Received 23 January 2006, revised 17 March 2006, accepted 7 April 2006 doi:10.1111/j.1742-4658.2006.05264.x The pcd1 mutant of pea lacks heme oxygenase HO activity required for the synth
Trang 1in the pcd1 mutant of pea
Philip J Linley1,*, Martin Landsberger1,†, Takayuki Kohchi2, Jon B Cooper1and Matthew J Terry1
1 School of Biological Sciences, University of Southampton, UK
2 Graduate School of Biostudies, Kyoto University, Sakyo, Japan
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
phyto-chromes 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 pro-cesses The phytochromes are now known to be
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: mjt@soton.ac.uk
http://www.sbs.soton.ac.uk/
Present address
*Graduate School of Biostudies, Kyoto
University, Sakyo, Kyoto 606–8502, Japan
†AG Molekulare Kardiologie, Klinik fu¨r
Innere Medizin B, Universita¨t Greifswald,
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).
(Received 23 January 2006, revised
17 March 2006, accepted 7 April 2006)
doi:10.1111/j.1742-4658.2006.05264.x
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 demonstra-ted 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 struc-tural 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
Abbreviations
BV, biliverdin IXa; EST, expressed sequence tag; GST, glutathione S-transferase; HO, heme oxygenase; pcd1, phytochrome chromophore-deficient 1 mutant; PFB, phytochromobilin.
Trang 2widespread in nature, and phytochrome-like proteins
have also been identified in most photosynthetic
organ-isms and even many nonphotosynthetic bacteria [3]
The phytochromes are photoreversible
chromo-proteins that, in plants, utilize the linear tetrapyrrole
chromophore, phytochromobilin (PFB), which is
cova-lently bound to an apoprotein of approximately
120 kDa [4,5] The phytochrome chromophore is
syn-thesized 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
bio-chemical 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
mem-brane 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
phyto-chrome chromophore is thought to be 3E-PFB, but
evidence for an isomerase that accomplishes this
reac-tion is currently lacking
Mutants that are unable to synthesize the
phyto-chrome chromophore have proved to be important in
developing our understanding of the role of the
phyto-chromes 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
phyto-chrome chromophore-deficient 1 (pcd1) mutant of pea
[19] This mutant was isolated from an
EMS-mutagen-esis screen and has pale yellow-green foliage and
elon-gated 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
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 degra-dation mutant in a legume species may be an import-ant resource in the study of nodulation and nitrogen fixation Root nodules contain exceedingly high con-centrations 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 specific-ally in legumes, we have characterized the pcd1 mutant
at the molecular level and demonstrated that the PCD1gene 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 par-tial cDNA sequence The parpar-tial cDNA showed more than 70% nucleotide sequence identity with the corres-ponding 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 pri-mer in combination with gene specific pripri-mers PsGSP1 and PsGSP2, respectively (see Experimental proce-dures) 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 molecu-lar mass of 25 937 Da (see Fig 1A) The HO1 RNA transcript was found to be approximately 1.5 kb inclu-ding 5¢- and 3¢-untranslated regions (data not shown) Complete sequences for plant HO1s from Arabidop-sis, tomato and rice have been reported previously [10,17,24] A sequence alignment of the regions enco-ding 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,
Trang 3black 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
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 con-served in comparison to HO1 genes from rice, tomato and AtHO3 and AtHO4 In AtHO1 the first and sec-ond 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 cod-ing region The pattern of bands hybridizcod-ing to the HO1 probe was consistent with the presence of only one HO1-like sequence in pea (data not shown) Sev-eral attempts were also made with new PCR primers optimized to different conserved regions of plant HOs,
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) Diagram-matic alignment of the genomic DNA and cDNA sequences of PsHO1 indicating the location of intron-exon boundaries The posi-tion of the mutaposi-tion in pcd1 causing premature chain terminaposi-tion 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 tran-sit peptide domains were removed based on predictions using Chlo-roP Sequences were aligned by C LUSTAL W 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.
Trang 4including HO2-specific sequences, to amplify
addi-tional 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
evolu-tionary constraints as the regions encoding the
cata-lytic 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)
Interest-ingly, while only single examples of HO2-like
sequences have been found in each species,
Arabidop-sis, 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
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 Fig-ure 3A shows that PsHO1 expression increased approxi-mately 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 tis-sue 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 PsHO1expression 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
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.
Trang 5induction 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
(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
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 gluta-thione S-transferase (B) Absorption spectra following the convers-ion 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 l M Data shown are the mean and standard error
of 2–3 independent measurements Inset: Lineweaver–Burk plot of the same data.
Fig 3 Light regulation of PsHO1 expression Graphs showing
den-sitometric quantification of relative band intensities of PsHO1
tran-scripts 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
far-red 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.
Trang 6digested with thrombin and the mature PsHO1
pro-tein further purified prior to use (see Experimental
procedures for details) The yield of PsHO1 protein
was routinely in the range 6.5–9 mgÆL)1 culture We
measured HO activity of PsHO1 by following
conver-sion 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
sub-stantially 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
non-specifically We therefore analysed the reaction
prod-ucts 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)1Æmg
pro-tein)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
desferrox-amine was omitted with a rate of only 4.0 nmol BV
IXa h)1Æmg protein)1 or 8.4% of the complete
reac-tion Using the data shown in Fig 4C, we determined
the kinetic constants for the HO reaction from a
Line-weaver-Burk plot (Fig 4C, insert) The Vmax value for
the complete reaction was estimated as 63.3 nmol BV
IXa h)1Æmg protein)1 with a Km for heme of 3.1 lm
When the assays were performed in the absence of
ascorbate or desferroxamine a loss of true Michaelis-Menten 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 associ-ated with heme binding, are conserved across all sequences A predicted structure for the PsHO1 pro-tein 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 pub-lished 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 iden-tified 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 hypothes-ized that the space adjacent to the bound heme might accommodate an ascorbate molecule in a suitable posi-tion to participate in the HO reacposi-tion We therefore attempted to model ascorbate into our PsHO1 struc-ture 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,
Table 1 Effect of assay components on activity of PsHO1
Reac-tions were performed for 10 min using 10 lM heme as substrate
and other reaction components as described in Experimental
proce-dures 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)1Æmg protein)1) % complete
Trang 7within 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
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 muta-tion 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 chomophore-deficient mutants in other species, gradually recover their ability to respond to phytochrome-mediated pho-tomorphogenic signals New internodes on 3 week-old pcd1 plants respond normally to end-of-day far-red (EOD-FR) treatments [19] indicating that phyB func-tion in mature pcd1 plants is no longer compromised This suggests that a minimum level of PFB must accu-mulate in pcd1 plants to permit the formation of holo-phytochrome 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 Ara-bidopsis, 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
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,
Synecho-cystis ho-1 and Corynebacterium diptheriae HmuO (see
Experimen-tal procedures for details) (A) Overall fold of PsHO1 including
position of the bound heme molecule (B) Close up of heme
bind-ing 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.
Trang 8family derived from gene duplication events
postspeci-ation Despite several attempts with numerous primer
combinations no further HO sequences could be
isola-ted from pea Southern blot results supporisola-ted the
pres-ence of only PsHO1 as an HO1-like sequpres-ence 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
under-taken 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
sig-nals from the chloroplast to nucleus reflecting the
pro-motion of chloroplast development and division under
prolonged white light [36] This contrasts to the strong
photoreceptor-mediated induction of some key
tetra-pyrrole-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
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 experi-ments on the kinetics of this response, and the use of phytochrome-deficient mutants, will be needed to con-firm 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 pos-sess the surfeit of plastids present in leaf tissue, chro-mophore 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 maxi-mum activity of AtHO1 and are consistent with a plas-tid-localized enzyme [11] The kinetic parameters for the PsHO1 reaction were also similar to those previ-ously 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 approxi-mately 10)7m as required by the cell [39] In Arabid-opsis, 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 chloro-plasts [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 partic-ularly important for the maximum activity of algal [9]
Trang 9and 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
con-served there was a significant difference within the
act-ive 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
mechan-ism 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
hyaluro-nate lyase from Streptococcus [45] all utilize an Arg
residue to bind to one of the oxygen atoms of
ascor-bate either via a salt bridge or, in the case of ascorascor-bate
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
ascor-bate 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
ani-mal HOs, Tyr231 is a Phe in aniani-mal 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
con-tain a number of residues that prevent ascorbate
bind-ing 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
resi-due (F203) that both prevent ascorbate binding All of
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 crystalliza-tion trials to obtain experimentally determined struc-tural 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 concen-trations 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 pro-tein 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 concentra-tions 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 sug-gested 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 23C in 16 h white light (250 lmolÆm)2Æs)1) photoperiods Plants grown for analy-sis of PsHO1 expression were germinated in the dark (22C) for 5 days and transferred to continuous light treatments at 22C for the period indicated Broad band
Trang 10white light (400–700 nm) was provided by fluorescent
tubes at 320 lmolÆm)2Æs)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
Sci-entific Inc.; model E-30LED) as described previously [53]
Red light (R) had a fluence rate of 75 lmolÆm)2Æs)1;
far-red 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 lmolÆm)2Æs)1 and
blue light (B) was 9.2 lmolÆm-2Æs-1 Plants for the
isola-tion 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
isola-ted 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 Shot TOP10F¢
competent cells (Invitrogen Ltd) The PsHO1 cDNA ends
were amplified by RACE (SMARTTM RACE kit, BD
Bio-sciences Clontech, Palo Alto, CA, USA) using the
Univer-sal 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
PsHO1 sequence was amplified from total genomic DNA
of P sativum cv Solara, cv Torsdag and pcd1 using
prim-ers 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
sep-arate RNA samples denatured at 65C 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 [a32
P]-dCTP using random hexanucleotide priming (Rediprime II
kit, Amersham Biosciences) Membranes were
prehybrid-ized and hybridprehybrid-ized in the presence of 50% (v⁄ v)
forma-mide at 42C and, following hybridization, were washed to
a final stringency 0.2· NaCl ⁄ Cit, 0.1% SDS at 42 C The
PsHO1probe 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
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 per-formed with a digital imaging system (Alpha Innotech Corp, San Leandro, CA, USA) using the Alphaease soft-ware 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 65C for 20 min in 400 lL
2· SDS sample buffer (62.5 mm Tris ⁄ HCl pH 6.8 contain-ing 10% (w⁄ v) glycerol, 2% (w ⁄ v) SDS, 5% (v ⁄ v) 2-merca-ptoethanol and 0.002% (w⁄ v) bromophenol blue) Samples were then centrifuged at top speed for 10 min at 4C in a bench-top microcentrifuge, diluted four-fold in sample buf-fer 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 anti-body raised to AtHO1 [10] with a goat antirabbit IgG-alkaline 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 gluta-thione 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 lgÆmL)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 sam-ple was concentrated using Centricon YM-10 centrifugal filter units (Millipore UK Ltd, Watford, UK) and the glutathione elution agent was removed by addition of phos-phate 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