Anatomia de folha e eixo da inflorescência: contribuições à taxonomia de Rapateaceae

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Anatomia de folha e eixo da inflorescência: contribuições à taxonomia de Rapateaceae

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Plasticity of stomatal distribution pattern and stem tracheid dimensions in Podocarpus lambertii: an ecological study

Leaf and wood plasticity are key elements in the survival of widely distributed plant species. Little is known, however, about variation in stomatal distribution in the leaf epidermis and its correlation with the dimensions of conducting cells in wood. This study aimed at testing the hypothesis that Podocarpus lambertii, a conifer tree, possesses a well-defined pattern of stomatal distribution, and that this pattern can vary together with the dimensions of stem tracheids as a possible strategy to survive in climatically different sites. Leaves and wood were sampled from trees growing in a cold, wet site in south-eastern Brazil and in a warm, dry site in north-eastern Brazil. Stomata were thoroughly mapped in leaves from each study site to determine a spatial sampling strategy. Stomatal density, stomatal index and guard cell length were then sampled in three regions of the leaf: near the midrib, near the leaf margin and in between the two. This sampling strategy was used to test for a pattern and its possible variation between study sites. Wood and stomata data were analysed together via principal component analysis. The following distribution pattern was found in the south-eastern leaves: the stomatal index was up to 25 higher in the central leaf region, between the midrib and the leaf margin, than in the adjacent regions. The inverse pattern was found in the north-eastern leaves, in which the stomatal index was 10 higher near the midrib and the leaf margin. This change in pattern was accompanied by smaller tracheid lumen diameter and length. Podocarpus lambertii individuals in sites with higher temperature and lower water availability jointly regulate stomatal distribution in leaves and tracheid dimensions in wood. The observed stomatal distribution pattern and variation appear to be closely related to the placement of conducting tissue in the mesophyll.

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model "diploid embryogenic callus protoplast + diploid mesophyll-derived protoplast". Protoplasts were isolated from embryogenic calli of 'Pera' and 'Westin' sweet orange cultivars (Citrus sinensis) and from young leaves of 'Fremont', Nules', and 'Thomas' mandarins (C. reticulata), and 'Nova' tangelo [C. reticulata x (C. paradisi x C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when 'Pera' sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of 'Pera' sweet orange + 'Fremont' mandarin, 3 'Pera' sweet orange + 'Nules' mandarin, and 2 'Pera' sweet orange + 'Nova' tangelo, and all the diploid regenerated plants showed the 'Pera' sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96 h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.

Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo

(Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo). The present work characterized and compared the anatomical structures of the leaves of Bactris gasipaes (Arecaceae) plants grown under different cultivation conditions (in vitro, ex vitro and in vivo) with the goal of identifying the origins of the difficulties encountered in acclimatizing micro-plants. The Quant program was used to determine leaf tissue thicknesses and areas, and histochemical tests were performed on leaf sections and analyzed using light microscopy. Stomatal and trichome densities were determined using the epidermal impression method and by scanning electronic microscopy. Our results indicated that there were no discernible alterations of the anatomical characteristics of the leaves of micro-plants cultivated under differing conditions and that the thickening of the mesophyll and the vascular fibers indicated adaptive responses to ex vitro conditions. As such, the observed difficulties in acclimatizing peach palm micro-plants to ex vitro conditions cannot be attributed to plant anatomical characteristics acquired during in vitro cultivation.

Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences

The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.

Differenzielle Expression von Tubulin-Genen bei der Entwicklung von Blattzellen der Gerste (Hordeum vulgare L.)

Die Morphogenese einer Pflanzenzelle wird in großem Maße durch die Dynamik kortikaler Mikrotubuli (MT) bestimmt, die auf die Zellwandsynthese Einfluß nehmen. In dieser Arbeit wurden die Transkriptmengen der alpha-Tubulin-Isotypen und des gamma-Tubulin während der Entwicklung des Gerstenblattes analysiert, um Zusammenhänge zu bereits beschriebenen Umwandlungen im kortikalen MT-Cytoskelett der Mesophyllzellen aufzudecken. Erstmals konnte bei einer höheren Pflanze die Genexpression auf RNA-Ebene innerhalb einer Tubulin-Multigenfamilie im Verlauf der Blattentwicklung umfassend dargestellt werden.Es wurden blattspezifische cDNA-Bibliotheken erstellt und mittels RT-PCR homologe DNA-Gensonden für die Screeningprozesse der cDNA-Bibliotheken hergestellt. cDNA-Sequenzen von alpha-, beta-, und gamma-Tubulin konnten isoliert werden. Weitere, weniger abundante alpha-Tubulin-Sequenzen wurden während zusätzlicher Screeningrunden über PCR-Ausschluß häufig vertretener, bereits bekannter Isotypen isoliert.Die cDNA-Sequenzen von insgesamt fünf verschiedenen Isotypen des alpha-Tubulin konnten aufgeklärt werden, drei Isotypen wiesen bis zu fünf im nicht kodierenden 3´-Bereich verkürzte Varianten auf, die aber in ihrer Anzahl deutlich unterrepräsentiert waren. Die abgeleiteten Aminosäuresequenzen umfassten bei drei Isotypen 451 Aminosäuren (AS), zwei Isotypen waren im C-Terminus um eine bzw. um zwei AS kürzer. Die fünf alpha-Tubulin-Isotypen wiesen charakteristische Expressionsmuster auf, die in drei Klassen unterteilbar waren. Die Isotypen HVATUB1 und HVATUB5 (MT-Band-Isotypen) hatten den maximalen Gehalt in Blattbereichen, in denen auch hauptsächlich Mesophyllzellen mit kortikalen MT-Bänderungen vorkommen, wobei HVATUB5 den am schwächsten exprimierte Isotyp darstellte. HVATUB3 (Random-MT-Isotyp) zeigte die stärksten Expressionsraten. Die im Meristem und meristemnahen Bereichen bereits recht hohe Abundanz erreichte erst nach der Zellstreckungszone in einer Blattzone das Maximum, in dem hauptsächlich Mesophyllzellen mit zerstreut angeordneten MT anzutreffen sind. Die Isotypen HVATUB2 und HVATUB4 (MImax-Isotypen) waren in mitotisch aktiven, basalen Blattbereichen dominant.Die cDNA-Sequenz vom gamma-Tubulin der Gerste, HVGTUB, wurde ermittelt; die abgeleitete Aminosäuresequenz bestand aus 469 AS. Das Auftreten einer im nicht kodierenden 3´-Bereich kürzeren Variante konnte erstmals bei pflanzlichem gamma-Tubulin beschrieben werden. Southernblot-Analysen ließen darauf schließen, daß gamma-Tubulin nur als Einzelkopie im Genom der Gerste vorkommt. gamma-Tubulin wurde im mitosereichen Meristem der Blattbasis am stärksten exprimiert. Da die Abnahme der Transkriptmenge weitaus langsamer verlief als die Abnahme der Zellteilungsaktivität, ist anzunehmen, daß gamma-Tubulin neben der Erfüllung von mitose- und zellteilungsspezifischen Funktionen auch eine Rolle im Zusammenhang mit der Dynamik des kortikalen MT-Cytoskeletts spielt. Einen ersten Schritt zur Aufklärung der Genfamilie des beta-Tubulin bei Gerste stellt die Isolierung drei verschiedener cDNA-Sequenzen von beta-Tubulin dar.

Morphological and physiological responses of apple trees under photoselective colored nets

In recent years and thanks to innovative technological advances in supplemental lighting sources and photo-selective filters, light quality manipulation (i.e. spectral composition of sunlight) have demonstrated positive effects on plant performance in ornamentals and vegetable crops. However, this aspect has been much less studied in fruit trees due to the difficulty of conditioning the light environment of orchards. The aim of the present PhD research was to study the use of different colored nets with selective light transmission in the blue (400 – 500 nm), red (600 – 700 nm) and near infrared (700 – 1100 nm) wavelengths as a tool to the light quality management and its morphological and physiological effects in field-grown apple trees. Chapter I provides a review the current status on physiological and technological advances on light quality management in fruit trees. Chapter II shows the main effect of colored nets on morpho-anatomical (stomata density, mesophyll structure and leaf mass area index) characteristics in apple leaves. Chapter III provides an analysis about the effect of micro-environmental conditions under colored nets on leaf stomatal conductance and leaf photosynthetic capacity. Chapter IV describes a study approach to evaluate the impact of colored nets on fruit growth potential in apples. Summing up results obtained in the present PhD dissertation clearly demonstrate that light quality management through photo-selective colored nets presents an interesting potential for the manipulation of plant morphological and physiological traits in apple trees. Cover orchards with colored nets might be and alternative technology to address many of the most important challenges of modern fruit growing, such as: the need for the efficient use of natural resources (water, soil and nutrients) the reduction of environmental impacts and the mitigation of possible negative effects of global climate change.

Nitrogen metabolism and remobilization during senescence

Senescence is a highly organized and well‐regulated process. As much as 75% of total cellular nitrogen may be located in mesophyll chloroplasts of C3‐plants. Proteolysis of chloroplast proteins begins in an early phase of senescence and the liberated amino acids can be exported to growing parts of the plant (e.g. maturing fruits). Rubisco and other stromal enzymes can be degraded in isolated chloroplasts, implying the involvement of plastidial peptide hydrolases. Whether or not ATP is required and if stromal proteins are modified (e.g. by reactive oxygen species) prior to their degradation are questions still under debate. Several proteins, in particular cysteine proteases, have been demonstrated to be specifically expressed during senescence. Their contribution to the general degradation of chloroplast proteins is unclear. The accumulation in intact cells of peptide fragments and inhibitor studies suggest that multiple degradation pathways may exist for stromal proteins and that vacuolar endopeptidases might also be involved under certain conditions. The breakdown of chlorophyll‐binding proteins associated with the thylakoid membrane is less well investigated. The degradation of these proteins requires the simultaneous catabolism of chlorophylls. The breakdown of chlorophylls has been elucidated during the last decade. Interestingly, nitrogen present in chlorophyll is not exported from senescencing leaves, but remains within the cells in the form of linear tetrapyrrolic catabolites that accumulate in the vacuole. The degradation pathways for chlorophylls and chloroplast proteins are partially interconnected.

Expression and distribution of a vacuolar aquaporin in young and mature leaf tissues of Brassica napus in relation to water fluxes

Recently, it has been shown that water fluxes across biological membranes occur not only through the lipid bilayer but also through specialized water-conducting proteins, the so called aquaporins. In the present study, we investigated in young and mature leaves of Brassica napus L. the expression and localization of a vacuolar aquaporin homologous to radish γ-tonoplast intrinsic protein/vacuolar-membrane integral protein of 23 kDa (TIP/VM 23). In-situ hybridization showed that these tonoplast aquaporins are highly expressed not only in developing but also in mature leaves, which export photosynthates. No substantial differences could be observed between different tissues of young and mature leaves. However, independent of the developmental stage, an immunohistochemical approach revealed that the vacuolar membrane of bundle-sheath cells contained more protein cross-reacting with antibodies raised against radish γ-TIP/VM 23 than the mesophyll cells. The lowest labeling was detected in phloem cells. We compared these results with the distribution of plasma-membrane aquaporins cross-reacting with antibodies detecting a domain conserved among members of the plasma-membrane intrinsic protein 1 (PIP1) subfamily. We observed the same picture as for the vacuolar aquaporins. Furthermore, a high density of gold particles labeling proteins of the PIP1 group could be observed in plasmalemmasomes of the vascular parenchyma. Our results indicate that γ-TIP/VM 23 and PIP1 homologous proteins show a similar expression pattern. Based on these results it is tempting to speculate that bundle-sheath cells play an important role in facilitating water fluxes between the apoplastic and symplastic compartments in close proximity to the vascular tissue.

Influence of Chilling Stress on the Intercellular Distribution of Assimilatory Sulfate Reduction and Thiols in Zea mays

Abstract: The effect of chilling on the intercellular distribution of mRNAs for enzymes of assimilatory sulfate reduction, the activity of adenosine 5′-phosphosulfate reductase (APR), and the level of glutathione was analysed in leaves and roots of maize (Zea mays L). At 25 °C the mRNAs for APR, ATP sulfurylase, and sulfite reductase accumulated in bundle-sheath only, whereas the mRNA for O-acetylserine sulfhydrylase was also detected in mesophyll cells. Glutathione was predominantly detected in mesophyll cells; however, oxidized glutathione was equally distributed between the two cell types. Chilling at 12 °C induced oxidative stress which resulted in increased concentrations of oxidized glutathione in both cell types and a prominent increase of APR mRNA and activity in bundle-sheath cells. After chilling, mRNAs for APR and sulfite reductase, as well as low APR activity, were detected in mesophyll cells. In roots, APR mRNA and activity were at higher levels in root tips than in the mature root and were greatly increased after chilling. These results demonstrate that chilling stress affected the levels and the intercellular distribution of mRNAs for enzymes of sulfate assimilation.

Assimilatory Sulfate Reduction in C3, C3-C4, and C4 Species of Flaveria

The activity of the enzymes catalyzing the first two steps of sulfate assimilation, ATP sulfurylase and adenosine 5'-phosphosulfate reductase (APR), are confined to bundle sheath cells in several C4 monocot species. With the aim to analyze the molecular basis of this distribution and to determine whether it was a prerequisite or a consequence of the C4 photosynthetic mechanism, we compared the intercellular distribution of the activity and the mRNA of APR in C3, C3-C4, C4-like, and C4 species of the dicot genusFlaveria. Measurements of APR activity, mRNA level, and protein accumulation in six Flaveria species revealed that APR activity, cysteine, and glutathione levels were significantly higher in C4-like and C4 species than in C3 and C3-C4 species. ATP sulfurylase and APR mRNA were present at comparable levels in both mesophyll and bundle sheath cells of C4 speciesFlaveria trinervia. Immunogold electron microscopy demonstrated the presence of APR protein in chloroplasts of both cell types. These findings, taken together with results from the literature, show that the localization of assimilatory sulfate reduction in the bundle sheath cells is not ubiquitous among C4 plants and therefore is neither a prerequisite nor a consequence of C4photosynthesis.

Drought stress and carbon assimilation in a warming climate: Reversible and irreversible impacts

Abstract Global change is characterized by increased {CO2} concentration in the atmosphere, increasing average temperature and more frequent extreme events including drought periods, heat waves and flooding. Especially the impacts of drought and of elevated temperature on carbon assimilation are considered in this review. Effects of extreme events on the subcellular level as well as on the whole plant level may be reversible, partially reversible or irreversible. The photosynthetically active biomass depends on the number and the size of mature leaves and the photosynthetic activity in this biomass during stress and subsequent recovery phases. The total area of active leaves is determined by leaf expansion and senescence, while net photosynthesis per leaf area is primarily influenced by stomatal opening (stomatal conductance), mesophyll conductance, activity of the photosynthetic apparatus (light absorption and electron transport, activity of the Calvin cycle) and {CO2} release by decarboxylation reactions (photorespiration, dark respiration). Water status, stomatal opening and leaf temperature represent a "magic triangle" of three strongly interacting parameters. The response of stomata to altered environmental conditions is important for stomatal limitations. Rubisco protein is quite thermotolerant, but the enzyme becomes at elevated temperature more rapidly inactivated (decarbamylation, reversible effect) and must be reactivated by Rubisco activase (carbamylation of a lysine residue). Rubisco activase is present under two forms (encoded by separate genes or products of alternative splicing of the pre-mRNA from one gene) and is very thermosensitive. Rubisco activase was identified as a key protein for photosynthesis at elevated temperature (non-stomatal limitation). During a moderate heat stress Rubisco activase is reversibly inactivated, but during a more severe stress (higher temperature and/or longer exposure) the protein is irreversibly inactivated, insolubilized and finally degraded. On the level of the leaf, this loss of photosynthetic activity may still be reversible when new Rubisco activase is produced by protein synthesis. Rubisco activase as well as enzymes involved in the detoxification of reactive oxygen species or in osmoregulation are considered as important targets for breeding crop plants which are still productive under drought and/or at elevated leaf temperature in a changing climate.

Development and Light Response of Leaves of Metasequoia and Close Relatives

Metasequoia glyptostroboides is a useful nearest living relative (NLR) of the Eocene fossil Metasequoia. Research on modern Metasequoia might give us some clues about its fossil counterpart. During this study the leaf anatomy of Metasequoia, Glyptostrobus, Sequoia and Taxodium was investigated with light microscopy and transmission electron microscopy. Metasequoia exhibits several characteristics of typical sciaphilic plants, such as slightly arched outer cell walls in the adaxial epidermal cells, strongly arched outer cell walls in the abaxial epidermal cells, mesophyll composed of spongy cells, chloroplasts with well-developed grana not only in mesophyll cells but in both the adaxial and abaxial epidermis. Based on comparison of leaf morphology and anatomy, we conclude that Metasequoia is best adapted to low light intensities, Sequoia and Taxodium are intermediate, and Glyptostrobus is adapted to higher light intensities. The effects of light intensity on mesophyll plastids of Metasequoia leaves were studied with trees grown under different light intensities. Metasequoia had the ability to synthesize chlorophyll under complete darkness and was stressed under high light. These characteristics would provide adaptive advantages for Metasequoia to adapt to low intensity, low angle, polar light at their Eocene high latitude paleoenvironments, particularly during the polar spring when light levels are exceedingly low. It provides evidence to explain why Metasequoia was the dominant tree species in Eocene high latitudes. The thesis is written as an article to be submitted to the American Journal of Botany.

Late Miocene palaeoflora and palaeoclimate of Lincang (China)

The Miocene Lincang leaf assemblage is used in this paper as proxy data to reconstruct the palaeoclimate of southwestern Yunnan (SW China) and the evolution of monsoon intensity. Three quantitative methods were chosen for this reconstruction, i.e. Leaf Margin Analysis (LMA), Climate Leaf Analysis Multivariate Program (CLAMP), and the Coexistence Approach (CA). These methods, however, yield inconsistent results, particularly for the precipitation, as also shown in European and other East Asian Cenozoic floras. The wide range of the reconstructed climatic parameters includes the Mean Annual Temperature (MAT) of 18.5-24.7 °C and the Mean Annual Precipitation (MAP) of 1213-3711 mm. Compared with the modern Lincang climate (MAT, 17.3 °C; MAP, 1178.7 mm), the Miocene climate is slightly warmer, wetter and has a higher temperature seasonality. A detailed comparison on the palaeoclimatic variables with the coeval Late Miocene Xiaolongtan flora from the eastern part of Yunnan allows us to investigate the development and interactions of both South Asian and East Asian monsoons during the Late Miocene in southwest China, now under strong influence of these monsoon systems. Our results suggest that the monsoon climate has already been established in southwest Yunnan during the Late Miocene. Furthermore, our results support that both Southeast Asian and East Asian monsoons co-occurred in Yunnan during the Late Miocene.