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Cycloheximide treatment of cotton ovules alters the abundance of specific classes of mRNAs and generates novel ESTs for microarray expression profiling

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Abstract

Fibres of cotton (Gossypium hirsutum L.) are single elongated epidermal cells that start to develop on the outer surface of cotton ovules on the day of anthesis. Little is known about the control of fibre initiation and development. As a first step towards discovering important genes involved in fibre initiation and development using a genomics approach, we report technical advances aimed at reducing redundancy and increasing coverage for anonymous cDNA microarrays in this study. Cotton ovule cDNA libraries (both normalised and un-normalised) from around the time of fibre initial formation have been prepared and partially characterised by sequencing. Re-association-based normalisation partially reduced library redundancy and increased representation of novel sequences. However, another library generated from in vitro cultured cotton ovules treated with the protein synthesis inhibitor, cycloheximide, showed a significantly altered gene representation including a greater proportion of protein phosphorylation genes, transport genes and transcription factors and a much reduced proportion of protein synthesis genes than were identified in the conventional types of libraries. Over 10,000 expressed sequence tag (EST) clones randomly selected from the three libraries were printed on microarray slides and used to assess gene expression in tissue cultured ovules with and without cycloheximide treatment. The microarray results showed that cycloheximide had a dramatic effect in modifying the pattern of the gene expression in cultured ovules, affecting the same types of genes identified in the preliminary analysis on relative EST abundance in the different ovule cDNA libraries. Cycloheximide clearly provided a simple and useful method for enriching novel gene sequences for genomic studies.

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Acknowledgements

This work was partially funded by the Cotton Research and Development Corporation of Australia and Cotton Seed Distributors, Wee Waa, NSW, Australia. The authors would like to thank Todd Collins, Adriane Machado and Pinghua He for excellent technical assistance; Dr. Iain Wilson for help in printing our microarray slides and many helpful suggestions on the microarray experiments; Dr Tony Arioli for bridging the CSIRO and Bayer groups on sequence analysis; Dr. Fei Zhang, Michael James, Leahka Henry and Gavin Kennedy for bioinformatics assistance with the sequence analysis and database management; Dr. Bob Anderssen for insightful discussions on sequence redundancy data analysis; and Warren Muller for helpful discussions on the statistical analysis of the microarray data. Over 7000 originally anonymous cDNA clones were converted to ESTs with the assistance of the Arizona Genomics Institute funded by the NSF Plant Genome Program#9872630 and#0211700. This research was carried out in compliance with the laws governing recombinant DNA research in Australia under OGTR permit NLRD-774. Upon request, all novel materials described in this publication will be made available in a timely manner for non-commercial research purposes, subject to the requisite permission from any third-party owners of all parts of the material. Obtaining any permission will be the responsibility of the requestor. The sequences described in this paper have been lodged with Genbank as Accessions DT455583-461485 (ON ESTs); DT461486-469116 (CHX ESTs) and DT526800-527666 (OCF ESTs).

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Authors and Affiliations

  1. CSIRO Plant Industry, GPO Box 1600, Canberra, ACT, 2601, Australia

    Yingru Wu, Danny J. Llewellyn & Elizabeth S. Dennis

  2. Bioinformatics Team, Bayer CropScience, Rue Pierre Fontaine, 91058, Evry Cedex, France

    Sophie Rozenfeld & Aurelie Defferrard

  3. CSIRO Mathematical and Information Sciences, GPO Box 664, Canberra, ACT, 2601, Australia

    Katya Ruggiero

  4. Department of Ecology, Evolution and Organismal Biology, Bessey Hall, Iowa State University, Ames, IA, 50011, USA

    Joshua A. Udall

  5. Arizona Genomics Institute, Department of Plant Sciences, University of Arizona, Forbes Building, 303, PO Box 210036, Tucson, AZ, 85721-0036, USA

    HyeRan Kim

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  1. Yingru Wu
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Correspondence to Danny J. Llewellyn.

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Communicated by M.-A. Grandbastion

Upon request, all novel materials described in this publication will be made available in a timely manner for non-commercial research purposes, subject to the requisite permission from any third-party owners of all parts of the material. Obtaining any permission will be the responsibility of the requestor.

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Wu, Y., Rozenfeld, S., Defferrard, A. et al. Cycloheximide treatment of cotton ovules alters the abundance of specific classes of mRNAs and generates novel ESTs for microarray expression profiling. Mol Genet Genomics 274, 477–493 (2005). https://doi.org/10.1007/s00438-005-0049-9

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