Functional Characterization of Vacuolar and Plastidic sugar transporter genes within the “Major Facilitator Superfamily” of Arabidopsis thaliana Funktionelle Charakterisierung von Genen für vakuoläre und plastidäre Zuckertransporter aus der “Major Facilitator Superfamily“ in Arabidopsis thaliana Den naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Sirisha Aluri aus Ramachandrapuram, Indien
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Functional Characterization of Vacuolar and Plastidic sugar transporter genes within the “Major
Facilitator Superfamily” of Arabidopsis thaliana
Funktionelle Charakterisierung von Genen für vakuoläre und plastidäre Zuckertransporter aus der
“Major Facilitator Superfamily“ in Arabidopsis thaliana
Den naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg zur
Erlangung des Doktorgrades
vorgelegt von Sirisha Aluri aus Ramachandrapuram, Indien
Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 11.12.2006 Vorsitzender der Promotionskommission: Prof. Dr. E. Bänsch Erstberichterstatter: PD Dr. Michael Büttner Zweitberichterstatter: Prof. Dr. Norbert Sauer
Acknowledgements I express my profound respect and gratitude to Prof. Dr. Norbert Sauer, for confiding
me the doctoral position at the Department of Molecular Plant Physiology and for being the
official reviewer, and have always been warm and supportive.
I convey my deepest gratitude to PD Dr. Michael Büttner, my supervisor, who has
always been kind, and produced enormous patience while I was at work, and bestowed full
support. He suggested the idea of this work and gave me an opportunity to work on a doctoral
thesis. He has constantly directed me to remain focused on achieving the set targets. His
observations and comments helped me to move forward with investigation in depth. His
inspiring ideas contributed so much to this uphill task.
I express my indebtedness to my colleagues Ms. Barbara Hannich and Mr. Constantin
von Schweinichen for their help in all ways through all means on and off the work and also
for large-scale scientific discussions.
I express my sincere gratitude to Prof. Dr. Petra Dietrich for her outstanding
discussions. I am grateful to PD Dr. Ruth Stadler and Dr. Stefan Hoth for their help,
especially with confocal laser scanning microscopy.
My sincere thanks to MS Sabine Schneider and Dr. Matthias Weider for their
invaluable comments and useful discussions. I specially thank all other colleagues at the
institute for their cooperation.
I wish to thank Ms. Christa Helmers and Ms. Walburga Summersammer, institute
secretaries, for their kind and helpful support in all administrative activities.
I express my sincere appreciation to Ms. Gudrun Steingräber, Ms. Rebecca Günther,
Ms. Silke Opplet and Ms. Angelica Wolf for their invaluable technical assistance which was
of timely help.
I would like to thank Gues H. and Monika V. for their assistance.
I just can’t verbalize my heartfelt feelings towards- my parents (Mrs & Mr. Rama
Brahmam V. Pakalapati), my inlaws (Mrs & Mr Ravi Kumar Aluri) for their care and support,
my sisters and brother-in-laws (Mrs & Mr. Nagesh Nadina and Mrs & Mr. Krishna Mohan
Devineni), my brothers (Satish Kumar and Siva Prasad) for their affection and encouragement
and our children Sreeja, Sravya, Chathurya and Baalu, talking to whom is a great refreshment.
This work was financially supported by DFG (SPP) and in part by AFGN.
Affectionately dedicated to my husband Mr. Naresh Kumar Aluri and to my
daughter Poorna Kusuma (Chitteelu)
Table of Contents
Table of Contents
Abbreviations........................................................................................................................... iv
2.1.1 Isolation and cloning of the AtVGT1 cDNA ........................................................... 11 2.1.2 Heterologous expression of AtVGT1 in Saccharomyces cerevisiae........................ 12
2.1.2.1 Substrate transport assay in transgenic yeast cells ........................................... 12 2.1.2.2 Growth complementation by AtVGT1............................................................. 13
2.1.3 Subcellular localization of AtVGT1......................................................................... 13 2.1.3.1 Cloning of AtVGT1 cDNA for GFP fusion ...................................................... 14 2.1.3.2 Expression of an AtVGT1 cDNA-GFP fusion construct in Yeast ................... 14 2.1.3.3 Transient expression of AtVGT1-GFP fusion in Arabidopsis protoplasts........ 15
2.1.4 AtVGT1 transport assay in isolated vacuoles of transgenic yeast .......................... 16 2.1.4.1 Isolation and stabilization of yeast vacuoles .................................................... 16 2.1.4.2 Sugar transport assay with isolated yeast vacuoles.......................................... 17 2.1.4.3 Sugar uptake into vacuoles of transgenic yeasts .............................................. 17 2.1.4.4 pH dependence of AtVGT1.............................................................................. 19
2.1.5 Analysis of AtVGT1-expression by reporter plants................................................. 19 2.1.6 Isolation and analysis of T-DNA insertion mutants of AtVGT1 ............................. 20
2.1.6.1 PCR analysis of AtVGT1-T-DNA insertion lines............................................. 20 2.1.6.2 Analysis of Homozygous AtVGT1 T-DNA insertion lines .............................. 22
2.2 Functional characterization of AtVGT2 .......................................................................... 24 2.2.1 Isolation and cloning of AtVGT2 cDNA ................................................................. 24 2.2.2 Expression of AtVGT2 cDNA in yeast................................................................... 25
2.2.2.1 Growth complementation tests......................................................................... 25 2.2.2.2 Substrate transport assay in transgenic yeast cells ........................................... 25
2.2.3 Subcellular localization of AtVGT2 ....................................................................... 26 2.2.3.1 Cloning of AtVGT2 cDNA for GFP fusion ...................................................... 26 2.2.3.2 Expression of AtVGT2-GFP fusion in yeast .................................................... 26 2.2.3.3 Transient expression of AtVGT2-GFP fusion in Arabidopsis protoplasts ....... 27
2.2.4 Expression of AtVGT2 gene in Planta ..................................................................... 28 2.2.5 Generation of Antibodies against AtVGT2............................................................. 29
2.2.5.1 Cloning for MBP-AtVGT2 fusion protein ....................................................... 30 2.2.6 Identification and analysis of AtVGT2 T-DNA insertion mutants .......................... 30
2.2.6.1 Isolation of homozygousT-DNA insertion lines for AtVGT2 .......................... 31 2.2.6.3 Analysis of homozygous T-DNA insertion lines for AtVGT2 ......................... 32
2.3 Generation and analysis of Atvgt1/Atvgt2 double mutants............................................. 32 2.3.1 Generation of Atvgt1/Atvgt2 double mutants .......................................................... 33 2.3.2 Analysis of Atvgt1/Atvgt2 double mutants .............................................................. 33
2.4 Functional Characterization of AtXYL3.......................................................................... 38 2.4.1 Subcellular localization of AtXYL3 ....................................................................... 38
2.4.1.1 Isolation and cloning of AtXYL3 cDNA ......................................................... 39 2.4.1.2 Transient expression of XYL3-GFP fusion in Arabidopsis protoplasts ........... 39
2.4.2 Generation of antibodies against AtXYL3.............................................................. 40
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Table of Contents
2.4.2.1 Cloning for MBP-AtXYL3 fusion ................................................................... 40 2.4.2.2 Western blot with isolated plastidic membrane proteins ................................. 40 2.4.2.5 Expression of AtXYL3-GFP fusion in yeast.................................................... 41
2.4.3 Analysis of AtXYL3 expression by GUS reporter plants ......................................... 41 2.4.3.1 Isolation and cloning of AtXYL3 promoter ..................................................... 41 2.4.3.2 Analysis of transgenic Arabidopsis plants for GUS expression ...................... 41
2.4.4 Isolation and analysis of T-DNA insertion mutants of AtVGT1 ............................. 42 2.4.4.1 PCR analysis of AtXYL3 T-DNA insertion lines.............................................. 43 2.4.4.2 Analysis of homozygous AtXYL3 T-DNA insertion line ................................. 44 2.4.4.3 Analysis of Atxyl3 mutants grown under continuous light .............................. 45
4.1.3 Vectors .................................................................................................................... 58 4.1.3.1 Empty vectors................................................................................................... 58 4.1.3.2 Vectors with inserts.......................................................................................... 58 4.1.4.2 Oligonucleotides used for cloning and sequencing of AtVGT2 ....................... 61 4.1.4.3 Oligonucleotides used for cloning and sequencing of AtXYL3 ........................ 61
4.1.5 Culturemedia ........................................................................................................... 62 4.1.5.1 Bacterial culture media..................................................................................... 62 4.1.5.2 Yeast culture media.......................................................................................... 62 4.1.5.3 Soil composition and media used to grow plants ............................................. 62
4.1.6 Solutions.................................................................................................................. 63 4.1.7 Other Chemicals and Enzymes ............................................................................... 67 4.1.8 Secondary antibody ................................................................................................. 68 4.1.9 Materials used ......................................................................................................... 68 4.1.10 Machines ............................................................................................................... 69
4.2 Methods.......................................................................................................................... 69 4.2.1. Culturing the organisms used................................................................................. 69
4.2.1.1. Microbial cultures (Bacteria and Yeast).......................................................... 69 4.2.1.2 Growing Arabidopsis plants............................................................................. 70 4.2.2.1 Stock cultures ................................................................................................... 70 4.2.2.2 Isolation and purification of DNA from E.coli ................................................ 70 4.2.2.3 Isolation of DNA from Arabidopsis thaliana .................................................. 70 4.2.2.4 Isolation of mRNA........................................................................................... 71 4.2.2.5 RNA preparation for gene chip analysis .......................................................... 71 4.2.2.6 Determination of DNA and/or mRNA concentration ...................................... 71 4.2.2.7 DNA purification and precipitation.................................................................. 72 4.2.2.8 Analysis of DNA sequence .............................................................................. 72 4.2.2.9 Annealing and 5’ phosphorylation of oligonucleotides ................................... 72 4.2.2.10 Sample preparation for HPLC analysis.......................................................... 73 4.2.2.11 Isolation of protoplasts from Arabidopsis thaliana........................................ 73 4.2.2.12 PEG transfection ............................................................................................ 73
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Table of Contents
4.2.2.13 Isolation of vacuoles from Arabidopsis thaliana ........................................... 74 4.2.2.14 Yeast transformation ...................................................................................... 74 4.2.2.15 Isolation of soluble proteins from S. cerevisiae ............................................. 75 4.2.2.16 Western blot analysis ..................................................................................... 75 4.2.2.17 Transport assay with yeast cells ..................................................................... 76 4.2.2.18 Isolation of vacuoles from Saccharomyces cerevisiae................................... 76 4.2.2.19 Uptake experiments with vacuoles................................................................. 77 4.2.2.20 Isolation of plastidic membrane fraction........................................................ 77 4.2.2.21 Embedding the plant material in Technovit ................................................... 77
0.5% SDS). Acid-washed glass beads (0.45-0-50 mm) were added to the suspension to the
level of the meniscus and the entire solution vortexed for 5 times of 20 sec each (the cell
suspension was cooled on ice for 1 min between each cycle of vortexing). The cell extract was
recovered via centrifugation (3,000g for 2 minutes at 4°C) into a fresh tube, centrifuged again
at 12,000g for 5 mins at 4°C for clarification, protease inhibitor (2PI) was added and the
extract was stored at -20°C until further use. In some cases, the starting cultures, as well as the
subsequent steps were scaled up to 100X.
4.2.2.16 Western blot analysis
Following SDS PAGE, proteins were transferred to Nitrocellulose (Amersham
Bioscience, Germany) in transfer buffer (25 mM Tris, 192 mM Glycin, 20% methanol).
Transfer was performed at a constant voltage of 400V for 20 mins. After completion of
transfer, the membrane was washed briefly in water and stained for 1 min in Ponceau S. The
membrane was then incubated in Blocking buffer (5% skim milk powder TBST Buffer) for 30
mins at RT. Primary antibody was diluted in blocking buffer and added to the membrane;
incubated at RT for one hour or o/N at 4°C. The membrane was then washed for two times
with blocking buffer, for a minimum of 15 min. The secondary antibody (Anti-Rabbit IgG-
75
Materials and Methods
Peroxidase conjugate) was added to the membrane after diluted by a factor of 4000 with
blocking buffer and incubated for 1 hr at RT. The membrane was washed for two times with
blocking buffer and incubated for 15 min in the same. Detection was performed using
Lumilight western blotting substrates.
4.2.2.17 Transport assay with yeast cells
To characterize the transport function, putative AtXYL genes were heterologously
expressed in yeast strain EBY VW 4000 and uptake experiments were carried out with
radioactively labelled sugars. In brief, Yeast cells harbouring the cDNA in sense as well as in
antisense orientation were grown oN in CAA medium at 29°C until an OD600 of 1.0-1.5. Cells
were harvested by centrifuging for 5 min at 3500 rpm and washed for the first time with water
and then with 25 mM Sodium Phosphate buffer pH 5.0. Cells were resuspended at the end in
25 mM Sodium Phosphate buffer to obtain an OD600 of 20 and stored on ice until use.
To measure the uptake, 1 ml of cells were taken in 25 ml Erlenmeyer flask and
incubated for 1 min at 29°C while shaking. 10mM sugar of 0.02 µCi was added to cells and
100 µl aliquots pipetted on to Nitrocellulose membrane of pore size 0.8 µm at definite time
intervals (15 sec, 1, 2, 5 and 10 mins). Vacuum was applied to suck the buffer and the
membrane was placed in 4 ml Scintillation cocktail after washing for 2 times with sodium
phosphate buffer. Radioactivity was measured in Scintillation counter.
4.2.2.18 Isolation of vacuoles from Saccharomyces cerevisiae
Yeast cells were grown to OD600 of 1 or below. The cells were sedimented and washed
with distilled water for two times by centrifuging the culture at 4500g for 5 min. Each pellet
resuspended in 30 ml of spheroplasting buffer and incubated at 29°C for 1hr. Spheroplasts
were harvested and washed twice with 1M Sorbitol by centrifugation at 2200g for 5min.
Pellet was resuspended in 12% Ficoll buffer of pH 6.9. Spheroplasts were lysed osmotically
in addition to mechanical stress by using the dounce homogeniser. Cell debris was removed
by centrifuging the homogenate at 2200g for 10 min. Supernatant was transferred to an
ultracentrifuge tube, over-layered with fresh 12% Ficoll and centrifuged for 1hr at 60,000g
using sw28 rotor of ultracentrifuge. Vacuoles were harvested from the surface of 12% Ficoll,
just by scooping with a spatula and resuspended in appropriate volume of 2X Buffer C.
76
Materials and Methods
4.2.2.19 Uptake experiments with vacuoles
Uptake measurements with isolated vacuoles were carried out basically as in the case
of whole cells after few modifications. Nitrocellulose membrane of 0.2 µm was used instead
of 0.8 µm. Initial sugar concentration was set to 100 µM with 0.1 µCi. 50 µg of vacuolar
protein (diluted to 100 µl with 2X bufferC) was used as uptake mix for each time point along
with 4 mM ATP and 4 mM MgSO4. Radioactive substrate was added after 5 min
preincubation of uptake mix at 29°C. 100 µl aliquots were pipetted onto nitrocellulose
membrane at each time point and vacuoles were washed for 2 times with 2X Buffer C of pH
7.9. Vacuum was applied very slowly to remove the excess buffer and unused radioactive
substrate. Nitrocellulose membrane was added to scintillation cocktail and the radioactivity
was measured.
4.2.2.20 Isolation of plastidic membrane fraction
Leaf tissue was added to isolation buffer at a ratio of 1:4 (w/v) and homogenized in
dounce homogenizer for 2 min. The homogenate was filtered through 4 layers of nylon mesh
(50 µm). The filtrate was layered over isolation buffer containing 40% Percoll (v/v). The
intact chloroplasts were pelleted after centrifugation at 4000g for 5 min. The chloroplasts
were lysed osmotically by incubating in hypotonic lysis buffer for 15 min. The suspension
was centrifuged at 105,000g for 1 hr, and the resultant pellet was used as membrane protein
fraction and the supernatant as stromal fraction.
4.2.2.21 Embedding the plant material in Technovit
The GUS stained plant material of interest was dehydrated by incubating in 90%
ethanol for 30 min and 2 times in 100% ethanol, incubated for 1 hr each time. The plant
material was then infiltrated by incubating in 100% ethanol: preparing solution (v/v) for 2 hr
at RT. To embed, the plant material was taken in a 0.2 ml Eppendorf cup and embedding
solution was added and incubated for 4 hr to let the embedding solution polymerize.
77
78
Summary
5. Summary
A new subfamily of the monosaccharide transporter genes, consisting of three
members At3g03090 (AtVGT1), At5g17010 (AtVGT2) and At5g59250 (AtXYL3), was
identified within the Major Facilitator Superfamily. The cDNAs of all three genes were
cloned and used for further analysis. For the highly homologous members AtVGT1 and
AtVGT2, vacuolar localization was demonstrated by expression of the GFP fusions in yeast
as well as in Arabidopsis protoplasts. The functional expression of AtVGT1 in yeast and
substrate transport assays with vacuoles revealed that AtVGT1 is a vacuolar H+/glucose
antiporter. This is the first identified and functionally analyzed plant vacuolar sugar
transporter. The analysis of GUS reporter plants showed AtVGT1 promoter activity only in
pollen, while results from our RT-PCRs as well as the Genevestigator microarray database
indicate, that AtVGT1 is expressed in most tissues at a low basic level. Analysis of AtVGT1 T-
DNA insertion mutants revealed the important role of this gene in seed germination and
determination of flowering time, since 20% of the mutant seeds failed to germinate and the
bolting process was delayed by 9 to 14 days. An important osmotic function of AtVGT1-
mediated glucose-transport was proposed.
AtVGT2, which also localized to the vacuole, is expressed in most of the tissues,
throughout the plant development. However, Atvgt2 T-DNA mutants did not show visible
phenotypes. Atvgt1/Atvgt2 double-mutant plants were analyzed to investigate a possible
functional compensation of the loss of AtVGT2 by AtVGT1. In addition to the seed
germination and bolting phenotypes observed in Atvgt1 mutants, in Atvgt1/Atvgt2 plants
lignification of the cell wall was impaired, and increased cell elongation with impaired
cessation of the internode elongation was observed. Together, these effects resulted in a weak
floral stem and fewer branches, thus leading to lower fresh weight. In addition, these plants
also showed delayed rosette development and impaired silique development.
The third and more distant member of this family, AtXYL3 has an extended N-
terminal sequence, predicted to be cTP. The plastidic localization of this protein was
determined by transient expression of a GFP fusion in Arabidopsis protoplasts. Since AtXYL3
is not expressed in yeast, a functional analysis of this transporter homolog remains to be
elucidated using a different system. Tissue specific expression analyzed by GUS-reporter
plants revealed that AtXYL3 is expressed in most tissues. Analysis of Atxyl3 mutants showed
that disruption of this gene leads to advanced vegetative plant development, whereas silique
79
Summary
and seed development is defective. Under continuous light the enhanced vegetative plant
development was even more pronounced. This suggests an important role of AtXYL3 in
diating glucose fluxes across the plastidic envelope, and in transient starch metabolism.
80
Zusammenfassung
6. Zusammenfassung
Innerhalb der „Major Facilitator Superfamily“ wurde eine neue Unterfamilie von
Monosaccharid-Transportern, bestehend aus den drei Mitgliedern At3g03090 (AtVGT1),
At5g17010 (AtVGT2) and At5g59250 (AtXYL3), identifiziert. Von allen drei Genen wurde die
cDNA kloniert und zu weiteren Analysen herangezogen. Für die hoch homologen Mitglieder
AtVGT1 und AtVGT2 konnte durch Expression eines GFP Fusionsproteins sowohl in Hefe,
als auch in Arabidopsis Protoplasten eine vakuoläre Lokalisation beobachtet werden. Die
funktionelle Expression von AtVGT1 in Hefe und folgende Aufnahmemessungen mit
isolierten Vakuolen zeigten, dass es sich bei AtVGT1 um einen vakuolären H+/Glucose
Antiporter handelt. Hiermit konnte zum ersten Mal ein pflanzlicher vakuolärer Zucker-
Transporter identifiziert und funktionell analysiert werden. Bei der Analyse von GUS
Reporterpflanzen wurde AtVGT1 Promotoraktivität ausschließlich im Pollen beobachtet,
wohingegen RT-PCR Experimente und Microarray Daten aus der Genevestigator Datenbank
auf eine niedrige, aber gleichmäßige Expression in allen Geweben hindeuten. Die
Untersuchung von AtVGT1 T-DNA Insertionslinien weist auf eine wichtige Rolle von
AtVGT1 bei der Samenkeimung und Blühinduktion hin, da 20% der Samen der KO-Mutante
nicht keimten und sich der Beginn der Blütensprossbildung um 9-14 Tage verzögerte. Diese
Ergebnisse deuten daraufhin, dass der AtVGT1-vermittelte Glukose-Transport eine wichtige
Funktion bei der Osmoregulation einnimmt.
Für AtVGT2 konnte ebenfalls eine vakuoläre Lokalisation gezeigt werden. Obwohl
AtVGT2 während der gesamten Entwicklung in fast allen Geweben exprimiert wird, zeigen
Atvgt1 T-DNA Mutanten keinen sichtbaren Phänotyp. Aus diesem Grund wurden
Atvgt1/Atvgt2 Doppelmutanten analysiert, um festzustellen, ob der Verlust von AtVGT2
möglicherweise durch AtVGT1 kompensiert werden kann. Zusätzlich zu dem in der Atvgt1
Mutante beobachteten Phänotyp bezüglich Samenkeimung und Sprossbildung zeigten sich in
der Doppelmutante weitere Auswirkungen. So war zum einen die Lignifizierung der Zellwand
beeinträchtigt, zum anderen wurde eine verstärkte Zell-Elongation verbunden mit
verlängerten Internodien im Stengel beobachtet. Insgesamt führten diese Effekte zu einer
verminderten Stabilität des Stengels und weniger Seitentrieben, was ein geringeres
Gesamtgewicht zur Folge hatte. Zusätzlich zeigten diese Pflanzen auch eine verzögerte
Entwicklung der Blattrosette und Störungen bei der Schotenbildung.
81
Zusammenfassung
AtXYL3, das dritte und weiter entfernt verwandte Mitglied dieser Familie, besitzt
einen verlängerten N-Terminus, der eine vorhergesagte Chloroplasten Lokalisierungssequenz
enthält. Die vermutete plastidäre Lokalisierung dieses Proteins konnte durch transiente
Expression eines GFP Fusionsproteins in Arabidopsis Protoplasten bestätigt werden. Da
AtXYL3 nicht in Hefe exprimiert wird, steht die funktionelle Analyse in einem anderen
System noch aus. Die Analyse der gewebsspezifischen Expression von AtXYL3 durch GUS
Reporterpflanzen zeigte, dass AtXYL3 in den meisten Geweben exprimiert wird. Bei der
Untersuchung von Atxyl3 Mutanten wurde beobachtet, dass die Ausschaltung diese Gens zu
einem erhöhten Wachstum der vegetativen Teile führt, wobei Schoten- und
Samenentwicklung gestört sind. Im Dauerlicht war dieses erhöhte Wachstum noch weiter
ausgeprägt. Dies deutet auf eine wichtige Rolle von AtXYL3 beim Fluss von Glukose durch
die Plastidenhülle und beim transienten Stärkemetabiolismus hin.
82
References
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Appendix
8. Appendix
The plasmid maps that are generated in the present thesis were displayed under this
section which also included the amino acid sequence of the three transporters of newly
identified monosaccharide family and the tissue specific expression patterns (Genevestogator
microarray database).
Figure A1: Plasmid map of pSO114. PCR fragment of the AtVGT1 cDNA was ligated into E.coli/yeast shuttle
vector NEV-E, as described in § 2.1.1 and used for transport measurements in yeast and for complementation of
yeast hxt mutant.
Figure A2: Plasmid map of pSA115. The modified ORF of AtVGT1 cDNA PCR fragment was ligated into mcs
of pGEM-T easy vector as described in § 2.1.3.1 and used for further cloning into pEX tag GFP2 and pSO35e
vectors
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Appendix
Figure A3: Plasmid pSA110. AtVGT1 cDNA was ligated into pEX tag-GFP2 vector in between plasma
membrane ATPase promoter and GFP ORF via NcoI cloning site and used to determine the subcellular
localization of AtVGT1 in yeast as decribed in §2.1.3.2.
Figure A4: Plasmid map of pSA120. AtVGT1 cDNA ligated into NcoI cloning site of pSO35e vector, inbetween
CaMV 35s promoter and NOS terminator as described in § 2.1.3.3 and used for transient expression in
Arabidopsis protoplasts.
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Appendix
Figure A5: Plasmid map of pSA103: AtVGT1 promoter was cloned infront of the GUS reporter gene over
HindIII/NcoI cloning sites as described in § 2.1.5 and AtVGT1 promoter-GUS reporter gene_NOS Terminator
cassette was transferred to plant vector pGPTV-BAR.
Figure A6: Plamid map of pSA104. The AtVGT1 promoter-GUS reporter_NOS terminator cassette from
pSA103 was ligated to pGPTV-BAR vector over Xma1/EcoR1 cloning sites and used to generate transgenic
Arabidopsis plants via Agrobacterium mediated transfer.
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Appendix
Figure A7: pSA101 plasmid map. PCR fragment of the AtVGT1 promoter was cloned into pAF1 vector infront
of the GFP ORF over the Sph1/NcoI cloning sites.
Figure A8: Plasmid map of pSA102. The AtVGT1 promoter-GFP cassette from the pSA101 plasmid was cloned
into pGPTV-BAR vector infront of the NOS-Terminator over XmaI/SacI cloning sites.
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Appendix
Figure A9: pSA218 plasmid map. AtVGT2 cDNA (BbsI (EcoRI compatible)) was ligated into NEV vector over
EcoR1 cloning site and used for the functional expression AtVGT2 in yeast.
Figure A10: Plasmid map of pSA217. PCR fragment of the modified ORF of AtVGT2 cDNA (NcoI/BbsI) was
ligated into pGEM-T easy vector as described in § 2.2.3.1 and after sequence verification was used for further
cloning into pEX-Tag-GFP2 and pSO35e vectors.
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Figure A11: Plasmid pSA219. The modified ORF of AtVGT2 (along with 5’ NcoI cloning site, the 3’ primer of
AtVGT2 cDNA has BbsI (NcoI compatible) restriction site) ligated into pEXtag-GFP2 vector over NcoI cloning
site, in between pMA1 promoter and GFP ORF and was used for expression of AtVGT2-GFP fusion in yeast as
described in § 2.2.3.2.
Figure A12: Plasmid map of pSA220. AtVGT2 cDNA (*no stop) was cloned over NcoI/BbsI (NcoI compatible)
into pSO35e vector, inbetween CaMV35s promoter and NOS terminator as described in § 2.2.3.3 and was used
for transient expression of AtVGT2-GFP fusion in Arabidopsis protoplasts.
Amino acid sequence of AtXYL3 in comparison to the other members of the newly identified
monosaccharide transporter family: Amino acid sequence, represented in block was the predicted
cTP and those represented in block letters were used to raise anti AtXYL3 antibodies. The identical
regions were represented against pale grey back ground. Completely diverse regions were represented
against dark grey back ground.
Figure A21: pSA305 plasmid map. A 285 bp N-terminal cDNA sequence of AtXYL3 was cloned into mcs of
pMALc2 vector and the resultant plasmid pSA305 was used to generate MBP-AtVGT2 fusion protein.
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Appendix
Figure A22: Plasmid map of pSA322. AtXYL3 promoter was cloned into pAF6 vector over XbaI/NcoI cloning
sites inform of the GUS reporter gene.
Figure A23: pSA323 plasmid map. AtXYL3 promoter-GUS cassette from pSA322 was cloned into pGPTV-
BAR vector and was used to generate transgenic Arabidopsis plants expressing GUS reporter gene under the
control of AtXYL3 promoter.
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Appendix
Figure A24: Plasmid map of pSA324. PCR fragment of the AtXYL3 promoter was ligated into pAF1 vector
infront of the GFP ORF over XbaI/NcoI cloning sites.
Figure A25: Plasmid map of pSA325. AtXYL3 promoter-GFP cassette from pSA324 plasmid was cloned into
pGPTV-BAR vector infront of the NOS-Terminator over XbaI/SacI cloning sites.
108
Appendix
0
1000
2000
3000
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5000
6000
7000ca
llus
cell
susp
ensi
on
seed
ling
coty
ledo
ns
hypo
coty
l
radi
cle
inflo
resc
ence
flow
er
carp
el
peta
l
sepa
l
stam
en
pedi
cel
siliq
ue
seed
embr
yo
stem
node
shoo
t ape
x
caul
ine
leaf
rose
tte
juve
nile
leaf
adul
t lea
f
petio
le
sene
scen
t lea
f
root
s
prim
ary
root
late
ral r
oot
root
hai
r
root
tip
elon
gatio
n zo
ne
Figure A26: Genevestigator microarray analysis for AtVGT1 gene showing highest level of expression in stamen and basal level expression in all the other tissues except in root
hair and root tip.
109
Appendix
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callu
s
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coty
ledo
nshy
poco
tyl
radi
cle
inflo
resc
ence
flow
er
carp
elpe
tal
sepa
lst
amen
pedi
cel
siliq
uese
ed
embr
yost
em
node
shoo
t ape
xca
ulin
e le
af
rose
tteju
veni
le le
af
adul
t lea
f
petio
lese
nesc
ent l
eaf
root
spr
imar
y ro
ot
late
ral r
oot
root
hai
rro
ot ti
p
elon
gatio
n zo
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Figure A27: Genevestigator microarray analysis for AtVGT2 gene which, significantly expressed in most of the developmental stages.
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Appendix
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500
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cell
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seed
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coty
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coty
l
radi
cle
inflo
resc
ence
flow
er
carp
el
peta
l
sepa
l
stam
en
polle
n
pedi
cel
siliq
ue
seed
stem
node
shoo
t ape
x
caul
ine
leaf
rose
tte
juve
nile
leaf
adul
t lea
f
petio
le
sene
scen
t lea
f
hypo
coty
l
xyle
m
cork
root
s
late
ral r
oot
root
tip
elon
gatio
n zo
ne
Figure A28: Genevestigator microarray analysis of AtXYL3 gene significanty expression in most of the plant tissues with highest level of expression in cotyledons.
111
Curriculum Vitae
Personal Data
Name (Family) ALURI
First and Middle names Sirisha
Date of Birth 15.06.1977
Nationality Indian
Place of Birth Ramachandrapuram, India
Marital Status / Sex Married / Female
Academics and Professional Experience
06/1982 – 04/1992 Primary and Secondary School Education
S.V.N.H. School, Vidayanagar, A.P., India.
06/1992 – 03/1994 Intermediate Education
Government Junior College, Eluru, A.P., India.
05/1994 – 04/1996 Preparatory course for the university entrance examination
Sri Helapuri Residential College, Eluru, A.P., India.
06/1996 – 04/1999 Bachelor of Science,
Andhra University, Visakhapatnam, A.P., India.
09/1999 – 10/2001 Masters in Biochemistry
University of Madras, Chennai, T.N., India.
11/2001 – 04/2002 Junior Biochemist
S. V. Diagnostic Laboratory, Hyderabad, A.P., India.
07/2002 – 10/2002 Research Assistant
Max-Planck Institute for Polymer Research, Mainz, Germany.
11/2002 – 04/2003 Research Assistant
Department of Molecular Plant Physiology, University of Erlangen,
Germany.
05/2003 – Till date Doctorial Thesis on Functional Characterization of Vacuolar and
Plastidic sugar transporters within the Major Facilitator Super family of
Arabidopsis thaliana
Department of Molecular Plant Physiology, University of Erlangen,