title: Nitrogen starvation, salt and heat stress in coffee (Coffea arabica L.): identification and validation of new genes for qPCR normalization.
abstract: Abiotic stresses are among the most important factors that affect food production. One important step to face these environmental challenges is the transcriptional modulation. Quantitative real-time PCR is a rapid, sensitive, and reliable method for the detection of mRNAs and it has become a powerful tool to mitigate plant stress tolerance; however, suitable reference genes are required for data normalization. Reference genes for coffee plants during nitrogen starvation, salinity and heat stress have not yet been reported. We evaluated the expression stability of ten candidate reference genes using geNorm PLUS, NormFinder, and BestKeeper softwares, in plants submitted to nitrogen starvation, salt and heat stress. EF1, EF1α, GAPDH, MDH, and UBQ10 were ranked as the most stable genes in all stresses and software analyses, while RPL39 and RPII were classified as the less reliable references. For reference gene validation, the transcriptional pattern of a Coffea non-symbiotic hemoglobin (CaHb1) was analyzed using the two new recommended and the most unstable gene references for normalization. The most unstable gene may lead to incorrect interpretation of CaHb1 transcriptional analysis. Here, we recommend two new reference genes in Coffea for use in data normalization in abiotic stresses: MDH and EF1.
authors.affiliation: Laboratório de Biotecnologia Vegetal, Instituto Agronômico do Paraná, Londrina, PR, Brazil. keniadecarvalho@gmail.com
authors.forename: Kenia
authors.initials: K
authors.lastname: de Carvalho
authors.forename: João Carlos
authors.initials: JC
authors.lastname: Bespalhok Filho
authors.forename: Tiago Benedito
authors.initials: TB
authors.lastname: dos Santos
authors.forename: Silvia Graciele Hülse
authors.initials: SG
authors.lastname: de Souza
authors.forename: Luiz Gonzaga Esteves
authors.initials: LG
authors.lastname: Vieira
authors.forename: Luis Filipe Protasio
authors.initials: LF
authors.lastname: Pereira
authors.forename: Douglas Silva
authors.initials: DS
authors.lastname: Domingues
mesh_terms: D040503:Coffea / Q000235:genetics* / Q000378:metabolism; D005511:Food Handling; D020869:Gene Expression Profiling / Q000379:methods; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006358:Hot Temperature; D009584:Nitrogen / Q000378:metabolism*; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D012965:Sodium Chloride; D012984:Software; D013312:Stress, Physiological
chemical_list: D018749:RNA, Plant; D012965:Sodium Chloride; D009584:Nitrogen