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The plant respiratory chain: Redox responses and catalytic definition of alternative pathways

Geisler, Daniela LU (2007)
Abstract (Swedish)
Popular Abstract in Swedish

Växternas respirationskedja innehåller protonpumpande enzymer, som konserverar energi i form av en protongradient, samt alternativa icke-protonpumpande enzymer. I det mitokondriella innermembranet finns ett protonpumpande NADH dehydrogenas, komplex I, och alternativa typ II NAD(P)H dehydrogenaser som kan oxidera NADH och NADPH från matrixen eller cytosolen. I Arabidopsis thaliana finns det 7 genhomologer för typ II NAD(P)H dehydrogenaser som kan delas in i 3 familjer; nda familjen innehåller två homologer, ndb familjen innehåller 4 homologer och det finns en ndc gen. Genom att producera alla 7 proteiner i Escherichia coli, kunde det visas att NDB1 och NDB2 binder kalcium. En punktmutation... (More)
Popular Abstract in Swedish

Växternas respirationskedja innehåller protonpumpande enzymer, som konserverar energi i form av en protongradient, samt alternativa icke-protonpumpande enzymer. I det mitokondriella innermembranet finns ett protonpumpande NADH dehydrogenas, komplex I, och alternativa typ II NAD(P)H dehydrogenaser som kan oxidera NADH och NADPH från matrixen eller cytosolen. I Arabidopsis thaliana finns det 7 genhomologer för typ II NAD(P)H dehydrogenaser som kan delas in i 3 familjer; nda familjen innehåller två homologer, ndb familjen innehåller 4 homologer och det finns en ndc gen. Genom att producera alla 7 proteiner i Escherichia coli, kunde det visas att NDB1 och NDB2 binder kalcium. En punktmutation demonstrerade att ett EF-hand motiv binder kalcium i NDB1 proteinet. Dessutom karakteriserades NDB1 som kalciumberoende NADPH-specifik dehydrogenas. NDB2 och NDB4 är specifika för NADH och enbart aktiviteten av NDB2 stimuleras av kalcium.



NADH och NADPH är viktiga reduktionsekvivalenter i cellen. Unga A. thaliana plantor i flytande kulturer behandlades antingen med ammonium eller nitrat, som har olika reduktionsbehov för assimilering. Detta inducerade motsatta effekter på genexpression av icke-protonpumpande enzymer. Ammonium ledde till induktion av icke-protonpumpande enzymer, medan nitrat undertryckte genexpression i samma familjer. Detta tyder på att icke-protonpumpande respirationsenzymer kan vara involverade i redoxbalansering.



Ett sätt att förändra reduktionsgraden av respirationskedjan är genom användning av inhibitorer som kan inducera oxidativ stress. Inhibering av komplex III med antimycin A ledde till minskad kapacitet för oxidering av matrix NADH genom den icke-protonpumpande vägen i isolerade mitokondrier. Detta kan vara en mekanism för att undvika överreduktion av det mitokondriella innermembranet.



En metod för permeabilisering av membran med hjälp av alamethicin har anpassats för användning på hela celler i en cellsuspension. Därmed kan man studera mitokondriella och cytosoliska enzymaktiviteter i sin ursprungliga miljö. Dessutom kunde vi förbättra metoden för isolering av mitokondrier från A. thaliana, växtforskningens viktigaste modellorganism. (Less)
Abstract
The respiratory chain of plants contains both proton-pumping enzymes that conserve energy and non-proton-pumping alternative enzymes that bypass the sites of energy conservation. In addition to the proton-pumping complex I, plant mitochondria contain alternative type II NAD(P)H dehydrogenases, which allow oxidation of NADH and NADPH from the matrix and the cytosol. The seven gene homologues of type II NAD(P)H dehydrogenases in Arabidopsis thaliana group into three families, including two nda homologues, four ndb homologues and one ndc gene. By producing the seven type II NAD(P)H dehydrogenases in Escherichia coli, we could show that NDB1 and NDB2 bind calcium. A single amino acid substitution identified an EF hand motif as the... (More)
The respiratory chain of plants contains both proton-pumping enzymes that conserve energy and non-proton-pumping alternative enzymes that bypass the sites of energy conservation. In addition to the proton-pumping complex I, plant mitochondria contain alternative type II NAD(P)H dehydrogenases, which allow oxidation of NADH and NADPH from the matrix and the cytosol. The seven gene homologues of type II NAD(P)H dehydrogenases in Arabidopsis thaliana group into three families, including two nda homologues, four ndb homologues and one ndc gene. By producing the seven type II NAD(P)H dehydrogenases in Escherichia coli, we could show that NDB1 and NDB2 bind calcium. A single amino acid substitution identified an EF hand motif as the calcium-binding site of NDB1. Furthermore, NDB1 was characterised as a calcium-dependent NADPH-specific dehydrogenase, while NDB4 and NDB2 were found to be NADH-specific dehydrogenases, with only the latter enzyme being stimulated by calcium.



NADH and NADPH are major reducing equivalents in the cell. Supplying A. thaliana seedlings with ammonium or nitrate, which have different reductant demands for assimilation, induced opposite effects on transcription of non-proton-pumping respiratory enzymes. Ammonium led to broad inductions in alternative pathways, also reflected at the enzyme level, whereas nitrate suppressed gene expression in the same families. This points to a role of non-proton-pumping respiratory enzymes in cellular redox balancing. Respiratory inhibitors can elevate the reduction state of the inner membrane, thereby inducing oxidative stress. In potato, inhibition of complex III by antimycin A resulted in a decrease of internal NADH oxidation via the alternative dehydrogenase in isolated mitochondria, which could potentially counteract over-reduction.



A method for permeabilisation of membranes by alamethicin was adapted for use on intact plant suspension cells. This allows in situ studies on mitochondrial and cytosolic enzymes in their native environment. Furthermore, the mitochondrial purification method for A. thaliana, the major plant model organism, was improved. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Prof. Macherel, David, France
organization
publishing date
type
Thesis
publication status
published
subject
keywords
enzymology, Proteins, Kärlväxters fysiologi, Physiology of vascular plants, Växtekologi, Plant ecology, Växtbiokemi, Plant biochemistry, respiratory chain, plant mitochondria, nitrogen, NADPH dehydrogenase, NADH dehydrogenase, alternative oxidase, calcium, Proteiner, enzymologi, Bioenergetics, Bioenergetik, Lipids, steroids, membranes, Lipider, steroider, mebran
pages
102 pages
publisher
Lund University Department of Cell and Organism Biology Sölvegatan 35B SE-223 62 Lund
defense location
Bioliogihuset, Hörsalen, Sölvegatan 35
defense date
2007-10-05 09:30
ISBN
978-91-85067-33-6
language
English
LU publication?
yes
id
ff8f02ff-287c-466b-9c95-ec971b29c051 (old id 598842)
date added to LUP
2007-11-13 07:42:10
date last changed
2016-09-19 08:45:06
@misc{ff8f02ff-287c-466b-9c95-ec971b29c051,
  abstract     = {The respiratory chain of plants contains both proton-pumping enzymes that conserve energy and non-proton-pumping alternative enzymes that bypass the sites of energy conservation. In addition to the proton-pumping complex I, plant mitochondria contain alternative type II NAD(P)H dehydrogenases, which allow oxidation of NADH and NADPH from the matrix and the cytosol. The seven gene homologues of type II NAD(P)H dehydrogenases in Arabidopsis thaliana group into three families, including two nda homologues, four ndb homologues and one ndc gene. By producing the seven type II NAD(P)H dehydrogenases in Escherichia coli, we could show that NDB1 and NDB2 bind calcium. A single amino acid substitution identified an EF hand motif as the calcium-binding site of NDB1. Furthermore, NDB1 was characterised as a calcium-dependent NADPH-specific dehydrogenase, while NDB4 and NDB2 were found to be NADH-specific dehydrogenases, with only the latter enzyme being stimulated by calcium.<br/><br>
<br/><br>
NADH and NADPH are major reducing equivalents in the cell. Supplying A. thaliana seedlings with ammonium or nitrate, which have different reductant demands for assimilation, induced opposite effects on transcription of non-proton-pumping respiratory enzymes. Ammonium led to broad inductions in alternative pathways, also reflected at the enzyme level, whereas nitrate suppressed gene expression in the same families. This points to a role of non-proton-pumping respiratory enzymes in cellular redox balancing. Respiratory inhibitors can elevate the reduction state of the inner membrane, thereby inducing oxidative stress. In potato, inhibition of complex III by antimycin A resulted in a decrease of internal NADH oxidation via the alternative dehydrogenase in isolated mitochondria, which could potentially counteract over-reduction.<br/><br>
<br/><br>
A method for permeabilisation of membranes by alamethicin was adapted for use on intact plant suspension cells. This allows in situ studies on mitochondrial and cytosolic enzymes in their native environment. Furthermore, the mitochondrial purification method for A. thaliana, the major plant model organism, was improved.},
  author       = {Geisler, Daniela},
  isbn         = {978-91-85067-33-6},
  keyword      = {enzymology,Proteins,Kärlväxters fysiologi,Physiology of vascular plants,Växtekologi,Plant ecology,Växtbiokemi,Plant biochemistry,respiratory chain,plant mitochondria,nitrogen,NADPH dehydrogenase,NADH dehydrogenase,alternative oxidase,calcium,Proteiner,enzymologi,Bioenergetics,Bioenergetik,Lipids,steroids,membranes,Lipider,steroider,mebran},
  language     = {eng},
  pages        = {102},
  publisher    = {ARRAY(0xab4aeb0)},
  title        = {The plant respiratory chain: Redox responses and catalytic definition of alternative pathways},
  year         = {2007},
}