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Title: Transcriptome-wide identification of reference genes for expression analysis of soybean responses to drought stress along the day

Author
item GOMES, JULIANA - Embrapa
item RODRIGUES, FABIANA - Embrapa
item FUGANTI-PAGLIARINI, RENATA - Embrapa
item NAKAYAMA, THIAGO - Embrapa
item REIS, RAFAELA - Embrapa
item FARIAS, JOSE - Embrapa
item Harmon, Frank
item NEPOMUCENO, ALEX - Embrapa

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/7/2015
Publication Date: 9/7/2015
Citation: Gomes, J.M., Rodrigues, F.A., Fuganti-Pagliarini, R., Nakayama, T.J., Reis, R., Farias, J.B., Harmon, F.G., Nepomuceno, A.L. 2015. Transcriptome-wide identification of reference genes for expression analysis of soybean responses to drought stress along the day. PLoS One. 10(9):e0139051.

Interpretive Summary: Gene expression in many plants changes in response to internal signals, like those from the circadian clock, and external signals, like drought stress. The study of this type of gene expression is important to understand the behavior and adaptation of many crop to the local climate. Accurate quantification of gene expression requires so called reference genes with stable expression in many conditions and at any time along the day that can be used in normalization calculations. The object of this work was to identify novel reference genes that will be useful for the normalization of gene expression in studies of soybean under both drought stress and diurnal conditions. The approach was to mine several genome-wide gene expression studies for candidate reference genes. This computational analysis identified the genes FYVE, NUDIX, Golgin-84 and CYST a good candidates for reference genes. Experimental validation and stability analysis in soybean plants exposed to drought stress showed that each of these genes is more stably expressed than conventionally used housekeeping genes (ELF1-ß and ß-actin). We also demonstrate these novel reference genes work well to study the expression of the drought-inducible soybean DREB5 gene.

Technical Abstract: The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expression quantification in combined studies on drought stress and diurnal oscillations. Recently, water deficit responses have been associated with circadian clock oscillations at the transcription level, revealing the existence of hitherto unknown processes and increasing the demand for studies on plant responses to drought stress and its oscillation during the day. We performed data mining from a transcriptome-wide background using microarrays and RNA-seq databases to select an unpublished set of candidate reference genes, specifically chosen for the normalization of gene expression in studies on soybean under both drought stress and diurnal oscillations. Experimental validation and stability analysis in soybean plants submitted to drought stress and sampled during a 24 h time course showed that four of these newer reference genes (FYVE, NUDIX, Golgin-84 and CYST) indeed exhibited greater expression stability than the conventionally used housekeeping genes (ELF1-ß and ß-actin) under these conditions. We also demonstrated the effect of using reference candidate genes with different stability values to normalize the relative expression data from a drought-inducible soybean gene (DREB5) evaluated in different periods of the day.