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Der programmierte Zelltod (PCD) stellt einen wichtigen Prozess bei der Entwicklung, Seneszenz und Pathogenabwehr von Pflanzen und Tieren dar. Zum Wohle des Gesamtorganismus kommt es zum genetisch regulierten und gezielten Selbstmord einzelner Zellen. Bei Pflanzen ist PCD vor allem im Rahmen der „hypersensitiven Abwehrreaktion“ (HR) von enormer Wichtigkeit. Sie schützen sich vor schädlichen Eindringlingen, indem infizierte Pflanzenzellen durch PCD gezielt getötet werden. So wird eine räumliche Barriere zwischen Wirt und Pathogen geschaffen und die systemische Ausbreitung von biotrophen Pathogenen verhindert. Die Induktion von PCD erfolgt über komplexe Signalwege. Als Signalsubstanzen spielen reaktive Sauerstoffspezies (ROS), vor allem H2O2, eine wesentliche Rolle. Der Transport von H2O2 über Zellmembranen hinweg erfolgt über Aquaporine. Da intrazelluläre H2O2-Konzentrationen darüber entscheiden wie vital eine Zelle ist, ist die räumliche und zeitliche Kontrolle dieses H2O2-Transports unabdingbar. Als potentieller Regulator der Kanalfunktion der Aquaporine wurde das Protein AQUAPORIN INTERACTOR 1 (AQI1) isoliert. Bei AQI1 handelt es sich um ein pflanzliches Protein mit Homologie zu Aminoacylase 1 aus Säugern. Über Aminoacylasen ist bekannt, dass sie die Hydrolyse acylierter Aminosäuren katalysieren. Die physiologische Funktion dieser Enzyme ist bisher jedoch kaum verstanden. In dieser Arbeit erfolgte erstmals die Charakterisierung einer Aminoacylase (AQI1) aus Pflanzen. Es wurde die physiologische Funktion von AQI1 als Regulator von Aquaporinen, sowie die zugrunde liegenden molekularen Mechanismen analysiert. Hierbei konnte gezeigt werden, dass das pflanzliche Protein AQI1, neben der Deacetylierung von Aminosäuren, eine weitere Aufgabe erfüllen kann. Durch Protein-Protein Interaktion nimmt AQI1 Einfluss auf die Kanalaktivität von Aquaporinen. AQI1 kann den H2O2-Einstrom, sowie den H2O-Einstrom durch Aquaporine inhibieren. Dies erfolgt vermutlich in Form einer Blockade der Aquaporinpore. Durch diese Funktion ist AQI1 wesentlich an der Zelltodregulation in Pflanzen beteiligt. Im Zuge der HR kommt es zur vermehrten Akkumulation von AQI1. Dies verhindert den Eintritt toxischer Mengen H2O2 in die Nachbarzellen, um so die lokale Kontrolle des PCD zu gewährleisten. Zudem scheint AQI1 an der Regulation von Seneszenzprozessen beteiligt zu sein. Es konnte gezeigt werden, dass AQI1 einem Altersgradienten folgend, in jungem Blattgewebe stark akkumuliert, wohingegen es in älterem Blattgewebe abgebaut wird. Durch diese altersabhängige Akkumulation könnte AQI1 zur Vitalität von Blättern beitragen, indem es in jungen Blättern den Eintritt zu hoher H2O2-Konzentrationen in die Zellen verhindert. Programmed cell death (PCD) is an important process during development, senescence and pathogen defence in plants and in animals. It is a genetically regulated and targeted cell suicide of single cells, for benefit of the whole organism. In plants, PCD is of great importance, especially in the course of the “hypersensitive response” (HR). For protecting themselves against harmful intruders, infected plant cells are directly deposed of by PCD. The developing local lesions act as a barrier between host plant and pathogen. This prevents the systemic expansion of biotrophic pathogens within the whole plant. The induction of PCD involves complex signal transduction pathways. Reactive oxygen species (ROS), in particular H2O2, play an important role as signal molecules during PCD. The transport of H2O2 across cell membranes is conducted by aquaporins. As the vitality of cells depends on intracellular H2O2-levels, a spatiotemporal control of this H2O2-transport is indispensable. AQUAPORIN INTERACTOR 1 (AQI1) was isolated as a potential regulator of the channel function of aquaporins. AQI1 is a plant protein with sequence homology to the mammal aminoacylase 1. It is known, that aminoacylases catalyse the hydrolysis of acyl-amino acids. However, the physiological function of these enzymes is still unclear. This study represents the first characterisation of an aminoacylase (AQI1) in plants. The physiological function of AQI1 as a regulator of aquaporins, as well as the underlying molecular mechanisms, have been analysed. In addition to deacetylation of amino acids, a second function of the protein AQI1 was discovered. AQI1 interferes with the channel activity of aquaporins by protein-protein interaction. In this way, AQI1 is able to inhibit the H2O2-, and to a certain extent also the H2O-influx, through aquaporins. Probably, this happens by blocking the aquaporinpore. Due to this function, AQI1 is a major component in cell death regulation in plants. During the „hypersensitive response“ (HR), which is induced as a result of pathogen attack, AQI1 accumulates to high levels to prevent the influx of toxic amounts of H2O2 into neighbouring cells. This ensures a local control of PCD. In addition, AQI1 seems to be involved in regulation of senescence processes. It could be demonstrated, that AQI1 accumulates in a gradient from juvenile to senescent leaves, due to degradation in older tissues. By this age-dependent accumulation, AQI1 could contribute to the vitality of leaves, by preventing the influx of excessive amounts of H2O2 into the cell.

Nos travaux sur la dynamique endomembranaire des aquaporines PIP indiquent que celle-ci est déterminée par des mécanismes généraux et spécifiques. Ainsi, le signal d’export hors du RE de l’isoforme AtPIP2;1 se trouve bien être un motif di-acide, mais dont la séquence DVE ne peut subir de substitution par aucune autre combinaison de résidus acides. De plus, le cyclage de cette isoforme entre la PM et les compartiments intracellulaires suit le modèle établi pour la famille des PIN bien que les effets de l’auxine sur le cyclage d’AtPIP2;1 semblent s’exercer à un niveau qui n’a pas été décrit. Par ailleurs, deux de nos études suggèrent, par des approches indépendantes, que l’endocytose ne ferait pas intervenir la clathrine sous stress salin. Nous projetons de vérifier cette hypothèse par des approches génétiques. La dissection d’un mécanisme d’endocytose indépendant de la clathrine chez les plantes serait de toute évidence originale. Le projet de recherche que je souhaiterais développer continuera à porter sur les aquaporines, et plus particulièrement les PIP, comme objets moléculaires. Il portera sur la découverte des mécanismes moléculaires qui gouvernent leur trafic endomembranaire et les régulations qui contrôlent leur localisation en réponse aux contraintes de l’environnement.

Aquaporins are channel proteins present in the plasma membrane and most of intracellular compartments of plant cells. This review focuses on recent insights into the cellular function of plant aquaporins, with an emphasis on the subfamily of Plasma membrane Intrinsic Proteins (PIPs). Whereas PIPs mostly serve as water channels, novel functions associated with their ability to transport carbon dioxide and hydrogen peroxide are emerging. Phosphorylation of PIPs was found to play a central role in the mechanisms that determine their gating and subcellular dynamics. Dynamic tracking of single aquaporin molecules in native plant membranes and the search for cell signaling intermediates acting upstream of aquaporins are now used to dissect their cellular regulation by hormonal and environmental stimuli.

International audience; In plants, aquaporins play a crucial role in regulating root water transport in response to environmental and physiological cues. Controls achieved at the post-translational level are thought to be of critical importance for regulating aquaporin function. To investigate the general molecular mechanisms involved, we performed, using the model species Arabidopsis, a comprehensive proteomic analysis of root aquaporins in a large set of physiological contexts. We identified nine physiological treatments that modulate root hydraulics in time frames of minutes (NO and H2O2 treatments), hours (mannitol and NaCl treatments, exposure to darkness and reversal with sucrose, phosphate supply to phosphate-starved roots), or days (phosphate or nitrogen starvation). All treatments induced inhibition of root water transport except for sucrose supply to dark-grown plants and phosphate resupply to phosphate-starved plants, which had opposing effects. Using a robust label-free quantitative proteomic methodology, we identified 12 of 13 plasma membrane intrinsic protein (PIP) aquaporin isoforms, 4 of the 10 tonoplast intrinsic protein isoforms, and a diversity of post-translational modifications including phosphorylation, methylation, deamidation, and acetylation. A total of 55 aquaporin peptides displayed significant changes after treatments and enabled the identification of specific and as yet unknown patterns of response to stimuli. The data show that the regulation of PIP and tonoplast intrinsic protein abundance was involved in response to a few treatments (i.e. NaCl, NO, and nitrate starvation), whereas changes in the phosphorylation status of PIP aquaporins were positively correlated to changes in root hydraulic conductivity in the whole set of treatments. The identification of in vivo deamidated forms of aquaporins and their stimulus-induced changes in abundance may reflect a new mechanism of aquaporin regulation. The overall work provides deep insights into the in vivo post-translational events triggered by environmental constraints and their possible role in regulating plant water status.

Genetics of root hydraulics in Arabidopsis thaliana . Genomic, physiological and breeding approaches for enhancing drought resistance in crops. Environmental Workshop of Universidad Internacional de Andalucia

Aquaporins and plant adaptation to a changing environment. Environmental Workshop of Universidad Internacional de Andalucia: Genomic, physiological and breeding approaches for enhancing drought resistance in crops

Coordinated post-translational responses of aquaporins to abiotic and nutritional stimuli in Arabidopsis roots . 7th European Plant Science Organisation (EPSO) Conference

Control of Arabidopsis leaf hydraulics involves light-dependent phosphorylation of aquaporins in veins. International Symposium on Plant Photobiology

Plant aquaporin endomembrane trafficking and dynamics. 13th International Workshop on Plant Membrane Biology

Emerging functions of aquaporins in Arabidopsis. 13th International Worshop on Plant Membrane Biology

Die vorliegende Arbeit behandelte drei Themen: 1) Klärung der umstrittenen Wasserleitfähigkeit von PfAQP, 2) Ammoniakleitfähigkeit von Aquaglyceroporinen am Beispiel von hAQP8 und PfAQP und 3) H2O2-Permeabilität von Aquaporinen und Aquaglyceroporinen. Durch den Einsatz verschiedener Polyole (Saccharose, Sorbitol, PEG300 und Glycerol) konnte die Wasserleitfähigkeit von PfAQP und EcGlpF geklärt werden. EcGlpF, wie viele Aquaglyceroporine, kann Glycerol aber praktisch kein Wasser transportieren. Die Wasserleitfähigkeit von PfAQP ist von dem eingesetzten Polyol abhängig. In der großen cyclischen Saccharose kann PfAQP sowohl Wasser als auch Glycerol gut leiten. Dagegen hemmt Sorbitol den Wassertransport (IC50 = 0,81 M) und erniedrigt die Glycerolpermeabilität um 80 %. Der Hemmeffekt von Sorbitol beruht nicht auf das Eindringen und Verschließen des Kanals sondern auf die Interaktion mit dem Einmündungsbereich. Glycerol wird durch PfAQP besser transportiert als Harnstoff, auch wenn beide Solute in unterschiedlichen Verhältnissen gemischt sind. Somit trifft die Molenbruch-Anomalie für die Aquaporine unter den herrschenden Testbedingungen nicht zu. Es konnte experimentell ein Protokoll zur Beladung von Hefezellen mit dem pH-sensitiven Fluoresceindiacetat (FDA) etabliert werden, mit dessen Hilfe die Ammoniakleitfähigkeit von Aquaporinen und Aquaglyceroporinen gemessen werden kann. Damit wurde die Ammoniakpermeabilität von hAQP8 und PfAQP gemessen. Beide Aquaporine erhöhten die Ammoniakpermeabilität um das drei- bzw. zweifache gegenüber den Kontrollzellen. Im letzten Teil wurde die H2O2-Leitfähigkeit beider AQP-Subfamilien gegenübergestellt. Die schnell wasserleitenden Isoformen (hAQP1, rAQP1 und hAQP8) sind in der Lage H2O2 schneller zu transportieren als die glycerolleitenden hAP9 und PfAQP. Die Mutante rAQP1 H180A besitzt eine 4mal größere ar/R-Region als der Wildtyp. Um den gleichen Faktor erhöhte sich die H2O2-Leitfähigkeit dieser Mutante. Im Gegensatz dazu verringerte sich die H2O2-Leitfähigkeit von Mutanten, in denen die ar/R-Region verkleinert wurde wie z. B. hAQP8 I200H und rAQP1 H180F.

UMR AGAP équipe Diversité, adaptation et amélioration de la vigne (DAAV); Hydraulic conductance and water transport in plants may be affected by environmental factors, which in turn regulate leaf gas exchange, plant growth and yield. In this study, we assessed the combined effects of radiation and water regimes on leaf stomatal conductance (g(s)), petiole specific hydraulic conductivity (K-petiole) and anatomy (vessel number and size); and leaf aquaporin gene expression of field-grown grapevines at the Agroscope Research Station (Leytron, Switzerland). Chasselas vines were subjected to two radiation (sun and shade) levels combined with two water (irrigated and water-stressed) regimes. The sun and shade leaves received similar to 61.2 and 1.48 mol m(-2) day(-1) of photosynthetically active radiation, respectively, during a clear-sky day. The irrigated vines were watered weekly from bloom to veraison whereas the water-stressed vines did not receive any irrigation during the season. Water stress reduced gs and Kpetiole relative to irrigated vines throughout the season. The petioles from water-stressed vines showed fewer large-sized vessels than those from irrigated vines. The shaded leaves from the irrigated vines exhibited a higher Kpetiole than the sun leaves at the end of the season, which was partially explained by a higher number of vessels per petiole and possibly by the upregulation of some of the aquaporins measured in the leaf. These results suggest that not only plant water status but also the light environment at the leaf level affected leaf and petiole hydraulics.

To help understand leaf hydraulic conductance (K-leaf) modulation under high irradiance, well-watered poplars (Populus trichocarpa Torr. & Gray ex Hook and Populus nigra L.) were studied diurnally at molecular and ecophysiological scales. Transcriptional and translational modulations of plasma membrane intrinsic protein (PIP) aquaporins were evaluated in leaf samples during diurnal time courses. Among the 15 poplar PIP genes, a subset of two PIP1s and seven PIP2s are precociously induced within the first hour of the photoperiod concomitantly with a K-leaf increase. Since expression patterns were cyclic and reproducible over several days, we hypothesized that endogenous signals could be involved in PIP transcriptional regulation. To address this question, plants were submitted to forced darkness during their subjective photoperiod and compared with their control counterparts, which showed that some PIP1s and PIP2s have circadian regulation while others did not. Promoter analysis revealed that a large number of hormone, light, stress response and circadian elements are present. Finally, involvement of aquaporins is supported by the reduction of K-leaf by HgCl2 treatment.

International audience; The water status of plant leaves is dependent on both stomatal regulation and water supply from the vasculature to inner tissues. The present review addresses the multiple physiological and mechanistic facets of the latter process. Inner leaf tissues contribute to at least a third of the whole resistance to water flow within the plant. Physiological studies indicated that leaf hydraulic conductance (K leaf) is highly dependent on the anatomy, development and age of the leaf and can vary rapidly in response to physiological or environmental factors such as leaf hydration, light, temperature, or nutrient supply. Differences in venation pattern provide a basis for variations in K leaf during development and between species. On a short time (hour) scale, the hydraulic resistance of the vessels can be influenced by transpiration-induced cavitations, wall collapses, and changes in xylem sap composition. The extravascular compartment includes all living tissues (xylem parenchyma, bundle sheath, and mesophyll) that transport water from xylem vessels to substomatal chambers. Pharmacological inhibition and reverse genetics studies have shown that this compartment involves water channel proteins called aquaporins (AQPs) that facilitate water transport across cell membranes. In many plant species, AQPs are present in all leaf tissues with a preferential expression in the vascular bundles. The various mechanisms that allow adjustment of K leaf to specific environmental conditions include transcriptional regulation of AQPs and changes in their abundance, trafficking, and intrinsic activity. Finally, the hydraulics of inner leaf tissues can have a strong impact on the dynamic responses of leaf water potential and stomata, and as a consequence on plant carbon economy and leaf expansion growth. The manipulation of these functions could help optimize the entire plant performance and its adaptation to extreme conditions over short and long time scales.

Aquaporins (AQPs) are membrane channels belonging to the major intrinsic proteins family and are known for their ability to facilitate water movement. While in Populus trichocarpa, AQP proteins form a large family encompassing fifty-five genes, most of the experimental work focused on a few genes or subfamilies. The current work was undertaken to develop a comprehensive picture of the whole AQP gene family in Populus species by delineating gene expression domain and distinguishing responsiveness to developmental and environmental cues. Since duplication events amplified the poplar AQP family, we addressed the question of expression redundancy between gene duplicates. On these purposes, we carried a meta-analysis of all publicly available Affymetrix experiments. Our in-silico strategy controlled for previously identified biases in cross-species transcriptomics, a necessary step for any comparative transcriptomics based on multispecies design chips. Three poplar AQPs were not supported by any expression data, even in a large collection of situations (abiotic and biotic constraints, temporal oscillations and mutants). The expression of 11 AQPs was never or poorly regulated whatever the wideness of their expression domain and their expression level. Our work highlighted that PtTIP1;4 was the most responsive gene of the AQP family. A high functional divergence between gene duplicates was detected across species and in response to tested cues, except for the root-expressed PtTIP2;3/PtTIP2;4 pair exhibiting 80% convergent responses. Our meta-analysis assessed key features of aquaporin expression which had remained hidden in single experiments, such as expression wideness, response specificity and genotype and environment interactions. By consolidating expression profiles using independent experimental series, we showed that the large expansion of AQP family in poplar was accompanied with a strong divergence of gene expression, even if some cases of functional redundancy could be suspected.

International audience; Aquaporins (AQPs) belong to the Major Intrinsic Protein family that conducts water and other small solutes across biological membranes. This study aimed to identify and characterize AQP genes in the primary root axis of two oak species, Quercus petraea and Quercus robur. Nine putative AQP genes were cloned, and their expression was profiled in different developmental root zones by real-time PCR. A detailed examination of the predicted amino acid sequences and subsequent phylogenetic analysis showed that the isolated AQPs could be divided into two subfamilies, which included six plasma membrane intrinsic proteins (PIPs) and three tonoplast intrinsic proteins (TIPs). We characterized the anatomical features of the roots and defined three developmental root zones: the immature, transition and mature zones. Expression analysis of the AQPs was performed according to these root developmental stages. Our results showed that the expression of PIP2;3 and TIP1 was significantly higher in Quercus petraea compared with Quercus robur in the three root zones. However, PIP2;1 and TIP2;1 were found to be differentially expressed in the mature zone of the two oak species. Of the nine AQP genes identified and analyzed, we highlighted four genes that might facilitate a deeper understanding of how these two closely related tree species adapted to different environments.

Objective: Fluorescence-guided surgery with 5-aminolevulinic acid (5-ALA) enables more complete resections of contrast-enhancing malignant gliomas in adults. Aquaporins (AQP) are a family of membrane proteins that provide a major pathway for water transport in mammals. Prior studies have suggested that[for full text, please go to the a.m. URL], 63. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit der Japanischen Gesellschaft für Neurochirurgie (JNS)

Leaf hydraulic conductance (Kleaf) takes a significant part in plant water relations. In walnut leaves, Kleaf was stimulated by light and tightly related to accumulation of JrPIP2s aquaporin transcripts. However, the light effect on Kleaf value is not systematically related to aquaporin regulation. Here we investigated the relationship between light, Kleaf and transcript levels of aquaporin in five species (Juglans regia, Fagus sylvitica, Quercus robur, Salix alba and Populus tremula) differing by the response of their Kleaf to light. Only for walnut leaves, we showed that light-increased Kleaf value is closely related to higher stimulation of both PIP1s and PIP2s aquaporins. To further investigate the involvement of aquporins (JrPIP1s and JrPIP2) in the light Kleaf modulation, 8 new full length aquaporins have been identified in walnut leaves and their expression pattern was monitored. All the aquaporin tested was turned up to be upregulated under light condition and downregulated under darkness. Moreover, we showed that the Kleaf response to light is quality-dependant, since it was reduced of 65% in the absence of blue light. Interstingly, this Kleaf reduction was correlated with a high downregulation of almost all aquaporins tested. To give an insight into the early molecular events involved in the light-induced Kleaf regulation, a large-scale transcriptomic analysis consisting of the cDNA-AFLP procedure was carried out on walnut leaves, kept at different light conditions. We obtained a total of 12,000 transcript-derived fragments (TDFs) by cDNA-AFLP with 128 primer pairs. Reliable sequences were obtained for 187 of these TDFs, and functions were attributed to 93 TDFs through BLAST searches in GenBank databases. Most of the 93 TDFs corresponded to genes encoding proteins involved in cellular regulation (58%). Leaves exposed to light showed changes in the Ca2+-signaling pathway, the ubiquitin-proteasome pathway, vesicle trafficking process and the expression of multiple transcription factors and protein regulators. To progress in understanding of a potential role for calcium signalling in light-modulated Kleaf, Kleaf values and transcript accumulation of 10 JrPIPs were monitored in leaves perfused with either a blocker of calcium channels [LaCl3] or a CaM antagonist [W7]. Compared to control, these Ca2+ -effectors led to a significant reduction in Kleaf and transcripts accumulation of almost all JrPIPs under light conditions. Our results indicate that Ca2+ /calmodulin complex may transduce the light signal required for stimulation of Kleaf and its correlated aquaporin expression.; La conductivité hydraulique foliaire (KF) est une composante majeure du transport d’eau dans toute la plante. Dans les feuilles de noyer, la KF est stimulée à la lumière et est étroitement liée à l’accroissement du taux des transcrits d’aquaporines JrPIP2s. Par ailleurs, la corrélation entre la stimulation de la KF et des transcrits d’aquaporines à la lumière, n’est pas générale et dépend de l’espèce. Ici, nous étudions cette corrélation chez cinq espèces forestières (Juglans regia, Fagus sylvitica, Quercus robur, Salix alba et Populus tremula) différant par leur réponse à la lumière. Nous démontrons seulement chez le noyer (Juglans regia), la contribution des deux familles d’aquaporines PIP1s et PIP2s. Afin de mieux comprendre le rôle des JrPIP1s et JrPIP2 dans la réponse à la lumière, nous avons isolé 8 nouvelles isoformes dans les feuilles de noyer et nous avons étudié leurs profils d’expression sur une cinétique lumière. Toutes les isoformes étudiées sont accumulées à la lumière et réprimées à l’obscurité. De plus, la KF est dépendante de la qualité de lumière. Elle est réduite de 65% en absence de lumière bleue. Cette diminution serait liée à l’inhibition des transcrits d’aquaporines. Afin de caractériser les mécanismes moléculaires précoces impliqués dans la modulation de KF par la lumière, l’approche globale cDNA-AFLP a été menée sur des feuilles de noyer sous différentes conditions d’éclairement. Nous obtenons 12000 transcrits différentiels dérivés (TDFs) générés par les 128 couples d’amorces. Parmi les 187 séquences obtenues, 93 d’entre elles ont une fonction putative. Leur classification fonctionnelle montre que les gènes relatifs à la régulation cellulaire représentent environ 58% des TDFs identifiés. Les feuilles exposées à la lumière, montrent des changements dans les voies de : signalisation calcique, protéolyse, trafic vésiculaire et l’expression de divers facteurs de transcription et protéines de régulation. Pour mieux comprendre le rôle potentiel de la signalisation calcique dans la modulation de la KF par la lumière, nous avons étudié l’effet d’un inhibiteur des canaux calciques [LaCl3] et d’un antagoniste de calmoduline [W7] sur la KF et les transcrits des 10 JrPIPs. Comparées aux feuilles témoins, les inhibiteurs calciques provoquent une réduction de la KF et de la majorité des JrPIPs étudiées à la lumière. Nos résultats confirment l’implication du complexe Ca2+ /calmoduline dans la transduction du signal lumineux responsable de la stimulation de la KF et des transcrits d’aquaporines chez le noyer.

Aquaporins (AQPS) are a family of small proteins forming water channels across cell membranes.AQPs 0, 1, 2, 4, 5, 6 and 8 are strictly water channel whereas AQPs 3, 7, 9 and 10 allow transport ofwater and glycerol. We have studied their expression in the epidermis, the outer-most water-impermeablelayer of the skin. In melanocytes, dendritic cells responsible for pigmentation, only AQP1 is expressed, invitro and ex vivo. We have shown that keratinocytes, principal cells of the epidermis, express AQPs 3 and10 in proliferation, whereas differentiated keratinocytes express AQPs 3 and 9. The localisation of AQP3to plasma membrane of keratinocytes was previously reported from the basal layer to the spinous layer ofthe skin. We localised AQP9 in the fully differentiated keratinocytes of the granular layer, where there is ahigh glycerol and low water content. So we think that AQP9 likely functions as a glycerol transporter.Unlike other authors, we were unable to identify a link between tumorous proliferation and the expressionof aquaporins., Les aquaporines (AQPs) sont des petites protéines formant des canaux hydriques àtravers les membranes cellulaires. Les AQPs 0, 1, 2 ,4 ,5 ,6 et 8 assurent le transport sélectif de l’eautandis que les AQPs 3, 7, 9 et 10 permettent également le passage du glycérol. Nous avons étudié leurexpression dans l’épiderme, la couche supérieure de notre peau supposée imperméable. Dans lesmélanocytes, des cellules dendritiques responsables de la pigmentation, seule l’AQP1 est exprimée.Nous avons montré que les kératinocytes, les cellules majoritaires de l’épiderme, expriment enprolifération les AQPs 3 et 10, alors que les kératinocytes différenciés expriment les AQPs 3 et 9. Lalocalisation de l'AQP3 a été précédemment rapportée à la membrane plasmique des kératinocytes, de lacouche basales à la couche épineuse. Nous avons localisé l'AQP9 dans les kératinocytes différenciés dela couche granuleuse au contenu riche en glycérol et réduit en eau. De fait nous pensons que l'AQP9 ysert de transporteur de glycérol. Enfin contrairement à d'autres auteurs, nous n’avons pu mettre enévidence de lien entre prolifération tumorale et expression des aquaporines.

To gain insights into the natural variation of root hydraulics and its molecular components, genotypic differences related to root water transport and plasma membrane intrinsic protein (PIP) aquaporin expression were investigated in 13 natural accessions of Arabidopsis (Arabidopsis thaliana). The hydraulic conductivity of excised root systems (Lpr) showed a 2-fold variation among accessions. The contribution of aquaporins to water uptake was characterized using as inhibitors mercury, propionic acid, and azide. The aquaporin-dependent and -independent paths of water transport made variable contributions to the total hydraulic conductivity in the different accessions. The distinct suberization patterns observed among accessions were not correlated with their root hydraulic properties. Real-time reverse transcription-polymerase chain reaction revealed, by contrast, a positive overall correlation between Lpr and certain highly expressed PIP transcripts. Root hydraulic responses to salt stress were characterized in a subset of five accessions (Bulhary-1, Catania-1, Columbia-0, Dijon-M, and Monte-Tosso-0 [Mr-0]). Lpr was down-regulated in all accessions except Mr-0. In Mr-0 and Catania-1, cortical cell hydraulic conductivity was unresponsive to salt, whereas it was down-regulated in the three other accessions. By contrast, the five accessions showed qualitatively similar aquaporin transcriptional profiles in response to salt. The overall work provides clues on how hydraulic regulation allows plant adaptation to salt stress. It also shows that a wide range of root hydraulic profiles, as previously reported in various species, can be observed in a single model species. This work paves the way for a quantitative genetics analysis of root hydraulics. Fil: Sutka, Moira Romina. Institut de Biologie Intégrative des Plantes; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Li, Guowei. Institut de Biologie Intégrative des Plantes; Francia Fil: Boudet, Julie. Institut de Biologie Intégrative des Plantes; Francia Fil: Boursiac, Yann. Institut de Biologie Intégrative des Plantes; Francia Fil: Doumas, Patrick. Centre National de la Recherche Scientifique. Institut de Recherche pour le Développement; Francia Fil: Maurel, Christophe. Institut de Biologie Intégrative des Plantes; Francia

International audience

This study consideres the molecular control of cell expansion in poplar root apex. The study was focused on the regulation of the TIP1 aquaporins expression under two levels of water deficit. A conceptual framework combining transcript density analysis (quantitative PCR) at a high spatial resolution and a fluid mechanics formalism was established to describe the regulation of gene expression in time and space along the root apex. Two contrasting growth status were both studied: root growth rate is either restored (after three days of moderate stress, 80 mmol kg-1, 100 g L-1 polyethylene glycol PEG 3500 g mol-1) or root growth rate is reduced (after 3 days of high stress, 250 mosmol kg-1, 200 g L-1 PEG). Kinematic analysis revealed a differential sensitivity of the relative elemental growth rate (REGR) according to the stress level and to the coordinate along the apex. At the molecular level, we showed that growth reduction was associated with a shift of maximal densities of transcripts towards the first millimeters of the apex, where cell expansion was maintained. Meanwhile the induction/repression levels were never stronger than in the control condition underlying that a high transcript density does not mean a high transcriptional induction. Focused on the combination of two dynamic processes, cell expansion and gene expression, my thesis showed that the conclusions issued from the analyses of these processes are influenced by the way time, space and age are considered, Cette étude porte sur le contrôle moléculaire de l'expansion cellulaire dans l'apex de la racine du peuplier. L'étude a été focalisée sur la régulation de l'expression des aquaporines TIP1s sous deux niveaux de déficit hydrique. Un cadre conceptuel, combinant analyse de la densité de transcrits à haute résolution spatiale et un formalisme de la mécanique des fluides, a été établi pour décrire la régulation de l'expression des gènes dans le temps et l'espace, le long de l'apex. Deux états de croissance contrastés ont été étudiés : un où la croissance de l'apex de la racine est rétablie (après 3 jours de stress modéré, 80 mosmol kg-1, 100 g L-1 de polyéthylène glycol PEG à 3500 g mol-1) et un où elle est réduite (après 3 jours de stress fort, 250 mosmol kg-1, 200 g L-1 de PEG). L'analyse cinématique a révélé une sensibilité différentielle de la vitesse d'allongement relatif (REGR) selon le niveau de stress et la position le long de l'apex. Au niveau moléculaire, nous montrons que lorsque la croissance est réduite, les pics de densité de transcrits des TIP1s sont plus forts et décalés dans les premiers millimètres de l'apex, zone où la croissance des cellules est maintenue. Cependant les niveaux d'induction/répression n'étaient jamais plus élevés que dans la condition témoin, soulignant qu'une forte densité de transcrits n'est pas forcément synonyme d'une forte induction de l'expression. Focalisé sur la combinaison de deux processus dynamiques, l'expansion cellulaire et l'expression des gènes, cette étude a mis en évidence que les conclusions tirées de l'analyse de ces processus sont influencées par la façon dont le temps, l'espace sont considérés

Diplôme : Dr. d'Université; Cette étude porte sur le contrôle moléculaire de l’expansion cellulaire dans l’apex de la racine du peuplier. L’étude a été focalisée sur la régulation de l’expression des aquaporines TIP1s sous deux niveaux de déficit hydrique. Un cadre conceptuel, combinant analyse de la densité de transcrits (PCR quantitative) à haute résolution spatiale et un formalisme de la mécanique des fluides, a été établi pour décrire la régulation de l’expression des gènes dans le temps et l’espace, le long d’apex. Deux états de croissance contrastés ont été étudiés : un où la croissance de l'apex de la racine est rétablie (après 3 jours de stress modéré, 80 mosmol kg-1, 100 g L-1 de polyéthylène glycol PEG à 3500 g mol-1) et un où elle est réduite (après 3 jours de stress fort, 250 mosmol kg-1, 200 g L-1 de PEG). L'analyse cinématique a révélé une sensibilité différentielle de la vitesse d’allongement relatif (REGR) selon le niveau de stress et la position le long de l’apex. Au niveau moléculaire, nous montrons que lorsque la croissance est réduite, l’expression de la plupart des TIP1s est induite précocement dans le temps et l’espace, générant un décalage des pics de densité de transcrits dans les premiers millimètres de l’apex, zone où la croissance des cellules est maintenue. Ce décalage était accompagné, pour certaines TIP1s, d’une augmentation de la densité de transcrits. Cependant les niveaux d’induction/répression n’étaient jamais plus élevés que dans la condition témoin, soulignant qu’une forte densité de transcrits n’est pas forcément synonyme d’une forte induction de l’expression. En effet le temps d’induction, rarement pris en compte, a un impact fort sur l’accumulation des transcrits. Parmi les patrons de densité de transcrits, nous avons détecté que seul celui de PtTIP1;4 coïncidait avec le profil de REGR quel que soit le traitement. De plus, la régulation de son expression apparait suivre un patron temporel similaire dans tous les traitements, bien que la taille de la zone de croissance varie. Ces deux arguments (profil de densité et régulation d’expression) soutiennent l’hypothèse d’un lien entre cette aquaporine et l’expansion cellulaire. Focalisé sur la combinaison de deux processus dynamiques, l’expansion cellulaire et l’expression des gènes, ma thèse a mis en évidence que les conclusions tirées de l’analyse de ces processus sont influencées par la façon dont le temps, l’espace et l’âge sont considérés.

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The effects of abscisic acid (ABA) on aquaporin content, root hydraulic conductivity (Lpr), whole plant hydraulic conductance, and leaf growth are controversial. We addressed these effects via a combination of experiments at different scales of plant organization and tested their consistency via a model. We analyzed under moderate water deficit a series of transformed maize (Zea mays) lines, one sense and three antisense, affected in NCED (for 9-cis-epoxycarotenoid dioxygenase) gene expression and that differed in the concentration of ABA in the xylem sap. In roots, the mRNA expression of most aquaporin PIP (for plasma membrane intrinsic protein) genes was increased in sense plants and decreased in antisense plants. The same pattern was observed for the protein contents of four PIPs. This resulted in more than 6-fold differences between lines in Lpr under both hydrostatic and osmotic gradients of water potential. This effect was probably due to differences in aquaporin activity, because it was nearly abolished by a hydrogen peroxide treatment, which blocks the water channel activity of aquaporins. The hydraulic conductance of intact whole plants was affected in the same way when measured either in steady-state conditions or via the rate of recovery of leaf water potential after rewatering. The recoveries of leaf water potential and elongation upon rehydration differed between lines and were accounted for by the experimentally measured Lpr in a model of water transfer. Overall, these results suggest that ABA has long-lasting effects on plant hydraulic properties via aquaporin activity, which contributes to the maintenance of a favorable plant water status.

absent

Article recommandé par Faculty of 1000 Biology; Aquaporins form a family of water and solute channel proteins and are present in most living organisms. In plants, aquaporins play an important role in the regulation of root water transport in response to abiotic stresses. In this work, we investigated the role of phosphorylation of plasma membrane intrinsic protein (PIP) aquaporins in the Arabidopsis thaliana root by a combination of quantitative mass spectrometry and cellular biology approaches. A novel phosphoproteomics procedure that involves plasma membrane purification, phosphopeptide enrichment with TiO(2) columns, and systematic mass spectrometry sequencing revealed multiple and adjacent phosphorylation sites in the C-terminal tail of several AtPIPs. Six of these sites had not been described previously. The phosphorylation of AtPIP2;1 at two C-terminal sites (Ser(280) and Ser(283)) was monitored by an absolute quantification method and shown to be altered in response to treatments of plants by salt (NaCl) and hydrogen peroxide. The two treatments are known to strongly decrease the water permeability of Arabidopsis roots. To investigate a putative role of Ser(280) and Ser(283) phosphorylation in aquaporin subcellular trafficking, AtPIP2;1 forms mutated at either one of the two sites were fused to the green fluorescent protein and expressed in transgenic plants. Confocal microscopy analysis of these plants revealed that, in resting conditions, phosphorylation of Ser(283) is necessary to target AtPIP2;1 to the plasma membrane. In addition, an NaCl treatment induced an intracellular accumulation of AtPIP2;1 by exerting specific actions onto AtPIP2;1 forms differing in their phosphorylation at Ser(283) to induce their accumulation in distinct intracellular structures. Thus, the present study documents stress-induced quantitative changes in aquaporin phosphorylation and establishes for the first time a link with plant aquaporin subcellular localization.

Mes travaux portent sur l'étude du développement et de la réponse des plantes à des stress environnementaux, à l'aide d'approches de protéomique membranaire. Les travaux de ma thèse ont démontré que la sensibilité à l'auxine de la H+-ATPase plasmique change au cours du développement de la plante. A l'issue de ma thèse, j'ai entrepris l'étude de mutants de développement d'Arabidopsis par le classement de leurs profils d'électrophorèse bidimensionnelle, ce qui a permis d'interpréter plusieurs phénotypes mutants. Par la suite, j'ai participé à l'étude du protéome de la membrane plasmique d'Arabidopsis et optimisé la desciption du protéome hydrophobe de cette membrane. J'ai ensuite utilisé cette approche pour étudier la régulation post-traductionnelle d'une famille de protéines membranaires facilitant le transport d'eau, les aquaporines, en réponse à des stress environnementaux. La découverte de la méthylation de certaines aquaporines et de multiples sites de phosphorylation sont des résultats originaux. Mon projet de recherche sera centré sur l'étude du rôle de ces modifications par le biais de i) l'identification de méthyltransférases d'aquaporines, ii) la caractérisation de protéines en interaction avec les domaines modifiés des aquaporines iii) la recherche de conditions environnementales susceptibles de moduler l'activité des aquaporines par méthylation et phosphorylation.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Die vorliegende Arbeit zeigt die Korrelation zwischen räumlichem und zeitlichem Expressionsmuster von dem Aquaporin NtAQP1 und seiner Funktion im Wasserhaushalt in planta. Immunologische in situ-Studien deuteten auf eine NtAQP1-Protein-Akkumulation in der Wurzelexodermis und -endodermis, im Cortex, in der Nähe der Leitbündel, im Xylemparenchym und in Zellen der Atemhöhle hin. Das Aquaporin wurde auch in longitudinalen Zellreihen der Petiolen in erhöhten Mengen gefunden. Expressionsstudien mit transgenen Pflanzen (Ntaqp1-Promotor::gus oder ::luc) bestätigten die NtAQP1-Akkumulation in der Wurzel, dem Spross und den Petiolen, lokalisierten dessen Expression aber auch in Pollen, Adventivwurzel und Blatthaaren. Die Ntaqp1-Expression wurde während Wachstumsprozessen wie Sprossorientierung nach Gravistimulation oder Photostimulation, Samenkeimung, aber auch während der vergleichsweise schnellen circadianen Blattbewegung induziert. Die Expression wurde weiterhin durch Phytohormone, im Speziellen durch Gibberellinsäure (GA) und osmotischen Stress stimuliert. Weitere Analysen hoben eine diurnale und sogar circadiane Expression von Ntaqp1 in Wurzeln und Petiolen hervor. Die funktionelle Analyse des Aquaporins wurde mittels reverser Genetik und biophysikalischen Studien durchgeführt. Die Antisense-Technik wurde benutzt, um die NtAQP1-Expression in Tabakpflanzen zu reduzieren. Die Antisense (AS)-Pflanzen zeigten eine starke Verringerung der Ntaqp1-mRNA, eine weniger ausgeprägte Verminderung der hoch homologen NtPIP1a-mRNA und keinen Effekt auf die Expression anderer Aquaporin-Genfamilien (PIP2, TIP). Die Funktion von NtAQP1 auf zellulärer Ebene wurde mit einer hierfür neuentwickelten Apparatur untersucht. Der experimentelle Aufbau ermöglichte die Aufzeichnung der osmotisch induzierten Protoplasten-Volumenzunahme. Die Reduktion von NtAQP1 durch die Antisense-Expression verminderte die zelluläre Wasserpermeabilität um mehr als 50 %. Die Funktion von NtAQP1 in der Gesamtpflanze wurde z.B. durch die "High-pressure flow meter" Methode bestimmt. Diese Messungen ergaben eine Reduktion der hydraulischen Wurzelleitfähigkeit pro Wurzeloberflächeneinheit (KRA) der Wurzeln der AS-Linien um mehr als 50 %. Die KRA wies eine starke diurnale und circadiane Schwankung auf, mit einem Maximum in der Mitte der Lichtperiode, ähnlich dem Verlauf des Expressionsmusters von Ntaqp1 in Wurzeln. Unter gut gewässerten Bedingungen ergaben Gaswechsel-, Spross- (Ystem) und Blatt- Wasserpotenial (Yleaf)-Messungen unterschiedliche Werte in AS- und Kontrollpflanzen. In wasserlimitierender Umgebung zeigten AS-Pflanzen jedoch ein stärker negativeres Y als Kontrollpflanzen, obwohl eine weitere Abnahme der Transpiration in AS-Pflanzen beobachtet werden konnte. Quantitative Analysen belegten eine stärker ausgeprägte Welkreaktion in den AS- als in den Kontrollpflanzen. Quantitative Studien der Blattbewegung von AS- verglichen mit Kontrollpflanzen hoben eine drastische Reduktion in Geschwindigkeit und Ausmaß der Reaktion hervor. Folgende Schlussfolgerungen konnten gezogen werden. NtAQP1 wurde an Orten mit erwartet hohem Wasserfluss von und zum Apoplasten oder Symplasten exprimiert. Außerdem deuteten das spezifische Verteilungsmuster und die zeitliche Expression von NtAQP1 in Petiolen und dem sich biegenden Spross auf eine Beteiligung in der transzellulären Wasserbewegung hin. Die Reduktion von NtAQP1 durch die Antisense-Expression verringerte die zelluläre Pos. Die NtAQP1-Funktion erhöht also eindeutig die Membranwasserpermeabilität von Tabak-Wurzelprotoplasten. Die Abnahme der spezifischen hydraulischen Wurzelleitfähigkeit (KRA) befand sich in der gleichen Größenordnung wie die Verringerung der mittleren zellulären Wasserpermeabilität. Dies weist darauf hin, dass die Aquaporin-Expression essentiell für die Aufrechterhaltung der natürlichen Wurzelleitfähigkeit ist. Die Verringerung von KRA in AS -Pflanzen könnte der erste sichere Beweis dafür sein, dass der Weg der Wasseraufnahme von der Wurzeloberfläche in das Xylem den Übergang über Membranen einschließt. Die Reduktion von NtAQP1 resultierte in einem Wasserstresssignal, das ein Schließen der Stomata zur Folge hatte. NtAQP1 scheint an der Vermeidung von Wasserstress in Tabak beteiligt zu sein. NtAQP1 spielt eine essentielle Rolle bei schnellen Pflanzenbewegungen und der transzellulären Wasserverschiebung., The presented work shows the analysis of the correlation between the spatial and temporal expression pattern of NtAQP1 and its function in water relation in planta. In situ immunological studies indicated NtAQP1-protein accumulation in the root exodermis and endodermis, in the cortex, close to vascular bundles, in the xylem parenchyma and in cells of the stomatal cavities. The aquaporin was also found to be abundant in longitudinal cell-rows in the petioles. Expression studies with generated transgenic plants (Ntaqp1-promoter::gus or luc) confirmed the Ntaqp1 accumulation in the root, stem and petioles but also revealed further localization in pollen grains, adventitious roots and leaf glandular hairs. Ntaqp1-expression was induced during growth processes, like stem bending after gravistimulation or photostimulation, seed germination and hypocotyl elongation as well as during the comparatively fast circadian leaf movement. The expression was further stimulated by phytohormones, especially gibberellic acid (GA) and osmotic stress. Further analysis displayed a diurnal and even circadian expression of Ntaqp1 in roots and petioles. The functional analysis of the aquaporin was accomplished by reverse genetics and biophysical studies. The antisense technique was used to reduce NtAQP1-expression in tobacco plants. The antisense (AS) plants exhibited a severe reduction of Ntaqp1-mRNA, less reduction of the highly homologous NtPIP1a RNA and no effect on expression of other aquaporin family genes (PIP2, TIP). The function of NtAQP1 at the cellular level was investigated by a newly developed experimental setup to record the osmotically induced increase in protoplast volume. The reduction of NtAQP1 by the antisense expression decreased the overall cellular waterpermeability Pos for more than 50 %. Function of NtAQP1 at the whole plant level was e.g. measured by the “high-pressure flow meter method”. Those measurements revealed that the root hydraulic conductivity per unit root surface area (KRA) of roots from the AS-lines was reduced by more than 50 %. KRA displayed a strong diurnal and circadian variation with a maximum in the middle of the light period, similar to the expression pattern of Ntaqp1 in roots. Gas exchange-, stem (Ystem) and leaf (Yleaf) water potential measurement gave dissimilar values in AS and control plants under well-watered conditions. Under a water-limiting environment the Y of AS-plants remained at more negative water values, even though a further decrease in transpiration of AS-plants was detected. Quantitative analysis displayed a much stronger wilting reaction in the AS than in the control plants. Quantitative studies of the leaf movement in AS compared to control plants exhibited a dramatic reduction in velocity and also in the extent of the process. The following conclusions can be drawn. NtAQP1 was expressed at sites of anticipated high water fluxes from and to the apoplast or symplast. Additionally, the specific distribution pattern and temporal expression of NtAQP1 in petioles and the bending stem strongly indicate a role in transcellular movement of water. The reduction of NtAQP1 by the antisense expression decreased the overall cellular Pos. Conclusively, NtAQP1-function increases membrane water permeability of tobacco root protoplasts. The decrease of the specific root hydraulic conductivity (KRA) was in the same order of magnitude as the mean cellular water permeability reduction, indicating that aquaporin expression is essential in maintaining a natural root hydraulic conductance. Reduction of KRA in AS plants might be the first definitive proof that the pathway of water uptake from the root surface to the xylem involves passage across membranes. The absence of NtAQP1 resulted in a water stress signal, causing a certain stomatal closure. NtAQP1 seems to contribute to water stress avoidance in tobacco. NtAQP1 plays an essential role in fast plant movements and transcellular water shift.

Proteins of the MIP family are membrane proteins that facilitate water or/and solute transport across biological membranes. The members of this family have been classified in three subgroups according to their sequence similarity and function: the aquaporins contains water selective transport proteins (AQP1, AQP2,...), the aquaglyceroporins are water and solute permeable proteins such as AQP3, and the glycerol facilitators are impermeable to water. We have shown that the glycerol permeability of red blood cell membrane is due to the presence of AQP3. In order to determine parts of the proteins involved in the selectivity, we have constructed chimeras between AQP2 and AQP3. Permeability measurements of oocyte expressing AQP3-AQP2 Cter showed that the C terminal extremity is critical for the transport of water but not for the glycerol one. Comparing the water and glycerol transports of oocytes at various stages of maturation, we have shown that an endogenous channel or transporter was present in immature oocytes. Our interest was then focused on the determination of the oligomerization status of an aquaglyceroporin: AQP3, in different membranes. The known tetrameric structure of AQP1 was observed by velocity sedimentation on sucrose gradient, in contrast, AQP3 was found in much slighter fraction in monomeric and dimeric forms. The dimer seems to be very resistant to SDS and hydrophilic reducing agent treatment. Due to a plausible sensitivity of AQP3 to the non-denaturant detergents used, we could not exclude its tetrameric form in the membrane. The results obtained with electronic microscopy on AQP1 and AQP3 expressing oocyte membrane cryofracture showed that AQP1 and AQP3 IMP sizes were not different enough to suggest another membrane organization., La famille des protéines MIPs est composée de canaux hydriques et de facilitateurs de glycérol. Parmi les canaux hydriques, certains sont sélectivement perm&ables à l'eau, les aquaporines (AQP1, AQP2,...), et les autres sont également perméables aux petits solutés comme le glycérol: les aquaglycéroporines, dont AQP3 fait partie. Nous avons montré dans un premier temps que la perméabilité au glycérol du globule rouge était due à la présence d'AQP3. Dans l'intention de mieux connaître quels étaient les éléments protéiques impliqués dans la sélectivité des protéines MIPs, nous avons construit des chimères entre AQP2 et AQP3. L'une d'entre elle, AQP3-AQP2 Cter, après expression dans l'ovocyte de Xénope, a permis de montrer que la partie C terminale cytoplasmique est impliquée dans le transport d'eau mais pas dans le transport de glycérol d'AQP3. Nous avons également montré que les ovocytes de xénope non matures possédaient des perméabilités à l'eau et au glycérol supérieurs à celles des ovocytes matures suggérant l'expression d'un canal ou d'un transporteur endogène. Enfin, nous avons envisagé de déterminer pour la première fois la structure quaternaire d'une aquaglycéroporine: AQP3. Alors qu'AQP1 dévoile sa forme tétramérique sur gradient de saccharose, après solubilisation en conditions non dénaturantes, AQP3 sédimente dans des fractions plus légères sous forme d'un monomère et d'un dimère très résistant au SDS et aux agents réducteurs hydrophiles. Nous n'avons pu conclure quant à son organisation dans les membranes du fait de son éventuelle sensibilité spécifique aux détergents non dénaturants utilisés. Nous avons donc engagé des études de microscopie électronique de cryofractures de membranes d'ovocytes exprimant AQP1 et AQP3. Nous en avons déduit que la taille d'AQP3 n'était pas suffisamment différente de celle d'AQP1 pour suggérer une organisation membranaire également différente.

Die Methodik und Technik der Gaswechselmessung von Pflanzen wurde für den Modellorganismus Arabidopsis thaliana optimiert und für Untersuchungen zur Beteiligung des Aquaporins PIP1b an Wassertransportvorgängen während der Stomaöffnung verwendet. Die Messungen der Transpirationsraten von PIP1b-Antisense-Pflanzen ergaben keine Hinweise auf Veränderungen des zeitlichen Verlaufs der Stomaöffnung. Die Wasserpermeabilitäten von Schließzell-Plasmamembranen scheinen somit nicht von der Expression des Aquaporins PIP1b beeinflußt zu sein. - Gaswechselmessungen an Nicotiana tabacum NtAQP1-Antisense-Pflanzen zeigten eine Verringerung der Transpirationsraten im Licht und eine geringere Grundtranspiration im Dunkeln. Dies deutet auf eine Beteiligung von NtAQP1 am Wassertransport hin. - Ausgewählte Arabidopsis thaliana-Mutanten wurden hinsichtlich ihrer stomatären Antwort auf Rot- und Rot-/Blaulicht-Bestrahlung analysiert. Hierfür wurde ein Doppelbestrahlungs-Protokoll entwickelt. Vergleiche mit den Wildtypen ergaben signifikante Unterschiede bei der Phytochrom-Mutante phyA-103, der Abscisinsäure-Mutante aba3-2 und der Auxin-resistenten Mutante axr1-3. Ferner zeigte die Mutante npq1-2 nicht die beschriebene Abweichung der stomatären Antwort auf Blaulicht. - Die Expressionsmuster eines PIP1b-GFP-Reportergens in transgenen Arabidopsis thaliana-Pflanzen wurden analysiert. Hohe Promotor-Aktivitäten konnten in meristematischen Bereichen von Wurzel und Sproß, in Elementen der Leitbündel, in jungen Kotyledonen und in Staubblättern beobachtet werden. Es zeigte sich eine enge Korrelation zwischen PIP1b-Promotoraktivität und Streckungswachstum. - Eine Sequenzanalyse des NtAQP1-Promotors ergab Übereinstimmungen mit spezifischen Bindungsmotiven von MYB-ähnlichen Transkriptionsfaktoren. Mit Promotor-Reportergenen konnte die Beteiligung eines dieser Sequenzmotive an der GA- und ABA-induzierten Aktivierung des NtAQP1-Promotors gezeigt werden. Zur Analyse der Phytohormon-Wirkungen auf deletierte Promotorbereiche wurde ein duales Vektorsystem entwickelt und bei der transienten Transformation von BY2-Protoplasten eingesetzt. - Die Expression eines GFP::NtAQP1-Fusionsgens in BY2-Zellen zeigte die subzelluläre Lokalisation des Aquaporins in der Zytoplasmamembran. Ferner wurde Fusionsprotein in Vesikel-ähnlichen Strukturen beobachtet., Gas exchange measurement of plants was optimized for the model organism Arabidopsis thaliana. Consequently, studies on the participation of aquaporin PIP1b in water transport processes became possible. The transpiration rates obtained for PIP1b anti-sense plants revealed no differences in the time course of stomatal opening. Thus, water permeabilities of guard cell plasma membranes seem not to be influenced by the extend of PIP1b expression. - Gas exchange measurement of the Nicotiana tabacum NtAQP1 anti-sense plants showed a reduced transpiration in response to light and a reduced basal transpiration in the dark. This indicates the participation of NtAQP1 in water movement. - Selected Arabidopsis thaliana mutants were analyzed with regard to their stomatal response to red, red/blue light irradiation. For that, a dual beam protocol was developed. A comparison of the mutant lines to the wild-type revealed significant differences for the phytochrome A (phyA-103), the abscisic acid (aba3-2) and the auxin resistent (axr1-3) mutants. Furthermore, the mutant line npq1-2 did not show the abnormal stomatal response to blue light reported previously. - The expression patterns of a PIP1b-GFP-reporter gene in transgenic Arabidopsis thaliana plants were analyzed. High promotor activities were obtained in areas of meristematic tissue in roots and shoot, in vascular elements, in young cotyledons and in stamina. A close correlation between PIP1b-promoter activity and cell elongation was obvious. - Sequence analysis of the NtAQP1 promoter discovered DNA-motifs that correspond to specific binding sites of MYB-related transcriptional activators. By using promotor-reporter constructs, the involvement of one of this motifs in a GA- and ABA-induced increase of NtAQP1-promoter activity could be shown. In order to elucidate the effects of phytohormones on deleted promoter elements a dual vector system has been created, which was employed in the transient transfection of BY2-protoplasts. - The expression of a GFP::NtAQP1 translational fusion in BY2-cells revealed a subcellular location in the cytoplasmic membrane. Furthermore, the protein-fusion was recorded in small vesicle-like structures.

The endodermis represents the main barrier to extracellular diffusion in plant roots, and it is central to current models of plant nutrient uptake. Despite this, little is known about the genes setting up this endodermal barrier. In this study, we report the identification and characterization of a strong barrier mutant, schengen3 (sgn3). We observe a surprising ability of the mutant to maintain nutrient homeostasis, but demonstrate a major defect in maintaining sufficient levels of the macronutrient potassium. We show that SGN3/GASSHO1 is a receptor-like kinase that is necessary for localizing CASPARIAN STRIP DOMAIN PROTEINS (CASPs)—major players of endodermal differentiation—into an uninterrupted, ring-like domain. SGN3 appears to localize into a broader band, embedding growing CASP microdomains. The discovery of SGN3 strongly advances our ability to interrogate mechanisms of plant nutrient homeostasis and provides a novel actor for localized microdomain formation at the endodermal plasma membrane. DOI: http://dx.doi.org/10.7554/eLife.03115.001, eLife digest Plant roots forage in the soil for minerals and water, but they must also provide a barrier that stops these nutrients leaking back out of the plant and stops microbes invading and causing disease. The endodermis—an inner layer of cells that surrounds the veins that run along the middle of a root—acts as such a barrier in young roots. Polymers that repel water are deposited between the cells in the roots of almost all vascular plants—which include ferns, conifers, and flowering plants—to form a band around the endodermis called the ‘Casparian strip’. This strip seals off the young roots and stops water moving through the gaps between plant cells, but still allows minerals, nutrients, and water to be transported through the root cells and into the plant. However, the importance of this structure has yet to be tested due to the lack of mutant plants without a Casparian strip. Pfister et al. now report that deleting the gene that encodes a protein called SCHENGEN3 in the model plant Arabidopsis thaliana causes the Casparian strip to be interrupted by irregularly sized holes. This protein is normally found at high levels in the root endodermis, where it is embedded into the cell membranes. Pfister et al. also showed that without the SCHENGEN3 protein, other proteins called CASPs—that normally mark out a stripe around the root cells where the Casparian strip will form—only accumulated in discontinuous patches. Further experiments revealed that deleting the gene for SCHENGEN3 does not cause general problems in delivering the CASP proteins to the cell membrane; instead, it specifically stops the CASP proteins from forming a single, uninterrupted stripe. Unexpectedly, disrupting the Casparian strip did not appear to hinder many of the functions of a root. The mutant plants could still take up water and nutrients, and the leaves of mutant plants had normal levels of many essential minerals—with the exception of potassium. The level of this mineral was much lower in mutant plants without the SCHENGEN3 protein. Pfister et al. suggest that in plants that lack an intact Casparian strip, potassium is continuously leaked from the root into the soil. These findings reveal that in Arabidopsis, at least, the Casparian strip might not be as important as once thought for helping the plant to take up and accumulate water and nutrients. Further work is now needed to uncover the as yet unknown backup systems that might be able to compensate for the loss of this structure. DOI: http://dx.doi.org/10.7554/eLife.03115.002

s of the Annual Main Meeting of the Society of Experimental Biology, 28th June - 1st July, Glasgow, UK; International audience; The effects of abscisic acid (ABA) on aquaporin content, root hydraulic conductivity (Lpr), whole plant hydraulic conductance, and leaf growth are controversial. We addressed these effects via a combination of experiments at different scales of plant organization and tested their consistency via a model. We analyzed under moderate water deficit a series of transformed maize (Zea mays) lines, one sense and three antisense, affected in NCED (for 9-cis-epoxycarotenoid dioxygenase) gene expression and that differed in the concentration of ABA in the xylem sap. In roots, the mRNA expression of most aquaporin PIP (for plasma membrane intrinsic protein) genes was increased in sense plants and decreased in antisense plants. The same pattern was observed for the protein contents of four PIPs. This resulted in more than 6-fold differences between lines in Lpr under both hydrostatic and osmotic gradients of water potential. This effect was probably due to differences in aquaporin activity, because it was nearly abolished by a hydrogen peroxide treatment, which blocks the water channel activity of aquaporins. The hydraulic conductance of intact whole plants was affected in the same way when measured either in steady-state conditions or via the rate of recovery of leaf water potential after rewatering. The recoveries of leaf water potential and elongation upon rehydration differed between lines and were accounted for by the experimentally measured Lpr in a model of water transfer. Overall, these results suggest that ABA has long-lasting effects on plant hydraulic properties via aquaporin activity, which contributes to the maintenance of a favorable plant water status.

National audience

Oligodendrozyten-Vorläuferzellen (OPCs) sind hoch motile Zellen, die für die Myelinbildung im Zentralnervensystem verantwortlich sind. Diese Arbeit untersucht Volumendynamiken der OPC Zellkörper während der Migration mit Hilfe der Raster-Ionenleitfähigkeitsmikroskopie (SICM). Im ersten Teil der Arbeit wurde die Scangeschwindigkeit sowie der Scanbereich des SICMs vergrößert sowie ein Korrekturalgorithmus entwickelt, der Überschätzungen der Zellhöhe an steilen Membranabschnitten ausgleicht. Die Untersuchung von migrierenden OPCs ergab vor einer Beschleunigung der Migration frontale Volumenzunahmen sowie rückseitige Volumenabnahmen. Um herauszufinden, ob die Volumenänderungen durch Wasserflüsse durch Aquaporine (AQPs) erfolgen, wurde die Expression von AQP immunzytochemisch analysiert. Obwohl AQP3 als hochexprimierter Wasserkanal identifiziert wurde, konnte die Migration - im Gegensatz zu einigen Krebszellen – nicht durch den AQP3-Blocker Kupfersulfat beeinflusst werden. Oligodendrocyte progenitor cells (OPCs) are highly motile cells responsible for the myelin formation in the central nervous system. This thesis investigates volume dynamics of OPC cell bodies during their migration obtained with scanning ion conductance microscopy (SICM). To this aim methodological improvements of SICM were performed, by improving scanning speed and range as well as introducing a correction algorithm for overestimations of sample height at sloped membrane sections. The results of the investigations of migrating OPCs with SICM support the hypothesis that water fluxes during migration lead to a volume gain at the frontal soma part and volume loss at the rear soma end prior to acceleration. To test whether water fluxes in migrating OPCs involve aquaporins (AQPs) a screening for several AQPs was performed, finding predominantly AQP3. However, in contrast to cancer cells, migration in OPCs was not affected by treatment with the AQP3 blocking agent copper sulphate.

Environmental and hormonal signaling pathways targeting or mediated by plant aquaporins. 27. International Conference on Arabidopsis Research (ICAR 2016)