Reference genes for transcript quantification in Gracilaria tenuistipitata under drought stress

Gracilaria tenuistipitata is a red macroalga often found in intertidal environments, where it is frequently under drought stress due to tidal water scarcity. In order to be able to study the molecular mechanisms involved in G. tenuistipitata resistance to dehydration, we have done a dehydration assay, tested different RNA extraction protocols, and selected new reference genes for reverse transcription quantitative PCR (RT-qPCR). After testing four different methods, we found that an adapted TRIzol method resulted in the highest quantities of pure RNA from less biomass. This method yielded an average of 783.6 ng mg(-1) of clean RNA from 25 mg of fresh tissue in only 1.5 h. Additionally, DNAse I treatment and silica spin column purification were done prior to RT-qPCR. Of the nine putative reference genes tested for expression stability under control and drought-like conditions, a combination of four genes: beta-GALACTOSIDASE B (GLB), TRANSCRIPTION INITIATION FACTOR IIB (GTF2B), EUKARYOTIC ELONGATION FACTOR 2 (EEF2), and beta-TUBULIN (TUB) was selected. This allowed us to measure the transcription level of THIOREDOXIN (TRX), OXYGEN-EVOLVING ENHANCER 1 (OEE), and NITRATE REDUCTASE 2 (NIA2). TRX was upregulated after 1-h dehydration and kept higher transcriptional levels even 4 h later. In contrast, OEE1 transcriptional levels were slowly downregulated, and NIA2 transcriptional levels were downregulated 1 h after dehydration but returned to regular levels 4 h after. This suggests that 40 % dehydration of G. tenuistipitata induced transcriptional responses without changing the relative growth rate or affecting the thalli mortality.