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.

Two distinct plant respiratory physiotypes might exist which correspond to fast-growing and slow-growing species

The origin of the carbon atoms in CO2 respired by leaves in the dark of several plant species has been studied using 13C/12C stable isotopes. This study was conducted using an open gas exchange system for isotope labeling that was coupled to an elemental analyser and further linked to an isotope ratio mass spectrometer (EA-IRMS) or coupled to a gas chromatography-combustion-isotope ratio mass spectrometer (GC-C-IRMS). We demonstrate here that the carbon, which is recently assimilated during photosynthesis, accounts for nearly ca. 50% of the carbon in the CO2 lost through dark respiration after illumination in fast-growing and cultivated plants and trees and, accounts for only ca. 10% in slow-growing plants. Moreover, our study shows that fast- growing plants, which had the largest percentages of newly fixed carbon of leaf-respired CO2 , were also those with the largest shoot/root ratios, whereas slow-growing plants showed the lowest shoot/root values.

Prediction of crude protein and classification of the growth stage of wheat plant samples from NIR spectra

Near-infrared spectroscopy (NIRS) was used to analyse the crude protein content of dried and milled samples of wheat and to discriminate samples according to their stage of growth. A calibration set of 72 samples from three growth stages of wheat (tillering, heading and harvest) and a validation set of 28 samples was collected for this purpose. Principal components analysis (PCA) of the calibration set discriminated groups of samples according to the growth stage of the wheat. Based on these differences, a classification procedure (SIMCA) showed a very accurate classification of the validation set samples : all of them were successfully classified in each group using this procedure when both the residual and the leverage were used in the classification criteria. Looking only at the residuals all the samples were also correctly classified except one of tillering stage that was assigned to both tillering and heading stages. Finally, the determination of the crude protein content of these samples was considered in two ways: building up a global model for all the growth stages, and building up local models for each stage, separately. The best prediction results for crude protein were obtained using a global model for samples in the two first growth stages (tillering and heading), and using a local model for the harvest stage samples.

Clonal growth and demography of a hemicryptophyte alpine plant: Leontopodium alpinum Cassini

Clonally reproducing hemicryptophytic rosette plants are common in the alpine belt. However, their demography, and indirectly their growth and reproductive strategy in these harsh conditions, was rarely studied. We analysed the morphology, clonal reproduction and demography of one such species, Leontopodium alpinum, in two populations of the Swiss Alps. The species forms small colonies of 1-5 (maximum 30) sterile rosettes with a few flowering stalks. After flowering, the apical meristem dies and one or two new axillary buds grow below the previous rosette in the following year, developing into short rhizomes (<2 cm), which decay after four years. The new stalk produces sterile rosettes before flowering after two to four years, depending on climatic conditions. The apical meristem often dies during the sterile stage, and is replaced by a new axillary bud. Levkovitch matrices on two stages (sterile and flowering rosettes) showed that rosette survival and clonal reproduction maintain long-lived populations (λ = 0.96). Elasticities indicated that a change in the survival of sterile rosettes had the strongest effect on population dynamics, and this stage lasts, on average, 6.8 years at 2480 m. Altogether, L. alpinum is following Tomlinson's architectural model. This growth form appears perfectly adapted to harsh alpine conditions: the clonal ramification ensures longevity to genets and the semelparous behaviour of the rosettes allows an efficient flowering, whatever the climatic conditions. L. alpinum appears to follow a common growth model among rosette possessing hemicryptophytes in the alpine belt.

Background, current status and future prospects of transgenic crop plant development

Les plantes transgèniques són una part integral de l’agricultura contemporània. Durant l’any 2006 més de noranta milions d’hectàrees de plantes transgèniques van ser cultivades en vint-i-un països. Des de la comercialització de la primera planta transgènica el 1996 els nivells d’adopció d’aquests cultius han augmentat anualment amb percentatges de dos dígits. El desenvolupament i la comercialització de les plantes transgèniques van lligats estretament al comerç mundial, a la globalització, a la disponibilitat de suficient menjar, a la protecció del medi ambient i del consumidor i a la propietat intel·lectual. En aquest article exposem els avenços més recents i les tendències actuals en el desenvolupament dels cultius transgènics i de la seva utilització. També ens fem ressò d’alguns assumptes no científics que s’han de solucionar abans que aquests cultius arribin al màxim del seu potencial, proporcionant una agricultura més sostenible i ecològica. Finalment, ressaltarem la importància de com les plantes transgèniques poden contribuir en la disponibilitat de menjar i en la millora de la pobresa en els països en vies de desenvolupament.

Plant-derived phenolics inhibit the accrual of structurally characterised protein and lipid oxidative modifications

Epidemiological data suggest that plant-derived phenolics beneficial effects include an inhibition of LDL oxidation. After applying a screening method based on 2,4-dinitrophenyl hydrazine- protein carbonyl reaction to 21 different plant-derived phenolic acids, we selected the most antioxidant ones. Their effect was assessed in 5 different oxidation systems, as well as in other model proteins. Mass-spectrometry was then used, evidencing a heterogeneous effect on the accumulation of the structurally characterized protein carbonyl glutamic and aminoadipic semialdehydes as well as for malondialdehyde-lysine in LDL apoprotein. After TOF based lipidomics, we identified the most abundant differential lipids in Cu++-incubated LDL as 1-palmitoyllysophosphatidylcholine and 1-stearoyl-sn-glycero-3-phosphocholine. Most of selected phenolic compounds prevented the accumulation of those phospholipids and the cellular impairment induced by oxidized LDL. Finally, to validate these effects in vivo, we evaluated the effect of the intake of a phenolic-enriched extract in plasma protein and lipid modifications in a well-established model of atherosclerosis (diet-induced hypercholesterolemia in hamsters). This showed that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake. that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake.

Preferential reproduction mode of hermaphrodite papaya plant (Carica papaya L; Caricaceae)

This research was done to study the reproductive system of papaya hermaphrodite plant based on the histochemical nature of pollen grain, stigma receptivity, in vivo pollen grain germination and pollen:ovule ratio. In the histochemical analysis, pollen grains were stained by using Sudan IV and I2KI solution ; the stigma receptivity was assessed by alpha-naphthtyl acetate solution in closed and opened flowers. Pollen germination and pollen tube growing were examined in flower buds near anthesis with 0.1% aniline blue. To estimate the pollen:ovule ratio , anthers from each flower bud were dissected and all pollen grains were counted; ovules were dissected from ovaries and were counted under stereomicroscope. The results indicated that papaya pollen grains are of lipidic nature; the stigmas were receptive before the opening and until 48 hours after opening; the pollen grains germinated and emitted polinic tube before flower opening and the pollen:ovule ratio indicated the predominance of autogamous reproductive system. These results indicate that hermaphrodite papaya trees is preferentially of optional autogamous with cleistogamy.

Sour orange bud regeneration and in vitro plant development related to culture medium composition and explant type

In order to evaluate the formation of adventitious buds and in vitro regeneration of sour orange plants (Citrus aurantium L.) two organogenesis-inducing experiments were conducted. In the first experiment, the induction and in vitro regeneration of adventitious buds were tested on epicotyl and internodal segments under the influence of BAP or KIN associated with NAA. The second experiment evaluated the in vitro regeneration of sour orange plants related to different explant types (epicotyls segments, internodal segments of in vitro germinated plantlets and internodal segments of greenhouse cultivated plants). Data collected on both experiments included the percentage of responsive explants (explants that formed buds), and the number of buds per explant. The addition of BAP showed the best organogenic response. In vitro germinated epicotyl segments and internodal segments are recommended as explants for sour orange in vitro organogenesis. Rooting of regenerated shoots was achieved without the need of auxin in the medium.

Anaerobic treatment of deinking pulp plant effluents

Anaerobic treatment as a first biological stage in wastewater treatment is nowadays a well-established technology in recycled paper processing mills using closed water circuits. Today further developed high-rate processes and especially high-tower reactors are also able to handle lower organic loads and become therefore feasible for deinking pulp plant effluents. The interest in the anaerobic method is based on a positive energy balance in form of biogas production and low biomass yield from the process. The anaerobic treatment method was researched and its suitability for the deinking pulp plant effluents was tested experimentally at Stora Enso Maxau mill. In the theory, the deinking pulp process is introduced and the effluents from the deinking process are characterized. The anaerobic treatment is brought up in depth in terms of its use for the deinking effluents, and different kind of reactor types are presented. In addition, other wastewater treatment methods are shortly introduced with the focus on tertiary treatment. Static biodegradability tests were carried out for the wastewaters both anaerobically and aerobically. Based on the results, the deinking effluents can be degraded anaerobically, and inhibition to the methanogenic bacteria was not noticed. In the aerobic static test a good performance of the existing wastewater treatment plant at Maxau mill was proved. Later on pilot trials with sequential anaerobic-aerobic treatment were carried out for the deinking effluents. The anaerobic reactor used was a so called internal circulation reactor. The results confirmed that the combination of the anaerobic treatment and the aerobic activated sludge process is a suitable method for deinking wastewaters with a COD reduction as good as with a two stage aerobic method. When combined with the outstanding quality of the produced biogas and the cost savings acquired from the lower sludge production, the anaerobic treatment was found to be an especially favorable treatment method.

Plant growth regulators to manipulate oat stands

Selostus: Kasvunsääteet vaikuttavat kauran kasvuston rakenteeseen ja tasoon

Intra-plant variation for progress of cell division in developing oat grains : a preliminary study

Selostus: Esitutkimus kauran röyhyn sisäisestä vaihtelusta jyvän täyttymisen käynnistyessä

Potassium fertilization for pineapple: effects on plant growth and fruit yield

A field experiment with pineapple (Smooth Cayenne) was carried out on an Ultisol located in the city of Agudos (22º30'S; 49º03'W), in the state of São Paulo, Brazil, with the objective of investigating the effects of rates and sources of potassium fertilizer on plant growth and fruit yield. The experiment was a complete factorial design (4x3) with four rates (0, 175, 350, and 700 kg ha-1 of K2O) and three combinations of K sources (100% KCl, 100% K2SO4, and 40% K2SO4 + 60% KCl). Plant growth and fruit yield were evaluated. Biomass accumulation of pineapple plants was impaired by chlorine added with potassium chloride. Fruit yield increased with potassium fertilization. At high rates of K application, fertilization with K2SO4 showed better results than with KCl. Detrimental effects of KCl were associated with excess of chlorine.

Potassium fertilization for pineapple: effects on soil chemical properties and plant nutrition

A field experiment was carried out on an Ultisol located at the city of Agudos (22º30'S; 49º03'W), in the state of São Paulo, Brazil, in order to determine the effects of rates and sources of potassium fertilizer on nutritional status of 'Smooth Cayenne' pineapple and on some soil chemical properties. The experiment was a complete factorial design with four rates (0, 175, 350, and 700 kg ha-1 of K2O) and three combinations of K sources (100% KCl, 100% K2SO4 and 40% K2SO4 + 60% KCl). Soil samples were taken from the depths 0-20 cm, 20-40 cm and 40-60 cm at planting and 14 months after. Nutritional status of pineapple plants was assessed by means of tissue analysis. Soil K availability increased with application of K fertilizer, regardless of K sources. Soil chlorine and Cl concentration in pineapple leaves increased with application of KCl or K2SO4+KCl. Plant uptake of potassium was shaped by soil K availability and by the application rates of K fertilizer, independently of K sources.

Tree cover at fine and coarse spatial grains interacts with shade tolerance to shape plant species distributions across the Alps

The role of competition for light among plants has long been recognized at local scales, but its potential importance for plant species' distribution at larger spatial scales has largely been ignored. Tree cover acts as a modulator of local abiotic conditions, notably by reducing light availability below the canopy and thus the performance of species that are not adapted to low-light conditions. However, this local effect may propagate to coarser spatial grains. Using 6,935 vegetation plots located across the European Alps, we fit Generalized Linear Models (GLM) for the distribution of 960 herbs and shrubs species to assess the effect of tree cover at both plot and landscape grain sizes (~ 10-m and 1-km, respectively). We ran four models with different combinations of variables (climate, soil and tree cover) for each species at both spatial grains. We used partial regressions to evaluate the independent effects of plot- and landscape-scale tree cover on plant communities. Finally, the effects on species' elevational range limits were assessed by simulating a removal experiment comparing the species' distribution under high and low tree cover. Accounting for tree cover improved model performance, with shade-tolerant species increasing their probability of presence at high tree cover whereas shade-intolerant species showed the opposite pattern. The tree cover effect occurred consistently at both plot and landscape spatial grains, albeit strongest at the former. Importantly, tree cover at the two grain sizes had partially independent effects on plot-scale plant communities, suggesting that the effects may be transmitted to coarser grains through meta-community dynamics. At high tree cover, shade-intolerant species exhibited elevational range contractions, especially at their upper limit, whereas shade-tolerant species showed elevational range expansions at both limits. Our findings suggest that the range shifts for herb and shrub species may be modulated by tree cover dynamics.