Modeling lignin biosynthesis: a pathway to renewable chemicals.

Plant biomass contains lignin that can be converted into high-value-added chemicals, fuels, and materials. The precise genetic manipulation of lignin content and composition in plant cells offers substantial environmental and economic benefits. However, the intricate regulatory mechanisms governing lignin formation challenge the development of crops with specific lignin profiles. Mathematical models and computational simulations have recently been employed to gain fundamental understanding of the metabolism of lignin and related phenolic compounds. This review article discusses the strategies used for modeling plant metabolic networks, focusing on the application of mathematical modeling for flux network analysis in monolignol biosynthesis. Furthermore, we highlight how current challenges might be overcome to optimize the use of metabolic modeling approaches for developing lignin-engineered plants.

Cell wall response of field grown Populus to Septoria infection.

Due to its ability to spread quickly and result in tree mortality, Sphaerulina musiva (Septoria) is one of the most severe diseases impacting Populus. Previous studies have identified that Septoria infection induces differential expression of phenylpropanoid biosynthesis genes. However, more extensive characterization of changes to lignin in response to Septoria infection is lacking. To study the changes of lignin due to Septoria infection, four field grown, naturally variant Populus trichocarpa exhibiting visible signs of Septoria infection were sampled at health, infected, and reaction zone regions for cell wall characterization. Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and acid hydrolysis were applied to identify changes to the cell wall, and especially lignin. FTIR and subsequent principal component analysis revealed that infected and reaction zone regions were similar and could be distinguished from the non-infected (healthy) region. NMR results indicated the general trend that infected region had a higher syringyl:guaiacyl ratio and lower p-hydroxybenzoate content than the healthy regions from the same genotype. Finally, Klason lignin content in the infected and/or reaction zone regions was shown to be higher than healthy region, which is consistent with previous observations of periderm development and metabolite profiling. These results provide insights on the response of Populus wood characteristics to Septoria infection, especially between healthy and infected region within the same genotype.

Multi-omic characterization of bifunctional peroxidase 4-coumarate 3-hydroxylase knockdown in Brachypodium distachyon provides insights into lignin modification-associated pleiotropic effects.

A bifunctional peroxidase enzyme, 4-coumarate 3-hydroxylase (C3H/APX), provides a parallel route to the shikimate shunt pathway for the conversion of 4-coumarate to caffeate in the early steps of lignin biosynthesis. Knockdown of C3H/APX (C3H/APX-KD) expression has been shown to reduce the lignin content in Brachypodium distachyon. However, like many other lignin-modified plants, C3H/APX-KDs show unpredictable pleiotropic phenotypes, including stunted growth, delayed senescence, and reduced seed yield. A system-wide level understanding of altered biological processes in lignin-modified plants can help pinpoint the lignin-modification associated growth defects to benefit future studies aiming to negate the yield penalty. Here, a multi-omic approach was used to characterize molecular changes resulting from C3H/APX-KD associated lignin modification and negative growth phenotype in Brachypodium distachyon. Our findings demonstrate that C3H/APX knockdown in Brachypodium stems substantially alters the abundance of enzymes implicated in the phenylpropanoid biosynthetic pathway and disrupt cellular redox homeostasis. Moreover, it elicits plant defense responses associated with intracellular kinases and phytohormone-based signaling to facilitate growth-defense trade-offs. A deeper understanding along with potential targets to mitigate the pleiotropic phenotypes identified in this study could aid to increase the economic feasibility of lignocellulosic biofuel production.

Proteomic and metabolic disturbances in lignin-modified Brachypodium distachyon.

Lignin biosynthesis begins with the deamination of phenylalanine and tyrosine (Tyr) as a key branch point between primary and secondary metabolism in land plants. Here, we used a systems biology approach to investigate the global metabolic responses to lignin pathway perturbations in the model grass Brachypodium distachyon. We identified the lignin biosynthetic protein families and found that ammonia-lyases (ALs) are among the most abundant proteins in lignifying tissues in grasses. Integrated metabolomic and proteomic data support a link between lignin biosynthesis and primary metabolism mediated by the ammonia released from ALs that is recycled for the synthesis of amino acids via glutamine. RNA interference knockdown of lignin genes confirmed that the route of the canonical pathway using shikimate ester intermediates is not essential for lignin formation in Brachypodium, and there is an alternative pathway from Tyr via sinapic acid for the synthesis of syringyl lignin involving yet uncharacterized enzymatic steps. Our findings support a model in which plant ALs play a central role in coordinating the allocation of carbon for lignin synthesis and the nitrogen available for plant growth. Collectively, these data also emphasize the value of integrative multiomic analyses to advance our understanding of plant metabolism.

Phylogenetic Occurrence of the Phenylpropanoid Pathway and Lignin Biosynthesis in Plants.

The phenylpropanoid pathway serves as a rich source of metabolites in plants and provides precursors for lignin biosynthesis. Lignin first appeared in tracheophytes and has been hypothesized to have played pivotal roles in land plant colonization. In this review, we summarize recent progress in defining the lignin biosynthetic pathway in lycophytes, monilophytes, gymnosperms, and angiosperms. In particular, we review the key structural genes involved in p-hydroxyphenyl-, guaiacyl-, and syringyl-lignin biosynthesis across plant taxa and consider and integrate new insights on major transcription factors, such as NACs and MYBs. We also review insight regarding a new transcriptional regulator, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, canonically identified as a key enzyme in the shikimate pathway. We use several case studies, including EPSP synthase, to illustrate the evolution processes of gene duplication and neo-functionalization in lignin biosynthesis. This review provides new insights into the genetic engineering of the lignin biosynthetic pathway to overcome biomass recalcitrance in bioenergy crops.

Targeting hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase for lignin modification in Brachypodium distachyon.

BACKGROUND: Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) is a central enzyme of the so-called "esters" pathway to monolignols. As originally envisioned, HCT functions twice in this pathway, to form coumaroyl shikimate and then, in the "reverse" direction, to convert caffeoyl shikimate to caffeoyl CoA. The discovery of a caffeoyl shikimate esterase (CSE) that forms caffeic acid directly from caffeoyl shikimate calls into question the need for the reverse HCT reaction in lignin biosynthesis. Loss of function of HCT gives severe growth phenotypes in several dicot plants, but less so in some monocots, questioning whether this enzyme, and therefore the shikimate shunt, plays the same role in both monocots and dicots. The model grass Brachypodium distachyon has two HCT genes, but lacks a classical CSE gene. This study was therefore conducted to evaluate the utility of HCT as a target for lignin modification in a species with an "incomplete" shikimate shunt. RESULTS: The kinetic properties of recombinant B. distachyon HCTs were compared with those from Arabidopsis thaliana, Medicago truncatula, and Panicum virgatum (switchgrass) for both the forward and reverse reactions. Along with two M. truncatula HCTs, B. distachyon HCT2 had the least kinetically unfavorable reverse HCT reaction, and this enzyme is induced when HCT1 is down-regulated. Down regulation of B. distachyon HCT1, or co-down-regulation of HCT1 and HCT2, by RNA interference led to reduced lignin levels, with only modest changes in lignin composition and molecular weight. CONCLUSIONS: Down-regulation of HCT1, or co-down-regulation of both HCT genes, in B. distachyon results in less extensive changes in lignin content/composition and cell wall structure than observed following HCT down-regulation in dicots, with little negative impact on biomass yield. Nevertheless, HCT down-regulation leads to significant improvements in biomass saccharification efficiency, making this gene a preferred target for biotechnological improvement of grasses for bioprocessing.

Plant Phenylalanine/Tyrosine Ammonia-lyases.

Aromatic amino acid deaminases are key enzymes mediating carbon flux from primary to secondary metabolism in plants. Recent studies have uncovered a tyrosine ammonia-lyase that contributes to the typical characteristics of grass cell walls and contributes to about 50% of the total lignin synthesized by the plant. Grasses are currently preferred bioenergy feedstocks and lignin is the most important limiting factor in the conversion of plant biomass to liquid biofuels, as well as being an abundant renewable carbon source that can be industrially exploited. Further research on the structure, evolution, regulation, and biological function of functionally distinct ammonia-lyases has multiple implications for improving the economics of the agri-food and biofuel industries.

Lignin biosynthesis: old roads revisited and new roads explored.

Lignin is a major component of secondarily thickened plant cell walls and is considered to be the second most abundant biopolymer on the planet. At one point believed to be the product of a highly controlled polymerization procedure involving just three potential monomeric components (monolignols), it is becoming increasingly clear that the composition of lignin is quite flexible. Furthermore, the biosynthetic pathways to the major monolignols also appear to exhibit flexibility, particularly as regards the early reactions leading to the formation of caffeic acid from coumaric acid. The operation of parallel pathways to caffeic acid occurring at the level of shikimate esters or free acids may help provide robustness to the pathway under different physiological conditions. Several features of the pathway also appear to link monolignol biosynthesis to both generation and detoxification of hydrogen peroxide, one of the oxidants responsible for creating monolignol radicals for polymerization in the apoplast. Monolignol transport to the apoplast is not well understood. It may involve passive diffusion, although this may be targeted to sites of lignin initiation/polymerization by ordered complexes of both biosynthetic enzymes on the cytosolic side of the plasma membrane and structural anchoring of proteins for monolignol oxidation and polymerization on the apoplastic side. We present several hypothetical models to illustrate these ideas and stimulate further research. These are based primarily on studies in model systems, which may or may not reflect the major lignification process in forest trees.

4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase.

Lignin biosynthesis is evolutionarily conserved among higher plants and features a critical 3-hydroxylation reaction involving phenolic esters. However, increasing evidence questions the involvement of a single pathway to lignin formation in vascular plants. Here we describe an enzyme catalyzing the direct 3-hydroxylation of 4-coumarate to caffeate in lignin biosynthesis as a bifunctional peroxidase that oxidizes both ascorbate and 4-coumarate at comparable rates. A combination of biochemical and genetic evidence in the model plants Brachypodium distachyon and Arabidopsis thaliana supports a role for this coumarate 3-hydroxylase (C3H) in the early steps of lignin biosynthesis. The subsequent efficient O-methylation of caffeate to ferulate in grasses is substantiated by in vivo biochemical assays. Our results identify C3H as the only non-membrane bound hydroxylase in the lignin pathway and revise the currently accepted models of lignin biosynthesis, suggesting new gene targets to improve forage and bioenergy crops.

Combining loss of function of FOLYLPOLYGLUTAMATE SYNTHETASE1 and CAFFEOYL-COA 3-O-METHYLTRANSFERASE1 for lignin reduction and improved saccharification efficiency in Arabidopsis thaliana.

BACKGROUND: Downregulation of genes involved in lignin biosynthesis and related biochemical pathways has been used as a strategy to improve biofuel production. Plant C1 metabolism provides the methyl units used for the methylation reactions carried out by two methyltransferases in the lignin biosynthetic pathway: caffeic acid 3-O-methyltransferase (COMT) and caffeoyl-CoA 3-O-methyltransferase (CCoAOMT). Mutations in these genes resulted in lower lignin levels and altered lignin compositions. Reduced lignin levels can also be achieved by mutations in the C1 pathway gene, folylpolyglutamate synthetase1 (FPGS1), in both monocotyledons and dicotyledons, indicating a link between the C1 and lignin biosynthetic pathways. To test if lignin content can be further reduced by combining genetic mutations in C1 metabolism and the lignin biosynthetic pathway, fpgs1ccoaomt1 double mutants were generated and functionally characterized. RESULTS: Double fpgs1ccoaomt1 mutants had lower thioacidolysis lignin monomer yield and acetyl bromide lignin content than the ccoaomt1 or fpgs1 mutants and the plants themselves displayed no obvious long-term negative growth phenotypes. Moreover, extracts from the double mutants had dramatically improved enzymatic polysaccharide hydrolysis efficiencies than the single mutants: 15.1% and 20.7% higher than ccoaomt1 and fpgs1, respectively. The reduced lignin and improved sugar release of fpgs1ccoaomt1 was coupled with changes in cell-wall composition, metabolite profiles, and changes in expression of genes involved in cell-wall and lignin biosynthesis. CONCLUSION: Our observations demonstrate that additional reduction in lignin content and improved sugar release can be achieved by simultaneous downregulation of a gene in the C1 (FPGS1) and lignin biosynthetic (CCOAOMT) pathways. These improvements in sugar accessibility were achieved without introducing unwanted long-term plant growth and developmental defects.

Development and commercialization of reduced lignin alfalfa.

Reducing lignin content in forage legumes can improve digestibility and, correspondingly, animal performance, and alfalfa (Medicago sativa) is the first genetically engineered crop commercialized for improved forage digestibility. Lignin reduction was achieved by downregulating the gene encoding caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), and development of the commercial product, branded as HarvXtra, required the coordination of two research institutions and two companies, and more than 15 years of research and field trials. Lignin modification has positive impacts on forage management. Future developments will likely stack lignin modification with additional forage quality traits.

Role of bifunctional ammonia-lyase in grass cell wall biosynthesis.

L-Phenylalanine ammonia-lyase (PAL) is the first enzyme in the biosynthesis of phenylpropanoid-derived plant compounds such as flavonoids, coumarins and the cell wall polymer lignin. The cell walls of grasses possess higher proportions of syringyl (S)-rich lignins and high levels of esterified coumaric acid compared with those of dicotyledonous plants, and PAL from grasses can also possess tyrosine ammonia-lyase (TAL) activity, the reason for which has remained unclear. Using phylogenetic, transcriptomic and in vitro biochemical analyses, we identified a single homotetrameric bifunctional ammonia-lyase (PTAL) among eight BdPAL enzymes in the model grass species Brachypodium distachyon. (13)C isotope labelling experiments along with BdPTAL1-downregulation in transgenic plants showed that the TAL activity of BdPTAL1 can provide nearly half of the total lignin deposited in Brachypodium, with a preference for S-lignin and wall-bound coumarate biosynthesis, indicating that PTAL function is linked to the characteristic features of grass cell walls. Furthermore, isotope dilution experiments suggest that the pathways to lignin from L-phenylalanine and L-tyrosine are distinct beyond the formation of 4-coumarate, supporting the organization of lignin synthesis enzymes in one or more metabolons.

Redefining Agricultural Residues as Bioenergy Feedstocks.

The use of plant biomass is a sustainable alternative to the reduction of CO2 emissions. Agricultural residues are interesting bioenergy feedstocks because they do not compete with food and add extra value to the crop, which might help to manage these residues in many regions. Breeding crops for dual production of food and bioenergy has been reported previously, but the ideal plant features are different when lignocellulosic residues are burnt for heat or electricity, or fermented for biofuel production. Stover moisture is one of the most important traits in the management of agricultural waste for bioenergy production which can be modified by genetic improvement. A delayed leaf senescence or the stay-green characteristic contributes to higher grain and biomass yield in standard, low nutrient, and drought-prone environments. In addition, the stay-green trait could be favorable for the development of dual purpose varieties because this trait could be associated with a reduction in biomass losses and lodging. On the other hand, the stay-green trait could be detrimental for the management of agricultural waste if it is associated with higher stover moisture at harvest, although this hypothesis has been insufficiently tested. In this paper, a review of traits relevant to the development of dual purpose varieties is presented with particular emphasis on stover moisture and stay-green, because less attention has been paid to these important traits in the literature. The possibility of developing new varieties for combined production is discussed from a breeding perspective.

The cell biology of lignification in higher plants.

BACKGROUND: Lignin is a polyphenolic polymer that strengthens and waterproofs the cell wall of specialized plant cell types. Lignification is part of the normal differentiation programme and functioning of specific cell types, but can also be triggered as a response to various biotic and abiotic stresses in cells that would not otherwise be lignifying. SCOPE: Cell wall lignification exhibits specific characteristics depending on the cell type being considered. These characteristics include the timing of lignification during cell differentiation, the palette of associated enzymes and substrates, the sub-cellular deposition sites, the monomeric composition and the cellular autonomy for lignin monomer production. This review provides an overview of the current understanding of lignin biosynthesis and polymerization at the cell biology level. CONCLUSIONS: The lignification process ranges from full autonomy to complete co-operation depending on the cell type. The different roles of lignin for the function of each specific plant cell type are clearly illustrated by the multiple phenotypic defects exhibited by knock-out mutants in lignin synthesis, which may explain why no general mechanism for lignification has yet been defined. The range of phenotypic effects observed include altered xylem sap transport, loss of mechanical support, reduced seed protection and dispersion, and/or increased pest and disease susceptibility.

Agente comunitário de saúde: percepções na estratégia da saúde da família / Community health agent: family health strategy perceptions / Agente comunitario de salud: percepciones en la estrategia de salud de la familia

O estudo teve como objetivo compreender o significado de ser um Agente Comunitário de Saúde-ACS em umPrograma Saúde da Família. Pesquisa descritiva, qualitativa, com inspiração fenomenológica que buscou a essência dodiscurso do ACS do município de Sete Lagoas-MG. As entrevistas foram gravadas com sete ACS a partir da questãonorteadora: Qual a sua percepção como Agente Comunitário de Saúde na Estratégia Saúde da Família? A análise compreensivadas falas possibilitou a construção de cinco categorias: ACS, elo entre a equipe multiprofissional e a comunidade;valorização profissional; papel do ACS na equipe multiprofissional e sua contribuição para a mudança do modelo deatenção básica; formação profissional; condições de trabalho insalubres. Evidenciou-se que os Agentes de Saúde daFamília têm conhecimento e consciência do seu papel dentro da equipe multiprofissional, sentem-se despreparados paraexercer todas as suas atribuições, desvalorizados em relação à comunidade e a outros membros da equipe. Percebem osriscos ocupacionais da profissão e a falta de ações para minimização destes, além da baixa remuneração.

This study's objective was to understand the meaning of being a Community Health Agent (ACS) in aFamily Health Program (PSF). Descriptive, qualitative, with phenomenological inspiration research that sought the essenceof the ACS? speech, and all them work in the city of Sete Lagoas-MG. The interviews were recorded with 7 ACS, guided bythe question ?what is your perception about family health strategy, as a Community Health Agent?? The comprehensiveanalysis of speech allowed the construction of five categories: ACS, link between the team and the community; professionalevaluation; the ACS role in the team and their contribution to the change of basic care model; training; unhealthy workconditions. It showed that the Agents of Family Health are aware and conscious of their role within the team, feelunprepared to exercise all their tasks, devalued in relation to community and other team members. Also, they perceive theoccupational hazards of the profession and lack of actions to minimize them, beyond the low payment.

El estudio tuvo como objetivo comprender el significado de ser un agente comunitario de salud - ACS en unPrograma de Salud de la Familia (PSF). Investigación descriptiva, cualitativa, con inspiración fenomenológica que buscó laesencia del discurso del ACS en la ciudad de Sete Lagoas-MG. Las entrevistas fueron grabadas con siete ACS a partir decuestión norteadora: ¿cuál es su percepción como un Agente Comunitario de Salud en la Estrategia de Salud de la familia? Elanálisis comprensivo de los discursos posibilitó la construcción de cinco categorías: ACS, enlace entre el equipo multiprofesionaly la comunidad; valorización profesional; el papel del ACS en el equipo multiprofesional y su contribución para la mudanzadel modelo de atención básica; formación profesional; condiciones de trabajo insalubres. Se demostró que el Agente de Saludde la Familia tiene conocimiento y conciencia de su papel dentro del equipo multiprofesional, se sienten despreparados paraejercer todas sus atribuciones, desvalorizados en relación a la comunidad y a otros miembros del equipo. Percibieron losriesgos ocupacionales de la profesión y la falta de acciones para minimización de estos, además de baja remuneración.

Changes in phenolic concentrations during recurrent selection for resistance to the Mediterranean corn borer (Sesamia nonagrioides Lef.).

Recurrent selection has been reported as successful for improving maize resistance against corn borers. This study was conducted to determine if phenolics concentration in maize changes during recurrent selection to improve stalk resistance to the Mediterranean corn borer. Three cycles of selection [EPS12(S)C0, ESP12(S)C2, and EPS12(S)C3] from the maize synthetic population EPS12 and test crosses to inbred lines A639, B93, and EP42 were field grown and artificially infested with Mediterranean corn borer larvae, and the pith tissues were sampled for biochemical analyses. Two major simple phenolic acids [p-coumaric (p-CA) and trans-ferulic (E-FA) acids] were identified in free and cell-wall fractions, whereas four isomers of diferulic acid (DFA) (8-5'l, 5-5', 8-o-4', and 8-5' benzofuran form) were present in the cell-wall bound fraction. The selection cycles EPS12(S)C0 and EPS12(S)C3 showed less damage and higher cell wall phenolics concentrations than the cycle EPS12(S)C2. In addition, higher concentrations of total DFAs were associated with shorter tunnel length and lower numbers of larvae per stem. The current study shows new and concrete evidence that the cell-wall bound phenolics could have a determinative role in the resistance to the Mediterranean corn borer, although future development of recurrent and divergent selection cycles will clarify this point.

Síndrome de Klinefelter associada a lupus eritematoso sitêmico: interferência dos esteróides sexuais / Kinefelter's syndrome associated to systemic lupus erythematous: interference of sexual steroids

A associação de síndrome de Klinefelter e lupus eritematoso sistêmico (LES) é muito rara, com cerca de 21 casos descritos na literatura, Relatamos um caso com desenvolvimento de pericardite, artrite, anemia e alopécia. O uso irregular de testosterona resultou em níveis séricos subnormais deste hormônio, que coincidiram com a abertura do quadro de LES, bem como com duas recidivas, O caso está em consonância com a literatura, que fortalece a hipótese de que os esteróides sexuais tenham um papel relevante sobre o desencadeamento de doenças autoimunes, especialmente o LES.

Asma, Embarazo y Anestesia

Se analizan la fisiopatología del asma y sus implicaciones durante el embarazo. Se destaca el incremento tanto en la prevalencia como en la mortalidad del asma en la última década planteándose la necesidad de reconsiderar su manejo, considerando algunos fármacos relativamente contraindicados y otros que pueden ser utilizados con seguridad durante el embarazo. La anestesia peridural es la técnica de elección en éstas pacientes. Sin embargo, las complicaciones respiratorias son sensiblemente iguales tanto con anestesia general como con anestesia regional especialmente cuando logramos niveles peridurales y/o espinales relativamente altos: T6-4 (T8-6: Niveles óptimos para cesárea)