Cyanogenic glucosides in grapevine: polymorphism, identification and developmental patterns

Twelve grapevine (Vitis vinifera L.) cultivars were surveyed for 'cyanide potential' (i.e. the total cyanide measured in beta-glucosidase-treated crude, boiled tissue extract) in mature leaves. Two related cultivars (Carignan and Ruby Cabernet) had mean cyanide potential (equivalent to 110 mgHCNkg-1fr.wt) ca. 25-fold greater than that of the other 10 cultivars, and so the trait is polymorphic in the species. In boiled leaf extracts of Carignan and Ruby Cabernet, free cyanide constituted a negligible fraction of the total cyanide potential because beta-glucosidase treatment was required to liberate the major cyanide fraction - which is therefore bound in glucosylated cyanogenic compound(s) (or cyanogenic glucosides). In addition, cyanide was liberated from ground leaf tissue of Ruby Cabernet but not Sultana (a cultivar with low cyanide potential). Hence, the high cyanide potential in Ruby Cabernet leaves is coupled with endogenous beta-glucosidase(s) activity and this cultivar may be considered 'cyanogenic'. A method was developed to detect and identify cyanogenic glucosides using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). Two putative cyanogenic glucosides were found in extracts from leaves of Carignan and Ruby Cabernet and were identified as the epimers prunasin and sambunigrin. Cyanide potential measured at three times over the growing season in young and mature leaves, petioles, tendrils, flowers, berries, seeds and roots of Ruby Cabernet was substantially higher in the leaves compared with all other tissues. This characterisation of cyanogenic glucoside accumulation in grapevine provides a basis for gauging the involvement of the trait in interactions of the species with its pests and pathogens.     

Cyanogenic and non-cyanogenic pyridone glucosides from Acalypha indica (Euphorbiaceae)

Seven cyanopyridone derivatives and one corresponding seco compound have been isolated from a methanolic extract of the inflorescences and leaves of Acalypha indica L. (Euphorbiaceae). The absolute configuration of the main cyanogenic glucoside acalyphin, (−)-(5R,6S)-5-cyano-5-β-d-glucopyranosyloxy-6-hydroxy-4-methoxy-1-methyl-2(5,6-dihydro)-pyridone, was deduced from an X-ray crystallographic study. In addition, the 6R-epimer of acalyphin, epiacalyphin, and the corresponding pair of N-demethyl derivatives were isolated. The corresponding amide of acalyphin and a 1′,2′-glucosyl-fused epiacalyphin amide were isolated from air-dried material. Structural elucidation was performed by means of ¹H and ¹³C NMR-spectra, chiroptical methods such as CD-spectroscopy and optical rotation. Two further corresponding derivatives, an aromatized compound and an open-chain structure, were isolated from the aqueous phase.     

Iridoid glucosides of Paederota lutea and the relationships between Paederota and Veronica

In a chemical investigation of the water soluble compounds in Paederota lutea eight known iridoids were isolated together with a new one with a 8,9-double bond, namely paederotoside (10-O-benzoyl-6'-O-alpha-arabino(1-->6)-beta-glucopyranosyl arborescosidic acid) and the 6-hydroxy-flavone glucoside 4'-O-methylscutellarein 7-O-beta-glucopyranoside. The known iridoid glucosides were 8-epiloganic acid, gardoside, aucubin, catalpol and the 6-O-esters of catalpol: veronicoside, catalposide, amphicoside and verproside. The compounds isolated show that Paederota has a glycoside content almost identical to that of a general Veronica species, and this is in good agreement with the results from recent investigations of the phylogeny of Veronica and its closest relatives, where Paederota is placed as a sister-group next to Veronica. In an analysis of the iridoid glucosides from some of these relatives, it is shown that Veronica, Paederota, Picrorhiza and Veronicastrum are all characterized by containing 6-O-esters of catalpol. Some less closely related taxa namely: Lagotis, Wulfenia, Plantago, Aragoa and Globularia instead contain 10-O-esters of catalpol or aucubin.     

Cyanogenesis in plants and arthropods

Cyanogenic glucosides are phytoanticipins known to be present in more than 2500 plant species. They are regarded as having an important role in plant defense against herbivores due to bitter taste and release of toxic hydrogen cyanide upon tissue disruption, but recent investigations demonstrate additional roles as storage compounds of reduced nitrogen and sugar that may be mobilized when demanded for use in primary metabolism. Some specialized herbivores, especially insects, preferentially feed on cyanogenic plants. Such herbivores have acquired the ability to metabolize cyanogenic glucosides or to sequester them for use in their own defense against predators. A few species of arthropods (within diplopods, chilopods and insects) are able to de novo biosynthesize cyanogenic glucosides and some are able to sequester cyanogenic glucosides from their food plant as well. This applies to larvae of Zygaena (Zygaenidae). The ratio and content of cyanogenic glucosides is tightly regulated in Zygaena filipendulae, and these compounds play several important roles in addition to defense in the life cycle of Zygaena. The transfer of a nuptial gift of cyanogenic glucosides during mating of Zygaena has been demonstrated as well as the involvement of hydrogen cyanide in male attraction and nitrogen metabolism. As more plant and arthropod species are examined, it is likely that cyanogenic glucosides are found to be more widespread than formerly thought and that cyanogenic glucosides are intricately involved in many key processes in the life cycle of plants and arthropods.     

Unexpected heterogeneity due to recessive and de novo dominant mutations of GJB2 in an Iranian family with nonsyndromic hearing loss: implication for genetic counseling

Mutations in the GJB2 gene are the most common cause of nonsyndromic autosomal recessive sensorineural hearing loss (HL). A few mutations in GJB2 have also been reported to cause dominant nonsyndromic HL. Here we report a large inbred family including two individuals with nonsyndromic sensorineural hearing loss. A dominant GJB2 mutation, c.551G>A (p.R184Q), was detected in the proband, yet his parents were negative for the mutation. The second affected person had heterozygous c.35delG mutation, which was inherited from his father. Large deletions of the GJB6 gene were not detected in this family. This study highlights the importance of mutation analysis in all affected cases within a pedigree.     

Cyanogenesis in glucosinolate-producing plants: Carica papaya and Carica quercifolia

(R)-2-(beta-D-Glucopyranosyloxy)-2-phenylacetonitrile (prunasin) was isolated from Carica papaya L. and C. quercifolia (A. St.-Hil.) Hieron. (syn. C. hastata Brign.). Earlier reported presence of cyclopentanoid cyanohydrin glycosides in C. papaya could not be confirmed, and no cyclopentanoid amino acids could be detected in extracts of C. papaya and C. quercifolia. Conversion of [2,3,4,5,6-3H]phenylalanine into tritiated prunasin was demonstrated in both species. On the other hand, when the plants were administered [2-14C]-2-(2'cyclopentenyl)glycine, extracted, and the extracts hydrolyzed with beta-glucosidase (Helix pomatia), formation of labelled cyanide was not observed. The absence of cyclopentanoids, which are typical for the Passifloraceae, and the inability of Carica species to utilize 2-(2'-cyclopentenyl)glycine as a precursor of cyanogenic glycosides are in agreement with the relative phylogenetic position of the Caricaceae and the Passifloraceae. Carica species are thus rare examples of taxa in which glucosinolates and cyanogenic glycosides co-occur, both types of natural products being derived from the same amino acid, phenylalanine.     

飞机草入侵种群与原产地种群生长性状的差异

飞机草(Chromolaena odorata)是我国热带地区危害严重的外来入侵植物,为揭示适应进化对其成功入侵的贡献,在同质种植园中,比较研究了飞机草10个入侵地种群与12个原产地种群生长性状的差异,为排除奠基者效应的可能影响,进一步比较了飞机草10个入侵地种群与其原产地可能的祖先种群间的差异。结果表明,飞机草10个入侵地种群的基茎、株高、分枝数、生物量和比叶面积均显著高于12个原产地种群;与可能的祖先种群相比,飞机草10个入侵种群的生物量、分枝数和比叶面积仍更高。这些结果表明,在长期的入侵过程中飞机草通过进化提高了资源向生长的分配,支持增强竞争能力的进化假说。     

Conservation between animals and plants of the cis-acting element involved in the unfolded protein response

Using Arabidopsis thaliana, we identified the cis-element involved in the plant unfolded protein response (UPR). In transgenic plants, tunicamycin stimulated expression of a reporter gene under the control of the BiP promoter and promoter analysis identified a 24 bp sequence crucial to this induction. When fused with a minimal promoter, a hexamer of this sequence was sufficient for induction of a reporter gene in protoplasts treated with tunicamycin or dithiothreitol. Induction rate equivalent to original promoter was observed when the assay was conducted in transgenic plants. This 24 bp sequence contained two elements also responsible for the UPR in animals. Either of these elements was sufficient for the plant UPR, indicating conservation between animals and plants of cis-elements involved in the UPR.     

Modelling O2 and O2 unidirectional fluxes in plants. III: Fitting of experimental data by a simple model

Photosynthetic assimilation of CO₂ in plants results in the balance between the photochemical energy developed by light in chloroplasts, and the consumption of that energy by the oxygenation processes, mainly the photorespiration in C₃ plants. The analysis of classical biological models shows the difficulties to bring to fore the oxygenation rate due to the photorespiration pathway. As for other parameters, the most important key point is the estimation of the electron transport rate (ETR or J), i.e. the flux of biochemical energy, which is shared between the reductive and oxidative cycles of carbon. The only reliable method to quantify the linear electron flux responsible for the production of reductive energy is to directly measure the O₂ evolution by ¹⁸O₂ labelling and mass spectrometry. The hypothesis that the respective rates of reductive and oxidative cycles of carbon are only determined by the kinetic parameters of Rubisco, the respective concentrations of CO₂ and O₂ at the Rubisco site and the available electron transport rate, ultimately leads to propose new expressions of biochemical model equations. The modelling of ¹⁸O₂ and ¹⁶O₂ unidirectional fluxes in plants shows that a simple model can fit the photosynthetic and photorespiration exchanges for a wide range of environmental conditions. Its originality is to express the carboxylation and the oxygenation as a function of external gas concentrations, by the definition of a plant specificity factor Sp that mimics the internal reactions of Rubisco in plants. The difference between the specificity factors of plant (Sp) and of Rubisco (Sr) is directly related to the conductance values to CO₂ transfer between the atmosphere and the Rubisco site. This clearly illustrates that the values and the variation of conductance are much more important, in higher C₃ plants, than the small variations of the Rubisco specificity factor. The simple model systematically expresses the reciprocal variations of carboxylation and oxygenation exchanges illustrated by a “mirror effect”. It explains the protective sink effect of photorespiration, e.g. during water stress. The importance of the CO₂ compensation point, in classical models, is reduced at the benefit of the crossing points Cx and Ox, concentration values where carboxylation and oxygenation are equal or where the gross O₂ uptake is half of the gross O₂ evolution. This concept is useful to illustrate the feedback effects of photorespiration in the atmosphere regulation. The constancy of Sp and of Cx for a great variation of P under several irradiance levels shows that the regulation of the conductance maintains constant the internal CO₂ and the ratio of photorespiration to photosynthesis (PR/P). The maintenance of the ratio PR/P, in conditions of which PR could be reduced and the carboxylation increased, reinforces the hypothesis of a positive role of photorespiration and its involvement in the plant-atmosphere co-evolution.     

Biosynthesis of 1-deoxynojirimycin in Commelina communis: a difference between the microorganisms and plants

1-Deoxynojirimycin is a glycosidase-inhibitory alkaloid obtained from several plants and microorganisms. Administration experiments using [1-(13C)] glucose in the higher plant Commelina communis and 13C NMR spectroscopic analyses of products suggested that 1-deoxynojirimycin was biosynthesized through a different route compared with that in Streptomyces and Bacilli microorganisms.     

Specific accumulation and revised structures of acridone alkaloid glucosides in the tips of transformed roots of Ruta graveolens

The root tips of Ruta graveolens (common rue) show strong autofluorescence of acridone alkaloids, which are characteristic secondary metabolites of this plant. To study the specific distribution and accumulation of acridone alkaloids in various root segments of Ruta graveolens, root material was harvested from genetically transformed root cultures and extracts were investigated by chromatographic techniques and HPLC-(1)H NMR spectroscopy. The cells of the elongation and differentiation zones contained acridone glucosides and large amounts of acridone alkaloids, mainly rutacridone. Gravacridondiol glucoside was identified as the dominant secondary compound of the root tips and its structure revised by means of spectroscopic methods. In addition, minor acridones, including the structurally revised gravacridontriol glucoside and unknown natural products, were found in the root tip.     

Jasmonate: A decision maker between cell death and acclimation in the response of plants to singlet oxygen

Under stress conditions that bring about excessive absorption of light energy in the chloroplasts, the formation of singlet oxygen (¹O₂) can be strongly enhanced, triggering programmed cell death. However, the ¹O₂ signaling pathway can also lead to acclimation to photooxidative stress, when ¹O₂ is produced in relatively low amounts. This acclimatory response is associated with a strong downregulation of the jasmonate biosynthesis pathway and the maintenance of low jasmonate levels, even under high light stress conditions that normally induce jasmonate synthesis. These findings suggest a central role for this phytohormone in the orientation of the ¹O₂ signaling pathway toward cell death or acclimation. This conclusion is confirmed here in an Arabidopsis double mutant obtained by crossing the ¹O₂-overproducing mutant ch1 and the jasmonate-deficient mutant dde2. This double mutant was found to be constitutively resistant to ¹O₂ stress and to display a strongly stimulated growth rate compared with the single ch1 mutant. However, the involvement of other phytohormones, such as ethylene, cannot be excluded.     

The structure and evolution of cis-regulatory regions: the shavenbaby story

In this paper, we provide a historical account of the contribution of a single line of research to our current understanding of the structure of cis-regulatory regions and the genetic basis for morphological evolution. We revisit the experiments that shed light on the evolution of larval cuticular patterns within the genus Drosophila and the evolution and structure of the shavenbaby gene. We describe the experiments that led to the discovery that multiple genetic changes in the cis-regulatory region of shavenbaby caused the loss of dorsal cuticular hairs (quaternary trichomes) in first instar larvae of Drosophila sechellia. We also discuss the experiments that showed that the convergent loss of quaternary trichomes in D. sechellia and Drosophila ezoana was generated by parallel genetic changes in orthologous enhancers of shavenbaby. We discuss the observation that multiple shavenbaby enhancers drive overlapping patterns of expression in the embryo and that these apparently redundant enhancers ensure robust shavenbaby expression and trichome morphogenesis under stressful conditions. All together, these data, collected over 13 years, provide a fundamental case study in the fields of gene regulation and morphological evolution, and highlight the importance of prolonged, detailed studies of single genes.     

Structural organization of de novo purine biosynthesis enzymes in plants: 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase cDNAs from Vigna aconitifolia

Nodules of tropical legumes generally export symbiotically fixed nitrogen in the form of ureides that are produced by oxidation of de novo synthesized purines. To investigate the regulation of de novo purine biosynthesis in these nodules, we have isolated cDNA clones encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase from a mothbean (Vigna aconitifolia) nodule cDNA library by complementation of Escherichia coli purE and purC mutants, respectively. Sequencing of these clones revealed that the two enzymes are distinct proteins in mothbean, unlike in animals where both activities are associated with a single bifunctional polypeptide. As is the case in yeast, the mothbean AIR carboxylase has a N-terminal domain homologous to the eubacterial purK gene product. This PurK-like domain appears to facilitate the binding of CO₂ and is dispensable in the presence of high CO₂ concentrations. Because the expression of the mothbean PurE cDNA clone in E. coli apparently generates a truncated polypeptide lacking at least 140 N-terminal amino acids, this N-terminal region of the enzyme may not be essential for its CO₂-binding activity.     

Convergent origins and rapid evolution of spliced leader trans-splicing in Metazoa: Insights from the Ctenophora and Hydrozoa

Replacement of mRNA 5′ UTR sequences by short sequences trans-spliced from specialized, noncoding, spliced leader (SL) RNAs is an enigmatic phenomenon, occurring in a set of distantly related animal groups including urochordates, nematodes, flatworms, and hydra, as well as in Euglenozoa and dinoflagellates. Whether SL trans-splicing has a common evolutionary origin and biological function among different organisms remains unclear. We have undertaken a systematic identification of SL exons in cDNA sequence data sets from non-bilaterian metazoan species and their closest unicellular relatives. SL exons were identified in ctenophores and in hydrozoan cnidarians, but not in other cnidarians, placozoans, or sponges, or in animal unicellular relatives. Mapping of SL absence/presence obtained from this and previous studies onto current phylogenetic trees favors an evolutionary scenario involving multiple origins for SLs during eumetazoan evolution rather than loss from a common ancestor. In both ctenophore and hydrozoan species, multiple SL sequences were identified, showing high sequence diversity. Detailed analysis of a large data set generated for the hydrozoan Clytia hemisphaerica revealed trans-splicing of given mRNAs by multiple alternative SLs. No evidence was found for a common identity of trans-spliced mRNAs between different hydrozoans. One feature found specifically to characterize SL-spliced mRNAs in hydrozoans, however, was a marked adenosine enrichment immediately 3′ of the SL acceptor splice site. Our findings of high sequence divergence and apparently indiscriminate use of SLs in hydrozoans, along with recent findings in other taxa, indicate that SL genes have evolved rapidly in parallel in diverse animal groups, with constraint on SL exon sequence evolution being apparently rare.     

Differences between the pathogenic processes induced by Steinernema and Heterorhabditis (Nemata: Rhabditida) in Pseudaletia unipuncta (Insecta: Lepidoptera)

Larvae of Pseudaletia unipuncta are moderately susceptible to infections caused by entomopathogenic nematodes, being a desirable host to study pathogenic processes caused by Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema glaseri and their associated bacteria. The ability of the infective stage of these nematodes to invade hosts is quite different. S. carpocapsae invades the highest number of insects and presents the highest penetration rate, followed by H. bacteriophora. Regression analysis between the number of insects parasitized and the number of IJs counted per insect, over time, showed a high correlation for S. carpocapsae whereas for H. bacteriophora it was low. Dose-response was most evident at a concentration below 100 IJs per insect on H. bacteriophora, whereas on S. carpocapsae it was found for doses ranging from 100 to 2,000 IJs. Student's t test analysis of dose-response showed parallel, yet unequal, slopes for both strains of H. bacteriophora, whereas distinct regressions were obtained for S. carpocapsae and S. glaseri, thus, evidencing each species develop a distinct pathogenic process. Insects injected with Photorhabdus luminescens died within 50 h after injection, whereas those treated with X. nematophila died much later. Moreover, the mortality in insects exposed to H. bacteriophora complex and injected with P. luminescens was close, but insects injected with bacteria died faster. Insect mortality in treatments with complexes S. carpocapsae and S. glaseri was significantly higher than that which was observed in insects injected with symbiotic bacteria.     

Who is in control of the stickleback immune system: interactions between Schistocephalus solidus and its specific vertebrate host

The cestode Schistocephalus solidus is a frequent parasite of three-spined sticklebacks and has a large impact on its host's fitness. Selection pressure should therefore be high on stickleback defence mechanisms, like an efficient immune system, and also on parasite strategies to overcome these. Even though there are indications for manipulation of the immune system of its specific second intermediate host by the cestode, nothing is yet known about the chronology of specific interactions of S. solidus with the stickleback immune system. We here expected sticklebacks to first mount an innate immune response directly post-exposure to the parasite to clear the infection at an early stage and after an initial lag phase to upregulate adaptive immunity. Most interestingly, we did not find any upregulation of the specific lymphocyte-mediated immune response. Also, the pattern of activation of the innate immune system did not match our expectations: the proliferation of monocytes followed fluctuating kinetics suggesting that the parasite repeatedly installs a new surface coat not immunogenic to the host. Furthermore, the respiratory burst activity, which has the potential to clear an early S. solidus infection, was upregulated very late during infection, when the parasite was too big to be cleared but ready for transmission to its final host. We here suggest that the late activation of the innate immune system interferes with the neuroendocrine system, which mediates reduced predation avoidance behaviour and so facilitates the transmission to the final host.