Reference gene selection for gene expression analysis in Coffea arabica L. under ABA and gibberellin treatments

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) requires stable reference genes (RGs) to ensure accurate target gene normalization. Plant growth regulators like abscisic acid (ABA) and gibberellins (GA₃) play critical roles in plant reproductive transitions, yet validated RGs for Coffea arabica under these specific treatments remain scarce. This study aimed to evaluate the expression stability of eight candidate RGs in coffee leaves subjected to various hormonal concentrations.

The stability of CaACT, CaGAPDH, CaEFA1, CaTUB, CaAP47, CaMDH, CaRPL39, and CaUBQ2 was evaluated in leaf tissues of Coffea arabica during the reproductive stage under different ABA and GA₃ treatments using five statistical algorithms: geNorm, NormFinder, BestKeeper, Delta-Ct, and RefFinder. The expression profiles of the candidate genes varied among treatments, with CaEFA1, CaGAPDH, CaMDH, and CaACT showing higher and more stable expression levels, whereas CaTUB and CaAP47 were consistently ranked as the least stable genes. Pearson correlation analysis revealed high correlations among CaRPL39, CaUBQ2, CaMDH, CaGAPDH, CaEFA1, and CaACT, while CaAP47 and CaTUB showed the lowest correlation coefficients. Validation analysis using the flowering-related gene CaTFL1 demonstrated that normalization with stable RGs (CaACT and CaMDH) produced more consistent expression profiles and narrower confidence intervals than normalization using unstable RGs.

CaACT and CaMDH are the most suitable RGs for RT-qPCR normalization in leaf tissues of Coffea arabica during the reproductive stage under ABA and GA₃ treatments. These findings provide a reliable basis for future studies involving hormonal regulation and reproductive development in coffee plants and reinforce the importance of validating RGs for specific experimental conditions.