An initial reaction of the phytosterol biosynthesis is the CYP51G-catalyzed 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 Δ14,15 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.

CYP710A participates in sterol biosynthesis by catalyzing sterol C22 desaturation reaction. Among the four CYP710A members of Arabidopsis, CYP710A1, CYP710A2, and CYP710A4 were biochemically characterized (Morikawa et al., 2006; Arnqvist et al., 2008). In in vitro assays, CYP710A1, CYP710A2, and CYP710A4 catalyzed the conversion of β-sitosterol to stigmasterol (Figure 2). Moreover, CYP710A2 also converted campesterol to crinosterol and 24-epi-campesterol to brassicasterol (Figure 2). However, based on gene over-expression studies in Arabidopsis, the in planta functions of CYP710A1 and CYP710A4 in stigmasterol biosynthesis, and CYP710A2 in brassicasterol and crinosterol biosynthesis were established (Morikawa et al., 2006; Arnqvist et al., 2008). Further substantiating these observations, cyp710a2 null mutant did not accumulate brassicasterol and crinosterol (Morikawa et al., 2006). Similar to the higher plants, Physcomitrella patens also engaged CYP710A for sterol C22 desaturation, signifying evolutionary conservation of the CYP710A’s function in sterol biosynthesis (Morikawa et al., 2009).

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 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).

The biosynthetic enzymes for the cyclopamine, a steroidal alkaloid that showed promising anti-neoplastic activities, were identified from Veratrum californicum (Lin et al., 2010; Augustin et al., 2015). These enzymes included three P450 family members (CYP90B27, CYP90G1, and CYP94N1) that were recently identified by exploring transcriptome (RNA-seq) data of V. californicum (Figure 2). These P450s were biochemically characterized by employing a protein expression platform based on Sf9 insect cells (Augustin et al., 2015). CYP90B27 being a cholesterol 22-hydroxylase, catalyzed the conversion of cholesterol to 22(R)-hydroxy-cholesterol. However, CYP94N1 converted 22(R)-hydroxy-cholesterol to 22-hydroxy-cholesterol-26-al following sequential two-step oxidations at the C26 position. CYP90G1 was designated as 22-hydroxy-26-amino-cholesterol 22-oxidase that converted 22-hydroxy-26-amino-cholesterol to 22-keto-26-amino-cholesterol. Similar to CYP90G1, CYP90B27 was also found to oxidize C22 hydroxyl to C22 ketone, however, only with a lower efficiency than the CYP90G1 (Augustin et al., 2015).

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 (Miettinen et al., 2017). 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.

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 (Miettinen et al., 2017). The first evidence for the CYP716s participation in triterpene biosynthesis appeared from a gene co-expression study in Arabidopsis that suggested a co-regulated 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 three-step 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).

Besides CYP716A subfamily members, CYP716C, CYP716E, CYP716S, and CYP716Y subfamily members were also known to oxidize pentacyclic triterpene scaffolds (Figures 1, 3–5). Tomato CYP716E26 and Centella asiatica CYP716E41 were biochemically characterized as C6β-hydroxylases that accepted α/β-amyrin and oleanolic acid/ursolic acid/maslinic acid as substrates, respectively (Miettinen et al., 2017; Yasumoto et al., 2017). Though CYP716E41 did not oxidize amyrin skeleton (Miettinen et al., 2017), whether CYP716E26 can oxidize oleanolic acid/ursolic acid/maslinic acid remains to be tested. C. asiatica CYP716C11 was found to catalyze C2α hydroxylation of oleanolic acid, 6β-hydroxy-oleanolic acid, and ursolic acid (Miettinen et al., 2017). However, P. grandiflorus CYP716S5 catalyzed C12-C13α epoxidation of β-amyrin and oleanolic acid. Moreover, Bupleurum falcatum CYP716Y1 is a C16α-hydroxylase that acted on β-amyrin and α-amyrin scaffolds (Moses et al., 2014b; Miettinen et al., 2017).

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 (Geisler et al., 2013). 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 anti-microbial 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 (Geisler et al., 2013).

Legumes accumulate hemolytic and non-hemolytic triterpene saponins (Moses et al., 2014c; Ghosh, 2016). Hederagenin, zanhic acid, and medicagenic acid scaffolds-derived saponins with oxidation at the C28 position are classified as hemolytic; however, soyasapogenols-derived saponins with oxidation at the C24 position are non-hemolytic (Carelli et al., 2011). In addition, hemolytic saponins also bear oxygen functionality at the C2 and C23 positions as compared to oxygen functionality at the C22 position in non-hemolytic saponins. Besides, CYP716A12 with β-amyrin C28-oxidase activity, CYP72A, CYP88D, and CYP93E subfamily members were known for the biosynthesis of the triterpene saponins in legumes (Seki et al., 2008; Moses et al., 2014c; Biazzi et al., 2015).

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 non-hemolytic 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; Fukushima et al., 2013; 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.

CYP72A subfamily members are distributed well across the angiosperm species and were not found in gymnosperm, moss, and fern species (Prall et al., 2016). However, the biochemical function of most of the angiosperm CYP72As remains to be known. To date, CYP72As of M. truncatula and Glycyrrhiza uralensis were biochemically characterized and were found to be associated with the legume-specific triterpene saponin biosynthesis. So far, four CYP72As of M. truncatula (CYP72A61, CYP72A63, CYP72A67, and CYP72A68) and one CYP72A of G. uralensis (CYP72A154) were reported to partake in oleanane triterpene scaffold oxidation (Figures 1, 3; Seki et al., 2011; Fukushima et al., 2013; Biazzi et al., 2015). M. truncatula CYP72A61, a C22-hydroxylase of the non-hemolytic saponin biosynthesis pathway, converted 24-hydroxy-β-amyrin (the product of the CYP93E) to soyasapogenol B. However, CYP72A67 and CYP72A68 were found to be C2β-hydroxylase (oleanolic acid/hederagenin C2β-hydroxylase) and C23-oxidase (oleanolic acid/hederagenin/2β-hydroxy-hederagenin C23-oxidase) of the hemolytic saponin pathway, respectively (Fukushima et al., 2013; Biazzi et al., 2015). Conversely, G. uralensis CYP72A154 is a C30-oxidase that catalyzed three-step oxidation of 11-oxo-β-amyrin to glycyrrhetinic acid, an intermediate for the biosynthesis of glycyrrhizin (Seki et al., 2011). Glycyrrhizin, a triterpene saponin derived from the underground parts of the Glycyrrhiza (licorice) plants has various pharmaceutical properties and is being used worldwide as a natural sweetener (Seki et al., 2008). CYP72A154 also reacted on β-amyrin and converted it to 30-hydroxy-β-amyrin. Similarly, M. truncatula CYP72A63 was found to possess C30-oxidase activity and converted β-amyrin to 11-deoxoglycyrrhetinic acid following a three-step oxidation (Seki et al., 2011). Besides legumes, other characterized CYP72As are from Catharanthus roseus (CYP72A1 and CYP72A224) that participate in indole alkaloid biosynthesis (Irmler et al., 2000; Salim et al., 2013; Miettinen et al., 2014).

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).

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.

A chemical mutagenesis approach in diploid oat species, Avena strigosa led to the identification of ten independent saponin-deficient (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-amyrin-modifying 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.

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.

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.

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.

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 root-rot 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 (Field et al., 2011). 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 (Field et al., 2011). 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 (Field et al., 2011).