Identification of Syn-Pimara-7,15-Diene Synthase Reveals Functional Clustering of Terpene Synthases Involved in Rice Phytoalexin/Allelochemical Biosynthesis

Abstract

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Accordingly, the committed step in biosynthesis of these natural products is catalyzed by the class I terpene synthase that converts syn-copalyl diphosphate to the corresponding polycyclic hydrocarbon intermediate syn-pimara-7,15-diene. Here, a functional genomics approach was utilized to identify a syn-copalyl diphosphate specific 9b-pimara-7,15-diene synthase (OsDTS2). To our knowledge, this is the first identified terpene synthase with this particular substrate stereoselectivity and, by comparison with the previously described and closely related ent-copalyl diphosphate specific cassa-12,15-diene synthase (OsDTC1), provides a model system for investigating the enzymatic determinants underlying the observed difference in substrate specificity. Further, OsDTS2 mRNA in leaves is up-regulated by conditions that stimulate phytoalexin biosynthesis but is constitutively expressed in roots, where momilactones are constantly synthesized as allelochemicals. Therefore, transcription of OsDTS2 seems to be an important regulatory point for controlling production of these defensive compounds. Finally, the gene identified here as OsDTS2 has previously been mapped at 14.3 cM on chromosome 4. The class II terpene synthase producing syn-copalyl diphosphate from the universal diterpenoid precursor geranylgeranyl diphosphate was also mapped to this same region. These genes catalyze sequential cyclization steps in momilactone biosynthesis and seem to have been evolutionarily coupled by physical linkage and resulting cosegregation. Further, the observed correlation between physical proximity and common metabolic function indicates that other such class I and class II terpene synthase gene clusters may similarly catalyze consecutive reactions in shared biosynthetic pathways.Plants produce a vast and diverse array of low-molecular weight organic compounds, the overwhelming majority of which are secondary metabolites with nonessential, yet important, functions such as defense . For example, phytoalexins are produced in response to microbial infections and exhibit antimicrobial activity (VanEtten et al., 1994), while allelochemicals are secreted to the rhizosphere and suppress the growth of neighboring plants (Bais et al., 2004). Often found serving in such roles are terpenoids, which are particularly abundant in plant metabolism and form the largest class of natural products, exhibiting wide diversity in chemical structure and biological function . Much of the structural variation within this class arises from the diverse carbon backbones formed by terpene synthases (cyclases). These divalent metal dependent enzymes carry out complex electrophilic cyclizations/ rearrangements to create these diverse skeletal structures from relatively simple acyclic precursors (Davis and Croteau, 2000). Notably, production of a specific backbone structure either dictates, or at least severely restricts, the metabolic fate of that particular molecule. Thus,...

Gene Clusters for Secondary Metabolic Pathways: An Emerging Theme in Plant Biology

“…These clusters are diverse in organization and function and all appear to have evolved independently (Field and Osbourn, 2008;Frey et al, 2009;Osbourn and Field, 2009;Swaminathan et al, 2009). The four clusters from cereals are all required for the synthesis of preformed or stress-induced compounds implicated in plant defense (Papadopoulou et al, 1999;Gierl and Frey, 2001;Wilderman et al, 2004;Shimura et al, 2007). The function of the Arabidopsis thalianol cluster is not known, but the high level of conservation of this cluster across different Arabidopsis accessions suggests that it is also like to have an important function in ecological interactions (Field and Osbourn, 2008).…”

“…Unlike operons, the genes within these clusters are transcribed separately. The five plant clusters are for the synthesis of cyclic hydroxamic acids in maize (Frey et al, 1997(Frey et al, , 2003(Frey et al, , 2009von Rad et al, 2001;Jonczyk et al, 2008), triterpenes in oat (Avena sativa) and Arabidopsis (the avenacin and thalianol clusters, respectively; Papadopoulou et al, 1999;Haralampidis et al, 2001;Qi et al, 2004Qi et al, , 2006Field and Osbourn, 2008;Mylona et al, 2008;Mugford et al, 2009), and diterpenes in rice (Oryza sativa; the momilactone and phytocassane clusters; Sakamoto et al, 2004;Wilderman et al, 2004;Shimura et al, 2007;Swaminathan et al, 2009). These clusters are diverse in organization and function and all appear to have evolved independently (Field and Osbourn, 2008;Frey et al, 2009;Osbourn and Field, 2009;Swaminathan et al, 2009).…”

“…This will be revealed through further analysis of fully sequenced plant genomes. The maize and rice gene clusters were defined using a combination of molecular biology, biochemistry, and reverse genetics (Frey et al, 1997(Frey et al, , 2003(Frey et al, , 2009von Rad et al, 2001;Sakamoto et al, 2004;Wilderman et al, 2004;Shimura et al, 2007;Jonczyk et al, 2008;Swaminathan et al, 2009). Analysis of the two rice clusters was aided by the fact that expression of these gene clusters is elicitor inducible and by the availability of rice genome sequence information.…”

Momilactone A and B as Allelochemicals from MossHypnum plumaeforme: First Occurrence in Bryophytes

“…The first cyclization from GGDP produces syn-copalyl diphosphate (syn-CDP) by the action of syn-CDP synthase (syn-CPS), and the second cyclization converts syn-CDP into synpimara-7,15-diene by the action of syn-pimara-7,15-diene synthase (OsDTS2). 19,20) These two synthases involved in the biosynthesis of momilactones have been identified using rice genome information. 19,20) Recently, we isolated ent-16 -hydroxykaurene and ent-kaurene synthase (PpCPS/KS) from the moss P. patens and analyzed its function.…”

“…19,20) These two synthases involved in the biosynthesis of momilactones have been identified using rice genome information. 19,20) Recently, we isolated ent-16 -hydroxykaurene and ent-kaurene synthase (PpCPS/KS) from the moss P. patens and analyzed its function. PpCPS/KS is a bifunctional cyclase that synthesizes the ent-kaurene skeleton directly from GGDP, similarly to fungal CPS/KS, with both ent-CPS and KS activity.…”