Triterpene Structural Diversification by Plant Cytochrome P450 Enzymes

Cytochrome P450 monooxygenases (P450s) represent the largest enzyme family of the plant metabolism. Plants typically devote about 1% of the protein-coding genes for the P450s to execute primary metabolism and also to perform species-specific specialized functions including metabolism of the triterpenes, isoprene-derived 30-carbon compounds. Triterpenes constitute a large and structurally diverse class of natural products with various industrial and pharmaceutical applications. P450-catalyzed structural modification is crucial for the diversification and functionalization of the triterpene scaffolds. In recent times, a remarkable progress has been made in understanding the function of the P450s in plant triterpene metabolism. So far, ∼80 P450s are assigned biochemical functions related to the plant triterpene metabolism. The members of the subfamilies CYP51G, CYP85A, CYP90B-D, CYP710A, CYP724B, and CYP734A are generally conserved across the plant kingdom to take part in plant primary metabolism related to the biosynthesis of essential sterols and steroid hormones. However, the members of the subfamilies CYP51H, CYP71A,D, CYP72A, CYP81Q, CYP87D, CYP88D,L, CYP93E, CYP705A, CYP708A, and CYP716A,C,E,S,U,Y are required for the metabolism of the specialized triterpenes that might perform species-specific functions including chemical defense toward specialized pathogens. Moreover, a recent advancement in high-throughput sequencing of the transcriptomes and genomes has resulted in identification of a large number of candidate P450s from diverse plant species. Assigning biochemical functions to these P450s will be of interest to extend our knowledge on triterpene metabolism in diverse plant species and also for the sustainable production of valuable phytochemicals.


INTRODUCTION
Triterpenes, 30-carbon compounds originated from the 5-carbon isoprene units, constitute a large and structurally diverse class of natural products (Hill and Connolly, 2017). The majority of the triterpene structural diversity is revealed within the plant kingdom. Plants often biosynthesize triterpenes in response to the developmental cues and environmental stimuli (Phillips et al., 2006;Misra et al., 2014;Moses et al., 2015a,b). Although, the actual biological function of most of the plant triterpenes is yet to be revealed, some triterpenes are known for their roles in plant developmental processes and defense response. Anti-microbial triterpene glycosides (avenacins) that accumulate constitutively in roots of the oat plant conferred defense against the root-infecting fungal pathogens (Papadopoulou et al., 1999). However, a few triterpenes were suggested to play crucial function during plant growth and organ development, e.g., lupeol in nodulation (Delis et al., 2011), β-amyrin in nodulation (Confalonieri et al., 2009), and root development (Kemen et al., 2014), thalianol in plant growth and development (Field and Osbourn, 2008), β-amyrin/dihydro-lupeol in root growth and flowering (Krokida et al., 2013), and marneralderived triterpene(s) in shoot and root development, flowering and embryogenesis (Go et al., 2012). Owing to the potent bioactivities, triterpenes have commercial applications in pharmaceutical, food and cosmetic sectors (Laszczyk, 2009;Sawai and Saito, 2011;Salvador et al., 2012;Moses et al., 2013). Plant-origin triterpenes are being used as dietary supplements and over-the-counter drugs and, moreover, some semi-synthetic triterpene derivatives are undergoing clinical trials (Sheng and Sun, 2011;Moses et al., 2013). For these reasons, triterpene biosynthesis and accumulation processes in plants are studied to a great extent and efforts were also being made to generate alternate sources for the sustainable production of the industrially relevant triterpenes (Moses et al., 2013;Dai et al., 2014;Luo et al., 2015;Arendt et al., 2017;Reed et al., 2017).
2,3-Oxidosqualene is a common biosynthetic precursor for the diverse triterpene skeletons produced in plants. Oxidosqualene cyclases (OSCs) catalyze the first diversifying step for triterpene biosynthesis by converting 2,3-oxidosqualene to a variety of cyclic triterpene scaffolds (Misra et al., 2014;Thimmappa et al., 2014;Ghosh, 2016). In majority of the cases, the cyclic triterpene scaffolds originated from the OSC-catalyzed reactions undergo a plethora of scaffold-, regio-, and stereo-specific oxidations catalyzed by the cytochrome P450 monooxygenases (P450s), leading to triterpene scaffold decoration with various functional groups such as hydroxyl, carbonyl, carboxyl, and epoxy moieties. Moreover, P450-mediated addition of oxygen functionality makes triterpene scaffolds subsequently accessible to the UDP-glycosyltransferases (UGTs) and acyltransferases (ATs) for the generation of the glycosylated (i.e., saponins) and acylated triterpenes Mugford et al., 2013;Moses et al., 2014a). In recent years, a notable progress has been made in understanding the biochemical functions of the P450s involved in plant triterpene metabolism. Together with the genetic screening of the mutant lines impaired with triterpene biosynthesis, the availability of the genomic and transcriptomic resources led to the identification of a number of P450s that are involved in plant triterpene structural modifications (Qi et al., 2006;Carelli et al., 2011;Augustin et al., 2015;Biazzi et al., 2015;Miettinen et al., 2017;Misra et al., 2017). This article highlights the diverse roles of the plant P450s in triterpene scaffold modifications.

BIOSYNTHETIC PATHWAY LEADING TO THE TRITERPENE STRUCTURAL DIVERSITY
To date, >23,000 triterpene structures are known from the natural sources. These are build-up on >100 structural scaffolds ranging from acyclic to hexacyclic structures (Hill and Connolly, 2017). Among these, tetracyclic and pentacyclic scaffolds represent the major triterpene classes (Ghosh, 2016). Triterpene scaffolds are decorated with various functional groups such as hydroxyl, carbonyl, carboxyl, epoxy, alkyl, acyl, malonyl, and glycosyl leading to a huge structural diversity Moses et al., 2014a;Thimmappa et al., 2014).
Triterpenes are generally produced from the acyclic 30carbon precursors squalene in bacteria and 2,3-oxidosqualene in eukaryotes. However, a few exceptions were also known. Green algae produce botryococcene and their methylated derivatives from farnesyl pyrophosphate (Jiang et al., 2016). Moreover, some bacteria use 2,3-oxidosqualene as biosynthetic precursor for the sterol (Bode et al., 2003;Wei et al., 2016). In eukaryotes, 2,3-oxidosqualene also serves a common precursor for the biosynthesis of the sterols and steroid hormones that play primary function during growth and development of the organisms (Benveniste, 2004).
The conversion of 2,3-oxidosqualene into diverse cyclic triterpenes by OSCs, the class II terpene synthases is the first diversifying step of the triterpene biosynthetic pathway, and also marks the branch point for the biosynthesis of the sterols and steroid hormones (Thimmappa et al., 2014;Ghosh, 2016). Unlike animals and fungi genomes that generally encode a single OSC (lanosterol synthase) for membrane sterol and steroid hormone biosynthesis, the higher plant genomes encode several OSCs (e.g., 9 and 13 in rice and Arabidopsis, respectively) for the biosynthesis of the sterol (cycloartenol synthase and lanosterol synthase) and triterpene (e.g., β-amyrin synthase) scaffolds (Sawai and Saito, 2011;Thimmappa et al., 2014;Ghosh, 2016). So far, about 100 OSCs are identified from the plants and biochemically characterized to reveal their product specificities. More than two-third of these OSCs were found to have product specificity for single triterpene (mono-functional OSCs). Other OSCs converted 2,3-oxidosqualene into triterpene products, ranging from 2 to 23 in number (multi-functional OSCs). Subsequent of the OSC-mediated cyclization, triterpene and sterol scaffolds undergo a plethora of structural modifications catalyzed by the P450s, ATs, and UGTs leading to scaffold-, regio-, and stereo-specific oxidation, methylation, acetylation, malonylation, and glycosylation . The reactions catalyzed by the P450s were found to be extremely diverse in nature, including oxidation, desaturation, and C-C bond cleavage. P450-catalyzed reactions of the plant triterpene pathways are discussed in the following sections.

CYP51G-Sterol 14α-Demethylase
An initial reaction of the phytosterol biosynthesis is the CYP51Gcatalyzed 14α-demethylation of obtusifoliol (Bak et al., 1997;Kim et al., 2005). CYP51G catalyzes the conversion of 14α-methyl group of obtusifoliol into 14α-carboxyaldehyde, involving two consecutive oxidation reactions and finally, the elimination of the 14α-aldehyde group as formic acid with concomitant formation of 14,15 double bond into the sterol scaffold (Shyadehi et al., 1996;Bak et al., 1997;Kim et al., 2005;Waterman and Lepesheva, 2005; Figure 2). CYP51 function remained conserved in fungi and animals as 14α-demethylase of sterol precursors such as lanosterol, dihydrolanosterol, and eburicol. In animals and fungi, CYP51 members are designated as CYP51A and ERG11/CYP51F, respectively. Moreover, protozoan and bacterial genomes also encoded sterol 14α-demethylase, designated as CYP51E and CYP51B, respectively. The bacterial genomes possibly gained CYP51 members through horizontal transfer from the eukaryotic genomes (Rezen et al., 2004). The biochemical and phenotypic analysis of the Arabidopsis CYP51G1 loss-of-function mutants established an essential role of the CYP51G1 in plant growth and development (Kim et al., 2005). Similarly, CYP51A and ERG11 functions are essential for the survival of the animals and fungi, respectively (Bard et al., 1993). CYP51 is the primary target of the anti-fungal (azoles) drugs and agricultural fungicides. Moreover, CYP51 is also a potential target for the development of anti-trypanosomal chemotherapy.

P450 in Brassinosteroids Biosynthesis
The biosynthesis and catabolism of brassinosteroids (BRs), the poly-hydroxylated steroid hormones that mediate plant growth and development, involve P450s-catalyzed regio-and stereo-specific oxidation of the campesterol scaffold at the C2, C3, C6, C22, C23, and C26 positions. The P450 family members (CYP72C, CYP85A, CYP90B-D, CYP724A,B, and CYP734A) that catalyze the consecutive biochemical reactions of the BRs pathway, were identified (Figure 2). The genetic screening of the mutant lines and biochemical analysis of the corresponding enzymes established the role of the CYP90B and CYP724B subfamily members in catalyzing the conversion of campesterol to 22-hydroxy-campesterol in Arabidopsis, rice, and tomato (Choe et al., 2001;Ohnishi et al., 2006c;Sakamoto et al., 2006). Likewise, Arabidopsis CYP90C1 and CYP90D1 have been implicated in BRs biosynthesis pathway as steroid C23-hydroxylases (Ohnishi et al., 2006b). Besides, Arabidopsis and tomato CYP85A subfamily members were known for the C6 oxidation activities in BR biosynthesis. Arabidopsis CYP85A1 and tomato ortholog catalyzed C6 oxidation of 6-deoxocastasterone leading to castasterone. However, Arabidopsis CYP85A2 and tomato CYP85A3 were responsible for the C6 oxidation of 6-deoxocastasterone leading to castasterone as well as the conversion of castasterone to brassinolide by Baeyer-Villiger oxidation (Shimada et al., 2001;Nomura et al., 2005). Arabidopsis CYP90A1 and CYP724A1 were also associated with the BRs biosynthetic pathway; however, their exact biochemical functions remain to be clarified (Szekeres et al., 1996;Bak et al., 2011). Moreover, genetic screening of the Arabidopsis activation tagged lines revealed the role FIGURE 1 | Phylogenetic analysis of the P450s known to catalyze structural modifications on steroidal and triterpene scaffolds. Complete amino acid sequences of the P450s were analyzed by MEGA6. Sequence alignment was carried out following ClustalW analysis and phylogenetic tree was built using the Neighbor Joining Method. Evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Agrobacterium tumefaciens CYP104A1 was included as out group.
of the CYP72C1 and CYP734A1 in BRs catabolism (Neff et al., 1999;Takahashi et al., 2005). CYP734A1 catalyzed C26 hydroxylation of bioactive brassinosteroids such as castasterone and brassinolide. However, the biochemical function of the CYP72C1 is yet to be known (Turk et al., 2003). Unlike Arabidopsis-specific CYP72C1, the function of the CYP734A1 appears to be conserved in other plants, as revealed for the tomato ortholog CYP734A7 (Ohnishi et al., 2006a).
Recently, a member of the CYP716A subfamily (CYP716A113v1) was identified from the basal eudicot Aquilegia coerulea and suggested to take part in steroidal saponin biosynthesis . CYP716A113v1 was found to hydroxylate the steroidal saponin precursor cycloartenol; however, the exact regio-and stereo-chemistry of the oxidation reaction remain to be known.
Eudicot CYP716A, CYP716C, CYP716E, CYP716S, and CYP716Y in Pentacyclic Triterpene Biosynthesis CYP716s are classified under the clan CYP85. They were evolved early with the land plants and were found in the genomes of the bryophytes, lycopods, ferns, gymnosperms, and angiosperms, however, not in monocot (Nelson and Werck-Reichhart, 2011). Although, the function of the lower plant CYP716As is yet to be clarified, the majority of the eudicot CYP716s was found to participate in pentacyclic triterpene scaffold modifications. A recent report of kingdom-wide phylogenetic analysis of CYP716s, collected from >200 plant species indicated that, in eudicots, CYP716 family evolved specifically toward triterpene biosynthesis . The first evidence for the CYP716s participation in triterpene biosynthesis appeared from a gene co-expression study in Arabidopsis that suggested a coregulated expression of CYP716A1 and CYP716A2 with PEN3, an OSC (Ehlting et al., 2008). However, for the first time, the genetic and biochemical evidences were gathered following characterization of a Medicago truncatula mutant line deficient in hemolytic saponin biosynthesis with a lesion in CYP716A12 gene (Carelli et al., 2011). It was found that CYP716A12 partakes in M. truncatula hemolytic saponin biosynthesis by catalyzing the sequential three-step oxidation at the C28 position of β-amyrin, leading to the formation of oleanolic acid (Carelli et al., 2011;Fukushima et al., 2011). Besides, in vitro and in vivo assays using heterologous system also revealed the ability of the CYP716A12 in converting α-amyrin to ursolic acid and lupeol to betulinic acid, following three-step oxidation at the C28 position (Carelli et al., 2011;Fukushima et al., 2011). Subsequent to these initial reports, several CYP716s were identified and biochemically characterized from the plants, mostly by exploring transcriptomic and genomic resources (Misra et al., 2017;Miettinen et al., 2017;Tamura et al., 2017a). To date, about 20 CYP716As are biochemically characterized for the amyrin/lupeol C28-oxidase activities (Figures 1, 3-5). A majority of these CYP716As catalyzed sequential threestep oxidation of the amyrin/lupeol scaffolds, leading to the consecutive formation of the hydroxyl, aldehyde, and carboxyl moieties at the C28 position (Carelli et al., 2011;Fukushima et al., 2011;Misra et al., 2017;Tamura et al., 2017a). A few plant CYP716As also target a carbon atom of the amyrin skeletons other than the C28. These are Arabidopsis CYP716A2 for C22α hydroxylation, Artemisia annua CYP716A14v2 for C3 oxidation, and Aquilegia coerulea CYP716A111 and Platycodon grandiflorus CYP716A141 for C16β hydroxylation (Moses et al., 2015b;Yasumoto et al., 2016;Miettinen et al., 2017;Tamura et al., 2017b).

Monocot CYP51H in Pentacyclic Triterpene Biosynthesis
The CYP51 family is conserved across algae to higher plants (Nelson and Werck-Reichhart, 2011). Unlike the conserved function of the CYP51G subfamily members as sterol 14α-demethylase, CYP51H subfamily appears to be specifically recruited for the pentacyclic triterpene scaffold modification in monocots. The CYP51H subfamily members seem to be restricted to the monocots such as oats and rice (Qi et al., 2006). Although the oat CYP51H10 was biochemically and functionally characterized, rice counterpart is yet to be analyzed . It might be hypothesized that CYP51H emerged in monocots to compensate the loss of the CYP716 family from monocots in maintaining triterpene structural diversity.
In a forward genetic screen for the avenacin-deficient oat mutants (sad mutants), CYP51H10 (sad2) was found to be dispensable for essential sterols biosynthesis. However, CYP51H10 was indispensable for the production of antimicrobial oleanane-triterpene saponins (avenacins) that conferred disease resistance in oats toward the root-infecting fungal pathogens (Qi et al., 2006). CYP51H10 catalyzed both C12-C13β epoxidation of the C ring and C16β hydroxylation of the D ring of β-amyrin leading to the formation of 12,13β-epoxy-16β-hydroxy-β-amyrin, an intermediate of the avenacin biosynthetic pathway (Figure 3; Kunii et al., 2012;Geisler et al., 2013). Moreover, molecular modeling and docking studies suggested that C16 hydroxylation of the D-ring is likely followed by C12-C13 epoxidation of the C-ring .
The members of the CYP93 family comprising of five subfamilies (A-E) were found in several plant genomes (Nelson and Werck-Reichhart, 2011). The majority of the CYP93 family members were associated with the flavonoid metabolism (Ayabe and Akashi, 2006). However, CYP93E subfamily members appear to be restricted to the legumes for the triterpene saponin biosynthesis (Moses et al., 2014c). The first characterized member of this subfamily is the Glycine max CYP93E1 (Shibuya et al., 2006). This enzyme is a C24-hydroxylase that converted β-amyrin and sophoradiol to 24-hydroxy-β-amyrin and soyasapogenol B, respectively, the intermediates for the biosynthesis of the legume-specific soyasaponins of nonhemolytic class. CYP93E1 also represents the first characterized P450 of the triterpene biosynthetic pathway. To date, eight additional CYP93Es (E2-E9) were identified from eight legume species; all of them showed C24-hydroxylase activity (Figure 3), suggesting the conserved role of the CYP93E in legume-specific triterpene saponin biosynthesis (Seki et al., 2008; Moses et al., 2014c). Despite the high degree of amino acid conservation (>80% identity), CYP93Es of different legumes exhibited a large variation in β-amyrin C24 hydroxylation efficiency in yeast (Saccharomyces cerevisiae). The highest activity was obtained for the Phaseolus vulgaris CYP93E9 that showed 61-fold higher activity than the Medicago truncatula CYP93E2 (Moses et al., 2014c). However, whether this large variation in C24 hydroxylation efficiency was due to the differential protein expression in heterologous host or the actual difference in the catalytic efficiency of the CYP93Es needs to be tested.
CYP88D6 is another enzyme of the G. uralensis glycyrrhizin biosynthesis pathway (Seki et al., 2008). CYP88D6 catalyzed sequential two-step oxidation of β-amyrin at C11 to produce 11-oxo-β-amyrin, the substrate for CYP72A154. Other characterized members of this family (CYP88A3 and CYP88A4) have been shown to be involved in the biosynthesis of the plant hormone gibberellins (Helliwell et al., 2001).

CYP71D and CYP87D in Pentacyclic Triterpene Biosynthesis
The clan CYP71 represents the largest set of the plant P450s. The families and subfamilies within the clan CYP71 diverged remarkably during plant evolution (Nelson et al., 2004;Nelson and Werck-Reichhart, 2011). Beside CYP93E subfamily, CYP71A, CYP71D, CYP81Q, and CYP705A subfamilies of the clan CYP71 were also found to take part in plant triterpene metabolism (Figure 1). Among these, Lotus japonicus CYP71D353 had been shown to oxidize lupane-type pentacyclic triterpene scaffold (Krokida et al., 2013). CYP71D353 catalyzed conversion of dihydro-lupeol to 20-hydroxy-lupeol following hydroxylation at the C20 position. CYP71D353 also converted 20-hydroxy-lupeol to 20-hydroxy-betulinic acid in a sequential three-step oxidation at the C28 position (Figure 5). CYP71D353 still remains the only known member of the CYP71D subfamily with a role in triterpene biosynthesis. Some other CYP71D subfamily members were known to take part in monoterpene and flavonoid hydroxylation (Haudenschild et al., 2000;Latunde-Dada et al., 2001).
Besides CYP88D and CYP716s, CYP87D is another subfamily under the CYP85 clan with a role in pentacyclic triterpene scaffold modification (Figure 1). To date, CYP87D16 of Maesa lanceolata is the only known member of the CYP87D subfamily associated with the pentacyclic triterpene scaffold modification (Moses et al., 2015a). CYP87D16 participates in triterpene saponin pathway by catalyzing C16α hydroxylation of β-amyrin. The same biochemical activity was also reported for the B. falcatum CYP716Y1 that showed only 27% homology with the CYP87D16 at the protein level (Moses et al., 2014b). So far, CYP87D16 and CYP716Y1 represent the only known examples of the P450s that belong to the separate P450 families and, however, possess the same biochemical function, suggesting that C16α hydroxylation activity might have evolved independently in different plant species.

P450 IN BIOSYNTHESIS OF OTHER TRITERPENE CLASSES
A few P450s of Arabidopsis were identified and biochemically characterized for their involvement in monocyclic (marnerol and marneral) and tricyclic (arabidiol and thalianol) triterpenes metabolism (Figures 1, 5). Arabidopsis CYP71A16 was found to hydroxylate both marnerol and marneral at the C23 position, when expressed in yeast along with an OSC, marneral synthase (MRN1) Castillo et al., 2013). Moreover, the in planta role of CYP71A16 in triterpene oxidation was also confirmed following analysis of the Arabidopsis null mutants (mro1-1 and mro1-2) and over-expression lines . CYP71A16 co-expressed and clustered in Arabidopsis genome with the MRN1. Based on similar experimental approach, the roles of Arabidopsis CYP708A2 and CYP705A5 in hydroxylation of thalianol to 7β-hydroxy-thalianol and in desaturation of 7β-hydroxy-thalianol, respectively, were also reported (Field and Osbourn, 2008;Castillo et al., 2013). CYP708A2 and CYP705A5 catalyzed two consecutive reactions of the thalianol pathway (Figure 5). These genes co-expressed and clustered in the Arabidopsis genome with the OSC thalianol synthase (Field and Osbourn, 2008).

UNDERSTANDING IN PLANTA FUNCTION OF THE P450 OF THE TRITERPENE PATHWAY
The biochemical function of majority of the steroidal scaffold-modifying plant P450s was substantiated based on characterization of the null mutants or gene over-expression lines of the model plant Arabidopsis. This was achievable due to the available genomic resources including functional mutants and efficient genome manipulation tools for Arabidopsis. This approach ascertained the general roles of CYP51G, CYP85A, CYP90B-D, CYP710A, CYP724A,B, and CYP734A across the plant kingdom for the metabolism of the primary sterols and steroid hormones (Kim et al., 2005;Morikawa et al., 2006;Ohnishi et al., 2006b;Sakamoto et al., 2006;Arnqvist et al., 2008). However, several steroidal compounds (e.g., steroidal saponins and steroidal alkaloids) and majority of the triterpene compounds are biosynthesised in species-specific manner (Thimmappa et al., 2014;Augustin et al., 2015;Miettinen et al., 2017). The biochemical functions of the P450s of these speciesspecific pathways were assigned mostly using heterologous expression host, including the model plants (Arabidopsis and tobacco), model microbe (yeast) and insect cell lines. Although biochemical characterization of about 60 P450s of the plant triterpene pathway (excluding steroidal scaffolds) has been completed following expression in heterologous host, only a handful of them were analyzed to determine the in planta function in triterpene biosynthesis (Carelli et al., 2011;Geisler et al., 2013;Moses et al., 2015b;Misra et al., 2017). The in planta functions of the triterpene-modifying P450s are highlighted in the following sections.

Oat CYP51H10
A chemical mutagenesis approach in diploid oat species, Avena strigosa led to the identification of ten independent saponindeficient (sad) mutants that either could not produce saponins in root or had reduced level (Papadopoulou et al., 1999). Saponin deficiency in sad mutant resulted in compromised disease resistance to a variety of root-infecting fungal pathogens. One of these oat mutants, i.e., sad2 was mutated in CYP51H10 gene (Qi et al., 2006). The physiological and biochemical analysis of sad2 provided a conclusive evidence for the involvement of CYP51H10 in root avenacins biosynthesis (Qi et al., 2006;Geisler et al., 2013). sad2 mutant accumulated high level of β-amyrin, confirming the biochemical function of CYP51H10 as β-amyrinmodifying enzyme (Qi et al., 2006;Kunii et al., 2012;Geisler et al., 2013). Moreover, the accumulation of abnormally high level of β-amyrin in sad2 triggered a 'superhairy' root phenotype due to the high rate of transformation of epidermal cells into root hair cells as compared to non-hair cells (Kemen et al., 2014). This observation suggested an important role of β-amyrin in root development.

Medicago CYP716A12 and CYP72A67
The roles of CYP716A12 and CYP72A67 in triterpene pathway were precisely determined based on in-depth analysis of M. truncatula mutant lines (Carelli et al., 2011;Biazzi et al., 2015). CYP716A12 and CYP72A67 loss-of-function mutants were developed following activation tagging and/or targeting induced local lesions in genomes (TILLING) approaches (Porceddu et al., 2008). CYP716A12 loss-of-function iha mutants could not produce hemolytic sapogenins (e.g., hederagenin, bayogenin, medicagenic, zanhic acid) with a C28 carboxylation (Carelli et al., 2011). This study confirmed the in planta function of CYP716A12 as a β-amyrin C28-oxidase. In accordance with the specific role of CYP716A12 in hemolytic saponin pathway, non-hemolytic saponins (e.g., soyasaponins) could be detected in iha mutants, similar to the wild type counterpart (Carelli et al., 2011). Interestingly, iha mutants showed severe growth retardation and altered gene expression related to the secondary metabolism and hormonal pathways, suggesting an important role of the hemolytic saponin biosynthesis pathway in plant growth processes (Carelli et al., 2011).
Moreover, genetic and biochemical analysis of the CYP72A67 TILLING mutants provided substantial evidences for its specific role in hemolytic saponin pathway by catalyzing C2 oxidation of oleanolic acid/hederagenin (Biazzi et al., 2015). Hemolytic sapogenins with C2 hydroxylation (bayogenin, medicagenic, zanhic acid) were completely absent in CYP72A67 mutants. However, sapogenins (gypsogenin, gypsogenic acid, and 16α-hydroxy gypsogenic acid) that lacked a C2 hydroxylation could be detected in CYP72A67 mutants (Biazzi et al., 2015). Interestingly, an alteration in nodulation pattern was observed in CYP72A67 mutant as compared with the wild-type plants, suggesting a potential role for the saponins in regulation of nodulation.

Artemisia CYP716A14v2
Heterologous expression of A. annua CYP716A14v2 in yeast assigned its biochemical function as amyrin C3-oxidase. CYP716A14v2 converted α-amyrin, β-amyrin, and δ-amyrin to α-amyrone, β-amyrone, and δ-amyrone, respectively (Moses et al., 2015b). To access whether CYP716A14v2 catalyzes the same biochemical reaction in planta, transgenic A. annua plants were generated following RNA interference (RNAi) that resulted in silencing of the CYP716A14v2 transcript expression (Moses et al., 2015b). The metabolite analysis of the RNAi and control plants revealed reduced levels of the α-amyrone and β-amyrone in CYP716A14v2-silenced plants as compared to the control, confirming the in planta role of CYP716A14v2 as amyrin C3-oxidase.

Cucumber CYP88L2/CYP81Q58 and Sweet Basil CYP716A252/CYP716A253
The physiological function of cucumber (C. sativus) and sweet basil (Ocimum basilicum) P450s of the tetracyclic and pentacyclic triterpene pathways, respectively, was determined following transient gene silencing approaches. The in planta role of CYP88L2 and CYP81Q58 in cucurbitacin biosynthesis was probed by silencing their transcript expression in the cotyledons using a transient RNAi system. The down-regulation of CYP88L2 and CYP81Q58 transcripts resulted in decreased level of cucurbitacin in the cotyledons, confirming their involvement in cucurbitacin biosynthesis (Shang et al., 2014). Similarly, the in planta function of sweet basil CYP716A252 and CYP716A253 in the biosynthesis of the medicinally important pentacyclic triterpenes (ursolic acid and oleanolic acid) was clarified following a virus induced gene silencing (VIGS) approach (Misra et al., 2017). The down-regulation of CYP716A252 and CYP716A253 expression in sweet basil leaves resulted in reduced level of ursolic acid and oleanolic acid, suggesting that both of these amyrin C28-oxidases are required for the biosynthesis of ursolic acid and oleanolic acid in sweet basil leaves. However, a major contribution of the CYP716A253 in elicitor-mediated accumulation of ursolic acid and oleanolic acid in sweet basil leaves was revealed. These studies in cucumber and sweet basil, suggested that transient gene silencing assays can be useful to probe the in planta function of P450s for the biosynthesis of the species-specific metabolites in non-model plants.
Arabidopsis CYP71A16, CYP705A1, CYP705A5, and CYP708A2 The in planta function of two P450s (CYP705A5 and CYP708A2) of the Arabidopsis thalianol gene cluster was precisely determined following biochemical analysis of the RNAi or T-DNA insertion lines (Field and Osbourn, 2008). CYP705A5 and CYP708A2 co-expressed with the OSC thalianol synthase and catalyzed two consecutive biosynthetic steps of the thalianol pathway ( Figure 5). CYP708A2 mutant line lacked thalian-diol (7β-hydroxy-thalianol) and, however, accumulated increased level of thalianol (Field and Osbourn, 2008). These observations confirmed the physiological role of CYP708A2 as a thalianol hydroxylase. Similarly, metabolite analysis of the CYP705A5 mutant lines revealed increased level of thalian-diol and absence of desaturated thalian-diol, confirming the in planta role of CYP705A5 in thalian-diol desaturation. Interestingly, CYP708A2 over-expression line that accumulated higher level of thalian-diol had dwarf phenotype and longer roots than the wild type, suggesting a crucial role of thalian-diol in plant growth and development.
The physiological role of Arabidopsis CYP705A1 in volatile (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) production in roots has been well documented (Sohrabi et al., 2015). CYP705A1 catalyzed cleavage of the prenyl side chain of arabidiol to produce volatile DMNT and a non-volatile 14-apo-arabidiol (Figure 5). CYP705A1 and arabidiol synthase clustered in the Arabidopsis genome. These genes co-expressed in roots, and also responded to the jasmonic acid treatment and pathogen infection (Sohrabi et al., 2015). The gene knockout line cyp705a1-1 could not emit DMNT and showed compromised defense toward the rootrot pathogen Pythium irregulare. Moreover, DMNT inhibited in vitro spore germination and growth of P. irregulare, suggesting that CYP705A1-mediated cleavage of arabidiol is an important defense mechanism of Arabidopsis.
The function of Arabidopsis CYP71A16 that constitutes the marneral cluster along with the OSC marneral synthase (MRN1) was also determined following biochemical analysis of the T-DNA mutant and over-expression lines . Unlike the wild-type plants, CYP71A16 T-DNA mutant lines accumulated marnerol, an alcohol spontaneously produced from marneral. Moreover, hydroxylated marnerol derivatives were detected in CYP71A16 and MRN1 over-expression lines . These results clearly indicated the physiological role of CYP71A16 in marneral/marnerol oxidation. Interestingly, Arabidopsis plants that over-expressed CYP71A16 and MRN1, had pronounced dwarf phenotype, suggesting a detrimental effect of the marneral pathway intermediates on plant growth and development .

CONCLUSION
So far, about eighty P450s were assigned specific functions to the plant triterpene metabolism (including steroidal scaffolds). Considering a huge structural diversity of the plant triterpenes, it is quite obvious that several other P450s with uncharacteristic biochemical features are yet to be known. With the availability of the large scale genomic and transcriptomic sequence information (The 1000 plants 1 ; Medicinal Plant Genomics Resource 2 ), an increasing number of P450s are expected to be assigned function in the plant triterpene metabolism. The future challenges are functional characterization of the P450s having unusual biochemical properties, and utilization of the P450s for the plant improvement program and for the production of valuable phytochemicals.
Nevertheless, notable advances in understanding the roles of the P450s in plant triterpene metabolism for the general as well as species-specific functions have been made. Usually CYP51G, CYP85A, CYP90B-D, CYP710A, CYP724B, and CYP734A subfamily members are found to metabolize sterols and steroid hormones that mediate primary functions in plants. However, CYP51H, CYP71A,D CYP72A, CYP81Q, CYP87D, CYP88D,L, CYP93E, CYP705A, CYP708A, and CYP716A,C,E,S,U,Y subfamily members are generally found to metabolize triterpenes that showed species-specific distribution in plants. The physiological roles of the P450s in metabolism of the sterols and steroid hormones were mostly revealed based on gene function analysis in the model plants like Arabidopsis, tomato, and rice. Although, a number of P450s were identified from non-model plants and biochemically characterized following protein expression in heterologous hosts, including model plants (Arabidopsis and tobacco) and microbe (S. cerevisiae), the in planta function of a limited number of these P450s was determined. Therefore, extending the functional genomics tools (e.g., VIGS, RNAi, CRISPR/Cas9) to the nonmodel plants is crucial to know the biochemical function of the species-specific P450s and also to establish the biological roles of the triterpene specialized metabolites in plants.

AUTHOR CONTRIBUTIONS
SG collected literatures and wrote the manuscript.

ACKNOWLEDGMENTS
The author thanks Director, CSIR-CIMAP for providing research facilities. CSIR and Department of Biotechnology are acknowledged for funding research on triterpene natural products under the Young Scientist Project (P81 EMR) and IYBA Project (BT/08/IYBA/2014-13), respectively. The author regrets that all literatures could not be cited because of space constraint. Institutional communication number for this article is CIMAP/PUB/2017/34.