Selection and Validation of Reference Genes for Accurate RT-qPCR Data Normalization inCoffeaspp. under a Climate Changes Context of Interacting Elevated [CO2] and Temperature.
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-qPCR expression studies using a set of 24 cDNAs from leaves of three coffee genotypes (CL153, Icatu, and IPR108), grown under 380 or 700 μL CO2L-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, classifiedMDHas the gene with the highest mRNA stability to a global use, followed byACTandS15, whereas α-TUBandCYCLshowed 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 thein silicoaggregation 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 ofCoffeaspp. in a context of climate changes and global warming.
Plant-Environment Interactions and Biodiversity Lab (PlantStress&Biodiversity), Linking Landscape, Environment, Agriculture and Food, Departmento de Recursos Naturais, Ambiente e Território, Instituto Superior de Agronomia, Universidade de Lisboa (ULisboa)Oeiras, Portugal; Programa de Pós-Graduação em Genética e Melhoramento, Centro de Ciências Agrárias e Engenharias, Universidade Federal do Espírito SantoAlegre, Brazil.
Madlles Q
MQ
Martins
Plant-Environment Interactions and Biodiversity Lab (PlantStress&Biodiversity), Linking Landscape, Environment, Agriculture and Food, Departmento de Recursos Naturais, Ambiente e Território, Instituto Superior de Agronomia, Universidade de Lisboa (ULisboa) Oeiras, Portugal.
Ana S
AS
Fortunato
Plant-Environment Interactions and Biodiversity Lab (PlantStress&Biodiversity), Linking Landscape, Environment, Agriculture and Food, Departmento de Recursos Naturais, Ambiente e Território, Instituto Superior de Agronomia, Universidade de Lisboa (ULisboa)Oeiras, Portugal; Setor Fisiologia Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual Norte Fluminense-Darcy RibeiroRio de Janeiro, Brazil.
Weverton P
WP
Rodrigues
Departmento de Ciências Agrárias e Biológicas, Centro Universitário Norte do Espírito Santo, Universidade Federal Espírito Santo São Mateus, Brazil.
Fábio L
FL
Partelli
Setor Fisiologia Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual Norte Fluminense-Darcy Ribeiro Rio de Janeiro, Brazil.
Eliemar
E
Campostrini
GeoBioTec, Departmento de Ciências da Terra, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa Monte da Caparica, Portugal.
Fernando C
FC
Lidon
Departmento de Biologia Vegetal, Universidade Federal Viçosa Viçosa, Brazil.
Fábio M
FM
DaMatta
Plant-Environment Interactions and Biodiversity Lab (PlantStress&Biodiversity), Linking Landscape, Environment, Agriculture and Food, Departmento de Recursos Naturais, Ambiente e Território, Instituto Superior de Agronomia, Universidade de Lisboa (ULisboa)Oeiras, Portugal; GeoBioTec, Departmento de Ciências da Terra, Faculdade de Ciências e Tecnologia, Universidade NOVA de LisboaMonte da Caparica, Portugal.
José C
JC
Ramalho
Plant-Environment Interactions and Biodiversity Lab (PlantStress&Biodiversity), Linking Landscape, Environment, Agriculture and Food, Departmento de Recursos Naturais, Ambiente e Território, Instituto Superior de Agronomia, Universidade de Lisboa (ULisboa)Oeiras, Portugal; GeoBioTec, Departmento de Ciências da Terra, Faculdade de Ciências e Tecnologia, Universidade NOVA de LisboaMonte da Caparica, Portugal.
Ana I
AI
Ribeiro-Barros
climate changes; coffee; global warming; heat stress; increased air [CO2]; normalization of transcriptomic studies; quantitative real-time PCR; reference genes