As aquaporinas são proteínas de membrana presentes em quase todos os órgãos e tecidos de animais e plantas, onde desempenham funções que vão além do transporte de água, transportando também moléculas como ureia, ácido bórico, ácido silícico, amônia, dióxido de carbono e arsênio. Em plantas, as aquaporinas podem ser classificadas de acordo com suas sequências de aminoácidos em cinco subfamílias: proteínas intrínsecas da membrana plasmática (PIPs), proteínas intrínsecas de tonoplastos (TIPs), proteínas intrínsecas do tipo nodulina 26 (NIPs), proteínas intrínsecas pequenas (SIPs) e proteínas intrínsecas não caracterizadas (XIPs). Dados genômicos determinam o número de genes de aquaporinas em 33 para arroz, 35 para Arabidopsis, 71 para algodão e 66 para soja. Dentre as principais culturas utilizadas como alimento, o arroz é particularmente eficiente no acúmulo do semimetal altamente tóxico e carcinogênico arsênio (As), representando um risco significativo para a saúde humana Assim, o principal objetivo deste trabalho é elucidar o papel das aquaporinas no transporte de As em arroz. Utilizando cultivares que apresentam susceptibilidade diferencial ao arsênio, foi analisada a expressão dos genes de aquaporinas em resposta ao tratamento com arsenito em diferentes condições. Para a caracterização dos genes de aquaporinas diferencialmente expressos, foram realizados ensaios de complementação funcional em leveduras. Nossos resultados indicam que membros das subfamílias NIP, TIP, PIP e SIP podem estar envolvidos no transporte e metabolismo de As em arroz, dentre estes, quatro podem estar envolvidos no transporte de As para dentro da célula e seis membros podem estar envolvidos no transporte de As para os vacúolos, fazendo com que essas proteínas sejam candidatas a estratégias de melhoramento genético e fitorremediação. / Aquaporins are membrane proteins present in almost all organs and tissues of animals and plants, where they perform functions that go beyond water transport, also transporting molecules such as urea, boric acid, silicic acid, ammonia, carbon dioxide and arsenic. In plants, aquaporins can be classified according to their ami-no acid sequences into five subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26 like intrinsic proteins (NIPs), small intrinsic proteins (SIPs) and uncharacterized intrinsic proteins (XIPs). Genomic data set the number of aquaporin genes in 33 for rice, 35 for Arabidopsis, 71 for cotton and 66 for soybean. Among the main crops used as food, rice is particularly effi-cient in the accumulation of the highly toxic and carcinogenic metalloid arsenic, thus representing a significant risk to human health. Therefore, the main goal of this work is to elucidate the role of aquaporins in the transport of arsenic in rice. Using cultivars with differential susceptibility to arsenic, the expression of aquaporin genes in response to the arsenite treatment under different conditions was ana-lyzed. For the characterization of differentially expressed aquaporin genes, func-tional complementation assays were performed in yeast cells. Our results indicate that members of the subfamilies NIP, TIP, PIP and SIP may be involved in the transport and metabolism of arsenic in rice, of these, four may be involved in the transport of As into the cell and six members may be involved in transporting As to the vacuoles, making these proteins candidates to genetic improvement strategies and phytoremediation.

As aquaporinas são proteínas de membrana presentes em quase todos os órgãos e tecidos de animais e plantas, onde desempenham funções que vão além do transporte de água, transportando também moléculas como ureia, ácido bórico, ácido silícico, amônia, dióxido de carbono e arsênio. Em plantas, as aquaporinas podem ser classificadas de acordo com suas sequências de aminoácidos em cinco subfamílias: proteínas intrínsecas da membrana plasmática (PIPs), proteínas intrínsecas de tonoplastos (TIPs), proteínas intrínsecas do tipo nodulina 26 (NIPs), proteínas intrínsecas pequenas (SIPs) e proteínas intrínsecas não caracterizadas (XIPs). Dados genômicos determinam o número de genes de aquaporinas em 33 para arroz, 35 para Arabidopsis, 71 para algodão e 66 para soja. Dentre as principais culturas utilizadas como alimento, o arroz é particularmente eficiente no acúmulo do semimetal altamente tóxico e carcinogênico arsênio (As), representando um risco significativo para a saúde humana Assim, o principal objetivo deste trabalho é elucidar o papel das aquaporinas no transporte de As em arroz. Utilizando cultivares que apresentam susceptibilidade diferencial ao arsênio, foi analisada a expressão dos genes de aquaporinas em resposta ao tratamento com arsenito em diferentes condições. Para a caracterização dos genes de aquaporinas diferencialmente expressos, foram realizados ensaios de complementação funcional em leveduras. Nossos resultados indicam que membros das subfamílias NIP, TIP, PIP e SIP podem estar envolvidos no transporte e metabolismo de As em arroz, dentre estes, quatro podem estar envolvidos no transporte de As para dentro da célula e seis membros podem estar envolvidos no transporte de As para os vacúolos, fazendo com que essas proteínas sejam candidatas a estratégias de melhoramento genético e fitorremediação. / Aquaporins are membrane proteins present in almost all organs and tissues of animals and plants, where they perform functions that go beyond water transport, also transporting molecules such as urea, boric acid, silicic acid, ammonia, carbon dioxide and arsenic. In plants, aquaporins can be classified according to their ami-no acid sequences into five subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26 like intrinsic proteins (NIPs), small intrinsic proteins (SIPs) and uncharacterized intrinsic proteins (XIPs). Genomic data set the number of aquaporin genes in 33 for rice, 35 for Arabidopsis, 71 for cotton and 66 for soybean. Among the main crops used as food, rice is particularly effi-cient in the accumulation of the highly toxic and carcinogenic metalloid arsenic, thus representing a significant risk to human health. Therefore, the main goal of this work is to elucidate the role of aquaporins in the transport of arsenic in rice. Using cultivars with differential susceptibility to arsenic, the expression of aquaporin genes in response to the arsenite treatment under different conditions was ana-lyzed. For the characterization of differentially expressed aquaporin genes, func-tional complementation assays were performed in yeast cells. Our results indicate that members of the subfamilies NIP, TIP, PIP and SIP may be involved in the transport and metabolism of arsenic in rice, of these, four may be involved in the transport of As into the cell and six members may be involved in transporting As to the vacuoles, making these proteins candidates to genetic improvement strategies and phytoremediation.

As aquaporinas são proteínas de membrana presentes em quase todos os órgãos e tecidos de animais e plantas, onde desempenham funções que vão além do transporte de água, transportando também moléculas como ureia, ácido bórico, ácido silícico, amônia, dióxido de carbono e arsênio. Em plantas, as aquaporinas podem ser classificadas de acordo com suas sequências de aminoácidos em cinco subfamílias: proteínas intrínsecas da membrana plasmática (PIPs), proteínas intrínsecas de tonoplastos (TIPs), proteínas intrínsecas do tipo nodulina 26 (NIPs), proteínas intrínsecas pequenas (SIPs) e proteínas intrínsecas não caracterizadas (XIPs). Dados genômicos determinam o número de genes de aquaporinas em 33 para arroz, 35 para Arabidopsis, 71 para algodão e 66 para soja. Dentre as principais culturas utilizadas como alimento, o arroz é particularmente eficiente no acúmulo do semimetal altamente tóxico e carcinogênico arsênio (As), representando um risco significativo para a saúde humana Assim, o principal objetivo deste trabalho é elucidar o papel das aquaporinas no transporte de As em arroz. Utilizando cultivares que apresentam susceptibilidade diferencial ao arsênio, foi analisada a expressão dos genes de aquaporinas em resposta ao tratamento com arsenito em diferentes condições. Para a caracterização dos genes de aquaporinas diferencialmente expressos, foram realizados ensaios de complementação funcional em leveduras. Nossos resultados indicam que membros das subfamílias NIP, TIP, PIP e SIP podem estar envolvidos no transporte e metabolismo de As em arroz, dentre estes, quatro podem estar envolvidos no transporte de As para dentro da célula e seis membros podem estar envolvidos no transporte de As para os vacúolos, fazendo com que essas proteínas sejam candidatas a estratégias de melhoramento genético e fitorremediação. / Aquaporins are membrane proteins present in almost all organs and tissues of animals and plants, where they perform functions that go beyond water transport, also transporting molecules such as urea, boric acid, silicic acid, ammonia, carbon dioxide and arsenic. In plants, aquaporins can be classified according to their ami-no acid sequences into five subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26 like intrinsic proteins (NIPs), small intrinsic proteins (SIPs) and uncharacterized intrinsic proteins (XIPs). Genomic data set the number of aquaporin genes in 33 for rice, 35 for Arabidopsis, 71 for cotton and 66 for soybean. Among the main crops used as food, rice is particularly effi-cient in the accumulation of the highly toxic and carcinogenic metalloid arsenic, thus representing a significant risk to human health. Therefore, the main goal of this work is to elucidate the role of aquaporins in the transport of arsenic in rice. Using cultivars with differential susceptibility to arsenic, the expression of aquaporin genes in response to the arsenite treatment under different conditions was ana-lyzed. For the characterization of differentially expressed aquaporin genes, func-tional complementation assays were performed in yeast cells. Our results indicate that members of the subfamilies NIP, TIP, PIP and SIP may be involved in the transport and metabolism of arsenic in rice, of these, four may be involved in the transport of As into the cell and six members may be involved in transporting As to the vacuoles, making these proteins candidates to genetic improvement strategies and phytoremediation.

Hydraulic conductance of roots of the grapevine cultivar, Chardonnay, varies diurnally, peaking at 1400 h. The diurnal amplitude of hydraulic conductance between 600 and 1400 h was not altered when potted grapevines were water-stressed by withholding water for 8 days. However, the diurnal change was greatly reduced for water-stressed Grenache. If the diurnal change in root hydraulic conductance is a result of changes in aquaporin gene expression or activity, it suggests that aquaporins respond differently in water-stressed Chardonnay and Grenache roots. Both Chardonnay and Grenache demonstrated a reduction in hydraulic conductance in response to water stress, with Grenache exhibiting a larger reduction. Suberisation of the roots increased in response to water stress, with complete suberisation of the endodermis occurring closer to the root tip of Grenache compared to the more drought sensitive Chardonnay. The drought sensitive rootstock, 101-14 (V. riparia × V. rupestris) demonstrated a similar reduction in hydraulic conductance to Chardonnay, while drought tolerant 1103 Paulsen (V. berlandieri × V. rupestris) had a non-significant reduction when water-stressed compared to the large reduction observed for drought tolerant Grenache. Therefore, in this study the degree of reduction in hydraulic conductance did not relate to the drought tolerance of the four varieties examined. The impact of partial drying (watering only half the root system) on hydraulic conductance also differed between Chardonnay and Grenache. There was no change in the conductance of the whole root system of Chardonnay due to an increase in conductance of the roots in the wet half which compensated for the reduction on the dry side. In contrast, Grenache did suffer a reduction measured over the whole root system due to a much larger reduction on the dry side compared to Chardonnay. There was an increase in hydraulic conductance on the wet side but this could not compensate for the large reduction on the dry side. Two aquaporins (VvPIP1;1 and VvPIP2;2) were cloned from the roots of grapevine cultivar Chardonnay. The genes were expressed in Xenopus oocytes to determine their osmotic permeability. As has been shown in a number of plant species, VvPIP1;1 was only slightly permeable to water, whereas VvPIP2;2 did transport water. However, when VvPIP1;1 was injected into the oocytes with VvPIP2;2, there was a substantial increase in the osmotic permeability. There was no significant variation in the diurnal expression of VvPIP2;2, whereas VvPIP1;1 showed a peak in expression at 1000 h prior to the peak in hydraulic conductance and peaked again at 1800 h. VvPIP2;2 did not vary in transcript level in response to water stress or rewatering in Chardonnay or Grenache roots. The level of VvPIP1;1 doubled in water stressed Chardonnay roots and declined again when the vines were rewatered 24 h previously. This response to water stress did not occur in Grenache roots. The roots used were from the apical 5 cm. Similar roots were used to measure the water permeability of the cortical cell membranes using the cell pressure probe. Changes in cell membrane permeability in response to water stress corresponded to changes in VvPIP1;1 expression. An experiment to determine if shoot topping had an effect on root hydraulic conductance revealed a significant 50% decline. This response was also observed in soybean (Glycine max L.) and maize (Zea mays L.). A range of experiments have been performed to determine the reason for the decline. Possibilities included a response to final leaf area and reduced transpirational demand; loss of a carbohydrate sink; or hormonal signals such as abscisic acid, auxin and ethylene. At this stage the nature of the positive or negative signal that causes the change in root hydraulic conductance remains elusive. However, the signal did cause a reduction in the transcript level of VvPIP1;1, indicating the involvement of aquaporins in the response. The root hydraulic conductance of grapevines is variable and dependent on factors such as time of day, water-stress, transpiration rate and unknown signals from the shoot. A proportion of this variability is due to changes in aquaporin number or activity. There are also genotypic differences which may be beneficial for future breeding efforts to improve water use efficiency of grapevines. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311202

Hydraulic conductance of roots of the grapevine cultivar, Chardonnay, varies diurnally, peaking at 1400 h. The diurnal amplitude of hydraulic conductance between 600 and 1400 h was not altered when potted grapevines were water-stressed by withholding water for 8 days. However, the diurnal change was greatly reduced for water-stressed Grenache. If the diurnal change in root hydraulic conductance is a result of changes in aquaporin gene expression or activity, it suggests that aquaporins respond differently in water-stressed Chardonnay and Grenache roots. Both Chardonnay and Grenache demonstrated a reduction in hydraulic conductance in response to water stress, with Grenache exhibiting a larger reduction. Suberisation of the roots increased in response to water stress, with complete suberisation of the endodermis occurring closer to the root tip of Grenache compared to the more drought sensitive Chardonnay. The drought sensitive rootstock, 101-14 (V. riparia × V. rupestris) demonstrated a similar reduction in hydraulic conductance to Chardonnay, while drought tolerant 1103 Paulsen (V. berlandieri × V. rupestris) had a non-significant reduction when water-stressed compared to the large reduction observed for drought tolerant Grenache. Therefore, in this study the degree of reduction in hydraulic conductance did not relate to the drought tolerance of the four varieties examined. The impact of partial drying (watering only half the root system) on hydraulic conductance also differed between Chardonnay and Grenache. There was no change in the conductance of the whole root system of Chardonnay due to an increase in conductance of the roots in the wet half which compensated for the reduction on the dry side. In contrast, Grenache did suffer a reduction measured over the whole root system due to a much larger reduction on the dry side compared to Chardonnay. There was an increase in hydraulic conductance on the wet side but this could not compensate for the large reduction on the dry side. Two aquaporins (VvPIP1;1 and VvPIP2;2) were cloned from the roots of grapevine cultivar Chardonnay. The genes were expressed in Xenopus oocytes to determine their osmotic permeability. As has been shown in a number of plant species, VvPIP1;1 was only slightly permeable to water, whereas VvPIP2;2 did transport water. However, when VvPIP1;1 was injected into the oocytes with VvPIP2;2, there was a substantial increase in the osmotic permeability. There was no significant variation in the diurnal expression of VvPIP2;2, whereas VvPIP1;1 showed a peak in expression at 1000 h prior to the peak in hydraulic conductance and peaked again at 1800 h. VvPIP2;2 did not vary in transcript level in response to water stress or rewatering in Chardonnay or Grenache roots. The level of VvPIP1;1 doubled in water stressed Chardonnay roots and declined again when the vines were rewatered 24 h previously. This response to water stress did not occur in Grenache roots. The roots used were from the apical 5 cm. Similar roots were used to measure the water permeability of the cortical cell membranes using the cell pressure probe. Changes in cell membrane permeability in response to water stress corresponded to changes in VvPIP1;1 expression. An experiment to determine if shoot topping had an effect on root hydraulic conductance revealed a significant 50% decline. This response was also observed in soybean (Glycine max L.) and maize (Zea mays L.). A range of experiments have been performed to determine the reason for the decline. Possibilities included a response to final leaf area and reduced transpirational demand; loss of a carbohydrate sink; or hormonal signals such as abscisic acid, auxin and ethylene. At this stage the nature of the positive or negative signal that causes the change in root hydraulic conductance remains elusive. However, the signal did cause a reduction in the transcript level of VvPIP1;1, indicating the involvement of aquaporins in the response. The root hydraulic conductance of grapevines is variable and dependent on factors such as time of day, water-stress, transpiration rate and unknown signals from the shoot. A proportion of this variability is due to changes in aquaporin number or activity. There are also genotypic differences which may be beneficial for future breeding efforts to improve water use efficiency of grapevines. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311202

No description available.

Les aquaporines (AQPs) sont de petites protéines membranaires permettant le passage facilité de l’eau, du glycérol et de certains solutés à travers les membranes biologiques. Elles jouent d’importants rôles de transport transmembranaires ou transcellulaires dans diverses cellules telles que les cellules rénales, mais aussi dans les kératinocytes de l'épiderme. L’épiderme est un épithélium pluristratifié en constant renouvellement. Le calcium extracellulaire joue un rôle important dans le mécanisme de différenciation des kératinocytes.Dans ce travail, nous avons montré que la différenciation induite par le calcium de kératinocytes humains s'accompagne de l’adressage de l’aquaporine-3 (AQP3) du réticulum endoplasmique, vers les membranes plasmiques. Pour étudier la cinétique et les bases moléculaires de cette régulation, notre objectif était de produire des clones stables d'une lignée de kératinocytes humains en culture (HaCat) exprimant une AQP3 fluorescente. Malgré plusieurs tentatives, je n'ai pas pu obtenir ces clones stables. J'ai alors choisi un autre modèle de cellules épithéliales en culture; les cellules MDCK. Nous avons produit deux lignées stables de MDCK exprimant des aquaporines fluorescentes: l'AQP3-GFP et l'AQP2-mCherry. De manière intéressante, dans les cellules MDCK, l'AQP3 -GFP reproduit la régulation de son adressage par le calcium observée dans les kératinocytes humains; dans des cellules MDCK cultivées en présence de 0,15mM de Ca2+, l’AQP3-GFP est localisée dans le réticulum endoplasmique, tandis qu’à 1,5mM de Ca2+ extracellulaire, celle-ci est localisée aux membranes plasmiques. Dans les mêmes conditions, l'AQP2-mCherry conserve une localisation intracellulaire. Par des expériences de « calcium switch », nous avons étudié la cinétique du trafic cellulaire de l'AQP3 et montré que l'adressage de l’AQP3 à la membrane plasmique en réponse au calcium est lent (6h minimum) et semble dépendant non seulement de la différenciation cellulaire, mais aussi de l'établissement de la polarité cellulaire. A l’aide d’inhibiteurs de la PLC et de la PKC, nous avons montré l’implication de cette voie de signalisation, qui dépend du calcium, dans le trafic de l’AQP3. De plus, l'adressage membranaire de l'AQP3 est dépendant du cytosquelette d’actine.En conclusion, nous montrons pour la première fois une régulation du trafic intracellulaire d'une aquaporine par le calcium au cours de la différenciation et de l'établissement de la polarité cellulaire de cellules épithéliales. Cette régulation permet probablement l'hydratation de l'épiderme humain, sans remettre en cause la barrière de perméabilité que constitue la peau. / The aquaporins (AQPs) are small membrane proteins forming water channels and transporters for smal solutes like glycerol. The AQPs play important roles in transmembrane or transcellular transports in various cells, like kidney cells, but also in skin epidermis keratinocytes. The skin epidermis is a pluristratified epithelium, undergoing continuous renewal. Extracellular calcium plays an important role in the differentiation of keratinocytes.In this work, we demonstrate that during calcium-induced differentiation of human keratinocytes, aquaporin-3 (AQP3) is translocated from the endoplasmic reticulum to plasma membranes. In order to study the kinetics and the molecular bases of this regulation, our goal was to produce stable clones of a human keratinocyte cell line (HaCat) expressing a fluorescent AQP3. Despite several trials, i was not able to obtain such clones. Thus i pursued with another epithelial cell line: MDCK cells. We have produced two lines of MDCK cells stably expressing fluorescent AQPs: AQP3-GFP and AQP2mCherry. Interestingly in MDCK cells, AQP3-GFP reproduced the regulated intracellular trafficking observed in human keratinocytes; in MDCK cells grown in a medium containing 0.15 mM Ca2+,, AQP3-GFP was localized in the endoplasmic reticulum. After extracellular Ca2+ was raised to 1.5 mM, AQP3-GFP was seen in plasma membranes. In the same conditions, AQP2-mCherry remained intracellular throughtout the experiment. With calcium-switch experiments, when have then studied the kinetics of AQP3 trafficking. We have shown that targeting of AQP3 to plasma membranes is a slow process (at least 6h) and seems dependent not only of cell differentiation, but also on the establishment of cell polarity. Using inhibitors of PLC and PKC, we have shown the implication of this signalling pathway, which is dependent on calcium, in AQP3 trafficking. In addition we found that plasma membrane expression of AQP3 is dependent on actin cytoskeleton.In conclusion, we show for the first time a regulation of intracelluar trafficking of an aquaporin in calcium-induced differentiation and after establishment of epithelial cell polarity. This regulation likely allows human skin epidermis hydration whithout compromising the permeability barrier of skin.

Les modèles actuels de changements climatiques prédisent une réduction globale des précipitations, limitant ainsi la disponibilité en eau des plantes et induisant une augmentation de la fréquence des stress hydriques. Un stress hydrique modéré peut conduire à la fermeture des stomates ce qui diminue l'activité photosynthétique des plantes, et dans les cas les plus extrêmes, induit l’embolie du xylème causée par la rupture de la colonne d'eau. Afin de limiter ces effets délétères, les plantes ajustent en permanence leurs résistances hydrauliques. Les feuilles sont considérées comme la principale résistance aux flux d'eau. La compréhension des mécanismes de régulation de la résistance hydraulique foliaire est donc un enjeu fondamental. Parmi les facteurs influençant potentiellement la résistance hydraulique foliaire, les aquaporines (AQPs) pourraient jouer un rôle prépondérant. Les membres de la famille des PIP (Plasma membrane Intrinsic Protein) sont des AQPs agissant comme des pores permettant un passage sélectif de l'eau au travers du plasmalemme. Ce travail visait à caractériser la conductance hydraulique foliaire (Kleaf l'inverse de la résistance) et à étudier la contribution potentielle des PIP dans cinq espèces tempérées ainsi que chez l’arbre modèle: le peuplier. Les espèces pionnières ont des valeurs de Kleaf élevées, mais l'augmentation de Kleaf en réponse à la lumière est beaucoup plus faible que chez les espèces non-pionnières. Chez le Noyer (Juglans regia), la lumière bleue est responsable de la plupart de l'augmentation de Kleaf et de la modulation transcriptionnelle des PIP. En situation de disponibilité en eau et de lumière non limitante, la Kleaf du peuplier augmente au cours de la journée pour atteindre son maximum à midi. Cette augmentation de Kleaf se produit simultanément avec la transcription et l'accumulation de protéines PIP en réponse à l’éclairement. Cependant, l'expression des gènes PIP est en partie sous influence d’une régulation endogène circadienne. L’influence des AQPs dans la valeur élevée de Kleaf est démontrée par son inhibition en présence d’HgCl2. / Climate changes models predict a global reduction of rainfalls hence limiting plants water availability and increase water stress occurrence. A moderate water stress can lead to stomata closure thereby decreasing the plants photosynthetic activity and in extreme cases, xylem embolism due to the water column breakage. In order to limit such detrimental effects, plants permanently adjust their hydraulic resistances. Leaves are considered as the main bottleneck in plants water fluxes, thus understanding leaf resistance to water flux regulation is fundamental. Within known factors that can potentially influence leaf hydraulic resistance, the aquaporins (AQPs) might have a central role. Plasma membrane Intrinsic Protein (PIP) family members are AQPs acting as pores that selectively allow water to flow through cell membranes. This work aimed at characterizing leaf hydraulic conductance (Kleaf the inverse of resistance) and investigate the potential contribution of PIPs in five temperate tree species and in tree model: Poplar. Pioneer species have high Kleaf values, but the Kleaf increase in response to light is much lower than for non-pioneer ones. In walnut (Juglans regia) blue light is responsible of most of the Kleaf increase and PIP transcript modulation. Under non-limiting light and water availability, poplar leaf hydraulic conductance increases diurnally with a maximum reached at midday. This increase of Kleaf occurs in parallel with PIP transcript and protein accumulation in response to high irradiance. PIP gene expression is also under endogenous circadian regulation to some extent. The involvement of AQPs is demonstrated by the reduction of Kleaf using HgCl2.

Gram-negative bacterial cells are surrounded by a cell membrane which protects the cell and controls the transport of nutrients and waste products in and out of the cells at a fast rate. This rapid transport of nutrients and wastes through the cell membrane is made possible by channel proteins called porins. Various types of porins present in the cell membrane have specific functions depending on their selectivity towards different nutrients, and channel proteins selective towards water are called aquaporins. These proteins restrict the passage of all entities except water molecules and they provide a fast transport rate of water molecules at 109 molecules/second per channel. The high selectivity of porins has led to their incorporation into synthetic systems, and one example is the addition of porins to separations membranes in order to enhance their performance in terms of selectivity and permeability, in a field called biomimetics. The concept of incorporating aquaporins into synthetic membranes has been studied for the last 10 years in order to enhance the water permeability and selectivity of membranes for water purification; however, there are still limitations such as high costs, difficulties in fabrication of aquaporins, their alignment into synthetic membrane assembly, low stability, and limitations on number of aquaporin molecules that can be introduced into synthetic membranes limit their applicability. In recent years, concurrent with the work on aquaporin-based biomimetic membranes, there has been an increase in the study of synthesizing molecules with similar structure-function relationships of aquaporins. These artificial channels attempt to mimic the high-water permeability and selectivity of aquaporins, while being synthesized using simple chemistry, being solvent compatible, and requiring less space on the membrane surface which helps to incorporate more channels into the membrane assembly. The objectives of this study were to first incorporate aquaporins into synthetic nanofiltration membranes without chemical alteration them to prevent flattening or denaturing of aquaporins; then, the second objective was to install functional groups on aquaporins and align them in the direction of water flow; lastly, the third objective was to synthesize artificial channels in order to overcome the issues with aquaporin stability, alignment, and efficient packing of water channels onto the membrane surface. For the first objective, aquaporins were treated with a polysaccharide, gum Arabic, and incorporated into an amphiphilic polymer, polyvinyl alcohol with alkyl side chains (PVA-alkyl), in order to simulate the natural housing of lipid bilayer for aquaporins and to protect them from denaturing. Long alkyl chains provided the hydrophobic component, while PVA provided the hydrophilic component of the amphiphilic polymer. Membranes modified with aquaporins displayed lower flux declines and higher flux recoveries after reverse flow filtration, along with improved rejection values for both protein and salt solutions as compared to PBI and PBI-PVA-alkyl membranes. However, there was leakage of ions between channels. Therefore, in order to improve the rejection of protons, ions and other impurities, the channels were aligned with the direction of water flow. Functional groups were installed on Aquaporins using site-directed mutagenesis for covalent attachment to the polymer matrix so that the proteins could be immobilized to the membranes and aligned in the direction of the flow. Aquaporin constructs were modified to bear affinity tags or unique amino acids at the N-terminus of the aquaporin molecule, which was used to facilitate directional immobilization. Each aquaporin monomer was modified with a unique amino acid Cys group at the N-terminus right after the first Met, and due to the aquaporin tetrameric nature, these Cys groups became four anchors for attachment. The presence of these four Cys anchors per aquaporin tetramer was used to attach on the membrane surface in alignment with the feed water flow direction. Membranes modified with mutated aquaporins showed consistently higher salt rejection values of ~70% irrespective of feed concentration, along with higher flux recoveries and lower flux declines. Commercial NF-270 membranes provide a monovalent salt (NaCl) rejection of ~50% and divalent salt (MgCl2) rejection of 97%. Also, approximate coverage of membrane surface with attached aquaporins was calculated using simulation studies. Simulation studies showed that immobilized aquaporins with PVA-alkyl provided a diffusion rate equivalent to 64% coverage on the membrane surface. This showed that aquaporins didn’t cover the entire surface area of the membrane. However, immobilized aquaporins were responsible for the rejection of a portion of ions passing through the membrane. In order to overcome the limitations of aquaporin incorporation into polymer membranes, artificial organic frameworks were added as surface modification on PBI membranes. Organic frameworks were synthesized as derivatives of hybrid bisamides. The series of bisamides 1-4 consist of 6-amino-pyridine-2-dicarboxylic acid, 6-hydroxymethyl-pyridine-2-carboxylic acid and ethylenediamine, trimethylenediamine, putrescine, and cadaverine depending on the length of carbon chain. These frameworks are amphiphilic in nature and have strong chemical attachment due to the presence of amines and carboxylic acids into each building block. These molecules were introduced into the membrane matrix using carbodiimide chemistry. FTIR results showed the attachment of these bisamide molecules onto the surface of a modified PBI membrane. Also, modified membranes showed a reduced molecular weight cut off (MWCO) for neutral organic molecules. Overall, membranes modified with aquaporins have shown a potential to provide consistently high salt rejections with increasing feed solutions. Also, preliminary results have shown that bisamide molecules can be attached onto the membrane surface as organic frameworks and have a potential to be an alternative for aquaporins based biomimetic membranes.

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