The regulatory function of isoprenyl diphosphate synthases in terpene biosynthesis

Terpenes are essential secondary metabolites they are important defensive compounds of plants and insects. The biosynthesis of terpenes starts with the formation isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP). In the next step of biosynthesis, isoprenyl diphosphate synthases (IDS) condense DMADP with a varying number of IDP molecules to produce longer prenyl diphosphates, like geranyl- (GDP), farnesyl- (FDP) and geranylgeranyl diphosphate (GGDP), which are substrates for terpene synthases that produce the various terpene skeletons. IDS have a critical role in terpene biosynthesis since they are located at a central point where the pathway branches towards products of different sizes. The published findings of the present dissertation begin with the description of a new LC-MS/MS based method for identification of the product specificity of IDS and the quantification of total IDS activity. This was the methodical basis for investigations on the regulatory function of IDS1 after overexpression in Picea glauca. Here it was shown that overexpression is only functional in needles. Overexpression increases the total IDS-activity. Despite the increases in terpene precursors the defensive oleoresin was unaltered. Instead large amounts of geranylgeranyl fatty acid esters were formed that were shown to have defensive properties against the needle feeding insect Lymantria monacha. Also insect employ terpenes to defend themselve. One example here is the horseradish leaf beetle Phaedon cochleariae, which has pairs of dorsal glands, that excrete the terpene chrysomelidial in case of an attack. Here IDS1 was identified to produce either GDP or FDP, depending on the metal ion cofactor. With an RNAi approach it was revealed that IDS1 is indeed a key enzyme in chrysomelidial biosynthesis. RNAi mediated transcript suppression, reduced the total IDS enzyme activity, the amount of intermediates in the larvae and the volume of the defensive secretions

Probing the specificity of CYP112 in bacterial gibberellin biosynthesis

Biosynthesis of the gibberellin A (GA) plant hormones evolved independently in plant-associated fungi and bacteria. While the relevant enzymes have distinct evolutionary origins, the pathways proceed via highly similar reactions. One particularly complex transformation involves combined demethylation and γ-lactone ring formation, catalyzed in bacteria by the cytochrome P450 CYP112 in three individual steps, which involves large structural changes in the transition from substrate to product, with further divergence in the recently demonstrated use of two separate mechanistic routes. Here the substrate specificity of the isozyme from Erwinia tracheiphila, EtCYP112, was probed via UV-Vis spectral binding studies and activity assays with alternate substrates from the GA biosynthetic pathway. EtCYP112 tightly binds its native substrate GA12 and the intermediates GA15 and GA24, as well as the methylated derivatives of GA12 and GA15. It however only poorly binds methylated GA24, its GA9 final product and the C-20 carboxylate side-product GA25. These distinct affinities are consistent with the known reactivity of EtCYP112. However, while it binds to the immediately preceding pathway metabolite GA12-aldehyde and even earlier oxygenated ent-kaurene precursors, EtCYP112 only reacts with GA12-aldehyde, and not the earlier ent-kaurene derived metabolites. But even with GA12-aldehyde conversion is limited to the first two steps and the final combined demethylation and γ-lactone ring forming reaction is not catalyzed. Thus, CYP112 has evolved specificity at the catalytic rather than substrate binding level to enable its role in GA biosynthesis

18O2 labeling experiments illuminate the oxidation of ent-kaurene in bacterial gibberellin biosynthesis

Bacteria can produce gibberellin plant hormones. While the bacterial biosynthetic pathway is similar to that of plants, the individual enzymes are very distantly related and arose via convergent evolution. The cytochromes P450 (CYPs) that catalyze the multi-step oxidation of the alkane precursor ent-kaurene (1) to ent-kauren-19-oic acid (5), are called ent-kaurene oxidase (KO), and in plants are from the CYP701 family, and share less than 19% amino acid sequence identity with those from bacteria, which are from the phylogenetically distinct CYP117 family. Here the reaction series catalyzed by CYP117 was examined by 18O2 labeling experiments, the results indicate successive hydroxylation of 1 to ent-kauren-19-ol (2) and then ent-kauren-19,19-diol (3) and most likely an intervening dehydration to ent-kauren-19-al (4) prior to the concluding hydroxylation to 5. Accordingly, the bacterial and plant KOs converged on catalysis of the same series of reactions, despite their independent evolutionary origin

Investigating the Phylogenetic Range of Gibberellin Biosynthesis in Bacteria

Certain plant-associated microbes can produce gibberellin (GA) phytohormones, as first described for the rice fungal pathogen Gibberella fujikuroi and, more recently, for bacteria, including several rhizobia and the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola. The relevant enzymes are encoded by a biosynthetic operon that exhibits both a greater phylogenetic range and scattered distribution among plant-associated bacteria. Here, the phylogenetic distribution of this operon was investigated. To demonstrate conserved functionality, the enzymes encoded by the disparate operon from X. translucens pv. translucens, along with those from the most divergent example, found in Erwinia tracheiphila, were biochemically characterized. In both of these phytopathogens, the operon leads to production of the bioactive GA4. Based on these results, it seems that this operon is widely dedicated to GA biosynthesis. However, there is intriguing variation in the exact product. In particular, although all plant pathogens seem to produce bioactive GA4, rhizobia generally only produce the penultimate hormonal precursor GA9. This is suggested to reflect their distinct interactions with plants, because production of GA4 counteracts the jasmonic-acid-mediated defense response, reflecting the importance of wounds as the entry point for these phytopathogens, whereas such suppression presumably is detrimental in the rhizobial symbiotic relationship

Diverging Mechanisms: Cytochrome‐P450‐Catalyzed Demethylation and γ‐Lactone Formation in Bacterial Gibberellin Biosynthesis

Biosynthesis of the gibberellin (GA) plant hormones evolved independently in plants and microbes, but the pathways proceed by similar transformations. The combined demethylation and γ‐lactone ring forming transformation is of significant mechanistic interest, yet remains unclear. The relevant CYP112 from bacteria was probed by activity assays and 18O2‐labeling experiments. Notably, the ability of tert‐butyl hydroperoxide to drive this transformation indicates use of the ferryl‐oxo (Compound I) from the CYP catalytic cycle for this reaction. Together with the confirmed loss of C20 as CO2, this necessitates two catalytic cycles for carbon–carbon bond scission and γ‐lactone formation. The ability of CYP112 to hydroxylate the δ‐lactone form of GA15, shown by the labeling studies, is consistent with the implied use of a further oxygenated heterocycle in the final conversion of GA24 into GA9, with the partial labeling of GA9, thus demonstrating that CYP112 partitions its reactants between two diverging mechanisms

An operon for production of bioactive gibberellin A4 phytohormone with wide distribution in the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola

• Phytopathogens have developed elaborate mechanisms to attenuate the defense response of their host plants, including convergent evolution of complex pathways for production of the gibberellin (GA) phytohormones, which were actually first isolated from the rice fungal pathogen Gibberella fujikuroi. The rice bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) has been demonstrated to contain a biosynthetic operon with cyclases capable of producing the universal GA precursor ent-kaurene. Genetic (knock-out) studies indicate that the derived diterpenoid serves as a virulence factor for this rice leaf streak pathogen, serving to reduce the jasmonic acid (JA) mediated defense response. • Here the function of the remaining genes in the Xoc operon are elucidated and the distribution of the operon in X. oryzae investigated in over 100 isolates. • The Xoc operon leads to production of the bioactive GA4, an additional step beyond production of the penultimate precursor GA9 mediated by the homologous operons recently characterized from rhizobia. Moreover, this GA biosynthetic operon was found to be widespread in Xoc (\u3e90%), but absent in the other major oryzae pathovar. • These results indicate selective pressure for production of GA4 in the distinct lifestyle of Xoc, and the importance of GA to both fungal and bacterial pathogens of rice

Bark beetle attack history does not influence the induction of terpene and phenolic defenses in mature Norway spruce (Picea abies) trees by the bark beetle-associated fungus Endoconidiophora polonica

Terpenes and phenolics are important constitutive and inducible conifer defenses against bark beetles and their associated fungi. In this study, the inducible defenses of mature Norway spruce (Picea abies) trees with different histories of attack by the spruce bark beetle, Ips typographus were tested by inoculation with the I. typographusassociated fungus Endoconidiophora polonica. We compared trees that had been under previous attack with those under current attack and those that had no record of attack. After fungal inoculation, the concentrations of mono-, sesqui-, and diterpenes in bark increased 3- to 9-fold. For the phenolics, the flavan-3-ols, catechin, and gallocatechin, increased significantly by 2- and 5-fold, respectively, while other flavonoids and stilbenes did not. The magnitudes of these inductions were not influenced by prior bark beetle attack history for all the major compounds and compound classes measured. Before fungal inoculation, the total amounts of monoterpenes, diterpenes, and phenolics (constitutive defenses) were greater in trees that had been previously attacked compared to those under current attack, possibly a result of previous induction. The transcript levels of many genes involved in terpene formation (isoprenyl diphosphate synthases and terpene synthases) and phenolic formation (chalcone synthases) were significantly enhanced by fungal inoculation suggesting de novo biosynthesis. Similar inductions were found for the enzymatic activity of isoprenyl diphosphate synthases and the concentration of their prenyl diphosphate products after fungal inoculation. Quantification of defense hormones revealed a significant induction of the jasmonate pathway, but not the salicylic acid pathway after fungal inoculation. Our data highlight the coordinated induction of terpenes and phenolics in spruce upon infection by E. polonica, a fungal associate of the bark beetle I. typographus, but provide no evidence for the priming of these defense responses by prior beetle attack.Zwillenberg-Tietz Foundation and the Max Planck Society.https://www.frontiersin.org/journals/plant-sciencedm2022Forestry and Agricultural Biotechnology Institute (FABI)Zoology and Entomolog

Unraveling a Tangled Skein: Evolutionary Analysis of the Bacterial Gibberellin Biosynthetic Operon

Gibberellin (GA) phytohormones are ubiquitous regulators of growth and developmental processes in vascular plants. The convergent evolution of GA production by plant-associated bacteria, including both symbiotic nitrogen-fixing rhizobia and phytopathogens, suggests that manipulation of GA signaling is a powerful mechanism for microbes to gain an advantage in these interactions. Although orthologous operons encode GA biosynthetic enzymes in both rhizobia and phytopathogens, notable genetic heterogeneity and scattered operon distribution in these lineages, including loss of the gene for the final biosynthetic step in most rhizobia, suggest varied functions for GA in these distinct plant-microbe interactions. Therefore, deciphering GA operon evolutionary history should provide crucial evidence toward understanding the distinct biological roles for bacterial GA production. To further establish the genetic composition of the GA operon, two operon-associated genes that exhibit limited distribution among rhizobia were biochemically characterized, verifying their roles in GA biosynthesis. This enabled employment of a maximum parsimony ancestral gene block reconstruction algorithm to characterize loss, gain, and horizontal gene transfer (HGT) of GA operon genes within alphaproteobacterial rhizobia, which exhibit the most heterogeneity among the bacteria containing this biosynthetic gene cluster. Collectively, this evolutionary analysis reveals a complex history for HGT of the entire GA operon, as well as the individual genes therein, and ultimately provides a basis for linking genetic content to bacterial GA functions in diverse plant-microbe interactions, including insight into the subtleties of the coevolving molecular interactions between rhizobia and their leguminous host plants