Functional Characterization of Monoterpenoid Indole Alkaloid (MIA) Biosynthetic Genes in Catharanthus roseus

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) are known to be among the most important source of natural drugs used in various cancer chemotherapies. MIAs are derived by combining the iridoid secologanin with tryptamine to form the central precursor strictosidine that is then converted to most known MIAs, such as catharanthine and vindoline that dimerize to form anticancer vinblastine and vincristine. While their assembly is still poorly understood, the complex multistep pathways involved occur in several specialized cell types within leaves that are regulated by developmental and environmental cues. The organization of MIA pathways is also coupled to secretory mechanisms that allow the accumulation of catharanthine in the waxy leaf surface, separated from vindoline found within leaf cells. While the spatial separation of catharanthine and vindoline provides an explanation for the low levels of dimeric MIAs found in the plants, the secretion of catharanthine to the leaf surface is shown to be part of plant defense mechanisms against fungal infection and insect herbivores. The transcriptomic databases of Catharanthus roseus and various MIA producing plants are facilitating bioinformatic approaches to identify novel MIA biosynthetic genes. Virus-induced gene silencing (VIGS) is being used to screen these candidate genes for their involvement in iridoid biosynthesis pathway, especially in the identification of 7-deoxyloganic acid 7-hydroxylase (CrDL7H) shown by the accumulation of its substrate, 7-deoxyloganic acid and decreased level of secologanin along with catharanthine and vindoline. VIGS can also confirm the biochemical function of genes being identified, such as in the glucosylation of 7-deoxyloganetic acid by CrUGT8 shown by decreased level of secologanin and MIAs within silenced plants. Silencing of other iridoid biosynthetic genes, loganic acid O-methyltransferase (LAMT) and secologanin synthase (SLS) also confirm the metabolic route for iridoid biosynthesis in planta through 7-deoxyloganic acid, loganic acid, and loganin intermediates. This route is validated by high substrate specificity of CrUGT8 for 7-deoxyloganetic acid and CrDL7H for 7-deoxyloganic acid. Further localization studies of CrUGT8 and CrDL7H also show that these genes are preferentially expressed within Catharanthus leaves rather than in epidermal cells where the last two steps of secologanin biosynthesis occur.

Identification and characterization of a Catharanthus roseus mutant altered in monoterpenoid indole alkaloid biosynthesis

The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is the only source for several pharmaceutically valuable monoterpenoid indole alkaloids (MIAs), including the powerful antihypertensive ajmalicine and the antineoplastic agents vincristine and vinblastine. While biosynthesis of MIA precursors has been elucidated, conversion of the common MIA precursor strictosidine to MIAs of different families, for example ajmalicine, catharanthine or vindoline, remains uncharacterized. Deglycosylation of strictosidine by the key enzyme Strictosidine beta-glucosidase (SGD) leads to a pool of uncharacterized reaction products that are diverted into the different MIA families, but the downstream reactions are uncharacterized. Screening of 3600 EMS (ethyl methane sulfonate) mutagenized C. roseus plants to identify mutants with altered MIA profiles yielded one plant with high ajmalicine, and low catharanthine and vindoline content. RNA sequencing and comparative bioinformatics of mutant and wildtype plants showed up-regulation of SGD and the transcriptional repressor Zinc finger Catharanthus transcription factor (ZCT1) in the mutant line. The increased SGD activity in mutants seems to yield a larger pool of uncharacterized SGD reaction products that are channeled away from catharanthine and vindoline towards biosynthesis of ajmalicine when compared to the wildtype. Further bioinformatic analyses, and crossings between mutant and wildtype suggest a transcription factor upstream of SGD and ZCT1 to be mutated, leading to up-regulation of Sgd and Zct1. The crossing experiments further show that biosynthesis of the different MIA families is differentially regulated and highly complex. Three new transcription factors were identified by bioinformatics that seem to be involved in the regulation of Zct1 and Sgd expression, leading to the high ajmalicine phenotype. Increased cathenamine reductase activity in the mutant converts the pool of SGD reaction products into ajmalicine and its stereoisomer tetrahydroalstonine. The stereochemistry of ajmalicine and tetrahydroalstonine biosynthesis in vivo and in vitro was further characterized. In addition, a new clade of perakine reductase-like enzymes was identified that reduces the SGD reaction product vallesiachotamine in a stereo-specific manner, characterizing one of the many reactions immediately downstream of SGD that determine the different MIA families. This study establishes that RNA sequencing and comparative bioinformatics, in combination with molecular and biochemical characterization, are valuable tools to determine the genetic basis for mutations that trigger phenotypes, and this approach can also be used for identification of new enzymes and transcription factors.

Chemical, biochemical, and molecular characterization of a low vindoline Catharanthus roseus mutant.

The Madagascar periwinkle (Catharanthus roseus) is the sole source of the anticancer drug vinblastine, which is formed via the coupling of monoterpenoid indole alkaloids (MIAs) catharanthine and vindoline. A mutant line of C. roseus (M2-1865) with an altered MIA profile was identified in a screen of 4000 M2 lines generated by ethylmethanesulfonate (EMS) chemical mutagenesis. While this line did not accumulate vinblastine due to reduced levels of vindoline within the leaves, significant levels of 2,3-epoxide derivatives of tabersonine accumulated on the leaf surface. Detailed nucleotide, amino acid, and enzyme activity analyses of tabersonine 3-reductase in the M2-1865 line showed that a single amino acid substitution (H189Y) diminished the biochemical activity of T3R by 95%. Genetic crosses showed the phenotype to be recessive, exhibiting standard Mendelian single-gene inheritance. The usefulness of EMS mutagenesis in elucidating MIA biosynthesis is highlighted by the results of this study.

Power outage, and other poems

Contents - At the Starry Night Cafe AIbino Buffalo Monteverdi In The Rain Lust Litter Poem After The Chinese Cleere's Pub Treasure Island Nipple Periwinkle Talkeetna Beached Whale Spending Christmas in the land of Enchantment Easter Poem For Emily Dickinson Second Hand Gifts The Power Outage October In The Observation Car Westerly

Growth optimization procedures for the phytopathogen Xylella fastidiosa

For the first time, growth curves are shown for the phytopathogen Xylella fastidiosa on traditional growth media such as PW (periwinkle wilt), BCYE (buffered charcoal yeast extract), and on new ones such as GYE (glutamate yeast extract) and PYE (phosphate yeast extract) that were developed in this work. The optimal growth conditions on solid and liquid media as well as their measurements are presented, by using total protein content and turbidity determinations. The results demonstrated that yeast extract provided sufficient nutrients for X. fastidiosa, since the cells grew well on PYE medium.

Development of a scar marker for Pierce's Disease strains of Xylella fastidiosa

O objetivo deste trabalho foi desenvolver um oligonucleotídeo iniciador para reação em cadeia da polimerase (PCR) específico para as estirpes de Xylella fastidiosa que causam o mal de Pierce (PD) em videira (Vitis vinifera). Amplificações de DNA de 23 diferentes hospedeiros, usando o conjunto de oligonucleotídeos REP1-R (5'-IIIICGICGIATCCIGGC-3') e REP 2 (5'-ICGICTTATCI GGCCTAC-3') utilizando o programa: 94 ºC/2 min; 35 X (94 ºC/1 min, 45 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/5 min, produziu um fragmento de 630 pb que diferenciou as estirpes de videiras dos demais. Entretanto, padrões de bandeamento REP não são considerados confiáveis para detecção devido ao par de oligonucleotídeos REP 1 e REP 2 corresponderem a seqüências repetitivas encontradas por todo o genoma bacteriano. Desse modo, o produto amplificado de 630 pb foi eluído do gel de agarose, purificado e seqüenciado. A informação da seqüência nucleotídica foi usada para identificar e sintetizar um oligonucleotídeo específico para o isolado de X. fastidiosa causadora do mal de Pierce denominado Xf-1 (5'-CGGGGGTGTAGGAGGGGTTGT-3'), que foi utilizado juntamente com o oligonucleotídeo REP-2 nas condições 94 ºC/2 min; 35 X (94 ºC/1 min, 62 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/10 min. Os DNAs das estirpes de X. fastidiosa de outros hospedeiros [amêndoa (Prumus amygdalus), citros (Citrus spp.), café (Coffea arabica), olmo (Ulmus americana), amora (Morus rubra), carvalho (Quercus rubra), vinca (Catharantus roseus), ameixa (Prunus salicina) e ragweed (Ambrosia artemisiifolia)] e de bactérias Gram negativas e positivas foram submetidos a amplificação com o conjunto de oligonucleotídeos Xf-1/REP 2. Um fragmento, de aproximadamente 350 pb, foi amplificado apenas com o DNA de X. fastidiosa isolada de videira.

Strain variability in the DNA immigration control region (ICR) of Xylella fastidiosa

The genome of the bacterium Xylella fastidiosa contains four ORFs (XF2721, XF2725, XF2739 and XF0295) related to the restriction modification type I system, ordinarily named R-M. This system belongs to the DNA immigration control region (ICR). Each CIRF is related to different operon structures, which are homologues among themselves and with subunit Hsd R from the endonuclease coding genes. In addition, these ORFs are highly homologous to genes in Pseudomonas aeruginosa, Methylococcus capsulatus str. Bath, Legionella pneumophila, Helicobacter pylori, Xanthomonas oryzae pv. Oryzae and Silicibacter pomeroyi, as well as to genes from X. fastidiosa strains that infect grapevine, almond and oleander plants. This study was carried out on R-M ORFs from forty-three X. fastidiosa strains isolated from citrus, coffee, grapevine, periwinkle, almond and plum trees, in order to assess the genetic diversity of these loci through PCR-RFLP. PCR-RFLP analysis of the four ORFs related to the R-M system from these strains enabled the detection of haplotypes for these loci. When the haplotypes were defined, wide genetic diversity and a large range of similar strains originating from different hosts were observed. This analysis also provided information indicating differences in population genetic structures, which led to detection of different levels of gene transfer among the groups of strains. (c) 2005 Elsevier SAS. All rights reserved.

Comparative analyses of the complete genome sequences of Pierce's disease and citrus variegated chlorosis strains of Xylella fastidiosa

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X.fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X.fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X.fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X.fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.

Coffee leaf scorch caused by a strain of Xylella fastidiosa from citrus

Citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS) are two economically important diseases in Brazil caused by the bacterium Xylella fastidiosa. Strains of the bacterium isolated from the two plant hosts are very closely related, and the two diseases share sharpshooter insect vectors. In order to determine if citrus strains of X. fastidiosa could infect coffee and induce CLS disease, plant inoculations were performed. Plants of coffee, Coffea arabica 'Mundo Novo', grafted on Coffea canephora var, robusta 'Apuatao 2258' were mechanically inoculated with triply cloned strains of X. fastidiosa isolated from diseased coffee and citrus. Three months postinoculation, 5 of the 10 plants inoculated with CLS-X. fastidiosa and 1 of the 10 plants inoculated with CVC-X. fastidiosa gave positive enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR). Eight months postinoculation, another six plants inoculated with CVC-X. fastidiosa gave positive PCR results. The two X. fastidiosa strains were isolated from the inoculated plants and showed the same characteristics as the original clones by microscopy, ELISA, and PCR. None of the plants inoculated with sterile periwinkle wilt (PW) medium as controls gave positive reactions in diagnostic tests, and none developed disease symptoms. Six months postinoculation, seven plants inoculated with CLS-X. fastidiosn and eight inoculated with CVC-X. fastidiosa began to develop characteristic CLS symptoms, including apical and marginal leaf scorch, defoliation, and reductions of internode length, leaf size, and plant height, terminal clusters of small chlorotic and deformed leaves, and lateral shoot dieback. We have demonstrated that X, fastidiosa from citrus plants is pathogenic for coffee plants. This has important consequences for the management of CLS disease and has implications for the origin of citrus variegated chlorosis disease.

Análise filogenética de linhagens de Xylella fastidiosa de diferentes hospedeiros baseada na sequencia da região intergênica 16S-23S rDNA

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Identification and genetic diversity in phytoplasmas associated with diseases of cassava and other agronomic relevant crops in south-east Asia and Latin America

Identification and genetic diversity of phytoplasmas infecting tropical plant species, selected among those most agronomically relevant in South-east Asia and Latin America were studied. Correlation between evolutionary divergence of relevant phytoplasma strains and their geographic distribution by comparison on homologous genes of phytoplasma strains detected in the same or related plant species in other geographical areas worldwide was achieved. Molecular diversity was studied on genes coding ribosomal proteins, groEL, tuf and amp besides phytoplasma 16S rRNA. Selected samples infected by phytoplasmas belonging to diverse ribosomal groups were also studied by in silico RFLP followed by phylogenetic analyses. Moreover a partial genome annotation of a ‘Ca. P. brasiliense’ strain was done towards future application for epidemiological studies. Phytoplasma presence in cassava showing frog skin (CFSD) and witches’ broom (CWB) diseases in Costa Rica - Paraguay and in Vietnam – Thailand, respectively, was evaluated. In both cases, the diseases were associated with phytoplasmas related to aster yellows, apple proliferation and “stolbur” groups, while only phytoplasma related to X-disease group in CFSD, and to hibiscus witches’ broom, elm yellows and clover proliferation groups in CWB. Variability was found among strains belonging to the same ribosomal group but having different geographic origin and associated with different disease. Additionally, a dodder transmission assay to elucidate the role of phytoplasmas in CWB disease was carried out, and resulted in typical phytoplasma symptoms in periwinkle plants associated with the presence of aster yellows-related strains. Lethal wilt disease, a severe disease of oil palm in Colombia that is spreading throughout South America was also studied. Phytoplasmas were detected in symptomatic oil palm and identified as ‘Ca. P. asteris’, ribosomal subgroup 16SrI-B, and were distinguished from other aster yellows phytoplasmas used as reference strains; in particular, from an aster yellows strain infecting corn in the same country.

Experimental evaluation of seaweeds as a vector for microplastics into marine food webs, supplementary material

The ingestion of microplastics has been shown for a great variety of marine organisms. However, benthic marine mesoherbivores such as the common periwinkle Littorina littorea have been largely disregarded in studies about the effects of microplastics on the marine biota, probably because the pathway for microplastics to this functional group of organisms was not obvious. In laboratory experiments we showed that the seaweed Fucus vesiculosus retains suspended microplastics on its surface. The numbers of microplastics that adhered to the algae correlated with the concentrations of suspended particles in the water. In choice feeding assays L. littorea did not distinguish between algae with adherent microplastics and clean algae without microplastics, indicating that the snails do not recognize solid nonfood particles in the submillimeter size range as deleterious. In periwinkles that were feeding on contaminated algae, microplastics were found in the stomach and in the gut. However, no microplastics were found in the midgut gland, which is the principle digestive organ of gastropods. Microplastics in the fecal pellets of the periwinkles indicate that the particles do not accumulate rapidly inside the animals but are mostly released with the feces. Our results provide the first evidence that seaweeds may represent an efficient pathway for microplastics from the water to marine benthic herbivores.

Wave-induced changes in seaweed toughness entail plastic modifications in snail traits maintaining consumption efficacy, link to supplementary material

1. Environmental stress can influence species traits and performance considerably. Using a seaweed-snail system from NW (Nova Scotia) and NE (Helgoland) Atlantic rocky shores, we examined how physical stress (wave exposure) modulates traits in the seaweed Fucus vesiculosus and indirectly in its main consumer, the periwinkle Littorina obtusata. 2. In both regions, algal tissue toughness increased with wave exposure. Reciprocal-transplant experiments showed that tissue toughness adjusts plastically to the prevailing level of wave exposure. 3. Choice experiments tested the feeding preference of snails from sheltered, exposed, and very exposed habitats for algae from such wave exposures. Snails from exposed and very exposed habitats consumed algal tissues at similar rates irrespective of the exposure of origin of the algae. However, snails from sheltered habitats consumed less algal tissues from very exposed habitats than tissues from sheltered and exposed habitats. Choice assays using reconstituted algal food (triturated during preparation) identified high thallus toughness as the explanation for the low preference of snails from sheltered habitats for algae from very exposed habitats. 4. Ultrastructural analyses of radulae indicated that rachidian teeth were longest and the number of cusps in lateral teeth (grazing-relevant traits) was highest in snails from very exposed habitats, suggesting that radulae are best suited to rupture tough algal tissues in such snails. 5. No-choice feeding experiments revealed that these radular traits are also phenotypically plastic, as they adjust to the toughness of the algal food. 6. Synthesis. This study indicates that the observed plasticity in the feeding ability of snails is mediated by wave exposure through phenotypic plasticity in the tissue toughness of algae. Thus, plasticity in consumers and their resource species may reduce the potential effects of physical stress on their interaction.

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.