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ISSN 0128-7680

Home / Regular Issue / JST Vol. 47 (2) May. 2024 / JTAS-2879-2023


Overexpression of amiR2937 and amiR854e in Transgenic Arabidopsis thaliana Indirectly Impacts the Photosynthesis Performances by Targeting Specific Target Transcripts in the MEP Pathway

Tuan Aini Nadirah Che-Wan-Ngah, Muhamad Hafiz Che Othman and Ismanizan Ismail

Pertanika Journal of Science & Technology, Volume 47, Issue 2, May 2024


Keywords: amiRNAs, Arabidopsis thaliana, gene-silencing, post-transcriptional regulation, sesquiterpenoid and phytol biosynthesis

Published on: 30 May 2024

Artificial miRNAs (amiRNAs) are artificial small RNAs engineered to silence specific plant mRNA transcripts. They are generated by expressing a functional microRNA (miRNA) with modified sequences in planta. Two miRNAs, miR2937 and miR854e, were selected based on their predicted target transcript, GGPS2 (geranylgeranyl pyrophosphate synthase 2) and TPS13 (terpenoid synthase 13). In the methylerythritol phosphate pathways, GGPS2 and TPS13 enzymes play a role in synthesizing sesquiterpenes, triterpenes, diterpenoids, carotenoids, gibberellins, and chlorophyll, respectively. Therefore, in this study, these two miRNAs were overexpressed in Arabidopsis thaliana in single and co-overexpression to analyze the change in the abundance of phytol and trans-beta-lone compounds. Through real-time quantitative polymerase chain reaction (RT-qPCR) analysis, a fold-up regulation of amiR2937 and amiR854e was observed in both transgenic plants harboring single and double constructs. Meanwhile, the GGPS2 and TPS13 enzymes showed a decreasing pattern in all transgenic plants, indicating that the miRNAs had successfully suppressed the target transcripts. Solid-phase microextraction-gas chromatography-mass spectrometry analysis revealed that the number of phytols was decreased in all transgenic plants but was significant in plants harboring construct miR854e. Meanwhile, there is an increasing pattern of trans-beta-ionone in all transgenic plants compared to wild-type plants. Consistently, with the decrease in phytol content, soil plant analysis development value, and total chlorophyll content, the photosynthesis rate decreased in the transgenic plants compared to the wild type. Indeed, the overexpression of these two miRNAs affects the production of target transcript and changes the plant development.

  • Akula, R., & Ravishankar, G. A. (2011). Influence of abiotic stress signals on secondary metabolites in plants. Plant Signaling and Behavior, 6(11), 1720–1731.

  • Ameres, S. L., & Zamore, P. D. (2013). Diversifying microRNA sequence and function. Nature Reviews Molecular Cell Biology, 14, 475–488.

  • Arteaga-Vázquez, M., Caballero-Pérez, J., & Vielle-Calzada, J. P. (2006). A family of microRNAs present in plants and animals. The Plant Cell, 18(12), 3355–3369.

  • Bahi, A., Al Mansouri, S., Al Memari, E., Al Ameri, M., Nurulain, S. M., & Ojha, S. (2014). β-Caryophyllene, a CB2 receptor agonist produces multiple behavioral changes relevant to anxiety and depression in mice. Physiology and Behavior, 135, 119–124.

  • Beck, G., Coman, D., Herren, E., Águila Ruiz-Sola, M., Rodríguez-Concepción, M., Gruissem, W., & Vranová, E. (2013). Characterization of the GGPP synthase gene family in Arabidopsis thaliana. Plant Molecular Biology, 82, 393–416.

  • Bent, A. (2006). Arabidopsis thaliana floral dip transformation method. In K. Wang (Ed.), Agrobacterium protocols: Methods in molecular biology (Vol. 343, pp. 87–104). Humana Press.

  • Berthelot, K., Estevez, Y., Deffieux, A., & Peruch, F. (2012). Isopentenyl diphosphate isomerase: A checkpoint to isoprenoid biosynthesis. Biochimie, 94(8), 1621–1634.

  • Câmara, J. S., Arminda Alves, M., & Marques, J. C. (2006). Development of headspace solid-phase microextraction-gas chromatography-mass spectrometry methodology for analysis of terpenoids in Madeira wines. Analytica Chimica Acta, 555(2), 191–200.

  • Carbonell, A., Takeda, A., Fahlgren, N., Johnson, S. C., Cuperus, J. T., & Carrington, J. C. (2014). New generation of artificial microRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Physiology, 165(1), 15–29.

  • Chaves, M. M., Flexas, J., & Pinheiro, C. (2009). Photosynthesis under drought and salt stress: Regulation mechanisms from whole plant to cell. Annals of Botany, 103(4), 551–560.

  • Chen, Q., Fan, D., & Wang, G. (2015). Heteromeric geranyl (geranyl) diphosphate synthase is involved in monoterpene biosynthesis in Arabidopsis flowers. Molecular Plant, 8(9), 1434–1437.

  • Chen, X. (2004). A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science, 303(5666), 2022–2025.

  • Chowdhury, R. R., & Ghosh, S. K. (2012). Phytol-derived novel isoprenoid immunostimulants. Frontiers in Immunology, 3, 49.

  • De Bolle, M., Beyers, W., De Clercq, B., & De Fruyt, F. (2012). General personality and psychopathology in referred and nonreferred children and adolescents: An investigation of continuity, pathoplasty, and complication models. Journal of Abnormal Psychology, 121(4), 958–970.

  • de Menezes Patrício Santos, C. C., Salvadori, M. S., Mota, V. G., Costa, L. M., de Almeida, A. A. C., de Oliveira, G. A. L., Costa, J. P., de Sousa, D. P., de Freitas, R. M., & de Almeida, R. N. (2013). Antinociceptive and antioxidant activities of phytol in vivo and in vitro models. Neuroscience Journal, 2013, 949452.

  • Ditengou, F. A., Müller, A., Rosenkranz, M., Felten, J., Lasok, H., van Doorn, M. M., Legué, V., Palme, K., Schnitzler, J.-P., & Polle, A. (2015). Volatile signaling by sesquiterpenes from ectomycorrhizal fungi reprogrammes root architecture. Nature Communications, 6, 6279.

  • Feng, L., Raza, M. A., Li, Z., Chen, Y., Khalid, M. H. Bin, Du, J., Liu, W., Wu, X., Song, C., Yu, L., Zhang, Z., Yuan, S., Yang, W., & Yang, F. (2019). The influence of light intensity and leaf movement on photosynthesis characteristics and carbon balance of soybean. Frontiers in Plant Science, 9, 1952.

  • Grant-Downton, R., Trionnaire, G. L., Schmid, R., Rodriguez-Enriquez, J., Hafidh, S., Mehdi, S., Twell, D., & Dickinson, H. (2009). MicroRNA and tasiRNA diversity in mature pollen of Arabidopsis thaliana. BMC Genomics, 10, 643.

  • Huang, M., Sanchez-Moreiras, A. M., Abel, C., Sohrabi, R., Lee, S., Gershenzon, J., & Tholl, D. (2012). The major volatile organic compound emitted from Arabidopsis thaliana flowers, the sesquiterpene (E)-β-caryophyllene, is a defense against a bacterial pathogen. New Phytologist, 193(4), 997–1008.

  • Jin, W., Wang, J., Liu, C.-P., Wang, H.-W., & Xu, R.-M. (2020). Structural basis for pri-miRNA recognition by Drosha. Molecular Cell, 78(3), 423-433.e5.

  • Kang, J., Liu, C., & Kim, S.-H. (2013). Environmentally sustainable textile and apparel consumption: The role of consumer knowledge, perceived consumer effectiveness and perceived personal relevance. International Journal of Consumer Studies, 37(4), 442–452.

  • Kasajima, I., Ide, Y., Ohkama-Ohtsu, N., Hayashi, H., Yoneyama, T., & Fujiwara, T. (2004). A protocol for rapid DNA extraction from Arabidopsis thaliana for PCR analysis. Plant Molecular Biology Reporter, 22, 49–52.

  • Kozomara, A., & Griffiths-Jones, S. (2014). MiRBase: Annotating high-confidence microRNAs using deep sequencing data. Nucleic Acids Research, 42(D1), D68–D73.

  • Kwon, S. C., Baek, S. C., Choi, Y.-G., Yang, J., Lee, Y-S., Woo, J.-S., & Kim, V. N. (2019). Molecular basis for the single-nucleotide precision of primary microRNA processing. Molecular Cell, 73(3), 505-518.e5.

  • Lange, B. M., & Ghassemian, M. (2003). Genome organization in Arabidopsis thaliana: A survey for genes involved in isoprenoid and chlorophyll metabolism. Plant Molecular Biology, 51, 925-948.

  • Laskovics, F. M., & Poulter, C. D. (1981). Prenyltransferase: Determination of the binding mechanism and individual kinetic constants for farnesylpyrophosphate synthetase by rapid quench and isotope partitioning experiments. Biochemistry, 20(7), 1893-1901.

  • Leonard, E., Ajikumar, P. K., Thayer, K., Xiao, W.-H., Mo, J. D., Tidor, B., Stephanopoulos, G., & Prather, K. L. J. (2010). Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control. PNAS, 107(31), 13654-13659.

  • Li, T., Hasegawa, T., Yin, X., Zhu, Y., Boote, K., Adam, M., Bregaglio, S., Buis, S., Confalonieri, R., Fumoto, T., Gaydon, D., Marcaida, M., Nakagawa, H., Oriol, P., Ruane, A. C., Ruget, F., Singh, B., Singh, U., Tang, L., … Bouman, B. (2015). Uncertainties in predicting rice yield by current crop models under a wide range of climatic conditions. Global Change Biology, 21(3), 1328–1341.

  • Liang, Y., Urano, D., Liao, K.-L., Hedrick, T. L., Gao, Y., & Jones, A. M. (2017). A non-destructive method to estimate the chlorophyll content of Arabidopsis seedlings. Plant Methods, 13, 26.

  • Lim, S.-Y., Meyer, M., Kjonaas, R. A., & Ghosh, S. K. (2006). Phytol-based novel adjuvants in vaccine formulation: I. assessment of safety and efficacy during stimulation of humoral and cell-mediated immune responses. Journal of Immune Based Therapies and Vaccines, 4, 6.

  • Ling, Q., Huang, W., & Jarvis, P. (2011). Use of a SPAD-502 meter to measure leaf chlorophyll concentration in Arabidopsis thaliana. Photosynthesis Research, 107, 209–214.

  • Liu, Z., Wang, J., Cheng, H., Ke, X., Sun, L., Zhang, Q. C., & Wang, H.-W. (2018). Cryo-EM structure of human dicer and its complexes with a pre-miRNA substrate. Cell, 173(5), 1191-1203.e12.

  • Martin, R. C., Liu, P.-P., Goloviznina, N. A., & Nonogaki, H. (2010). MicroRNA, seeds, and Darwin?: Diverse function of miRNA in seed biology and plant responses to stress. Journal of Experimental Botany, 61(9), 2229–2234.

  • McCourt, P., & Benning, C. (2010). Arabidopsis: A rich harvest 10 years after completion of the genome sequence. Plant Journal, 61(6), 905–908.

  • Nauš, J., Prokopová, J., Řebíček, J., & Špundová, M. (2010). SPAD chlorophyll meter reading can be pronouncedly affected by chloroplast movement. Photosynthesis Research, 105, 265–271.

  • Parida, A. K., Mittra, B., Das, A. B., Das, T. K., & Mohanty, P. (2005). High salinity reduces the content of a highly abundant 23-kDa protein of the mangrove Bruguiera parviflora. Planta, 221, 135–140.

  • Picaud, S., Olsson, M. E., Brodelius, M., & Brodelius, P. E. (2006). Cloning, expression, purification, and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale. Archives of Biochemistry and Biophysics, 452(1), 17–28.

  • Ramirez-Estrada, K., Vidal-Limon, H., Hidalgo D., Moyano, E., Golenioswki, M., Cusidó, R. M., & Palazon, J. (2016). Elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories. Molecules, 21(2), 182.

  • Ro, D.-K., Paradise, E. M., Quellet, M., Fisher, K. J., Newman, K. L., Ndungu, J. M., Ho, K. A., Eachus, R. A., Ham, T. S., Kirby, J., Chang, M. C. Y., Withers, S. T., Shiba, Y., Sarpong, R., & Keasling, J. D. (2006). Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature, 440, 940–943.

  • Ruppel, N. J., Kropp, K. N., Davis, P. A., Martin, A. E., Luesse, D. R., & Hangarter, R. P. (2013). Mutations in GERANYLGERANYL DIPHOSPHATE SYNTHASE 1 affect chloroplast development in Arabidopsis thaliana (Brassicaceae). American Journal of Botany, 100(10), 2074–2084.

  • Ryu, K.-R., Choi, J.-Y., Chung, S., & Kim, D.-H. (2011). Anti-scratching behavioral effect of the essential oil and phytol isolated from Artemisia princeps Pamp. in mice. Planta Medica, 77(1), 22–26.

  • Sapir-Mir, M., Mett, A., Belausov, E., Tal-Meshulam, S., Frydman, A., Gidoni, D., & Eya, Y. (2008). Peroxisomal localization of Arabidopsis isopentenyl diphosphate isomerases suggests that part of the plant isoprenoid mevalonic acid pathway is compartmentalized to peroxisomes. Plant Physiology, 148(3), 1219–1228.

  • Saravanavel, R., Ranganathan, R., & Anantharaman, P. (2011). Effect of sodium chloride on photosynthetic pigments and photosynthetic characteristics of Avicennia officinalis seedlings. Recent Research in Science and Technology, 3, 177-180.

  • Tholl, D. (2015). Biosynthesis and biological functions of terpenoids in plants. In J. Schrader & J. Bohlmann (Eds.), Biotechnology of isoprenoids: Advances in biochemical engineering/biotechnology (Vol. 148, pp. 63–106). Springer.

  • Tholl, D., & Lee, S. (2011). Terpene specialized metabolism in Arabidopsis thaliana. The Arabidopsis Book, 9, e0143.

  • Wang, J., Lee, J. E., Riemondy, K., Yu, Y., Marquez, S. M., Lai, E. C., & Yi, R. (2020). XPO5 promotes primary miRNA processing independently of RanGTP. Nature Communications, 11, 1845.

  • Yu, Z.-X., Wang, L.-J., Zhao, B., Shan, C.-M., Zhang, Y.-H., Chen, D.-F., & Chen, X.-Y. (2015). Progressive regulation of sesquiterpene biosynthesis in arabidopsis and patchouli (Pogostemon cablin) by the MIR156-targeted SPL transcription factors. Molecular Plant, 8(1), 98–110.