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JXB Advance Access originally published online on September 5, 2008
Journal of Experimental Botany 2008 59(13):3661-3674; doi:10.1093/jxb/ern214
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Expression profile analysis of genes involved in cell wall regeneration during protoplast culture in cotton by suppression subtractive hybridization and macroarray

Xiyan Yang, Lili Tu, Longfu Zhu, Lili Fu, Ling Min and Xianlong Zhang*

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China

* To whom correspondence should be addressed: E-mail: xlzhang{at}mail.hzau.edu.cn

The molecular mechanisms underlying cell wall biosynthesis are poorly understood. In this study, microscopic analysis showed that protoplasts generated a new cell wall within 48 h after transfer to a wall-regeneration medium. To identify genes related to cell wall biosynthesis in cotton, suppression subtractive hybridization was used to visualize differential gene expression at seven time points within the first 48 h. In total, 412 differentially expressed sequence tags (ESTs; >3-fold) were identified, and 210 unigenes were sequenced successfully. As confirmed by reverse-transcription PCR (RT-PCR) and real-time quantitative reverse-transcription PCR (QRT-PCR) analysis, the selected genes displayed complex expression patterns during cell wall regeneration from protoplasts and included most previously published cell-wall-associated genes. ESTs similar to cell-wall-protein genes, such as proline-rich protein (PRPL), glycine-rich protein (GRP), extension (EPR1), fasciclin-like arabinogalactan protein (FLA2), and expensing-like protein (EXLA and EXLB), which might participate in primary cell wall or secondary cell wall construction and modification, were up-regulated during cell wall regeneration from protoplasts. Sucrose synthase, an important enzyme in the sugar signalling pathway, played important roles in cellulose biosynthesis. Our findings also highlighted the function of some transcription factors during cell wall regeneration from protoplasts, including the squamosa promoter binding protein-like 14 (SPL14), NAC, Gbiaa-re, MYB, WRKY, swellmap 1 (SMP1), RAD5, and zinc finger family protein, as well as the enrichment of Ca2+-calmodulin signal molecules. On the basis of the gene expression profiles, a model of cell wall regeneration from protoplasts derived from cotton suspension cultures is proposed.

Key words: Cell wall regeneration, cotton, expression pattern, suppression subtractive hybridization

Received 11 May 2008; Revised 27 July 2008 Accepted 30 July 2008


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