Regulation of Ferulate-5-Hydroxylase Expression in Arabidopsis in the Context of Sinapate Ester Biosynthesis1

Sinapic acid is an intermediate in syringyl lignin biosynthesis in angiosperms, and in some taxa serves as a precursor for soluble secondary metabolites. The biosynthesis and accumulation of the sinapate esters sinapoylglucose, sinapoylmalate, and sinapoylcholine are developmentally regulated in Arabidopsis and other members of the Brassicaceae. The FAH1 locus of Arabidopsis encodes the enzyme ferulate-5-hydroxylase (F5H), which catalyzes the rate-limiting step in syringyl lignin biosynthesis and is required for the production of sinapate esters. Here we show that F5H expression parallels sinapate ester accumulation in developing siliques and seedlings, but is not rate limiting for their biosynthesis. RNA gel-blot analysis indicated that the tissue-specific and developmentally regulated expression of F5H mRNA is distinct from that of other phenylpropanoid genes. Efforts to identify constructs capable of complementing the sinapate ester-deficient phenotype of fah1 mutants demonstrated that F5H expression in leaves is dependent on sequences 3′ of the F5H coding region. In contrast, the positive regulatory function of the downstream region is not required for F5H transcript or sinapoylcholine accumulation in embryos.

Comparison of the Ability of Partially N-Acetylated Chitosans and Chitooligosaccharides to Elicit Resistance Reactions in Wheat Leaves1

Chitin, a linear polysaccharide composed of (1→4)-linked 2-acetamido-2-deoxy-β-d-glucopyranose (GlcNAc) residues, and chitosan, the fully or partially N-acetylated, water-soluble derivative of chitin composed of (1→4)-linked GlcNAc and 2-amino-2-deoxy-β-d-glucopyranose (GlcN), have been proposed as elicitors of defense reactions in higher plants. We tested and compared the ability of purified (1→4)-linked oligomers of GlcNAc (tetramer to decamer) and of GlcN (pentamer and heptamer) and partially N-acetylated chitosans with degrees of acetylation (DA) of 1%, 15%, 35%, 49%, and 60% and average degrees of polymerization between 540 and 1100 to elicit phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, lignin deposition, and microscopically and macroscopically visible necroses when injected into the intercellular spaces of healthy, nonwounded wheat (Triticum aestivum L.) leaves. Purified oligomers of (1→4)-linked GlcN were not active as elicitors, whereas purified oligomers of (1→4)-linked GlcNAc with a degree of polymerization ≥ 7 strongly elicited POD activities but not PAL activities. Partially N-acetylated, polymeric chitosans elicited both PAL and POD activities, and maximum elicitation was observed with chitosans of intermediate DAs. All chitosans but not the chitin oligomers induced the deposition of lignin, the appearance of necrotic cells exhibiting yellow autofluorescence under ultraviolet light, and macroscopically visible necroses; those with intermediate DAs were most active. These results suggest that different mechanisms are involved in the elicitation of POD activities by GlcNAc oligomers, and of PAL and POD activities by partially N-acetylated chitosan polymers and that both enzymes have to be activated for lignin biosynthesis and ensuing necrosis to occur.

Purification and Characterization of Peroxidases Correlated with Lignification in Poplar Xylem1

Lignin is an integral cell wall component of all vascular plants. Peroxidases are widely believed to catalyze the last enzymatic step in the biosynthesis of lignin, the dehydrogenation of the p-coumaryl alcohols. As the first stage in identifying lignin-specific peroxidase isoenzymes, the classical anionic peroxidases found in the xylem of poplar (Populus trichocarpa Trichobel) were purified and characterized. Five different poplar xylem peroxidases (PXP 1, PXP 2, PXP 3–4, PXP 5, and PXP 6) were isolated. All five peroxidases were strongly glycosylated (3.6% to 4.9% N-glucosamine), with apparent molecular masses between 46 and 54 kD and pI values between pH 3.1 and 3.8. Two of the five isolated peroxidases (PXP 3–4 and PXP 5) could oxidize the lignin monomer analog syringaldazine, an activity previously correlated with lignification in poplar. Because these isoenzymes were specifically or preferentially expressed in xylem, PXP 3–4 and PXP 5 are suggested to be involved in lignin polymerization.

Developmental Expression and Substrate Specificities of Alfalfa Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase in Relation to Lignification1

The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze functionally identical reactions in these two pathways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of the two enzymes increase from the first to the sixth internode in stems of alfalfa (Medicago sativa L.), preceding the deposition of lignin. Alfalfa CCOMT is highly similar at the amino acid sequence level to the CCOMT from parsley, although it contains a six-amino acid insertion near the N terminus. Transcripts encoding both COMT and CCOMT are primarily localized to vascular tissue in alfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalyzes O-methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It has low activity against the free acids. COMT expressed in E. coli is active against both caffeic and 5-hydroxyferulic acids, with preference for the latter compound. Surprisingly, very little extractable O-methyltransferase activity versus 5-hydroxyferuloyl CoA is present in alfalfa stem internodes, in which relative O-methyltransferase activity against 5-hy-droxyferulic acid increases with increasing maturity, correlating with increased lignin methoxyl content.

Suppression of O-Methyltransferase Gene by Homologous Sense Transgene in Quaking Aspen Causes Red-Brown Wood Phenotypes1

Homologous sense suppression of a gene encoding lignin pathway caffeic acid O-methyltransferase (CAOMT) in the xylem of quaking aspen (Populus tremuloides Michx.) resulted in transgenic plants exhibiting novel phenotypes with either mottled or complete red-brown coloration in their woody stems. These phenotypes appeared in all independent transgenic lines regenerated with a sense CAOMT construct but were absent from all plants produced with antisense CAOMT. The CAOMT sense transgene expression was undetectable, and the endogenous CAOMT transcript levels and enzyme activity were reduced in the xylem of some transgenic lines. In contrast, the sense transgene conferred overexpression of CAOMT and significant CAOMT activity in all of the transgenic plants' leaves and sclerenchyma, where normally the expression of the endogenous CAOMT gene is negligible. Thus, our results support the notion that the occurrence of sense cosuppression depends on the degree of sequence homology and endogene expression. Furthermore, the suppression of CAOMT in the xylem resulted in the incorporation of a higher amount of coniferyl aldehyde residues into the lignin in the wood of the sense plants. Characterization of the lignins isolated from these transgenic plants revealed that a high amount of coniferyl aldehyde is the origin of the red-brown coloration—a phenotype correlated with CAOMT-deficient maize (Zea mays L.) brown-midrib mutants.

Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases.

The fah1 mutant of Arabidopsis is defective in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. Earlier results indicated that the FAH1 locus encodes ferulate-5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase (P450) of the general phenylpropanoid pathway. We have cloned the gene encoding this P450 by T-DNA tagging and have confirmed the identity of the cloned gene by complementation of the mutant phenotype. F5H shows 34% amino acid sequence identity with the avocado ripening-induced P450 CYP71A1 and 32% identity with the flavonoid-3',5'-hydroxylases of Petunia hybrida. In contrast, it shares much less homology with cinnamate-4-hydroxylase, a P450 that catalyzes the hydroxylation of cinnamic acid three steps earlier in the general phenylpropanoid pathway. Since the highest degree of identity between F5H and previously sequenced P450s is only 34%, F5H identifies a new P450 subfamily that has been designated CYP84.

Metabolismo secundário e ligninas de espécies de Piper

O estudo químico das folhas e dos frutos de P. richardiaefolium resultou no isolamento de oito lignanas, sendo duas lignanas furofurânicas (sesamina e kobusina), quatro lignanas dibenzilbutirolactônicas (hinokinina, kusunokinina, arctigenina e haplomirfolina), duas lignanas dibenzilbutirolactólicas (cubebina e 3,4- dimetoxi-3,4-desmetilenodioxicubebina), dois cinamatos de bornila (ferulato de bornila e cumarato de bornila) e na identificação de duas amidas (piplartina e diidropiplartina). Das folhas de P. richardiaefolium foi extraído e analisado o óleo volátil. As estruturas das substâncias isoladas foram identificadas através de métodos espectroscópicos (RMN de 1H e de 13C e espectrometria de massas). O estudo de análise de componentes principais (PCA) das espécies Piper (P. truncatum - k 616, P. richardiaefolium - k 290, P. richardiaefolium - k 350, P. richardiaefolium - k 593, P. truncatum - k 597, P. pseudopotifolium - k 598, P. richardiaefolium - k 854, P. richardiaefolium - k 610, P. truncatum - k 112, P. pseudopotifolium - k 211 e P. cernuum - k 137) permitiu agrupar as espécies em dois grandes grupos e quatro subgrupos em relação à similaridade entre elas. Ligninas do caule de seis espécies de Piper foram extraídas utilizando o método de degradação de Klason e método de Bjorkman, e analisadas por métodos espectroscópicos (IV, RMN de 1H e de 13C). O método de degradação por oxidação por nitrobenzeno foi o escolhido para determinar a relação entre os monolignóis siringila e guaiacila. Os principais metabólitos das espécies estudadas foram comparados com os tipos de ligninas das mesmas espécies e os resultados sugeriram uma independência entre as vias biossintéticas de ligninas e lignanas.

Painéis de partículas homogêneas cimento-bagaço de cana-de-açúcar curados por carbonatação acelerada

O presente estudo teve como objetivo produzir e avaliar o desempenho de painéis de partículas homogêneas de cimento-bagaço de cana-de-açúcar curados por carbonatação acelerada. Para atingir os resultados foram realizados ensaios de caracterizações morfológica e físico-química das partículas de bagaço de cana-de-açúcar, bem como ensaio de termometria para identificar a compatibilidade da matéria prima (bagaço) com o cimento. Os painéis de partículas cimento-bagaço produzidos foram submetidos a dois processos de cura distintos: 1- cura por 48 h em câmara climática, seguida por 24 h em ambiente com concentração de 15% ±0.6 de CO2, seguida por 24 dias em ambiente saturado ao ar; 2- cura em câmara climática por 48 h, seguida por 25 dias em ambiente saturado ao ar. Ao final dos 28 dias de cura e após ensaio de envelhecimento acelerado de imersão e secagem foram realizadas as caracterizações físico-mecânicas seguindo as recomendações das normativas DIN: 310; 322 e 323, bem como caracterização microestrutural e de condutividade térmica do painel de partículas cimento-bagaço. Os resultados obtidos indicaram que os painéis de partículas cimento-bagaço curados por carbonatação acelerada apresentaram melhor desempenho físico-mecânico quando comparados aos painéis não carbonatados, pois a carbonatação melhorou a interface entre as partículas e a matriz cimentícia, proporcionando maior adesividade entre as fases. E, além disso, reduziu o pH do meio alcalino em que as partículas de bagaço de cana-de-açúcar estão inseridas, minimizando o processo de degradação da lignina, celulose e hemicelulose.

Uma nova metodologia para a síntese de modelos de lignina a partir de reações de inserção O-H entre fenóis de α-aril diazocetonas

A biomassa lignicelulósica tem sua estrutura composta por celulose, hemicelulose e lignina. Dentre essas, a lignina tem se mostrado interessante por ser uma fonte precursora sustentável de fragmentos aromáticos antes obtidos apenas de combustíveis fósseis. Sua estrutura é composta por resíduos de fenilpropanóides p-hidroxibenzeno (H), guaiacil (G) e siringil (S) unidas por ligações C–C e C–O–C em que a ligação β–O–4 é a predominante (mais de 50%). Devido à sua complexidade estrutural e conformacional, a clivagem de suas ligações é pouco seletiva e a caracterização dos fragmentos resultantes é complexa. Uma estratégia comumente empregada para evitar esses desafios é o uso de modelos mais simples. Entretanto, poucas metodologias são reportadas na literatura para a sua síntese e a maioria delas envolve o emprego de halocetonas. O presente trabalho desenvolveu duas novas metodologias promissoras para síntese desses oligômeros, contendo ligação β–O–4 por meio da química de diazo: (a) reação de inserção O–H entre fenol e α–aril diazocetonas, e (b) compostos α–diazo β-cetoéster. Ademais, a utilização de monômeros contendo a função fenol e diazocetona no mesmo anel permitiria a síntese de cadeias de diversos tamanhos em uma única etapa. Como ponto de partida para o estudo, limitou-se à síntese de dímeros, visando entender a reação de inserção O–H. Os produtos desejados foram obtidos em rendimentos de 27–51% após catálise com Cu(hfac)2. Por fim, os modelos de lignina propriamente ditos foram sintetizados após simples adição aldólica e redução em rendimentos globais de 51–78%. Os estudos envolvendo a inserção de fenol em α–diazo β-cetoéster mostraram resultados promissores, corroborando para uma nova estratégia sintética para a obtenção de modelos de lignina. Novos estudos em nosso laboratório estão sendo desenvolvidos para se obter resultados mais conclusivos.

Hydrothermal Carbons from Hemicellulose-Derived Aqueous Hydrolysis Products as Electrode Materials for Supercapacitors

Acid pretreatment of lignocellulosic biomass, required for bioethanol production, generates large amounts of by-products, such as lignin and hydrolyzed hemicellulose fractions, which have found so far very limited applications. In this work, we demonstrate how the recovered hemicellulose hydrolysis products can be effectively utilized as a precursor for the synthesis of functional carbon materials through hydrothermal carbonization (HTC). The morphology and chemical structure of the synthesized HTC carbons are thoroughly characterized to highlight their similarities with glucose-derived HTC carbons. Furthermore, two routes for introducing porosity within the HTC carbon structure are presented: i) silica nanoparticle hard-templating, which is shown to be a viable method for the synthesis of carbonaceous hollow spheres; and ii) KOH chemical activation. The synthesized activated carbons (ACs) show an extremely high porosity (pore volume≈1.0 cm3 g−1) mostly composed of micropores (90 % of total pore volume). Because of their favorable textural properties, the ACs are further tested as electrodes for supercapacitors, yielding very promising results (300 F g−1 at 250 mA g−1) and confirming the high suitability of KOH-activated HTC carbons derived from spruce and corncob hydrolysis products as materials for electric double layer supercapacitors.

Physical and chemical evaluation of furniture waste briquettes

Furniture waste is mainly composed of wood and upholstery foam (mostly polyurethane foam). Both of these have a high calorific value, therefore, energy recovery would be an appropriate process to manage these wastes. Nevertheless, the drawback is that the energy content of these wastes is limited due to their low density mainly that of upholstery foam. Densification of separate foam presents difficulties due to its elastic character. The significance of this work lies in obtaining densified material by co-densification of furniture wood waste and polyurethane foam waste. Densification of furniture wood and the co-densification of furniture wood waste with polyurethane foam have been studied. On the one hand, the parameters that have an effect on the quality of the furniture waste briquettes have been analysed, i.e., moisture content, compaction pressure, presence of lignin, etc. The maximum weight percentage of polyurethane foam that can be added with furniture wood waste to obtain durable briquettes and the optimal moisture were determined. On the other hand, some parameters were analysed in order to evaluate the possible effect on the combustion. The chemical composition of waste wood was compared with untreated wood biomass; the higher nitrogen content and the concentration of some metals were the most important differences, with a significant difference of Ti content.

Potential Shifts in Canadian High Arctic Sedimentary Organic Matter Composition With Permafrost Active Layer Detachments

Increased temperature and precipitation in Arctic regions have led to deeper thawing and structural instability in permafrost soil. The resulting localized disturbances, referred to as active layer detachments (ALDs), may transport organic matter (OM) to more biogeochemically active zones. To examine this further, solid state cross polarization magic angle spinning 13C nuclear magnetic resonance (CPMAS NMR) and biomarker analysis were used to evaluate potential shifts in riverine sediment OM composition due to nearby ALDs within the Cape Bounty Arctic Watershed Observatory, Nunavut, Canada. In sedimentary OM near ALDs, NMR analysis revealed signals indicative of unaltered plant-derived material, likely derived from permafrost. Long chain acyclic aliphatic lipids, steroids, cutin, suberin and lignin occurred in the sediments, consistent with a dominance of plant-derived compounds, some of which may have originated from permafrost-derived OM released by ALDs. OM degradation proxies for sediments near ALDs revealed less alteration in acyclic aliphatic lipids, while constituents such as steroids, cutin, suberin and lignin were found at a relatively advanced stage of degradation. Phospholipid fatty acid analysis indicated that microbial activity was higher near ALDs than downstream but microbial substrate limitation was prevalent within disturbed regions. Our study suggests that, as these systems recover from disturbance, ALDs likely provide permafrost-derived OM to sedimentary environments. This source of OM, which is enriched in labile OM, may alter biogeochemical patterns and enhance microbial respiration within these ecosystems.

Morphomechanical Innovation Drives Explosive Seed Dispersal

How mechanical and biological processes are coordinated across cells, tissues, and organs to produce complex traits is a key question in biology. Cardamine hirsuta, a relative of Arabidopsis thaliana, uses an explosive mechanism to disperse its seeds. We show that this trait evolved through morphomechanical innovations at different spatial scales. At the organ scale, tension within the fruit wall generates the elastic energy required for explosion. This tension is produced by differential contraction of fruit wall tissues through an active mechanism involving turgor pressure, cell geometry, and wall properties of the epidermis. Explosive release of this tension is controlled at the cellular scale by asymmetric lignin deposition within endocarp b cells-a striking pattern that is strictly associated with explosive pod shatter across the Brassicaceae plant family. By bridging these different scales, we present an integrated mechanism for explosive seed dispersal that links evolutionary novelty with complex trait innovation.

Organic geochemistry on sediment profiles from the Arctic Ocean

During the ARCTIC '91-Expedition with RV 'Polarstern', several Multicorer and Kastenlot-cores were recovered along a profile crossing the eastern part of the Arctic Ocean. The investigated cores consist mainly of clayey-silty sediments, and some units with a higher sand content. In this thesis, detailed sedimentological and organic-geochemical investigations were performed. In part, the near surface sediments were AMS-14C dated making it possible to Interpret the results of the organic-geochemical investigations in terms of climatic changes (isotopic stage 2 to the Holocene). The more or less absence of foraminifers within the long cores prevented the development of an oxygen isotope stratigraphy. Only the results of core PS2174-5 from the Amundsen-Basin could be discussed in terms of the climatic change that could be dated back to oxygen isotope stage 7. Detailed organic-geochemical investigations in the central Arctic Ocean are rare. Therefore, several different organic-geochemical methods were used to obtain a wide range of data for the Interpretation of the organic matter. The high organic carbon content of the surface sediments is derived from a high input of terrigenous organic matter. The terrigenous organic material is most likely entrained within the sea-ice On the Siberian shelves and released during ice-drift over the Arctic Ocean. Other factors such as iceberg-transport and turbidites are also responsible for the high input of terrigenous organic matter. Due to the more or less closed sea-ice Cover, the Arctic Ocean is known as a low productivity system. A model shows, that only 2 % of the organic matter in central Arctic Ocean sediments is of a marine origin. The influence of the West-Spitsbergen current increases the marine organic matter content to 16 %. Short chain n-alkanes (C17 and C19) can be used as a marker of marine productivity in the Arctic Ocean. Higher contents of short chain n-alkanes exist in surface sediments of the Lomonosov-Ridge and the Makarov-Basin, indicating a higher marine productivity caused by a reduced sea-ice Cover. The Beaufort-Gyre and Transpolar-Drift drift Patterns could be responsible for the lower sea-ice distribution in this region. The sediments of Stage 2 and Stage 3 in this region are also dominated by a higher content of short chain-nalkanes indicating a comparable ice-drift Pattern during that time. The content and composition of organic carbon in the sediments of core PS2174-5 reflect glaciallinterglacial changes. Interglacial stages 7 and 5e show a low organic carbon content (C 0,5 %) and, as indicated by high hydrogen-indices, low CIN-ratios, higher content of n-alkanes (C17 and C19) and a higher opal content, a higher marine productivity. In the Holocene, a high content of foraminifers, coccoliths, ostracodes, and sponge spicules indicate higher surface-water productivity. Nevertheless, the low hydrogenindices reveal a high content of terrigenous organic matter. Therefore, the Holocene seems to be different from interglacials 7 and 5e. During the glacial periods (stages 6, upper 5, and 4), TOC-values are significantly higher (0.7 to 1.3 %). In addition, low hydrogen-indices, high CIN-ratios, low short chain n-alkanes and opal contents provide evidence for a higher input of terrigenous organic matter and reduced marine productivity. The high lignin content in core sections with high TOC-contents, substantiates the high input of terrigenous organic matter. Changes in the content and composition of the organic carbon is believed to vary with the fluctuations in sea-level and sea-ice coverage.

Biogeochemical parameters of sea water and bottom sediments near the Curieuse Island (Seychelles Islands)

Biogeochemical reef studies carried out in 1981 and 1984 found low concentration of total natural and anthropogenic hydrocarbons in inshore waters. Detection of lignin in marine and bottom sediments indicates that the land has major effect on makeup of organic matter there. Comparison of compositions of organic matter in sea water, suspended matter and bottom sediments indicated that it was altered rapidly by the reef community. Thus, in the inshore zone of the island, runoff from the land is important in supplying nutrients to the reef ecosystem alongside with transport of nutrients by deep waters. Concentrations of nutri¬ents (N, P) in the inshore zone are higher than in waters of the tropical part of the ocean. Nitrogen is the limiting element in development of phytoplankton in the inshore zone.