Abstract
Rice (Oryza sativa) produces numerous diterpenoid phytoalexins that are important in defense against pathogens. Surprisingly, despite extensive previous investigations, a major group of such phytoalexins, the abietoryzins, were only recently reported. These aromatic abietanes are presumably derived from ent-miltiradiene, but such biosynthetic capacity has not yet been reported in O. sativa. While wild rice has been reported to contain such an enzyme, specifically ent-kaurene synthase-like 10 (KSL10), the only characterized ortholog from O. sativa (OsKSL10), specifically from the well-studied cultivar (cv.) Nipponbare, instead has been shown to make ent-sandaracopimaradiene, precursor to the oryzalexins. Notably, in many other cultivars, OsKSL10 is accompanied by a tandem duplicate, termed here OsKSL14. Biochemical characterization of OsKLS14 from cv. Kitaake demonstrates that this produces the expected abietoryzin precursor ent-miltiradiene. Strikingly, phylogenetic analysis of OsKSL10 across the rice pan-genome reveals that from cv. Nipponbare is an outlier, whereas the alleles from most other cultivars group with those from wild rice, suggesting that these also might produce ent-miltiradiene. Indeed, OsKSL10 from cv. Kitaake exhibits such activity as well, consistent with its production of abietoryzins but not oryzalexins. Similarly consistent with these results is the lack of abietoryzin production by cv. Nipponbare. Although their equivalent product outcome might suggest redundancy, OsKSL10 and OsKSL14 were observed to exhibit distinct expression patterns, indicating such differences may underlie retention of these duplicated genes. Regardless, the results reported here clarify abietoryzin biosynthesis and provide insight into the evolution of rice diterpenoid phytoalexins.
Avoid common mistakes on your manuscript.
Introduction
Phytoalexins serve an important role in plant defense against pathogens (Ahuja et al. 2012). Their activity is of agricultural importance in crop plants, where such natural products have been intensively studied. As a critically important staple food crop, the arsenal of rice phytoalexins have been extensively investigated for many decades, with the first identified in the 1970s (Valletta et al. 2023). Despite these extended studies, a new group of rice phytoalexins were recently reported, with these abietoryzins further found in a large fraction of the investigated cultivars (Kariya et al. 2023).
Just as with most of the previously known rice phytoalexins (Murphy and Zerbe 2020), the abietoryzins are labdane-related diterpenoids. The biosynthesis of this super-family is initiated by class II diterpene cyclases (Peters 2010). These catalyze bicyclization of the general diterpenoid precursor geranylgeranyl diphosphate (GGPP), prototypically producing the eponymous labdadienyl/copalyl diphosphate (CPP), leading to designation of such enzymes as CPP synthases (CPSs). All land plants must contain a CPS that produces ent-CPP, as well as a subsequently acting class I diterpene synthase that yields ent-kaurene, for phytohormone biosynthesis (Wang et al. 2023). In angiosperms, this CPS and kaurene synthase (KS) have often given rise to expanded gene families mediating more specialized metabolism (Zi et al. 2014), with members of the latter typically termed KS-like (KSL).
Rice (O. sativa) contains two CPSs producing ent-CPP, with OsCPS1 required for gibberellin phytohormone biosynthesis (Sakamoto et al. 2004), while OsCPS2 is associated with more specialized metabolism (Otomo et al. 2004b; Prisic et al. 2004; Lu et al. 2018). There are then several rice KSLs that act on ent-CPP and produce distinct hydrocarbon backbones that characterize the previously known families of labdane-related diterpenoid phytoalexins (Toyomasu et al. 2020). In particular, OsKSL6 yields the ent-isokaurene precursor to the oryzalides and related phytoalexins (Kanno et al. 2006), OsKSL7 produces the ent-cassadiene precursor to the phytocassanes (Cho et al. 2004), and OsKSL10 yields the ent-sandaracopimaradiene precursor to the oryzalexins (Otomo et al. 2004a; Xu et al. 2007a).
Notably, none of the previously characterized OsKSLs reacts with ent-CPP to yield an abietane, as is required for abietoryzin biosynthesis (Fig. 1A). More specifically, the aromatic nature of the distal ‘C’-ring in this group of labdane-related diterpenoid phytoalexins would be most readily derived from ent-miltiradiene (ent-abieta-8,12-diene), whose cyclohexa-1,4-diene arrangement in this KSL formed ring leaves it planar and poised for such aromatization (Gao et al. 2009), which can occur spontaneously (Zi and Peters 2013). Intriguingly, it has been reported that KSL10 from wild rice (Oryza rufipogon) encodes an ent-miltiradiene synthase (Toyomasu et al. 2016). Here, identification of ent-miltiradiene synthases from domesticated rice is reported.
Results
While much of the previous work on rice KSLs was carried out with cv. Nipponbare, it has been observed that there are some differences in KSL activity between various subspecies and even cultivars (Xu et al. 2007b; Zhao et al. 2023). Given the emerging use of cv. Kitaake as a model system (Kim et al. 2013; Jain et al. 2019), particularly for investigation of labdane-related diterpenoid phytoalexins (Zhang et al. 2021; Li et al. 2022), an inventory of its KSL gene family was carried out, which revealed the presence of a new KSL. This was found adjacent to OsKSL10 (OsKit12g133500) and, building on previous numbering of the rice KSL family, was termed OsKSL14 (OsKit12g133400). Notably, OsKSL14 was found to be most closely related to OsKSL10 (Fig. 1B), and their proximity suggests these arose from tandem gene duplication. Consistent with the lack of previous recognition of this gene, OsKSL14 does not appear to be present in cv. Nipponbare.
The close phylogenetic relationship of OsKSL14 to OsKSL10 and, hence, the KSL10 from wild rice, along with lack of previously identified ent-miltiradiene synthases in domesticated rice, immediately led to the hypothesis that OsKSL14 might serve this function. Accordingly, OsKSL14 was cloned from cv. Kitaake and incorporated into a previously described modular metabolic engineering system, which enables KSL co-expression in E. coli that also produce CPP of defined stereochemical configuration (Cyr et al. 2007). Indeed, comparison of OsKSL14 with a previously described wild rice (O. rufipogon) OrKSL10 (Toyomasu et al. 2016) demonstrated that it also reacts with ent-CPP to produce ent-miltiradiene (Fig. 1C and Supplemental Fig. S1).
As the abietoryzins were reported to be widespread in rice (Kariya et al. 2023), a previously reported phylogenetically representative set of domesticated rice (Zhou et al. 2020), along with a broader set to include other species across the Oryza genus (Stein et al. 2018), were examined for the presence of OsKSL14. Consistent with a role in such prevalent biosynthesis, KSL14 appears to be present in all cultivars of O. sativa other than Nipponbare (although potentially as a partial pseudo-gene in one other case), as well as all species from the AA-genome clade of Oryza, but not in the BB-genome representative Oryza punctata. Such presence–absence variation analysis of KSL10 indicates this neighboring gene is similarly prevalent in O. sativa, although it also appears to be a partial pseudo-gene in three cultivars. There is only one cultivar (subspecies indica cv. Larha Mugad) where both seem to be missing. Notably, KSL10 also appears to be limited to the AA-genome clade of Oryza, as it is not present in O. punctata.
Intriguingly, while phylogenetic analysis of OsKSL14 generally matches subspecies (ssp.) assignment, that of OsKSL10 exhibited some conflict (Supplemental Fig. S2). Specifically, matching the accepted relationships within the Oryza genus (Wing et al. 2018), for OsKSL14 the alleles from cultivars within ssp. japonica subspecies, along with the associated minor ssp. basmati, group separately from those from the other major ssp. (indica) group and associated minor ssp. aus. By contrast, for OsKSL10, the allele from cv. Nipponbare is an outlier, with the other alleles more closely related to OrKSL10, suggesting that the previously reported activity of the Nipponbare allele (OsKSL10N) might be divergent.
To investigate the potentially more general ent-miltiradiene synthase activity of OsKSL10, the allele from cv. Kitaake (OsKSL10K) was cloned and its activity compared to that of OsKSL10N using the modular metabolic engineering system in E. coli. Indeed, while OsKSL10N reacts with ent-CPP to produce ent-sandaracopimaradiene as previously reported (Xu et al. 2007a), OsKSL10K was found to produce ent-miltiradiene instead (Fig. 1D and Supplemental Fig. S3). While clearly orthologous, OsKSL10N and OsKSL10K share less than 97% sequence identity at the amino acid level (Supplemental Fig. S4), and their numerous differences preclude ready identification of the particular residue(s) responsible for their distinct product outcomes.
Given that OsKSL10N has been reported to also react with syn-CPP to produce syn-labdatriene (Morrone et al. 2011), OsKSL10K and OrKSL10 as well as OsKSL14 were similarly examined. Intriguingly, each of these was found to also react with syn-CPP. However, these yield not only syn-labdatriene but also syn-stemodene, which is the major product of OsKSL11 (Morrone et al. 2006), now known to be the allele of OsKSL8 specific to ssp. indica (Toyomasu et al. 2016) and more accurately termed OsKSL8i (Fig. 1E and Supplemental Fig. S5).
Given the inducible nature of the abietoryzins (Kariya et al. 2023), the expression of OsKSL10 and OsKSL14 in response to MeJA in cv. Kitaake was analyzed via RT-qPCR. Although only OsKSL10 and not OsKSL14 mRNA was detectable prior to induction, consistent with roles in abietoryzin biosynthesis both exhibited increases in transcript levels following treatment with MeJA, with the highest expression found 24 h post-induction (Supplemental Fig. S6).
The biochemical results indicate that cv. Kitaake should produce abietoryzins but cannot make oryzalexins, while cv. Nipponbare should exhibit the opposite metabolite profile, which would be consistent with the previously reported lack of abietoryzins in this cultivar (Kariya et al. 2023). Indeed, targeted analysis of induced seedlings verifies that cv. Nipponbare produces at least oryzalexin C while cv. Kitaake does not, and suggests the opposite is true for the abietoryzins—i.e., at least some may be made by cv. Kitaake, although only after induction, but none by cv. Nipponbare (Supplemental Fig. S7).
Discussion
Consistent with the previously reported prevalence of abietoryzins (Kariya et al. 2023), here identification of not just a single but dual ent-miltiradiene synthases (OsKSL10 and OsKSL14) in most rice cultivars is reported. However, there are certain cultivars that lack any such activity, not least cv. Nipponbare, which does not contain OsKSL14 and whose copy of OsKSL10 exhibits divergent activity in producing ent-sandaracopimadiene instead, consistent with the previously reported lack of abietoryzins in this cultivar (Kariya et al. 2023). By contrast, cv. Kitaake seems to produce abeitoryzins but not orzyalexins, consistent with its lack of an ent-sandaracopimaradiene synthase. Given the apparently typical biochemical redundancy exhibited by OsKSL10 and OsKSL14, it is unclear what drove the spread of the gene duplication leading to these tandem KSLs. Regardless, the prevalent ent-miltiradiene synthase activity encoded by OsKSL10 and/or OsKSL14 suggests that biosynthesis of the only recently discovered abietoryzins arose early in evolution of the AA-genome clade of the Oryza genus and highlights the need for further investigation of these intriguing natural products.
Materials and methods
Unless otherwise stated all chemicals were obtained from Fisher Scientific. KSL sequences were obtained via BLAST searches with the known genes from cv. Nipponbare against the Gramene Oryza database (Tello-Ruiz et al. 2022). OsKSL14 and OsKSL10K were cloned from cv. Kitaake, while OsKSL10N from Nipponbare is that previously described (Xu et al. 2007a), and a gene (optimized for expression in Escherichia coli) was synthesized for the previously described OrKSL10 (Toyomasu et al. 2016). All genes were truncated to remove the N-terminal plastid targeting sequence and characterized via a previously described modular metabolic engineering system (Cyr et al. 2007). The resulting diterpene products were extracted and analyzed by gas chromatography with mass spectrometry (GC–MS) as previously described (Jia et al. 2022), with the addition of passage over silica gel to remove confounding polar metabolites from the extract prior to GC–MS analysis. Given the presence of putative splicing errors in the predicted coding sequences, genomic sequences were obtained for KSL10 and KSL14 and iteratively aligned by BLAST with the exons from the known examples. An error in the predicted junction between exons 10 and 11 in OsKSL10 was found by comparison of that predicted and cloned for cv. Kitaake, and the correction (shift of four nucleotides from exon 10 to 11) applied in this re-analysis. Similarly, frame-shifting mutations were also noted in the KSL10s predicted by this re-analysis for Oryza glumaepatula and Oryza glaberrima, so those originally found in Gramene were used instead. These coding sequences were used for the presented phylogenetic analyses, which were carried out using MEGA11 (Tamura et al. 2021). Gene expression levels were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using the primers listed in the supplemental information (Table S1), and 10-day (post-germination) cv. Kitaake seedlings induced with methyl jasmonate (MeJA) as previously described (Wang et al. 2011). For diterpenoid measurement, rice seedlings were grown, induced and extracted as described by Kariya et al. (2023), with analysis by liquid chromatography with mass spectrometry (LC–MS/MS) as previously described (Zhang et al. 2021), and the relevant diterpenoids identified using an oryzalexin C standard and the relative retention times and MS/MS detection reported by Kariya et al. (2023) for the abietoryzins.
Data availability
All data generated or analyzed in this study are available from the corresponding author upon reasonable request.
References
Ahuja I, Kissen R, Bones AM (2012) Phytoalexins in defense against pathogens. Trends Plant Sci 17:73–90
Cho E-M, Okada A, Kenmoku H, Otomo K, Toyomasu T, Mitsuhashi W, Sassa T, Yajima A, Yabuta G, Mori K, Oikawa H, Toshima H, Shibuya N, Nojiri H, Omori T, Nishiyama M, Yamane H (2004) Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor. Plant J 37:1–8
Cyr A, Wilderman PR, Determan M, Peters RJ (2007) A modular approach for facile biosynthesis of labdane-related diterpenes. J Am Chem Soc 129:6684–6685
Gao W, Hillwig ML, Huang L, Cui G, Wang X, Kong J, Yang B, Peters RJ (2009) A functional genomics approach to tanshinone biosynthesis provides stereochemical insights. Org Lett 11:5170–5173
Jain R, Jenkins J, Shu S, Chern M, Martin JA, Copetti D, Duong PQ, Pham NT, Kudrna DA, Talag J, Schackwitz WS, Lipzen AM, Dilworth D, Bauer D, Grimwood J, Nelson CR, Xing F, Xie W, Barry KW, Wing RA, Schmutz J, Li G, Ronald PC (2019) Genome sequence of the model rice variety KitaakeX. BMC Genomics 20:905
Jia Q, Brown R, Kollner TG, Fu J, Chen X, Wong GK, Gershenzon J, Peters RJ, Chen F (2022) Origin and early evolution of the plant terpene synthase family. Proc Natl Acad Sci USA 119:2100361119
Kanno Y, Otomo K, Kenmoku H, Mitsuhashi W, Yamane H, Oikawa H, Toshima H, Matsuoka M, Sassa T, Toyomasu T (2006) Characterization of a rice gene family encoding type-A diterpene cyclases. Biosci Biotechnol Biochem 70:1702–1710
Kariya K, Fujita A, Ueno M, Yoshikawa T, Teraishi M, Taniguchi Y, Ueno K, Ishihara A (2023) Natural variation of diterpenoid phytoalexins in rice: aromatic diterpenoid phytoalexins in specific cultivars. Phytochemistry 211:113708
Kim SL, Choi M, Jung KH, An G (2013) Analysis of the early-flowering mechanisms and generation of T-DNA tagging lines in Kitaake, a model rice cultivar. J Exp Bot 64:4169–4182
Li R, Zhang J, Li Z, Peters RJ, Yang B (2022) Dissecting the labdane-related diterpenoid biosynthetic gene clusters in rice reveals directional cross-cluster phytotoxicity. New Phytol 233:878–889
Lu X, Zhang J, Brown B, Li R, Rodriguez-Romero J, Berasategui A, Liu B, Xu M, Luo D, Pan Z, Baerson SR, Gershenzon J, Li Z, Sesma A, Yang B, Peters RJ (2018) Inferring roles in defense from metabolic allocation of rice diterpenoids. Plant Cell 30:1119–1131
Morrone D, Hillwig ML, Mead ME, Lowry L, Fulton DB, Peters RJ (2011) Evident and latent plasticity across the rice diterpene synthase family with potential implications for the evolution of diterpenoid metabolism in the cereals. Biochem J 435:589–595
Morrone D, Jin Y, Xu M, Choi SY, Coates RM, Peters RJ (2006) An unexpected diterpene cyclase from rice: functional identification of a stemodene synthase. Arch Biochem Biophys 448:133–140
Murphy KM, Zerbe P (2020) Specialized diterpenoid metabolism in monocot crops: biosynthesis and chemical diversity. Phytochemistry 172:112289
Otomo K, Kanno Y, Motegi A, Kenmoku H, Yamane H, Mitsuhashi W, Oikawa H, Toshima H, Itoh H, Matsuoka M, Sassa T, Toyomasu T (2004a) Diterpene cyclases responsible for the biosynthesis of phytoalexins, momilactones A, B, and oryzalexins A-F in rice. Biosci Biotechnol Biochem 68:2001–2006
Otomo K, Kenmoku H, Oikawa H, Konig WA, Toshima H, Mitsuhashi W, Yamane H, Sassa T, Toyomasu T (2004b) Biological functions of ent- and syn-copalyl diphosphate synthases in rice: key enzymes for the branch point of gibberellin and phytoalexin biosynthesis. Plant J 39:886–893
Peters RJ (2010) Two rings in them all: the labdane-related diterpenoids. Nat Prod Rep 27:1521–1530
Prisic S, Xu M, Wilderman PR, Peters RJ (2004) Rice contains two disparate ent-copalyl diphosphate synthases with distinct metabolic functions. Plant Physiol 136:4228–4236
Sakamoto T, Miura K, Itoh H, Tatsumi T, Ueguchi-Tanaka M, Ishiyama K, Kobayashi M, Agrawal GK, Takeda S, Abe K, Miyao A, Hirochika H, Kitano H, Ashikari M, Matsuoka M (2004) An overview of gibberellin metabolism enzyme genes and their related mutants in rice. Plant Physiol 134:1642–1653
Stein JC, Yu Y, Copetti D, Zwickl DJ, Zhang L, Zhang C, Chougule K, Gao D, Iwata A, Goicoechea JL, Wei S, Wang J, Liao Y, Wang M, Jacquemin J, Becker C, Kudrna D, Zhang J, Londono CEM, Song X, Lee S, Sanchez P, Zuccolo A, Ammiraju JSS, Talag J, Danowitz A, Rivera LF, Gschwend AR, Noutsos C, Wu CC, Kao SM, Zeng JW, Wei FJ, Zhao Q, Feng Q, El Baidouri M, Carpentier MC, Lasserre E, Cooke R, Rosa Farias DD, da Maia LC, Dos Santos RS, Nyberg KG, McNally KL, Mauleon R, Alexandrov N, Schmutz J, Flowers D, Fan C, Weigel D, Jena KK, Wicker T, Chen M, Han B, Henry R, Hsing YC, Kurata N, de Oliveira AC, Panaud O, Jackson SA, Machado CA, Sanderson MJ, Long M, Ware D, Wing RA (2018) Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza. Nat Genet 50:285–296
Tamura K, Stecher G, Kumar S (2021) MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol 38:3022–3027
Tello-Ruiz MK, Jaiswal P, Ware D (2022) Gramene: a resource for comparative analysis of plants genomes and pathways. Methods Mol Biol 2443:101–131
Toyomasu T, Miyamoto K, Shenton MR, Sakai A, Sugawara C, Horie K, Kawaide H, Hasegawa M, Chuba M, Mitsuhashi W, Yamane H, Kurata N, Okada K (2016) Characterization and evolutionary analysis of ent-kaurene synthase like genes from the wild rice species Oryza rufipogon. Biochem Biophys Res Commun 480:402–408
Toyomasu T, Shenton MR, Okada K (2020) Evolution of labdane-related diterpene synthases in cereals. Plant Cell Physiol 61:1850–1859
Valletta A, Iozia LM, Fattorini L, Leonelli F (2023) Rice phytoalexins: half a century of amazing discoveries; part i: distribution, biosynthesis, chemical synthesis, and biological activities. Plants (Basel) 12
Wang Q, Hillwig ML, Peters RJ (2011) CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice. Plant J 65:87–95
Wang Z, Nelson DR, Zhang J, Wan X, Peters RJ (2023) Plant (di)terpenoid evolution: from pigments to hormones and beyond. Nat Prod Rep 40:452–469
Wing RA, Purugganan MD, Zhang Q (2018) The rice genome revolution: from an ancient grain to Green Super Rice. Nat Rev Genet 19:505–517
Xu M, Wilderman PR, Morrone D, Xu J, Roy A, Margis-Pinheiro M, Upadhyaya NM, Coates RM, Peters RJ (2007a) Functional characterization of the rice kaurene synthase-like gene family. Phytochemistry 68:312–326
Xu M, Wilderman PR, Peters RJ (2007b) Following evolution’s lead to a single residue switch for diterpene synthase product outcome. Proc Natl Acad Sci USA 104:7397–7401
Zhang J, Li R, Xu M, Hoffmann RI, Zhang Y, Liu B, Zhang M, Yang B, Li Z, Peters RJ (2021) A (conditional) role for labdane-related diterpenoid natural products in rice stomatal closure. New Phytol 230:698–709
Zhao L, Oyagbenro R, Feng Y, Xu M, Peters RJ (2023) Oryzalexin S biosynthesis: a cross-stitched disappearing pathway. aBIOTECH 4:1–7
Zhou Y, Chebotarov D, Kudrna D, Llaca V, Lee S, Rajasekar S, Mohammed N, Al-Bader N, Sobel-Sorenson C, Parakkal P, Arbelaez LJ, Franco N, Alexandrov N, Hamilton NRS, Leung H, Mauleon R, Lorieux M, Zuccolo A, McNally K, Zhang J, Wing RA (2020) A platinum standard pan-genome resource that represents the population structure of Asian rice. Sci Data 7:113
Zi J, Mafu S, Peters RJ (2014) To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism. Annu Rev Plant Biol 65:259–286
Zi J, Peters RJ (2013) Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamiaceae. Org Biomol Chem 11:7650–7652
Acknowledgements
This work was supported by grants from the NIH (GM131885) and USDA (2020-67013-32557) to R.J.P.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
On behalf of all the authors, the corresponding author states there are no conflicts of interest.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Feng, Y., Weers, T. & Peters, R.J. Double-barreled defense: dual ent-miltiradiene synthases in most rice cultivars. aBIOTECH (2024). https://doi.org/10.1007/s42994-024-00167-3
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s42994-024-00167-3