Cecilie Cetti Hansen

David R Nelson

Birger Lindberg Møller

Daniele Werck-Reichhart

Plant cytochrome P450 plasticity and evolution

Keywords: land plants, green algae, diversification, promiscuity, neofunctionalization, convergent evolution, functionalities, plant metabolism

The superfamily of cytochrome P450 (CYP) enzymes plays key roles in plant evolution and metabolic diversification. This review provides a status on the CYP landscape within green algae and land plants. The 11 conserved CYP clans known from vascular plants are all present in green algae and several green algaespecific clans are recognized. Clan 71, 72, and 85 remain the largest CYP clans and include many taxaspecific CYP (sub)families reflecting emergence of linage-specific pathways. Molecular features and dynamics of CYP plasticity and evolution are discussed and exemplified by selected biosynthetic pathways. High substrate promiscuity is commonly observed for CYPs from large families, favoring retention of gene duplicates and neofunctionalization, thus seeding acquisition of new functions. Elucidation of biosynthetic pathways producing metabolites with sporadic distribution across plant phylogeny reveals multiple examples of convergent evolution where CYPs have been independently recruited from the same or different CYP families, to adapt to similar environmental challenges or ecological niches. Sometimes only a single or a few mutations are required for functional interconversion. A compilation of functionally characterized plant CYPs is provided online through the Plant P450 Database (erda.dk/public/vgrid/PlantP450/).

INTRODUCTION

Cytochromes P450 (CYPs) constitute a superfamily of hemethiolate enzymes that catalyze an impressive arsenal of reactions. CYPs are ancient enzymes, present in all kingdoms of life, and they have particularly diversified in the plant kingdom. The high number and functional diversity of plant CYPs provide a molecular basis for plant plasticity as CYPs are involved in the formation of membrane sterols, phytohormones and signaling molecules, biopolymers for structural support and for protection from desiccation, UV protectants, pigments, volatile organic compounds for biotic and abiotic interactions, and a range of lineage-specific specialized metabolites with diverse roles, e.g., in pest and herbivore interactions.

The CYPomes (the total number of CYPs in a given species) of plants are usually much larger than those found in animals or microorganisms. The CYPs started to diversify early in plant evolution, with 39 CYP genes detected in the green algae Chlamydomonas reinhardtii, and 71 in the moss Physcomitrium patens (previously Physcomitrella patens; [START_REF] Nelson | Plant cytochrome P450s from moss to poplar[END_REF][START_REF] Rensing | The moss Physcomitrium (Physcomitrella) patens: a model organism for non-seed plants[END_REF]. In some flowering plants (angiosperms), CYPomes constitute up to 1% of the protein-coding genes [START_REF] Nelson | A P450-centric view of plant evolution[END_REF]. This is exemplified by the 279 annotated full-length CYP genes in grape vine (Vitis vinifera) [START_REF] Ilc | Annotation, classification, genomic organization and expression of the Vitis vinifera CYPome[END_REF], 329 in rice (Oryza sativa; [START_REF] Nelson | A P450-centric view of plant evolution[END_REF], 443 in Eucalyptus grandis, and 741 in wheat (Triticum aestivum). Notably, when the total number of CYP sequences includes pseudogenes and short fragments, it increases significantly, reaching 579 and 798 in V. vinifera and E. grandis, respectively, illustrating the dynamic nature of CYPomes, with multiple gene duplications and losses.

CYPs are classified into families based on their amino acid sequence identities and phylogenetic clustering (Box 1). With the exponential number of CYP sequences becoming available from genomic and transcriptomic data without associated functional characterization, it is important that the global scientific community maintains and adheres to this nomenclature system to facilitate exchange of scientific knowledge. In 1990, CYP71A1 from avocado (Persea americana) was the first plant CYP to have its full sequence determined and named [START_REF] Bozak | Sequence analysis of ripening-related cytochrome P-450 cDNAs from avocado fruit[END_REF]. In 2018, almost 30 years later, the number of named CYPs in the plant kingdom passed 16 000 [START_REF] Nelson | Cytochrome P450 diversity in the tree of life[END_REF]. This number has kept steeply increasing, with over 32 000 plant CYPs distributed over 670 families named to date. Some CYP families are ubiquitous among kingdoms (CYP51) or land plants (e.g., CYP73 and CYP78), whereas others are lineage-specific (e.g., CYP702 and CYP708 in Brassicaceae).

The large number of plant CYP families reflects a diverse amino acid sequence space. BLAST alignments down to the low 20% identity range are observed between plant CYPs. Despite this, all CYP proteins share a common three-dimensional (3D) architecture dominated by the presence of a helixes and a smaller proportion of b sheets [START_REF] Lee | Structural insights into the evolutionary paths of oxylipin biosynthetic enzymes[END_REF][START_REF] Li | Modes of heme binding and substrate access for cytochrome P450 CYP74A revealed by crystal structures of allene oxide[END_REF][START_REF] Fujiyama | Structural insights into a key step of brassinosteroid biosynthesis and its inhibition[END_REF][START_REF] Gu | Crystal structure of CYP76AH1 in 4-PI-bound state from Salvia miltiorrhiza[END_REF][START_REF] Niu | Structural basis for plant lutein biosynthesis from alpha-carotene[END_REF][START_REF] Zhang | Structure and function of the cytochrome P450 monooxygenase cinnamate 4-hydroxylase from Sorghum bicolor[END_REF]. A unique feature is the cysteine-linked covalently bound heme group in the catalytic center. Initially described for mammalian CYP2, six substrate recognition sites (SRSs; [START_REF] Gotoh | Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences[END_REF]) that frame the distal heme region are key determinants of catalytic specificity. Substrateaccess channels may also contribute to the specificity of a CYP by controlling access of substrates to the active site. Microsomal CYPs are membrane-bound through an N-terminal membrane helix that restricts its mobility and provides an anchor point focusing the interaction with up-and downstream pathway partners, including the obligatory electron donor protein cytochrome P450 oxidoreductase (POR; [START_REF] Knudsen | Dynamic metabolic solutions to the sessile life style of plants[END_REF]. Isoforms of cytochrome b 5 may also serve as electron donors.

The plant CYPome is highly dynamic in evolutionary terms and the families, subfamilies, as well as the proportion of CYP genes belonging to each of them, can vary greatly between taxonomic groups. These differences translate into qualitative and quantitative differences in metabolite profiles, sometimes visually by morphological or developmental phenotypes, and by adaptation to specific ecological niches. Lineage-specific gene proliferation of paralogs is often termed ''blooms'' [START_REF] Feyereisen | Arthropod CYPomes illustrate the tempo and mode in P450 evolution[END_REF]. A CYP family bloom is, for example, found in O. sativa with 90 full-length CYP71s (88 CYP71 plus two CYP99 subfamily genes), some members of which are reported to be involved in diterpenoid phytoalexin biosynthesis (e.g., [START_REF] Wang | CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice[END_REF][START_REF] Wu | Parsing a multifunctional biosynthetic gene cluster from rice: biochemical characterization of CYP71Z6 & 7[END_REF][START_REF] Zhan | Selection of a subspecies-specific diterpene gene cluster implicated in rice disease resistance[END_REF]. Another example is the fern Cystopteris utahensis, where 44 different CYP981 transcripts were identified from 1kP transcriptomes [START_REF] Thousand | One thousand plant transcriptomes and the phylogenomics of green plants[END_REF], although none of them has been assigned a function.

The number of functionally characterized CYPs is steadily increasing, and more than 800 plant CYPs have been characterized by their ability to metabolize one or more substrates. These are collected in The Plant Cytochrome P450 Database (erda.dk/ public/vgrid/PlantP450/), which provides an extensive compilation of CYPs known to metabolize one or more substrates and includes references for further exploration. The classic CYP-catalyzed reaction is a monooxygenation of a carbon atom. The CYPmediated molecular oxygen activation can lead to the formation of alcohols, ketones, aldehydes, carboxylic acids, and epoxides, but other types of reactions, such as oxidative rearrangement of carbon skeletons (e.g., [START_REF] Nasomjai | Mechanistic insights into the cytochrome P450-mediated oxidation and rearrangement of littorine in tropane alkaloid biosynthesis[END_REF], oxidative C-C bond cleavage (e.g., [START_REF] Irmler | Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase[END_REF], dehydration (e.g., [START_REF] Hansen | Reconfigured cyanogenic glucoside biosynthesis in Eucalyptus cladocalyx involves a cytochrome P450 CYP706C55[END_REF], dealkylation (e.g., [START_REF] Bak | Cloning and expression in Escherichia coli of the obtusifoliol 14a-demethylase of Sorghum bicolor (L.) Moench, A cytochrome P450 orthologous to the sterol 14a-demethylases (CYP51) from fungi and mammals[END_REF], desaturation (e.g., Morikawa et al., 2006a), and decarboxylation (e.g., [START_REF] Koch | The primary sequence of cytochrome P450tyr, the multifunctional N-hydroxylase catalyzing the conversion of L-tyrosine to p-hydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench[END_REF] can also result from radicals or reactive oxygenated intermediates generated by CYPs. Formation of methylenedioxy bridges (e.g., [START_REF] Ikezawa | Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family, from cultured Coptis japonica cells[END_REF], lactone rings (e.g., [START_REF] Kim | Arabidopsis CYP85A2, a cytochrome P450, mediates the Baeyer-Villiger oxidation of castasterone to brassinolide in brassinosteroid biosynthesis[END_REF], and rings harboring several heteroatoms [START_REF] Klein | Two cytochrome P450 catalyze Sheterocyclizations in cabbage phytoalexin biosynthesis[END_REF], oxidative C-C and C-O phenol coupling reactions (e.g., [START_REF] Ikezawa | Molecular cloning and characterization of CYP80G2, a cytochrome P450 that catalyzes an intramolecular C-C phenol coupling of (S)-reticuline in magnoflorine 1260[END_REF][START_REF] El-Awaad | Bifunctional CYP81AA proteins catalyse identical hydroxylations but alternative regioselective phenol couplings in plant xanthone biosynthesis[END_REF], ring expansion (e.g., [START_REF] Nett | Discovery and engineering of colchicine alkaloid biosynthesis[END_REF], ring contraction (e.g., [START_REF] Helliwell | The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway[END_REF], formation of allene oxides (e.g., [START_REF] Howe | Cytochrome P450-dependent metabolism of oxylipins in tomato. Cloning and expression of allene oxide synthase and fatty acid hydroperoxide lyase[END_REF], N-oxygenations (e.g., [START_REF] Koch | The primary sequence of cytochrome P450tyr, the multifunctional N-hydroxylase catalyzing the conversion of L-tyrosine to p-hydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench[END_REF][START_REF] Sibbesen | Cytochrome P-450TYR is a multifunctional heme-Thiolate enzyme catalyzing the conversion of L-tyrosine to phydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench[END_REF], isomerization reactions as observed in oxime metabolism [START_REF] Halkier | The biosynthesis of cyanogenic glucosides in higher plants[END_REF][START_REF] Clausen | The bifurcation of the cyanogenic glucoside and glucosinolate biosynthetic pathways[END_REF], and aryl migration reactions as observed in isoflavonoid biosynthesis [START_REF] Akashi | Cloning and functional expression of a cytochrome P450 cDNA encoding 2hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice[END_REF] are other outcomes of CYP activity. We refer to other reviews for a detailed description of catalytic mechanisms Box 1. The CYP nomenclature system

In the cross-kingdom nomenclature system [START_REF] Nelson | The cytochrome P450 homepage[END_REF], CYPs with more than 40% amino acid sequence identity are listed within the same family, and CYPs that share more than 55% amino acid sequence identity are grouped into the same subfamily. Plant CYPs are assigned to families with the numbers 51, 71-99, 701-999, and from 7001 onward. Subfamilies are designated by a letter after the number. CYP families are further separated into clans defined by the deepest distinct branching phylogenetic clades that are reproducible in multiple phylogenetic trees.

Family creep may stretch the 40% boundaries for family classification when a new sequence phylogenetically groups with an existing CYP family, but is slightly below the identity threshold [START_REF] Nelson | A P450-centric view of plant evolution[END_REF]. Accordingly, on a few occasions, family merger has occurred where CYP83, CYP99, and CYP726 have been absorbed into the large CYP71 family, CYP723 has merged into the CYP89 family, and CYP705 is now viewed as a Brassicales-specific subfamily of CYP712. The old names are retained for historical reasons and to avoid confusion in the literature. On the contrary, family split has also occurred a couple of times, where addition of more sequences to the named collection resulted in a phylogenetic split. Consequently, CYP72B and CYP709A were renamed CYP734 and CYP735, respectively. Family split has also occurred for the CYP90B subfamily, which now groups with CYP724, but CYP90Bs have retained their original names to avoid confusion.

A new challenge for the nomenclature arises from adding evolutionarily distant CYPs. At more distant nodes, the CYP lineages converge to fewer ancestral sequences and this creates a naming problem. For example, two or more angiosperm CYP families may diverge from one gymnosperm family and each be >40% identical to that phylogenetic distinct gymnosperm family. Accordingly, the gymnosperm family must be given a new name and this breaks the 40% rule. The large fern-specific family CYP981 is an example where members of CYP981 are >40% identical to CYP92, CYP93, and CYP736 [START_REF] Thodberg | The fern CYPome: fernspecific cytochrome P450 family involved in convergent evolution of chemical defense[END_REF].

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of unusual plant CYP reactions [START_REF] Mizutani | Unusual P450 reactions in plant secondary metabolism[END_REF][START_REF] Zhang | Expansion of chemical space for natural products by uncommon P450 reactions[END_REF]. Generally, the mechanism of CYP attack is the same, and most of the unusual CYP reactions only diverge in a subsequent rearrangement of a reaction intermediate or a labile product [START_REF] Guengerich | Unusual cytochrome P450 enzymes and reactions[END_REF].

The diverse sets of reactions catalyzed by CYPs all contribute to expansion of the chemical space and decoration of carbon backbones with oxygen providing activated sites enabling further expansion of the chemical space by, for example, O-methylation, O-glycosylation and O-acylation. In this review, we provide an update on plant CYP families within green algae and land plants, and discuss molecular features and dynamics of CYP plasticity and evolution, highlighted along with specific examples from various biosynthetic pathways representing intriguing evolutionary events.

CYP FAMILIES THROUGH PLANT EVOLUTION

The plant lineage Viridiplantae covers green algae, land plants, and a recently defined new phylum of marine algae, the Prasinodermophyta, established to be the earliest diverging phylum within Viridi-plantae [START_REF] Li | The genome of Prasinoderma coloniale unveils the existence of a third phylum within green plants[END_REF]. Chlorophyte and charophyte algae are collectively known as green algae. Chlorophytes comprise 12 known algal classes, while charophytes comprise a paraphyletic assemblage of five algal classes in the division Streptophyta (charophyte algae and land plants) [START_REF] Guiry | How many species of algae are there?[END_REF][START_REF] Becker | Snow ball earth and the split of Streptophyta and Chlorophyta[END_REF]. The origin of land plants (embryophytes) may be traced back to terrestrial charophyte algae [START_REF] Delwiche | The evolutionary origin of a terrestrial flora[END_REF][START_REF] Harholt | Why plants were terrestrial from the beginning[END_REF]. Changes in biochemistry, accompanied with innovations in anatomy and signaling, have been a prerequisite for successful evolution of land plants [START_REF] Rensing | How plants conquered land[END_REF]. Appearance of some CYP families during plant evolution is directly linked to the occurrence of specific metabolite classes, such as carotenoids, phytohormones, oxygenated lipids, or phenylpropanoids. Given their prevalent occurrences in diverse plant metabolic pathways, CYPs can be used to infer the development of metabolic systems throughout plant evolution [START_REF] Nelson | A P450-centric view of plant evolution[END_REF][START_REF] Cannell | Multiple metabolic innovations and losses are associated with major transitions in land plant evolution[END_REF]. This section provides a status on occurrence of plant CYP families from chlorophyte algae to angiosperms, as an update to the 2011 review by Nelson and Werck-Reichhart. Catalytic functions of conserved CYP families dating back to green algae and CYP families with recently elucidated functions are emphasized.

Occurrence of CYP families

Many green algae CYP sequences and CYP genes from the newly sequenced unicellular marine algae Prasinoderma coloniale have been identified and named within the past decade. The P. coloniale genome [START_REF] Li | The genome of Prasinoderma coloniale unveils the existence of a third phylum within green plants[END_REF] contains 13 CYP genes in nine families (CYP51, CYP97, CYP710, CYP745, CYP746, CYP800, CYP804, CYP7196, and CYP7200). These may represent a sample of CYPs from the earliest green plants before chlorophytes and charophytes diverged. Chlorophyte algae have 1199 CYP sequences named from 117 species distributed in 275 CYP families, and charophyte algae have 1320 sequences named from 62 species distributed in 115 CYP families (supplemental data 1 and 2). A resource of named CYPs from green algae is provided in supplemental data 2. Algal CYPomes are smaller than those of vascular plants (tracheophytes) and represent up to 0.2% of protein-coding genes in some algal species. The chlorophyte Volvox carteri has 19 CYP genes, Trentepohlia annulata has 58, and most chlorophytes fall in between these sizes, like C. reinhardtii with 39 CYP genes. In the partially annotated genome of the charophyte Penium margaritaceum, 46 CYP genes have been recorded so far.

In green algae, CYPs are highly diversified. Green algae is the plant group featuring the highest number of CYP families, the majority of which are algae specific (Figure 1, supplemental data 3). To date, the number of algal CYP families has not shown signs of saturation and there are probably many more to be discovered. Only 41 CYP families are shared between chlorophyte and charophyte algae (about 12%), showing the significant divide between the two groups. Fungal CYP55 (nitric oxide synthase) is also present in chlorophytes, but not in charophytes, which, together with high sequence identity, might indicate lateral transfer of CYP55 after the two algal clades split.

Figure 2 shows the phylogenetic distribution of CYP families in green algae and land plants. CYP clans 51,74,97,710,711,and 746 The total number of known CYP families is divided into families that are shared between two or several taxonomic groups and CYP families that are specific to a given taxon. The estimated numbers of species in the nine taxa are: chlorophytes 13 000; charophytes 9000; hornworts 200-250; liverworts 7500; mosses 12 000; lycophytes 1300; ferns 10 500; gymnosperms 1000; angiosperms 300 000 [START_REF] Cox | Moss diversity: a molecular phylogenetic analysis of genera[END_REF][START_REF] Villarreal | A synthesis of hornwort diversity: patterns, causes and future work[END_REF][START_REF] Konrat | Early Land Plants Today (ELPT): how many liverwort species are there?[END_REF][START_REF] Guiry | How many species of algae are there?[END_REF][START_REF] Christenhusz | The number of known plants species in the world and its annual increase[END_REF][START_REF] Ppg | A community-derived classification for extant lycophytes and ferns[END_REF]. The number of CYP families found in land plants has shown signs of saturation, whereas more CYP families from green algae are expected to be identified. *The newly defined phylum within Viridiplantae, the Prasinodermophyta [START_REF] Li | The genome of Prasinoderma coloniale unveils the existence of a third phylum within green plants[END_REF], is not included in the figure .   1246 Molecular Plant 14, 1244-1265, August 2 2021 ª The Author 2021.

Molecular Plant

Plant cytochrome P450

Werck-Reichhart, 2011), but annotation of more algal sequences has changed this picture, revealing three green-algae-specific CYP families in the 51 clan, four in the 710 clan, no less than 46 in the 711 clan, and possibly two in the 746 clan (supplemental data 1). These clans are still represented by a single CYP family in land plants, and clans 74 and 97 remain single-family clans within Viridiplantae. Remarkably, classification of the many green algal sequences shows that all CYP clans known from land plants are present in green algae. In addition to the conserved clans, green algae possesses 14 algae-specific clans.

Families common to chlorophytes and charophytes that are conserved throughout land plants are CYP51, CYP74, CYP97, [START_REF] Nelson | The cytochrome P450 homepage[END_REF] and 105 of the green algae sequences were obtained from [START_REF] Cooper | Green algal transcriptomes for phylogenetics and comparative genomics[END_REF]. Amino acid sequences (954) were aligned using Clustal Omega and phylogeny was inferred using the neighbor-joining method. A high-resolution phylogenetic tree with tip labels is available in supplemental Figure 1. and CYP710. A couple of CYP families appeared in green algae and are conserved in some land plants but were lost during evolution (supplemental data 1). For example, the CYP746 family (clan 746) is present in algae, moss, liverworts, and a few ferns, but not in hornworts or seed plants (spermatophytes). This is the only example of a CYP clan that is present in early-diverging plant lineages and lost upon evolution of vascular plants. The CYP765 family (clan 72) is conserved from green algae to gymnosperms and lost in angiosperms.

Mining of the 1kP transcriptomes and genomes of early land plants revealed many taxaspecific families (Figure 1; supplemental data 1 and 2), some of which show great abundance, such as the fern-specific CYP981 family (clan 71), which is the richest CYP family in this taxon [START_REF] Thodberg | The fern CYPome: fernspecific cytochrome P450 family involved in convergent evolution of chemical defense[END_REF]. Other CYP families are shared between a few taxa (supplemental data 1). For example, family CYP825 (clan 71) is found in liverworts and hornworts, and families CYP7020 (clan 7020), CYP7332, and CYP7333 (both clan 71) are found in ferns and lycophytes. The new 7020 clan is present only in ferns and lycophytes. Other CYP families show yet a broader distribution, but are still limited to a few neighboring taxa. This applies to CYP947 family members (clan 85) that are spotted only in ferns, lycophytes, and gymnosperms. A list of named CYPs, including protein sequences from bryophytes, lycophytes, ferns, and gymnosperms, is available in supplemental data 2.

Functionalities of conserved CYP families

The CYP families CYP51, CYP710, CYP97, CYP74, and CYP86 are conserved from green algae and throughout land plants, carrying out essential catalytic reactions in metabolism, and five families (CYP51, CYP710, CYP97, CYP74, and CYP704) are present across several kingdoms. CYP704 is, however, not found in green algae. The families tend to be maintained as a single copy or at low copy numbers, although retention of duplicates and recruitment to a specialized pathway also occur.

CYP51G functions as an obtusifoliol 14a-demethylase in the biosynthesis of membrane sterols (e.g., [START_REF] Bak | Cloning and expression in Escherichia coli of the obtusifoliol 14a-demethylase of Sorghum bicolor (L.) Moench, A cytochrome P450 orthologous to the sterol 14a-demethylases (CYP51) from fungi and mammals[END_REF]. This essential CYP51 function is conserved in other kingdoms, like fungi and animals, but with enzymes using lanosterol as substrate. Grasses (family Poaceae) have evolved a new subfamily, CYP51H, with eight genes in O. sativa and six in oat (Avena strigosa). The CYP51H10 gene (also known as SAD2) from A. strigosa has been characterized. It catalyzes epoxidation and hydroxylation of b-amyrin in the formation of the toxin avenacin [START_REF] Qi | A different function for a member of an ancient and highly conserved cytochrome P450 family: from essential sterols to plant defense[END_REF][START_REF] Kunii | b-Amyrin oxidation by oat CYP51H10 expressed heterologously in yeast cells: the first example of CYP51-dependent metabolism other than the 14-demethylation of sterol precursors[END_REF][START_REF] Geisler | Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants[END_REF].

CYP710A is a sterol C-22 desaturase, also contributing to the formation of membrane sterols in land plants (Morikawa et al., 2006a). Occurrence of sterols containing a double bond at C-22 in the sterol side chain is a characteristic of land plant and fungi sterol profiles [START_REF] Morikawa | Cytochrome P450 subfamily CYP710A genes encode sterol C-22 desaturase in plants[END_REF], and CYP710 shares a common ancestor with its functional homologs in fungi (CYP61). Subfamily CYP710A is specific to Streptophyta, while subfamily CYP710B is present in green algae. CYP710 genes are also found in other algae and in some protists, documenting that the family is very ancient.

The CYP97 family includes key enzymes in the biosynthesis of carotenoid pigments, which have vital roles in photosynthesis and photoprotection. CYP97 genes are not restricted to the plant kingdom and have also been identified in several protists. Some protists have plastids acquired by tertiary symbiosis of green algae [START_REF] Keeling | The endosymbiotic origin, diversification and fate of plastids[END_REF], and they may have obtained CYP97 genes by such symbiosis. Plant CYP97s catalyze ring hydroxylation of a-and b-carotenes and their monohydroxylated derivatives, a function that is conserved from green algae and through land plants [START_REF] Kim | The evolution and function of carotenoid hydroxylases in Arabidopsis[END_REF][START_REF] Liang | Functional identification of two types of carotene hydroxylases from the green alga Dunaliella bardawil rich in lutein[END_REF]. Neofunctionalization of a CYP97 for production of the acyclic diterpene plaunotol, known for its anti-gastric-ulcer properties, has been reported for Croton stellatopilosus. Here, CsCYP97C27 catalyzes C18-hydroxylation of geranylgeraniol to form plaunotol [START_REF] Sintupachee | Molecular cloning, bacterial expression and functional characterisation of cytochrome P450 monooxygenase, CYP97C27, and NADPH-cytochrome P450 reductase, CPR I, from Croton stellatopilosus Ohba[END_REF].

The plant CYP74 family has previously been dated back to liverworts [START_REF] Nelson | A P450-centric view of plant evolution[END_REF], but investigation of more algae and early land plant datasets shows presence of CYP74 members in hornworts, charophytes, and freshwater chlorophytes. CYP74 genes have also been identified in some animal species, such as cnidarians. CYP74s may thus predate the split between animals and plants or the cnidarian copies must be a result of lateral transfer. The rarity of CYP74 outside the plants favors a lateral transfer otherwise many gene losses would be required. Members of the plant CYP74 family are involved in the metabolism of oxylipins, including jasmonic acid, which functions in cell signaling and defense [START_REF] Wasternack | The oxylipin pathways: biochemistry and function[END_REF][START_REF] Farmer | Jasmonates: what ALLENE OXIDE SYNTHASE does for plants[END_REF]. They are atypical CYPs, not requiring electron donation and molecular oxygen, and use fatty acid hydroperoxides as substrates. Land plants typically contain a few CYP74 genes from different subfamilies. CYP74As usually function as allene oxide synthases (AOSs) to form 12-oxo-phytodienoic acid, a precursor of the essential plant signaling compounds jasmonic acid and derivatives. Other CYP74s also metabolize fatty acid hydroperoxides, but act as hydroperoxide lyases (HPLs), divinyl ether synthases (DESs), or epoxyalcohol synthases (EASs). The charophyte Klebsormidium flaccidum contains a single CYP74 (CYP74Z1), which functions as an AOS [START_REF] Koeduka | Biochemical characterization of allene oxide synthases from the liverwort Marchantia polymorpha and green microalgae Klebsormidium flaccidum provides insight into the evolutionary divergence of the plant CYP74 family[END_REF]. The presence of a single CYP74 has also been observed in the charophytes Klebsormidium subtile, Coleochaete scutata, and Entransia fimbriata. In addition to angiosperms, AOS, HPL, and DES activities have been identified in bryophytes and lycophytes [START_REF] Scholz | Biosynthesis of allene oxides in Physcomitrella patens[END_REF][START_REF] Koeduka | Biochemical characterization of allene oxide synthases from the liverwort Marchantia polymorpha and green microalgae Klebsormidium flaccidum provides insight into the evolutionary divergence of the plant CYP74 family[END_REF][START_REF] Gorina | Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: molecular cloning and identification of divinyl ether synthases CYP74M1 and CYP74M3[END_REF]. These activities are scattered on various CYP74 subfamilies, suggesting that the activities have arisen multiple times in plant evolution. Along this line, a single or few mutations have been shown sufficient to partly or fully change AOS to HPL activity [START_REF] Lee | Structural insights into the evolutionary paths of oxylipin biosynthetic enzymes[END_REF][START_REF] Toporkova | Determinants governing the CYP74 catalysis: conversion of allene oxide synthase into hydroperoxide lyase by site-directed mutagenesis[END_REF], DES to AOS activity [START_REF] Toporkova | Structurefunction relationship in the CYP74 family: conversion of divinyl ether synthases into allene oxide synthases by site-directed mutagenesis[END_REF], or AOS to EAS activity [START_REF] Toporkova | Catalysis by allene oxide synthases (CYP74A and CYP74C): alterations by the Phe/Leu mutation at the SRS-1 region[END_REF], showing that only a few amino acid changes are needed for a diverse set of functional interconversions.

Presence of CYP86 members across the plant lineage has also been broadened and now dates back to chlorophytes. Notably, this family has more subfamilies than the other conserved CYP families (supplemental data 1). CYP86s catalyze oxygenation of fatty acids at the omega position [START_REF] Bak | Cytochromes p450[END_REF], forming hydroxylated fatty acids, but also more oxidized derivatives such as dicarboxylic acids. Thereby, they generate a broad range of precursors for formation of waterproof biopolymers like cutin and suberin, essential for water transport or protection from water loss. CYP86 in green algae were thus most likely required to protect spores from desiccation, and they represented a critical asset for plant terrestrialization. Besides, CYP86s have also been reported to positively regulate the plant immune system, with high impact on defense against pathogens both in A. thaliana and sea island cotton (Gossypium barbadense) [START_REF] Xiao | Arabidopsis CYP86A2 represses Pseudomonas syringae type III genes and is required for cuticle development[END_REF]Wang et al., 2020a). CYP704, which is ancestral to CYP86 and is also described as a fatty acid u-hydroxylase in angiosperms [START_REF] Dobritsa | CYP704B1 is a long-chain fatty acid omega-hydroxylase essential for sporopollenin synthesis in pollen of Arabidopsis[END_REF], is present in land plants, protists, and red algae but was lost in green algal lineages.

CYP families with recently elucidated functionalities

Despite numerous CYPs being characterized in recent years, several CYP families are still without known function. On the positive side, functions have been assigned to some members of CYP families that were listed as orphan in the 2011 review. These include family CYP716 from the clan 85 and families CYP712, CYP736, and CYP750 from the 71 clan. In addition, the role of CYP711s (711 clan) and CYP722s (clan 85) in strigolactone biosynthesis is now better understood.

Molecular Plant

Plant cytochrome P450 involved in brassinosteroid metabolism, resulting in activities contributing to biosynthesis of triterpenoids with a defense function in land plants. Several CYP716As catalyze a threestep C28 oxidation of b-amyrin, a-amyrin, and/or lupeol, but oxygenation of various carbon atoms in the triterpene backbone has also been reported (e.g., [START_REF] Fukushima | ª The Author 2021. 1259 Plant cytochrome P450 Molecular Plant subfamily members are multifunctional oxidases in triterpenoid biosynthesis[END_REF][START_REF] Miettinen | The ancient CYP716 family is a major contributor to the diversification of eudicot triterpenoid biosynthesis[END_REF]. A few CYP712Ks were found to oxygenate the triterpenoid friedelin at C29, resulting in formation of polpunoic acid in the biosynthesis of celastrol [START_REF] Bicalho | CYP712K4 catalyzes the C-29 oxidation of friedelin in the Maytenus ilicifolia quinone methide triterpenoid biosynthesis pathway[END_REF][START_REF] Hansen | Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol[END_REF]. Some members of the CYP736 family oxygenate small, often volatile compounds: CYP736A2 from Lotus japonicus converts valine-and isoleucine-derived oximes to their corresponding a-hydroxynitriles in cyanogenic glucoside biosynthesis [START_REF] Takos | Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway[END_REF], CYP736A107 from Sorbus aucuparia and CYP736A163 from Malus 3 domestica are biphenyl 4-hydroxylases [START_REF] Sircar | Biphenyl 4-hydroxylases involved in aucuparin biosynthesis in rowan and apple are cytochrome P450 736A proteins[END_REF], and CYP736A167 from Santalum album hydroxylates sesquiterpenes like santalene and bergamotene [START_REF] Celedon | Heartwood-specific transcriptome and metabolite signatures of tropical sandalwood (Santalum album) reveal the final step of (Z)santalol fragrance biosynthesis[END_REF]. CY736A304 and CYP736A305 from Nicotiana attenuata produce 17-hydroxygeranyllinalool for defense against herbivores [START_REF] Li | Controlled hydroxylations of diterpenoids allow for plant chemical defense without autotoxicity[END_REF]. CYP750B1 catalyzes hydroxylation of the monoterpene sabinene in thujone biosynthesis [START_REF] Gesell | The gymnosperm cytochrome P450 CYP750B1 catalyzes stereospecific monoterpene hydroxylation of (+)-sabinene in thujone biosynthesis in western redcedar[END_REF]. CYP749A1 has been linked to the arising tolerance to herbicides in upland cotton (Gossypium hirsutum), but its activity in plant metabolism is still unknown [START_REF] Thyssen | The P450 gene CYP749A16 is required for tolerance to the sulfonylurea herbicide trifloxysulfuron sodium in cotton (Gossypium hirsutum L.)[END_REF].

Strigolactones are involved in the symbiotic relation between land plants and soil arbuscular mycorrhiza and function as endogenous plant hormones [START_REF] Moreno | Plant apocarotenoids: from retrograde signaling to interspecific communication[END_REF]. In land plants, CYP711As (also known as MAX1) oxygenate the intermediate carlactone in strigolactone biosynthesis [START_REF] Abe | Carlactone is converted to carlactonoic acid by MAX1 in Arabidopsis and its methyl ester can directly interact with AtD14 in vitro[END_REF][START_REF] Zhang | Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis[END_REF][START_REF] Zhang | The tomato MAX1 homolog, SlMAX1, is involved in the biosynthesis of tomato strigolactones from carlactone[END_REF][START_REF] Yoneyama | Conversion of carlactone to carlactonoic acid is a conserved function of MAX1 homologs in strigolactone biosynthesis[END_REF][START_REF] Mori | Chemical identification of 18-hydroxycarlactonoic acid as an LjMAX1 product and in planta conversion of its methyl ester to canonical and noncanonical strigolactones in Lotus japonicus[END_REF]. Most of the functionally characterized CYP711As convert carlactone to carlactonoic acid, but Selaginella moellendorffii CYP711A17v1 and CYP711A17v3 and O. sativa CYP711A2 further catalyze the BC-ring closure proceeding via 18-hydroxy carlactonoic acid and resulting in 4-deoxyorobanchol. In addition, hydroxylation of 4-deoxyorobanchol to orobanchol has been shown for Os-CYP711A3 [START_REF] Zhang | Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis[END_REF][START_REF] Yoneyama | Conversion of carlactone to carlactonoic acid is a conserved function of MAX1 homologs in strigolactone biosynthesis[END_REF]. CYP722Cs act downstream of the most common CYP711A reaction and convert carlactonoic acid to orobanchol, or to the stereoisomer 5-doxystrigol in some plants such as tree cotton (Gossypium arboreum) [START_REF] Wakabayashi | Direct conversion of carlactonoic acid to orobanchol by cytochrome P450 CYP722C in strigolactone biosynthesis[END_REF][START_REF] Wakabayashi | CYP722C from Gossypium arboreum catalyzes the conversion of carlactonoic acid to 5-deoxystrigol[END_REF].

In some other cases, such as for CYP715 or CYP720A, activities in hormonal pathways have been suggested by observed physiological roles in flower development as revealed by null mutants, but no substrate was assigned [START_REF] Liu | A conserved cytochrome P450 evolved in seed plants regulates flower maturation[END_REF][START_REF] Chaturvedi | CYP720A1 function in roots is required for flowering time and systemic acquired resistance in the foliage of Arabidopsis[END_REF]. Some conifer-specific CYP720Bs, however, oxygenate several diterpene backbones [START_REF] Ro | Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is a multifunctional, multisubstrate cytochrome P450 monooxygenase[END_REF][START_REF] Hamberger | Evolution of diterpene metabolism: Sitka spruce CYP720B4 catalyzes multiple oxidations in resin acid biosynthesis of conifer defense against insects[END_REF][START_REF] Geisler | Modularity of conifer diterpene resin acid biosynthesis: P450 enzymes of different CYP720B clades use alternative substrates and converge on the same products[END_REF], which might constitute a lead to the family activity in hormone metabolism. The functions of CYP78s that were already known to control organ growth have been extended to cell fate maintenance and control of defense against a broad spectrum of pathogens, still without identification of the signal generated [START_REF] Maeda | The rice CYP78A gene BSR2 confers resistance to Rhizoctonia solani and affects seed size and growth in Arabidopsis and rice[END_REF][START_REF] Poretska | AMP1 and CYP78A5/7 act through a common pathway to govern cell fate maintenance in Arabidopsis thaliana[END_REF].

Only very few CYPs from taxa-specific CYP families in early land plants have been functionally characterized. These include CYP788A1 from the lycophyte S. moellendorffii [START_REF] Weng | Convergent evolution of syringyl lignin biosynthesis via distinct pathways in the lycophyte Selaginella and flowering plants[END_REF], CYP964A1 and CYP970A14 from the moss Calohypnum plumiforme [START_REF] Mao | Genomic evidence for convergent evolution of gene clusters for momilactone biosynthesis in land plants[END_REF], and CYP981F5 from the fern Phlebodium aureum [START_REF] Thodberg | The fern CYPome: fernspecific cytochrome P450 family involved in convergent evolution of chemical defense[END_REF]. Notably, these CYPs enable the species to produce compounds already known from angiosperms through similar or alternative metabolic routes (discussed further below).

EVOLUTION OF THE PHENYLPROPANOID PATHWAY MIRRORS LAND PLANT EVOLUTION

The CYP73, CYP98, and CYP84 families involved in the phenylpropanoid metabolic network eminently reflect some important evolutionary events in plant metabolism, both in terms of functions in the biosynthesis of key metabolites important for terrestrialization of plants and as illustrative examples of lineage-specific gene duplication and functional specialization.

Phenylpropanoids play key roles in the evolution of terrestrial plants, functioning as building blocks for structural biopolymers such as lignin, suberin, cutin, and sporopollenin, ensuring mechanical support, water retention and transport, but also serving as UV protectants, antioxidants, and defense compounds. Members of the CYP73/CYP98 clade are present in charophytes, possibly representative of their common ancestor (Renault et al., 2017b), but both CYP73 and CYP98 simultaneously emerged 450 mya when bryophytes colonized land. They show very different duplication and diversification modes along land plant evolution.

CYP73

The second step in the core phenylpropanoid pathway is catalyzed by CYP73 (C4H) and is a para-hydroxylation of cinnamic acid (Figure 3). The CYP73 family is a low-copy-number family under high purifying selection with usually one to five CYP73 genes detected in plant genomes. In seed plants, a conserved gene duplication has occurred prior to the split between gymnosperms and angiosperms giving rise to an additional CYP73 (also called class II CYP73) (Renault et al., 2017b). While retained in many species, it was lost in most gymnosperms and in Brassicaceae. A second fixed gene duplication occurred in monocots. Functional analysis of the three CYP73 members from Brachypodium distachyon demonstrated highly efficient cinnamic acid hydroxylase activity for all of them. The class I and II enzymes, however, differ in the length of the N-terminal membrane anchor, where the class II CYP73 has an elongated anchor causing altered membrane topology. The exact physiological consequences of this feature are still unknown, but it might affect enzyme mobility and interaction with redox or pathway partners. Current data thus indicate that fixation of gene duplicates does not necessarily depend on altered catalytic activities but may result from other functional aspects leading to fitness increase. This argues that CYP localization, topology, but also expression pattern analyses in the cause of plant ontogeny need to be investigated to guide proper assessments of their physiological functions.

Molecular Plant

CYP98

At the entry of the lignin branch of the phenylpropanoid network, CYP98 members catalyze 3 0 -hydroxylation of p-coumaroyl-shikimate (Figure 3). They also efficiently catalyze hydroxylation of pcoumaroyl-quinate for the formation of chlorogenic acid. Coffea canephora, which accumulates chlorogenic acid as a major soluble phenolic, has two CYP98s. One of them (CcCYP98A36) shows a clear preference for p-coumaroyl-shikimate, and the other (CcCYP98A35) has specialized toward production of chlorogenic acid while maintaining activity with p-coumaroylshikimate [START_REF] Mahesh | Functional characterization of two pcoumaroyl ester 3'-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis[END_REF].

Despite its essential role in lignin biosynthesis in vascular plants, the appearance of CYP98 predates the occurrence of the lignin biopolymer. Genome mining for CYP98 members across the plant kingdom identified single CYP98 copies in bryophytes, lycophytes, ferns, and gymnosperms, whereas angiosperm genomes often contain more than one gene copy (Alber et al., 2019). Despite absence of lignin in moss, the P. patens CYP98 is essential for plant 3D-development. Null mutation of PpCYP98A34 leads to defects in gametophore development, in the biosynthesis of the major soluble phenolic derivative caffeoyl-threonate, and in the formation of the cuticle polymer (Renault et al., 2017a). Thus, already in early land plants, the canonical CYP98 has been required for plant aerial growth and for the formation of essential biopolymers. Early on, it used the p-coumaroyl shikimate ester as a main substrate for phenolic ring meta-hydroxylation forming a regulatory shikimate-sensing loop for the biochemical regulation of the lignin pathway [START_REF] Kriegshauser | Function of the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE is evolutionarily conserved in embryophytes[END_REF].

Phylogenetic analysis shows several CYP98 gene duplications within various angiosperm linages that have occurred independently [START_REF] Alber | Evolution of coumaroyl conjugate 3-hydroxylases in land plants: lignin biosynthesis and defense[END_REF]. Consistent with relaxed selection on one of the duplicates, promiscuity consistently increases after gene duplication favoring acquisition of new functions. For example, T. aestivum (monocot, Poales), Arabidopsis thaliana (eudicot, Brassicales), and Populus trichocarpa (eudicot, Malpighiales) all contain three CYP98s, where the duplicate members have arisen independently in the different linages and where some of the additional CYP98 copies have functionally diversified with distinct outcomes in the different species (Figure 4) [START_REF] Morant | Catalytic activity, duplication and evolution of the CYP98 cytochrome P450 family in wheat[END_REF][START_REF] Matsuno | Evolution of a novel phenolic pathway for pollen development[END_REF][START_REF] Alber | Evolution of coumaroyl conjugate 3-hydroxylases in land plants: lignin biosynthesis and defense[END_REF]. The physiological implications for duplicate retention are best understood for A. thaliana.

The three A. thaliana CYP98s are named CYP98A3, CYP98A8, and CYP98A9. A retrotransposition of the canonical CYP98 occurred early during evolution of Brassicales and a later tandem duplication after divergence of the genus Aethionema gave rise to AtCYP98A8 and AtCYP98A9 [START_REF] Matsuno | Evolution of a novel phenolic pathway for pollen development[END_REF][START_REF] Liu | Evolutionary interplay between sister cytochrome P450 genes shapes plasticity in plant metabolism[END_REF]. AtCYP98A3 has maintained the original p-coumaroylshikimate 3 0 -hydroxylation activity, whereas AtCYP98A8 and AtCYP98A9 have functionally diversified and have been recruited to an anther-specific phenolamide pathway to produce di(hydroxyferuloyl)-sinapoylspermidine, which is a major (A) Proposed CYP98 gene duplication and functional divergence in T. aestivum [START_REF] Morant | Catalytic activity, duplication and evolution of the CYP98 cytochrome P450 family in wheat[END_REF], A. thaliana [START_REF] Matsuno | Evolution of a novel phenolic pathway for pollen development[END_REF][START_REF] Liu | Evolutionary interplay between sister cytochrome P450 genes shapes plasticity in plant metabolism[END_REF], and P. trichocarpa [START_REF] Alber | Evolution of coumaroyl conjugate 3-hydroxylases in land plants: lignin biosynthesis and defense[END_REF]. Boxes with a thick frame represent the essential paralog dedicated to the synthesis of lignin precursors and that is expressed in lignified tissues. An, ancestor; D, duplication; R, retrotransposition; WGD, whole-genome duplication. Plant cytochrome P450 constituent of the pollen coat and helps in structuring the pollen wall [START_REF] Matsuno | Evolution of a novel phenolic pathway for pollen development[END_REF][START_REF] Xu | ABORTED MICROSPORES acts as a master regulator of pollen wall formation in Arabidopsis[END_REF]. In the diversification process, the original shikimate ester hydroxylase activity was lost. Interestingly, the new pathway seems to have emerged from a minor promiscuous activity in the common ancestor still detectable in AtCYP98A3. Homology modeling predicts increased flexibility and enlarged substrate-access channel and active-site cavity in AtCYP98A8 and AtCYP98A9 compared with AtCYP98A3, enabling metabolism of the larger spermidineconjugated substrates. The production of di(hydroxyferuloyl)sinapoylspermidine from tri-coumaroyl-spermidine is dependent on both 3 0 -and 5 0 -hydroxylations of the three phenol rings. At-CYP98A9 has 3 0 -hydroxylase activity, whereas AtCYP98A8 possesses 3 0 -as well as 5 0 -hydroxylase activities in vitro. However, mutant studies show that both CYP98s are necessary to achieve normal levels of di(hydroxyferuloyl)-sinapoylspermidine [START_REF] Matsuno | Evolution of a novel phenolic pathway for pollen development[END_REF][START_REF] Liu | Evolutionary interplay between sister cytochrome P450 genes shapes plasticity in plant metabolism[END_REF]. In addition, AtCYP98A9 overexpression lines point to a role in the metabolism of soluble flavonoids, and naringenin 3 0 -hydroxylase activity was observed with CYP98A9 in vitro [START_REF] Liu | Evolutionary interplay between sister cytochrome P450 genes shapes plasticity in plant metabolism[END_REF]. Evolution of the CYP98A8/ CYP98A9 cluster within Brassicales is complex: some genera have lost the CYP98A9 ortholog, whereas their CYP98A8 ortholog has maintained dual 3 0 -and 5 0 -hydroxylase activity on spermidine conjugates.

In other plant species, duplication and neofunctionalization of CYP98 genes have resulted in the ability to carry out hydroxylations in rosmarinic acid biosynthesis. This applies to CYP98A6 from Lithospermum erythrorhizon [START_REF] Matsuno | CYP98A6 from Lithospermum erythrorhizon encodes 4coumaryl-4'-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis[END_REF], CYP98A14 from Coleus blumei [START_REF] Eberle | cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from Coleus blumei involved in rosmarinic acid biosynthesis[END_REF], and CYP98A112 and CYP98A113 from Phavelia campanularia [START_REF] Levsh | Independent evolution of rosmarinic acid biosynthesis in two sister families under the Lamiids clade of flowering plants[END_REF], all species belong to the Lamiids clade. Phylogenetics combined with biochemical analyses showed that rosmarinic acid biosynthesis has evolved independently at least twice in the Lamiids [START_REF] Levsh | Independent evolution of rosmarinic acid biosynthesis in two sister families under the Lamiids clade of flowering plants[END_REF]. Hydroxylation at the 3 and 3 0 position of 4-coumaroyl-4 0 -hydroxyphenyllactic acid is needed to form rosmarinic acid. Although LeCYP98A14 catalyzes hydroxylation at both the 3 and 3 0 position (but most efficiently at the 3 position), PcCYP98A112, PcCYP98A113, and CbCYP98A6 carry out monohydroxylations. Although rosmarinic acid is mainly known from species belonging to the Lamiids, the compound sporadically appears throughout land plant phylogeny, as early as hornworts and ferns [START_REF] Petersen | Evolution of rosmarinic acid biosynthesis[END_REF]. CYP98 members are present in all the plant lineages currently known to accumulate rosmarinic acid, thus more independent CYP98 specialization events are expected to be associated to the occurrence of rosmarinic acid throughout the plant kingdom.

CYP84

In the series of reactions required for the formation of lignin monomers, a conserved CYP84 (F5H) catalyzes the 5-hydroxylation of coniferaldehyde, the branch point toward S lignin precursors [START_REF] Meyer | Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis[END_REF][START_REF] Humphreys | New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase[END_REF]. In A. thaliana, the 5-hydroxylase CYP84A1 has a paralog, AtCYP84A4, originating from a gene duplication event after the divergence of the Arabidopsis genus [START_REF] Weng | Assembly of an evolutionary new pathway for a-pyrone biosynthesis in Arabidopsis[END_REF]. AtCYP84A4 has neofunctionalized to 3-hydroxylate p-coumaraldehyde. It forms caffealdehyde and thereby establishes a new branch toward production of arabidopyrones, a group of Arabidopsis-specific compounds. Noteworthy, AtCYP84A1 shows weak pcoumaraldehyde 3-hydroxylase activity in vitro, although with a Km value more than 100 times higher than for coniferaldehyde [START_REF] Weng | Convergent evolution of syringyl lignin biosynthesis via distinct pathways in the lycophyte Selaginella and flowering plants[END_REF][START_REF] Weng | Assembly of an evolutionary new pathway for a-pyrone biosynthesis in Arabidopsis[END_REF]. The CYP84 family is only present in angiosperms. However, S lignin is also found in the lycophyte order Selaginellales. In S. moellendorffii, a lineage-specific CYP788A1 has been documented to catalyze formation of S lignin via an alternative route [START_REF] Weng | Independent origins of syringyl lignin in vascular plants[END_REF][START_REF] Weng | Convergent evolution of syringyl lignin biosynthesis via distinct pathways in the lycophyte Selaginella and flowering plants[END_REF] (Figure 3). Both CYP84 and CYP788 belong to the 71 clan.

MULTIPLICITY OF SUBSTRATES

Increasing numbers of studies report CYPs with multiple physiological substrates or demonstrate previously overlooked substrate promiscuity. The degree of observed promiscuity is dependent on the number of tested compounds and the range of compound classes tested. Such studies are thus never expected to be exhaustive.

Plant CYP substrate promiscuity can translate into cascade oxidations on a same carbon with various outcomes (e.g., [START_REF] Bak | Cloning and expression in Escherichia coli of the obtusifoliol 14a-demethylase of Sorghum bicolor (L.) Moench, A cytochrome P450 orthologous to the sterol 14a-demethylases (CYP51) from fungi and mammals[END_REF][START_REF] Helliwell | Arabidopsis ent-kaurene oxidase catalyzes three steps of gibberellin biosynthesis[END_REF][START_REF] Cankar | A chicory cytochrome P450 monooxygenase CYP71AV8 for the oxidation of (+)-valencene[END_REF], multiple attacks at different positions to catalyze consecutive steps in a same pathway as found in a large diversity of pathways (e.g., [START_REF] Holton | Cloning and expression of cytochrome P450 genes controlling flower colour[END_REF][START_REF] Eberle | cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from Coleus blumei involved in rosmarinic acid biosynthesis[END_REF][START_REF] Matsuno | Evolution of a novel phenolic pathway for pollen development[END_REF][START_REF] Ralston | Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum)[END_REF][START_REF] Bathe | CYP76 oxidation network of abietane diterpenes in Lamiaceae reconstituted in yeast[END_REF][START_REF] Zabel | A single cytochrome P450 oxidase from Solanum habrochaites sequentially oxidizes 7-epi-zingiberene to derivatives toxic to whiteflies and various microorganisms[END_REF][START_REF] Rodriguez-Lopez | Two bi-functional cytochrome P450 CYP72 enzymes from olive (Olea europaea) catalyze the oxidative C-C bond cleavage in the biosynthesis of secoxy-iridoids -flavor and quality determinants in olive oil[END_REF][START_REF] Nett | Discovery and engineering of colchicine alkaloid biosynthesis[END_REF], or in multifunctional enzymes sometimes using completely different catalytic mechanisms [START_REF] Dang | Dual catalytic activity of a cytochrome P450 controls bifurcation at a metabolic branch point of alkaloid biosynthesis in Rauwolfia serpentina[END_REF][START_REF] Sibbesen | Cytochrome P-450TYR is a multifunctional heme-Thiolate enzyme catalyzing the conversion of L-tyrosine to phydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench[END_REF]. However, it is most commonly encountered as conversion of multiple natural or man-made compounds showing similar structural and/or physicochemical properties. The latter is usually referred to as ''permissiveness'' and may be further sub-divided into enzymes utilizing multiple physiological relevant substrates and enzymes being able to metabolize other compounds than its evident physiological relevant substrate. Permissiveness is our main focus in this review since it is considered an important driver of enzyme evolution [START_REF] Aharoni | The 'evolvability' of promiscuous protein functions[END_REF]. The main structural features controlling CYP's permissiveness are illustrated in Figure 5A. It should be mentioned that product promiscuity can be also observed, for example, upon scaffold formation and resulting from intermediate rearrangement and/or associated with the presence of different reductases (e.g., [START_REF] Li | Functional genomic analysis of alkaloid biosynthesis in Hyoscyamus niger reveals a cytochrome P450 involved in littorine rearrangement[END_REF][START_REF] Kellner | Discovery of a P450catalyzed step in vindoline biosynthesis: a link between the aspidosperma and eburnamine alkaloids[END_REF][START_REF] Kilgore | CYP96T1 of Narcissus sp. aff. Pseudonarcissus catalyzes formation of the para-para' C-C phenol couple in the Amaryllidaceae alkaloids[END_REF]. Altogether, substrate and product promiscuity allows the formation of complex metabolic networks and production of a series of differently oxygenated molecules with a reduced set of CYPs.

Many terpenoid CYPs show broad substrate range

CYPs metabolizing highly hydrophobic substrates (e.g., terpenoids) have a tendency to show a higher substrate range, possibly because their access channels and active sites are essentially lined by hydrophobic residues and hence are less selective, at least with regard to hydrophobic compounds. This is, for example, illustrated by AtCYP706A3, a CYP with two narrow hydrophobic substrate-access channels and a small hydrophobic substrate-binding site, which was demonstrated to metabolize more than 20 different mono-and sesquiterpenes and their oxidation products, both in vitro and in A. thaliana flowers as a defense response against florivores [START_REF] Boachon | A promiscuous CYP706A3 reduces terpene volatile emission from Arabidopsis flowers, affecting florivores and the floral microbiome[END_REF]. Another

Molecular Plant

Plant cytochrome P450 example is the multifunctional CYP720B4 from Sitka spruce (Picea sitchensis) belonging to the conifer-specific CYP720B subfamily. PsCYP720B4 catalyze consecutive C18 oxidations on eight diterpene backbones when expressed in yeast, generating products that are also observed in the diterpenoid profile of P. sitchensis [START_REF] Hamberger | Evolution of diterpene metabolism: Sitka spruce CYP720B4 catalyzes multiple oxidations in resin acid biosynthesis of conifer defense against insects[END_REF].

Promiscuous tendencies are also observed for CYPs with conserved functions. They can be found in the phenolic pathways as mentioned above for AtCYP98A3 and AtCYP84A1, but are also reported for hormonal metabolism. Ent-kaurene oxidases (KO, CYP701 members) oxidize ent-kaurene at C19 to form kaurenoic acid in the pathway toward gibberellins, a function that is conserved across land plants [START_REF] Miyazaki | The CYP701B1 of Physcomitrella patens is an ent-kaurene oxidase that resists inhibition by uniconazole-P[END_REF][START_REF] Nelson | A P450-centric view of plant evolution[END_REF]. Testing of multiple substrates for A. thaliana CYP701A3 (AtKO) and O. sativa CYP701A6 (OsKO2) showed that AtCYP701A3 can oxygenate several labdane-related diterpene substrates, although with lower affinity and efficiency than for ent-kaurene, whereas OsCYP701A6 is more specific for ent-kaurene and ent-isokaurene [START_REF] Morrone | Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis[END_REF][START_REF] Mafu | Probing the promiscuity of ent-kaurene oxidases via combinatorial biosynthesis[END_REF]. It was speculated that the metabolic background of labdane-related diterpenes in O. sativa played a role in determining the specificity of OsCYP701A6 for this conserved biosynthetic step [START_REF] Mafu | Probing the promiscuity of ent-kaurene oxidases via combinatorial biosynthesis[END_REF]. O. sativa harbors five CYP701A genes. One of the additional copies, CYP701A8 (OsKOL4), has diversified to hydroxylate the C3 of the diterpenes sandaracopimaridiene and cassadiene, which are expected intermediates in the biosynthesis of oryzalexin and phytocassane phytoalexins, respectively [START_REF] Wang | CYP701A8: a rice ent-kaurene oxidase paralog diverted to more specialized diterpenoid metabolism[END_REF][START_REF] Wu | Picking sides: distinct roles for CYP76M6 and CYP76M8 in rice oryzalexin biosynthesis[END_REF]. CYP701A8 is still able to metabolize ent-kaurene, maintaining some promiscuity after neofunctionalization.

Multisubstrate functionalities can shape metabolism as nonlinear biosynthetic pathways, as illustrated by CYP76AHs in (A) Structural and chemical properties of the active site (green) and the access channel(s) (blue) are determinants of the range of substrates that can be metabolized. Flexible residues located opposite to the main access channel may allow for opening and enhanced access to the active site. New interaction partners and availability of different substrates also contribute to evolution toward new functionalities. (B) Permissive enzymes are more likely to provide starting points for functional divergences, which may be selected for if they confer an immediate physiological advantage.

forskolin biosynthesis in Plectranthus barbatus (often referred to as Coleus forskohlii) [START_REF] Pateraki | Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii[END_REF], or as an hourglass-shaped biosynthetic network as reported in maize (Zea mays) involving CYP71Zs and CYP81As [START_REF] Ding | Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity[END_REF]. In the latter case, three tandemly associated CYP71Z genes form a resilient cluster encoding versatile enzymes that contribute to several sesquiterpenoid and diterpenoid defense pathways (zealexins, kauralexins, dolabralexins and costic acid), mainly forming carboxylic acids [START_REF] Mafu | Discovery, biosynthesis and stress-related accumulation of dolabradiene-derived defenses in maize[END_REF][START_REF] Ding | Multiple genes recruited from hormone pathways partition maize diterpenoid defences[END_REF][START_REF] Ding | Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity[END_REF]. A different cluster of three genes codes for permissive CYP81A enzymes catalyzing diverse oxygenation and desaturation reactions more focused on sesquiterpene acidic derivatives to generate the diversity of zealexins [START_REF] Ding | Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity[END_REF]. Permissiveness thereby allows for formation of an efficient, diversified arsenal of defensive compounds.

CYP promiscuity as a seed for new functions

Promiscuity may facilitate the ability of plants to adapt to environmental challenges such as the formation of deterrents to new herbivores and pests and thereby may represent an evolutionary advantage. For these reasons, promiscuous CYPs can be speculated to show faster functional diversification to acquire new specialized functions, with promiscuous activities being selected for when they provide a physiological advantage (Figure 5B). In this context, exploring promiscuity of CYPs can provide insight into underlying mechanisms for evolution of phytochemical profiles. Examples are provided by the above-mentioned CYP98 enzymes in the phenolic metabolism, but further examples can be found in other pathways.

Sesquiterpene lactones are characteristic metabolites in Asteraceae. The most well known, artemisinin from Artemisia annua, is species specific and used as an anti-malarial drug. In contrast, costunolide appeared earlier in evolution and is widespread in Asteraceae. In costunolide biosynthesis, germacrene A oxidases (GAOs, CYP71AV members) catalyze the multistep conversion of germacrene A to germacrene A acid [START_REF] Nguyen | Biochemical conservation and evolution of germacrene A oxidase in Asteraceae[END_REF][START_REF] Nguyen | Catalytic plasticity of germacrene A oxidase underlies sesquiterpene lactone diversification[END_REF][START_REF] Cankar | A chicory cytochrome P450 monooxygenase CYP71AV8 for the oxidation of (+)-valencene[END_REF][START_REF] Eljonaidi | Cytochrome P450s from Cynara cardunculus L. CYP71AV9 and CYP71BL5, catalyze distinct hydroxylations in the sesquiterpene lactone biosynthesis pathway[END_REF], while amorphadiene synthase (CYP71AV1) catalyzes the equivalent step in artemisinin biosynthesis [START_REF] Ro | Production of the antimalarial drug precursor artemisinic acid in engineered yeast[END_REF][START_REF] Teoh | Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin[END_REF]. Investigation of substrate promiscuity of GAOs showed that they can convert several sesquiterpenes, including amorphadiene, to the corresponding acids, which suggests that amorphadiene synthase activity was embedded in an ancestral GAO holding the catalytic plasticity for gaining new physiological functions [START_REF] Nguyen | Biochemical conservation and evolution of germacrene A oxidase in Asteraceae[END_REF][START_REF] Nguyen | Catalytic plasticity of germacrene A oxidase underlies sesquiterpene lactone diversification[END_REF][START_REF] Cankar | A chicory cytochrome P450 monooxygenase CYP71AV8 for the oxidation of (+)-valencene[END_REF][START_REF] Eljonaidi | Cytochrome P450s from Cynara cardunculus L. CYP71AV9 and CYP71BL5, catalyze distinct hydroxylations in the sesquiterpene lactone biosynthesis pathway[END_REF]. Conversely, the recently evolved species-specific CY-P71AV1 from A. annua gained more stringent specificity when recruited for a distinct metabolic pathway.

CYP promiscuity can also provide dual functionalities and favor the emergence of herbicide resistance. This is illustrated by the above-mentioned AtCYP706A3. This permissive CYP, the physiological function of which is to reduce the emission of volatile terpenoids in A. thaliana and to generate terpene oxides to protect flowers from florivores, also metabolizes volatile and hydrophobic herbicides from the class of dinitroanilines. As a result, shifting the expression of AtCYP706A3 from flowers to the vegetative tissues confers an increased tolerance to dinitroanilines [START_REF] Abdollahi | Innate promiscuity of the CYP706 family of P450 enzymes provides a suitable context for the evolution of dinitroaniline resistance in weed[END_REF]. A similar dual functionality has been observed for promiscuous monoterpenol metabolizing CYP76s that confer a tolerance to another class of herbicides, the phenylureas (H€ ofer et al., 2014). In this light, it is striking that members of the CYP81A subfamily have been recently shown to metabolize a broad range of herbicides, thereby conferring herbicide resistance to monocotyledonous weeds [START_REF] Dimaano | Cytochrome P450-mediated herbicide metabolism in plants: current understanding and prospects[END_REF][START_REF] Han | Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action[END_REF], while CYP81As from maize were demonstrated to catalyze diverse oxidation reactions on acidic sesquiterpenes to form zealexin antibiotics [START_REF] Ding | Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity[END_REF].

REPEATED RECRUITMENT OF CYPs FROM THE SAME FAMILY

Several compounds show a patchy distribution across plant phylogeny, and multiple examples of parallel evolution are known where plants independently have acquired the ability to produce the same compound. Some CYPs with the same catalytic function are not orthologs and CYPs may be independently and repeatedly recruited from the same CYP family (discussed here) or from diverse CYP families (discussed in the next section) to catalyze the same reaction. Parallel evolution involving CYPs from the same family have already been exemplified by the CYP98 and the CYP74 families, and more examples are discussed below.

Flavonoid 3 0 5 0 -hydroxylating CYP75 Several members of the CYP75 family hydroxylate the B ring of flavonoids at the 3 0 position (F3 0 H) or catalyze hydroxylation at both the 3 0 and 5 0 positions (F3 0 5 0 H). Most F3 0 5 0 Hs belong to the CYP75A subfamily, but some diverse CYP75B members, which are usually F3 0 H, have independently gained 5 0 -hydroxylase activity [START_REF] Seitz | Cloning, functional identification and sequence analysis of flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase cDNAs Molecular Plant 14, 1244-1265[END_REF][START_REF] Seitz | Multiple evolution of flavonoid 3',5'-hydroxylase[END_REF][START_REF] Lui | Convergent recruitment of 5'-hydroxylase activities by CYP75B flavonoid B-ring hydroxylases for tricin biosynthesis in Medicago legumes[END_REF]. F3 0 5 0 H activity has thus evolved from F3 0 H activity several times. Specific amino acids in especially SRS6 have been identified to be important for the 5 0 -hydroxylase activity [START_REF] Seitz | Identification of the molecular basis for the functional difference between flavonoid 3'hydroxylase and flavonoid 3',5'-hydroxylase[END_REF][START_REF] Seitz | Multiple evolution of flavonoid 3',5'-hydroxylase[END_REF][START_REF] Lui | Convergent recruitment of 5'-hydroxylase activities by CYP75B flavonoid B-ring hydroxylases for tricin biosynthesis in Medicago legumes[END_REF]. Pollinator attraction has been suggested to be a driving force behind gain of F3 0 5 0 H activity as it affords production of purple to blue anthocyanins in flower petals [START_REF] Seitz | Cloning, functional identification and sequence analysis of flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase cDNAs Molecular Plant 14, 1244-1265[END_REF][START_REF] Seitz | Multiple evolution of flavonoid 3',5'-hydroxylase[END_REF].

Amino acid-metabolizing CYP79

All functionally characterized CYP79 family members catalyze a multistep reaction converting an amino acid (or in some cases a chain-elongated amino acid) into a corresponding oxime [START_REF] Kahn | Substrate specificity of the cytochrome P450 enzymes CYP79A1 and CYP71E1 involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench[END_REF][START_REF] Sibbesen | Cytochrome P-450TYR is a multifunctional heme-Thiolate enzyme catalyzing the conversion of L-tyrosine to phydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench[END_REF]. The catalysis proceeds via two consecutive N-hydroxylations followed by a dehydration and decarboxylation [START_REF] Gleadow | Cyanogenic glycosides: synthesis, physiology, and phenotypic plasticity[END_REF]. The oximes can be released as volatiles (Irmisch et al., 2013a(Irmisch et al., , 2013b;;[START_REF] Luck | CYP79D enzymes contribute to jasmonic acidinduced formation of aldoximes and other nitrogenous volatiles in two Erythroxylum species[END_REF] or further metabolized to, for example, glucosinolates or cyanogenic glucosides [START_REF] Gleadow | Cyanogenic glycosides: synthesis, physiology, and phenotypic plasticity[END_REF] (Figure 6A). In spite of the complex catalytic process catalyzed by the CYP79s, phylogenetic studies demonstrate that substrate preference toward specific amino acids cannot be assigned to separate subfamilies. Likewise, the CYP79s giving rise to oximes used for cyanogenic glucoside biosynthesis are scattered throughout the CYP79 phylogeny and cannot be identified based solely on phylogenetic analyses. For example, CYP79A125 from Eucalyptus cladocalyx and CYP79D16 from Prunus dulcis belong to different CYP subfamilies but both catalyze the conversion of phenylalanine into phenylacetaldoxime in cyanogenic glucoside biosynthesis [START_REF] Hansen | Reconfigured cyanogenic glucoside biosynthesis in Eucalyptus cladocalyx involves a cytochrome P450 CYP706C55[END_REF][START_REF] Thodberg | Elucidation of the amygdalin pathway reveals the metabolic basis of bitter and sweet almonds (Prunus dulcis)[END_REF]. A few changes in a CYP79 amino acid sequence is sometimes enough to change substrate specificity. A study investigating glucosinolate accumulation in two Boechera stricta populations with distinct chemical profiles identified two amino acid positions (134 and 536) in glucosinolate CYP79Fs that showed accelerated evolution [START_REF] Prasad | A gain-of-function polymorphism controlling complex traits and fitness in nature[END_REF]. Individually, as well as in combination, the G134L and P536K mutations enabled the mainly methionine-metabolizing BsCYP79F4v1 (BCMA2) to increase conversion of valine in vitro. In one of the B. stricta populations, the closely related BsCYP79F3 (BCMA1-MT) had also acquired the ability to metabolize isoleucine. Many CYP79s are specific toward a certain amino acid in planta, but sometimes show promiscuous substrate abilities when assayed in vitro. In contrast, some CYP79Ds from Populus species metabolize a few amino acids, and the corresponding aldoximes are present in the volatile blend of the species (Irmisch et al., 2013a(Irmisch et al., , 2013b)). These examples illustrate the functional plasticity of the CYP79 family. Some modern ferns produce cyanogenic glucosides, but genes encoding CYP79s are not present in this taxonomic group. Instead, a flavin-containing monooxygenase was recently found to convert phenylalanine to phenylacetaldoxime in the ferns P. aureum and Pterium aquilinum [START_REF] Thodberg | A flavin-dependent monooxygenase catalyzes the initial step in cyanogenic glycoside synthesis in ferns[END_REF].

Terpenoid CYP76

CYP76 is one of the families characterized by multiple duplications and losses in different lineages illustrated by a large number of lineage-specific subfamilies and resulting in completely different subfamily sets in major lineages (e.g., gymnosperms, dicots, or monocots) (Figure 7). This bursting protein diversification translates into a number of lineage-specific pathways, such as phytocassane and oryzalexin classes of diterpenoid phytoalexins in monocots (CYP76M subfamily; Wang et al., 2012a), labdanetype diterpenoids in Lamiales (CYP76AH/AK subfamilies; [START_REF] Scheler | Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast[END_REF], or tyrosine-derived betalain pigments in Caryophyllales (CYP76AD subfamily; [START_REF] Timoneda | The evolution of betalain biosynthesis in Caryophyllales[END_REF]. Within dicots, emergence of new CYP76 subfamilies is repeatedly associated with species-specific adaptive functions of monoterpenol 1254 Molecular Plant 14, 1244-1265, August 2 2021 ª The Author 2021. metabolism (e.g., H€ ofer et al., 2014;[START_REF] Miettinen | The secoiridoid pathway from Catharanthus roseus[END_REF][START_REF] Ilc | Monoterpenol oxidative metabolism: role in plant adaptation and potential applications[END_REF]. So far, CYP76s from the subfamilies C, B, F, and T were found involved in the formation of oxygenated monoterpenol derivatives, catalyzing cascade oxygenations of linalool, geraniol, and other monoterpenols [START_REF] Collu | Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis[END_REF][START_REF] Wang | Cloning and functional analysis of geraniol 10-hydroxylase, a cytochrome P450 from Swertia mussotii Franch[END_REF][START_REF] Ginglinger | Gene coexpression analysis reveals complex metabolism of the monoterpene alcohol linalool in Arabidopsis flowers[END_REF][START_REF] Boachon | CYP76C1 (cytochrome P450)mediated linalool metabolism and the formation of volatile and soluble linalool oxides in Arabidopsis flowers: a strategy for defense against floral antagonists[END_REF][START_REF] Ilc | A grapevine cytochrome P450 generates the precursor of wine lactone, a key odorant in wine[END_REF]. For example, the linage-specific CYP76C1 from A. thaliana and CYP76F14 from V. vinifera both oxygenate linalool to 8carboxylinalool via the intermediates 8-hydroxylinalool and 8oxolinalool [START_REF] Boachon | CYP76C1 (cytochrome P450)mediated linalool metabolism and the formation of volatile and soluble linalool oxides in Arabidopsis flowers: a strategy for defense against floral antagonists[END_REF][START_REF] Ilc | A grapevine cytochrome P450 generates the precursor of wine lactone, a key odorant in wine[END_REF] as a result of parallel evolution.

Steroid hydroxylating CYP90

The spiroketal steroid diosgenin is sporadically distributed in monocots and dicots. A comparison of diosgenin biosynthesis in Paris polyphylla (monocot, Liliales) and Trigonella foenum-graecum (dicot, Fabales) shows that the CYPs have been independently recruited for the biosynthesis of diosgenin from cholesterol [START_REF] Christ | Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants[END_REF]. The biosynthesis is initiated by two hydroxylations at C16 and C22 followed by a cyclization. PpCYP90G4 and TfCYP90B50 arose from the canonical brassinosteroid CYP90s by two independent gene duplications events, and both catalyze 16,22-dihydroxylation (Figure 6B). Investigation of the brassinosteroid C22-hydroxylases, PpCYP90B52 and TfCYP90B51, by transient expression in N. benthamiana, revealed formation of minor amounts of 16,22-dihydroxylated cholesterol, suggesting that minor side-activity seeded repeated emergence of the dihydroxylase activity for diosgenin biosynthesis. Mutation of nine amino acid residues that were differentially conserved between the diosgenin CYP90s and brassinosteroid CYP90s resulted in increased 16,22-dihydroxylase activity but did not obliterate C22-monohydroxylation activity of the brassinosteroid CYP90s [START_REF] Christ | Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants[END_REF].

REPEATED RECRUITMENT OF CYPs FROM DIFFERENT FAMILIES

While repeated recruitment of CYPs from the same family has occurred for the first multistep reaction in cyanogenic glucoside and diosgenin biosynthesis, CYPs from distinct families have been recruited for the downstream biosynthetic steps. Other compounds with scattered distribution across plant phylogeny also demonstrate recruitment of distinct CYP families for catalysis of identical biosynthetic steps.

Cyanogenic glucosides and volatile nitriles

In cyanogenic glucoside biosynthesis, the amino acid-derived oxime produced by a CYP79 (or an FMO in ferns) is converted into an a-hydroxynitrile, which is subsequently glucosylated to produce the cyanogenic glucoside [START_REF] Gleadow | Cyanogenic glycosides: synthesis, physiology, and phenotypic plasticity[END_REF]. In several cyanogenic plant species, conversion of the oxime to the a-hydroxynitrile is catalyzed by a multifunctional CYP71 [START_REF] Bak | Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a 1258[END_REF][START_REF] Kahn | Isolation and reconstitution of cytochrome P450ox and in vitro reconstitution of the entire biosynthetic pathway of the cyanogenic glucoside dhurrin from sorghum[END_REF] (Figure 6A). Nomenclature-wise, such a CYP may also be named a CYP83. Similar to the CYP79s, the CYP71s involved in cyanogenic glucoside biosynthesis do not form a monophyletic group. In L. japonicas, a CYP from a distinct family, CYP736A2, catalyzes the multistep conversion of two aliphatic oximes to the corresponding a-hydroxynitriles [START_REF] Takos | Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway[END_REF]. In E. cladocalyx, two CYPs have been recruited to complete the catalytic sequence of reactions to form an a-hydroxynitrile from an oxime [START_REF] Hansen | Reconfigured cyanogenic glucoside biosynthesis in Eucalyptus cladocalyx involves a cytochrome P450 CYP706C55[END_REF]. First, EcCYP706C55 converts phenylacetaldoxime to Plant cytochrome P450

Molecular Plant

phenylacetonitrile, which is subsequently hydroxylated by EcCYP71B103 to form the a-hydroxynitrile mandelonitrile. In the fern P. aureum, CYP981F5 was recently identified to hydroxylate phenylacetonitrile in cyanogenic glucoside biosynthesis [START_REF] Thodberg | The fern CYPome: fernspecific cytochrome P450 family involved in convergent evolution of chemical defense[END_REF]. P. trichocarpa and Fallopia sachalinensis also produce phenylacetonitrile, which is released upon herbivore attack [START_REF] Irmisch | Herbivore-induced poplar cytochrome P450 enzymes of the CYP71 family convert aldoximes to nitriles which repel a generalist caterpillar[END_REF][START_REF] Yamaguchi | Cytochrome P450 CYP71AT96 catalyses the final step of herbivore-induced phenylacetonitrile biosynthesis in the giant knotweed, Fallopia sachalinensis[END_REF]. Conversion of phenylacetaldoxime to phenylacetonitrile is catalyzed by CYP71B40v3 and CYP71B41v2 in P. trichocarpa [START_REF] Irmisch | Herbivore-induced poplar cytochrome P450 enzymes of the CYP71 family convert aldoximes to nitriles which repel a generalist caterpillar[END_REF] and by CYP71AT96 in F. sachalinensis [START_REF] Yamaguchi | Cytochrome P450 CYP71AT96 catalyses the final step of herbivore-induced phenylacetonitrile biosynthesis in the giant knotweed, Fallopia sachalinensis[END_REF]. Numerous members of the large CYP71 family have been characterized, and this family covers a wide range of substrates and catalytic functions, some of which are also referred to in this review. Fewer members of the CYP736 and CYP706 families have been functionally

characterized, but it is striking that both families show activities toward small, often volatile compounds. CYP736 functions are mentioned above and the CYP706 family oxygenates various monoterpenes, sesquiterpenes, and flavonoids [START_REF] Luo | Molecular cloning and functional identification of (+)-dcanadiene-8-hydroxylase, a cytochrome P450 mono-oxygenase (CYP706B1) of cotton sesquiterpene biosynthesis[END_REF][START_REF] Cankar | Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis)[END_REF][START_REF] Liu | Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches[END_REF][START_REF] Boachon | A promiscuous CYP706A3 reduces terpene volatile emission from Arabidopsis flowers, affecting florivores and the floral microbiome[END_REF].

Specialized steroids

In diosgenin biosynthesis, the step after the PpCYP90G4/ TfCYP90B50 hydroxylations and subsequent cyclization is an end-of-tail-hydroxylation followed by another cyclization to form the spiroketal moiety. P. polyphylla and T. foenum-graecum have recruited various CYP families for this purpose [START_REF] Christ | Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants[END_REF]. In P. polyphylla, diosgenin production was achieved by the combined action of CYP90G4 with CYP94D108, CYP94D109, or et al., 2012a), OsCYP76M6 (Wang et al., 2012a;[START_REF] Wu | Picking sides: distinct roles for CYP76M6 and CYP76M8 in rice oryzalexin biosynthesis[END_REF], OsCYP76M7 [START_REF] Swaminathan | CYP76M7 Is an ent-Cassadiene C11a-hydroxylase defining a second multifunctional diterpenoid biosynthetic gene cluster in rice[END_REF], OsCYP76M8 (Wang et al., 2012a;[START_REF] Wu | Picking sides: distinct roles for CYP76M6 and CYP76M8 in rice oryzalexin biosynthesis[END_REF], OsCYP76M10 (Wang et al., 2012a), TpCYP76AA25 [START_REF] Gesell | The gymnosperm cytochrome P450 CYP750B1 catalyzes stereospecific monoterpene hydroxylation of (+)-sabinene in thujone biosynthesis in western redcedar[END_REF], BvCYP76AD1 [START_REF] Hatlestad | The beet R locus encodes a new cytochrome P450 required for red betalain production[END_REF], BvCYP76AD5 [START_REF] Sunnadeniya | Tyrosine Hydroxylation in Betalain Pigment Biosynthesis Is Performed by Cytochrome P450 Enzymes in Beets (Beta vulgaris)[END_REF], BvCYP76AD6 [START_REF] Polturak | Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants[END_REF]; [START_REF] Sunnadeniya | Tyrosine Hydroxylation in Betalain Pigment Biosynthesis Is Performed by Cytochrome P450 Enzymes in Beets (Beta vulgaris)[END_REF]), SmCYP76AH1 ((Guo et al., 2013); [START_REF] Zi | Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamiaceae[END_REF], SmCYP76AH3 [START_REF] Guo | Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones[END_REF], and SmCYP76AK1 [START_REF] Guo | Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones[END_REF]. All full-length CYP76 sequences from the listed species have been used to generate the tree. Amino acid sequences were aligned using MUSCLE and phylogeny was inferred by maximum likelihood analysis. A phylogenetic tree with tip labels is available in supplemental Figure 2.

1256 Molecular Plant 14, 1244-1265, August 2 2021 ª The Author 2021.

CYP72A616. In T. foenum-graecum, CYP90B50 together with either CYP82J17 or CYP72A613 catalyzed diosgenin formation (Figure 6B). PpCYP90G4 co-expressed with PpCYP94D108 and TfCYP90B50 co-expressed with TfCYP82J17 resulted in the highest diosgenin production as monitored by transient expression in N. benthamiana. The CYP94 family is known to metabolize jasmonate conjugates essential for jasmonic acid regulation (e.g., [START_REF] Heitz | Cytochromes P450 CYP94C1 and CYP94B3 catalyze two successive oxidation steps of plant hormone jasmonoyl-isoleucine for catabolic turnover[END_REF][START_REF] Widemann | Sequential oxidation of jasmonoyl-phenylalanine and jasmonoyl-isoleucine by multiple cytochrome P450 of the CYP94 family through newly identified aldehyde intermediates[END_REF] but is also reported as having activity toward other hydrophobic substrates [START_REF] Tijet | Functional expression in yeast and characterization of a clofibrate-inducible plant cytochrome P-450 (CYP94A1) involved in cutin monomers synthesis[END_REF][START_REF] Le Bouquin | Cloning and functional characterization of CYP94A2, a medium chain fatty acid hydroxylase from Vicia sativa[END_REF][START_REF] Augustin | Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells[END_REF]. The CYP82 family shows broad catalytic activities toward various flavones, coumarins, alkaloids, and terpenes (e.g., [START_REF] Lee | Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis[END_REF][START_REF] Dang | Dual catalytic activity of a cytochrome P450 controls bifurcation at a metabolic branch point of alkaloid biosynthesis in Rauwolfia serpentina[END_REF]Zhao et al., 2018b;[START_REF] Rajniak | Biosynthesis of redox-active metabolites in response to iron deficiency in plants[END_REF]. Several CYP72 members are known to metabolize hydrophobic isoprene-derived compounds such as steroids, other triterpene backbones, and diterpenes (e.g., [START_REF] Umemoto | Two cytochrome P450 monooxygenases catalyze early hydroxylation steps in the potato steroid glycoalkaloid biosynthetic pathway[END_REF][START_REF] He | CYP72A enzymes catalyse 13hydrolyzation of gibberellins[END_REF][START_REF] Liu | The cytochrome P450 CYP72A552 is key to production of hederagenin-based saponins that mediate plant defense against herbivores[END_REF], and also to catalyze various hydroxylation, oxidation, and ringopening steps leading to iridoid derivatives in indole alkaloid biosynthesis [START_REF] Irmler | Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase[END_REF][START_REF] Salim | Virus-induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid-7hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis[END_REF][START_REF] Yang | Bifunctional cytochrome P450 enzymes involved in camptothecin biosynthesis[END_REF][START_REF] Rodriguez-Lopez | Two bi-functional cytochrome P450 CYP72 enzymes from olive (Olea europaea) catalyze the oxidative C-C bond cleavage in the biosynthesis of secoxy-iridoids -flavor and quality determinants in olive oil[END_REF].

Furanocoumarins

Furanocoumarins are tricyclic phenolic derivatives present in a few non-related plant families [START_REF] Pathak | The presently known distribution of furocoumarins (psoralens) in plants[END_REF]. Hydroxylation of xanthotoxin to 5-hydroxy-xanthotoxin in the biosynthesis of the linear furanocoumarin isopimpinellin is catalyzed by CYP71AZ1 in Ammi majus and CYP71AZ6 in parsnip (Pastinaca sativa; both Apiaceae family) [START_REF] Krieger | The CYP71AZ P450 subfamily: a driving factor for the diversification of coumarin biosynthesis in apiaceous plants[END_REF], whereas CYP82A64 catalyzes the same reaction in Citrus paradisi (Rutaceae) [START_REF] Limones-Mendez | Convergent evolution leading to the appearance of furanocoumarins in citrus plants[END_REF]. Both CYP families cover broad catalytic activities, as mentioned earlier in the review.

Momilactones

Momilactone diterpenoids are well-known phytoalexins from O. sativa but are also reported to occur in the mosses C. plumiforme and Pseudoleskeella papillosa (Zhao et al., 2018a). CYPs involved in momilactone biosynthesis have been identified for O. sativa [START_REF] Shimura | Identification of a biosynthetic gene cluster in rice for momilactones[END_REF][START_REF] Wang | CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice[END_REF][START_REF] De La Pena | Rerouting plant terpene biosynthesis enables momilactone pathway elucidation[END_REF] and C. plumiforme [START_REF] Mao | Genomic evidence for convergent evolution of gene clusters for momilactone biosynthesis in land plants[END_REF]. In O. sativa, CYP99A3 catalyzes consecutive oxygenations of the momilactone precursor syn-pimaradiene at the C19 methyl group to form syn-pimaradienoic acid [START_REF] Wang | CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice[END_REF], and CYP76M8 has been shown to hydroxylate multiple labdane diterpenes, including C6-hydroxylation of synpimaradiene (Wang et al., 2012a). A more recent study coexpressed the terpene synthases OsKSL4 and OsCPS4 together with OsCYP99A3 and OsCYP76M8 in N. benthamiana and observed accumulation of syn-pimaradiene with oxidations at C6 and/or C19, some of which might be catalyzed by endogenous N. benthamiana oxidases (De La Pena and Sattely, 2020). However, co-expression of each of the OsCYPs with the terpene synthases confirmed previously reported OsCYP activities on syn-pimaradiene. Co-expression of the dehydrogenase OsMAS with the two terpene synthases and two CYPs in N. benthamiana resulted in formation of the lactone ring. Accumulation of momilactone A and small amounts of other oxidation products were also observed. Other CYP-catalyzed steps required for momilactone production are oxygenation at C3 and C20. Further addition of C20-hydroxylating OsCYP76M14 and the promiscuous OsCYP701A8 resulted in production of momilactone B. Another recent study showed involvement of Os-CYP99A3, OsCYP76M8, and OsCYP701A8 in production of momilactone A using engineered Escherichia coli and in vitro assays [START_REF] Kitaoka | Interdependent evolution of biosynthetic gene clusters for momilactone production in rice[END_REF]. In C. plumiforme, moss-specific CYP families have been recruited for momilactone production. CpCYP970A14 (71 clan) converts syn-pimaradiene to syn-pimaradienoic acid similarly to OsCYP99A3, and CpCYP964A1 (85 clan) was found to hydroxylate C3 of syn-pimaradiene [START_REF] Mao | Genomic evidence for convergent evolution of gene clusters for momilactone biosynthesis in land plants[END_REF]. Co-expression of the CpCYPs in N. benthamiana together with the bifunctional terpene synthase CpDTC resulted in 3OH-syn-pimaradienolide and some momilactone A, the latter compound possibly resulting from endogenous N. benthamiana enzyme activity. Interestingly, in both plant species, the genes encoding biosynthetic enzymes for momilactone production are localized within gene clusters [START_REF] Shimura | Identification of a biosynthetic gene cluster in rice for momilactones[END_REF][START_REF] Mao | Genomic evidence for convergent evolution of gene clusters for momilactone biosynthesis in land plants[END_REF][START_REF] Kitaoka | Interdependent evolution of biosynthetic gene clusters for momilactone production in rice[END_REF].

CONCLUDING REMARKS AND FUTURE PERSPECTIVES

In this review, we highlight the immense numbers and functionalities of CYPs present in the plant lineage. While reports on functional characterization of CYPs from seed plants have increased dramatically in recent years, some families, including some of the most conserved, remain functionally uncharacterized. Their deorphanization is expected to reveal new branches in plant hormone pathways and possibly overlooked classes of signaling compounds. Moreover, only a very few CYPs from more recently sequenced green algae and basal land plant species have been investigated, which leaves much to explore to fully apprehend the diversity of plant CYP activities.

The current surge of open access sequence information and rise in transcriptome and genome sequence data provides new opportunities to understand the molecular events leading to the emergence of new functions. Most notably, the number of sequenced genomes from extant charophytes keeps increasing [START_REF] Hori | Klebsormidium flaccidum genome reveals primary factors for plant terrestrial adaptation[END_REF][START_REF] Nishiyama | The Chara genome: secondary complexity and implications for plant terrestrialization[END_REF][START_REF] Cheng | Genomes of subaerial Zygnematophyceae provide insights into land plant evolution[END_REF][START_REF] Wang | Genomes of early-diverging streptophyte algae shed light on plant terrestrialization[END_REF][START_REF] Jiao | The Penium margaritaceum genome: hallmarks of the origins of land plants[END_REF] and now offers the opportunity to disclose events resulting in the evolution of land plants in context with the acquisition of CYPs with new functionalities. In addition, useful information on metabolic pathways involving orphan CYPs is expected to arise from parallel analyses with other enzyme families (e.g., terpene synthases, glycosyltransferases, dehydrogenases, methyl transferases).

Beyond catalytic activities, CYP's physiological functions are often not investigated or are limited to studies of specific facets of their roles in plant development, growth, or fitness. All aspects of plant ontogeny and the exposures to varying growth conditions, abiotic and biotic (negative and positive) conditions, ecological niches, including metabolism and interference of man-made exogenous compounds such as pollutants and pesticides, need to be considered to integrate the complex role of CYPs in different ecological systems with plant metabolic networks and physiology.

A previously largely overlooked characteristic emerging within the last years is the promiscuity of some plant CYP enzymes. This feature clearly favors functional diversification and emergence of new functions in gene duplicates. Because duplications result in relaxation of substrate specificity, it also clearly relates to the evolution of blooms within some CYP families playing an important role in the flexibility, adaptability, and resilience of plants' specialized metabolism. Thorough exploration of individual plant CYP substrate ranges is expected to unravel additional functionalities, and resulting overlaps or crosstalks between pathways and adaptive functions. Such knowledge would also obviously serve to expand the use of CYPs in synthetic biology approaches aimed at developing environmentally benign production systems for the future [START_REF] Møller | Solar-driven synthesis of bioactive natural products[END_REF].

In addition to the structures of atypical CYP74s, the crystal structures of five plant CYP proteins representative of three clans are now accessible in public repository [START_REF] Fujiyama | Structural insights into a key step of brassinosteroid biosynthesis and its inhibition[END_REF][START_REF] Gu | Crystal structure of CYP76AH1 in 4-PI-bound state from Salvia miltiorrhiza[END_REF][START_REF] Niu | Structural basis for plant lutein biosynthesis from alpha-carotene[END_REF][START_REF] Zhang | Structure and function of the cytochrome P450 monooxygenase cinnamate 4-hydroxylase from Sorghum bicolor[END_REF]. This is an important asset for modeling other CYP protein structures, so as to understand ligand binding in the active sites and to engineer CYP activities. With the availability of an increasing number of sequences, reference structures, and characterized activities, it is now possible to envisage modeling approaches to predict CYP substrates and activities. Systematic studies taking into consideration the molecular fingerprint of compounds and the sequences of the SRSs may also be used to predict substrate candidates for CYP families for which no substrate has yet been found. However, it is necessary to keep in mind that, in addition to the physiological substrate(s), additional compounds may gain significant docking scores. In this context, the role of the access channel in the filtration of potential substrates is still poorly understood and has to be further considered.

Other central questions to be explored regard the importance of substrate(s) availability in the plant tissue for directing fluxes; possible diverging activities in different tissues; supramolecular organization of the pathway enzymes, including how this organization compartmentalizes or interconnects different pathways; and regulatory factors for driving metabolic fluxes.

In conclusion, the multiplicity of CYPs in plants in combination with their functionality and promiscuity provides an excellent molecular background for development of metabolic diversity and plasticity required by plants to adapt to different and rapidly changing growth conditions and environmental challenges. More studies into the substrate space of individual CYPs may provide essential knowledge to understand the evolution-specific functional characteristics. Knowledge of enzyme promiscuity may also guide engineering of desired specificities for, e.g., optimized biotechnological applications, and may help to predict and manage metabolism of pesticides, other encountered exogenous chemicals, and pollutants. We hope that The Plant Cytochrome P450 Database will facilitate search for hitherto functionally characterized CYPs for further investigations, biotechnological applications, and the discovery of CYPs with unknown functions.

Figure 1 .

 1 Figure2shows the phylogenetic distribution of CYP families in green algae and land plants.CYP clans 51, 74, 97, 710, 711, and 746 were previously known as single-family clans(Nelson and

Figure 2 .

 2 Figure 2. Phylogeny of CYP families within green algae and land plants.

  Many eudicot CYP716s have been shown to metabolize triterpenes. This family possibly evolved from a duplication of genes 1248 Molecular Plant 14, 1244-1265, August 2 2021 ª The Author 2021.

Figure 3 .

 3 Figure 3. The phenylpropanoid metabolic network underlying the biosynthesis of H, G, and S-lignin monomers. SmCYP788A1 reactions are labeled in green and green arrows indicate the pathway specific to Selaginella. PAL, phenylalanine ammonia-lyase; 4CL, 4coumarate-CoA ligase; HCT, hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase; CSE, caffeoyl shikimate esterase; CCoAMT, caffeoyl-CoA O-methyltransferase; COMT, caffeic acid O-methyltransferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase.

Figure 4 .

 4 Figure 4. CYP98 gene evolution and functional diversity.

  (B) Products of diverse CYP98 reactions. The introduced hydroxyl groups are colored blue. Molecular Plant 14, 1244-1265, August 2 2021 ª The Author 2021. 1251

1252

  Molecular Plant 14, 1244-1265, August 2 2021 ª The Author 2021.

Figure 5 .

 5 Figure 5. Promiscuity as a driver for evolving new functionalities.

Figure 6 .

 6 Figure 6. Identical or similar CYP reactions catalyzed by diverse CYP families.(A) Biosynthetic pathway for production of amino acid-derived oximes, nitriles, and cyanogenic glucosides. (B) Suggested diosgenin biosynthesis according to[START_REF] Christ | Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants[END_REF]. Another possible intermediate toward the first ring formation could also be a dihydroxylation of C22.

Figure 7 .

 7 Figure 7. Dynamics of the CYP76 family.Subfamily CYP76C is specific to Brassicales, CYP76AH/AK is specific to Lamiales, CYP76AD is specific to Caryophyllales, and subfamily CYP76AA is specific to gymnosperms. Subfamily CYP76M is specific to monocots and has particularly expanded in O. sativa (green), T. aestivum and Saccharum officinarum. Subfamily CYP76F members are present in several angiosperm species but have particularly expanded in Citrus sinensis and E. grandis. In contrast, subfamily CYP76G tend to be maintained at a single or low copy number in most rosids and asterids. CYP76s marked by an asterisk are functionally characterized: AtCYP76C1[START_REF] H€ Ofer | Dual function of the cytochrome P450 CYP76 family from Arabidopsis thaliana in the metabolism of monoterpenols and phenylurea herbicides[END_REF][START_REF] Boachon | CYP76C1 (cytochrome P450)mediated linalool metabolism and the formation of volatile and soluble linalool oxides in Arabidopsis flowers: a strategy for defense against floral antagonists[END_REF], AtCYP76C2(H€ ofer et al., 2014), AtCYP76C3[START_REF] Ginglinger | Gene coexpression analysis reveals complex metabolism of the monoterpene alcohol linalool in Arabidopsis flowers[END_REF], AtCYP76C4(H€ ofer et al. 2013; H€ ofer et al., 2014), VvCYP76F12 and VvCYP76F14[START_REF] Ilc | A grapevine cytochrome P450 generates the precursor of wine lactone, a key odorant in wine[END_REF], OsCYP76M2(Wang et al., 2012a), OsCYP76M5(Wang et al., 2012a), OsCYP76M6(Wang et al., 2012a;[START_REF] Wu | Picking sides: distinct roles for CYP76M6 and CYP76M8 in rice oryzalexin biosynthesis[END_REF], OsCYP76M7[START_REF] Swaminathan | CYP76M7 Is an ent-Cassadiene C11a-hydroxylase defining a second multifunctional diterpenoid biosynthetic gene cluster in rice[END_REF], OsCYP76M8(Wang et al., 2012a;[START_REF] Wu | Picking sides: distinct roles for CYP76M6 and CYP76M8 in rice oryzalexin biosynthesis[END_REF], OsCYP76M10(Wang et al., 2012a), TpCYP76AA25[START_REF] Gesell | The gymnosperm cytochrome P450 CYP750B1 catalyzes stereospecific monoterpene hydroxylation of (+)-sabinene in thujone biosynthesis in western redcedar[END_REF], BvCYP76AD1[START_REF] Hatlestad | The beet R locus encodes a new cytochrome P450 required for red betalain production[END_REF], BvCYP76AD5[START_REF] Sunnadeniya | Tyrosine Hydroxylation in Betalain Pigment Biosynthesis Is Performed by Cytochrome P450 Enzymes in Beets (Beta vulgaris)[END_REF], BvCYP76AD6[START_REF] Polturak | Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants[END_REF];[START_REF] Sunnadeniya | Tyrosine Hydroxylation in Betalain Pigment Biosynthesis Is Performed by Cytochrome P450 Enzymes in Beets (Beta vulgaris)[END_REF]), SmCYP76AH1 ((Guo et al., 2013);[START_REF] Zi | Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamiaceae[END_REF], SmCYP76AH3[START_REF] Guo | Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones[END_REF], and SmCYP76AK1[START_REF] Guo | Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones[END_REF]. All full-length CYP76 sequences from the listed species have been used to generate the tree. Amino acid sequences were aligned using MUSCLE and phylogeny was inferred by maximum likelihood analysis. A phylogenetic tree with tip labels is available in supplemental Figure2.

Molecular Plant 14, 1244-1265, August 2 2021 ª The Author 2021. 1247 Plant cytochrome P450

Molecular Plant 14, 1244-1265, August 2 2021 ª The Author 2021. 1253 Plant cytochrome P450

ACKNOWLEDGMENTS

We thank Drs. Charles Francis Delwiche and Endymion Cooper for early access to green algal transcriptomes supported by NSF, grant DEB-1036506. We also thank Dr. Elizabeth H.J. Neilson for comments on early versions of the manuscript, Dr. Franc ¸ois Andre ´for providing the protein model structures in Figure 5, and Jakob Bo Cetti Hansen for help coding The Plant Cytochrome P450 Database. The authors declare no

FUNDING C.C.H. is supported by a PhD fellowship provided through a Villum Foundation Young Investigator Program fellowship granted to Elizabeth H.J. Neilson (grant number 13167). This work was supported by the VILLUM Center for Plant Plasticity (VKR023054) (B.L.M.); a European Research Council Advanced Grant (ERC-2012-ADG_20120314), and the Novo Nordisk Foundation Distinguished Investigator 2019 Grant (NNF 0054563, The Black Holes in the Plant Universe). B.L.M. acknowledges the continued strong external support from Danish Foundations and the European Research Council throughout his career.

SUPPLEMENTAL INFORMATION

Supplemental information is available at Molecular Plant Online.

AUTHOR CONTRIBUTIONS