Phosphate nutrition in the Ricinus communis L. seedling [Elektronische Ressource] : role of the phosphate transporter and acid phosphatase / vorgelegt von Tran Dang Khoa

K Narayanaswami Aiyar

Sujets

• Biologie

Informations

• Publié par: universitat_bayreuth
• Publié le: 01 janvier 2007
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Phosphate nutrition in the Ricinus communis L.
seedling:
Role of the phosphate transporter and acid
phosphatase






Dissertation zur Erlangung des Doktorgrades
an der Fakultät für Biologie, Chemie und Geowissenschaften
der Universität Bayreuth


vorgelegt von
Tran Dang Khoa
aus Viet nam



Bayreuth, 2006


Vollständiger Abdruck der von der Fakultät für Biologie, Chemie und Geowissenschaften
der Universität Bayreuth zur Erlangung des akademischen Grades eines Doktors der
Naturwissenschaften genehmigten Dissertation.





























Tag der Einreichung: 15. Nov 2006

Tag des wissenschaftlichen Kolloquiums: 23. Februar 2007


Prüfungsausschuss:
Prof. Dr. E. Komor 1. Gutachter
Prof. Dr. W. Schumann 2. Gutachter
Prof. Dr. Y. Kuzyakov
Prof. Dr. E. Steudle
Prof. Dr. G. Gebauer Vorsitzender

























For my Family


Contents i





Contents


1 Introduction..................................................................................................1
1.1 Morphological plant responses to Pi starvation.................................................1
1.2 Biochemical and genetic responses to Pi starvation .........................................2
1.2.1 Pi transport mechanism in plants ..............................................................2
1.2.2 Pi regulated gene expression....................................................................3
1.2.3 Regulation of plant phosphate transporters...............................................6
1.2.4 Acid phosphatases ....................................................................................7
1.3 Phosphates in germinating seeds. ..................................................................10
Aims of the present study............................................................................................12
2 Material and Methods ................................................................................13
2.1 Material............................................................................................................13
2.1.1 Chemicals................................................................................................13
2.1.2 Enzymes and kits13
2.1.3 Plasmid vectors .......................................................................................13
2.1.4 Plant materials and growth conditions.....................................................14
2.1.5 Bacterial strains14
2.1.6 Yeast strains............................................................................................15
2.1.7 Oligonucleotide Primers ..........................................................................15
2.1.8 Sequence analysis softwares and online sequences ..............................16
2.2 Methods...........................................................................................................17
2.2.1 Determination of phosphorus ..................................................................17
2.2.2 Application of radiotracers .......................................................................17
2.2.3 Autoradiography and tracer detection .....................................................18
2.2.4 Isolation of DNA and Southern Blot analysis...........................................19
2.2.5 RNA and Northern Blot analysis19
2.2.6 cDNA synthesis by reverse transcriptase polymerase chain reaction (RT-
PCR)........................................................................................................20
2.2.7 Polymerase chain reaction (PCR) ...........................................................20
2.2.7.1 Standard PCR .....................................................................................20
2.2.7.2 Screening bacterial colonies with PCR...............................................21
2.2.7.3 Design of specific and degenerate primers .........................................21
2.2.7.4 Cloning of amplified products ..............................................................22
2.2.8 RNA Ligase Mediated Rapid amplification of cDNA Ends (RLM-RACE).22
2.2.8.1 5´ RACE ..............................................................................................22
2.2.8.2 3´ RACE .............................................................................................23
2.2.9 Nonradioactive RNA probe synthesis......................................................23
2.2.10 In situ hybridization..................................................................................25
2.2.10.1 Fixation and embedding sample25
2.2.10.2 Pre-hybridization and hybridization..................................................25
2.2.11 Expression of RcPT1 in yeast mutants....................................................27
2.2.11.1 Transformation of yeast...................................................................27
2.2.11.2 Acid phosphatase activity test and yeast growth experiments ........28 Contents ii

322.2.11.3 Uptake of P in yeast ......................................................................28
2.2.12 Expression of RcPS1 recombinant protein in E.coli using pET system...28
2.2.13 Protein analysis .......................................................................................29
2.2.13.1 Purification of recombinant protein ..................................................29
2.2.13.2 Protein extraction from plants..........................................................29
2.2.13.3 SDS-PAGE ......................................................................................29
2.2.13.4 Coomassie staining of protein gel....................................................30
2.2.13.5 Antibody Production.........................................................................30
2.2.13.6 Western blot.....................................................................................31
2.2.13.7 Immunolocalization..........................................................................31
3 Results........................................................................................................33
3.1 Characterization of Pi translocation in Ricinus communis L. seedlings...........33
3.1.1 Translocation of phosphate from the cotyledons to the hypocotyl33
3.1.2 Phosphate fluxes after incubation of the roots with phosphate buffer .....35
3.2 Effect of phosphate deficiency on germination and growth of Ricinus plants..37
3.2.1 Influence of exogenous Pi on Ricinus seedlings during germination ......37
3.2.2 Effect of Pi deficiency on plant growth and phosphate concentration .....40
3.3 Cloning and functional characterization of RcPT1, a phosphate transporter ..42
3.3.1 Cloning of RcPT1, a phosphate transporter ............................................42
3.3.2 Deduced peptide sequence of RcPT1 showing a structure of a
transmembrane protein ...........................................................................43
3.3.3 Genomic organization of RcPT1 gene.....................................................48
3.3.4 Yeast functional complementation and phosphate uptake properties .....49
3.3.5 Expression of phosphate transporter RcPT1 during germination of
seedlings .................................................................................................52
3.3.6 Western blot analysis of the RcPT1 protein in seedling ..........................54
3.3.7 RcPT1 transcript induction under phosphate starvation in plants ...........55
3.3.8 In Situ hybridization of RcPT1 .................................................................56
3.3.9 Immunolocalization of the RcPT1 protein................................................57
3.4 Acid phosphatases ..........................................................................................59
3.4.1 Cloning of RcPS1, a novel acid phosphatase and computational
sequence analysis...................................................................................59
3.4.2 Genomic organization of RcPS1 gene ....................................................64
3.4.3 Expression of recombinant in E.coli ............................................65
3.4.4 Expression pattern of RcPS1 in seedling organs during germination .....66
3.4.5 RcPS1 transcript induction under phosphate starvation in plant .............68
3.4.6 Localization of RcPS1 transcript in cotyledon and leaf............................68
4 Discussion..................................................................................................72
4.1 Phosphate homeostasis in Ricinus seedlings .................................................72
4.1.1 Phosphate uptake via cotyledons............................................................72
4.1.2 Phosphate uptake via roots.....................................................................73