Proteome and Transcriptional Analysis of Arabidopsis thaliana Sperm Cells

Ajaraporn Sriboonlert

Research output: ThesisDoctoral Thesis

Abstract

Angiosperm sexual reproduction is unique and remarkable. Unlike for other living organisms, fertilisation involves the fusion of two sperm cells to two cell types of female gametophyte, the egg and central cell. This fertilization process is called double fertilisation. Fusion of the egg and the sperm yields a zygote whereas the interaction of the central cell and the sperm gives the nutritive endosperm. This fertilization process has been studied extensively for many years. However, despite these studies, relatively little is known at the molecular level about either of the plant gametes. This is primarily due to the difficulties in plant gamete isolation. Both plant gametes reside within enclosed tissues, pollen grains and embryo sacs. In this study, sperm isolation techniques were successfully developed for Brassica oleracea and Arabidopsis thaliana which ultimately utilised fluorescence-activated cell sorting (FACS) to obtain pure cell samples. Proteomic studies utilising two-dimensional protein electrophoresis and mass spectrometry (MS) were carried out on both semi-purified and FACS-purified sperm cells. In parallel with the proteomic studies a bioinformatics approach was taken which used sperm transcriptome data of maize, Plumbago zeylanica, rice and tobacco to identify homologues in Arabidopsis. Transcriptional analyses, RT-PCR and GFP translational fusion experiments were used to investigate these Arabidopsis sperm cell-expressed gene candidates. As a result, two sperm cell-expressed genes (At1g10090 and At5g39650) were identified and these are being analysed to confirm their functions in the reproductive process. Moreover, the sperm cell-expressed gene candidates derived from the bioinformatics were also screened for roles in plant reproduction by a reverse genetics approach (Arabidopsis T-DNA insertion mutagenesis plant screening) and eight genes were identified. In addition, for the first time, sperm cell size dimorphism was identified for Arabidopsis in this study utilising a GFP-labelled sperm line and confocal microscopy. Overall the techniques for sperm cell-expressed gene candidate selection were proven to be effective and will certainly facilitate further sperm cell-specific gene identification studies. Further the Arabidopsis sperm purification technique successfully developed in this project will surely be useful for any plant sperm cell studies in the future.
LanguageEnglish
QualificationPh.D.
Awarding Institution
  • University of Bath
Supervisors/Advisors
  • Doughty, James, Supervisor
Award date1 Sep 2008
StatusUnpublished - Sep 2008

Fingerprint

proteome
Arabidopsis thaliana
spermatozoa
cells
fertilization (reproduction)
Arabidopsis
germ cells
genes
bioinformatics
proteomics
flow cytometry
Plumbago zeylanica
purification methods
plant reproduction
embryo sac
isolation techniques
zygote
Brassica oleracea
dimorphism
sexual reproduction

Keywords

  • sperm cell
  • Arabidopsis

Cite this

Proteome and Transcriptional Analysis of Arabidopsis thaliana Sperm Cells. / Sriboonlert, Ajaraporn.

2008.

Research output: ThesisDoctoral Thesis

Sriboonlert, A 2008, 'Proteome and Transcriptional Analysis of Arabidopsis thaliana Sperm Cells', Ph.D., University of Bath.
@phdthesis{f4f1f6d345b546d1b4d821657684b8e7,
title = "Proteome and Transcriptional Analysis of Arabidopsis thaliana Sperm Cells",
abstract = "Angiosperm sexual reproduction is unique and remarkable. Unlike for other living organisms, fertilisation involves the fusion of two sperm cells to two cell types of female gametophyte, the egg and central cell. This fertilization process is called double fertilisation. Fusion of the egg and the sperm yields a zygote whereas the interaction of the central cell and the sperm gives the nutritive endosperm. This fertilization process has been studied extensively for many years. However, despite these studies, relatively little is known at the molecular level about either of the plant gametes. This is primarily due to the difficulties in plant gamete isolation. Both plant gametes reside within enclosed tissues, pollen grains and embryo sacs. In this study, sperm isolation techniques were successfully developed for Brassica oleracea and Arabidopsis thaliana which ultimately utilised fluorescence-activated cell sorting (FACS) to obtain pure cell samples. Proteomic studies utilising two-dimensional protein electrophoresis and mass spectrometry (MS) were carried out on both semi-purified and FACS-purified sperm cells. In parallel with the proteomic studies a bioinformatics approach was taken which used sperm transcriptome data of maize, Plumbago zeylanica, rice and tobacco to identify homologues in Arabidopsis. Transcriptional analyses, RT-PCR and GFP translational fusion experiments were used to investigate these Arabidopsis sperm cell-expressed gene candidates. As a result, two sperm cell-expressed genes (At1g10090 and At5g39650) were identified and these are being analysed to confirm their functions in the reproductive process. Moreover, the sperm cell-expressed gene candidates derived from the bioinformatics were also screened for roles in plant reproduction by a reverse genetics approach (Arabidopsis T-DNA insertion mutagenesis plant screening) and eight genes were identified. In addition, for the first time, sperm cell size dimorphism was identified for Arabidopsis in this study utilising a GFP-labelled sperm line and confocal microscopy. Overall the techniques for sperm cell-expressed gene candidate selection were proven to be effective and will certainly facilitate further sperm cell-specific gene identification studies. Further the Arabidopsis sperm purification technique successfully developed in this project will surely be useful for any plant sperm cell studies in the future.",
keywords = "sperm cell, Arabidopsis",
author = "Ajaraporn Sriboonlert",
year = "2008",
month = "9",
language = "English",
school = "University of Bath",

}

TY - THES

T1 - Proteome and Transcriptional Analysis of Arabidopsis thaliana Sperm Cells

AU - Sriboonlert,Ajaraporn

PY - 2008/9

Y1 - 2008/9

N2 - Angiosperm sexual reproduction is unique and remarkable. Unlike for other living organisms, fertilisation involves the fusion of two sperm cells to two cell types of female gametophyte, the egg and central cell. This fertilization process is called double fertilisation. Fusion of the egg and the sperm yields a zygote whereas the interaction of the central cell and the sperm gives the nutritive endosperm. This fertilization process has been studied extensively for many years. However, despite these studies, relatively little is known at the molecular level about either of the plant gametes. This is primarily due to the difficulties in plant gamete isolation. Both plant gametes reside within enclosed tissues, pollen grains and embryo sacs. In this study, sperm isolation techniques were successfully developed for Brassica oleracea and Arabidopsis thaliana which ultimately utilised fluorescence-activated cell sorting (FACS) to obtain pure cell samples. Proteomic studies utilising two-dimensional protein electrophoresis and mass spectrometry (MS) were carried out on both semi-purified and FACS-purified sperm cells. In parallel with the proteomic studies a bioinformatics approach was taken which used sperm transcriptome data of maize, Plumbago zeylanica, rice and tobacco to identify homologues in Arabidopsis. Transcriptional analyses, RT-PCR and GFP translational fusion experiments were used to investigate these Arabidopsis sperm cell-expressed gene candidates. As a result, two sperm cell-expressed genes (At1g10090 and At5g39650) were identified and these are being analysed to confirm their functions in the reproductive process. Moreover, the sperm cell-expressed gene candidates derived from the bioinformatics were also screened for roles in plant reproduction by a reverse genetics approach (Arabidopsis T-DNA insertion mutagenesis plant screening) and eight genes were identified. In addition, for the first time, sperm cell size dimorphism was identified for Arabidopsis in this study utilising a GFP-labelled sperm line and confocal microscopy. Overall the techniques for sperm cell-expressed gene candidate selection were proven to be effective and will certainly facilitate further sperm cell-specific gene identification studies. Further the Arabidopsis sperm purification technique successfully developed in this project will surely be useful for any plant sperm cell studies in the future.

AB - Angiosperm sexual reproduction is unique and remarkable. Unlike for other living organisms, fertilisation involves the fusion of two sperm cells to two cell types of female gametophyte, the egg and central cell. This fertilization process is called double fertilisation. Fusion of the egg and the sperm yields a zygote whereas the interaction of the central cell and the sperm gives the nutritive endosperm. This fertilization process has been studied extensively for many years. However, despite these studies, relatively little is known at the molecular level about either of the plant gametes. This is primarily due to the difficulties in plant gamete isolation. Both plant gametes reside within enclosed tissues, pollen grains and embryo sacs. In this study, sperm isolation techniques were successfully developed for Brassica oleracea and Arabidopsis thaliana which ultimately utilised fluorescence-activated cell sorting (FACS) to obtain pure cell samples. Proteomic studies utilising two-dimensional protein electrophoresis and mass spectrometry (MS) were carried out on both semi-purified and FACS-purified sperm cells. In parallel with the proteomic studies a bioinformatics approach was taken which used sperm transcriptome data of maize, Plumbago zeylanica, rice and tobacco to identify homologues in Arabidopsis. Transcriptional analyses, RT-PCR and GFP translational fusion experiments were used to investigate these Arabidopsis sperm cell-expressed gene candidates. As a result, two sperm cell-expressed genes (At1g10090 and At5g39650) were identified and these are being analysed to confirm their functions in the reproductive process. Moreover, the sperm cell-expressed gene candidates derived from the bioinformatics were also screened for roles in plant reproduction by a reverse genetics approach (Arabidopsis T-DNA insertion mutagenesis plant screening) and eight genes were identified. In addition, for the first time, sperm cell size dimorphism was identified for Arabidopsis in this study utilising a GFP-labelled sperm line and confocal microscopy. Overall the techniques for sperm cell-expressed gene candidate selection were proven to be effective and will certainly facilitate further sperm cell-specific gene identification studies. Further the Arabidopsis sperm purification technique successfully developed in this project will surely be useful for any plant sperm cell studies in the future.

KW - sperm cell

KW - Arabidopsis

M3 - Doctoral Thesis

ER -