Positive Darwinian selection is a driving force for the diversification of terpenoid biosynthesis in the genus Oryza

Background: Terpenoids constitute the largest class of secondary metabolites made by plants and display vast chemical diversity among and within species. Terpene synthases (TPSs) are the pivotal enzymes for terpenoid biosynthesis that create the basic carbon skeletons of this class. Functional divergence of paralogous and orthologous TPS genes is a major mechanism for the diversification of terpenoid biosynthesis. However, little is known about the evolutionary forces that have shaped the evolution of plant TPS genes leading to terpenoid diversity. Results: The orthologs of Oryza Terpene Synthase 1 (OryzaTPS1), a rice terpene synthase gene involved in indirect defense against insects in Oryza sativa, were cloned from six additional Oryza species. In vitro biochemical analysis showed that the enzymes encoded by these OryzaTPS1 genes functioned either as (E)-β-caryophyllene synthases (ECS), or (E)-β-caryophyllene & germacrene A synthases (EGS), or germacrene D & germacrene A synthases (DAS). Because the orthologs of OryzaTPS1 in maize and sorghum function as ECS, the ECS activity was inferred to be ancestral. Molecular evolutionary detected the signature of positive Darwinian selection in five codon substitutions in the evolution from ECS to DAS. Homology-based structure modeling and the biochemical analysis of laboratory-generated protein variants validated the contribution of the five positively selected sites to functional divergence of OryzaTPS1. The changes in the in vitro product spectra of OryzaTPS1 proteins also correlated closely to the changes in in vivoblends of volatile terpenes released from insect-damaged rice plants. Conclusions: In this study, we found that positive Darwinian selection is a driving force for the functional divergence of OryzaTPS1. This finding suggests that the diverged sesquiterpene blend produced by the Oryza species containing DASmay be adaptive, likely in the attraction of the natural enemies of insect herbivores

Localization of sesquiterpene formation and emission in maize leaves after herbivore damage

BACKGROUND: Maize (Zea mays L.) leaves damaged by lepidopteran herbivores emit a complex volatile blend that can attract natural enemies of the herbivores and may also have roles in direct defense and inter- or intra-plant signaling. The volatile blend is dominated by sesquiterpenes of which the majority is produced by two herbivore-induced terpene synthases, TPS10 and TPS23. However, little is known about the pattern of volatile emission within maize leaves. RESULTS: In this study, we restricted herbivore feeding to small sections of the maize leaf with the aim of determining the patterns of volatile sesquiterpene emission throughout the damaged leaf and in neighboring leaves. Sesquiterpene volatiles were released at high rates from damaged leaves, but at much lower rates from neighboring leaves. Release was restricted to the site of damage or to leaf sections located apical to the damage, but was not seen in sections basal to the damage or on the other side of the midrib. The emission pattern correlated well with the transcript pattern of the respective sesquiterpene synthase genes, tps10 and tps23, implying that biosynthesis likely occurs at the site of emission. The concentrations of jasmonic acid and its leucine derivative were also elevated in terpene-emitting tissues suggesting a role for jasmonates in propagating the damage signal. CONCLUSIONS: In contrast to other defense reactions which often occur systemically throughout the whole plant, herbivore-induced sesquiterpene production in maize is restricted to the wounding site and distal leaf parts. Since the signal mediating this reaction is directed to the leaf tip and cannot propagate parallel to the leaf axis, it is likely connected to the xylem. The increasing gradient of volatiles from the tip of the leaf towards the damage site might aid herbivore enemies in host or prey finding

De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug.

Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (1S,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication

The lactonase BxdA mediates metabolic specialisation of maize root bacteria to benzoxazinoids.

Root exudates contain specialised metabolites that shape the plant's root microbiome. How host-specific microbes cope with these bioactive compounds, and how this ability affects root microbiomes, remains largely unknown. We investigated how maize root bacteria metabolise benzoxazinoids, the main specialised metabolites of maize. Diverse and abundant bacteria metabolised the major compound in the maize rhizosphere MBOA (6-methoxybenzoxazolin-2(3H)-one) and formed AMPO (2-amino-7-methoxy-phenoxazin-3-one). AMPO forming bacteria were enriched in the rhizosphere of benzoxazinoid-producing maize and could use MBOA as carbon source. We identified a gene cluster associated with AMPO formation in microbacteria. The first gene in this cluster, bxdA encodes a lactonase that converts MBOA to AMPO in vitro. A deletion mutant of the homologous bxdA genes in the genus Sphingobium, did not form AMPO nor was it able to use MBOA as a carbon source. BxdA was identified in different genera of maize root bacteria. Here we show that plant-specialised metabolites select for metabolisation-competent root bacteria. BxdA represents a benzoxazinoid metabolisation gene whose carriers successfully colonize the maize rhizosphere and thereby shape the plant's chemical environmental footprint

A radiation of Psylliodes flea beetles on Brassicaceae is associated with the evolution of specific detoxification enzymes

Flea beetles of the genus Psylliodes have evolved specialized interactions with plant species belonging to several distantly related families, mainly Brassicaceae, Solanaceae, and Fagaceae. This diverse host use indicates that Psylliodes flea beetles are able to cope with different chemical defense metabolites, including glucosinolates, the characteristic defense metabolites of Brassicaceae. Here we investigated the evolution of host use and the emergence of a glucosinolate-specific detoxification mechanism in Psylliodes flea beetles. In phylogenetic analyses, Psylliodes species clustered into four major clades, three of which contained mainly species specialized on either Brassicaceae, Solanaceae, or Fagaceae. Most members of the fourth clade have broader host use, including Brassicaceae and Poaceae as major host plant families. Ancestral state reconstructions suggest that Psylliodes flea beetles were initially associated with Brassicaceae and then either shifted to Solanaceae or Fagaceae, or expanded their host repertoire to Poaceae. Despite a putative ancestral association with Brassicaceae, we found evidence that the evolution of glucosinolate-specific detoxification enzymes coincides with the radiation of Psylliodes on Brassicaceae, suggesting that these are not required for using Brassicaceae as hosts but could improve the efficiency of host use by specialized Psylliodes species.Funding was provided by the Max Planck Society, the International Max Planck Research School, and the Daimler & Benz Foundation (project number 32-01/14). Harald Letsch was funded by the Austrian Science Fund (FWF) project P32029-B. Part of the genetic results presented here were achieved in the frame of the German Barcode of Life, a project of the Humboldt Ring, grant funded by the German Federal Ministry for Education and Research (GBOL1: BMBF #01LI1101A/#01LI1501A).Peer reviewe