Cosentino, Cristian (2008)
Na+/H+ transporters of the halophyte Mesembryanthemum crystallinum L.
Technische Universität Darmstadt
Ph.D. Thesis, Primary publication
Abstract
The aim of this work was to understand the mechanisms of Na+ accumulation in the halophyte Mesembryanthemum crystallinum L. during NaCl induced transition from C3 photosynthesis to crassulacean acid metabolism (CAM). Under high salinity M. crystallinum is a strong salt includer accumulating high amounts of Na + in leaves. To understand the mechanisms of Na+ accumulation during NaCl adaptation Na+/H+ antiporters from leaves of M. crystallinum were cloned by RACE PCR. In silico analysis identified the five cloned antiporters as belonging to three different families of exchangers: NhaP/SOS1 family, represented by McSOS1; IT/NhaD, represented by McNhaD: IC-NHE/NHX, with McNHX1 and McNHX3 belonging to the vacuolar class I and McNHX2 to the endomembrane class II. McSOS1, McNhaD and McNHX1 are homologous to the Na+/H+ antiporters AtSOS1, AtNHX1-2 and AtNHD1 of Arabidopsis thaliana, which are located at the plasma membrane, tonoplast and plastidial membrane, respectively. Functional complementation tests in Saccharomyces cerevisiae revealed that McSOS1 and McNhaD can complement the Na+ sensitivity of a yeast mutant strain (ena1-4 nha1 nhx1). Out of the three cloned antiporters of the IC-NHE/NHX family only McHX1 was able to restore resistance to Hygromycin B in the yeast mutant strain nhx1 implying that only this antiporter functions at the vacuolar membrane. Real-time PCR analysis demonstrated that the expression level of McSOS1, McNhaD and McNHX1 increased under salt stress. This increase in expression level correlated with the accumulation of sodium in leaves suggesting a physiological role for the antiporters in Na+ compartmentation during adaptation to high salinity. In particular, analysis of salt accumulation on the cellular level revealed a high Na+ content not only in vacuoles but also in chloroplasts. Together with the observation that the cloned antiporter McNhaD is localized to the plastidial membrane this points to a hitherto unknown pathway of Na+ transport out of the cytosol. The integrated function of the Na+/H+ antiporter localized to the plasma membrane (McSOS1), the tonoplast (McNHX1) and the chloroplast membrane (McNhaD) will allow an immediate detoxification of the cytoplasm from Na+.
Item Type: |
Ph.D. Thesis
|
Erschienen: |
2008 |
Creators: |
Cosentino, Cristian |
Type of entry: |
Primary publication |
Title: |
Na+/H+ transporters of the halophyte Mesembryanthemum crystallinum L. |
Language: |
English |
Referees: |
Homann, PD Dr. Ulrike ; Thiel, Prof. Dr. Gerhard |
Date: |
15 October 2008 |
Place of Publication: |
Darmstadt |
Publisher: |
Technische Universität |
Refereed: |
31 July 2008 |
URL / URN: |
urn:nbn:de:tuda-tuprints-11617 |
Abstract: |
The aim of this work was to understand the mechanisms of Na+ accumulation in the halophyte Mesembryanthemum crystallinum L. during NaCl induced transition from C3 photosynthesis to crassulacean acid metabolism (CAM). Under high salinity M. crystallinum is a strong salt includer accumulating high amounts of Na + in leaves. To understand the mechanisms of Na+ accumulation during NaCl adaptation Na+/H+ antiporters from leaves of M. crystallinum were cloned by RACE PCR. In silico analysis identified the five cloned antiporters as belonging to three different families of exchangers: NhaP/SOS1 family, represented by McSOS1; IT/NhaD, represented by McNhaD: IC-NHE/NHX, with McNHX1 and McNHX3 belonging to the vacuolar class I and McNHX2 to the endomembrane class II. McSOS1, McNhaD and McNHX1 are homologous to the Na+/H+ antiporters AtSOS1, AtNHX1-2 and AtNHD1 of Arabidopsis thaliana, which are located at the plasma membrane, tonoplast and plastidial membrane, respectively. Functional complementation tests in Saccharomyces cerevisiae revealed that McSOS1 and McNhaD can complement the Na+ sensitivity of a yeast mutant strain (ena1-4 nha1 nhx1). Out of the three cloned antiporters of the IC-NHE/NHX family only McHX1 was able to restore resistance to Hygromycin B in the yeast mutant strain nhx1 implying that only this antiporter functions at the vacuolar membrane. Real-time PCR analysis demonstrated that the expression level of McSOS1, McNhaD and McNHX1 increased under salt stress. This increase in expression level correlated with the accumulation of sodium in leaves suggesting a physiological role for the antiporters in Na+ compartmentation during adaptation to high salinity. In particular, analysis of salt accumulation on the cellular level revealed a high Na+ content not only in vacuoles but also in chloroplasts. Together with the observation that the cloned antiporter McNhaD is localized to the plastidial membrane this points to a hitherto unknown pathway of Na+ transport out of the cytosol. The integrated function of the Na+/H+ antiporter localized to the plasma membrane (McSOS1), the tonoplast (McNHX1) and the chloroplast membrane (McNhaD) will allow an immediate detoxification of the cytoplasm from Na+. |
Alternative Abstract: |
Alternative abstract | Language |
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Ziel der vorliegenden Arbeit war die Untersuchung von Mechanismen, die an der Na+-Akkumulation in der Halophyte Mesembryanthemum crystallinum L. während der NaCl induzierten Umstellung von C3-Photosynthese zum Crassulaceen-Säure-Metabolismus (CAM). Unter hoher Salzbelastung reichert M. crystallinum große Mengen an Na+ in den Blättern an. Um den Mechanismus der Na+-Akkumulation während der Salzanpassung zu verstehen wurden Na+/H+-Antiporter aus Blättern von M. crystallinum mittels RACE PCR kloniert. Die in silico Analyse der fünf klonierten Antiporter erlaubte die Zuordnung der Transportproteine zu drei verschiedenen Proteinfamilien: McSOS1 gehört zur NhaP/SOS1-Familie; McNhaD ist ein Transporter der IT/NhaD-Familie; McNHX1 (vakuoläre Membranen, Klasse I), McNHX2 (endosomale Membranen, Klasse II) und McNHX3 (vakuoläre Membranen, Klasse I) gehören zur IC-NHE/NHX-Familie. McSOS1, McNhad und McNhx1 sind homolog zu den Na+/H+ Antiportern AtSOS1, AtNhx1-2 und AtNHD1 in Arabidopsis thaliana, die an der Plasmamembran, am Tonoplasten und an der plastidären Membran lokalisiert sind. Funktionale Komplementationsexperimente mit Saccaromyces cerevisiae zeigen, dass McSOS1 und McNhaD die Na+ Sensitivität der Hefemutante ena1-4 nha1 nhx1 komplementieren können. Von den drei klonierten Antiportern der IC-NHE/NHX Familie konnte nur McHX1 die Hygromycin B-Resistenz in der Hefemutante nhx1 wieder herstellen. Dies legt nahe, dass nur dieser Antiporter eine Funktion an der vakuolären Membran einnimmt. Real-time PCR Analysen zeigten, dass die Expression von McSOS1, McNhaD und McNHX1 unter Salzstress ansteigt. Dieser Anstieg war mit der Akkumulation von Salz in Blätter korreliert und deutet auf eine physiologische Rolle dieser Antiporter in der Na+-Kompartimentierung während der Anpassung an hohe Na+-Konzentrationen hin. Die Analyse der Salzakkumulation auf zellulärer Ebene zeigt eine hohe Salzkonzentration nicht nur in Vakuolen, sondern auch in Chloroplasten. Die hohe Na+-Konzentration in Chloroplasten und die Lokalisation des klonierten Antiporters McNhaD an einer der plastidären Membranen weißt auf einen bisher unbekannten Weg für den Na+-Export aus dem Cytosol hin. Das Zusammenspiel der Na+/H+ Antiporter an der Plasmamembran (McSOS1), am Tonoplasten (McNHX1) und an der Chloroplastenmembran (McNhaD) gewährleistet eine schnelle Na+-Detoxifizierung des Cytosols. | German |
|
Classification DDC: |
500 Science and mathematics > 580 Plants (botany) |
Divisions: |
10 Department of Biology |
Date Deposited: |
31 Oct 2008 11:27 |
Last Modified: |
15 Jan 2019 08:33 |
PPN: |
|
Referees: |
Homann, PD Dr. Ulrike ; Thiel, Prof. Dr. Gerhard |
Refereed / Verteidigung / mdl. Prüfung: |
31 July 2008 |
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