As moléculas orgânicas podem ser a principal entrada de nitrogênio para plantas em ambientes onde as fontes inorgânicas de nitrogênio são limitadas, como o ambiente epífitico. Estudos recentes têm mostrado que plantas de Vriesea gigantea, uma bromélia epífita formadora de tanque, possuem alta capacidade de absorver ureia, fazendo dela um excelente modelo para estudar o metabolismo de ureia. Entretanto, os processos de absorção e assimilação de ureia estão pouco caracterizados nessas plantas. Várias aquaporinas de plantas têm mostrado ser capazes de facilitar a difusão de ureia através das membranas. Três genes que codificam para aquaporina foliares, VgPIP1;2, VgPIP1;5 e VgTIP2, recentemente foram clonados a partir de plantas V. gigantea tratadas com ureia, sendo que as expressões de VgPIP1,5 e VgTIP2, foram induzidas por essa fonte nitrogenada. No entanto, não tinha sido testado funcionalmente se, de fato, essas aquaporinas seriam capazes de transportar ureia, amônio ou água através das membranas. Uma vez absorvida, a ureia precisa ser metabolizada. Sugere-se que a assimilação do N ocorra por meio da via GS/GOGAT, com prévia hidrólise da ureia pela enzima urease, fornecendo amônio e CO2. Contudo, nunca se analisou a relevância da urease nesse processo em V. gigantea. Dessa maneira, no presente trabalho o transporte de ureia, amônio e água através de VgPIP1;2, VgPIP1;5 e VgTIP2 foi determinado por meio de ensaios de absorção em ovócitos de Xenopus laevis (água e ureia) e de estudos de complementação em Saccharomyces cerevisiae (NH4+/NH3). Os resultados mostraram que, enquanto VgTIP2 facilita o transporte de água quando expresso isoladamente em ovócitos, VgPIP1;2 e VgPIP1;5 precisaram de ser co-expressos com aquaporinas do tipo PIP2 para serem corretamente transportadas para a membrana plasmática e atuem como canais de água. Além disso, VgTIP2 foi a única aquaporina capaz de facilitar a difusão de ureia através das membranas, enquanto que VgPIP1;2 parece ser capaz de transportar NH4+/NH3. Adicionalmente, a relevância da urease no processo de assimilação de ureia foi analisada por meio do perfil isotópico dos aminoácidos em plantas de V. gigantea tratadas com um inibidor da urease (cloranil) antes de fornecer ureia duplamente marcada com C13 e N15. Os experimentos foram conduzidos em plantas nas fases ontogenéticas, atmosférica e adulta-tanque devido a existência de diferenças metabólicas e morfológicas. Os resultados sugeriram que a atividade da urease é um passo limitante na conversão do N da ureia em amônio para sua assimilação. Adicionalmente, foi visto que a diminuição na atividade da urease afeta principalmente a formação de glutamina (Gln) em plantas atmosféricas, enquanto que em plantas adultas-tanque a transaminação é o principal processo prejudicado. A diferença de assimilação de ureia entre as fases ontogenéticas podem ser consequência de diferenças morfológicas associadas com estratégias para captar nutrientes. Além disso, apesar da diminuição da atividade da urease pela ação do inibidor, processos de assimilação direta (sem prévia hidrólise da ureia anterior) em plantas V. gigantea parecem improváveis de acontecer / Organic molecules can be the main input of nitrogen for plants in environments where inorganic nitrogen sources are limited, such as the epiphytic habitat. Recent studies have shown a high capacity of Vriesea gigantean, an epiphytic tank-forming bromeliad, to absorb urea by their leaves, making this bromeliad an excellent model to study urea metabolism. Nevertheless, urea uptake and assimilation processes are little characterized in these plants. Several plant aquaporins from different species are able to facilitate the diffusion of urea through the membranes. Three foliar aquaporin genes, VgPIP1;2, VgPIP1;5 and VgTIP2, have been recently cloned from urea-treated V. gigantea plants. The expression of VgPIP1;5 and VgTIP2 was specifically up-regulated by urea in the basal part of the leaves. Nevertheless, it had not been tested whether these aquaporins were in fact capable of facilitating the membrane diffusion of either urea, ammonium or water. Moreover, it was suggested that after urea absorption, this organic N compound is hydrolyzed by the urease enzyme into CO2 and NH4+ prior to NH4+ assimilation by the GS/GOGAT pathway. In the present project, urea, NH4+/NH3 and water diffusion through VgPIP1;2, VgPIP1;5 and VgTIP2 were determined by uptake studies in Xenopus laevis oocytes (urea and water)and complementation assay in Saccharomyces cerevisiae (NH4+/NH3). The results showed that while VgTIP2 facilitates water transport when expressed alone in oocytes, VgPIP1;2 and VgPIP1;5 needed to be co-expressed with a PIP2 aquaporin to be targeted to the plasma membrane and act as water channels. Moreover, VgTIP2 was the only aquaporin able to facilitate the diffusion of urea through the membrane, while VgPIP1;2 seems to be capable of transporting NH4+/NH3. Additionally, the urease relevance in the urea assimilation process was investigated through the analysis of the amino acid profile in V. gigantea plants kept under a urease inhibitor (chloranil) and supplied with labeled [13C]-[ 15N]2-urea. The experiments were conducted in atmosphheric and adult-tank ontogenetic stages of V. gigantea due to their metabolic and morphological differences. The results suggested that urease activity may be a limiting step in the conversion of N from urea to ammonium. Moreover, decreases in urease activity by chloranil impared the first steps in N assimilation, droping the pool of glutamine (Gln) in atmospheric plants. In adult-tank plants the transamination appeared to be adversely affected. Those differences in urea assimilation might be due to differences in the morphology and the nutrient capture strategies of the ontogenetic phases. Finally, direct urea assimilation process (without previous urea hydrolysis) in V. gigantea plants seems unlikely to occur

Luk Chun-yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 119-131). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.iv / LIST OF FIGURES --- p.x / LIST OF TABLES --- p.xii / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.6 / Chapter 2.1 --- Introduction --- p.7 / Chapter 2.2 --- Ammonia chemistry --- p.10 / Chapter 2.3 --- Ammonia metabolism and excretion --- p.11 / Chapter 2.3.1 --- Ammonia production --- p.11 / Chapter 2.3.2 --- Blood levels of ammonia --- p.12 / Chapter 2.3.3 --- Ammonia Excretion --- p.17 / Chapter 2.4 --- Urea metabolism and excretion --- p.23 / Chapter 2.4.1 --- Urea Chemistry --- p.23 / Chapter 2.4.2 --- Urea production in fishes --- p.24 / Chapter 2.4.3 --- Argininolysis --- p.25 / Chapter 2.4.4 --- Uricolysis --- p.26 / Chapter 2.4.5 --- Ornithine-urea Cycle (OUC) --- p.28 / Chapter 2.4.5.1 --- Tilapia inhabiting the highly alkaline Lake Magadi --- p.32 / Chapter 2.4.5.2 --- High Ambient Ammonia --- p.33 / Chapter 2.4.5.3 --- Air Exposure --- p.34 / Chapter 2.4.5.4 --- Toadfishes --- p.34 / Chapter 2.4.6 --- Blood urea concentration --- p.35 / Chapter 2.4.7 --- Urea excretion in fishes --- p.37 / Chapter 2.4.7.1 --- Branchial urea excretion in fishes --- p.37 / Chapter 2.4.7.2 --- Mechanisms of renal excretion in fishes --- p.40 / Chapter 2.5 --- Influence of environmental salinity on nitrogen excretion in teleosts --- p.42 / Chapter CHAPTER 3 --- BODY COMPOSITION AND UREA BIOSYNTHESIS OF SPAR US SARBA IN DIFFERENT SALINITIES --- p.46 / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Materials and Methods --- p.49 / Chapter 3.2.1 --- Experimental animals --- p.49 / Chapter 3.2.2 --- Tissue sampling --- p.49 / Chapter 3.2.3 --- Water chemistry analysis --- p.50 / Chapter 3.2.4 --- Hematological parameters --- p.50 / Chapter 3.2.5 --- Metabolite and electrolyte contents --- p.51 / Chapter 3.2.6 --- Hepatic enzymes activities --- p.51 / Chapter 3.2.6.1 --- Tissue preparation --- p.51 / Chapter 3.2.6.2 --- Carbamyl phosphate synthetases (CPSases; E.C. 2.7.2.5) --- p.52 / Chapter 3.2.6.3 --- Ornithine carbamoyl transferase (OCTase; E.C. 2.1.3.3) --- p.53 / Chapter 3.2.6.4 --- Argininosuccinate synthetase (ASS; E.C. 6.3.4.5) --- p.54 / Chapter 3.2.6.5 --- Argininosuccinate lyase (ASL; E.C. 4.3.2.1) --- p.54 / Chapter 3.2.6.6 --- Arginase (ARG; 3.5.3.1) --- p.55 / Chapter 3.2.6.7 --- Glutamate dehydrogenase (EC 1.4.1.3) --- p.55 / Chapter 3.2.6.8 --- Uricase (E.C. 1.7.3.3) --- p.56 / Chapter 3.2.6.9 --- Allantoinase --- p.57 / Chapter 3.2.6.10 --- Allantoicase --- p.57 / Chapter 3.2.7 --- Statistical analysis --- p.58 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- "Changes in hepatosmatic index, renal somatic index, muscle water and lipid content and hematological parametersin response to different salinity acclimation" --- p.59 / Chapter 3.3.2 --- Changes in serum chemistry in response to different salinity acclimation --- p.60 / Chapter 3.3.3 --- Changes in hepatic ornithine-urea cycle enzyme activitiesin response to different salinity acclimation --- p.61 / Chapter 3.3.4 --- Changes in GDHase and uricolytic enzyme activitiesin response to different salinity acclimation --- p.62 / Chapter 3.4 --- Discussion --- p.71 / Chapter 3.4.1 --- Hematological responses --- p.72 / Chapter 3.4.2 --- Muscle moisture content --- p.74 / Chapter 3.4.3 --- Circulating electrolyte levels --- p.75 / Chapter 3.4.4 --- Circulating metabolites levels --- p.77 / Chapter 3.4.5 --- Urea metabolism --- p.80 / Chapter 3.4.5.1 --- Ornithine-urea cycle enzymes --- p.80 / Chapter 3.4.5.2 --- Carbamoyl phosphate synthetase isozymes --- p.81 / Chapter 3.4.5.3 --- Uricolytic pathway and argininolysis --- p.85 / Chapter 3.4.5.4 --- Influence of salinity on urea metabolism --- p.86 / Chapter 3.4.6 --- Conclusion --- p.87 / Chapter CHAPTER 4 --- EFFECT OF SALINITY ON NITROGEN EXCRETION OF SPARUS SARBA --- p.88 / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.2 --- Materials and Methods --- p.91 / Chapter 4.2.1 --- Experimental animals --- p.91 / Chapter 4.2.2 --- Experimental protocol --- p.92 / Chapter 4.2.3 --- Determination of net ammonia and urea excretion rates --- p.94 / Chapter 4.2.4 --- Statistical analysis --- p.94 / Chapter 4.3 --- Results --- p.95 / Chapter 4.3.1 --- Net ammonia-N and urea-N excretion rates --- p.95 / Chapter 4.3.2 --- Changes in net ammonia-N and urea-N excretion ratesin response to abrupt hyposmotic exposure --- p.95 / Chapter 4.3.3 --- Changes in net ammonia-N and urea-N excretion rates after exposure to amiloride for 3 hours --- p.96 / Chapter 4.3.4 --- Changes in net urea-N excretion rates in response to elevated body urea levels --- p.96 / Chapter 4.3.5 --- Changes in net ammonia-N excretion rates in response to elevated body ammonia levels --- p.97 / Chapter 4.4 --- Discussion --- p.106 / Chapter 4.4.1 --- Influence of environmental salinity on net ammonia-N and urea-N excretion rates --- p.106 / Chapter 4.4.2 --- Effects of amiloride on nitrogen excretion --- p.109 / Chapter 4.4.3 --- Effect of increased body ammonia on ammonia excretion --- p.113 / Chapter 4.4.4 --- Changes in net urea-N excretion rates in response to elevated body urea levels --- p.113 / Chapter 4.5 --- Conclusion --- p.114 / Chapter CHAPTER 5 --- GENERAL CONCLUSION --- p.115 / references --- p.119