Cytochrome P450
From Purdue Genomics Database Facility
Jing-Ke Weng (wengj@purdue.edu) and Clint Chapple (chapple@purdue.edu) Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.
David R. Nelson (dnelson@utmem.edu) Department of Molecular Sciences, University of Tennessee, Memphis, TN 38163, USA.
Contents |
Summary
Cytochrome P450s (CYPs) are heme-thiolate proteins, most of which catalyze NADPH- and O2-dependent hydroxylation reactions. P450s form a vast superfamily of genes that have been found in bacteria, insects, fish, mammals, plants, and fungi (Chapple, 1998). The nomenclature of P450s is based on amino acid sequence similarity. It assigns proteins with more than 40% identity into the same family, and proteins with more than 55% identity into the same subfamily (Werck-Reichhart and Feyereisen, 2000). Genomes of higher plants contain large number of P450s. For example, the Arabidopsis thaliana genome encodes 246 full-length P450s, accounting for approximately 1% of Arabidopsis gene complement (Nelson et al., 2004). Plant P450s have been shown to participate in a variety of biochemical pathways, including those involved in the biosynthesis of plant natural products such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates, as well as plant growth regulators such as auxin, gibberellins, jasmonic acid, and brassinosteroids (Chapple, 1998).
Recent advance in whole genome sequencing projects of several plant species, including Chlamydomonas reinhardii, Physcomitrella patens, and Populus trichocarpa, together with the available genome sequences of Arabidopsis and rice, has allowed comparative studies of plant P450 superfamily in a deep evolutionary context, covering plant lineages from green algae to woody trees (Nelson, 2006). Here we report the identification and annotation of Cytochrome P450s from the genome of lycophyte Selaginella moellendorffii. We also conducted phylogenetic analysis of Selaginella P450s together with those from Physcomitrella and Arabidopsis, in an attempt to bridge the gap between mosses and flowering plants in previous studies. Through the window of P450s, we hope this study will shed light on the evolution of biochemical pathways in plants and how it contributed to their successful colonization on land.
Distribution of Selaginella P450s across major plant CYP clans
We identified 390 full-length P450s from the Selaginella moellendorffii genome assembly, among which 225 were considered to be the non-redundant P450s from one haplotype, whereas the rest 165 are putative allelic variants from the other haplotype. Considering that the genome size of Selaginella moellendorffii is slightly smaller than that of Arabidopsis thaliana, the P450 gene content (total P450 number/genome size) in Selaginella genome is comparable to that in Arabidopsis genome. It is noteworthy that Physcomitrella genome contains much less P450 gene content, approximately ten times less than that in Selaginella or Arabidopsis genome. Therefore, it’s tempting to speculate that there was a burst of plant P450 functions that coincided with the radiation of vascular plants.
All Selaginella P450s identified in this study can be sorted into the 9 plant CYP clans existing in the Arabidopsis genome and two more absent in Arabidopsis. Here, we compare the distribution of P450s across the major plant CYP clans in Physcomitrella, Selaginella, and Arabidopsis.
| CYP51 clan | CYP71 clan | CYP72 clan | CYP74 clan | CYP85 clan | CYP86 clan | CYP97 clan | CYP710 clan | CYP711 clan | CYP727 clan | CYP746 clan | total | |
| Selaginella non-redundant P450s | 1 | 132 | 30 | 10 | 34 | 13 | 3 | 1 | 1 | 0 | 0 | 225 |
| Arabidopsis P450s | 1 | 154 | 19 | 2 | 28 | 33 | 3 | 4 | 1 | 0 | 0 | 245 |
| Physcomitrella P450s | 1 | 41 | 4 | 3 | 5 | 10 | 3 | 2 | 0 | 1 | 1 | 71 |
Only the 225 non-redundant P450 gene set was used in the phylogenetic analysis described below.
CYP51 clan
Selaginella moellendorffii genome contains one CYP51 gene, which encodes obtusifoliol 14a-demethylase involved in the postsqualene sterol biosynthetic pathway. CYP51 is the only CYP family that is conserved in all eukaryotes. A recent study revealed that mutations in Arabidopsis CYP51G1 cause defects in membrane integrity and cell elongation, as well as seedling lethality, which demonstrated the essential role of CYP51 for normal plant growth (Kim et al., 2005).
Selaginella CYP51 clan P450 sequence page
CYP71 clan
CYP71 clan is the largest clan of plant P450s, which count for 62%, 59%, and 58% of the total P450s in the genome of Arabidopsis, Selaginella, and Physcomitrella respectively. The angiosperm CYP71 clan P450s have been shown to play important roles in multiple secondary metabolic pathways that lead into the biosynthesis of a vast array of secondary metabolites and polymers. Previous studies have shown that Chlamydomonas does not contain CYP71 clan P450, suggesting that CYP71 clan evolved only in land plants (Nelson, 2006).
Our phylogenetic analysis reveals that among all the CYP71 clan families, CYP73, CYP78, CYP98, CYP701, and CYP703 are the only five families that are conserved from mosses to flowering plants. CYP73 and CYP98 encode cinnamic acid 4-hydroxylase and p-coumaroylshikimate 3’-hydroxylase respectively, which catalyze the aromatic para- and the first meta-hydroxylation reactions in the phenylpropanoid pathway (Mizutani et al., 1997; Franke et al., 2002). Although CYP73 and CYP98 activities are usually referred as critical prerequisites for lignin biosynthesis, a hallmark for tracheophytes, CYP73 and CYP98 orthologs are also present in bryophytes that lack lignin biosynthesis. It is now known that hydoxycinnamic acids, products of phenylpropanoid metabolism, are key building blocks of biopolymer sporopollenin, which covers land plant gametophytes and protect them from UV radiation and dryness (Bubert et al., 2002). Therefore, the early evolution of CYP73 and CYP98 in land plants was probably driven by the selection advantage provided by sporopollenin that ensures the survival of plant gametophytes in terrestrial environment. In addition, CYP73 and CYP98 are also involved in lignan biosynthesis in bryophytes (Cullmann and Becker, 1999). Although the exact function of CYP78 is still not known, studies in an Arabidopsis activation tagging mutant with overexpressed CYP78A9 and a rice mutant plastochron1 with defective CYP78A11 suggested that CYP78 plays an important role in regulating postembryonic leaf primordial development at shoot apex (Ito and Meyerowitz, 2000; Miyoshi et al., 2004). The conservation of CYP78 family across land plants suggests that the regulatory mechanism mediated by CYP78 P450s is likely to be also conserved in all the land plants. Genetic and biochemical studies of Arabidopsis CYP701A3 demonstrated that it encodes ent-kaurene oxidase, which catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid (Helliwell et al., 1998; Helliwell et al., 1999). Interestingly, the gibberellin biosynthetic pathway seems to be only partially evolved in mosses, since Physcomitrella genome lacks CYP88 ortholog in the CYP85 clan. CYP88 encodes the ent-kaurenoic acid oxidase that functions in the steps immediately after CYP701, which will be further discussed. CYP703 has long been suggested to be a lauric acid hydroxylase involved in flower development (Imaishi et al., 1999). However, its exact role was not solved until recently. A study in the Arabidopsis CYP703A2 null mutant revealed that CYP703 catalyzes in-chain hydroxylation of lauric acid to provide building blocks for sporopollenin biosynthesis in pollen (Morant et al., 2007). Our phylogenetic analysis indicates that Selaginella contains two CYP703C members, closely related to Arabidopsis CYP703A2 and three Physcomitrella CYP703B members. Moreover, Selaginella further contains other three genes in the CYP703D and E subfamilies, which are more distantly related. We hypothesize that this subfunctionalization phenomenon in Selaginella CYP703 family may coincide with the independent origin of heterospory in lycophytes. The CYP703D and E members may be specifically involved in sporopollenin biosynthesis in megaspores, whereas CYP703C members, like their orthologs in moss and flowering plants, function only in male gametophytes. Indeed, Selaginella megaspores do contain wall structures, analogous to pollen wall (Morbelli et al., 2003).
Physcomitrella genome has 41 full-length CYP71 clan P450s, twelve of which fall into the five conserved families discussed above, and the rest 29 are sorted under 11 families. Physcomitrella CYP752 and CYP753 are rooted to a clade containing CYP77 and CYP89. Although named under new CYP families, Physcomitrella CYP752 and CYP753 are still likely to be orthologous to Selaginella and Arabidopsis CYP77 members, whereas Arabidopsis CYP89 is probably a newly derived family from CYP77. The functions of angiosperm CYP77 and CYP89 are still not known. Physcomitrella CYP754, CYP755, CYP756, CYP759, CYP760, together with Selaginella CYP782A1 form a clade which lacks Arabidopsis orthologs, indicating this clade of P450s are maintained in Physcomitrella and Selaginella, but might have been lost in angiosperms. Physcomitrella CYP761 family is an intriguing case. While CYP761A1, CYP761B1, CYP761C1 and CYP761D1 are clustered with Arabidopsis CYP75B1 that encodes a flavonoid 3’-hydroxylase (Schoenbohm et al., 2000), CYP761E1 and CYP761E2 are placed in a huge Selaginella specific clade and clustered with the Selaginella CYP795 members. CYP758 is highly diversified and is only restricted to Physcomitrella.
The 132 full-length CYP71 clan P450s from Selaginella genome are sorted under 22 families. Surprisingly, our phylogenetic analysis reveals that 11 families that are composed of 79 Selaginella CYP71 clan P450s form a huge clade that is almost purely Selaginella specific, except for CYP795, which has two Physcomitrella orthologs as mentioned above. This observation suggests that there has been a large-scale P450 gene duplication and neofunctionalization in lycophyte lineage in parallel to the P450 functional diversification in angiosperms. Therefore, this group of Selaginella P450s may represent an array of novel biochemical pathways that have been essential to the adaptation of lycophytes, but have never been uncovered through studies mainly focused on angiosperms. Indeed, a recent study in lignin biosynthesis in Selaginella identified a P450 from this clade, CYP788A1, to be a ferulic acid/coniferaldehyde/coniferyl alcohol 5-hydroxylase that is functionally analogous to angiosperm CYP84 in its syringyl lignin biosynthesis, a trait which has been considered to be independently evolved only in lycophyte and angiosperm lineages (Weng et al., 2008). Selaginella CYP781, CYP783, CYP784, and CYP785 are also Selaginella specific families, but may share ancestors with Physcomitrella CYP754, CYP755, CYP756, CYP759 and CYP760 families.
The 154 Arabidopsis CYP71 clan P450s fall into 20 families. Except for the five highly conserved families discussed above, all the rest 15 families are absent in both Selaginella and Physcomitrella. These Arabidopsis unique CYP71 clan families potentially represent biochemical pathways that mark the differences between flowering plants from plants from other lineages. Our data suggest that Arabidopsis CYP79 family might be a derived family from the CYP703 family. CYP79 are known to be responsible for catalyzing the conversion of amino acids to aldoximes (Halkier and Gershenzon, 2006). Characterization of cyp79B2/cyp79B3 double knockout mutant, together with in vitro biochemical data, showed that Arabidopsis CYP79B2 and CYP79B3 mediate conversion of tryptophan to indole-3-acetaloxime, which is involved both indole glucosinolate biosynthesis and the tryptophan-dependent auxin biosynthetic pathway (Hull et al., 2000; Zhao et al., 2002; Ljung et al., 2005). Independent mutant studies of Arabidopsis supershoot and bushy1 identified CYP79F1 that converts short chain methionine derivatives to oximes (Reintanz et al., 2001; Tantikanjana et al., 2001), whereas its homolog CYP79F2 was later found to have a distinct function that converts long chain methionine derivatives to oximes (Chen et al., 2003; Tantikanjana et al., 2004). CYP79F1 and CYP79F2 are therefore involved in the biosynthesis of short- and long-chain aliphatic glucosinolates respectively. CYP79A2 has been shown to be capable of converting L-phenylalanine to phenylacetaldoxime, a key step towards benzylglucosinolate biosynthesis (Wittstock and Halkier, 2000). Similar to the case in Selaginella, Arabidopsis genome also contains a huge clade of CYP71 clan P450s that seem to be angiosperm specific, which includes CYP71, CYP76, CYP81, CYP82, CYP83, CYP84, CYP93, CYP705, CYP706 and CYP712. CYP83A1 and CYP83B1, defined by Arabidopsis ref2 and sur2 mutant respectively, function redundantly in catalyzing the hydroxylation reaction on aldoximes to form aci-Nitro compounds, a step immediately after CYP79 in glucosinolate biosynthesis (Barlier et al., 2000; Hemm et al., 2003). The fact that both Physcomitrella and Selaginella lack orthologs of CYP79 and CYP83 indicates that glucosinolate biosynthesis might be a newly evolved pathway in angiosperms, probably only in dicots, since the rice genome also lacks CYP83 (Nelson et al., 2004). Furthermore, the tryptophan-dependent auxin biosynthetic pathway that requires the activity of CYP79 may also be absent in Physcomitrella and Selaginella. CYP76 and CYP706 are closely related families in our phylogenetic analysis. CYP76B6 from Catharanthus roseus has been characterized to be geraniol 10-hydroxylase, involved in terpenoid indole alkaloid biosynthesis (Collu et al., 2001), whereas CYP706B1 from cotton is a (+)-δ-cadinene-8-hydroxylase, which is also involved in sesquiterpene biosynthesis (Luo et al., 2001). CYP705 family is a highly diversified family in Arabidopsis, and is probably a Brassicaceae specific family (Nelson et al., 2004). CYP705A2 has recently been found to be embedded in a metabolic cluster in Arabidopsis genome, which contains genes involved in triterpene metabolism (Field and Osbourn, 2008). CYP705A2 encodes thalianol-diol desaturase that mediates conversion of thalian-diol to desaturated thalian-diol, the step immediately after CYP708A2 (a CYP85 clan P450) (Field and Osbourn, 2008). CYP71 is another large family in Arabidopsis genome that has been shown to be involved in pathogen defense. CYP71B15, defined by pad3 mutant, catalyze the conversion of dihydrocamalexic acid to camalexin, the last step in the biosynthesis of camalexin, a major phytoalexin in Arabidopsis (Zhou et al., 1999; Schuhegger et al., 2006). Characterization of Arabidopsis CYP71A13 mutants, together with in vitro enzyme activity data, suggests that CYP71A13 converts indole acetaldoxime to indole-3-acetonitrile, the step after CYP79 towards camalexin biosynthesis (Nafisi et al., 2007). CYP82 family lacks orthologs in rice genome, therefore it might be a family evolved only in dicots. Although the function of CYP82 family in Arabidopsis is currently unknown, CYP82E4 from tobacco has been identified to be a nicotine N-demethylase, that is responsible for conversion of nicotine to nornicotine (Siminszky et al., 2005). Arabidopsis contains 18 members in the CYP81 family, which may be involved in flavonoid metabolism, since studies of alfalfa CYP81E enzymes suggest that they possess regiospecific 2’ or 3’ hydroxylase activities towards isoflavones (Liu et al., 2003). CYP93D1 is the only member in Arabidopsis CYP93 family with its function currently unknown. However, characterizations of CYP93B1 from licorice, CYP93A1, CYP93C1, and CYP93E from soybean suggest CYP93 members may be involved in multiple steps in flavonoid metabolism (Akashi et al., 1998; Schopfer et al., 1998; Steele et al., 1999; Jung et al., 2000). The function of CYP712 family is to be solved.
Selaginella CYP71 clan P450 sequence page
CYP72 clan
Physcomitrella genome only has two families (CYP765 and CYP766) in the CYP72 clan. These genes lack angiosperm othologs, and may represent the ancestral state of the CYP72 clan that has diversified later in vascular plant lineages. Selaginella CYP774A1 is potentially an ortholog for Physcomitrella CYP765A1.
Arabidopsis genome contains seven families in the CYP72 clan, which are CYP72, CYP721, CYP734, CYP709, CYP735, CYP714 and CYP715.
Intensive biochemical and genetic studies have demonstrated that Arabidopsis CYP734A1 (the old CYP72B1) and CYP72C1 are brassinolide/castasterone 26-hydroxylases, which play important roles in the brassinosteroid catabolic pathway by inactivation of growth-promoting brassinosteroids. Transgenic plants with Ectopic expression of either one of the two genes led to phenotypes similar to those brassinosteroid biosynthetic and response mutants (Neff et al., 1999; Nakamura et al., 2005; Takahashi et al., 2005; Turk et al., 2005). Arabidopsis CYP72B1 protein level in the hypocotyls elongation zone increased as plant shifted from dark to far-red light (Turk et al., 2003). Therefore, CYP72B1 has been suggested to serve as a developmental switch from skotomorphogenesis to photomorphogenesis (Turk et al., 2003). The function of CYP72A and CYP721 members is currently unknown. Arabidopsis CYP709 family is the most closely related family to the clade that contains brassinosteroid 26-hydroxylases. A study of CYP709C1 from wheat has shown its ability to hydroxylate long chain fatty acids at the ω-1 and ω-2 positions in vitro (Kandel et al., 2005). However, the in vivo role of CYP709 is still lacking. Selaginella genome contains only one P450 (CYP773A1) that clustered to the root of this Arabidopsis clade containing CYP72, CYP721, CYP734, and CYP709, which may possess a potential role in brassinosteroid catabolism.
In vitro studies have suggested that Arabidopsis CYP735A1 and CYP735A2 encode cytokinin hydrolases that are required for trans-zeatin biosynthesis, a major isoprenoid cytokinin in Arabidopsis. Although cytokinin signaling has been reported even in mosses (Cove et al., 2006), neither Physcomitrella nor Selaginella contains clear orhtologs of cytokinin hydrolases, suggesting that the ability to synthesize trans-zeatin might have been a late addition to the plant biochemical repertoire.
Selaginella genome contains seven families in the CYP72 clan, which are CYP773, CYP774, CYP775, CYP776, CYP777, CYP778 and CYP779. Except CYP774 and CYP773 that have clear orthologs in Physcomitrella and Arabidopsis respectively, the other families are likely to have been evolved independently in the lycophyte lineage.
Selaginella CYP72 clan P450 sequence page
CYP74 clan
CYP74 family is the only family in CYP74 clan. In angiosperms, CYP74A and CYP74B P450s have distinct biochemical functions. Biochemical study of Arabidopsis CYP74A demonstrated that it is an allene oxide synthase involved in the oxylipin pathway leading to the biosynthesis of jasmonic acid and its methyl ester (Laudert et al., 1996). An Arabidopsis CYP74A knock-out mutant is male sterile and fails to accumulate jasmonic acid upon wounding (Park et al., 2002). CYP74B is a hydroperoxide lyase also involved in the oxylipin pathway, which can convert 13-hydroperoxide (the same substrate as CYP74A) into 12-oxo-cis-9-dodecenoic acid and hexanal (Bate et al., 1998). The activity of CYP74B is required for the biosynthesis of a group of plant C6 volatiles (also known as green leaf volatiles), which have been suggested to have roles in plant defense signaling (Duan et al., 2005).
CYP74 family is absent in the Chlamydomonas genome (Nelson, 2006), but is conserved in the genome of Physcomitrella, Selaginella and Arabidopsis, suggesting that oxylipin pathway might have been evolved only in land plants. The functional split between allene oxide synthase and hydroperoxide lyase seems to be ancient, with two groups of homologs from Physcomitrella or Selaginella clearly clustered with Arabidopsis CYP74A and CYP74B2 respectively. Interestingly, Selaginella CYP74 family contains much more genes than that of Physcomitrella or Arabidopsis in each of the two clades. This may infer a complexity in spatial or temporal regulation of the gene functions in this family, or the oxylipin pathway in Selaginella could be more complicated than that in other plant lineages, which may involve novel intermediates.
Selaginella CYP74 clan P450 sequence page
CYP85 clan
Higher plant CYP85 clan P450s are known to have several functions in various hormone metabolic or catabolic pathways, including brassinosteroid (BR), gibberellin (GA), and abscisic acid (ABA).
Several families from the CYP85 clan, namely CYP85, CYP90 and CYP724, haves been shown to be involved the biosynthesis of brassinosteroids in angiosperms. Genetic and biochemical studies of CYP85A1 and CYP85A2 from Arabidopsis, and CYP85A3 from tomato revealed that CYP85 is a bifunctional enzyme, which can mediate the conversion of castasterone to brassinolide as well as the C-6 oxidation of brassinosteroids (Shimada et al., 2001; Kim et al., 2005b; Nomura et al., 2005). Arabidopsis cyp85a1 and cyp85a2 double mutant show severe dwarf phenotypes, reminiscent of the phenotypes of those strong brassinosteroid-deficient mutants (Kwon et al., 2005; Nomura et al., 2005). Analysis of the Arabidopsis photomorphogenesis mutant cpd identified CYP90A1 to be a brassinosteroid 23α-hydroxylase that catalyzes the conversion from 6-deoxocathasterone to 6-deoxoteasterone (Szekeres et al., 1996). Arabidopsis dwarf4 mutant defines CYP90B1 that mediates multiple 22α-hydroxylation steps in brassinosteroid biosynthesis (Choe et al., 1998). In vitro studies further suggested campesterol as the best substrate for this enzyme (Fujita et al., 2006). Characterization of the Arabidopsis rot3 mutant led to the identification of CYP90C1 as a potential player in brassinosteroid biosynthesis (Kim et al., 1998). A recent more detailed study further showed that Arabidopsis CYP90C1 and CYP90D1 act redundantly in vivo, and are brassinosteroid 23α-hydroxylases that define a novel route of brassinosteroid biosynthesis from (22S,24R)-22-hydroxy-5a-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol (Ohnishi et al., 2006). Rice mutant dwarf11 contains a defective CYP724B1, which has been suggested to play a role in supplying 6-deoxotyphasterol and typhasterol in the brassinosteroid biosynthesis network in rice (Tanabe et al., 2005). CYP708A2 has been recently found to be thalianol hydroxylase that converts thalianol to thaliana-diol in triterpene metabolism, which belongs to a family only restricted in Brassicaceae (Field and Osbourn, 2008). Although their function currently unknown, Arabidopsis CYP87, CYP702 and CYP720 families are closely related with those involved in brassinosteroid biosynthesis. Compared to the complex functional diversification of P450s related to brassinosteroid biosynthesis in Arabidopsis, Selaginella only contains a single family (CYP90) in this clade, which potentially represents brassinosteroid 23α-hydroxylase orthologs. This observation suggests that the brassinosteroid 23α-hydroxylase activity might be the ancestral activity for this clade of P450s. Selaginella may have a brassinosteroid biosynthesis pathway less complicated than that in angiosperms, or it may have evolved a different set of P450s independently that can produce novel brassinosteroids not observed in angiosperms. Physcomitrella only contains four P450s in the CYP85 clan, but none of them are clustered with those brassinosteroid biosynthetic P450s, suggesting that the elaborate brassinosteroid signaling pathway might not be present in bryophytes.
Studies of maize mutant dwarf3, barley mutant grd5, together with results of enzyme assays using heterologously expressed CYP88s unequivocally demonstrated that angiosperm CYP88 members encode ent-kaurenoic acid oxidase that catalyze the three steps of GA biosynthetic pathway from ent-kaurenoic acid to GA12 (Helliwell et al., 2001). Selaginella contains clear orthologs of ent-kaurenoic acid that falls into two CYP88 subfamilies, whereas Physcomitrella contains none. It is noteworthy that Physcomitrella encodes ent-kaurene oxidase ortholog, another P450 (CYP701) involved in the upstream steps of GA biosynthesis. Therefore, it is likely that the GA biosynthetic pathway has only been partially evolved in moss, but elaborated in vascular plants. This hypothesis is supported by the recent finding that the angiosperm GID1-mediated GA perception mechanism is conserved in Selaginella but not in Physcomitrella (Hirano et al., 2007; Yasumura et al., 2007).
Fine control of physiologically active ABA level is essential for normal plant growth as well as response to abiotic stresses (Nambara and Marion-Poll, 2005). Arabidopsis CYP707 family encodes ABA 8’-hydroxylase that catalyzes the conversion of biologically active ABA into 8’-hydroxy ABA, followed by spontaneously cyclization to form biologically inactive phaseic acid (Kushiro et al., 2004; Saito et al., 2004). It has been suggested that the catabolic pathway defined by CYP707 is the major route for ABA catabolism and is indispensable for the proper control of seed dormancy and germination in Arabidopsis (Okamoto et al., 2006). Selaginella contains five P450s in CYP707 family, among which CYP707A20 and CYP70743 are clearly Arabidopsis CYP707A orthologs. Selaginella CYP707B1 and CYP707B2, as well as CYP707C1 are more distantly related, which may represent a novel group of ABA modifying P450s. Although ABA signaling in mosses has been well documented (Cove et al., 2006), Physcomitrella lacks obvious Arabidopsis CYP707 orthologs. If Physcomitrella also possesses an angiosperm-like ABA catabolic pathway, the Physcomitrella CYP762 and CYP763 members make the most likely candidates for ABA hydroxylases, since they immediately branch off from the CYP707A clade. Arabidopsis CYP722A1 seems also to be related to the CYP707 clade, with its function currently unknown.
CYP716 is the only family in the CYP85 clan that is conserved from moss to angiosperm. Interestingly, whereas Physcomitrella and Arabidopsis genome contains only one and two CYP716 members respectively, Selaginella genome contains twelve. The burst of P450s in the CYP716 family might have given Selaginella significant selection advantages during its evolution. Arabidopsis CYP718 is also closely clustered within the CYP716 clade. The function of CYP716 family is still to be resolved.
Besides the CYP85 clan families discussed above, Selaginella also contains a family, CYP780, which seems to be uniquely restricted to Selaginella. Similar to the Selaginella CYP716 family, CYP780 family is also highly diversified.
Selaginella CYP85 clan P450 sequence page
CYP86 clan
In Arabidopsis, the CYP86 clan is composed of four CYP families, CYP86, CYP94, CYP96 and CYP704. Our analysis revealed that whereas CYP86, CYP94 and CYP704 families are conserved in Physcomitrella, Selaginella and Arabidopsis, CYP96 seems to be a newly evolved family unique to angiosperms.
CYP86 clan P450s has long been associated with plant cuticle formation. Plant cuticles cover the above-ground epidermal surface of plant body, which serve as a barrier for water and other molecules. The evolution of cuticles in land plants is absolutely essential for their successful adaptation to the terrestrial environment. Cuticles are mainly composed of cutin (polyester of hydroxy-fatty acids and their derivatives) and cutan (hydrocarbon polymer). Both CYP86 and CYP94 members have been shown to be able to catalyze ω-hydroxylation reactions on long chain fatty acids, which are required for cutin biosynthesis (Benveniste et al., 1998; Tijet et al., 1998). An Arabidopsis CYP86A2 mutant att1 shows significantly reduced cutin content, increased permeability to water vapor, and enhanced susceptibility to Pseudomonas syringae (Xiao et al., 2004). Although CYP704 family is conserved through moss to higher plants, its function is currently unknown. A recent study in the Arabidopsis CYP96A15 mutant mah1 suggests that the Arabidopsis CYP96A15 is a midchain alkane hydroxylase that is responsible for the formation of secondary alcohols and ketones in stem cuticular wax. Our phylogenetic analysis suggests that the CYP96 functions probably evolved in vascular plants after lycophytes diverged, and the CYP96 family might have been derived from some ancestral CYP86 members.
Selaginella CYP86 clan P450 sequence page
CYP97 clan
Selaginella genome contains three CYP97 clan P450s that fall into the CYP97 family .They are clearly the orthologs from the CYP97A, CYP97B, and, CYP97C subfamilies. The three CYP97 subfamilies have been previously shown to be highly conserved throughout the plant kingdom, which can be found in the genome of Chlamydomonas, Physcomitrella, pine, rice, Arabidopsis and poplar (Nelson, 2006). In Arabidopsis, genetic and biochemical studies have shown that CYP97C1 and CYP97A3 are carotenoid ε- and β-ring hydroxylase respectively, which are indispensable for lutein biosynthesis (Tian et al., 2004; Kim and DellaPenna, 2006). Although its function currently unknown, CYP97B is likely to be involved in carotenoid metabolism as well. Our result is consistent with the hypothesis that the ability to metabolize carotenoids arose early during plant evolution and has been highly conserved thereafter.
Selaginella CYP97 clan P450 sequence page
CYP710 clan
Plant CYP710 clan consists of only one family, CYP710 family, which is closely related to the CYP61 family in fungi. Recent genetic and biochemical studies of CYP710A1 and CYP710A2 from Arabidopsis, and CYP710A11 from tomato revealed that CYP710 P450 encodes sterol C-22 desaturase, which is responsible for the biosynthesis of Δ22-sterols restricted only in plants and fungi (Morikawa et al., 2006). Arabidopsis T-DNA insertion mutant of CYP710A2 completely lack brassicasterol/crinosterol production, whereas transgenic Arabidopsis with overexpressed CYP710A1 and CYP710A11 produce dramatically increased level of stigmasterol (Morikawa et al., 2006).
CYP710 exists as multiple genes in Arabidopsis and Physcomitrella, but as a single gene in Selaginella.
Selaginella CYP710 clan P450 sequence page
CYP711 clan
CYP711 family is the only family in the CYP711 clan. CYP711 exists as single gene in Arabidopsis and Selaginella, but absent in Physcomitrella. It has been suggested that the moss lineage might have lost this family independently, since CYP711-like P450s (CYP743 and CYP744) can be found in Chlamydomonas genome (Nelson, 2006). The function of CYP711 is currently unknown.
Selaginella CYP711 clan P450 sequence page
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