Suppression subtractive hybridization for studying gene expression during aerial exposure and desiccation in fucoid algae

Abstract

Gene expression in Fucus vesiculosus L. from the Ria Formosa, Faro, Portugal was investigated by screening a cDNA library generated by suppression subtractive hybridization (SSH) for algae undergoing mild desiccation stress (60–70% tissue water content). The subtractive library was constructed from small amounts (1 μg) of total RNA using PCR-based techniques. Screening by reverse Northern analysis (cDNA Southerns) revealed that between 60% and 70% of clones randomly selected from the library were differentially regulated in desiccated and hydrated algae. Most genes could not be directly identified based on sequence homology with known gene sequences. However, several cDNAs for chloroplast-encoded transcripts were identified and shown to be up-regulated or differentially regulated in desiccated algae relative to hydrated controls. These included partial sequences for ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco; rbcL/rbcS), chloroplast coupling factor ATPase (atpH/atpI) and a photosystem I P700 chlorophyll a binding protein (psaA). The usefulness of reverse Northern analysis was confirmed with conventional Northern analysis of transcript abundance for Rubisco, which varied rapidly in response to light and the hydration status of the algae. These results show that SSH is a useful technique for analysing the responses of gene expression to environmental stress, and as a starting point for the subsequent identification of stress-responsive genes in macroalgae.