2006) 2594–2606 ª 2006 The Authors Journal compilation ª 2006 FEBS