Evaluation of different natural ingredients as satiety inductors

Dissertation to obtain a Master’s Degree in Chemical and Biochemical Engineering

Study of grapevine solute transporters involved in berry quality. A biochemical and molecular approach

Tese de Doutoramento em Biologia de Plantas

The design and development of heat extraction technologies for the utilisation of compost thermal energy

A composting Heat Extraction Unit (HEU) was designed to utilise waste heat from decaying organic matter for a variety of heating application The aim was to construct an insulated small scale, sealed, organic matter filled container. In this vessel a process fluid within embedded pipes would absorb thermal energy from the hot compost and transport it to an external heat exchanger. Experiments were conducted on the constituent parts and the final design comprised of a 2046 litre container insulated with polyurethane foam and kingspan with two arrays of qualpex piping embedded in the compost to extract heat. The thermal energy was used in horticultural trials by heating polytunnels using a radiator system during a winter/spring period. The compost derived energy was compared with conventional and renewable energy in the form of an electric fan heater and solar panel. The compost derived energy was able to raise polytunnel temperatures to 2-3°C above the control, with the solar panel contributing no thermal energy during the winter trial and the electric heater the most efficient maintaining temperature at its preset temperature of 10°C. Plants that were cultivated as performance indicators showed no significant difference in growth rates between the heat sources. A follow on experiment conducted using special growing mats for distributing compost thermal energy directly under the plants (Radish, Cabbage, Spinach and Lettuce) displayed more successful growth patterns than those in the control. The compost HEU was also used for more traditional space heating and hot water heating applications. A test space was successfully heated over two trials with varying insulation levels. Maximum internal temperature increases of 7°C and 13°C were recorded for building U-values of 1.6 and 0.53 W/m2K respectively using the HEU. The HEU successfully heated a 60 litre hot water cylinder for 32 days with maximum water temperature increases of 36.5°C recorded. Total energy recovered from the 435 Kg of compost within the HEU during the polytunnel growth trial was 76 kWh which is 3 kWh/day for the 25 days when the HEU was activated. With a mean coefficient of performance level of 6.8 calculated for the HEU the technology is energy efficient. Therefore the compost HEU developed here could be a useful renewable energy technology particularly for small scale rural dwellers and growers with access to significant quantities of organic matter

Estudos químico-agrícolas sôbre o enxofre

1. Analyses of soluble sulphates in 2 N ammonium chloride extracts of 24 samples of soils of the state of São Paulo, Brazil, S. A., showed a sulphur content varying from 0,0013 g per 100 g (found in the b layer of a genuine "terra roxa") to 0,007 g per 100 g of soil (b layer of a soil of depression without definite characteristics). (The results are expressed as elemental sulphur). Determinations of total sulphur in 56 samples of soils of the same state using the method of fusion with sodium carbonate and sodium nitrate revealed 0.007 g of elemental S per 100 g of soil as the lowest value (found in several soil types) and 0.096 g as the highest one (found in the b layer of an ar-quean soil). Apparently soluble sulphates accumulate in the upper layers and total sulphur does the opposite. It was found a strong correlation between total S and carbon content. 2. Under laboratory conditions, in a compost of fresh soil, powdered sulphur and apatite, it was verified after a three months period of incubation that the pH value lowered from 6.30 to 3.23; the citric acid solubility of apatite increased to 271.1 per cent of the original one. Lupinus sp. grown in soil manured with sulphur and apatite has showed fresh and dry weights higher than the plants in control pots; the results are significant at 5% level of probability; phosphorus content is also higher in the manured plants. It was observed a net influence of the apatite plus sulphur treatment on the weight of root nodosities that was four times greater than in the control plants. 3. Nearly five hundred determinations of S, N and P were carried out in 35 species of plants cultivated in the state of São Paulo. A great variation in the amounts of these elements was observed. As a general rule, the leaves contain more sulphur than the stems and roots show the lowest percentages. The conjunct roots and stem of guar (Cyamopsis psoraloides) revealed only 0.019 per cent sulphur; the leaves of kale showed the highest sulphur content, i. e., 2.114%. Apparently there is no correlation between the amounts of S, N and P. The ratio S/N increases from 0.006 (guar) to 0.485 (kale). The ratio S/P, always higher than the corresponding S/N, increases from 0.082 (guar) to 6.381 (older leaves of tomato plants). It is interesting to mention that several among the most important crops in the state of São Paulo namely, cotton, rice, coffee and sugar cane contain more sulphur than phosphorus. 4. Tomato plants cultivated in nutrient solution lacking sulphur showed the following visual symptons of deficiency : chlorosis first in the younger leaves and afterwards in all the leaves; anthocyanin pigments in the petioles and stems; absence of fruits; primary roots stunted and secondary ones longer than in the control plants; stems slender, hard, woody. The histological study of petioles suffering from sulphur deficiency revealed anthocyanin in the parenchyme layer instead of clo-rophyll pigments observed in normal petioles; in the chlorotic leaves the large chloroplasts present only the stroma but the small ones have a little amount of green pigments. Chemical analysis revealed in the abnormal plants : less sulphur and an increased proportion of phosphorus; older leaves contain more sulphur and less phosphorus than the younger ones probably due to physiological difficulties in translocation of sulphur bearing material; increased amount of total N attributed to accumulation of nitrates; marked decrease in ash, sugars and starch; increased proportion of crude fiber and dry material. In the plants suffering from sulphur deficiency photosyntetic rate decreased 34 per cent. 5. Tomato plants were succesfully cultivated in nutrient solution in absence of mineral sulphur but in presence of cysteine. The plants absorbed sulphur, under that form and were able to grow up quite well; the fruiting was normal. In this way rested cleary demonstrated the possibility of absorption of organic sulphur without previous mineralization and its utilization in the building up of protein molecules.

Alterações morfológicas e citológicas do cafeeiro (Coffea arabica L, var. Bourbon (B. Rodr.) Choussy) cultivado em solução nutritiva decorrentes das deficiências e excessos dos macronutrientes

The present work was carried out in order to study: (1) the symptoms of deficiency and excess of macronutrients (N, P, K, Ca, S, Mg) in the coffee plant (Coffea arabica L. var. Mundo novo); (2) the modifications induced by those treatments in the hystological make up of the leaves; (3) the effects of deficiency and excess on the growth and in the chemical composition of the plants. Young coffee plants were grown in nutrient solution, three treatments being used, namely: complete solution (HOAGLAND & ARNON, 1950), deficient solution, in which a giVen element was omitted, and solution with 3 times the concentration of the element under study. The main conclusions can be summaryzed as follows. 1. SYMPTOMS. Clear cut symptoms of malnutrition were observed in the treatments: -N, -P, +P, -Ca, -Mg, -S and +S; the signals - and + stared respectively for deficient and excess level. 2. HISTOLOGICAL EFFECTS. The most definite alterations took place in the treatments +P, -Mg and +S. Usually the characteristics of the chloroplasts were affected: loss of the green color and coalescence into irregular bodies.

Sintomas externos (morfológicos) e internos (anatômicos), observados em folhas de goiabeira (Psidium guajava L.), de plantas cultivadas em solução nutritiva com carência dos macronutrientes

The present work was carried out in order to study: (1) The symptoms of deficiency of macronutrients (N, P, K, Ca, Mg, S) in guava (Psidium guajava L.). (2) The modifications induced by those treatments in the histological make up of the leaves. Young guava plants were grown in nutrient solution two treatments being used, namely: complete solution (HOAGLAND and ARNON, 1950), deficient solution, in which a given element was ommitted. The main conclusions can be summaryzed as follows. a) Symptoms Clear cut symptoms of malnutrition were observed in the treatments: -N, -P, -Ca, -K, -Mg, and -S. The signal - stared respectively for deficient level. b) Histological effects Usually the characteristics of the chloroplasts were affected: loss of the green and coalescence into irregular bodies.

Inducible malondialdehyde pools in zones of cell proliferation and developing tissues in Arabidopsis.

Malondialdehyde (MDA) is a natural and widespread genotoxin. Given its potentially deleterious effects, it is of interest to establish the identities of the cell types containing this aldehyde. We used in situ chemical trapping with 2-thiobarbituric acid and mass spectrometry with a deuterated standard to characterize MDA pools in the vegetative phase in Arabidopsis thaliana. In leaves, MDA occurred predominantly in the intracellular compartment of mesophyll cells and was enriched in chloroplasts where it was derived primarily from triunsaturated fatty acids (TFAs). High levels of MDA (most of which was unbound) were found within dividing cells in the root tip cell proliferation zone. The bulk of this MDA did not originate from TFAs. We confirmed the localization of MDA in transversal root sections. In addition to MDA in proliferating cells near the root tip we found evidence for the presence of MDA in pericyle cells. Remodeling of non-TFA-derived MDA pools occurred when seedlings were infected with the fungus Botrytis cinerea. Treatment of uninfected seedlings with mediators of plant stress responses (jasmonic acid or salicylic acid) increased seedling MDA levels over 20-fold. In summary, major pools of MDA are associated with cell division foci containing stem cells. The aldehyde is pathogen-inducible in these regions and its levels are increased by cellular mediators that impact defense and growth.

Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight.

Transgenic Arabidopsis thaliana (L.) Heynh. plants expressing the three enzymes encoding the biosynthetic route to polyhydroxybutyrate (PHB) are described. These plants accumulated more than 4% of their fresh weight (approximately 40% of their dry weight) in the form of PHB in leaf chloroplasts. These very high producers were obtained and identified following a novel strategy consisting of a rapid GC-MS analysis of a large number of transgenic Arabidopsis plants generated using a triple construct, thus allowing the parallel transfer of all three genes necessary for PHB synthesis in a single transformation event. The level of PHB produced was 4-fold greater than previously published values, thus demonstrating the large potential of plants to produce this renewable resource. However, the high levels of the polymer produced had severe effects on both plant development and metabolism. Stunted growth and a loss of fertility were observed in the high-producing lines. Analysis of the metabolite composition of these lines using a GC-MS method that we have newly developed showed that the accumulation of high levels of PHB was not accompanied by an appreciable change in either the composition or the amount of fatty acids. Substantial changes were, however, observed in the levels of various organic acids, amino acids, sugars and sugar alcohols.

PIF3 is a repressor of chloroplast development.

The phytochrome-interacting factor PIF3 has been proposed to act as a positive regulator of chloroplast development. Here, we show that the pif3 mutant has a phenotype that is similar to the pif1 mutant, lacking the repressor of chloroplast development PIF1, and that a pif1pif3 double mutant has an additive phenotype in all respects. The pif mutants showed elevated protochlorophyllide levels in the dark, and etioplasts of pif mutants contained smaller prolamellar bodies and more prothylakoid membranes than corresponding wild-type seedlings, similar to previous reports of constitutive photomorphogenic mutants. Consistent with this observation, pif1, pif3, and pif1pif3 showed reduced hypocotyl elongation and increased cotyledon opening in the dark. Transfer of 4-d-old dark-grown seedlings to white light resulted in more chlorophyll synthesis in pif mutants over the first 2 h, and analysis of gene expression in dark-grown pif mutants indicated that key tetrapyrrole regulatory genes such as HEMA1 encoding the rate-limiting step in tetrapyrrole synthesis were already elevated 2 d after germination. Circadian regulation of HEMA1 in the dark also showed reduced amplitude and a shorter, variable period in the pif mutants, whereas expression of the core clock components TOC1, CCA1, and LHY was largely unaffected. Expression of both PIF1 and PIF3 was circadian regulated in dark-grown seedlings. PIF1 and PIF3 are proposed to be negative regulators that function to integrate light and circadian control in the regulation of chloroplast development.

Chloroplastic phosphoadenosine phosphosulfate metabolism regulates basal levels of the prohormone jasmonic acid in Arabidopsis leaves.

Levels of the enzymes that produce wound response mediators have to be controlled tightly in unwounded tissues. The Arabidopsis (Arabidopsis thaliana) fatty acid oxygenation up-regulated8 (fou8) mutant catalyzes high rates of alpha -linolenic acid oxygenation and has higher than wild-type levels of the alpha -linolenic acid-derived wound response mediator jasmonic acid (JA) in undamaged leaves. fou8 produces a null allele in the gene SAL1 (also known as FIERY1 or FRY1). Overexpression of the wild-type gene product had the opposite effect of the null allele, suggesting a regulatory role of SAL1 acting in JA synthesis. The biochemical phenotypes in fou8 were complemented when the yeast (Saccharomyces cerevisiae) sulfur metabolism 3'(2'), 5'-bisphosphate nucleotidase MET22 was targeted to chloroplasts in fou8. The data are consistent with a role of SAL1 in the chloroplast-localized dephosphorylation of 3'-phospho-5'-adenosine phosphosulfate to 5'-adenosine phosphosulfate or in a closely related reaction (e.g. 3',5'-bisphosphate dephosphorylation). Furthermore, the fou8 phenotype was genetically suppressed in a triple mutant (fou8 apk1 apk2) affecting chloroplastic 3'-phospho-5'-adenosine phosphosulfate synthesis. These results show that a nucleotide component of the sulfur futile cycle regulates early steps of JA production and basal JA levels.

Changes in anatomy and root cell ultrastructure of soybean genotypes under manganese stress

The deleterious effects of both Mn deficiency and excess on the development of plants have been evaluated with regard to aspects of shoot anatomy, ultrastructure and biochemistry, focusing mainly on the manifestation of visual symptoms. However, there is little information in the literature on changes in the root system in response to Mn supply. The objective of this study was to evaluate the effects of Mn doses (0.5, 2.0 and 200.0 μmol L-1) in a nutrient solution on the anatomy of leaves and roots of the Glycine max (L.) cultivars Santa Rosa, IAC-15 and IAC-Foscarin 31. Visual deficiency symptoms were first observed in Santa Rosa and IAC-15, which were also the only cultivars where Mn-toxicity symptoms were observed. Only in IAC-15, a high Mn supply led to root diameter thickening, but without alteration in cells of the bark, epidermis, exodermis and endodermis. The degree of disorganization of the xylem vessels, in particular the metaxylem, differed in the cultivars. Quantity and shape of the palisade parenchyma cells were influenced by both Mn deficiency and toxicity. A reduction in the number of chloroplasts was observed in the three Mn-deficient genotypes. The anatomical alterations in IAC-15 due to nutritional stress were greater, as expressed in extensive root cell cytoplasm disorganization and increased vacuolation at high Mn doses. The degree of changes in the anatomical and ultrastructural organization of roots and leaves of the soybean genotypes studied differed, suggesting the existence of tolerance mechanisms to different intensities of Mn deficiency or excess.

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.

A genetic and biochemical investigation of malondialdehyde production and turnover in Arabidopsis

Malondialdehyde (MDA) is a small reactive molecule which occurs ubiqui¬tous among eukaryotes. Interest in this molecule stems from the fact that it can be highly reactive. In green tissues of plants it is apparently formed pre¬dominantly by reactive oxygen species (ROS)-mediated non-enzymatic oxi¬dation (nLPO) of triunsaturated fatty acids (TFAs). MDA which is formed by nLPO is widely used as a disease marker and is regarded to be a cel-lular toxin. Surprisingly, sites of ROS production like mitochondria and chloroplasts possess membranes which are enriched in nLPO-prone polyun¬saturated fatty acids (PUFAs). In this work we showed that chloroplasts are the major site of MDA production in leaves of adult Arabidopsis thaliana plants, whereas analyses in seedlings revealed accumulation in meristematic tissues like the root tip, lateral roots and the apical meristem region. Char-acterizing the MDA pools in more detail, we could show that MDA in plants was predominantly present in a free, non-reactive enolate form. This might explain why it is tolerated in sites where its protonated form could poten¬tially damage the genome and proteome. Analyzing the biological fate of MDA in leaves using labeled MDA-isotopes. we were able to show that MDA is metabolized and used to assemble lipids. The major end-point metabolite was identified as 18:3-16:3-monqgalactosyldiacylglycerol (MGDG), which is the most abundant lipid in chloroplasts. We hypothesize that PUFAs in sites of ROS production, like at PS II in chloroplasts, might act as buffers pre¬venting damage of proteins, thereby generating molecules such as MDA. The MDA produced in this way appears predominantly in a non-reactive enolate form in the cell until it fulfills a biological function or until it is metabo¬lized in order to assemble polyunsaturated MGDGs. Additionally, nLPO has been reported to increase in pathogenesis and we challenged seedlings and adult plants with necrotrophic fungi. Monitoring MDA during the in¬fections, we found MDA pools in seedlings were highly inducible although they were tightly controlled in the leaves of adult plants. - Malondialdehyde (MDA) est une petite molecule réactive présente de manière ubiquitaire dans les eucaryotes. L'intérêt de cette molécule vient du fait que celle-ci pourrait être très réactive. Dans les tissus verts des plantes, la majorité du MDA est apparement formée par l'oxydation non-enzymatique (nLPO) des acides gras polyinsaturés (PUFAs) transmis par des espèces ac¬tives d'oxygène (ROS). Le MDA formé par nLPO est souvent utilisé comme marqueur de maladies et il est considéré comme une toxine cellulaire. Etonnament, les sites de production comme les mitochondries et les chloro- plastes sont riches en PUFAs qui sont sensibles à la nLPO. Dans cette thèse nous montrons que les chloroplastes répresentent le site de production de MDA dans les feuilles adultes d'Arabidopsis thaliana. Les analyses de MDA dans les plantules ont révélé que le MDA s'accumule dans les tissus meris- tematiques comme celles de la pointe de la racine, des racines latéralles et du meristème apical. Par la caractérisation du MDA présent nous avons pu montrer que la majorité du MDA était présent sous la forme d'un énolate non-réactif. Ceci pourrait expliquer pourquoi le MDA est toléré dans les sites où il pourrait casser le genome ou le protéome s'il est présent sous sa forme protonée. Les analyses du devenir du MDA dans les feuilles par des isotopes du MDA ont montré que celui-ci est metabolisé et utilisé pour assembler des lipides. Le lipide majoritairement métabolisé a été identifié comme étant le 18:3-16:3-monogalactosyldiacylglycerole (MGDG); le lipide le plus abondant dans les chloroplastes. Nous supposons que la présence des PUFAs dans les sites de production du ROS, tout comme le PS II dans les chloroplastes, pourrait jouer un rôle de tampon pour prevenir les protéines de différentes dégradations et ainsi générer des molécules telle que le MDA. La majorité du MDA produit par cette réaction est présente dans la cellule sous la forme d'énolate non-réactif, jusqu'au moment de son utilisation ou lorsqu'il serra metabolisé pour produire des MGDGs polyinsaturés. De plus, il a été décrit que nLPO pourait augmenter dans la pathogenèse, et nous avons testé des plantes adultes et des plantules en présence de champignons nécrotrophiques. L'observation du MDA pendant les infections a montré que les concentrations en MDA sont fortement induites dans les plantules mais contrôlées dans les plantes adultes.

Chemical defenses in Sacoglossan Opisthobranchs: Taxonomic trends and evolutionary implications

Sacoglossan sea slugs (Mollusca: Opisthobranchia) are one of the few groups of specialist herbivores in the marine environment. Sacoglossans feed suctorially on the cell sap of macroalgae, from which they 'steal' chloroplasts (kleptoplasty) and deterrent substances (kleptochemistry), retaining intracellularly both host plastids and chemicals. The ingested chloroplasts continue to photosynthesize for periods ranging from a few hours or days up to 3 months in some species. Shelled, more primitive sacoglossans feed only on the siphonalean green algal genus Caulerpa, and they do not have functional kleptoplasty. The diet of sacoglossans has radiated out from this ancestral food. Among the shell-less Plakobranchidae (=Elysiidae), the more primitive species feed on other siphonales (families Derbesiaceae, Caulerpaceae, Bryopsidaceae and Codiaceae) and fix carbon, while the more 'advanced' species within the Plakobranchidae and Limapontioidae have a more broad dietary range. Most of these 'advanced' species are unable to fix carbon because the chloroplasts of their food algae are mechanically disrupted during ingestion. Mesoherbivores are likely to be eaten if they live on palatable seaweeds, their cryptic coloration and form not always keeping them safe from predators. Sacoglossans prefer to live on and eat chemically defended seaweeds, and they use ingested algal chemicals as deterrents of potential predators. The most ancestral shelled sacoglossans (Oxynoidae) and some Plakobranchidae such as Elysia translucens, Thuridilla hopei and Bosellia mimetica have developed a diet-derived chemical defense mechanism. Oxynoids and Thuridilla hopei are able to biomodify the algal metabolites. However, the Plakobranchidae Elysia timida and E. viridis, together with Limapontioidea species, are characterized by their ability to de novo synthesize polypropionate metabolites. A whole analysis of kleptoplasty and chemical defenses in sacoglossans may offer a better understanding of the ecology and evolution of these specialized opisthobranchs. In this paper we summarize some of the latest findings, related mainly to Mediterranean species, and offer a plausible evolutionary scenario based on the biological and chemical trends we can distinguish in them.

Evolutionary insights on C4 photosynthetic subtypes in grasses from genomics and phylogenetics

In plants, an oligogene family encodes NADP-malic enzymes (NADP-me), which are responsible for various functions and exhibit different kinetics and expression patterns. In particular, a chloroplast isoform of NADP-me plays a key role in one of the three biochemical subtypes of C4 photosynthesis, an adaptation to warm environments that evolved several times independently during angiosperm diversification. By combining genomic and phylogenetic approaches, this study aimed at identifying the molecular mechanisms linked to the recurrent evolutions of C4-specific NADP-me in grasses (Poaceae). Genes encoding NADP-me (nadpme) were retrieved from genomes of model grasses and isolated from a large sample of C3 and C4 grasses. Genomic and phylogenetic analyses showed that 1) the grass nadpme gene family is composed of four main lineages, one of which is expressed in plastids (nadpme-IV), 2) C4-specific NADP-me evolved at least five times independently from nadpme-IV, and 3) some codons driven by positive selection underwent parallel changes during the multiple C4 origins. The C4 NADP-me being expressed in chloroplasts probably constrained its recurrent evolutions from the only plastid nadpme lineage and this common starting point limited the number of evolutionary paths toward a C4 optimized enzyme, resulting in genetic convergence. In light of the history of nadpme genes, an evolutionary scenario of the C4 phenotype using NADP-me is discussed.