Modulating Rice Grain Phenylalanine: Effects of Foliar Biostimulant Application

Document Type : Original Article

Authors

1 Department of Plant sciences and Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.

2 Plant Sciences and Biotechnology Department, Life Sciences and Biotechnology School, Shahid Beheshti University, Evin, Tehran, Iran, 1983963113.

3 Rice Research Institute of Amol, Iran, 91951-46191.

10.48308/pae.2026.242182.1127

Abstract

Phenylketonuria (PKU) requires a strict low-phenylalanine diet to prevent harmful accumulation in the body. Current dietary options, largely reliant on processed foods, are often unpalatable, expensive, and inaccessible in developing countries. This study aimed to naturally develop a rice strain with reduced phenylalanine levels, avoiding genetic modification, andoptimize biological stimulants for modulating phenylalanine biosynthesis. Field trials involved foliar application of cinnamic acid (0, 0.5, 1, and 1.5 g/L) and phenylalanine (0, 0.25, 0.5, and 1 g/L) on two local rice cultivars (Helal and Keshvari) during seed formation and filling, using a randomized complete block design with three replications. Key molecular traits measured included total protein content, phenylalanine ammonia-lyase (PAL) enzyme activity, and expression of genes involved in phenylalanine biosynthesis and catabolism (phenylpyruvate aminotransferase, arogenate dehydratase, and PAL). Results showed that phenylalanine application increased PAL gene expression, while cinnamic acid suppressed it. Although both stimulants significantly downregulated phenylalanine biosynthesis genes and reduced total protein content for both treatments, a decrease in total protein content occurred at the highest applied concentration. Notably, cinnamic acid significantly decreased phenylalanine levels in Keshvari (1 and 1.5 g/L), while increasing tyrosine and tryptophan concentrations at higher dosages. In addition, phenylalanine treatments at concentrations of 0.5 and 1 g/L led to decreased expression of the arogenate dehydratase gene. Moreover, the application of phenylalanine at 0.5 and 1 g/L reduced the expression of the phenylpyruvate aminotransferase gene in both rice cultivars studied. Cinnamic acid treatment can reduce phenylalanine in rice, offering a natural strategy for PKU-friendly diets.

Keywords

References

Ali, A., More, T.A. and Shaikh, Z., 2021. Artificial sweeteners and their health implications: a review. Biosciences Biotechnology Research Asia, 18(2), pp.227-237. Doi: http://dx.doi.org/10.13005/bbra/2910.
Ashe, K., Kelso, W., Farrand, S., Panetta, J., Fazio, T., De Jong, G. and Walterfang, M., 2019. Psychiatric and cognitive aspects of phenylketonuria: the limitations of diet and promise of new treatments. Frontiers in psychiatry, 10, p.561. Doi: https://doi.org/10.3389/fpsyt.2019.00561.
Aydaş, S.B., Ozturk, S. and Aslım, B., 2013. Phenylalanine ammonia lyase (PAL) enzyme activity and antioxidant properties of some cyanobacteria isolates. Food Chemistry, 136(1), pp.164-169. Doi: https://doi.org/10.1016/j.foodchem.2012.07.119.
Bahadur, A., Singh, D.P., Sarma, B.K. and Singh, U.P., 2012. Foliar application of l-phenylalanine and ferulic acids to pea plants: induced phenylalanine ammonia lyase activity and resistance against Erysiphe pisi. Archives of Phytopathology and Plant Protection, 45(4), pp.398-403. Doi: https://doi.org/10.1080/03235408.2011.587963.
Baziramakenga, R., Leroux, G.D., Simard, R.R. and Nadeau, P., 1997. Allelopathic effects of phenolic acids on nucleic acid and protein levels in soybean seedlings. Canadian Journal of Botany, 75(3), pp.445-450.Doi: https://doi.org/10.1139/b97-047.
Blau, N., 2016. Genetics of phenylketonuria: then and now. Human mutation, 37(6), pp.508-515. Doi: https://doi.org/10.1002/humu.22980.
Blount, J.W., Korth, K.L., Masoud, S.A., Rasmussen, S., Lamb, C. and Dixon, R.A., 2000. Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway. Plant physiology, 122(1), pp.107-116. Doi: https://doi.org/10.1104/pp.122.1.107.
Bubna, G.A., Lima, R.B., Zanardo, D.Y.L., Dos Santos, W.D., Ferrarese, M.D.L.L. and Ferrarese-Filho, O., 2011. Exogenous caffeic acid inhibits the growth and enhances the lignification of the roots of soybean (Glycine max). Journal of Plant Physiology, 168(14), pp.1627-1633. Doi: https://doi.org/10.1016/j.jplph.2011.03.005.
Chattopadhyay, S., Raychaudhuri, U. and Chakraborty, R., 2014. Artificial sweeteners–a review. Journal of food science and technology, 51(4), pp.611-621. Doi: https://doi.org/10.1007/s13197-011-0571-1.
Chen, Q., Man, C., Li, D., Tan, H., Xie, Y. and Huang, J., 2016. Arogenate dehydratase isoforms differentially regulate anthocyanin biosynthesis in Arabidopsis thaliana. Molecular plant, 9(12), pp.1609-1619. Doi: 10.1016/j.molp.2016.09.010 .
Cheng, F. and Cheng, Z., 2015. Research progress on the use of plant allelopathy in agriculture and the physiological and ecological mechanisms of allelopathy. Frontiers in Plant Science, 6, p.1020. Doi: https://doi.org/10.3389/fpls.2015.01020.
Cho, M.H., Corea, O.R., Yang, H., Bedgar, D.L., Laskar, D.D., Anterola, A.M., Moog-Anterola, F.A., Hood, R.L., Kohalmi, S.E., Bernards, M.A. and Kang, C., 2007. Phenylalanine biosynthesis in Arabidopsis thaliana: identification and characterization of arogenate dehydratases. Journal of Biological Chemistry, 282(42), pp.30827-30835. Doi: 10.1074/jbc.M702662200.
Corea, O.R., Ki, C., Cardenas, C.L., Kim, S.J., Brewer, S.E., Patten, A.M., Davin, L.B. and Lewis, N.G., 2012. Arogenate dehydratase isoenzymes profoundly and differentially modulate carbon flux into lignins. Journal of Biological Chemistry, 287(14), pp.11446-11459. Doi: https://doi.org/10.1074/jbc.M111.322164.
El-Azaz, J., de la Torre, F., Pascual, M.B., Debille, S., Canlet, F., Harvengt, L., Trontin, J.F., Ávila, C. and Cánovas, F.M., 2020. Transcriptional analysis of arogenate dehydratase genes identifies a link between phenylalanine biosynthesis and lignin biosynthesis. Journal of Experimental Botany, 71(10), pp.3080-3093. Doi: https://doi.org/10.1093/jxb/eraa099.
Fatahi Siahkamary, S., Rabiei, V., Shoor, M. and Nicola, S., 2025. Foliar application of L-phenylalanine, sodium selenate, and nitroxine biological fertilizer can improve antioxidant and phytochemical properties of goji berry (Lycium barbarum L.). Journal of Horticulture and Postharvest Research, pp.291-308. Doi: https://doi.org/10.22077/jhpr.2024.7895.1397.
Feduraev, P., Skrypnik, L., Riabova, A., Pungin, A., Tokupova, E., Maslennikov, P. and Chupakhina, G., 2020. Phenylalanine and tyrosine as exogenous precursors of wheat (Triticum aestivum L.) secondary metabolism through PAL-associated pathways. Plants, 9(4), p.476. Doi: https://doi.org/10.3390/plants9040476.
Ghalamboran, M.R., Kohnavard, A. and Hassani, S.B., 2023. Phenylalanine response in rice kernel under chitosan nanoparticles spraying. Acta Physiologiae Plantarum, 45(4), p.61. Doi: https://doi.org/10.1007/s11738-023-03538-3.
Haghighi, F., Talebpour, Z., Amini, V., Ahmadzadeh, A., & Farhadpour, M. (2015). A fast high performance liquid chromatographic (HPLC) analysis of amino acid phenylketonuria disorder in dried blood spots and serum samples, employing C18 monolithic silica columns and photo diode array detection. Analytical Methods, 7(18), 7560-7567.Doi: https://doi.org/10.1039/C5AY00745C.
Huke, R.E., 1982. Rice area by type of culture: South, Southeast, and East Asia. Int. Rice Res. Inst.
Hussain, I., Singh, N.B., Singh, A., Singh, H., Singh, S.C. and Yadav, V., 2017. Exogenous application of phytosynthesized nanoceria to alleviate ferulic acid stress in Solanum lycopersicum. Scientia horticulturae, 214, pp.158-164. Doi: https://doi.org/10.1016/j.scienta.2016.11.032.
Hussain, M.I. and Reigosa, M.J., 2011. A chlorophyll fluorescence analysis of photosynthetic efficiency, quantum yield and photon energy dissipation in PSII antennae of Lactuca sativa L. leaves exposed to cinnamic acid. Plant Physiology and Biochemistry, 49(11), pp.1290-1298. Doi: https://doi.org/10.1016/j.plaphy.2011.08.007.
IRRI, I., 2002. Standard evaluation system for rice. International Rice Research Institute, Philippine, pp.1-45.
Jan, R., Asaf, S., Numan, M., Lubna and Kim, K.M., 2021. Plant secondary metabolite biosynthesis and transcriptional regulation in response to biotic and abiotic stress conditions. Agronomy, 11(5), p.968. Doi: https://doi.org/10.3390/agronomy11050968.
Jorrín, J., López-Valbuena, R. and Tena, M., 1990. L-phenylalanine ammonia-lyase from sunflower hypocotyls: Modulation by cinnamic acids. Journal of plant Physiology, 136(4), pp.415-420. Doi: https://doi.org/10.1016/S0176-1617(11)80029-5.
Kapoor, R.T., Alyemeni, M.N. and Ahmad, P., 2021. Exogenously applied spermidine confers protection against cinnamic acid-mediated oxidative stress in Pisum sativum. Saudi Journal of Biological Sciences, 28(5), pp.2619-2625.Doi: https://doi.org/10.1016/j.sjbs.2021.02.052.
Kruger, N.J., 2009. The Bradford method for protein quantitation. The protein protocols handbook, pp.17-24. Doi: https://doi.org/10.1007/978-1-59745-198-7_4.
Levitt, J., 1980. Responses of plants to environmental stresses. Chilling, Freezing, and High Temperature Stress, 1, pp.345-447.Doi: https://doi.org/10.2503/jjshs.72.29.
Liu, J., Lefevere, H., Coussement, L., Delaere, I., De Meyer, T., Demeestere, K., Höfte, M., Gershenzon, J., Ullah, C. and Gheysen, G., 2024. The phenylalanine ammonia‐lyase inhibitor AIP induces rice defence against the root‐knot nematode Meloidogyne graminicola. Molecular Plant Pathology, 25(1), p.e13424. Doi: https://doi.org/10.1111/mpp.13424.
López-González, D., Bruno, L., Díaz-Tielas, C., Lupini, A., Aci, M.M., Talarico, E., Madeo, M.L., Muto, A., Sánchez-Moreiras, A.M. and Araniti, F., 2023. Short-term effects of trans-cinnamic acid on the metabolism of Zea mays L. Roots. Plants, 12(1), p.189. Doi: https://doi.org/10.3390/plants12010189.
MacDonald, M.J. and D’Cunha, G.B., 2007. A modern view of phenylalanine ammonia lyase. Biochemistry and Cell Biology, 85(3), pp.273-282. Doi: https://doi.org/10.1139/O07-018.
Maeda, H., Shasany, A.K., Schnepp, J., Orlova, I., Taguchi, G., Cooper, B.R., Rhodes, D., Pichersky, E. and Dudareva, N., 2010. RNAi suppression of Arogenate Dehydratase1 reveals that phenylalanine is synthesized predominantly via the arogenate pathway in petunia petals. The Plant Cell, 22(3), pp.832-849. Doi: https://doi.org/10.1105/tpc.109.073247.
Mohagheghian, E. and Ehsan Pour, A.A., 2021. Effect of Cinnamic acid on the activity of phenylalanine ammonialyase (PAL) and tyrosine ammonialyase (TAL) enzymes and some physiological characteristics of tobacco plant (Nicotiana rustica L.) under salinity stress in vitro calture. Cell and Tissue Journal, 12(2), pp.88-102. Doi: https://doi.org/10.52547/JCT.12.2.88.
Mohidem, N.A., Hashim, N., Shamsudin, R. and Che Man, H., 2022. Rice for food security: Revisiting its production, diversity, rice milling process and nutrient content. Agriculture, 12(6), p.741. Doi: https://doi.org/10.3390/agriculture12060741.
Oliva, M., Hatan, E., Kumar, V., Galsurker, O., Nisim-Levi, A., Ovadia, R., Galili, G., Lewinsohn, E., Elad, Y., Alkan, N. and Oren-Shamir, M., 2020. Increased phenylalanine levels in plant leaves reduces susceptibility to Botrytis cinerea. Plant Science, 290, p.110289. Doi: https://doi.org/10.1016/j.plantsci.2019.110289.
Peng, W., Wang, N., Wang, S., Wang, J. and Bian, Z., 2023. Effect of co‐treatment of microwave and exogenous l‐phenylalanine on the enrichment of flavonoids in Tartary buckwheat sprouts. Journal of the Science of Food and Agriculture, 103(4), pp.2014-2022. Doi: https://doi.org/10.1002/jsfa.12263.
Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic acids research, 29(9), pp.e45-e45. Doi: https://doi.org/10.1093/nar/29.9.e45.
Ping, H., Jie, M., Shujing, K., Sanfeng, L., Xianmei, W., Longjun, Z., Caolin, L., Rui, H., Huiying, H., Lianguang, S. and Yuchun, R., 2023. Chlorophyllide-a oxygenase 1 (OsCAO1) over-expression affects rice photosynthetic rate and grain yield. Rice Science, 30(2), p.87. Doi: 10.1016/j.rsci.2022.05.006.
Qian, Y., Lynch, J.H., Guo, L., Rhodes, D., Morgan, J.A. and Dudareva, N., 2019. Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants. Nature communications, 10(1), p.15.Doi: https://doi.org/10.1038/s41467-018-07969-2.
Rajaeian, S., Ehsanpour, A.A., and Toghyani, M.A., 2015. Changes in phenolic compound, TAL, PAL activity of Nicotiana rustica triggered by ethanolamine pretreatment under in vitro salt stress condition. Journal of Plant Biological Sciences, 7(26), pp.1-12.Doi: 20.1001.1.20088264.1394.7.26.2.4.
Reham, M.S., Khattab, M.E., Ahmed, S.S. and Kandil, M.A.M., 2016. Influence of foliar spray with phenylalanine and nickel on growth, yield quality and chemical composition of genoveser basil plant. African Journal of Agricultural Research, 11(16), pp.1398-1410.Doi: https://doi.org/10.5897/AJAR2015.10699.
Singh, D.P., Bahadur, A., Sarma, B.K., Maurya, S., Singh, H.B. and Singh, U.P., 2010. Exogenous application of L-phenylalanine and ferulic acid enhance phenylalanine ammonia lyase activity and accumulation of phenolic acids in pea (Pisum sativum) to offer protection against Erysiphe pisi. Archives Of Phytopathology and Plant Protection, 43(15), pp.1454-1462. Doi: https://doi.org/10.1080/03235400802536881.
Singh, P.K., Singh, R. and Singh, S., 2013. Cinnamic acid induced changes in reactive oxygen species scavenging enzymes and protein profile in maize (Zea mays L.) plants grown under salt stress. Physiology and Molecular Biology of Plants, 19(1), pp.53-59. Doi: https://doi.org/10.1007/s12298-012-0126-6.
Singh, U.P., Sarma, B.K. and Singh, D.P., 2003. Effect of plant growth-promoting rhizobacteria and culture filtrate of Sclerotium rolfsii on phenolic and salicylic acid contents in chickpea (Cicer arietinum). Current Microbiology, 46(2), pp.0131-0140. Doi: https://doi.org/10.1007/s00284-002-3834-2.
Singh, U.P., Sarma, B.K., Singh, D.P. and Bahadur, A., 2002. Plant growth-promoting rhizobacteria-mediated induction of phenolics in pea (Pisum sativum) after infection with Erysiphe pisi. Current Microbiology, 44(6), pp.396-400. Doi: https://doi.org/10.1007/s00284-001-0007-7.
Soltanizadeh, N. and Mirmoghtadaie, L., 2014. Strategies used in production of phenylalanine‐free foods for PKU management. Comprehensive Reviews in Food Science and Food Safety, 13(3), pp.287-299. Doi: https://doi.org/10.1111/1541-4337.12057.
Van Spronsen, F.J., Blau, N., Harding, C., Burlina, A., Longo, N. and Bosch, A.M., 2021. Phenylketonuria. Nature reviews Disease primers, 7(1), p.36. Doi: https://doi.org/10.1038/s41572-021-00267-0.
Vermerris, W. and Nicholson, R., 2008. The role of phenols in plant defense. In Phenolic compound biochemistry (pp. 211-234). Dordrecht: Springer Netherlands. Doi: https://doi.org/10.1007/978-1-4020-5164-7_6.
Wakasa, K. and Ishihara, A., 2009. Metabolic engineering of the tryptophan and phenylalanine biosynthetic pathways in rice. Plant biotechnology, 26(5), pp.523-533. Doi: https://doi.org/10.5511/plantbiotechnology.26.523.
Waterman, P.G. and Mole, S., 2019. Extrinsic factors influencing production of secondary metabolites in plants. In Insect-plant interactions (pp. 107-134). CRC press. Doi:
Wen, P.F., Chen, J.Y., Kong, W.F., Pan, Q.H., Wan, S.B. and Huang, W.D., 2005. Salicylic acid induced the expression of phenylalanine ammonia-lyase gene in grape berry. Plant Science, 169(5), pp.928-934. Doi: https://doi.org/10.1016/j.plantsci.2005.06.011.
Wisetkomolmat, J., Arjin, C., Hongsibsong, S., Ruksiriwanich, W., Niwat, C., Tiyayon, P., Jamjod, S., Yamuangmorn, S., Prom-U-Thai, C. and Sringarm, K., 2023. Antioxidant activities and characterization of polyphenols from selected Northern Thai rice husks: Relation with seed attributes. Rice Science, 30(2), pp.148-159. Doi: https://doi.org/10.1016/j.rsci.2023.01.007.
Yamada, T., Matsuda, F., Kasai, K., Fukuoka, S., Kitamura, K., Tozawa, Y., Miyagawa, H. and Wakasa, K., 2008. Mutation of a rice gene encoding a phenylalanine biosynthetic enzyme results in accumulation of phenylalanine and tryptophan. The Plant Cell, 20(5), pp.1316-1329. Doi: https://doi.org/10.1105/tpc.107.057455.
Yang, Q.Q., Zhang, C.Q., Chan, M.L., Zhao, D.S., Chen, J.Z., Wang, Q., Li, Q.F., Yu, H.X., Gu, M.H., Sun, S.S.M. and Liu, Q.Q., 2016. Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance. Journal of experimental botany, 67(14), pp.4285-4296. Doi: https://doi.org/10.1093/jxb/erw209.
Yang, W., Li, Y., Zhao, Q., Guo, Y. and Dong, Y., 2022. Intercropping alleviated the phytotoxic effects of cinnamic acid on the root cell wall structural resistance of faba bean and reduced the occurrence of Fusarium wilt. Physiologia Plantarum, 174(6), p.e13827. Doi: https://doi.org/10.1111/ppl.13827.
Yoo, H., Shrivastava, S., Lynch, J.H., Huang, X.Q., Widhalm, J.R., Guo, L., Carter, B.C., Qian, Y., Maeda, H.A., Ogas, J.P. and Morgan, J.A., 2021. Overexpression of arogenate dehydratase reveals an upstream point of metabolic control in phenylalanine biosynthesis. The Plant Journal, 108(3), pp.737-751. Doi: https://doi.org/10.1111/tpj.15467.
Yoo, H., Widhalm, J.R., Qian, Y., Maeda, H., Cooper, B.R., Jannasch, A.S., Gonda, I., Lewinsohn, E., Rhodes, D. and Dudareva, N., 2013. An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine: phenylpyruvate aminotransferase. Nature communications, 4(1), p.2833. Doi: https://doi.org/10.1038/ncomms3833.
Yuan, X., Tang, B., Wang, Y., Jiang, Y., He, J., Wang, G., Yang, P. and Wang, B., 2023. Inhibitory effects of peppermint extracts on the browning of cold-stored fresh-cut taro and the phenolic compounds in extracts. Frontiers in Sustainable Food Systems, 7, p.1191396. Doi: https://doi.org/10.3389/fsufs.2023.1191396.
Zhang, X. and Liu, C.J., 2015. Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoids. Molecular plant, 8(1), pp.17-27. Doi: 10.1016/j.molp.2014.11.001 .
Zulet-González, A., Barco-Antoñanzas, M., Gil-Monreal, M., Royuela, M. and Zabalza, A., 2020. Increased glyphosate-induced gene expression in the shikimate pathway is abolished in the presence of aromatic amino acids and mimicked by shikimate. Frontiers in Plant Science, 11, p.459. Doi: https://doi.org/10.3389/fpls.2020.00459.
Plant, Algae, and Environment
Volume 10, Issue 1 - Serial Number 1
DOi
March 2026
Pages 1-24

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