Dual factors required for cytochrome-P450-mediated hydrocarbon ring contraction in bacterial gibberellin phytohormone biosynthesis.

Cytochromes P450 (CYPs) are heme-thiolate monooxygenases that prototypically catalyze the insertion of oxygen into unactivated C-H bonds but are capable of mediating more complex reactions. One of the most remarked-upon alternative reactions occurs during biosynthesis of the gibberellin A (GA) phytohormones, involving hydrocarbon ring contraction with coupled aldehyde extrusion of ent-kaurenoic acid to form the first gibberellin intermediate. While the unusual nature of this reaction has long been noted, its mechanistic basis has remained opaque. Building on identification of the relevant CYP114 from bacterial GA biosynthesis, detailed structure-function studies are reported here, including development of in vitro assays as well as crystallographic analyses both in the absence and presence of substrate. These structures provided insight into enzymatic catalysis of this unusual reaction, as exemplified by identification of a key role for the "missing" acid from an otherwise highly conserved acid-alcohol pair of residues. Notably, the results demonstrate that ring contraction requires dual factors, both the use of a dedicated ferredoxin and absence of the otherwise conserved acidic residue, with exclusion of either limiting turnover to just the initiating and more straightforward hydroxylation. The results provide detailed insight into the enzymatic structure-function relationships underlying this fascinating reaction and support the use of a semipinacol mechanism for the unusual ring contraction reaction.

Oryzalexin S biosynthesis: a cross-stitched disappearing pathway.

Rice produces many diterpenoid phytoalexins and, reflecting the importance of these natural products in this important cereal crop plant, its genome contains three biosynthetic gene clusters (BGCs) for such metabolism. The chromosome 4 BGC (c4BGC) is largely associated with momilactone production, in part due to the presence of the initiating syn-copalyl diphosphate (CPP) synthase gene (OsCPS4). Oryzalexin S is also derived from syn-CPP. However, the relevant subsequently acting syn-stemarene synthase gene (OsKSL8) is not located in the c4BGC. Production of oryzalexin S further requires hydroxylation at carbons 2 and 19 (C2 and C19), presumably catalyzed by cytochrome P450 (CYP) monooxygenases. Here it is reported the closely related CYP99A2 and CYP99A3, whose genes are also found in the c4BGC catalyze the necessary C19-hydroxylation, while the closely related CYP71Z21 and CYP71Z22, whose genes are found in the recently reported chromosome 7 BGC (c7BGC), catalyze subsequent hydroxylation at C2α. Thus, oryzalexin S biosynthesis utilizes two distinct BGCs, in a pathway cross-stitched together by OsKSL8. Notably, in contrast to the widely conserved c4BGC, the c7BGC is subspecies (ssp.) specific, being prevalent in ssp. japonica and only rarely found in the other major ssp. indica. Moreover, while the closely related syn-stemodene synthase OsKSL11 was originally considered to be distinct from OsKSL8, it has now been reported to be a ssp. indica derived allele at the same genetic loci. Intriguingly, more detailed analysis indicates that OsKSL8(j) is being replaced by OsKSL11 (OsKSL8i), suggesting introgression from ssp. indica to (sub)tropical japonica, with concurrent disappearance of oryzalexin S production. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-022-00092-3.

Plant (di)terpenoid evolution: from pigments to hormones and beyond.

Covering: up to 2014-2022.Diterpenoid biosynthesis in plants builds on the necessary production of (E,E,E)-geranylgeranyl diphosphate (GGPP) for photosynthetic pigment production, with diterpenoid biosynthesis arising very early in land plant evolution, enabling stockpiling of the extensive arsenal of (di)terpenoid natural products currently observed in this kingdom. This review will build upon that previously published in the Annual Review of Plant Biology, with a stronger focus on enzyme structure-function relationships, as well as additional insights into the evolution of (di)terpenoid metabolism since generated.

Investigation of Acid-Base Catalysis in Halimadienyl Diphosphate Synthase Involved in Mycobacterium tuberculosis Virulence.

The devastating human pathogenMycobacterium tuberculosis (Mtb) is able to parasitize phagosomal compartments within alveolar macrophage cells due, in part, to the activity of its cell-surface lipids. Prominent among these is 1-tuberculosinyl-adenosine (1-TbAd), a derivative of the diterpenoid tuberculosinyl (halima-5,13-dienyl) diphosphate produced by the class II diterpene cyclase encoded by Rv3377c, termed here MtHPS. Given the demonstrated ability of 1-TbAd to act as a virulence factor for Mtb and the necessity for Rv3377c for its production, there is significant interest in MtHPS activity. Class II diterpene cyclases catalyze a general acid-base-mediated carbocation cascade reaction initiated by protonation of the terminal alkene in the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate and terminated by deprotonation of the final cyclized (and sometimes also rearranged) intermediate. Here, structure-guided mutagenesis was applied to characterize the various residues contributing to activation of the enzymatic acid, as well as identify the enzymatic base in MtHPS. Particularly given the ability of conservative substitution for the enzymatic base (Y479F) to generate an alternative product (labda-7,13-dienyl diphosphate) via deprotonation of an earlier unrearranged intermediate, further mutational analysis was carried out to introduce potential alternative catalytic bases. The results were combined with mechanistic molecular modeling to elucidate how these mutations affect the catalytic activity of this important enzyme. This not only provided detailed structure-function insight into MtHPS but also further emphasized the inert nature of the active site of MtHPS and class II diterpene cyclases more generally.

Rice diterpenoid phytoalexins are involved in defence against parasitic nematodes and shape rhizosphere nematode communities.

Rice diterpenoid phytoalexins (DPs) are secondary metabolites with a well known role in resistance to foliar pathogens. As DPs are also known to be produced and exuded by rice roots, we hypothesised that they might play an important role in plant-nematode interactions, and particularly in defence against phytoparasitic nematodes. We used transcriptome analysis on rice roots to analyse the effect of infection by the root-knot nematode Meloidogyne graminicola or treatment with resistance-inducing chemical stimuli on DP biosynthesis genes, and assessed the susceptibility of mutant rice lines impaired in DP biosynthesis to M. graminicola. Moreover, we grew these mutants and their wild-type in field soil and used metabarcoding to assess the effect of impairment in DP biosynthesis on rhizosphere and root nematode communities. We show that M. graminicola suppresses DP biosynthesis genes early in its invasion process and, conversely, that resistance-inducing stimuli transiently induce the biosynthesis of DPs. Moreover, we show that loss of DPs increases susceptibility to M. graminicola. Metabarcoding on wild-type and DP-deficient plants grown in field soil reveals that DPs significantly alter the composition of rhizosphere and root nematode communities. Diterpenoid phytoalexins are important players in basal and inducible defence against nematode pathogens of rice and help shape rice-associated nematode communities.

Deceptive Complexity in Formation of Cleistantha-8,12-diene.

A barley diterpene synthase (HvKSL4) was found to produce (14S)-cleistantha-8,12-diene (1). Formation of the nearly planar cyclohexa-1,4-diene configuration leaves the ring poised for aromatization, but necessitates a deceptively complicated series of rearrangements steered through a complex energetic landscape, as elucidated here through quantum chemical calculations and labeling studies.

Origin and early evolution of the plant terpene synthase family.

As a midsized gene family conserved more by lineage than function, the typical plant terpene synthases (TPSs) could be a valuable tool to examine plant evolution. TPSs are pivotal in biosynthesis of gibberellins and related phytohormones as well as in formation of the extensive arsenal of specialized plant metabolites mediating ecological interactions whose production is often lineage specific. Yet the origin and early evolution of the TPS family is not well understood. Systematic analysis of an array of transcriptomes and sequenced genomes indicated that the TPS family originated after the divergence of land plants from charophytic algae. Phylogenetic and biochemical analyses support the hypothesis that the ancestral TPS gene encoded a bifunctional class I and II diterpene synthase producing the ent-kaurene required for phytohormone production in all extant lineages of land plants. Moreover, the ancestral TPS gene likely underwent duplication at least twice early in land plant evolution. Together these two gave rise to three TPS lineages leading to the extant TPS-c, TPS-e/f, and the remaining TPS (h/d/a/b/g) subfamilies, with the latter dedicated to secondary rather than primary metabolism while the former two contain those genes involved in ent-kaurene production. Nevertheless, parallel evolution from the ent-kaurene­producing class I and class II diterpene synthases has led to roles for TPS-e/f and -c subfamily members in secondary metabolism as well. These results clarify TPS evolutionary history and provide context for the role of these genes in producing the vast diversity of terpenoid natural products observed today in various land plant lineages.

Production of the plant hormone gibberellin by rhizobia increases host legume nodule size.

Plant-associated microbes have evolved the ability to independently produce gibberellin (GA) phytohormones as a mechanism to influence their host. Indeed, GA was first discovered as a metabolite from the fungal rice pathogen Gibberella fujikuroi, which uses it as a virulence factor. Though some bacterial plant pathogens similarly use GA to promote infection, symbiotic nitrogen-fixing bacteria (rhizobia), which inhabit the root nodules of legumes, also can produce GA, suggesting a role in symbiosis. The bacterial GA biosynthetic operon has been identified, but in rhizobia this typically no longer encodes the final metabolic gene (cyp115), so that these symbionts can only produce the penultimate intermediate GA9. Here, we demonstrate that soybean (Glycine max) expresses functional GA 3-oxidases (GA3ox) within its nodules, which have the capability to convert GA9 produced by the enclosed rhizobial symbiont Bradyrhizobium diazoefficiens to bioactive GA4. This rhizobia-derived GA is demonstrated to cause an increase in nodule size and decrease in the number of nodules. The increase in individual nodule size correlates to greater numbers of bacterial progeny within a nodule, thereby providing a selective advantage to rhizobia that produce GA during the rhizobia-legume symbiosis. The expression of GA3ox in nodules and resultant nodulation effects of the GA product suggests that soybean has co-opted control of bioactive GA production, and thus nodule size, for its own benefit. Thus, our results suggest rhizobial GA biosynthesis has coevolved with host plant metabolism for cooperative production of a phytohormone that influences nodulation in a mutually beneficial manner.

Tanshinones: Leading the way into Lamiaceae labdane-related diterpenoid biosynthesis.

Tanshinones are the bioactive diterpenoid constituents of the traditional Chinese medicinal herb Danshen (Salvia miltiorrhiza), and are examples of the phenolic abietanes widely found within the Lamiaceae plant family. Due to the significant interest in these labdane-related diterpenoid natural products, their biosynthesis has been intensively investigated. In addition to providing the basis for metabolic engineering efforts, this work further yielded pioneering insights into labdane-related diterpenoid biosynthesis in the Lamiaceae more broadly. This includes stereochemical foreshadowing of aromatization, with novel protein domain loss in the relevant diterpene synthase, as well as broader phylogenetic conservation of the relevant enzymes. Beyond such summary of more widespread metabolism, formation of the furan ring that characterizes the tanshinones also has been recently elucidated. Nevertheless, the biocatalysts for the pair of demethylations remain unknown, and the intriguing potential connection of these reactions to the further aromatization observed in the tanshinones are speculated upon here.

Diterpene synthases from Leonurus japonicus elucidate epoxy-bridge formation of spiro-labdane diterpenoids.

Spiro-9,13-epoxy-labdane diterpenoids are commonly found in Leonurus species, particularly in Leonurus japonicus Houtt., which is a medicinal herb of long-standing use in Asia and in which such spiro-heterocycles are present in at least 38 diterpenoids. Here, through generation of a transcriptome and functional characterization of six diterpene synthases (diTPSs) from L. japonicus, including three class II diTPSs (LjTPS1, LjTPS3, and LjTPS4) and three class I diTPSs (LjTPS5, LjTPS6, and LjTPS7), formation of the spiro-9,13-epoxy-labdane backbone was elucidated, along with identification of the relevant diTPSs for production of other labdane-related diterpenes. Similar to what has been found with diTPSs from other plant species, while LjTPS3 specifically produces the carbon-9 (C9) hydroxylated bicycle peregrinol diphosphate (PPP), the subsequently acting LjTPS6 yields a mixture of four products, largely labda-13(16),14-dien-9-ol, but with substantial amounts of viteagnusin D and the C13-S/R epimers of 9,13-epoxy-labda-14-ene. Notably, structure-function analysis identified a critical residue in LjTPS6 (I420) in which single site mutations enable specific production of the 13S epimer. Indeed, extensive mutagenesis demonstrated that LjTPS6:I420G reacts with PPP to both specifically and efficiently produce 9,13S-epoxy-labda-14-ene, providing a specialized synthase for further investigation of derived diterpenoid biosynthesis. The results reported here provide a strong foundation for future studies of the intriguing spiro-9,13-epoxy-labdane diterpenoid metabolism found in L. japonicus.

Dissecting the labdane-related diterpenoid biosynthetic gene clusters in rice reveals directional cross-cluster phytotoxicity.

Rice (Oryza sativa) is a staple food crop and serves as a model cereal plant. It contains two biosynthetic gene clusters (BGCs) for the production of labdane-related diterpenoids (LRDs), which serve important roles in combating biotic and abiotic stress. While plant BGCs have been subject to genetic analyses, these analyses have been largely confined to the investigation of single genes. CRISPR/Cas9-mediated genome editing was used to precisely remove each of these BGCs, as well as simultaneously knock out both BGCs. Deletion of the BGC from chromosome 2 (c2BGC), which is associated with phytocassane biosynthesis, but not that from chromosome 4 (c4BGC), which is associated with momilactone biosynthesis, led to a lesion mimic phenotype. This phenotype is dependent on two closely related genes encoding cytochrome P450 (CYP) mono-oxygenases, CYP76M7 and CYP76M8, from the c2BGC. However, rather than being redundant, CYP76M7 has been associated with the production of phytocassanes, whereas CYP76M8 is associated with momilactone biosynthesis. Intriguingly, the lesion mimic phenotype is not present in a line with both BGCs deleted. These results reveal directional cross-cluster phytotoxicity, presumably arising from the accumulation of LRD intermediates dependent on the c4BGC in the absence of CYP76M7 and CYP76M8, further highlighting their interdependent evolution and the selective pressures driving BGC assembly.

Mining of the Catharanthus roseus Genome Leads to Identification of a Biosynthetic Gene Cluster for Fungicidal Sesquiterpenes.

Characterization of cryptic biosynthetic gene clusters (BGCs) from microbial genomes has been proven to be a powerful approach to the discovery of new natural products. However, such a genome mining approach to the discovery of bioactive plant metabolites has been muted. The plant BGCs characterized to date encode pathways for antibiotics important in plant defense against microbial pathogens, providing a means to discover such phytoalexins by mining plant genomes. Here is reported the discovery and characterization of a minimal BGC from the medicinal plant Catharanthus roseus, consisting of an adjacent pair of genes encoding a terpene synthase (CrTPS18) and cytochrome P450 (CYP71D349). These two enzymes act sequentially, with CrTPS18 acting as a sesquiterpene synthase, producing 5-epi-jinkoheremol (1), which CYP71D349 further hydroxylates to debneyol (2). Infection studies with maize revealed that 1 and 2 exhibit more potent fungicidal activity than validamycin. Accordingly, this study demonstrates that characterization of such cryptic plant BGCs is a promising strategy for the discovery of potential agrochemical leads. Moreover, despite the observed absence of 1 and 2 in C. roseus, the observed transcriptional regulation is consistent with their differential fungicidal activity, suggesting that such conditional coexpression may be sufficient to drive BGC assembly in plants.

Rice contains a biosynthetic gene cluster associated with production of the casbane-type diterpenoid phytoalexin ent-10-oxodepressin.

Diterpenoids play important roles in rice microbial disease resistance as phytoalexins, as well as acting in allelopathy and abiotic stress responses. Recently, the casbane-type phytoalexin ent-10-oxodepressin was identified in rice, but its biosynthesis has not yet been elucidated. Here ent-10-oxodepressin biosynthesis was investigated via co-expression analysis and biochemical characterisation, with use of the CRISPR/Cas9 technology for genetic analysis. The results identified a biosynthetic gene cluster (BGC) on rice chromosome 7 (c7BGC), containing the relevant ent-casbene synthase (OsECBS), and four cytochrome P450 (CYP) genes from the CYP71Z subfamily. Three of these CYPs were shown to act on ent-casbene, with CYP71Z2 able to produce a keto group at carbon-5 (C5), while the closely related paralogues CYP71Z21 and CYP71Z22 both readily produce a keto group at C10. Together these C5 and C10 oxidases can elaborate ent-casbene to ent-10-oxodepressin (5,10-diketo-ent-casbene). OsECBS knockout lines no longer produce casbane-type diterpenoids and exhibit impaired resistance to the rice fungal blast pathogen Magnaporthe oryzae. Elucidation of ent-10-oxodepressin biosynthesis and the associated c7BGC provides not only a potential target for molecular breeding, but also, gives the intriguing parallels to the independently assembled BGCs for casbene-derived diterpenoids in the Euphorbiaceae, further insight into plant BGC evolution, as discussed here.

Interdependent evolution of biosynthetic gene clusters for momilactone production in rice.

Plants can contain biosynthetic gene clusters (BGCs) that nominally resemble those found in microbes. However, while horizontal gene transmission is often observed in microbes, plants are limited to vertical gene transmission, implying that their BGCs may exhibit distinct inheritance patterns. Rice (Oryza sativa) contains two unlinked BGCs involved in diterpenoid phytoalexin metabolism, with one clearly required for momilactone biosynthesis, while the other is associated with production of phytocassanes. Here, in the process of elucidating momilactone biosynthesis, genetic evidence was found demonstrating a role for a cytochrome P450 (CYP) from the other "phytocassane" BGC. This CYP76M8 acts after the CYP99A2/3 from the "momilactone" BGC, producing a hemiacetal intermediate that is oxidized to the eponymous lactone by a short-chain alcohol dehydrogenase also from this BGC. Thus, the "momilactone" BGC is not only incomplete, but also fractured by the need for CYP76M8 to act in between steps catalyzed by enzymes from this BGC. Moreover, as supported by similar activity observed with orthologs from the momilactone-producing wild-rice species Oryza punctata, the presence of CYP76M8 in the other "phytocassane" BGC indicates interdependent evolution of these two BGCs, highlighting the distinct nature of BGC assembly in plants.

A pair of threonines mark ent-kaurene synthases for phytohormone biosynthesis.

All land plants (embryophytes) must contain an ent-kaurene synthase (KS), as the ability to produce this olefin from ent-copalyl diphosphate (ent-CPP) is required for phytohormone biosynthesis. These KSs have frequently given rise to other class I diterpene synthases that catalyze distinct reactions for more specialized plant metabolism. Indeed, the prevalence of such gene duplication and neofunctionalization has obscured phylogenetic assignment of function. Here a pair of threonines is found to be conserved in all land plant KS involved in phytohormone biosynthesis, and their role in enzyme function investigated. Surprisingly, these threonines are not required, nor even particularly important for efficient production of ent-kaurene from ent-CPP. In addition, these threonines do not seem to affect protein structure or stability. Moreover, the absence of codon bias and positioning within an intron do not support a role in transcription or translation either. Despite their lack of apparent function, this pair of threonines are nevertheless completely conserved in all embryophyte KS from phytohormone biosynthesis. Thus, regardless of exact role, this serves as a diagnostic mark for such KS, enabling more confident distinction of these critical enzymes.

Expansion within the CYP71D subfamily drives the heterocyclization of tanshinones synthesis in Salvia miltiorrhiza.

Tanshinones are the bioactive nor-diterpenoid constituents of the Chinese medicinal herb Danshen (Salvia miltiorrhiza). These groups of chemicals have the characteristic furan D-ring, which differentiates them from the phenolic abietane-type diterpenoids frequently found in the Lamiaceae family. However, how the 14,16-epoxy is formed has not been elucidated. Here, we report an improved genome assembly of Danshen using a highly homozygous genotype. We identify a cytochrome P450 (CYP71D) tandem gene array through gene expansion analysis. We show that CYP71D373 and CYP71D375 catalyze hydroxylation at carbon-16 (C16) and 14,16-ether (hetero)cyclization to form the D-ring, whereas CYP71D411 catalyzes upstream hydroxylation at C20. In addition, we discover a large biosynthetic gene cluster associated with tanshinone production. Collinearity analysis indicates a more specific origin of tanshinones in Salvia genus. It illustrates the evolutionary origin of abietane-type diterpenoids and those with a furan D-ring in Lamiaceae.

A (conditional) role for labdane-related diterpenoid natural products in rice stomatal closure.

Rice (Oryza sativa) is the staple food for over half the world's population. Drought stress imposes major constraints on rice yields. Intriguingly, labdane-related diterpenoid (LRD) phytoalexins in maize (Zea mays) affect drought tolerance, as indicated by the increased susceptibility of an insertion mutant of the class II diterpene cyclase ZmCPS2/An2 that initiates such biosynthesis. Rice also produces LRD phytoalexins, utilizing OsCPS2 and OsCPS4 to initiate a complex metabolic network. For genetic studies of rice LRD biosynthesis the fast-growing Kitaake cultivar was selected for targeted mutagenesis via CRISPR/Cas9, with an initial focus on OsCPS2 and OsCPS4. The resulting cps2 and cps4 knockout lines were further crossed to create a cps2x4 double mutant. Both CPSs also were overexpressed. Strikingly, all of the cv Kitaake cps mutants exhibit significantly increased susceptibility to drought, which was associated with reduced stomatal closure that was evident even under well-watered conditions. However, CPS overexpression did not increase drought resistance, and cps mutants in other cultivars did not alter susceptibility to drought, although these also exhibited lesser effects on LRD production. The results suggest that LRDs may act as a regulatory switch that triggers stomatal closure in rice, which might reflect the role of these openings in microbial entry.

Author Correction: Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.