Cytochrome P450s are evolutionarily conserved enzymes that are involved in the catalysis of numerous reactions, required for growth, development, defence [30] and secondary metabolism [31]. Prior to this study, no CYP450 genes had been identified, let alone characterized, in any species of the genus Fontainea. This is significant as CYP450 genes are likely to be important for future understanding of the biosynthetic pathways that produce medicinally significant diterpene esters, such as TT, which are unique to Fontainea. Towards that aim, we have identified and classified putative full-length CYP450 encoding genes in two species of Fontainea, F. picrosperma and F. venosa. Phylogenetic analysis allowed us to identify groups of genes for further evaluation. Moreover, their expression profiles in leaf and root tissues were investigated, with a particular focus on the CYP450 genes linked with diterpenoid biosynthesis, potentially involved in the production of TT.
We report 103 and 123 full-length CYP450 genes from F. picrosperma and F. venosa, respectively, that were classified into clans, which cumulatively consisted of 37 families and 67 subfamilies that fit into conformed plant-derived functions, most prominently with diterpenoid, flavonoid and other functions. An ortholog comparison showed that CYP450 genes of Fontainea species are largely unique when compared to other plant species of both Euphorbiaceae and non-Euphorbiaceae. In support of our metabolomics analysis, the S. lycopersicum and A. thaliana CYP450 showed low overall similarity to Fontainea species. This may be attributed to the unique biosynthesis of diterpenoid derivatives that are phorbol ester-specific, found in Fontainea and other members of the Euphorbiaceae family.
The total number of Fontainea CYP450 identified in this study was consistent with that found in other plant transcriptomics CYP450 research, including the total number of full-length sequences, clans, families and subfamilies [16, 32, 33]. For example, transcriptomic studies allowed the elucidation of 151 full-length CYP450 genes in Lonicera japonica [32], 118 full-length in Taxus chinensis [33] and 116 full-length in Salvia miltiorrhiza [16]. However, in S. miltorrhiza, the tissues used for transcriptomics included leaves, roots and flowers, while in L. japonica, flower and buds were used. Our study identified 127 and 125 partial-length CYP450 gene from F. picrosperma and F. venosa, respectively. To obtain the full-length sequence, additional RNA-seq from the stem, flower and fruit would be helpful, as well as from different stages of development. In addition, this could be complemented by genome sequencing.
If a genome is available for a species, it does provide an alternate mechanism for CYP450 gene identification, through genome-wide interrogation. In A. thaliana, this approach identified 246 genes that clustered into 9 clans and 47 families [21, 34], while a much larger number were identified from the soybean (G. max) and rice (Japonica) genomes, containing 332 and 355 CYP450 genes, respectively [35]. Far fewer were present in the legume (Medicago truncatula), where 151 putative CYP450 genes were identified, including 135 novel CYP450 [36]. We expect that once a genome is available for Fontainea, a more complete list of full-length CYP450 genes will be established.
Our results using these predictive protein characterisation analyses (i.e. molecular weight, cell localization, function) were in line with prior studies of CYP450 proteins. CYP450s are typically anchored on the surface of the endoplasmic reticulum [37] and some may target to the plastids or mitochondria [38]. It is common that animal CYPs are anchored to mitochondria, but there is no report of any plant CYP with mitochondrial localization [36] except maize, where 3 CYPs have been reported (Zea mays) [39]. In our study of F. picrosperma or F. venosa, no deduced CYP450 proteins were predicted to have mitochondrial targeting peptides. CYP74A1, CYP74B1 and CYP74B2 in F. picrosperma and F. venosa and CYP726A4 and CYP71B12 in F. picrosperma were found in the chloroplast. In other plants, such as Triticum araraticum, Z. mays all members of CYP74 and CYP701 were targeted to chloroplast [39].
The diversity of different CYP450s between F. picrosperma and F. venosa, and other species, likely contributes to the observed differences in their chemical profiles. In all plant species that have been researched to date, the largest CYP450 clan is CYP71 [22]. The families and subfamilies within the clan have diverged remarkably during plant evolution, many of which are known to be involved in secondary metabolite biosynthesis of flavonoids and alkaloids [40]. Similarly, the CYP71 family is the largest CYP450 clan in Fontainea (see Fig. 2). On the contrary, two CYP711 representatives were identified from F. picrosperma, but were absent in F. venosa, although CYP711 have been described in other plant species [24]. In our phylogenetic tree, the CYP74 clan is adjacent to CYP711 family, suggesting that Fontainea CYP711 may also function within the metabolism of oxylipins and strigolactone signals [24], as strigolactones have been identified as branching inhibition hormones in plants, and several CYP711 have been experimentally confirmed as strigolactones biosynthetic enzymes [41, 42]. We additionally found that F. venosa had more CYP97 genes compared to F. picrosperma; the CYP97 clan is involved in the hydroxylation of carotenoids [43]. Carotenoids are a group of widely distributed pigments derived from the ubiquitous isoprenoid biosynthetic pathway and play diverse roles in plant primary and secondary metabolism. Carotenoids contain two pigments, carotene and lutein, which absorb and transfer energy to protect chlorophyll [34]. We speculate that this may partially explain why F. venosa have darker leaves compared to F. picrosperma.
We found that Fontainea (F. picrosperma and F. venosa) CYP450 genes were more actively expressed in root tissue compared to leaf tissue (see Fig. 3). Among those significantly more highly expressed in the root have been associated with fertility reduction (CYP78A), UV stress tolerance (CYP84A), gibberellin metabolism (CYP714A) and jasmonic acid metabolism (CYP74A) [44–47]. Those significantly more highly expressed in the leaf include those previously associated with catalysing successive oxidation steps of the plant hormone jasmonoyl-isoleucine for catabolic turnover (CYP94), expression of ABA 8’-hydroxylase and affects ABA levels to control seed dormancy (CYP707A), hydroxylation of carotenoids (CYP97), biosynthesis of castasterone in the brassinosteroid biosynthetic pathway (CYP85A) and glucosinolate metabolism (CYP83) [37, 43, 48–50]. Some species variation existed in CYP450 homolog tissue expression (see Fig. 3B). This may be explained by the different growth and developmental stage of plants from which the tissue was sampled, as CYP450s are involved in the regulation of plant hormone metabolism, growth and development and hormones are involved in formation and development of flowers, leaves, stems and fruits [51].
Diterpenoids are one of the most widespread classes of secondary metabolites in higher plants, which are synthesized from basic isoprene units (C5H8) and further modified by various oxidoreductases, acyltransferases, dehydrogenases and glucosyltransferases [52]. CYP450-dependent oxidative modification is essential for the biosynthesis of diterpenes [52]. There are countless products formed in plants, among them diterpenoids are one of the most diverse groups, consisting of more than 12,000 metabolites [53] that have proven to be valuable as therapeutic drugs. According to previous research, CYP71D and CYP726A subfamilies are key CYP450s involved in diterpenoid biosynthesis [25, 53] and most of these diterpenoids can only be found in plants [54]. Our phylogenetic analysis of diterpenoid CYP71 clan members revealed that Fontainea have representatives within two different diterpenoid subfamilies, namely CYP71D and CYP726A.
F. picrosperma diterpenoid CYP450 genes are significantly more highly expressed in root tissue compared to leaf tissue. The expression of genes can be affected by the developmental stage of plants, environmental conditions, seasonal and diurnal effects as well as biotic and abiotic stress [55]. Therefore, future research should explore the expression of the identified CYP450 genes under these different scenarios and in additional tissues. In other Euphorbiacea, diterpenoid genes were found to be co-regulated in rhizome and hairy roots [17] or highly expressed in root tissue compared to leaf and flower [16]. Nonetheless, Fontainea CYP71D and CYP726A genes are excellent candidates for involvement in diterpenoid biosynthesis pathways, in particular, the biosynthesis of epoxy-tigliane diterpene esters, which are only found in species of Fontainea, although further experiments are required to confirm this hypothesis. Their identification allows for experimental analysis of their function, for example, in vitro expression of the proteins followed by activity detection, or by knock-in and knock-out can be completed. This may be followed by activity detection in vivo, depending on the availability of a robust experimental system. Also, the analysis of high TT producing F. picroserma, compared with low producers, will provide guidance about CYP450 (and other genes) that potentially regulate TT production.