Camptotheca acuminata 10‐hydroxycamptothecin O‐methyltransferase: an alkaloid biosynthetic enzyme co‐opted from flavonoid metabolism

The medicinal plant Camptotheca acuminata accumulates camptothecin, 10‐hydroxycamptothecin, and 10‐methoxycamptothecin as its major bioactive monoterpene indole alkaloids. Here, we describe identification and functional characterization of 10‐hydroxycamptothecin O‐methyltransferase (Ca10OMT), a member of the Diverse subclade of class II OMTs. Ca10OMT is highly active toward both its alkaloid substrate and a wide range of flavonoids in vitro and in this way contrasts with other alkaloid OMTs in the subclade that only utilize alkaloid substrates. Ca10OMT shows a strong preference for the A‐ring 7‐OH of flavonoids, which is structurally equivalent to the 10‐OH of 10‐hydroxycamptothecin. The substrates of other alkaloid OMTs in the subclade bear little similarity to flavonoids, but the 3‐D positioning of the 7‐OH, A‐ and C‐rings of flavonoids is nearly identical to the 10‐OH, A‐ and B‐rings of 10‐hydroxycamptothecin. This structural similarity likely explains the retention of flavonoid OMT activity by Ca10OMT and also why kaempferol and quercetin aglycones are potent inhibitors of its 10‐hydroxycamptothecin activity. The catalytic promiscuity and strong inhibition of Ca10OMT by flavonoid aglycones in vitro prompted us to investigate the potential physiological roles of the enzyme in vivo. Based on its regioselectivity, kinetic parameters and absence of 7‐OMT flavonoids in vivo, we conclude that the major and likely only substrate of Ca10OMTin vivo is 10‐hydroxycamptothecin. This is likely accomplished by Ca10OMT being kept spatially separated at the tissue levels from potentially inhibitory flavonoid aglycones, and flavonoid aglycones being rapidly glycosylated to non‐inhibitory flavonoid glycosides.     

Two polyketide synthases are necessary for 4‐hydroxy‐5‐methylcoumarin biosynthesis in Gerbera hybrida

Gerbera (Gerbera hybrida) is an economically important ornamental species and a model plant of the Asteraceae family for flower development and secondary metabolism. Gerberin and parasorboside, two bitter tasting glucosidic lactones, are produced in high amounts in nearly all gerbera tissues. Gerbera and its close relatives also produce a rare coumarin, 4‐hydroxy‐5‐methylcoumarin (HMC). Unlike most coumarins, 5‐methylcoumarins have been suggested to be derived through the acetate‐malonate pathway. All of these polyketide‐derived glucosylated molecules are considered to have a role in defense against herbivores and phytopathogens in gerbera. Gerbera expresses three genes encoding 2‐pyrone synthases (G2PS1–3). The enzymes are chalcone synthase‐like polyketide synthases with altered starter substrate specificity. We have shown previously that G2PS1 is responsible for the synthesis of 4‐hydroxy‐6‐methyl‐2‐pyrone (triacetolactone), a putative precursor of gerberin and parasorboside. Here we show that polyketide synthases G2PS2 and G2PS3 are necessary for the biosynthesis of HMC in gerbera, and that a reductase enzyme is likely required to complete the pathway to HMC. G2PS2 is expressed in the leaf blade and inflorescences of gerbera, while G2PS3 is strictly root specific. Heterologous expression of G2PS2 or G2PS3 in tobacco leads to the formation of 4,7‐dihydroxy‐5‐methylcoumarin, apparently an unreduced precursor of HMC, while ectopic expression in gerbera leads to HMC formation in tissues where nontransgenic tissue does not express the genes and does not accumulate the compound. Using protein modelling and site‐directed mutagenesis we identified the residues I203 and T344 in G2PS2 and G2PS3 to be critical for pentaketide synthase activity.     

Single mutations toggle the substrate selectivity of multifunctional Camptotheca secologanic acid synthases

Terpene indole alkaloids (TIAs) are plant-derived specialized metabolites with widespread use in medicine. Species-specific pathways derive various TIAs from common intermediates, strictosidine or strictosidinic acid, produced by coupling tryptamine with secologanin or secologanic acid. The penultimate reaction in this pathway is catalyzed by either secologanin synthase (SLS) or secologanic acid synthase (SLAS) according to whether plants produce secologanin from loganin or secologanic acid from loganic acid. Previous work has identified SLSs and SLASs from different species, but the determinants of selectivity remain unclear. Here, combining molecular modeling, ancestral sequence reconstruction, and biochemical methodologies, we identified key residues that toggle SLS and SLAS selectivity in two CYP72A (cytochrome P450) subfamily enzymes from Camptotheca acuminata. We found that the positions of foremost importance are in substrate recognition sequence 1 (SRS1), where mutations to either of two adjacent histidine residues switched selectivity; His131Phe selects for and increases secologanin production whereas His132Asp selects for secologanic acid production. Furthermore, a change in SRS3 in the predicted substrate entry channel (Arg/Lys270Thr) and another in SRS4 at the start of the I-helix (Ser324Glu) decreased enzyme activity toward either substrate. We propose that the Camptotheca SLASs have maintained the broadened activities found in a common asterid ancestor, even as the Camptotheca lineage lost its ability to produce loganin while the campanulid and lamiid lineages specialized to produce secologanin by acquiring mutations in SRS1. The identification here of the residues essential for the broad substrate scope of SLASs presents opportunities for more tailored heterologous production of TIAs.     

The foxtail millet (Setaria italica) terpene synthase gene family

Terpenoid metabolism plays vital roles in stress defense and the environmental adaptation of monocot crops. Here, we describe the identification of the terpene synthase (TPS) gene family of the panicoid food and bioenergy model crop foxtail millet (Setaria italica). The diploid S. italica genome contains 32 TPS genes, 17 of which were biochemically characterized in this study. Unlike other thus far investigated grasses, S. italica contains TPSs producing all three ent‐, (+)‐ and syn‐copalyl pyrophosphate stereoisomers that naturally occur as central building blocks in the biosynthesis of distinct monocot diterpenoids. Conversion of these intermediates by the promiscuous TPS SiTPS8 yielded different diterpenoid scaffolds. Additionally, a cytochrome P450 monooxygenase (CYP99A17), which genomically clustered with SiTPS8, catalyzes the C19 hydroxylation of SiTPS8 products to generate the corresponding diterpene alcohols. The presence of syntenic orthologs to about 19% of the S. italica TPSs in related grasses supports a common ancestry of selected pathway branches. Among the identified enzyme products, abietadien‐19‐ol, syn‐pimara‐7,15‐dien‐19‐ol and germacrene‐d ‐4‐ol were detectable in planta, and gene expression analysis of the biosynthetic TPSs showed distinct and, albeit moderately, inducible expression patterns in response to biotic and abiotic stress. In vitro growth‐inhibiting activity of abietadien‐19‐ol and syn‐pimara‐7,15‐dien‐19‐ol against Fusarium verticillioides and Fusarium subglutinans may indicate pathogen defensive functions, whereas the low antifungal efficacy of tested sesquiterpenoids supports other bioactivities. Together, these findings expand the known chemical space of monocot terpenoid metabolism to enable further investigations of terpenoid‐mediated stress resilience in these agriculturally important species.     

Auxin biosynthetic gene TAR2 is involved in low nitrogen‐mediated reprogramming of root architecture in Arabidopsis

In plants, the plasticity of root architecture in response to nitrogen availability largely determines nitrogen acquisition efficiency. One poorly understood root growth response to low nitrogen availability is an observed increase in the number and length of lateral roots (LRs). Here, we show that low nitrogen‐induced Arabidopsis LR growth depends on the function of the auxin biosynthesis gene TAR2 (tryptophan aminotransferase related 2). TAR2 was expressed in the pericycle and the vasculature of the mature root zone near the root tip, and was induced under low nitrogen conditions. In wild type plants, low nitrogen stimulated auxin accumulation in the non‐emerged LR primordia with more than three cell layers and LR emergence. Conversely, these low nitrogen‐mediated auxin accumulation and root growth responses were impaired in the tar2‐c null mutant. Overexpression of TAR2 increased LR numbers under both high and low nitrogen conditions. Our results suggested that TAR2 is required for reprogramming root architecture in response to low nitrogen conditions. This finding suggests a new strategy for improving nitrogen use efficiency through the engineering of TAR2 expression in roots.     

A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae

The genetic transformation of plant cells is critically dependent on the availability of efficient selectable marker gene. Sulfonamides are herbicides that, by inhibiting the folic acid biosynthetic pathway, suppress the growth of untransformed cells. Sulfonamide resistance genes that were previously developed as selectable markers for plant transformation were based on the assumption that, in plants, the folic acid biosynthetic pathway resides in the chloroplast compartment. Consequently, the Sul resistance protein, a herbicide‐insensitive dihydropteroate synthase, was targeted to the chloroplast. Although these vectors produce transgenic plants, the transformation efficiencies are low compared to other markers. Here, we show that this inefficiency is due to the erroneous assumption that the folic acid pathway is located in chloroplasts. When the RbcS transit peptide was replaced by a transit peptide for protein import into mitochondria, the compartment where folic acid biosynthesis takes place in yeast, much higher resistance to sulfonamide and much higher transformation efficiencies are obtained, suggesting that current sul vectors are likely to function due to low‐level mistargeting of the resistance protein to mitochondria. We constructed a series of optimized transformation vectors and demonstrate that they produce transgenic events at very high frequency in both the seed plant tobacco and the green alga Chlamydomonas reinhardtii. Co‐transformation experiments in tobacco revealed that sul is even superior to nptII, the currently most efficient selectable marker gene, and thus provides an attractive marker for the high‐throughput genetic transformation of plants and algae.     

Structural characterization of CalO2: a putative orsellinic acid P450 oxidase in the calicheamicin biosynthetic pathway

Although bacterial iterative Type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products remains poorly understood. Toward this goal, we report the X-ray structure determination at 2.5 A resolution and preliminary characterization of the putative orsellenic acid P450 oxidase (CalO2) involved in calicheamicin biosynthesis. These studies represent the first crystal structure for a P450 involved in modifying a bacterial iterative Type I polyketide product and suggest the CalO2-catalyzed step may occur after CalO3-catalyzed iodination and may also require a coenzyme A- (CoA) or acyl carrier protein- (ACP) bound substrate. Docking studies also reveal a putative docking site within CalO2 for the CLM orsellinic acid synthase (CalO5) ACP domain which involves a well-ordered helix along the CalO2 active site cavity that is unique compared with other P450 structures.     

Functional characterisation of two phytochelatin synthases in rice (Oryza sativa cv. Milyang 117) that respond to cadmium stress

• Cadmium (Cd) is one of the most toxic heavy metals and a non‐essential element to all organisms, including plants; however, the genes involved in Cd resistance in plants remain poorly characterised.
• To identify Cd resistance genes in rice, we screened a rice cDNA expression library treated with CdCl₂ using a yeast (Saccharomyces cerevisiae) mutant ycf1 strain (DTY167) and isolated two rice phytochelatin synthases (OsPCS5 and OsPCS15).
• The genes were strongly induced by Cd treatment and conferred increased resistance to Cd when expressed in the ycf1 mutant strain. In addition, the Cd concentration was twofold higher in yeast expressing OsPCS5 and OsPCS15 than in vector‐transformed yeast, and OsPCS5 and OsPCS15 localised in the cytoplasm. Arabidopsis thaliana plants overexpressing OsPCS5/‐15 paradoxically exhibited increased sensitivity to Cd, suggesting that overexpression of OsPCS5/‐15 resulted in toxicity due to excess phytochelatin production in A. thaliana.
• These data indicate that OsPCS5 and OsPCS15 are involved in Cd tolerance, which may be related to the relative abundances of phytochelatins synthesised by these phytochelatin synthases.

     

Sequestration and biosynthesis of cyanogenic glucosides in passion vine butterflies and consequences for the diversification of their host plants

The colorful heliconiine butterflies are distasteful to predators due to their content of defense compounds called cyanogenic glucosides (CNglcs), which they biosynthesize from aliphatic amino acids. Heliconiine larvae feed exclusively on Passiflora plants where ~30 kinds of CNglcs have been reported. Among them, some CNglcs derived from cyclopentenyl glycine can be sequestered by some Heliconius species. In order to understand the evolution of biosynthesis and sequestration of CNglcs in these butterflies and its consequences for their arms race with Passiflora plants, we analyzed the CNglc distribution in selected heliconiine and Passiflora species. Sequestration of cyclopentenyl CNglcs is not an exclusive trait of Heliconius, since these compounds were present in other heliconiines such as Philaethria, Dryas and Agraulis, and in more distantly related genera Cethosia and Euptoieta. Thus, it is likely that the ability to sequester cyclopentenyl CNglcs arose in an ancestor of the Heliconiinae subfamily. Biosynthesis of aliphatic CNglcs is widespread in these butterflies, although some species from the sara‐sapho group seem to have lost this ability. The CNglc distribution within Passiflora suggests that they might have diversified their cyanogenic profile to escape heliconiine herbivory. This systematic analysis improves our understanding on the evolution of cyanogenesis in the heliconiine–Passiflora system.     

Abundance of Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) and its parasitoids on vegetables and cassava plants in Burkina Faso (West Africa)

The whitefly Bemisia tabaci is a pest of many agricultural and ornamental crops worldwide and particularly in Africa. It is a complex of cryptic species, which is extremely polyphagous with hundreds of host plants identified around the world. Previous surveys in western Africa indicated the presence of two biotypes of the invasive MED species (MED‐Q1 and MED‐Q3) living in sympatry with the African species SSA and ASL. This situation constitutes one of the rare cases of local coexistence of various genetic entities within the B. tabaci complex. In order to study the dynamics of the distribution and abundance of genetic entities within this community and to identify potential factors that could contribute to coexistence, we sampled B. tabaci populations in Burkina Faso in 2015 and 2016 on various plants, and also their parasitoids. All four genetic entities were still recorded, indicating no exclusion of local species by the MED species. While B. tabaci individuals were found on 55 plant species belonging to eighteen (18) families showing the high polyphagy of this pest, some species/biotypes exhibited higher specificity. Two parasitoid species (Eretmocerus mundus and Encarsia vandrieschei) were also recorded with E. mundus being predominant in most localities and on most plants. Our data indicated that whitefly abundance, diversity, and rate of parasitism varied according to areas, plants, and years, but that parasitism rate was globally highly correlated with whitefly abundance suggesting density dependence. Our results also suggest dynamic variation in the local diversity of B. tabaci species/biotypes from 1 year to the other, specifically with MED‐Q1 and ASL species. This work provides relevant information on the nature of plant–B. tabaci‐parasitoid interactions in West Africa and identifies that coexistence might be stabilized by niche differentiation for some genetic entities. However, MED‐Q1 and ASL show extensive niche overlap, which could ultimately lead to competitive exclusion.