Momilactones are bioactive diterpenoids that contribute to plant defense against pathogens and allelopathic interactions between plants. Both cultivated and wild grass species ofOryzaandEchinochloa crus-galli(barnyard grass) produce momilactones using a biosynthetic gene cluster (BGC) in their genomes. The bryophyteCalohypnum plumiforme(formerlyHypnum plumaeforme) also produces momilactones, and the bifunctional diterpene cyclase geneCpDTC1/HpDTC1,which is responsible for the production of the diterpene framework, has been characterized. To understand the molecular architecture of the momilactone biosynthetic genes in the moss genome and their evolutionary relationships with other momilactone-producing plants, we sequenced and annotated theC. plumiformegenome. The data revealed a 150-kb genomic region that contains two cytochrome P450 genes, theCpDTC1/HpDTC1gene and the “dehydrogenase momilactone A synthase” gene tandemly arranged and inductively transcribed following stress exposure. The predicted enzymatic functions in yeast and recombinant assay and the successful pathway reconstitution inNicotiana benthamianasuggest that it is a functional BGC responsible for momilactone production. Furthermore, in a survey of genomic sequences of a broad range of plant species, we found that momilactone BGC is limited to the two grasses (OryzaandEchinochloa) andC. plumiforme, with no synteny among these genomes. These results indicate that while the gene cluster inC. plumiformeis functionally similar to that in rice and barnyard grass, it is likely a product of convergent evolution. To the best of our knowledge, this report of a BGC for a specialized plant defense metabolite in bryophytes is unique.
Momilactones, which are diterpenoid phytoalexins with antimicrobial and allelopathic functions, have been found only in rice and the moss Hypnum plumaeforme. Although these two evolutionarily distinct plant species are thought to produce momilactones as a chemical defence, the momilactone biosynthetic pathway in H. plumaeforme has been unclear. Here, we identified a gene encoding syn-pimara-7,15-diene synthase (HpDTC1) responsible for the first step of momilactone biosynthesis in the moss. HpDTC1 is a bifunctional diterpene cyclase that catalyses a two-step cyclization reaction of geranylgeranyl diphosphate to syn-pimara-7,15-diene. HpDTC1 transcription was up-regulated in response to abiotic and biotic stress treatments. HpDTC1 promoter-GUS analysis in transgenic Physcomitrella patens showed similar transcriptional responses as H. plumaeforme to the stresses, suggesting that a common response system to stress exists in mosses. Jasmonic acid (JA), a potent signalling molecule for inducing plant defences, could not activate HpDTC1 expression. In contrast, 12-oxo-phytodienoic acid, an oxylipin precursor of JA in vascular plants, enhanced HpDTC1 expression and momilactone accumulation, implying that as-yet-unknown oxylipins could regulate momilactone biosynthesis in H. plumaeforme. These results demonstrate the existence of an evolutionarily conserved chemical defence system utilizing momilactones and suggest the molecular basis of the regulation for inductive production of momilactones in H. plumaeforme.
ent-Kaurenoic acid (KA) is a key intermediate connected to a phytohormone gibberellin. To date, the general procedure for quantifying KA is by using traditional gas chromatography–mass spectrometry (GC–MS). In contrast, gibberellins, which are more hydrophilic than KA, can be easily quantified by liquid chromatography-tandem mass spectrometry (LC–MS/MS). In this study, we have established a new method to quantify KA by LC–MS/MS by taking advantage of a key feature of KA, namely the lack of fragmentation that occurs in MS/MS when electrospray ionization (ESI) is in the negative mode. Q1 and Q3 were adopted as identical channels for the multiple reaction monitoring of KA. The method was validated by comparing with the results obtained by selected ion monitoring in GC–MS. This new method could be applicable for the quantification of other hydrophobic compounds.
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