Transcriptome analysis of Arabidopsis microgametogenesis
David Honys
Laboratory of Pollen Biology, Institute of Experimental Botany AS CR, Rozvojová 135, 16502 Praha 6, Czech Republic
Tel.: +420-220 390 450
Fax: +420-220 390 461
e-mail: honys@ueb.cas.cz
Laboratory Homepage: http://www.ueb.cas.cz/laboratory_of_pollen_biology/default.htm
David Twell
Department of Biology, University of Leicester, Leicester LE1 7RH, United Kingdom
Tel: +44-116-2522281
Fax: +44-116-2522791
e-mail: twe@le.ac.uk
Twell Homepage: http://www.le.ac.uk/bl/staff/bltwe.htm
Pollen Research Group Homepage: http://www.le.ac.uk/biology/research/pollen/pollen.html
Department of Biology Homepage: http://www.le.ac.uk/biology/bl.htm
Aims
We aim to use transcriptome analysis to establish on a genome-wide scale the identity and regulatory clusters of genes that specify microgametogenesis from the haploid microspore to mature functional pollen in Arabidopsis.
Background
Pollen as the haploid male gametophyte plays a vital role in plant fertility and crop production through the ability to deliver the male gametes in fertilisation. Despite the obvious importance for plant fertility and crop production we have a very limited understanding of the regulatory mechanisms that have evolved to specify male gametophyte development and functions and less than 150 genes have been identified that are gametophytically expressed in the anther.The availability of functional genomic resources now provides the opportunity to undertake a comprehensive approach to describing cellular development in terms of the transcriptome. This approach is particularly powerful where the complete transcriptome of a single developing cell can be analysed. The male gametophyte is a uniquely accessible cell type for such studies, enabling RNA analysis from distinct purified cell populations during development.
Biological material and methods
Isolated microspores and pollen at 4 different developmental stages will be analysed. We will isolate spores from developmentally staged buds of Ler grown under defined growth conditions. Buds from several batches of 100 plants will be rapidly sorted into 4 groups according to developmental age, uninucleate microspores (UNM), bicellular pollen (BCP) tricellular pollen (TCP) and mature pollen (MPG). Spores will be released by gentle mechanical tissue disruption and purified by filtration and purification of spores. We are confident that our spore isolation procedures are rigorous since we could not detect even trace expression of highly abundant sporophytic transcripts such RbcS and Cab transcripts in microarray data from pollen RNA.
BioSource Information
Organism: Arabidopsis thaliana
Alias: Ler-0
Stock Code: NW20
Growth Conditions:
        Location Growth Room
        Atmosphere Normal
        Temperature 21°C
        Lighting (Source: 100 umol.m-2.s-1) 16h light
        Medium compost
Developmental Stage:
        Growth Stage: 5.10
        Genetic Variation: None
        Tissue: Developing spores, pollen
        Diseased: Normal
        Target Cell Type: Uninucleate microspores (UNM), Bicellular pollen (BCP), Tricellular pollen (TCP),                                                   Mature pollen (MPG)
Separation Technique: Filtration
References
Honys, D. & Twell, D.: Transcriptome analysis of developing haploid male gametophytes in Arabidopsis thaliana. Genome Biology 5: R85, 2004.
Honys, D. & Twell, D.: Comparative analysis of Arabidopsis pollen transcriptome. Plant Physiol. 132: 640-652, 2003.
Links to datasets for download at NASC:
http://affymetrix.arabidopsis.info/narrays/experimentpage.pl?experimentid=48 http://affymetrix.arabidopsis.info/narrays/experimentpage.pl?experimentid=6
Original raw data:
Uninucleate microspores (UNM):
  (2%) Trimmed mean (TM=100) normalized data
UNM11.cel UNM12.cel   Original cel files
Bicellular pollen (BCP):
  (2%) Trimmed mean (TM=100) normalized data
BCP11.cel BCP12.cel   Original cel files
Tricellular pollen (TCP):
  (2%) Trimmed mean (TM=100) normalized data
TCP11.cel TCP12.cel    Original cel files
Mature pollen (MPG):
  (2%) Trimmed mean (TM=100) normalized data
MPG11.cel MPG12.cel   Original cel files
dCHIP:   dCHIP-normalised expression data
Original cel files: