Characterization of three 2-hydroxy-acid dehydrogenases in the context of a biotechnological approach to short-circuit photorespiration

Photorespiration results from the incorporation of oxygen into ribulose-1,5-bisphosphate due to the failure of RuBisCO to properly discriminate between oxygen and carbon dioxide. This process lowers photosynthetic efficiency in that CO2 and ammonia should be re-assimilated with the concomitant consumption of both ATP and reducing power. Two recent approaches, aimed at decreasing the detrimental effects of photorespiration by introducing novel metabolic pathways into plant chloroplasts, show great promise. The goal of this work was to identify and biochemically characterize a single-gene glycolate dehydrogenase for use in further improving the synthetic pathways. Forward and reverse genetics were used to identify three candidate genes in Arabidopsis thaliana; At5g06580, At4g36400 and At4g18360. The proteins encoded by these genes were expressed in Escherichia coli, purified and characterized. Moreover, in silico analysis and the analysis of loss-of-function mutants yielded insights into the significance of these novel enzymatic activities in plant metabolism. AtD-LDH, encoded by At5g06580, is a homodimeric FAD-binding flavoprotein that catalyzes the cytochrome c- dependent oxidation of substrates. The enzyme has high activity with D- and L-lactate, D-2-hydroxybutyrate and D-glycerate, but of these only D-lactate and D-2-hydroxybutyrate are bound with high affinity. Knock-out mutants show impaired growth on medium containing methylglyoxal and D-lactate. Together, the data indicates a role for AtD-LDH in the mitochondrial intermembrane space where it oxidizes D-lactate to pyruvate in the final step of methylglyoxal detoxification. AtD-2HGDH, encoded by At4g36400, is a homodimeric FAD-binding flavoprotein. The enzyme only has activity with D-2-hydroxyglutarate and uses a synthetic electron acceptor in vitro. Metabolic analysis of knock-out mutants reveals high accumulation of D-2-hydroxyglutarate in plants exposed to long periods of extended darkness, confirming that this is the in vivo substrate for the enzyme. Co-expression analysis reveals that AtD-2HGDH is co-expressed with enzymes and transporters participating in the breakdown of lipids, branched-chain amino acids and chlorophyll, all pathways that converge in the production of propionyl-CoA. Together, the data suggest a role for AtD-2HGDH in the mitochondrial matrix where it oxidizes D-2-hydroxyglutarate, most probably originating from propionyl-CoA metabolism, to 2-oxoglutarate, using an electron transfer flavoprotein as an electron acceptor. Finally, AtGOX3, encoded by At4g18360, is a peroxisomal (S)-2-hydroxy-acid oxidase with specificity towards glycolate, L-lactate and L-2-hydroxybutyrate. AtGOX3 is almost exclusively expressed in roots where it might participate in either the metabolism of L-lactate produced during hypoxia, or glycolate produced from glycolaldehyde. In this work, the identification and thorough characterization of three novel enzymatic activities in the model plant A. thaliana are described. Moreover, novel plant metabolic pathways in which these enzymes participate were discovered. The biochemical characterization of these enzymes indicated that they are not suited for use in pathways aimed at decreasing photorespiration and thus, the search for a single-gene glycolate dehydrogenase should continue

Imaging plant germline differentiation within Arabidopsis flowers by light sheet microscopy

In higher plants, germline differentiation occurs during a relatively short period within developing flowers. Understanding of the mechanisms that govern germline differentiation lags behind other plant developmental processes. This is largely because the germline is restricted to relatively few cells buried deep within floral tissues, which makes them difficult to study. To overcome this limitation, we have developed a methodology for live imaging of the germ cell lineage within floral organs of Arabidopsis using light sheet fluorescence microscopy. We have established reporter lines, cultivation conditions, and imaging protocols for high-resolution microscopy of developing flowers continuously for up to several days. We used multiview imagining to reconstruct a three-dimensional model of a flower at subcellular resolution. We demonstrate the power of this approach by capturing male and female meiosis, asymmetric pollen division, movement of meiotic chromosomes, and unusual restitution mitosis in tapetum cells. This method will enable new avenues of research into plant sexual reproduction.Web of Science9art. no. e5254

Habitáculo para peixe zebra para imagens PET: controlo das condições ambientais e de sinais vitais

The zebrafi sh has emerged as an excellent animal model for studies in research in several areas of biomedicine because it has unique characteristics, such as low cost and simplicity of maintenance, short life cycle, sequenced genome, which facilitates its genetic manipulation, and some physiological mechanisms similar to human beings. In preclinical research, it is often necessary to evaluate certain metabolic processes that occur within the animal models under study. For this, molecular and functional imaging techniques are usually used, such as Positron Emission Tomography (PET) . However, this type of medical technique requires the animal to be alive, which, to a certain extent, entails an additional dificulty of morphological imaging systems. In the present work, a zebra sh enclosure was developed to allow the acquisition of PET images in vivo, while monitoring, in real time, the animal's well-being through the analysis of their heart rate. Thus, the zebrafi sh enclosure was designed, developed and built together with vital signs (infrared LED and photodiode) and water temperature (thermistor) monitoring system. Furthermore, the developed system was tested with zebra sh under controlled conditions, and videos of the zebrafi sh were simultaneously acquired once it is visually possible to quantify the heartbeat and operculum using computer vision methods. The range of values obtained by both methods for zebrafi sh heart rate was 80 - 130 bpm, as expected. Moreover, it has also been found that the movement of the operculum makes it challenging to measure the heart rate, but its efect can be attenuated. Thus, the developed system has the particularity of monitoring the water temperature and the zebra fi sh's vital signs, ensuring physiological stability during medical imaging exams.O peixe zebra tem vindo a demonstrar ser um excelente modelo animal para a investigação em diversas áreas da biomedicina, uma vez que apresenta características únicas, como o baixo custo, simplicidade de manutenção, curto ciclo de vida, genoma sequenciado, que facilita a sua manipulação genética e alguns mecanismos fisiológicos semelhantes aos do ser humano. Em investigação pré-clínica, muitas vezes, é necessário avaliar determinados processos metabólicos que ocorrem no interior dos modelos animais em estudo. Para isso, recorre-se usualmente a técnicas de imagem molecular e funcional, como por exemplo a Tomografia por Emissão de Positrões (PET do inglês Positron Emission Tomography). Contudo, este tipo de técnica médica exige que o animal esteja vivo, o que, em certa medida, acarreta uma dificuldade acrescida relativamente aos sistemas de imagem morfológica. No presente trabalho pretende-se desenvolver um habitáculo para peixes zebra que permita realizar a aquisição de imagens PET in vivo, enquanto se monitoriza, em tempo real, o bem-estar do animal, através da análise do batimento cardíaco do mesmo. Assim, o habitáculo do peixe zebra foi pensado, desenhado e construído em conjunto com o sistema de monitorização dos sinais vitais (LED infra vermelho e fotodíodo) e da temperatura da _agua (termístor). O sistema desenvolvido foi testado com peixes zebra em condições controladas e simultaneamente foram adquiridos vídeos dos peixes zebra, onde visualmente é possível quantificar o batimento do coração e dos opérculos por métodos de visão computacional. A gama de valores obtidos por ambos os métodos para o batimento cardíaco do peixe zebra foi de 80 - 130 bpm, conforme esperado. Além disso, também se verificou que o movimento do opérculo dificulta a medição do batimento cardíaco, contudo o seu efeito pode ser atenuado. Assim, o sistema desenvolvido tem a particularidade de monitorizar tanto a temperatura da agua como os sinais vitais do peixe zebra, garantindo a sua estabilidade fisiológica durante os exames de imagem médica.Mestrado em Engenharia Biomédic

Housing Environment and Farm Animals' Well-Being

This reprint contains articles from the Special Issue of Animals “Housing Environment and Farm Animals' Well-Being”, including original research, review, and communication related to livestock and poultry environmental management, air quality control, emissions mitigation, and assessment of animal health and well-being

Airfoil Optimisation with a Genetic Algorithm

In this Master Thesis, an airfoil optimisation using a Genetic Algorithm is developed. This project has been proposed by myself and done with the guidance and supervision of professor Manel Soria. The main idea of the project is to develop from scratch an algorithm capable of finding the optimal airfoil for specific flow conditions, such as the angle of attack, the Reynolds number, and the Mach number. The objective is to create a useful tool for aerospace engineering students so they can use it on their projects and designs during the college years. The work has a first theoretical part about Genetic Algorithms, in which the basic concepts needed to understand the current project are explained. Then, the implementation of the algorithm is fully explained and all the intern processes of the genetic algorithm can be consulted. Several validations of the code have also been made. The Genetic Algorithm created uses crossovers and mutations. The airfoil parametrisation used has been the PARSEC parametrisation and the computation of the aerodynamic coefficients is done with XFOIL. The whole code is written in C language and the analysis and graphs of the results are done with MATLAB and XFLR5. Finally, the algorithm is tested with two real design cases, an airfoil for a heavy lifter aircraft that participated in the Air Cargo Challenge 2017 in Stuttgart, and an airfoil for a glider that flew in the Paper Air Challenge 2015 in ESEIAAT, Terrasa. The results and improvements offered by the algorithm are compared with the results that the designers of these aircraft obtained manually during the design process

CHARACTERIZATION AND VALIDATION OF FLOWERING TIME GENES IN A DIVERSITY PANEL OF RICE (Oryza sativa L.)

In order to meet the needs of a growing population, rice production must be doubled by 2050 in comparison to 2005 production levels. However, rice production faces many challenges including rising temperatures, drought, and salinity stress. Flowering is one of the most heat sensitive periods of the rice life cycle, causing significant decreases to yield if temperatures are too high during the flowering process. As an agronomically important trait, days to flowering determines the climate rice varieties can grow in. Days to flowering is highly variable and quantitative in nature with over 30 genes known to control days to flowering. This study aimed to characterize genes controlling days to flowering through a genome wide association study (GWAS), analysis of genetic variation in known flowering time genes, and genome editing of two known flowering time genes. Genotyping of the material used in the GWAS was performed using a rice 7K SNP chip referred to as the Cornell-IR LD Rice Array (C7AIR). The C7AIR successfully distinguished between the five subgroups of Oryza sativa; however, due to all varieties genotyped being inbred, the amount of heterozygosity was low, causing problems with identifying where the heterozygous cluster should fall when creating the custom cluster file in GenomeStudio. The GWAS identified 5 candidate loci which contribute to days to flowering, including a region co-localizing with the known flowering time genes Hd3a and RFT1. The five genes analyzed for structural variation were highly conserved among the varieties observed with no nonsense, and few missense mutations being observed. Guide RNAs were designed to knockout the function of Hd3a and RFT1; however, the ribonucleoprotein (RNP) transfection was unsuccessful due to the need for optimizing the rice protoplast isolation protocol