Characterization and functional identification of a novel plant extradiol 4,5-dioxygenase involved in betalain pigment biosynthesis in Portulaca Grandiflora

RESUME Les bétalaïnes sont des pigments chromo-alcaloïdes violets et jaunes présents dans les plantes appartenant à l'ordre des Caryophyllales et dans les champignons des genres Amanita et Hygrocybe. Leur courte voie de biosynthèse est élucidée chimiquement depuis de nombreuses années, mais les enzymes impliquées dans cette biosynthèse chez les plantes ne sont toujours pas caractérisées. L'enzyme de la DOPA-dioxygénase d' Amanita muscaria a été identifiée (Girod et Zryd, 1991a), mais de nombreuses tentatives d'isolation d'un homologue chez les plantes ont échoué. Afin d'isoler les gènes spécifiques des bétalaïnes chez les plantes, nous avons construit des banques soustraites d'ADNc à partir d'ARN total de pétales immatures de Portulaca grandiflora (Pg) de génotypes jaunes et blancs, respectivement violets et blancs. Les clones couleur- spécifiques ont été détectés en premier par analyse Northem du RNA de pétales blancs et colorés. Les candidats positifs ont alors été soumis à une analyse de transcription au niveau des tiges colorées, vertes et des feuilles, afin d'établir leur expression spécifique. Deux ARNs messagers complets ont une expression corrélée avec l'accumulation des bétalaïnes dans les tissus. Le premier de ces clones, A.16, code pour une oxydase de l'acyl-Coenzyme A (ACX) putative, mais le domaine de liaison du FAD essentiel pour l'activité d'ACX est absent. Toutes nos tentatives pour démontrer sa fonction ont échoué. Le rôle de cette protéine dans la voie de synthèse des bétalaïnes reste inconnu. Le deuxième de ces clones spécifique aux bétalaïnes, L.6 (isolé par Zaiko, 2000), a été renommé DODA en raison de son homologie avec le domaine LigB (pfam02900) d'une 4,5-dioxygénase extradiol bactérienne. DODA a été identifié in silico comme une dioxygénase extradiol en raison de la conservation stricte, au niveau de sa séquence peptidique, des résidus catalytiques de LigB et de ceux liant le cofacteur fer. Une analyse de transfert Southem a montré que ce gène est unique dans Pg. L'expression transitoire de DODA par transformation biolistique dans des pétales blancs de Pg a produit des taches violettes ou jaunes dans des cellules transformées. Une analyse HPLC de ces taches a démontré leur identité avec les bétalaïnes présentes naturellement dans les pétales violets et jaunes de Pg, confirmant ainsi la complémentation par le gène Pg DODA de l'allèle récessif cc présent dans les pétales blancs de Pg. Des homologues de DODA (DOPA-dioxygénase) ont été identifiés dans de nombreuses espèces de plantes, y compris dans celles sans bétalaïne. L'alignement de ces homologues a permis l'identification d'un motif spécifique aux bétalaïnes à côté d'une histidine catalytique conservée. Ce motif [H-P-(S,A)-(N,D)-x-T-P] remplace le motif [H-N-L-R] conservé dans les plantes sans bétalaïne et le motif [H-N-L-x] présent dans tous les homologues bactériens et archaebactériens. Une modélisation tridimensionnelle préliminaire du site actif de Pg DODA et de son homologue dans la mousse Physcomitrella patens a montré l'importance de ce motif spécifique aux bétalaïnes pour l'accessibilité du substrat au site actif. L'analyse phylogénétique de DODA a confirmé l'évolution séparée de cette protéine chez les plantes à bétalaïnes par comparaison avec celle des plantes sans bétalaïne. Nous avons donc conclu que les bétalaïnes sont apparues par modification de l'affinité pour un substrat d'enzymes similaires à DODA, chez un ancêtre unique des Caryophyllales qui a perdu toute capacité de biosynthèse des anthocyanes. Finalement, Pg DODA n'a aucune similarité avec la protéine DODA d' Amanita muscaria, bien que celle-ci complémente aussi la pigmentation des pétales blancs de Pg. La biosynthèse des bétalaïnes est un exemple remarquable de convergence évolutive biochimique indépendante entre espèces de règnes différents. ABSTRACT Betalains are violet and yellow chromo-alkaloid pigments present in plants belonging to the order Caryophyllales and also in the fungal genera Amanita and Hygrocybe. Their short biosynthetic pathway is chemically well understood since many years, but enzymes involved in the plant pathway are still uncharacterized. The DOPA-dioxygenase from Amanita muscaria was identified (Girod and Zryd, 1991a), but numerous attempts to identify a plant homologue to the corresponding gene, failed. In order to isolate betalain-specific genes in plants, subtractive cDNA libraries were built with total RNA from white and yellow and respectively, violet immature petals from Portulaca grandiflora (Pg) genotypes. Colour-specific clones were first detected by Northern blot analysis using RNA from white and coloured petals. Positive candidates were submitted to further transcription analysis in coloured, green stems and leaves in order to assess their specific expression. Two full-length mRNAs showed a correlated expression with betalain accumulation in tissues. One of them, A.16, encodes a putative acyl-Coenzyme A oxidase (ACX), but missing the FAD binding domain essential for the ACX activity. Thus, all attempts to demonstrate its function failed. The role of this protein in the betalain biosynthesis pathway, if any, is still unknown. The second betalain-specific mRNA, L.6 (isolated by Zaiko, 2000) shows a homology with a LigB domain (pfam02900) from a bacterial extradiol 4,5-dioxygenase. It was then renamed DODA (DOPA-dioxygenase). DODA was identified in silico as a highly conserved extradiol dioxygenase due to the strict conservation of its peptidic sequence with LigB catalytic residues and iron-binding cofactor residues. Southern blot analysis showed that this gene is a single copy-gene in Pg. Transient expression of DODA protein through biolistic transformation of Pg white petals produced violet or yellow spots in individual cells. HPLC analysis of these spots showed an identity with betalain pigments present naturally in yellow and violet Pg petals, thus confirming the complementation of the recessive cc allele present in Pg white petals by Pg DODA gene. DODA homologues were identified in numerous plant species including those without betalain. Alignment of these homologues allowed the identification of a betalain-specific pattern beside a highly conserved catalytic histidine. This [H-P-(S,A)-(N,D)-x-T-P] pattern replaces a [H-N-L-R] pattern strictly conserved in non-betalain plants and a [H-N-L-x] pattern present in all bacterial and archaebacterial homologues. Preliminary three-dimensional modeling of the active site of Pg DODA and its Physcomitrella patens moss homologue revealed the importance of this betalain-specific pattern for the substrate accessibility to the DODA active site. DODA phylogenetic analysis confirmed the separate evolution of this protein in betalain-producing plants. We conclude that betalain pigments appeared in a unique ancestor of the Caryophyllales order in which anthocyanin biosynthetic pathway was impaired, by a modification of enzymes of the DODA family for substrate affinity. The Pg DODA protein has no sequence similarity with Amanita muscaria DODA, despite the fact that they both complement Pg white petals for their pigmentation. Betalain biosynthesis is an interesting example of independent biochemical evolutionary convergence between species from different kingdoms.

Relative humidity changes in a warmer climate

Key climate feedbacks due to water vapor and clouds rest largely on how relative humidity R changes in a warmer climate, yet this has not been extensively analyzed in models. General circulation models (GCMs) from the CMIP3 archive and several higher resolution atmospheric GCMs examined here generally predict a characteristic pattern of R trend with global temperature that has been reported previously in individual models, including increase around the tropopause, decrease in the tropical upper troposphere, and decrease in midlatitudes. This pattern is very similar to that previously reported for cloud cover in the same GCMs, confirming the role of R in controlling changes in simulated cloud. Comparing different models, the trend in each part of the troposphere is approximately proportional to the upward and/or poleward gradient of R in the present climate. While this suggests that the changes simply reflect a shift of the R pattern upward with the tropopause and poleward with the zonal jets, the drying trend in the subtropics is roughly three times too large to be attributable to shifts of subtropical features, and the subtropical R minima deepen in most models. R trends are correlated with horizontal model resolution, especially outside the tropics, where they show signs of convergence and latitudinal gradients become close to available observations for GCM resolutions near T85 and higher. We argue that much of the systematic change in R can be explained by the local specific humidity having been set (by condensation) in remote regions with different temperature changes, hence the gradients and trends each depend on a model’s ability to resolve moisture transport. Finally, subtropical drying trends predicted from the warming alone fall well short of those observed in recent decades. While this discrepancy supports previous reports of GCMs underestimating Hadley Cell expansion, our results imply that shifts alone are not a sufficient interpretation of changes.

The Role Of Edaphic Environment And Climate In Structuring Phylogenetic Pattern In Seasonally Dry Tropical Plant Communities.

Seasonally dry tropical plant formations (SDTF) are likely to exhibit phylogenetic clustering owing to niche conservatism driven by a strong environmental filter (water stress), but heterogeneous edaphic environments and life histories may result in heterogeneity in degree of phylogenetic clustering. We investigated phylogenetic patterns across ecological gradients related to water availability (edaphic environment and climate) in the Caatinga, a SDTF in Brazil. Caatinga is characterized by semiarid climate and three distinct edaphic environments - sedimentary, crystalline, and inselberg -representing a decreasing gradient in soil water availability. We used two measures of phylogenetic diversity: Net Relatedness Index based on the entire phylogeny among species present in a site, reflecting long-term diversification; and Nearest Taxon Index based on the tips of the phylogeny, reflecting more recent diversification. We also evaluated woody species in contrast to herbaceous species. The main climatic variable influencing phylogenetic pattern was precipitation in the driest quarter, particularly for herbaceous species, suggesting that environmental filtering related to minimal periods of precipitation is an important driver of Caatinga biodiversity, as one might expect for a SDTF. Woody species tended to show phylogenetic clustering whereas herbaceous species tended towards phylogenetic overdispersion. We also found phylogenetic clustering in two edaphic environments (sedimentary and crystalline) in contrast to phylogenetic overdispersion in the third (inselberg). We conclude that while niche conservatism is evident in phylogenetic clustering in the Caatinga, this is not a universal pattern likely due to heterogeneity in the degree of realized environmental filtering across edaphic environments. Thus, SDTF, in spite of a strong shared environmental filter, are potentially heterogeneous in phylogenetic structuring. Our results support the need for scientifically informed conservation strategies in the Caatinga and other SDTF regions that have not previously been prioritized for conservation in order to take into account this heterogeneity.

Modeling the skin pattern of fishes

Complicated patterns showing various spatial scales have been obtained in the past by coupling Turing systems in such a way that the scales of the independent systems resonate. This produces superimposed patterns with different length scales. Here we propose a model consisting of two identical reaction-diffusion systems coupled together in such a way that one of them produces a simple Turing pattern of spots or stripes, and the other traveling wave fronts that eventually become stationary. The basic idea is to assume that one of the systems becomes fixed after some time and serves as a source of morphogens for the other system. This mechanism produces patterns very similar to the pigmentation patterns observed in different species of stingrays and other fishes. The biological mechanisms that support the realization of this model are discussed.

Suppression Pattern of Neutral Pions at High Transverse Momentum in Au plus Au Collisions at root S(NN)=200 GeV and Constraints on Medium Transport Coefficients

For Au + Au collisions at 200 GeV, we measure neutral pion production with good statistics for transverse momentum, p(T), up to 20 GeV/c. A fivefold suppression is found, which is essentially constant for 5 < p(T) < 20 GeV/c. Experimental uncertainties are small enough to constrain any model-dependent parametrization for the transport coefficient of the medium, e. g., <(q) over cap > in the parton quenching model. The spectral shape is similar for all collision classes, and the suppression does not saturate in Au + Au collisions.

New prediction methods for CO2 evaporation inside tubes: Part I - A two-phase flow pattern map and a flow pattern based phenomenological model for two-phase flow frictional pressure drops

An updated flow pattern map was developed for CO2 on the basis of the previous Cheng-Ribatski-Wojtan-Thome CO2 flow pattern map [1,2] to extend the flow pattern map to a wider range of conditions. A new annular flow to dryout transition (A-D) and a new dryout to mist flow transition (D-M) were proposed here. In addition, a bubbly flow region which generally occurs at high mass velocities and low vapor qualities was added to the updated flow pattern map. The updated flow pattern map is applicable to a much wider range of conditions: tube diameters from 0.6 to 10 mm, mass velocities from 50 to 1500 kg/m(2) s, heat fluxes from 1.8 to 46 kW/m(2) and saturation temperatures from -28 to +25 degrees C (reduced pressures from 0.21 to 0.87). The updated flow pattern map was compared to independent experimental data of flow patterns for CO2 in the literature and it predicts the flow patterns well. Then, a database of CO2 two-phase flow pressure drop results from the literature was set up and the database was compared to the leading empirical pressure drop models: the correlations by Chisholm [3], Friedel [4], Gronnerud [5] and Muller-Steinhagen and Heck [6], a modified Chisholm correlation by Yoon et al. [7] and the flow pattern based model of Moreno Quiben and Thome [8-10]. None of these models was able to predict the CO2 pressure drop data well. Therefore, a new flow pattern based phenomenological model of two-phase flow frictional pressure drop for CO2 was developed by modifying the model of Moreno Quiben and Thome using the updated flow pattern map in this study and it predicts the CO2 pressure drop database quite well overall. (C) 2007 Elsevier Ltd. All rights reserved.

Resource use pattern analysis for predicting resource availability in opportunistic grids

This work presents a method for predicting resource availability in opportunistic grids by means of use pattern analysis (UPA), a technique based on non-supervised learning methods. This prediction method is based on the assumption of the existence of several classes of computational resource use patterns, which can be used to predict the resource availability. Trace-driven simulations validate this basic assumptions, which also provide the parameter settings for the accurate learning of resource use patterns. Experiments made with an implementation of the UPA method show the feasibility of its use in the scheduling of grid tasks with very little overhead. The experiments also demonstrate the method`s superiority over other predictive and non-predictive methods. An adaptative prediction method is suggested to deal with the lack of training data at initialization. Further adaptative behaviour is motivated by experiments which show that, in some special environments, reliable resource use patterns may not always be detected. Copyright (C) 2009 John Wiley & Sons, Ltd.

Spatial pattern of black spot incidence within citrus trees related to disease severity and pathogen dispersal

Guignardia citricarpa, the causal agent of citrus black spot, forms airborne ascospores on decomposing citrus leaves and water-spread conidia on fruits, leaves and twigs. The spatial pattern of diseased fruit in citrus tree canopies was used to assess the importance of ascospores and conidia in citrus black spot epidemics in Sao Paulo State, Brazil. The aggregation of diseased fruit in the citrus tree canopy was quantified by the binomial dispersion index (D) and the binary form of Taylor`s Power Law for 303 trees in six groves. D was significantly greater than 1 in 251 trees. The intercept of the regression line of Taylor`s Power Law was significantly greater than 0 and the slope was not different from 1, implying that diseased fruit was aggregated in the canopy independent of disease incidence. Disease incidence (p) and severity (S) were assessed in 2875 citrus trees. The incidence-severity relationship was described (R-2 = 88.7%) by the model ln(S) = ln(a) + bCLL(p) where CLL = complementary log-log transformation. The high severity at low incidence observed in many cases is also indicative of low distance spread of G. citricarpa spores. For the same level of disease incidence, some trees had most of the diseased fruit with many lesions and high disease severity, whereas other trees had most of the fruit with few lesions and low disease severity. Aggregation of diseased fruit in the trees suggests that splash-dispersed conidia have an important role in increasing the disease in citrus trees in Brazil.

Restoration of Pattern Recognition Receptor Costimulation to Treat Chromoblastomycosis, a Chronic Fungal Infection of the Skin

Chromoblastomycosis is a chronic skin infection caused by the fungus Fonsecaea pedrosoi. Exploring the reasons underlying the chronic nature of F. pedrosoi infection in a murine model of chromoblastomycosis, we find that chronicity develops due to a lack of pattern recognition receptor (PRR) costimulation. F. pedrosoi was recognized primarily by C-type lectin receptors (CLRs), but not by Toll-like receptors (TLRs), which resulted in the defective induction of proinflammatory cytokines. Inflammatory responses to F. pedrosoi could be reinstated by TLR costimulation, but also required the CLR Mincle and signaling via the Syk/CARD9 pathway. Importantly, exogenously administering TLR ligands helped clear F. pedrosoi infection in vivo. These results demonstrate how a failure in innate recognition can result in chronic infection, highlight the importance of coordinated PRR signaling, and provide proof of the principle that exogenously applied PRR agonists can be used therapeutically.

Interference pattern with a dark center in resonance fluorescence from two atoms driven by a squeezed vacuum field

We study the resonance fluorescence from two interacting atoms driven by a squeezed vacuum field and show that this system produces an interference pattern with a dark center. We discuss the role of the interatomic interactions in this process and find that the interference pattern results from an unequal population of the symmetric and antisymmetric states of the two-atom system. We also identify intrinsically nonclassical effects versus classical squeezed field effects, (C) 1998 Elsevier Science B.V. All rights reserved.

Spatial and temporal variation in distribution of mangroves in Moreton Bay, subtropical Australia: a comparison of pattern metrics and change detection analyses based on aerial photographs

An assessment of the changes in the distribution and extent of mangroves within Moreton Bay, southeast Queensland, Australia, was carried out. Two assessment methods were evaluated: spatial and temporal pattern metrics analysis, and change detection analysis. Currently, about 15,000 ha of mangroves are present in Moreton Bay. These mangroves are important ecosystems, but are subject to disturbance from a number of sources. Over the past 25 years, there has been a loss of more than 3800 ha, as a result of natural losses and mangrove clearing (e.g. for urban and industrial development, agriculture and aquaculture). However, areas of new mangroves have become established over the same time period, offsetting these losses to create a net loss of about 200 ha. These new mangroves have mainly appeared in the southern bay region and the bay islands, particularly on the landward edge of existing mangroves. In addition, spatial patterns and species composition of mangrove patches have changed. The pattern metrics analysis provided an overview of mangrove distribution and change in the form of single metric values, while the change detection analysis gave a more detailed and spatially explicit description of change. An analysis of the effects of spatial scales on the pattern metrics indicated that they were relatively insensitive to scale at spatial resolutions less than 50 m, but that most metrics became sensitive at coarser resolutions, a finding which has implications for mapping of mangroves based on remotely sensed data. (C) 2003 Elsevier Science B.V. All rights reserved.

Could the Leukocyte X Chromosome Inactivation Pattern Be Extrapolated to Hair Bulbs?

Background: The androgen receptor gene is located on the X chromosome with a polymorphic tract of CAG repeats that is inversely correlated to the receptor`s transactivation activity. A short CAG tract is associated with hyperandrogenic disorders. In women, one of the X chromosomes is inactivated and the X chromosome inactivation (XCI) pattern varies among tissues. Previous studies of hyperandrogenic disorders only evaluated XCI in leukocytes. Objective: To evaluate whether the XCI pattern in leukocytes could be extrapolated to those in hair bulbs. Material: A total of 58 healthy women were used for this study. DNA was extracted from leukocytes (n = 58 women) and pubic (n = 53 women) and scalp hair (n = 21 women). Methods: Hpa II digested and undigested DNA samples underwent fluorescence PCR GeneScan (R) analysis. Results: A significant and positive correlation of XCI was found between leukocytes and hair bulbs. However, individual comparisons showed that 13 and 19% of the women presented a different leukocyte XCI pattern in pubic hair and similar in leukocytes and hair bulbs of normal women indicating that leukocyte DNA is useful for XCI analysis. However, the XCI pattern could vary among tissues from the same subject, indicating that care should be taken when extrapolating individual leukocyte XCI patterns to other tissue. Copyright (C) 2010 S. Karger AG, Basel

Centrilobular Fibrosis: An Underrecognized Pattern in Systemic Sclerosis

Background: The impressive association of lung involvement and gastroesophageal reflux in scleroderma raises the possibility of a cause-effect relationship. Objectives: To determine clinical, radiological and histopathological features of systemic sclerosis (SSc) patients according the presence or absence of centrilobular fibrosis (CLF). Methods: Twenty-eight SSc patients with lung involvement were submitted to open lung biopsy and the specimens classified for the presence of CLF (bronchocentric distribution of the lesions and intraluminal matter according to the classification of idiopathic interstitial pneumonia). HRCT, pulmonary function tests and esophageal analysis were also performed. Subsequently, cyclophosphamide was introduced for the nonspecific interstitial pneumonia subgroup and antireflux treatment was intensified for isolated CLF patients. Results: Isolated CLF was found in 21% of the biopsies and also found associated to nonspecific interstitial pneumonia in 84% of these patients. The other 3 cases had usual interstitial pneumonia, pulmonary hypertension and respiratory bronchiolitis-associated interstitial lung disease. The histopathological analysis revealed that all 6 patients with isolated CLF had the bronchocentric distribution and intraluminal basophilic content, with foreign bodies detected in one third of them. The central distribution of lung involvement on HRCT was found in 67% of these patients with a consistent patchy distribution (100%). Ground glass (67%) and consolidation (33%) were the predominant patterns found. The constant clinical finding in all isolated CLF cases was dyspnea, esophageal abnormalities and a moderate lung impairment (FVC: 63.83 +/- 16.31%; DLCO: 61.66 +/- 18.84%). Lung function parameters in isolated CLF patients remained stable after 1 year of exclusively intensive antireflux treatment (FVC, p = 0.23; DLCO, p = 0.59). Conclusions: The novel description of CLF pattern in SSc lung disease with peculiar histological, tomographic and clinical features will certainly contribute to a more appropriate therapeutic approach. Copyright (C) 2008 S. Karger AG, Basel

Adrenalectomy counteracts the local modulation of astroglial fibroblast growth factor system without interfering with the pattern of 6-OHDA-induced dopamine degeneration in regions of the ventral midbrain

The present study investigated the effects of bilateral adrenalectomy (ADX) on the synthesis of basic fibroblast growth factor (bFGF, FGF-2) mRNA and on the expression of its FGF receptor subtype-2 (FGFR2) mRNA after a 6-hydroxydopamine (6-OHDA)-induced lesion of nigrostriatal dopamine system. In previous papers we have demonstrated that corticosterone increases FGF-2 immunoreactivity mainly in the astrocytes of the substantia nigra [Chadi, G., Rosen, L., Cintra, A., Tinner, B., Zoli, M., Pettersson, R.F., Fuxe, K., 1993b. Corticosterone increases FGF-2 (bFGF) immunoreactivity in the substantia nigra of the rat. Neuroreport 4, 783-786.] and that 6-OHDA injected in the ventral midbrain upregulates FGF-2 synthesis in reactive astrocytes in the ascending dopamine pathways [Chadi, G., Cao, Y., Pettersson, R.F., Fuxe, K., 1994. Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons. Neuroscience 61, 891-910.]. Rats were adrenalectomized and received a 6-OHDA stereotaxical injection in the ventral midbrain 2 days later. Seven days after the dopamine lesion, Western blot analysis showed a decreased level of tyrosine hydroxylase in the lesioned side of the midbrain, an event that was not altered by ADX or corticosterone replacement. Moreover, the degeneration of nigral dopamine neurons, which was confirmed by the disappearance of acidic FGF (FGF-1) mRNA and the decrement of tyrosine hydroxylase mRNA labeled nigral neurons, was not altered by ADX. The FGF-2 protein (23 kDa isoform but not 21 kDa fraction) levels increased in the lesioned side of the ventral midbrain. This elevation was counteracted by ADX, an effect that was fully reversed by corticosterone replacement. In situ hybridization revealed that ADX counteracted the elevated FGF-2 mRNA levels in putative glial cells of the ipsilateral pars compacta of the substantia nigra and in the ventral tegmental area. The ADX also counteracted the increased density and intensity of the astroglial FGF-2 immunoreactive profiles within the lesioned pars compacta of the substantia nigra and the ventral tegmental area as determined by stereology. The stereotaxical mechanical needle insertion triggered the expression of FGFR2 mRNA in putative glial cells, spreading to the entire ipsilateral ventral midbrain from the region of needle track, an occurrence that was partially reversed by ADX. In conclusion, bilateral ADX counteracted the increased astroglial FGF-2 synthesis in the dopamine regions of the ventral midbrain following a 6-OHDA-induced local lesion and interfered with FGF receptor regulation around injury. These findings give further evidence that adrenocortical hormones may regulate the astroglial FGF-2-mediated trophic mechanisms and wound repair events in the lesioned central nervous system. (c) 2007 Elsevier B.V. All rights reserved.