Cucurbitacin E and I in Iberis amara: Feeding inhibitors for Phyllotreta nemorum

Cucurbitacin E and cucurbitacin I have been isolated from green parts of Iberis amara and identified by TLC, UV and MS. It is shown that cucurbitacins act as feeding inhibitors for the flea beetle Phyllotreta nemorum. The most potent feeding inhibitors in green parts of I. amara towards P. nemorum are cucurbitacin E and I, and the concentrations of these compounds in the plant are found to be high enough to prevent feeding of the flea beetle.     

Cucurbitacine als stathmokinetische Wirkstoffe

Summary A synergism of colchicine and different cucurbitacins has been shown, to be a cause in the formation of ball-metaphases in Bryonia species. The bitter principles cucurbitacin L and I, dihydrocucurbitacin B, and tetrahydrocucurbitacin I, which were examined more closely with respect to their karyological effects, were shown to give rise to C-metaphases in Hordeum and Vicia at concentrations of about 0.016 per cent. On the one hand the number of mitotic stages is reduced, and on the other hand there is an increase of metaphase stages. Higher concentrations lead to pycnotic degenerations of nuclei and are toxic. When colchicine is applied together with cucurbitacins, ball-metaphases result. This could be demonstrated in plants lacking colchicine and cucurbitacins (Hordeum) when both substances were applied at the same time. Ball-metaphases also appear in plants containing cucurbitacins (Bryonia alba, B. dioica, Citrullus colocythis, Iberis amara) when they are treated with colchicine.     

New cucurbitacins from Phormium tenax and Marah oreganus

Cucurbitacins I and D and two new cucurbitacins, isocucurbitacin D and 3-epi-isocucurbitacin D, were isolated from Phormium tenax. A new cucurbitacin, dihydroisocucurbitacin B, was isolated from Marah oreganus. The acid sensitivity of the 2β-hydroxy-3-keto system found in cucurbitacin D was demonstrated.     

Divergence of defensive cucurbitacins in independent Cucurbita pepo domestication events leads to differences in specialist herbivore preference

Crop domestication and improvement often concurrently affect plant resistance to pests and production of secondary metabolites, creating challenges for isolating the ecological implications of selection for specific metabolites. Cucurbitacins are bitter triterpenoids with extreme phenotypic differences between Cucurbitaceae lineages, yet we lack integrated models of herbivore preference, cucurbitacin accumulation, and underlying genetic mechanisms. In Cucurbita pepo, we dissected the effect of cotyledon cucurbitacins on preference of a specialist insect pest (Acalymma vittatum) for multiple tissues, assessed genetic loci underlying cucurbitacin accumulation in diverse germplasm and a biparental F₂ population (from a cross between two independent domesticates), and characterized quantitative associations between gene expression and metabolites during seedling development. Acalymma vittatum affinity for cotyledons is mediated by cucurbitacins, but other traits contribute to whole-plant resistance. Cotyledon cucurbitacin accumulation was associated with population structure, and our genetic mapping identified a single locus, Bi-4, containing genes relevant to transport and regulation – not biosynthesis – that diverged between lineages. These candidate genes were expressed during seedling development, most prominently a putative secondary metabolite transporter. Taken together, these findings support the testable hypothesis that breeding for plant resistance to insects involves targeting genes for regulation and transport of defensive metabolites, in addition to core biosynthesis genes.     

Die cucurbitacine in Bryonia alba und Bryonia dioica

The cucurbitacins in roots of Bryonia dioica and B. alba have been investigated. Both species contain the cucurbitacins E, B, I, D, J, K and L, the dihydrocucurbitacins E and B, and tetrahydrocucurbitacin I. The detection of certain cucurbitacin aglycones depends upon the date of harvest, the duration of storage and the methods used for extraction.     

Cucurbitacin E and I target the JAK/STAT pathway and induce apoptosis in Sézary cells

Cutaneous T-cell lymphomas and leukemias (CTCLs) are a heterogeneous group of extranodal non-Hodgkin's lymphomas. These are characterized by an accumulation of malignant CD4⁺ T-lymphocytes in the skin, lymph nodes, and peripheral blood. Novel treatment options are needed for patients who progress to advanced stage disease. Cucurbitacin I has previously shown promising results in Sézary syndrome (Sz). A plethora of cucurbitacins, however, have not yet been tested in CTCL. Herein, we investigated the effect of cucurbitacin E and I in two CTCL cell lines. We show that both cucurbitacins decrease viability and cause apoptosis in these cell lines, although HuT-78 was more affected than SeAx (IC₅₀ of 17.38 versus 22.01 μM for cucurbitacin E and 13.36 versus 24.47 μM for cucurbitacin I). Moreover, both cucurbitacins decrease viability of primary cells of a Sz patient (56.46% for cucurbitacin E and 59.07% for cucurbitacin I). Furthermore, while JAK2 inhibition leads to decreased viability in SeAx cells (IC₅₀ of 9.98 and 29.15 μM for AZD1480 and ruxolitinib respectively), both JAK1 and JAK3 do not. This suggests that JAK2 has a preferential role in promoting survival. Western blotting in SeAx cells revealed that both cucurbitacins inhibit STAT3 activation (P < 0.0001), while only cucurbitacin I inhibits STAT5 activation (P = 0.05). This suggests that STAT3 plays a preferential role in the mechanism of action of these cucurbitacins. Nevertheless, a role of STAT5 and JAK2 cannot be excluded and should be explored further. This knowledge could contribute to the development of effective therapies for CTCL and other malignancies involving dysfunction of the JAK/STAT pathway.     

Induction by alkaloids and phenobarbital of Family 4 Cytochrome P450s in Drosophila : evidence for involvement in host plant utilization

In vertebrates, cytochrome P450s of the CYP2 and CYP3 families play a dominant role in drug metabolism, while in insects members of the CYP6 and CYP28 families have been implicated in metabolism of insecticides and toxic natural plant compounds. A degenerate 3^′ RACE strategy resulted in the identification of fifteen novel P450s from an alkaloid-resistant species of Drosophila. The strong (17.4-fold) and highly specific induction of a single gene (CYP4D10) by the toxic isoquinoline alkaloids of a commonly utilized host-plant (saguaro cactus) provides the first indication that members of the CYP4 family in insects may play an important role in the maintenance of specific insect-host plant relationships. Strong barbiturate inducibility of CYP4D10 and two other D. mettleri P450 sequences of the CYP4 family was also observed, suggesting a pattern of xenobiotic responsiveness more similar to those of several vertebrate drug-metabolizing enzymes than to putative vertebrate CYP4 homologs.     

Structural organization and classification of cytochrome P450 genes in flax (Linum usitatissimum L.)

Flax CYPome analysis resulted in the identification of 334 putative cytochrome P450 (CYP450) genes in the cultivated flax genome. Classification of flax CYP450 genes based on the sequence similarity with Arabidopsis orthologs and CYP450 nomenclature, revealed 10 clans representing 44 families and 98 subfamilies. CYP80, CYP83, CYP92, CYP702, CYP705, CYP708, CYP728, CYP729, CYP733 and CYP736 families are absent in the flax genome. The subfamily members exhibited conserved sequences, length of exons and phasing of introns. Similarity search of the genomic resources of wild flax species Linum bienne with CYP450 coding sequences of the cultivated flax, revealed the presence of 127 CYP450 gene orthologs, indicating amplification of novel CYP450 genes in the cultivated flax. Seven families CYP73, 74, 75, 76, 77, 84 and 709, coding for enzymes associated with phenylpropanoid/fatty acid metabolism, showed extensive gene amplification in the flax. About 59% of the flax CYP450 genes were present in the EST libraries.     

The transformation of 10α-cucurbita-5,24-dien-3β-ol into cucurbitacin C by seedlings of Cucumis sativus

Labelled 10α-cucurbita-5,24-dien-3β-ol, the simplest tetracyclic triterpene with a cucurbitane skeleton, was transformed into cucurbitacin C in Cucumis sativus seedlings. This transformation has been previously postulated, but this is the first time it has been demonstrated to operate in plant tissues. Two other potential precursors of cucurbitacins, cycloartenol and parkeol, were incubated under the same conditions. Cycloartenol gave only the expected phytosterols whereas parkeol was recovered unchanged.     

Identification of triterpene biosynthetic genes from Momordica charantia using RNA-seq analysis

Cucurbitaceae plants contain characteristic triterpenoids. Momordica charantia, known as a bitter melon, contains cucurbitacins and multiflorane type triterpenes, which confer bitter tasting and exhibit pharmacological activities. Their carbon skeletons are biosynthesized from 2,3-oxidosqualene by responsible oxidosqualene cyclase (OSC). In order to identify OSCs in M. charantia, RNA-seq analysis was carried out from ten different tissues. The functional analysis of the resulting four OSC genes revealed that they were cucurbitadienol synthase (McCBS), isomultiflorenol synthase (McIMS), β-amyrin synthase (McBAS) and cycloartenol synthase (McCAS), respectively. Their distinct expression patterns based on RPKM values and quantitative RT-PCR suggested how the characteristic triterpenoids were biosynthesized in each tissue. Although cucurbitacins were finally accumulated in fruits, McCBS showed highest expression in leaves indicating that the early step of cucurbitacins biosynthesis takes place in leaves, but not in fruits.

Abbreviations: OSC: oxidosqualene cyclase; RPKM: reads perkilobase of exon per million mapped reads     

Effects of age, sex, and dietary history on response to cucurbitacin in Acalymma vittatum

The chrysomelid Acalymma vittatum is stenophagous, subsisting almost entirely on plants in the Cucurbitaceae, which generally contain cucurbitacins. Cucurbitacins are extremely bitter tetracyclic triterpenoids that are toxic to most organisms. As do other diabroticite beetles, A. vittatum sequester cucurbitacins, which have been shown to act as phagostimulants and arrestants. Our results reveal, however, that for A. vittatum the response to cucurbitacin diminishes with continued sequestration. Colony-reared A. vittatum were fed only roots (as larvae) and foliage of either `Marketmore 76' (which contains a normal amount of cucurbitacin, `bitter') or `Marketmore 80' (a near isogenic line that contains no cucurbitacin, `non-bitter') cucumber. Over 1200 individual beetles from the day of adult emergence to 15 days following emergence were placed in choice and no-choice arenas containing potted cotyledons of the two cucumber varieties for 24 h. In choice tests, overall preference for the bitter cucumber cultivar was maintained, but degree of preference changed with age and became significantly less for beetles reared on bitter diets. Furthermore, in no-choice tests, age, sex, dietary history, and interactions among these variables all significantly affected the feeding response to cucurbitacin. For A. vittatum reared without cucurbitacin, total consumption of the bitter cultivar increased over time. For beetles reared with cucurbitacin, total foliage consumption of the bitter cultivar declined, within nine days, to equal that of the non-bitter cultivar. Feral A. vittatum, unexpectedly, consumed more of the non-bitter than the bitter cultivar in no-choice tests. Ecological and applied implications of this variation in response to cucurbitacin are discussed.     

Production of Bioactive Diterpenoids in the Euphorbiaceae Depends on Evolutionarily Conserved Gene Clusters

The Euphorbiaceae produce a diverse range of diterpenoids, many of which have pharmacological activities. These diterpenoids include ingenol mebutate, which is licensed for the treatment of a precancerous skin condition (actinic keratosis), and phorbol derivatives such as resiniferatoxin and prostratin, which are undergoing investigation for the treatment of severe pain and HIV, respectively. Despite the interest in these diterpenoids, their biosynthesis is poorly understood at present, with the only characterized step being the conversion of geranylgeranyl pyrophosphate into casbene. Here, we report a physical cluster of diterpenoid biosynthetic genes from castor (Ricinus communis), including casbene synthases and cytochrome P450s from the CYP726A subfamily. CYP726A14, CYP726A17, and CYP726A18 were able to catalyze 5-oxidation of casbene, a conserved oxidation step in the biosynthesis of this family of medicinally important diterpenoids. CYP726A16 catalyzed 7,8-epoxidation of 5-keto-casbene and CYP726A15 catalyzed 5-oxidation of neocembrene. Evidence of similar gene clustering was also found in two other Euphorbiaceae, including Euphorbia peplus, the source organism of ingenol mebutate. These results demonstrate conservation of gene clusters at the higher taxonomic level of the plant family and that this phenomenon could prove useful in further elucidating diterpenoid biosynthetic pathways.     

Structural organization,classification and phylogenetic relationship of cytochrome P450 genes in Citrus clementina and Citrus sinensis

The genus Citrus is an important fruit crop and nutritional source for the good health of humans. Cytochrome P450s represent about 1 % of the proteome and mediate diverse biochemical reactions pertaining to both primary and secondary metabolism. Analysis of Citrus genomic resources identified 296 plant cytochrome P450s (CYP) coding genes in Citrus clementina, 272 in double haploid (dh) Citrus sinensis, and 202 in C. sinensis. In C. clementina and dh C. sinensis, CYP genes are distributed into nine clans. In the three genomes, single intron containing CYP genes are predominant in the A-type families. Among non-A-type CYP families, multiple intron containing genes are predominant. More number of genes in CYP A-type families over non-A-type families is attributed to rapid evolution of A-type genes facilitated by their gene organization. Further, complex gene organization of non-A-type genes with the presence of multiple introns might have contributed to the slower evolvement of paralogs. Majority of introns (1,660) from three genomes showed canonical GT-AG splice sites. However, 33 introns showed non-conventional GC… PyAG splice sites and functionality of these splice sites is confirmed by the ESTs lacking this intron. Across the families, gene organization is conserved between the three genomes. In dh C. sinensis, 22 genes were identified to have alternate splicing. Examination of scaffolds in C. clementina revealed that majority of the Citrus CYP genes are solitary and a few of them are in clusters of 3–8 genes. PCR amplification of C. sinensis genomic DNA with gene-specific primers failed to amplify out-grouped genes Ccl-CYP706A16 and Ccl-CYP706B1, confirming that they are specific to C. clementina. Differential number of CYP genes observed between C. clementina and C. sinensis is attributed to the extent of variability between their parents representing ancestral taxa.     

Comparison of Cytochrome P450 Genes from Six Plant Genomes

Plants depend on cytochrome P450 (CYP) enzymes for nearly every aspect of their biology. In several sequenced angiosperms, CYP genes constitute up to 1% of the protein coding genes. The angiosperm sequence diversity is encapsulated by 59 CYP families, of which 52 families form a widely distributed core set. In the 20 years since the first plant P450 was sequenced, 3,387 P450 sequences have been identified and annotated in plant databases. As no new angiosperm CYP families have been discovered since 2004, it is now apparent that the sampling of CYP diversity is beginning to plateau. This review presents a comparison of 1,415 cytochrome P450 sequences from the six sequenced genomes of Vitis vinifera (grape), Carica papaya (papaya), Populus trichocarpa (poplar), Oryza sativa (rice), Arabidopsis thaliana (Arabidopsis or mouse ear’s cress) and Physcomitrella patens (moss). An evolutionary analysis is presented that tracks land plant P450 innovation over time from the most ancient and conserved sequences to the newest dicot-specific families. The earliest or oldest P450 families are devoted to the essential biochemistries of sterol and carotenoid synthesis. The next evolutionary radiation of P450 families appears to mediate crucial adaptations to a land environment. And, the newest CYP families appear to have driven the diversity of angiosperms in mediating the synthesis of pigments, odorants, flavors and order-/genus-specific secondary metabolites. Family-by-family comparisons allow the visualization of plant genome plasticity by whole genome duplications and massive gene family expansions via tandem duplications. Molecular evidence of human domestication is quite apparent in the repeated P450 gene duplications occurring in the grape genome.     

Human cytochromes P450 in health and disease

There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases—confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development.     

Cytochrome P450 Genes from the Sacred Lotus Genome

Cytochrome P450 monooxygenases (P450s) in the sacred lotus (Nelumbo nucifera) genome have been identified and named according to systematic P450 nomenclatures. Comparisons of these sequences with those in the papaya and grape CYPomes have indicated that gene blooms exist in the CYP89, CYP94, CYP96 and CYP714 families and that less dramatic expansions exist in the CYP71 and CYP72 families. Expansions in the CYP94 and CYP96 families may be associated with generation of the extremely hydrophobic leaf surfaces associated with the “lotus effect” in this water-adapted species, since these families are known to hydroxylate fatty acids and alkanes in the wax biosynthetic pathways of other plant species. Evolution of the CYP719 and CYP80 families may be associated with production of a number of benzylisoquinoline and aporphine alkaloids. Structures for anonaine and roemerine, two of the most abundant aporphine alkaloids in lotus leaves and seeds, contain methylenedioxy bridges that are known to be generated by members of the CYP719 family. With only one CYP719A22 gene existing in the lotus genome, it is likely that it is involved in making aporphine alkaloids. The fact that CYP719 has not previously been seen in angiosperm phylogeny below the order of Ranunculales suggests that its presence in lotus (in the Proteales) presents an evolutionary terminus prior to its loss in more recent eudicot species. With several CYP80 family genes existing in the lotus genome, there are multiple candidates for those involved in conducting benzylisoquinoline alkaloid synthesis.     

CYP86B1 Is Required for Very Long Chain ω-Hydroxyacid and α,ω-Dicarboxylic Acid Synthesis in Root and Seed Suberin Polyester

Suberin composition of various plants including Arabidopsis (Arabidopsis thaliana) has shown the presence of very long chain fatty acid derivatives C20 in addition to the C16 and C18 series. Phylogenetic studies and plant genome mining have led to the identification of putative aliphatic hydroxylases belonging to the CYP86B subfamily of cytochrome P450 monooxygenases. In Arabidopsis, this subfamily is represented by CYP86B1 and CYP86B2, which share about 45% identity with CYP86A1, a fatty acid ω-hydroxylase implicated in root suberin monomer synthesis. Here, we show that CYP86B1 is located to the endoplasmic reticulum and is highly expressed in roots. Indeed, CYP86B1 promoter-driven β-glucuronidase expression indicated strong reporter activities at known sites of suberin production such as the endodermis. These observations, together with the fact that proteins of the CYP86B type are widespread among plant species, suggested a role of CYP86B1 in suberin biogenesis. To investigate the involvement of CYP86B1 in suberin biogenesis, we characterized an allelic series of cyp86B1 mutants of which two strong alleles were knockouts and two weak ones were RNA interference-silenced lines. These root aliphatic plant hydroxylase lines had a root and a seed coat aliphatic polyester composition in which C22- and C24-hydroxyacids and α,ω-dicarboxylic acids were strongly reduced. However, these changes did not affect seed coat permeability and ion content in leaves. The presumed precursors, C22 and C24 fatty acids, accumulated in the suberin polyester. These results demonstrate that CYP86B1 is a very long chain fatty acid hydroxylase specifically involved in polyester monomer biosynthesis during the course of plant development.