The daisy-like flowers of pyrethrum (Tanacetum cinerariifolium) are used to extract pyrethrins, a botanical insecticide with a long history of safe and effective use. Pyrethrum flowers also contain ...other potential defense compounds, particularly sesquiterpene lactones (STLs), which represent problematic allergenic residues in the extracts that are removed by the pyrethrum industry. The STLs are stored in glandular trichomes present on the pyrethrum achenes, and have been shown to be active against herbivores, micro-organisms and in the below-ground competition with other plants. Despite these reported bioactivities and industrial significance, the biosynthetic origin of pyrethrum sesquiterpene lactones remains unknown. In the present study, we show that germacratrien-12-oic acid is most likely the central precursor for all sesquiterpene lactones present in pyrethrum. The formation of the lactone ring depends on the regio- (C6 or C8) and stereo-selective (α or β) hydroxylation of germacratrien-12-oic acid. Candidate genes implicated in three committed steps leading from farnesyl diphosphate to STL and other oxygenated derivatives of germacratrien-12-oic acid were retrieved from a pyrethrum trichome EST library, cloned, and characterized in yeast and in planta. The diversity and distribution of sesquiterpene lactones in different tissues and the correlation with the expression of these genes are shown and discussed.
a In the natural pesticides known as pyrethrins, which are esters produced in flowers of Tanacetum cinerariifolium (Asteraceae), the monoterpenoid acyl moiety is pyrethric acid or chrysanthemic acid.
...We show here that pyrethric acid is produced from chrysanthemol in six steps catalyzed by four enzymes, the first five steps occurring in the trichomes covering the ovaries and the last one occurring inside the ovary tissues.
Three steps involve the successive oxidation of carbon 10 (C10) to a carboxylic group by TcCHH, a cytochrome P450 oxidoreductase. Two other steps involve the successive oxidation of the hydroxylated carbon 1 to give a carboxylic group by TcADH2 and TcALDH1, the same enzymes that catalyze these reactions in the formation of chrysanthemic acid. The ultimate result of the actions of these three enzymes is the formation of 10-carboxychrysanthemic acid in the trichomes. Finally, the carboxyl group at C10 is methylated by TcCCMT, a member of the SABATH methyltransferase family, to give pyrethric acid. This reaction occurs mostly in the ovaries.
a Expression in N. benthamiana plants of all four genes encoding aforementioned enzymes, together with TcCDS, a gene that encodes an enzyme that catalyzes the formation of chrysanthemol, led to the production of pyrethric acid.
A microfluidic tongue-on-a-chip platform has been evaluated relative to the known sensory properties of various sweeteners. Analogous metrics of typical sensory features reported by human panels such ...as sweet taste thresholds, onset, and lingering, as well as bitter off-flavor and blocking interactions were deduced from the taste receptor activation curves and then compared. To this end, a flow cell containing a receptor cell array bearing the sweet and six bitter taste receptors was transiently exposed to pure and mixed sweetener samples. The sample concentration gradient across time was separately characterized by the injection of fluorescein dye. Subsequently, cellular calcium responses to different doses of advantame, aspartame, saccharine, and sucrose were overlaid with the concentration gradient. Parameters describing the response kinetics compared to the gradient were quantified. Advantame at 15 μM recorded a significantly faster sweetness onset of 5 ± 2 s and a longer lingering time of 39 s relative to sucrose at 100 mM with an onset of 13 ± 2 s and a lingering time of 6 s. Saccharine was shown to activate the bitter receptors TAS2R8, TAS2R31, and TAS2R43, confirming its known off-flavor, whereas addition of cyclamate reduced or blocked this saccharine bitter response. The potential of using this tongue-on-a-chip to bridge the gap with in vitro assays and taste panels is discussed.
Plants are at the basis of the food chain, but there is no such thing as a "free lunch" for herbivores. To promote reproductive success, plants evolved multi-layered defensive tactics to avoid or ...discourage herbivory. To the detriment of plants, herbivores, in turn, evolved intricate strategies to find, eat, and successfully digest essential plant parts to raise their own offspring. In this battle the digestive tract is the arena determining final victory or defeat as measured by growth or starvation of the herbivore. Earlier, specific molecular opponents were identified as proteases and inhibitors: digestive proteases of herbivores evolved structural motifs to occlude plant protease inhibitors, or alternatively, the insects evolved proteases capable of specifically degrading the host plant inhibitors. In response plant inhibitors evolved hyper-variable and novel protein folds to remain active against potential herbivores. At the level of protease regulation in herbivorous insects, it was shown that inhibition-insensitive digestive proteases are up-regulated when sensitive proteases are inhibited. The way this regulation operates in mammals is known as negative feedback by gut-luminal factors, so-called 'monitor peptides' that are sensitive to the concentration of active enzymes. We propose that regulation of gut enzymes by endogenous luminal factors has been an open invitation to plants to "hijack" this regulation by evolving receptor antagonists, although yet these plant factors have not been identified. In future research the question of the co-evolution of insect proteases and plant inhibitors should, therefore, be better approached from a systems level keeping in mind that evolution is fundamentally opportunistic and that the plant's fitness is primarily improved by lowering the availability of essential amino acids to an herbivore by any available mechanism.
Aphids induce many transcriptional perturbations in their host plants, but the signalling cascades responsible and the effects on plant resistance are largely unknown. Through a genome-wide ...association (GWA) mapping study in Arabidopsis thaliana, we identified WRKY22 as a candidate gene associated with feeding behaviour of the green peach aphid, Myzus persicae. The transcription factor WRKY22 is known to be involved in pathogen-triggered immunity, and WRKY22 gene expression has been shown to be induced by aphids. Assessment of aphid population development and feeding behaviour on knockout mutants and overexpression lines showed that WRKY22 increases susceptibility to M. persicae via a mesophyll-located mechanism. mRNA sequencing analysis of aphid-infested wrky22 knockout plants revealed the up-regulation of genes involved in salicylic acid (SA) signalling and down-regulation of genes involved in plant growth and cell-wall loosening. In addition, mechanostimulation of knockout plants by clip cages up-regulated jasmonic acid (JA)-responsive genes, resulting in substantial negative JA–SA crosstalk. Based on this and previous studies, WRKY22 is considered to modulate the interplay between the SA and JA pathways in response to a wide range of biotic and abiotic stimuli. Its induction by aphids and its role in suppressing SA and JA signalling make WRKY22 a potential target for aphids to manipulate host plant defences.
Data analysis for flow-based in-vitro receptomics array, like a tongue-on-a-chip, is complicated by the relatively large variability within and between arrays, transfected DNA types, spots, and cells ...within spots. Simply averaging responses of spots of the same type would lead to high variances and low statistical power. This paper presents an approach based on linear mixed models, allowing a quantitative and robust comparison of complex samples and indicating which receptors are responsible for any differences. These models are easily extended to take into account additional effects such as the build-up of cell stress and to combine data from replicated experiments. The increased analytical power this brings to receptomics research is discussed.
Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation ...reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12–0.16 μg h−1 g−1 fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.Chrysanthemyl diphosphate synthase (CDS) is known to catalyze the formation of the irregular terpenoid, chrysanthemyl diphosphate (CPP).
CDS also catalyzes the next step from CPP to chrysanthemol.
CDS is actually a chrysanthemol synthase (CHS) with bifunctional enzyme activity.
Identification of CHS increases our understanding in terpene biosynthesis and paves the way for engineering biosynthesis of the most widely used natural pesticide, pyrethrins.
Main conclusion
In flowers multiple secretory systems cooperate to deliver specialized metabolites to support specific roles in defence and pollination. The collective roles of cell types, enzymes, ...and transporters are discussed.
The interplay between reproductive strategies and defense mechanisms in flowering plants has long been recognized, with trade-offs between investment in defense and reproduction predicted. Glandular trichomes and secretory cavities or ducts, which are epidermal and internal structures, play a pivotal role in the secretion, accumulation, and transport of specialized secondary metabolites, and contribute significantly to defense and pollination. Recent investigations have revealed an intricate connection between these two structures, whereby specialized volatile and non-volatile metabolites are exchanged, collectively shaping their respective ecological functions. However, a comprehensive understanding of this profound integration remains largely elusive. In this review, we explore the secretory systems and associated secondary metabolism primarily in Asteraceous species to propose potential shared mechanisms facilitating the directional translocation of these metabolites to diverse destinations. We summarize recent advances in our understanding of the cooperativity between epidermal and internal secretory structures in the biosynthesis, secretion, accumulation, and emission of terpenes, providing specific well-documented examples from pyrethrum (
Tanacetum cinerariifolium
). Pyrethrum is renowned for its natural pyrethrin insecticides, which accumulate in the flower head, and more recently, for emitting an aphid alarm pheromone. These examples highlight the diverse specializations of secondary metabolism in pyrethrum and raise intriguing questions regarding the regulation of production and translocation of these compounds within and between its various epidermal and internal secretory systems, spanning multiple tissues, to serve distinct ecological purposes. By discussing the cooperative nature of secretory structures in flowering plants, this review sheds light on the intricate mechanisms underlying the ecological roles of terpenes in defense and pollination.
Plant defensive mimicry based on the aphid alarm pheromone (E)-β-farnesene (EβF) was previously shown to operate in Tanacetum cinerariifolium (Asteraceae) flowers. Germacrene D (GD), is another ...dominant volatile of T. cinerariifolium flowers and may modulate both defense and pollination. Here, we find that the increase in GD/EβF ratio at later developmental stages is correlated with the tissue distribution in the flower head: the total content of EβF and GD is similar, but GD accumulates comparatively more in the upper disk florets. Naphthol and N, N-dimethyl-p-phenylenediamine dihydrochloride (NADI)-stained purple ducts containing EβF and GD, were observed in the five petal lips of the corolla and two-lobed stigma of disk florets. By contrast in the peduncle, EβF accounts for nearly 80% of total terpenes, compared to 5% for GD. EβF is accumulated inside inner cortex cells and parenchyma cells of the pith in young peduncle. This is followed by the formation of terpene-filled axial secretory cavities parallel to the vascular bundles. In conclusion, the observed developmental and diurnal emissions of different EβF/GD ratios appear to be regulated by their tissue distribution.
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•EβF and GD are the dominant volatiles with similar contents in pyrethrum flowers.•GD accumulates comparatively more in the upper disk florets.•EβF and GD were observed in the five petal lips of the corolla and two-lobed stigma of disk florets.•In peduncle, EβF was accumulated in the cortex cells and further stored in the secretory ducts.
Terpenoids are important for plant survival and also possess biological properties that are beneficial to humans. Here, we describe the state of the art in terpenoid metabolic engineering, showing ...that significant progress has been made over the past few years. Subcellular targeting of enzymes has demonstrated that terpenoid precursors in subcellular compartments are not as strictly separated as previously thought and that multistep pathway engineering is feasible, even across cell compartments. These engineered plants show that insect behavior is influenced by terpenoids. In the future, we expect rapid progress in the engineering of terpenoid production in plants. In addition to commercial applications, such transgenic plants should increase our understanding of the biological relevance of these volatile secondary metabolites.
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