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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: 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 für Innere Medizin B, Universität 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). 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. 2594 FEBS Journal 273 (2006) 2594–2606 ª 2006 The Authors Journal compilation ª 2006 FEBS 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, FEBS Journal 273 (2006) 2594–2606 ª 2006 The Authors Journal compilation ª 2006 FEBS 2595 A heme oxygenase-deficient mutant of pea P. J. Linley et al. 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 2596 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, FEBS Journal 273 (2006) 2594–2606 ª 2006 The Authors Journal compilation ª 2006 FEBS 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 FEBS Journal 273 (2006) 2594–2606 ª 2006 The Authors Journal compilation ª 2006 FEBS 2597 A heme oxygenase-deficient mutant of pea P. J. Linley et al. 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 2598 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 FEBS Journal 273 (2006) 2594–2606 ª 2006 The Authors Journal compilation ª 2006 FEBS 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 mgÆL)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)1Æmg 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)1Æmg 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)1Æmg 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)1Æmg 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 distance (predicted to be 3.3 Å) to interact with the heme. Six residues in the protein, Glu96, Phe120, His207, Ile214, Tyr231 and Ser274 are also suitably placed, FEBS Journal 273 (2006) 2594–2606 ª 2006 The Authors Journal compilation ª 2006 FEBS 2599 A heme oxygenase-deficient mutant of pea P. J. Linley et al. within 2.4–3.8 Å, 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. 2600 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 FEBS Journal 273 (2006) 2594–2606 ª 2006 The Authors Journal compilation ª 2006 FEBS 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] FEBS Journal 273 (2006) 2594–2606 ª 2006 The Authors Journal compilation ª 2006 FEBS 2601 A heme oxygenase-deficient mutant of pea 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 lmolÆm)2Æs)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 FEBS Journal 273 (2006) 2594–2606 ª 2006 The Authors Journal compilation ª 2006 FEBS P. J. Linley et al. white 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 Scientific Inc.; model E-30LED) as described previously [53]. Red light (R) had a fluence rate of 75 lmolÆm)2Æs)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 lmolÆm)2Æs)1 and blue light (B) was 9.2 lmolÆm-2Æs-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 Shot 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 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 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. FEBS Journal 273 (2006) 2594–2606 ª 2006 The Authors Journal compilation ª 2006 FEBS 2603