Diversidade específica e de hábitat dos Cosmarium de parede lisa (Zygnemaphyceae) do estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A semente de Passiflora edulis Sims f. flavicarpa O. Deg. (Maracujá-Amarelo) durante a germinação e crescimento inicial da plântula: a dinâmica de lipídios, carboidratos e cloroplastos, e sua relevância nesse período

Pós-graduação em Ciências Biológicas (Biologia Vegetal) - IBRC

Cultivo de Eucalyptus urograndis em atmosfera enriquecida com CO2: mudanças no proteoma cloroplastidial

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A distinct tymovirus infecting Cassia hoffmannseggii in Brazil

Leaves of Cassia hoffmannseggii, a wild fabaceous species found in the Atlantic Forest, with a severe mosaic symptom were collected in Pernambuco State, Brazil. By transmission electron microscopy, two types of virus particles were found: the first was recognized as particles of a potyvirus, which was later identified as Cowpea aphid-borne mosaic virus; and the second was isometric and present in high concentration. The observation of vesicles at the periphery of chloroplasts suggested a tymovirus infection, which was confirmed by subsequent assays. A serological assay against several tymovirus antisera resulted in positive reaction of this tymo-like virus with an antiserum of Passion fruit yellow mosaic virus. By means of RT-PCR and using degenerated primers for the conserved region of RNA-dependent RNA polymerase (RdRp) gene of tymoviruses, a specific DNA fragment was amplified and sequenced. Based on this sequence, a specific forward primer was synthesized and successfully used to amplify the 3' terminal genome region, containing the partial RdRp gene and the complete coat protein (CP) sequences. The CP was 188 amino acids (aa) long, and the highest CP aa identity was observed with Kennedya yellow mosaic virus (61 %). Based on the current ICTV demarcation criterion, this isolate was considered as a distinct tymovirus and tentatively named as Cassia yellow mosaic-associated virus.

DEFICIENCY SYMPTOMS AND UPTAKE OF MICRONUTRIENTS BY CASTOR BEAN GROWN IN NUTRIENT SOLUTION

Castor bean is a nutrient-demanding species, but there is still little information on its micronutrient requirements. The objectives of this study were to evaluate the effects of levels of B (2.5, 12.5 and 25.0 mu mol L-1), Cu (0.05, 0.25 and 0.50 mu mol L-1), Mn (0.2, 1.0 and 2.0 mu mol L-1) and Zn (0.2, 1.0 and 2.0 mu mol L-1) in a nutrient solution on plant B, Cu, Mn and Zn concentrations and uptake, vegetative growth and fruit yield of castor bean "Iris", grown in greenhouse. The experiment was arranged in a completely randomized block design with three replicates. The first deficiency symptoms were observed for B, followed by Zn, Cu and Mn. The main changes in the cell ultrastructure due to lack of B were thickening of the cell walls and middle lamellae, distorted chloroplasts and tightly stacked thylakoids, besides the absence of starch grains. The Mn, Zn and Cu deficiencies led to disruption of chloroplasts, disintegration of thylakoids and absence of amyloplasts. The concentration and uptake of B, Cu, Mn, and Zn in castor bean plants increased with micronutrient supply in the solution. Fruit yield was drastically reduced by B and Mn deficiencies. On the other hand, the dry matter yield of the shoot and root of castor bean plants was not. In the treatment with full nutrient solution, the leaves accumulated 56 and 48 % of the total B and Mn taken up by the plants, respectively, and the seeds and roots 85 and 61 % of the total Cu and Zn taken up, respectively. This shows the high demand of castor bean Iris for B and Mn for fruit yield.

Effects of Natural Radiation, Photosynthetically Active Radiation and Artificial Ultraviolet Radiation-B on the Chloroplast Organization and Metabolism of Porphyra acanthophora var. brasiliensis (Rhodophyta, Bangiales)

We undertook a study of Porphyra acanthophora var. brasiliensis to determine its responses under ambient conditions, photosynthetically active radiation (PAR), and PAR+UVBR (ultraviolet radiation-B) treatment, focusing on changes in ultrastructure, and cytochemistry. Accordingly, control ambient samples were collected in the field, and two different treatments were performed in the laboratory. Plants were exposed to PAR at 60 mu mol photons m(-2) s(-1) and PAR+UVBR at 0.35 W m(-2) for 3 h per day during 21 days of in vitro cultivation. Confocal laser scanning microscopy analysis of the vegetative cells showed single stellate chloroplast in ambient and PAR samples, but in PAR+UVBR-exposed plants, the chloroplast showed alterations in the number and form of arms. Under PAR+UVBR treatment, the thylakoids of the chloroplasts were disrupted, and an increase in the number of plastoglobuli was observed, in addition to mitochondria, which appeared with irregular, disrupted morphology compared to ambient and PAR samples. After UVBR exposure, the formation of carpospores was also observed. Plants under ambient conditions, as well as those treated with PAR and PAR+UVBR, all showed different concentrations of enzymatic response, including glutathione peroxidase and reductase activity. In summary, the present study demonstrates that P. acanthophora var. brasiliensis shows the activation of distinct mechanisms against natural radiation, PAR and PAR+UVBR.

Deficiency symptoms and uptake of micronutrients by castor bean grown in nutrient solution

Castor bean is a nutrient-demanding species, but there is still little information on its micronutrient requirements. The objectives of this study were to evaluate the effects of levels of B (2.5, 12.5 and 25.0 µmol L-1), Cu (0.05, 0.25 and 0.50 µmol L-1), Mn (0.2, 1.0 and 2.0 µmol L-1) and Zn (0.2, 1.0 and 2.0 µmol L-1) in a nutrient solution on plant B, Cu, Mn and Zn concentrations and uptake, vegetative growth and fruit yield of castor bean "Iris", grown in greenhouse. The experiment was arranged in a completely randomized block design with three replicates. The first deficiency symptoms were observed for B, followed by Zn, Cu and Mn. The main changes in the cell ultrastructure due to lack of B were thickening of the cell walls and middle lamellae, distorted chloroplasts and tightly stacked thylakoids, besides the absence of starch grains. The Mn, Zn and Cu deficiencies led to disruption of chloroplasts, disintegration of thylakoids and absence of amyloplasts. The concentration and uptake of B, Cu, Mn, and Zn in castor bean plants increased with micronutrient supply in the solution. Fruit yield was drastically reduced by B and Mn deficiencies. On the other hand, the dry matter yield of the shoot and root of castor bean plants was not. In the treatment with full nutrient solution, the leaves accumulated 56 and 48 % of the total B and Mn taken up by the plants, respectively, and the seeds and roots 85 and 61 % of the total Cu and Zn taken up, respectively. This shows the high demand of castor bean Iris for B and Mn for fruit yield.

Role of xanthophyll and water-water cycles in the protection of photosynthetic apparatus in Arbutus unedo and Arabidopsis thaliana

Photosynthetic organisms have sought out the delicate balance between efficient light harvesting under limited irradiance and regulated energy dissipation under excess irradiance. One of the protective mechanisms is the thermal energy dissipation through the xanthophyll cycle that may transform harmlessly the excitation energy into heat and thereby prevent the formation of damaging active oxygen species (AOS). Violaxanthin deepoxidase (VDE) converts violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z) defending the photosynthetic apparatus from excess of light. Another important biological pathway is the chloroplast water-water cycle, which is referred to the electrons from water generated in PSII reducing atmospheric O2 to water in PSI. This mechanism is active in the scavenging of AOS, when electron transport is slowed down by the over-reduction of NADPH pool. The control of the VDE gene and the variations of a set of physiological parameters, such as chlorophyll florescence and AOS content, have been investigated in response to excess of light and drought condition using Arabidopsis thaliana and Arbutus unedo.. Pigment analysis showed an unambiguous relationship between xanthophyll de-epoxidation state ((A+Z)/(V+A+Z)) and VDE mRNA amount in not-irrigated plants. Unexpectedly, gene expression is higher during the night when xanthophylls are mostly epoxidated and VDE activity is supposed to be very low than during the day. The importance of the water-water cycle in protecting the chloroplasts from light stress has been examined through Arabidopsis plant with a suppressed expression of the key enzyme of the cycle: the thylakoid-attached copper/zinc superoxide dismutase. The analysis revealed changes in transcript expression during leaf development consistent with a signalling role of AOS in plant defence responses but no difference was found any in photosynthesis efficiency or in AOS concentration after short-term exposure to excess of light. Environmental stresses such as drought may render previously optimal light levels excessive. In these circumstances the intrinsic regulations of photosynthetic electron transport like xanthophyll and water-water cycles might modify metabolism and gene expression in order to deal with increasing AOS.

Harmful algae and their potential impacts on the coastal ecosystem: growth and toxin production dynamics

The main goal of the present thesis was to study some harmful algal species which cause blooms in Italian coastal waters, leading to consequences for human health, coastal ecosystem, fishery and tourism. In particular, in the first part of this thesis the toxicity of Adriatic strains of the raphidophyte Fibrocapsa japonica was investigated. Despite several hypotheses have been proposed for the toxic mechanism of the raphidophytes, especially for the species Chattonella antiqua and C. marina, which have been studied more extensively, just a few studies on the toxic effects of these species for different organisms were reported. Moreover, a careful reading of the literature evidenced as any ichthyotoxic events reported worldwide can be linked to F. japonica blooms. Although recently several studies were performed on F. japonica strains from the USA, Japan, Australia, New Zealand, the Netherlands, Germany, and France in order to characterize their growth and toxicity features, the work reported in this thesis results one of the first investigation on the toxic effects of F. japonica for different organisms, such as bacteria, crustaceans and fish. Mortality effects, together with haemolysis of fish erythrocytes, probably due to the relatively high amount of PUFAs produced by this species, were observed. Mortality for fish, however, was reported only at a high cell density and after a long exposition period (9-10 days); moreover a significant increase of H2O2 obtained in the tanks where sea basses were exposed to F. japonica was also relevant. This result may justify the absence of ichthyotoxic events in the Italian coasts, despite F. japonica blooms detected in these areas were characterized by high cell densities. This work reports also a first complete characterization of the fatty acids produced and extracellularly released by the Adriatic F. japonica, and results were also compared with the fatty acid profile of other strains. The absence of known brevetoxins in F. japonica algal extracts was also highlighted, leading to the hypothesis that the toxicity of F. japonica may be due to a synergic effect of PUFAs and ROS. Another microalgae that was studied in this thesis is the benthic dinoflagellate Ostreopsis cf. ovata. This species was investigated with the aim to investigate the effect of environmental parameters on its growth and toxicity. O. cf. ovata, in fact, shows different blooming periods along the Italian coasts and even the reported toxic effects are variable. The results of this work confirmed the high variability in the growth dynamic and toxin content of several Italian strains which were isolated in recent years along the Adriatic and Tyrrhenian Seas. Moreover, the effects of temperature and salinity on the behaviour of the different isolates are in good agreement with the results obtained from field surveys, which evidence as the environmental parameters are important factors modulating O. cf. ovata proliferation. Another relevant result that was highlighted is the anomaly in the production of palytoxin-like compounds reported by one of the studied isolate, in particular the one isolated in 2008 in Ancona (Adriatic Sea). Only this strain reported the absence of two (ovatoxin-b and –c) of the five ovatoxins so far known in the toxin profile and a different relative abundance of the other toxins. The last aspect that was studied in this thesis regards the toxin biosythesis. In fact, toxins produced (palytoxin-like compounds) or supposed to be produced (brevetoxin-like compounds) by O. cf. ovata and F. japonica, respectively, are polyketides, which are highly oxygenated compounds synthesized by complex enzymes known as polyketide synthase (PKS) enzymes. These enzymes are multi-domain complexes that structurally and functionally resemble the fatty acid synthases (FASs). This work reports the first study of PKS proteins in the dinoflagellates O. cf. ovata, C. monotis and in the raphidophyte F. japonica. For the first time some PKSs were identified in these species, confirming the presence of PKS proteins predicted by the in silico translation of the transcripts found in K. brevis also in other species. The identification of O. cf. ovata PKSs and the localization of the palytoxin-like compounds produced by this dinoflagellate in a similar location (chloroplast) as that observed for other dinoflagellate and cyanobacterial toxins provides some indication that these proteins may be involved in polyketide biosynthesis. However, their potential function as fatty acid synthases cannot be ruled out, as plant fatty acid synthesis also occurs within chloroplasts. This last hypothesis is also supported by the fact that in all the investigated species, and in particular in F. japonica, PKS proteins were present. Therefore, these results provide an important contribution to the study of the polyketides and of the involvement of PKS proteins in the toxin biosynthesis.

In vitro synthesis of the light-harvesting complex into artificial membrane systems

In green plants, the function of collecting solar energy for photosynthesis is fulfilled by a series of light-harvesting complexes (LHC). The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP), then imported into chloroplasts and assembled into photosynthetic thylakoid membranes. Knowledge about the regulation of the transport processes of LHCP is rather limited. Closely mimicking the in vivo situation, cell-free protein expression system is employed in this dissertation to study the reconstitution of LHCP into artificial membranes. The approach starts merely from the genetic information of the protein, so the difficult and time-consuming procedures of protein expression and purification can be avoided. The LHCP encoding gene from Pisum sativum was cloned into a cell-free compatible vector system and the protein was expressed in wheat germ extracts. Vesicles or pigment-containing vesicles were prepared with either synthetic lipid or purified plant leaf lipid to mimic cell membranes. LHCP was synthesized in wheat germ extract systems with or without supplemented lipids. The addition of either synthetic or purified plant leaf lipid was found to be beneficial to the general productivity of the expression system. The lipid membrane insertion of the LHCP was investigated by radioactive labelling, protease digestion, and centrifugation assays. The LHCP is partially protected against protease digestion; however the protection is independent from the supplemented lipids.

Structural analysis of the major lightharvesting complex II by electron paramagnetic resonance (EPR) : comparison between monomers and trimers

Der Haupt-Lichtsammenkomplex II (LHCII) höherer Pflanzen ist das häufigsternMembranprotein der Welt und in die chloroplastidäre Thylakoidmembran integriert. DerrnLHCII kann als Modellsystem genutzt werden, um die Funktionsweise vonrnMembranproteinen besser zu verstehen, da 96 % seiner Struktur kristallografisch aufgelöstrnist und er in rekombinanter Form in vitro rückgefaltet werden kann. Hierbei entsteht einrnvoll funktionaler Protein-Pigment.Komplex, der nahezu identisch mit der in vivo Varianternist.rnElektronenparamagnetischen Resonanz (EPR) Spektroskopie ist eine hoch sensitive undrnideal geeignete Methode, um die Strukturdynamik von Proteinen zu untersuchen. Hierzurnist eine ortsspezifische Markierung mit Spinsonden notwendig, die kovalent an Cysteinernbinden. Möglich wird dies, indem sorgfältig ausgewählte Aminosäuren gegen Cysteinerngetauscht werden, ohne dass die Funktionsweise des LHCII beeinträchtigt wird.rnIm Rahmen dieser Arbeit wurden die Stabilität des verwendeten Spinmarkers und diernProbenqualität verbessert, indem alle Schritte der Probenpräparation untersucht wurden.rnMithilfe dieser Erkenntnisse konnte sowohl die Gefahr einer Proteinaggregation als auchrnein Verlust des EPR Signals deutlich vermindert werden. In Kombination mit derrngleichzeitigen Etablierung des Q-Band EPR können nun deutlich geringer konzentrierternProben zuverlässig vermessen werden. Darüber hinaus wurde eine reproduzierbarernMethode entwickelt, um heterogene Trimere herzustellen. Diese bestehen aus einemrndoppelt markierten Monomer und zwei unmarkierten Monomeren und erlauben es, diernkristallografisch unvollständig aufgelöste N-terminale Domäne im monomeren undrntrimeren Assemblierungsgrad zu untersuchen. Die Ergebnisse konnten einerseits diernVermutung bestätigen, dass diese Domäne im Vergleich zum starren Proteinkern sehrrnflexibel ist und andererseits, dass sie in Monomeren noch mobiler ist als in Trimeren.rnZudem wurde die lumenale Schleifenregion bei unterschiedlichen pH Werten undrnvariierender Pigmentzusammensetzung untersucht, da dieser Bereich sehr kontroversrndiskutiert wird. Die Messergebnisse offenbarten, dass diese Region starre und flexiblerernSektionen aufweist. Während der pH Wert keinen Einfluss auf die Konformation hatte,rnzeigte sich, dass die Abwesenheit von Neoxanthin zu einer Änderung der Konformationrnführt. Weiterführende Analysen der strukturellen Dynamik des LHCII in einerrnLipidmembran konnten hingegen nicht durchgeführt werden, da dies eine gerichteternInsertion des rückgefalteten Proteins in Liposomen erfordert, was trotz intensiverrnVersuche nicht zum Erfolg führte.

Small-Scale Features of Marine Sediments and Their Importance to the Study of Deposit-Feeding

Techniques currently in use by sedimentologists for the study of marine sedment microfabric are of limited use for understandmg the relationship between sediment organic matter and mineral grains. In this article it is shown that by combining standard histological protocols for fixation and dehydration with petrological protocols for resin embedding and thin sectioning, very fine details of the sediment structure can be seen. Because of the ubiquitous presence of the organic matrix, organicmineral aggregates are not seen in situ. Other features of the sediment of importance to deposit-feeders, such as the presence of intact chloroplasts, can be observed through the use of epifluorescence illumination, while partially crossed polarizers help to delimit the grain boundaries. It is suggested that if these procedures can be combined with histological staining techniques, it may be possible to determine the potential food value of sedment on a scale equivalent to that perceived by infaunal deposit-feeders.