Chemotaxonomy of the Oleaceae: iridoids as taxonomic markers

The distribution and biosynthesis of iridoid glucosides in the Oleaceae is reviewed and five distinct biosynthetic pathways to iridoids have been identified in the family, deoxyloganic acid apparently being a common intermediate. Likewise, the distributions of caffeoyl phenylethanoid glycosides (CPGs), i.e. verbascoside and its analogues, as well as cornoside are listed. Iridoid glucoside data exist for 17 genera of Oleaceae and the occurrence of iridoids from the different biosynthetic pathways correlate extremely well with the phylogenetic classification inferred from recent chloroplast DNA sequence data. Thus the tribe Fontanesieae (Fontanesia) contains "normal" secoiridoids, Forsythieae (Abeliophyllum, Forsythia) contains cornoside and/or iridoids from the forsythide pathway, Myxopyreae (Myxopyrum, Nyctanthes) have iridoids from the myxopyroside pathway, and finally, the two tribes Jasmineae and Oleeae (the remaining genera) both contain iridoids from the oleoside pathway. Within Jasmineae, one group of Jasminum sp. is characterized by the presence of jasminin or similar compounds, while another group of Jasminum species and Menodora display derivatives of 10-hydroxyoleoside, compounds not present in the other group. CPGs are reported from about half of the species investigated. With regard to taxonomy at the order level, the chemical data might support a position within or close to Lamiales due to the common presence of CPGs, the iridoids being of less significance since they are of a type that are barely found elsewhere.     

The roles of foraging environment,host species,and host diet for a generalist pupal parasitoid

Even for parasitoids with a wide host range, not all host species are equally suitable, and host quality often depends on the plant the host feeds on. We compared oviposition choice and offspring performance of a generalist pupal parasitoid, Pteromalus apum (Retzius) (Hymenoptera: Pteromalidae), on two congeneric hosts reared on two plant species under field and laboratory conditions. The plants contain defensive iridoid glycosides that are sequestered by the hosts. Sequestration at the pupal stage differed little between host species and, although the concentrations of iridoid glycosides in the two plant species differ, there was no effect of diet on the sequestration by host pupae. The rate of successful parasitism differed between host species, depending on the conditions they were presented in. In the field, where plant‐associated cues are present, the parasitoid used Melitaea cinxia (L.) over Melitaea athalia (Rottemburg) (Lepidoptera: Nymphalidae), whereas more M. athalia were parasitised in simplified laboratory conditions. In the field, brood size, which is partially determined by rate of superparasitism, depended on both host and plant species. There was little variation in other aspects of offspring performance related to host or plant species, indicating that the two host plants are of equal quality for the hosts, and the hosts are of equal quality for the parasitoids. Corresponding to this, we found no evidence for associative learning by the parasitoid based on their natal host, so with respect to these host species they are truly generalist in their foraging behaviour.     

Iridoids in Hydrangeaceae

The content of glycosides in Kirengeshoma palmata and Jamesia americana (Hydrangeaceae) have been investigated. The former contains loganin and secoiridoids, including the alkaloid demethylalangiside. The latter contains no iridoids, but the known glucosides arbutin, picein and prunasin. In order to futher investigate the chemotaxonomy of the family Hydrangeaceae, the distribution of the iridoid and secoiridoid glucosides as well as the known biosynthetic pathways to these compounds have been reviewed. However, only a few genera of the family has been investigated. Loganin, secologanin, and derivatives of these are common. The genus Deutzia is characteristic in containing more structurally simple iridoids in which C-10 has been lost during biosynthesis. Such compounds have so far only been reported from the genus Mentzelia (Loasaceae). The taxonomic relationships between Hydrangeaceae and the closely related Cornaceae and Loasaceae is discussed and found to agree well with recent DNA sequence results.     

Iridoid Glycosides from Tabebuia avellanedae

Five novel iridoid glycosides, avellanedaesides A‐E ( 1  –  5 ) were isolated from the H₂O extract of Tabebuia avellanedae. Their structures were determined on the basis of NMR and MS analysis. Isolated compounds suppressed inflammatory cytokine, tumor‐necrosis factor‐α and interleukin‐1β production in cultured human myeloma THP‐1 cells co‐stimulated with lipopolysaccharide (LPS). In addition, the study revealed iridoid glycosides inhibited the activity of cytochrome CYP3A4 enzyme.     

13,14‐seco‐Withanolides from Physalis minima with Potential Anti‐inflammatory Activity

Four new 13,14‐seco‐withanolides, minisecolides A – D ( 1  –  4 ), together with three known analogues 5  –  7 , were isolated from the whole plants of Physalis minima. The structures of new compounds were determined on the basis of spectroscopic analysis, including ¹H‐, ¹³C‐NMR, 2D‐NMR (HMBC, HSQC, ROESY), and HR‐ESI‐MS. Evaluation of all isolates for their inhibitory effects on nitric oxide (NO) production was conducted on lipopolysaccaride‐activated RAW264.7 macrophages. Compounds 2 , 3 , 5 , and 6 showed inhibitory activities, especially for compound 5 with IC₅₀ value of 3.87 μm .     

Virus‐induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors

Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus‐based virus‐induced gene silencing (VIGS) approach, involving an Agrobacterium‐mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic‐mediated approach of inoculation of virus‐encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down‐regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45–90%), similar to results obtained via agro‐transformation. Thus, the biolistic‐based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium‐based approach, e.g. when difficulties arise with agro‐inoculations or when Agrobacterium‐free procedures are required to avoid plant defence responses.     

Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis

Phosphatidylcholine (PC) is an almost ubiquitous phospholipid in eukaryotic algae and plants but is not found in a few species, for example Chlamydomonas reinhardtii. We recently found that some species of the genus Chlamydomonas possess PC. In the universal pathway, PC is synthesized de novo by methylation of phosphatidylethanolamine (PE) or transfer of phosphocholine from cytidine diphosphate (CDP)‐choline to diacylglycerol. Phosphocholine, the direct precursor to CDP‐choline, is synthesized either by methylation of phosphoethanolamine or phosphorylation of choline. Here we analyzed the mechanism of PC biosynthesis in two species of Chlamydomonas (asymmetrica and sphaeroides) as well as in a red alga, Cyanidioschyzon merolae. Comparative genomic analysis of enzymes involved in PC biosynthesis indicated that C. merolae possesses only the PE methylation pathway. Radioactive tracer experiments using [³²P]phosphate showed delayed labeling of PC with respect to PE, which was consistent with the PE methylation pathway. In Chlamydomonas asymmetrica, labeling of PC was detected from the early time of incubation with [³²P]phosphate, suggesting the operation of phosphoethanolamine methylation pathway. Genomic analysis indeed detected the genes for the phosphoethanolamine methylation pathway. In contrast, the labeling of PC in C. sphaeroides was slow, suggesting that the PE methylation pathway was at work. These results as well as biochemical and computational results uncover an unexpected diversity of the mechanisms for PC biosynthesis in algae. Based on these results, we will discuss plausible mechanisms for the scattered distribution of the ability to biosynthesize PC in the genus Chlamydomonas.     

Chemotaxonomy of Plantago. Iridoid glucosides and caffeoyl phenylethanoid glycosides

Data for 34 species of Plantago (Plantaginaceae), including subgen. Littorella (= Littorella uniflora), have been collected with regard to their content of iridoid glucosides and caffeoyl phenylethanoid glycosides (CPGs). In the present work, 21 species were investigated for the first time and many known compounds were found together with three new iridoid glucosides. Of these, arborescoside and arborescosidic acid, both of the uncommon type with an 8,9-double bond, were present in several species, while 6-deoxymelittoside was found only in P. subulata. The known compounds deoxyloganic acid, caryoptoside and rehmannioside D were isolated from the genus for the first time. The earlier reported occurrence of sorbitol in the family was confirmed, and this compound was shown by NMR spectroscopy to be the main sugar in the three species investigated for this. The combined data show that CPGs are present in all species investigated. With regard to the iridoids, the distribution patterns showed a good correlation with the classification of Rahn. Thus, aucubin is typical for the whole genus, while bartsioside and catalpol as well as 5-substituted iridoids are each characteristic for a subgenus in the family. Finally, the close relationship between Plantago and Veronica suggested by chloroplast DNA sequence analysis. could be corroborated by the common occurrence of the rare 8,9-unsaturated iridoids in these two genera.     

Phytochemistry and the systematics and ecology of Loasaceae and Gronoviaceae (Loasales)

A screening for iridoid compounds of 78 of 315 species from all major groups in Gronoviaceae and Loasaceae has been carried out. The results were compared to the systematic concepts in the family and distribution and ecology of the taxa. Iridoids are present in at least some species of all genera screened. Some simple, monomeric compounds (e.g., loganin, sweroside) are found in all major groups of the two families and represent the basic iridoid inventory. Other compounds are restricted to certain taxonomic groups: nine-carbon iridoids (e.g., deutzioside) are restricted to Mentzelia (Loasaceae subfam. Mentzelioideae), hetero-oligomeric iridoids (e.g., tricoloroside methyl ester, acerifolioside) are restricted to two small groups in Loasa (Loasa ser. Macrospermae and ser. Floribundae, Loasaceae subfam. Loasoideae), and oleosides (e.g., 10-hydroxyoleoside dimethyl ester) are restricted to the large genus Caiophora sensu Weigend). The distribution of certain iridoid compounds thus confirms some of the generic limits. Iridoid phytochemistry does not correlate with systematic entities above the generic level nor does it in any way correlate with the morphological evolution of taxa. Conversely, the amount and complexity of iridoid compounds present in taxa correlate positively with the aridity of their habitat and the extent of mammalian herbivore pressure.     

Small‐molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function

Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin‐deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole‐3‐pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole‐3‐acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin‐containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4‐biphenylboronic acid (BBo) and 4‐phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild‐type Arabidopsis seedlings. Co‐treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (K_i) of BBo and PPBo were 67 and 56 nm , respectively. In addition, PPBo did not interfere with the auxin response of auxin‐marker genes when it was co‐treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.     

Genome‐guided investigation of plant natural product biosynthesis

The medicinal plant Madagascar periwinkle, Catharanthus roseus (L.) G. Don, produces hundreds of biologically active monoterpene‐derived indole alkaloid (MIA) metabolites and is the sole source of the potent, expensive anti‐cancer compounds vinblastine and vincristine. Access to a genome sequence would enable insights into the biochemistry, control, and evolution of genes responsible for MIA biosynthesis. However, generation of a near‐complete, scaffolded genome is prohibitive to small research communities due to the expense, time, and expertise required. In this study, we generated a genome assembly for C. roseus that provides a near‐comprehensive representation of the genic space that revealed the genomic context of key points within the MIA biosynthetic pathway including physically clustered genes, tandem gene duplication, expression sub‐functionalization, and putative neo‐functionalization. The genome sequence also facilitated high resolution co‐expression analyses that revealed three distinct clusters of co‐expression within the components of the MIA pathway. Coordinated biosynthesis of precursors and intermediates throughout the pathway appear to be a feature of vinblastine/vincristine biosynthesis. The C. roseus genome also revealed localization of enzyme‐rich genic regions and transporters near known biosynthetic enzymes, highlighting how even a draft genome sequence can empower the study of high‐value specialized metabolites.     

Trichobaris weevils distinguish amongst toxic host plants by sensing volatiles that do not affect larval performance

Herbivorous insects use plant metabolites to inform their host plant selection for oviposition. These host‐selection behaviours are often consistent with the preference–performance hypothesis; females oviposit on hosts that maximize the performance of their offspring. However, the metabolites used for these oviposition choices and those responsible for differences in offspring performance remain unknown for ecologically relevant interactions. Here, we examined the host‐selection behaviours of two sympatric weevils, the Datura (Trichobaris compacta) and tobacco (T. mucorea) weevils in field and glasshouse experiments with transgenic host plants specifically altered in different components of their secondary metabolism. Adult females of both species strongly preferred to feed on D. wrightii rather than on N. attenuata leaves, but T. mucorea preferred to oviposit on N. attenuata, while T. compacta oviposited only on D. wrightii. These oviposition behaviours increased offspring performance: T. compacta larvae only survived in D. wrightii stems and T. mucorea larvae survived better in N. attenuata than in D. wrightii stems. Choice assays with nicotine‐free, JA‐impaired, and sesquiterpene‐over‐produced isogenic N. attenuata plants revealed that although half of the T. compacta larvae survived in nicotine‐free N. attenuata lines, nicotine did not influence the oviposition behaviours of both the nicotine‐adapted and nicotine‐sensitive species. JA‐induced sesquiterpene volatiles are key compounds influencing T. mucorea females’ oviposition choices, but these sesquiterpenes had no effect on larval performance. We conclude that adult females are able to choose the best host plant for their offspring and use chemicals different from those that influence larval performance to inform their oviposition decisions.     

Convergent development of a parasitoid wasp on three host species with differing mass and growth potential

Koinobiont parasitoids develop in hosts that continue feeding and growing during the course of parasitism. Here, we compared development of a solitary koinobiont endoparasitoid, Meteorus pulchricornis Westmael (Hymenoptera: Braconidae), in second (L2) and fourth (L4) instars of three host species that are closely related (Lepidoptera: Noctuidae) but which exhibit large variation in growth potential. Two hosts, Mamestra brassicae L. and Spodoptera littoralis Boisduval, may reach 1 g or more when the caterpillars are fully mature, whereas Spodoptera exigua Hübner is much smaller with mature caterpillars rarely exceeding 200 mg. Parasitoid survival (to pupation) in the two host instars was much higher on the larger hosts than on S. exigua. However, other fitness correlates in M. pulchricornis were very similar in the three host species. Development time was fairly uniform in L2 and L4 hosts of the three host species, whereas wasps were larger in L4 than in L2 hosts. However, M. pulchricornis developmentally arrested each of the hosts differently. The mass of dying L2 and L4 hosts after parasitoid larval egression (i.e., when they emerge from the dying caterpillar) varied significantly, with S. littoralis being by far the largest and S. exigua the smallest. These results reveal that M. pulchricornis is able to adjust its own development in response to species‐specific differences in host resources.     

Molecular and physiological comparison of spoilage wine yeasts

Aims

Dekkera bruxellensis and Pichia guilliermondii are contaminating yeasts in wine due to the production of phenolic aromas. Although the degradation pathway of cinnamic acids, precursors of these phenolic compounds has been described in D. bruxellensis, no such pathway has been described in P. guilliermondii.

Methods and Results

A molecular and physiological characterization of 14 D. bruxellensis and 15 P. guilliermondii phenol‐producing strains was carried out. Both p‐coumarate decarboxylase (CD) and vinyl reductase (VR) activities, responsible for the production of volatile phenols, were quantified and the production of 4‐vinylphenol and 4‐ethylphenol were measured. All D. bruxellensis and some P. guilliermondii strains showed the two enzymatic activities, whilst 11 of the 15 strains of this latter species showed only CD activity and did not produce 4‐EP in the assay conditions. Furthermore, PCR products obtained with degenerated primers showed a low homology with the sequence of the gene for a phenyl acrylic acid decarboxylase activity described in Saccharomyces cerevisiae.

Conclusions

D. bruxellensis and P. guilliermondii may share a similar metabolic pathway for the degradation of cinnamic acids.

Significance and Impact of the Study

This is the first work that analyses the CD and VR activities in P. guilliermondii, and the results suggest that within this species, there are differences in the metabolization of cinnamic acids.     

Host selection by Ibalia leucospoides based on temporal variations of volatiles from the hosts’ fungal symbiont

Insect parasitoids locate hosts via reliable and predictable cues such as volatile emissions from hosts and/or host plants. For insects that depend on mutualistic organisms, such as many wood‐boring insects, symbiont‐derived semiochemicals may represent a source of such cues to be exploited by natural enemies. Ultimately, exploitation of these signals may increase fitness by optimizing foraging efficiency. Female parasitoids of Ibalia leucospoides use volatiles from the fungal symbiont Amylostereum areolatum of their host Sirex noctlio to find concealed host eggs and young larvae within the xylem. We hypothesize that the temporal pattern of fungal emissions may indicate not only the presence of host larvae but also be used as a cue that indicates host suitability and age. Such information would allow female parasitoids to discern more efficiently between hosts within ovipositor reach from those already buried too deep into the xylem and out of reach. In this context, we assessed the behaviour of I. leucospoides females to volatiles of A. areolatum in a ‘Y’‐tube olfactometer at regular intervals over 30 days. We concurrently examined the fungal volatiles by headspace sampling through solid‐phase microextraction (SPME) followed by gas chromatography mass spectrometry (GC‐MS). We observed that females were attracted to volatiles produced by two‐week‐old fungal cultures, a period that matches when older larvae are still within ovipositor reach. Four chemical compounds were detected: ethanol, acetone, acetaldehyde and the sesquiterpene 2,2,8‐trimethyltricyclo[6.2.2.01,6]dodec‐5‐ene, with each compounds’ relative abundance changing over time. Results are discussed in the context of parasitoids fitness. Future studies involving electrophysiology, different collection techniques and further behavioural assays will help in identifying the compounds that convey temporal information to female parasitoids and have the potential for being used in integrated pest management programmes.     

Accumulation pattern of endogenous cytokinins and phenolics in different organs of 1‐year‐old cytokinin pre‐incubated plants: implications for conservation

A better understanding of phytohormone physiology can provide an essential basis to coherently achieve a conservation drive/strategy for valuable plant species. We evaluated the distribution pattern of cytokinins (CKs) and phenolic compounds in different organs of 1‐year‐old greenhouse‐grown Tulbaghia simmleri pre‐treated (during micropropagation) with three aromatic CKs (benzyladenine = BA, meta‐topolin = mT, meta‐topolin riboside = mTR). The test species is highly valuable due to its medicinal and ornamental uses. Based on UHPLC‐MS/MS quantification, mT and mTR pre‐treated plants had the highest total CK, mostly resulting from the isoprenoid CK‐type, which occurred at highest concentrations in the roots. Although occurring in much lower concentrations when compared to isoprenoid CKs, aromatic CKs were several‐fold more abundant in the root of mT pre‐treated plants than with other treatments. Possibly related to the enhanced aromatic CKs, free bases and ribonucleotides, plants pre‐treated with mT generally displayed better morphology than the other treatments. A total of 12 bioactive phenolic compounds, including four hydroxybenzoic acids, five hydroxycinnamic acids and three flavonoids at varying concentrations, were quantified in T. simmleri. The occurrence, distribution and levels of these phenolic compounds were strongly influenced by the CK pre‐treatments, thereby confirming the importance of CKs in phenolic biosynthesis pathways.     

Comparison of spleen transcriptomes of two wild rodent species reveals differences in the immune response against Borrelia afzelii

Different host species often differ considerably in susceptibility to a given pathogen, but the causes of such differences are rarely known. The natural hosts of the tick‐transmitted bacterium Borrelia afzelii, which is one of causative agents of Lyme borreliosis in humans, include a variety of small mammals like voles and mice. Previous studies have shown that B. afzelii‐infected bank voles (Myodes glareolus) have about ten times higher bacterial load than infected yellow‐necked mice (Apodemus flavicollis), indicating that these two species differ in resistance. In this study, we compared the immune response to B. afzelii infection in these host species by using RNA sequencing to quantify gene expression in spleen. Gene set enrichment analysis (GSEA) showed that several immune pathways were down‐regulated in infected animals in both bank voles and yellow‐necked mice. Moreover, IFNα response was up‐regulated in B. afzelii‐infected yellow‐necked mice, while IL6 signaling and the complement pathway were down‐regulated in infected bank voles; differences in regulation of these three pathways between bank voles and yellow‐necked mice could thus contribute to the difference in resistance to B. afzelii between the species. This study provides knowledge of gene expression induced by a zoonotic pathogen in its natural host, and possible species‐specific regulation of immune responses associated with resistance.     

Biogeography and host‐related factors trump parasite life history: limited congruence among the genetic structures of specific ectoparasitic lice and their rodent hosts

Parasites and hosts interact across both micro‐ and macroevolutionary scales where congruence among their phylogeographic and phylogenetic structures may be observed. Within southern Africa, the four‐striped mouse genus, Rhabdomys, is parasitized by the ectoparasitic sucking louse, Polyplax arvicanthis. Molecular data recently suggested the presence of two cryptic species within P. arvicanthis that are sympatrically distributed across the distributions of four putative Rhabdomys species. We tested the hypotheses of phylogeographic congruence and cophylogeny among the two parasite lineages and the four host taxa, utilizing mitochondrial and nuclear sequence data. Despite the documented host‐specificity of P. arvicanthis, limited phylogeographic correspondence and nonsignificant cophylogeny was observed. Instead, the parasite–host evolutionary history is characterized by limited codivergence and several duplication, sorting and host‐switching events. Despite the elevated mutational rates found for P. arvicanthis, the spatial genetic structure was not more pronounced in the parasite lineages compared with the hosts. These findings may be partly attributed to larger effective population sizes of the parasite lineages, the vagility and social behaviour of Rhabdomys, and the lack of host‐specificity observed in areas of host sympatry. Further, the patterns of genetic divergence within parasite and host lineages may also be largely attributed to historical biogeographic changes (expansion‐contraction cycles). It is thus evident that the association between P. arvicanthis and Rhabdomys has been shaped by the synergistic effects of parasite traits, host‐related factors and biogeography over evolutionary time.