Characterization of Biosynthetic Pathways for the Production of the Volatile Homoterpenes DMNT and TMTT in Zea mays.

Plant volatiles not only have multiple defense functions against herbivores, fungi, and bacteria, but also have been implicated in signaling within the plant and toward other organisms. Elucidating the function of individual plant volatiles will require more knowledge of their biosynthesis and regulation in response to external stimuli. By exploiting the variation of herbivore-induced volatiles among 26 maize (Zea mays) inbred lines, we conducted a nested association mapping and genome-wide association study (GWAS) to identify a set of quantitative trait loci (QTLs) for investigating the pathways of volatile terpene production. The most significant identified QTL affects the emission of (E)-nerolidol, linalool, and the two homoterpenes (E)-3,8-dimethyl-1,4,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). GWAS associated a single nucleotide polymorphism in the promoter of the gene encoding the terpene synthase TPS2 with this QTL Biochemical characterization of TPS2 verified that this plastid-localized enzyme forms linalool, (E)-nerolidol, and (E,E)-geranyllinalool. The subsequent conversion of (E)-nerolidol into DMNT maps to a P450 monooxygenase, CYP92C5, which is capable of converting nerolidol into DMNT by oxidative degradation. A QTL influencing TMTT accumulation corresponds to a similar monooxygenase, CYP92C6, which is specific for the conversion of (E,E)-geranyllinalool to TMTT The DMNT biosynthetic pathway and both monooxygenases are distinct from those previously characterized for DMNT and TMTT synthesis in Arabidopsis thaliana, suggesting independent evolution of these enzymatic activities.

A small, differentially regulated family of farnesyl diphosphate synthases in maize (Zea mays) provides farnesyl diphosphate for the biosynthesis of herbivore-induced sesquiterpenes.

MAIN CONCLUSION: Of the three functional FPPS identified in maize, fpps3 is induced by herbivory to produce FDP important for the formation of the volatile sesquiterpenes of plant defense. Sesquiterpenes are not only crucial for the growth and development of a plant but also for its interaction with the environment. The biosynthesis of sesquiterpenes proceeds over farnesyl diphosphate (FDP), which is either used as a substrate for protein prenylation, converted to squalene, or to volatile sesquiterpenes. To elucidate the regulation of sesquiterpene biosynthesis in maize, we identified and characterized the farnesyl diphosphate synthase (FPPS) gene family which consists of three genes. Synteny analysis indicates that fpps2 and fpps3 originate from a genome duplication in an ancient tetraploid ancestor. The three FPPSs encode active enzymes that produce predominantly FDP from the isopentenyl diphosphate and dimethylallyl diphosphate substrates. Only fpps1 and fpps3 are induced by elicitor treatment, but induced fpps1 levels are much lower and only increased to the amounts of fpps3 levels in intact leaves. Elicitor-induced fpps3 levels in leaves increase to more than 15-fold of background levels. In undamaged roots, transcript levels of fpps1 are higher than those of fpps3, but only fpps3 transcripts are induced in response to herbivory by Diabrotica virgifera virgifera. A kinetic of transcript abundance in response to herbivory in leaves provided further evidence that the regulation of fpps3 corresponds to that of tps23, a terpene synthase, that converts FDP to the volatile (E)-ß-caryophyllene. Our study indicates that the differential expression of fpps1 and fpps3 provides maize with FDP for both primary metabolism and terpene-based defenses. The expression of fpps3 seems to coincide with the herbivore-induced emission of volatile sesquiterpenes that were demonstrated to be important defense signals.