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Background <p>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 (GA<sub>3</sub>) play critical roles in plant reproductive transitions, yet validated RGs for <i>Coffea arabica</i> 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.</p> Methods and results <p>The stability of <i>CaACT</i>, <i>CaGAPDH</i>, <i>CaEFA1</i>, <i>CaTUB</i>, <i>CaAP47</i>, <i>CaMDH</i>, <i>CaRPL39</i>, and <i>CaUBQ2</i> was evaluated in leaf tissues of <i>Coffea arabica</i> during the reproductive stage under different ABA and GA<sub>3</sub> treatments using five statistical algorithms: geNorm, NormFinder, BestKeeper, Delta-Ct, and RefFinder. The expression profiles of the candidate genes varied among treatments, with <i>CaEFA1</i>, <i>CaGAPDH</i>, <i>CaMDH</i>, and <i>CaACT</i> showing higher and more stable expression levels, whereas <i>CaTUB</i> and <i>CaAP47</i> were consistently ranked as the least stable genes. Pearson correlation analysis revealed high correlations among <i>CaRPL39</i>, <i>CaUBQ2</i>, <i>CaMDH</i>, <i>CaGAPDH</i>, <i>CaEFA1</i>, and <i>CaACT</i>, while <i>CaAP47</i> and <i>CaTUB</i> showed the lowest correlation coefficients. Validation analysis using the flowering-related gene <i>CaTFL1</i> demonstrated that normalization with stable RGs (<i>CaACT</i> and <i>CaMDH</i>) produced more consistent expression profiles and narrower confidence intervals than normalization using unstable RGs.</p> Conclusions <p><i>CaACT</i> and <i>CaMDH</i> are the most suitable RGs for RT-qPCR normalization in leaf tissues of <i>Coffea arabica</i> during the reproductive stage under ABA and GA<sub>3</sub> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月31日

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  • Lillian Magalhães Azevedo,
  • Robert Márquez-Gutiérrez,
  • Matheus Martins Daúde,
  • Horllys Gomes Barreto,
  • Renato Ribeiro de Lima,
  • Raphael Ricon de Oliveira,
  • Antonio Chalfun-Junior

摘要

Background

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.

Methods and results

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.

Conclusions

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.