The bark beetle Ips typographus carries numerous fungi that could be assisting the beetle in colonizing live Norway spruce (Picea abies) trees. Phenolic defenses in spruce phloem are degraded by the ...beetle's major tree-killing fungus Endoconidiophora polonica, but it is unknown if other beetle associates can also catabolize these compounds. We compared the ability of five fungi commonly associated with I. typographus to degrade phenolic compounds in Norway spruce phloem. Grosmannia penicillata and Grosmannia europhioides were able to degrade stilbenes and flavonoids faster than E. polonica and grow on minimal growth medium with spruce bark constituents as the only nutrients. Furthermore, beetles avoided medium amended with phenolics but marginally preferred medium colonized by fungi. Taken together our results show that different bark beetle-associated fungi have complementary roles in degrading host metabolites and thus might improve this insect's persistence in well defended host tissues.
•Ips typographus-associated fungi have the ability to degrade conifer phenolics.•Grosmannia penicillata degrades major spruce phenolics most rapidly.•Efficiency of phenolic degradation varies directly with fungal virulence.•Bark beetles avoid phenolics but prefer fungus-colonized medium.
Multiple lepidopteran larvae feed successfully on plants containing glucosinolates despite the diverse array of toxic and deterrent breakdown products, such as isothiocyanates (ITCs), formed upon ...plant damage. While much is known about how specialist lepidopterans metabolize and tolerate glucosinolates, there is little information about the metabolic fate of these plant defense compounds in specialized herbivores. Employing
C- and
C-labeled 4-methylsulfinylbutyl glucosinolate (glucoraphanin), we identified and quantified the major detoxification products of glucosinolates and ITCs in selected specialized and generalist larvae. While specialists prevented glucosinolate hydrolysis or diverted hydrolysis to form nitriles, hydrolysis in generalists proceeded to toxic ITCs, of which a portion were conjugated to glutathione. However, a large amount of ITCs remained unmodified, which may have led to the observed negative effects on growth and development. The performance of two generalist-feeding caterpillars,
(African cotton leafworm) and
(cabbage moth) on
Col-0 and various glucosinolate-deficient mutants was investigated from hatching until pupation. We found that glucosinolates negatively affected larval growth and development, but not survival, with aliphatic glucosinolates having stronger effects than indolic glucosinolates, and the combination of the two glucosinolate types being even more detrimental to growth and development. Curiously, last instar larvae grew better on wild type than on non-glucosinolate-containing plant lines, but this could not be attributed to a change in detoxification rate or feeding behavior. Glucosinolates thus appear to be effective defenses against generalist lepidopteran herbivores at least during most stages of larval development. Nevertheless, the reversal of negative effects in the oldest instar is intriguing, and further investigation of this phenomenon may shed light on how generalists adjust their physiology to feed on diets with many different types of plant defense compounds.
The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic ...compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range DXS activities were dynamically labeled with ¹³CO₂ in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-Cmethylerythritol-2,4-cyclodiphosphate (MEcDP), metabolicaUy isolated from the MEP pathway. DXS overexpression led to a 3-to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway.
The spatial distribution of plant defenses within a leaf may be critical in explaining patterns of herbivory. The generalist lepidopteran larvae, Helicoverpa armigera (the cotton bollworm), avoided ...the midvein and periphery of Arabidopsis thaliana rosette leaves and fed almost exclusively on the inner lamina. This feeding pattern was attributed to glucosinolates because it was not evident in a myrosinase mutant that lacks the ability to activate glucosinolate defenses by hydrolysis. To measure the spatial distribution of glucosinolates in A. thaliana leaves at a fine scale, we constructed ion intensity maps from MALDI-TOF (matrix assisted laser desorption/ionization-time of flight) mass spectra. The major glucosinolates were found to be more abundant in tissues of the midvein and the periphery of the leaf than the inner lamina, patterns that were validated by HPLC analyses of dissected leaves. In addition, there were differences in the proportions of the three major glucosinolates in different leaf regions. Hence, the distribution of glucosinolates within the leaf appears to control the feeding preference of H. armigera larvae. The preferential allocation of glucosinolates to the periphery may play a key role in the defense of leaves by creating a barrier to the feeding of chewing herbivores that frequently approach leaves from the edge.
The pea aphid (
), a phloem-sucking insect, has undergone a rapid radiation together with the domestication and anthropogenic range expansion of several of its legume host plants. This insect species ...is a complex of at least 15 genetically different host races that can all develop on the universal host plant
. However, each host race is specialized on a particular plant species, such as
,
, or
, which makes it an attractive model insect to study ecological speciation. Previous work revealed that pea aphid host plants produce a specific phytohormone profile depending on the host plant - host race combination. Native aphid races induce lower defense hormone levels in their host plant than non-native pea aphid races. Whether these changes in hormone levels also lead to changes in other metabolites is still unknown. We used a mass spectrometry-based untargeted metabolomic approach to identify plant chemical compounds that vary among different host plant-host race combinations and might therefore, be involved in pea aphid host race specialization. We found significant differences among the metabolic fingerprints of the four legume species studied prior to aphid infestation, which correlated with aphid performance. After infestation, the metabolic profiles of
and
plants infested with their respective native aphid host race were consistently different from profiles after infestation with non-native host races and from uninfested control plants. The metabolic profiles of
plants infested with their native aphid host race were also different from plants infested with non-native host races, but not different from uninfested control plants. The compounds responsible for these differences were putatively identified as flavonoids, saponins, non-proteinogenic amino acids and peptides among others. As members of these compound classes are known for their activity against insects and aphids in particular, they may be responsible for the differential performance of host races on native vs. non-native host plants. We conclude that the untargeted metabolomic approach is suitable to identify candidate compounds involved in the specificity of pea aphid - host plant interactions.
The pea aphid (Acyrthosiphon pisum Harris), a legume specialist, encompasses at least 11 genetically distinct sympatric host races. Each host race shows a preference for a certain legume species. Six ...pea aphid clones from three host races were used to localize plant factors influencing aphid probing and feeding behavior on four legume species. Aphid performance was tested by measuring survival and growth. The location of plant factors influencing aphid probing and feeding was determined using the electrical penetration graph (EPG) technique. Every aphid clone performed best on the plant species from which it was originally collected, as well as on Vicia faba. On other plant species, clones showed intermediate or poor performance. The most important plant factors influencing aphid probing and feeding behavior were localized in the epidermis and sieve elements. Repetitive puncturing of sieve elements might be relevant for establishing phloem feeding, since feeding periods appear nearly exclusively after these repetitive sieve element punctures. A combination of plant factors influences the behavior of pea aphid host races on different legume species and likely contributes to the maintenance of these races.
The accumulation of proanthocyanidins is regulated by a complex of transcription factors composed of R2R3 MYB, basic helix-loop-helix, and WD40 proteins that activate the promoters of biosynthetic ...genes. In poplar (genus Populus), MYB134 is known to regulate proanthocyanidin biosynthesis by activating key flavonoid genes. Here, we characterize a second MYB regulator of proanthocyanidins, MYB115. Transgenic poplar overexpressing MYB115 showed a highproanthocyanidin phenotype and reduced salicinoid accumulation, similar to the effects of MYB134 overexpression. Transcriptomic analysis of MYB115- and MYB134-overexpressing poplar plants identified a set of common up-regulated genes encoding proanthocyanidin biosynthetic enzymes and several novel uncharacterized MYB transcriptional repressors. Transient expression experiments demonstrated the capacity of both MYB134 and MYB115 to activate flavonoid promoters, but only in the presence of a basic helix-loop-helix cofactor. Yeast two-hybrid experiments confirmed the direct interaction of these transcription factors. The unexpected identification of dihydromyricetin in leaf extracts of both MYB115- and MYB134-overexpressing poplar led to the discovery of enhanced flavonoid B-ring hydroxylation and an increased proportion of prodelphinidins in proanthocyanidin of the transgenics. The dramatic hydroxylation phenotype of MYB115 overexpressors is likely due to the up-regulation of both flavonoid 3',5'-hydroxylases and cytochrome b₅. Overall, this work provides new insight into the complexity of the gene regulatory network for proanthocyanidin synthesis in poplar.
Terpenes are structurally diverse natural products involved in many ecological interactions. The pivotal enzymes for terpene biosynthesis, terpene synthases (TPSs), had been described only in plants ...and fungi in the eukaryotic domain. In this report, we systematically analyzed the genome sequences of a broad range of nonplant/nonfungus eukaryotes and identified putative TPS genes in six species of amoebae, five of which are multicellular social amoebae from the order of Dictyosteliida. A phylogenetic analysis revealed that amoebal TPSs are evolutionarily more closely related to fungal TPSs than to bacterial TPSs. The social amoeba Dictyostelium discoideum was selected for functional study of the identified TPSs. D. discoideum grows as a unicellular organism when food is abundant and switches from vegetative growth to multicellular development upon starvation. We found that expression of most D. discoideum TPS genes was induced during development. Upon heterologous expression, all nine TPSs from D. discoideum showed sesquiterpene synthase activities. Some also exhibited monoterpene and/or diterpene synthase activities. Direct measurement of volatile terpenes in cultures of D. discoideum revealed essentially no emission at an early stage of development. In contrast, a bouquet of terpenes, dominated by sesquiterpenes including β-barbatene and (E,E)-α-farnesene, was detected at the middle and late stages of development, suggesting a development-specific function of volatile terpenes in D. discoideum. The patchy distribution of TPS genes in the eukaryotic domain and the evidence for TPS function in D. discoideum indicate that the TPS genes mediate lineage-specific adaptations.
Flavan-3-ols including the monomeric catechin and the polymeric proanthocyanidins (PAs) are abundant phenolic metabolites in poplar (
spp.) previously described to protect leaves against pathogen ...infection. However, it is not known whether stems are also defended in this way. Here we investigated flavan-3-ol accumulation, activity, and the regulation of formation in black poplar (
) stems after infection by a newly described fungal stem pathogen,
, which forms canker-like lesions in stems. We showed that flavan-3-ol contents increased in
-infected black poplar stems over the course of infection compared to non-infected controls. Transcripts of leucoanthocyanidin reductase (
) and anthocyanidin reductase (
) genes involved in the last steps of flavan-3-ol biosynthesis were also upregulated upon fungal infection indicating
biosynthesis. Amending culture medium with catechin and PAs reduced the mycelial growth of
, suggesting that these metabolites act as anti-pathogen defenses in poplar
. Among the hormones, salicylic acid (SA) was higher in
-infected tissues compared to the non-infected controls over the course of infection studied, while jasmonic acid (JA) and JA-isoleucine (JA-Ile) levels were higher than controls only at the early stages of infection. Interestingly, cytokinins (CKs) were also upregulated in
-infected stems. Poplar saplings treated with CK showed decreased levels of flavan-3-ols and SA in stems suggesting a negative association between CK and flavan-3-ol accumulation. Taken together, the sustained upregulation of SA in correlation with catechin and PA accumulation suggests that this is the dominant hormone inducing the formation of antifungal flavan-3-ols during
infection of poplar stems.
Plant volatiles not only have multiple defense functions against herbivores, fungi, and bacteria, but also have been implicated in signaling within the plant and toward other organisms. Elucidating ...the function of individual plant volatiles will require more knowledge of their biosynthesis and regulation in response to external stimuli. By exploiting the variation of herbivore-induced volatiles among 26 maize (Zea mays) inbred lines, we conducted a nested association mapping and genome-wide association study (GWAS) to identify a set of quantitative trait loci (QTLs) for investigating the pathways of volatile terpene production. The most significant identified QTL affects the emission of (E)-nerolidol, linalool, and the two homoterpenes (E)-3,8-dimethyl-1,4,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). GWAS associated a single nucleotide polymorphism in the promoter of the gene encoding the terpene synthase TPS2 with this QTL. Biochemical characterization of TPS2 verified that this plastid-localized enzyme forms linalool, (E)-nerolidol, and (E,E)-geranyllinalool. The subsequent conversion of (E)-nerolidol into DMNT maps to a P450 monooxygenase, CYP92C5, which is capable of converting nerolidol into DMNT by oxidative degradation. A QTL influencing TMTT accumulation corresponds to a similar monooxygenase, CYP92C6, which is specific for the conversion of (E,E)-geranyllinalool to TMTT. The DMNT biosynthetic pathway and both monooxygenases are distinct from those previously characterized for DMNT and TMTT synthesis in Arabidopsis thaliana, suggesting independent evolution of these enzymatic activities.
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