Cloning and characterisation of the HMA3 gene and its promoter from Arabidopsis halleri (L.) O'Kane and Al'Shehbaz and Arabidopsis thaliana (L.) Heynhold [Elektronische Ressource] / by Toni Hoffmann

Toni Hoffmann

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• Biologie

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• Publié par: universitat_potsdam
• Publié le: 01 janvier 2007
• Nombre de lectures: 46
• Langue: English
• Poids de l'ouvrage: 13 Mo

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Max Planck InstituteofMolecularPlantPhysiology-ResearchGroup: MetalHomeostasis
Cloning and characterisation
of the HMA3 gene and its promoter from
Arabidopsishalleri (L.) O´KANE and AL´SHEHBAZ and
Arabidopsisthaliana (L.) HEYNHOLD
DISSERTATION
infulfilmentoftherequirements
fortheacademicdegreeof
doctorrerumnaturalis
(Dr. rer. nat.)
submittedtothe
FacultyofMathematicsandNaturalSciences
UniversityofPotsdam
by
ToniHoffmann
Potsdam,March2007This work is licensed under the Creative Commons Attribution-Noncommercial-No
Derivative Works 2.0 Germany License. To view a copy of this license, visit
http://creativecommons.org/licenses/by-nc-nd/2.0/de/ or send a letter to Creative
Commons, 171 Second Street, Suite 300, San Francisco, California, 94105, USA.










































Elektronisch veröffentlicht auf dem
Publikationsserver der Universität Potsdam:
http://opus.kobv.de/ubp/volltexte/2007/1525/
urn:nbn:de:kobv:517-opus-15259
[http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-15259] The work presented in this thesis was carried out between October 2002 and January 2006
in the Metal Homeostasis group of Dr. habil. Ute Krämer at the Max-Planck-Institute of
Molecular Plant Physiology, Potsdam, Germany.
st1 Examiner Prof. Dr. Bernd Müller-Röber, Institute of Biology and Biochemistry,
University of Potsdam, Potsdam, Germany
nd2 Examiner Dr. Lorraine E. Williams, School of Biological Sciences, University of
Southampton, Southampton, UK
rd3 Examiner Dr. Pierre Berthomieu, UMR de Biochimie et Physiologie Moléculaire
des Plantes, Centre National de la Recherche Scientifique, Université
Montpellier 2, France“Within walnuts and trees and other plants
vast treasures will be found,
which lie hidden there and well guarded”
Leonardo da Vinci in ’Prophecies’, 1293Danksagung
ie vorliegende Arbeit entstand am Max Planck Institut für Molekulare Pflanzenphysiologie
inPotsdaminderMetallhomöostase ArbeitsgruppevonDr.UteKrämer.Dr.UteKrämer
gilt an erster Stelle mein besonderer Dankfür dieBereitstellung des interessantenThemas,
die geduldige Betreuung, ihre stetige Diskussionbereitschaft und dafür, dass sie mir die Möglichkeit
gab,dieseArbeitzuvollenden.
Weiterhin möchte ich mich bei Prof. Dr. Mark Stitt und Prof. Dr. Bernd Müller Röber dafür be
danken,dasssiealsBetreuerundGutachtermeinerDoktorarbeitzurVerfügungstandenundmirviele
hilfreicheRatschlägegaben.WeiterhinbedankeichmichbeiDr.LorraineE.WilliamsundDr.Pierre
BerthomieufürdieexterneBegutachtungmeinerArbeit.
Für die ausgesprochen freundliche und gute Arbeitsatmosphäre, für die Hilfsbereitschaft bei fach
lichenundpraktischenFragenmöchteichmichbeiallenehemaligenundderzeitigenMitgliedernder
Arbeitsgruppe von Dr. Ute Krämer bedanken. Im Speziellen danke ich Toralf Senger, Marc Haniken
ne, Ina Talke und Len Krall für viele anregende Diskussionen zu den verschiedensten Themen sowie
KatrinVoigtundStéphanieArrivaultfürihrenerfrischendenHumor.
Ebenso danke ich allen Mitarbeitern des Max Planck Institutes für Molekulare Pflanzenphysiolo
gie für das überaus angenehme familiäre Arbeitsklima und insbesondere dem „Green Team“ für die
gärtnerischeBetreuungderPflanzenexperimente.
VonganzemHerzendankeichmeinenEltern,ohnederenuneingeschränkteUnterstützungmein
Studium und diese Arbeit nicht möglich gewesen wären. Und schließlich danke ich Claudia, für die
verlässlicheundliebevollePersondiesieistsowieihrerFamilie.
UndnatürlichauchanalleungenanntengutenFreunde:Dankeschön!
DSummary
Being living systems unable to adjust their location to changing environmental conditions, plants
display homeostatic networks that have evolved to maintain transition metal levels in a very narrow
concentration range in order to avoid either deficiency or toxicity. Hence, plants possess a broad
repertoire of mechanisms for the cellular uptake, compartmentation and efflux, as well as for the
chelationoftransitionmetalions.
A small number of plants are hypertolerant to one or a few specific transition metals. Some
metal tolerant plants are also able to hyperaccumulate metal ions. The Brassicaceae family member
Arabidopis halleri ssp. halleri (L.) O´KANE and AL´SHEHBAZ is a hyperaccumulator of zinc (Zn),
anditiscloselyrelatedtothenon hypertolerantandnon hyperaccumulatingmodelplant Arabidopsis
thaliana(L.)HEYNHOLD.Thecloserelationshiprenders A.halleriapromisingemergingmodelplant
for the comparative investigation of the molecular mechanisms behind hypertolerance and hyper
accumulation. Among several potential candidate genes that are probably involved in mediating the
zinc hypertolerantandzinc hyperaccumulatingtraitis AhHMA3. TheAhHMA3geneishighlysimilar
toAtHMA3(AGInumber: At4g30120)inA.thaliana,anditsencodedproteinbelongstotheP typeIB
ATPasefamilyofintegralmembranetransporterproteinsthattransporttransitionmetals. Incontrast
to the low AtHMA3 transcript levels in A. thaliana, the gene was found to be constitutively highly
expressedacrossdifferentZntreatmentsin A.halleri,especiallyinshoots.
Inthisstudy,thecloningandcharacterisationoftheHMA3geneanditspromoterfromArabidopsis
halleri (L.) O´KANE and AL´SHEHBAZ and Arabidopsis thaliana (L.) HEYNHOLD is described. Het
erologously expressed AhHMA3 mediated enhanced tolerance to Zn and to a much lesser degree
to cadmium (Cd) but not to cobalt (Co) in metal sensitive mutant strains of budding yeast. It is
demonstrated that the genome of A. halleri contains at least four copies of AhHMA3, AhHMA3 1 to
AhHMA3 4. A copy specific real time RT PCR indicated that an AhHMA3 1 related gene copy is the
sourceoftheconstitutivelyhightranscriptlevelinA.halleriandnotagenecopysimilartoAhHMA3 2
or AhHMA3 4.
InaccordancewiththeenhancedAtHMA3mRNAtranscriptlevelinA.thalianaroots,anAtHMA3
promoter GUS gene construct mediated GUS activity predominantly in the vascular tissues of roots
andnotinshoots. However,theobserved AhHMA3 1 and AhHMA3 2 promoter mediatedGUSactiv-
ityin A. thalianaor A. halleriplantsdidnotreflecttheconstitutivelyhighexpressionof AhHMA3in
shoots of A. halleri. It is suggested that other factors e.g. characteristic sequence inserts within the
firstintronofAhHMA3 1 mightenableaconstitutivelyhighexpression. Moreover,theunknownpro
moteroftheAhHMA3 3 genecopycouldbethesourceoftheconstitutivelyhighAhHMA3transcript
levels in A. halleri. In that case, the AhHMA3 3 sequence is predicted to be highly homologous to
AhHMA3 1.The lack of solid localisation data for the AhHMA3 protein prevents a clear functional assign
ment. The provided data suggest several possible functions of the AhHMA3 protein: Like AtHMA2
and AtHMA4 it might be localised to the plasma membrane and could contribute to the efficient
translocation of Zn from root to shoot and/or to the cell to cell distribution of Zn in the shoot. If
localisedtothevacuolarmembrane, thenaroleinmaintainingalowcytoplasmiczincconcentration
byvacuolarzincsequestrationispossible. Inaddition,AhHMA3mightbeinvolvedinthedeliveryof
zincionstotrichomesandmesophyllleafcellsthataremajorzincstoragesitesin A.halleri.