Reference gene validation and differential expression of Psy and ACCase in oil palm with contrasting oil and carotene profiles at different ripening stages
Keywords:
carotenoid biosynthesis, Elaeis guineensis, housekeeping genesAbstract
Reliable normalization and accurate quantification are essential for gene expression studies in oil palm mesocarp. This study assessed three candidate reference genes (Actin, GAPDH, and EF1α) for stability using four algorithms (ΔCt, BestKeeper, NormFinder, and GeNorm). Actin was consistently identified as the most stable single housekeeping gene (HKG) (stability value 0.021), while the combination of GAPDH and EF1α provided the most stable normalization pair (stability value 0.017), supporting their use as internal controls for normalization. Normalized expression analysis of two key biosynthetic genes, acetyl-CoA carboxylase (ACCase) and phytoene synthase (Psy), was conducted in contrasting phenotypes differing in oil yield and carotene content. The high-yielding, high-carotene genotype displayed lower ΔCt values for both ACCase (4.67 vs. 6.19) and Psy (4.75 vs. 5.26), corresponding to 2.86-fold and 1.42-fold higher expression, respectively. These expression differences aligned closely with phenotypic outcomes, as the superior traits achieved a 40.8% increase in oil yield and a 155.3% increase in carotene content. Expression patterns showed stage-specific specialization: ACCase was upregulated during the unripe stage, enhancing fatty acid biosynthesis, while Psy was most differentially expressed during ripening, coinciding with carotenoid accumulation. Together, these findings suggest that transcript abundance of oil and carotenoid pathways may be coordinated in elite oil palm and provide a framework for linking gene expression with metabolic output, which may inform future efforts to improve oil yield and quality.
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Aberlenc-Bertossi, F., Chabrillange, N., Duval, Y., & Tregear, J. (2008). Contrasting globulin and cysteine proteinase gene expression patterns reveal fundamental developmental differences between zygotic and somatic embryos of oil palm. Tree Physiology, 28(8), 1157–1167. https://doi.org/10.1093/treephys/28.8.1157
Afifi, E. H., John Martin, J. J., Wang, Q., Li, X., Liu, X., Zhou, L., Li, R., Fu, D., Li, Q., Ye, J., & Cao, H. (2025). Fatty acid and lipid metabolism in oil palm: From biochemistry to molecular mechanisms. International Journal of Molecular Sciences, 26(6), 2531. https://doi.org/10.3390/ijms26062531
Afiq, M. A. M., Shaharuddin, N. A., & Zubaidah, R. (2019). Identification of reliable reference genes for gene expression studies of oil palm plantlets using NormFinder and BestKeeper algorithms. Journal of Oil Palm Research, 31(2), 204–211. https://doi.org/10.21894/jopr.2019.0010
Aithal, M. G. S., & Rajeswari, N. (2015). Validation of housekeeping genes for gene expression analysis in glioblastoma using quantitative real-time polymerase chain reaction. Brain Tumor Research and Treatment, 3(1), 24. https://doi.org/10.14791/btrt.2015.3.1.24
Andersen, C. L., Jensen, J. L., & Ørntoft, T. F. (2004). Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research, 64(15), 5245–5250. https://doi.org/10.1158/0008-5472.CAN-04-0496
Antoniassi, R., Wilhelm, A. E., Guedes, A. M. M., & Faria-Machado, A. F. (2024). Bioactive compounds and composition of Elaeis oleifera mesocarp oil extracted by hydraulic pressing. Grasas y Aceites, 75(1), e536. https://doi.org/10.3989/gya.0646241.2194
Apriyanto, A., Compart, J., Zimmermann, V., Alseekh, S., Fernie, A. R., & Fettke, J. (2022). Indication that starch and sucrose are biomarkers for oil yield in oil palm (Elaeis guineensis Jacq.). Food Chemistry, 393, 133361. https://doi.org/10.1016/j.foodchem.2022.133361
Batool, K., Jabeen, S., Khawar, H., et al. (2024). qRT-PCR data normalization by the identification of expression analysis of the most stable and the least stable housekeeping genes (HKGs) in Covid-19. History of Medicine, 10(2), 542–556. https://doi.org/10.17720/24095834.v10.2.2024.40
Bocian, J., Jabłoński, B., & Nadolska-Orczyk, A. (2025). Validated reference genes for normalization of RT-qPCR in developing organs of wheat to study developmentally/spatio-temporally expressed family genes. Scientific Reports, 15, 1–11. https://doi.org/10.1038/s41598-025-08295-6
Budiani, A. (2014). Ekspresi gen penyandi ACCase subunit biotin karboksilase dari mesokarp buah kelapa sawit pada Escherichia coli. Menara Perkebunan, 82(1), 32–38. https://doi.org/10.22302/iribb.jur.mp.v74i1.119
Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J., & Wittwer, C. T. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4), 611–622. https://doi.org/10.1373/clinchem.2008.112797
Chan, P. L., Rose, R. J., Abdul Murad, A. M., et al. (2014). Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture. PLOS ONE, 9(6), e99774. https://doi.org/10.1371/journal.pone.0099774
Chang, S., Puryear, J., & Cairney, J. (1993). A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter, 11(2), 113–116. https://doi.org/10.1007/BF02670468
Esteva-Socias, M., Artiga, MJ., Bahamonde, O. et al. (2019). In search of an evidence-based strategy for quality assessment of human tissue samples: report of the tissue Biospecimen Research Working Group of the Spanish Biobank Network. Journal of Translational Medicine, 17, 370. https://doi.org/10.1186/s12967-019-2124-8
Expósito-Rodríguez, M., Borges, A. A., Borges-Pérez, A., & Pérez, J. A. (2008). Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biology, 8, 131. https://doi.org/10.1186/1471-2229-8-131
Forero, D. C., Hormaza, P. & Romero, H. (2012). Phenological growth stages of African oil palm (Elaeis guineensis). Annals of Applied Biology. 160, 56-65. https://doi.org/10.1111/j.17447348.2011.00520.x
Grätz, C., Bui, M. L. U., Thaqi, G., Kirchner, B., Loewe, R. P., & Pfaffl, M. W. (2022). Obtaining reliable RT‐qPCR results in molecular Diagnostics—MIQE goals and pitfalls for transcriptional biomarker discovery. Life, 12(3), 386. https://doi.org/10.3390/life12030386
Kozera, B., & Rapacz, M. (2013). Reference genes in real-time PCR. Journal of Applied Genetics, 54(4), 391–406. https://doi.org/10.1007/s13353-013-0173-x
Kwan, Y. M., Meon, S., Ho, C. L., & Wong, M. Y. (2016). Selection of reference genes for quantitative real-time PCR normalization in Ganoderma-infected oil palm (Elaeis guineensis) seedlings. Australasian Plant Pathology, 45(3), 261–268. https://doi.org/10.1007/s13313-016-0417-4
Lieb, V. M., Kerfers, M. R., Kronmüller, A., Esquivel, P., Alvarado, A., Jiménez, V. M., Schmarr, H.-G., Carle, R., Schweiggert, R. M., & Steingass, C. B. (2017). Characterization of mesocarp and kernel lipids from Elaeis guineensis Jacq., Elaeis oleifera [Kunth] Cortés, and their interspecific hybrids. Journal of Agricultural and Food Chemistry, 65(18), 3617–3626. https://doi.org/10.1021/acs.jafc.7b00604
Masura, S. S., Rasid, O. A. & Shaharuddin, N. A. (2022). Identification of oil palm root-specific genes through mining of RNA-seq data and RT-qPCR analysis. Journal of Oil Palm Research, 34(2), 261–275. https://doi.org/ 10.21894/jopr.2021.0036
Moebes, M., Kuhlmann, H., Demidov, D., & Lermontova, I. (2022). Optimization of quantitative reverse transcription PCR method for analysis of weakly expressed genes in crops based on rapeseed. Frontiers in Plant Science, 13, 954976. https://doi.org/10.3389/fpls.2022.954976
Morcillo, F., Vaissayre, V., Serret, J., Avallone, S., Domonhedo, H., Jacob, F. & Dussert, S. (2021). Natural diversity in the carotene, tocochromanol and fatty acid composition of crude palm oil. Food Chemistry, 365, 130638. https://doi.org/10.1016/j.foodchem.2021.130638
Pfaffl, M. W., Tichopad, A., Prgomet, C., & Neuvians, T. P. (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnology Letters, 26(6), 509–515. https://doi.org/10.1023/b:bile.0000019559.84305.47
Plotka, M., Wozniak, M., & Kaczorowski, T. (2017) Quantification of plasmid copy number with single colour droplet digital PCR. PLoS ONE 12(1): e0169846. https://doi.org/10.1371/ journal.pone.0169846
Puteh, A. Q., Shahrome, A. A. M., Razali, M. H. H., & Sulaiman, A. (2022). Comparative study of carotenoids content in ripe and unripe oil palm fresh fruit bunches. International Journal of Integrated Engineering, 14(9), 240–246. https://doi.org/10.30880/ijie.2022.14.09.030
Putranto, R. A., Syaputra, I., & Budiani, A. (2016). Differential gene expression in oil palm varieties susceptible and tolerant to Ganoderma. 6th Indonesian Biotechnology Conference, 233–243.
Rasid, O. A., Wan Nur Syuhada, W. S., Hanin, A. N., et al. (2008). RT-PCR amplification and cloning of partial DNA sequence coding for oil palm (Elaeis oleifera) Phytoene synthase gene. Asia-Pacific Journal of Molecular Biology and Biotechnology, 16(1), 17–24.
Robinson, L. J., Munro, J., Grimsey, N., & Poulsen, R. (2025). Stability of commonly used housekeeping genes for gene expression studies differs in human osteoarthritic chondrocytes from males compared to females. Osteoarthritis and Cartilage, 33(10), 1471–1482.
Salas, J. J., García-Gonzalez, D. L., & Aparicio, R. (2006). Volatile compound biosynthesis by green leaves from an Arabidopsis thaliana hydroperoxide lyase knockout mutant. Journal of Agricultural and Food Chemistry, 54(21), 8199–8205. https://doi.org/10.1021/jf061493f
Shakeri, M. S. (2022). Comparison of DNA standards for real-time PCR-based quantification of Lactobacillus acidophilus in dairy products. Journal of Microbiology, Biotechnology and Food Sciences, 11(4), e3738. https://doi.org/10.55251/jmbfs.3738
Silver, N., Best, S., Jiang, J., & Thein, S. L. (2006). Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Molecular Biology, 7, 33. https://doi.org/10.1186/1471-2199-7-33
Sinta, M. M., Riyadi, I., Kresnawaty, I., Siregar, H. A., & Saptari, R. T. (2025, May). Pengaruh teknik preparasi terhadap hasil analisis karoten pada buah kelapa sawit klonal [Poster presentation]. PTKS 2025, 16-17 Juli 2025, Yogyakarta, Indonesia.
Syuhada, W. S. W. N., Rasid, O. A., & Parveez, G. K. A. (2021). Molecular cloning of Elaeis guineensis phytoene synthase (EgPSY) and its expression in mesocarp tissues. Journal of Oil Palm Research, 33(3), 447–457. https://doi.org/10.21894/jopr.2020.0113
Ting, N. C., Sherbina, K., Khoo, J. S., Kamaruddin, K., Chan, P. L., Chan, K. L., Halim, M. A. A., Sritharan, K., Yaakub, Z., Mayes, S., Massawe, F., Chang, P. L., Nuzhdin, S. V., Sambanthamurthi, R., & Singh, R. (2020). Expression of fatty acid and triacylglycerol synthesis genes in interspecific hybrids of oil palm. Scientific Reports, 10(1), 10355.
https://doi.org/10.1038/s41598-020-73170-5
Tranbarger, T. J., Dussert, S., Joët, T., Argout, X., Summo, M., Champion, A., Cros, D., Omore, A., Nouy, B., & Morcillo, F. (2011). Regulatory mechanisms underlying oil palm fruit mesocarp maturation, ripening, and functional specialization in lipid and carotenoid metabolism. Plant Physiology, 156(2), 564–584. https://doi.org/10.1104/pp.111.175141
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., & Speleman, F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3(7), research0034.1. https://doi.org/10.1186/gb-2002-3-7-research0034
Welsch, R., Arango, J., Bär, C., Salazar, B., Al-Babili, S., Beltrán, J., Chavarriaga, P., Ceballos, H., Tohme, J., & Beyer, P. (2010). Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene. Plant Cell, 22(10), 3348–3356. https://doi.org/10.1105/tpc.110.077560
Wong, Y. C., Kwong, Q. B., Lee, H. L., Ong, C. K., Mayes, S., Chew, F. T., Appleton, D. R., & Kulaveerasingam, H. (2014). Expression comparison of oil biosynthesis genes in oil palm mesocarp tissue using custom array. Microarrays, 3(4), 263-281. https://doi.org/ 10.3390/microarrays3040263
Yeap, W. C., Loo, J. M., Wong, Y. C., & Kulaveerasingam, H. (2014). Evaluation of suitable reference genes for qRT-PCR gene expression normalization in reproductive, vegetative tissues and during fruit development in oil palm. Plant Cell, Tissue and Organ Culture, 116(1), 55–66. https://doi.org/10.1007/ s11240-013-0382-3
Zhang, R., Liu, X., Cheng, Y., Martin, J. J. J., Li, X., Zhou, L., Fu, D., Li, Q., Li, R. Cao, H. (2025). Multi-omic analysis to develop key genes involved in carotenoid synthesis and establish indicative molecular markers for oil palm fruit colour. Industrial Crops and Products, 225, 120487. https://doi.org/10.1016/j.indcrop.2025.120487
Zhou, X., Rao, S., Wrightstone, E., Sun, T., Lui, A. C. W., Welsch, R., & Li, L. (2022). Phytoene synthase: The key rate-limiting enzyme of carotenoid biosynthesis in plants. Frontiers in Plant Science, 13, Article 884720. https://doi.org/10.3389/fpls.2022.884720
Zuhar, L. M., Madihah, A. Z., Ahmad, S. A., Zainal, Z., Idris, A.S., & Shaharuddin, N. A. (2021). Identification of oil palm’s consistently upregulated genes during early infections of Ganoderma boninense via RNA-seq technology and real-time quantitative PCR. Plants, 10(10), 2026. https://doi.org/ 10.3390/plants10102026
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Copyright (c) 2026 Fauziatul Fitriyah, Ikram Maulana, Masna Maya Sinta, Rizka Tamania Saptari, Irma Kresnawaty, Heri Andriwan Siregar, Imron Riyadi
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