Selection and validation of reference genes for accurate RT-qPCR data normalization in Coffea spp. under a climate changes context of interacting elevated [CO2] and temperature

This work was supported by by national funds from Fundacao para a Ciencia e a Tecnologia through the projects PTDC/AGR-PRO/3386/2012, the research units UID/AGR/04129/2013 (LEAF) and UID/GEO/04035/2013 (GeoBioTec), as well through the grant SFRH/BPD/47563/2008 (AF) co-financed through the POPH program subsidized by the European Social Fund. Brazilian funding from CAPES (grants PDSE: 000427/2014-04, WR; 0343/2014-05, MM), CNN and Fapemig (fellowships to FD, FP, and EC) are also greatly acknowledged.World coffee production has faced increasing challenges associated with ongoing climatic changes. Several studies, which have been almost exclusively based on temperature increase, have predicted extensive reductions (higher than half by 2,050) of actual coffee cropped areas. However, recent studies showed that elevated [CO2 ] can strongly mitigate the negative impacts of heat stress at the physiological and biochemical levels in coffee leaves. In addition, it has also been shown that coffee genotypes can successfully cope with temperatures above what has been traditionally accepted. Altogether, this information suggests that the real impact of climate changes on coffee growth and production could be significantly lower than previously estimated. Gene expression studies are an important tool to unravel crop acclimation ability, demanding the use of adequate reference genes. We have examined the transcript stability of 10 candidate reference genes to normalize RT-qPCRexpression studies using a set of 24 cDNAs from leaves of three coffee genotypes (CL153, Icatu, and IPR108), grown under 380 or 700 µL CO2 L−1, and submitted to increasing temperatures from 25/20° C (day/night) to 42/34° C. Samples were analyzed according to genotype, [CO2 ], temperature, multiple stress interaction ([CO2 ], temperature) and total stress interaction (genotype, [CO2 ], and temperature). The transcript stability of each gene was assessed through a multiple analytical approach combining the Coeficient of Variation method and three algorithms (geNorm, BestKeeper, NormFinder). The transcript stability varied according to the type of stress for most genes, but the consensus ranking obtained with RefFinder, classified MDH as the gene with the highest mRNA stability to a global use, followed by ACT and S15, whereas α-TUB and CYCL showed the least stable mRNA contents. Using the coffee expression profiles of the gene encoding the large-subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RLS), results from the in silico aggregation and experimental validation of the best number of reference genes showed that two reference genes are adequate to normalize RT-qPCR data. Altogether, this work highlights the importance of an adequate selection of reference genes for each single or combined experimental condition and constitutes the basis to accurately study molecular responses of Coffea spp. in a context of climate changes and global warming.publishersversionpublishe