Synthesis of a kinetin and graphene oxide nanoensemble and its effects on defense responses of cotton against Verticillium dahliae

N. R. Amrahov; U. A. Hasanova; M. Levy; G. S. Aliyeva; G. A. Aghazada; R. B. Mammadova; S. A. Alizade; F. H. Rzayev; E. K. Gasimov; S. S. Hasanova; N. A. Malikova; Z. M. Mammadov

doi:10.15421/0226077

N. R. Amrahov Baku State University
U. A. Hasanova Baku State University
M. Levy The Hebrew University of Jerusalem
G. S. Aliyeva Baku State University
G. A. Aghazada Baku State University
R. B. Mammadova Genetic Resources Institute
S. A. Alizade Genetic Resources Institute
F. H. Rzayev Azerbaijan Medical University
E. K. Gasimov Azerbaijan Medical University
S. S. Hasanova Baku State University
N. A. Malikova Baku State University
Z. M. Mammadov Baku State University

Keywords: phytohormone delivery, carbon nanomaterials, plant–pathogen interaction, Verticillium dahliae, cotton genotypes, non-covalent functionalization.

Abstract

In this study, a kinetin-graphene oxide nanoensemble (KN+GO) was synthesized via non-covalent interactions . Its structure and morphology were characterized by transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, and powder X-ray diffraction (XRD), confirming the successful immobilization of kinetin on graphene oxide. The loading efficiency of kinetin on GO was determined by UV–Vis spectroscopy. The effects of the KN+GO nanoensemble on the physiological and biochemical responses of two cotton genotypes ( Gossypium hirsutum L. TM-1 and G . barbadense L. Pima 3-79 ) were investigated during infection with Verticillium dahliae . Morphometric traits, seedling survival, antioxidant enzyme activities (catalase, peroxidase, and polyphenol oxidase), nitric oxide and proline levels, and photosynthetic pigment composition were analyzed. Pathogen infection induced oxidative stress and significantly altered the growth and biochemical parameters in both genotypes. Free kinetin exhibited genotype-dependent effects, whereas the KN+GO nanoensemble consistently enhanced stress tole r ance, particularly under pathogen pressure. In the TM-1 plants , KN+GO markedly improved seedling survival, i n creased antioxidant enzyme activity, promoted proline accumulation, and stabilized photosynthetic pigment levels. In the Pima 3-79 plants , the nanoensemble mainly supported survival and redox balance, although the responses were less pronounced. These findings demonstrate that graphene oxide–based kinetin nanoensembles mo d ulate cotton defense responses in a genotype- and stress-dependent manner, highlighting their potential as nano-enabled phytohormone delivery systems for improving resistance to fungal pathogens.

References

Alizada, S., Aliyeva, K., Mammadova, R., Bayramli, O., & Moghanloo, B. S. (2024). Salinity, Verticillium wilt tolerance and genetic diversity analysis of upland cotton genotypes. Advances in Biology and Earth Sciences, 9(2), 242–252.

Amil-Ruiz, F., Blanco-Portales, R., Munoz-Blanco, J., & Caballero, J. L. (2011). The strawberry plant defense mechanism: A molecular review. Plant and Cell Physiology, 52(11), 1873–1903.

Baghirov, M. B., Muradov, M., Eyvazova, G., Mammadyarova, S., Gahramanli, L., Aliyeva, G., Huseynov, E., & Abdullayev, M. (2023). Features of structure and optical properties GO and a GO/PVA composite subjected to gamma irradiation. RSC Advances, 13(50), 35648–35658.

Barnaby, S. N., Frayne, S. H., Fath, K. R., & Banerjee, I. A. (2011). Growth of Se nanoparticles on kinetin assemblies and their biocompatibility studies. Soft Materials, 9(4), 313–334.

Bellin, D., Asai, S., Delledonne, M., & Yoshioka, H. (2013). Nitric oxide as a mediator for defense responses. Molecular Plant-Microbe Interactions, 26(3), 271–277.

Boctor, F. N. (1971). An improved method for colorimetric determination of proline with isatin. Analytical Biochemistry, 43(1), 66–70.

Camejo, D., Guzmán-Cedeño, Á., & Moreno, A. (2016). Reactive oxygen species, essential molecules, during plant–pathogen interactions. Plant Physiology and Biochemistry, 103, 10–23.

Chance, B., & Maehly, A. C. (1955). Assay of catalases and peroxidases. Methods of Biochemical Analysis, 1, 357–424.

Chen, J., Peng, H., Wang, X., Shao, F., Yuan, Z., & Han, H. (2014). Graphene oxide exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal conidia by intertwining and membrane perturbation. Nanoscale, 6(3), 1879–1889.

Choi, J., Choi, D., Lee, S., Ryu, C. M., & Hwang, I. (2011). Cytokinins and plant immunity: Old foes or new friends? Trends in Plant Science,16(7), 388–394.

Chou, H. M., Bundock, N., Rolfe, S. A., & Scholes, J. D. (2000). Infection of Arabidopsis thaliana leaves with Albugo candida (white blister rust) causes a reprogramming of host metabolism. Molecular Plant Pathology, 1(2), 99–113.

Chun, J., Wan, M., Guo, H., Zhang, Q., Feng, Y., Tang, Y., Zhu, B., Sang, Y., Jing, S., Chen, T., & Zeng, Z. (2024). Cytokinin-mediated enhancement of potato growth and yield by Verticillium dahliae effector VDAL under low temperature stress. BMC Plant Biology, 24(1), 1115.

Dadd-Daigle, P., Kirkby, K., Chowdhury, P. R., Labbate, M., & Chapman, T. A. (2021). The Verticillium wilt problem in Australian cotton. Australasian Plant Pathology, 50(2), 129–135.

Di, X., Takken, F. L., & Tintor, N. (2016). How phytohormones shape interactions between plants and the soil-borne fungus Fusarium oxysporum. Frontiers in Plant Science, 7, 170.

Ermakov, A. I., Arasimovich, V. V., Yarosh, N. P., Peruanskiy, Y. V., Lukovnikova, G. A., & Ikonnikova, M. I. (1987). Methods of biochemical investigation of plants. Agropromizdat, Leningrad (in Russian).

Fatehi, S. F., Oraei, M., Gohari, G., Akbari, A., & Faramarzi, A. (2022). Proline-functionalized graphene oxide nanoparticles (GO–Pro NPs) mitigate salt-induced adverse effects on morpho-physiological traits and essential oils constituents in Moldavian balm (Dracocephalum moldavica L.). Journal of Plant Growth Regulation, 41(7), 2818–2832.

Fernández-Gómez, D., Villegas-Moreno, J., Kumar-Tiwari, D., Gómez-Romero, M., & Juárez-Cisneros, G. (2023). Effect of multi-walled carbon nanotubes functionalized with kinetin on the development of Avena sativa. Microscopy and Microanalysis: the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada, 29(S1), 42–43.

Gao, W., Long, L., Zhu, L. F., Xu, L., Gao, W. H., Sun, L. Q., Liu, L. L., & Zhang, X. L. (2013). Proteomic and virus-induced gene silencing (VIGS) analyses reveal that gossypol, brassinosteroids, and jasmonic acid contribute to the resistance of cotton to Verticillium dahliae. Molecular and Cellular Proteomics, 12(12), 3690–3703.

Göre, M. E. (2007). Vegetative compatibility and pathogenicity of Verticillium dahliae isolates from the aegean region of Turkey. Phytoparasitica, 35(3), 222–231.

Hajiyeva, S., Hasanova, U., Gakhramanova, Z., Israyilova, A., Ganbarov, K., Gasimov, E., & Maharramov, A. (2019). The role of diazacrown ether in the enhancement of the biological activity of silver nanoparticles. Turkish Journal of Chemistry, 43(6), 1711–1721.

Hersh, M. H., Vilgalys, R., & Clark, J. S. (2012). Evaluating the impacts of multiple generalist fungal pathogens on temperate tree seedling survival. Ecology, 93(3), 511–520.

Hidayah, N. M. S., Liu, W. W., Lai, C. W., Noriman, N. Z., Khe, C. S., Hashim, U., & Lee, H. C. (2017). Comparison on graphite, graphene oxide and reduced graphene oxide: Synthesis and characterization. AIP Conference Proceedings, 1892(1), 150002.

Huseynzada, A., Hasanova, U., & Gakhramanova, Z. (2023). Graphene oxide-modified dihydropyrimidines. Azerbaijan Chemical Journal, 3, 142–151.

Joost, O., Bianchini, G., Bell, A. A., Benedict, C. R., & Magill, C. W. (1995). Differential induction of 3-hydroxy-3-methylglutaryl CoA reductase in two cotton species following inoculation with Verticillium. Molecular Plant-Microbe Interactions, 8(6), 880–885.

Kabiri, S., Degryse, F., Tran, D. N. H., da Silva, R. C., McLaughlin, M. J., & Losic, D. (2017). Graphene oxide: A new carrier for slow release of plant micronutrients. ACS Applied Materials and Interfaces, 9(49), 43325–43335.

Kalidas, N., Gopal, P. M., & Kavimani, V. (2025). Development and characterization of silicon nitride and synthesized graphene oxide reinforced Cu/GO/xSi3N4 composites. Silicon, 17(1), 141–153.

Kużniak, E., & Skłodowska, M. (2005). Fungal pathogen-induced changes in the antioxidant systems of leaf peroxisomes from infected tomato plants. Planta, 222(1), 192–200.

Laohavisit, A., Wakatake, T., Ishihama, N., Mulvey, H., Takizawa, K., Suzuki, T., & Shirasu, K. (2020). Quinone perception in plants via leucine-rich-repeat receptor-like kinases. Nature, 587(7832), 92–97.

Lee, I. R., Lui, E. Y., Chow, E. W., Arras, S. D., Morrow, C. A., & Fraser, J. A. (2013). Reactive oxygen species homeostasis and virulence of the fungal pathogen Cryptococcus neoformans requires an intact proline catabolism pathway. Genetics, 194(2), 421–433.

Liu, W. Z., Kong, D. D., Gu, X. X., Gao, H. B., Wang, J. Z., Xia, M., Gao, Q., Tian, L. L., Xu, Z. H., Bao, F., Hu, Y., Ye, N. S., Pei, Z. M., & He, Y. K. (2013). Cytokinins can act as suppressors of nitric oxide in Arabidopsis. Proceedings of the National Academy of Sciences, 110(4), 1548–1553.

Lutter, F., Brenner, W., Krajinski-Barth, F., & Safavi-Rizi, V. (2024). Nitric oxide and cytokinin cross-talk and their role in plant hypoxia response. Plant Signaling and Behavior, 19(1), 2329841

Magro, M. R., Vella, D. A., & Cassar, G. (2025). Synthesis of graphene oxide: A refined approach. Carbon Trends, 20, 100509.

Mammadova, R. B., Akparov, Z. I., Alızade, S. A., Amrahov, N. R., & Abdulalıyeva, G. S. (2025). Verticillium wilt effect on fiber production and its quality traits in upland cotton (Gossypium hirsutum L.). SABRAO Journal of Breeding and Genetics, 57(5), 2015–2023.

Martínez-Medina, A., Pescador, L., Terrón-Camero, L. C., Pozo, M. J., & Romero-Puertas, M. C. (2019). Nitric oxide in plant–fungal interactions. Journal of Experimental Botany, 70(17), 4489–4503.

Maryum, Z., Luqman, T., Nadeem, S., Khan, S. M. U. D., Wang, B., Ditta, A., & Khan, M. K. R. (2022). An overview of salinity stress, mechanism of salinity tolerance and strategies for its management in cotton. Frontiers in Plant Science, 13, 907937.

Maxwell, S. A., & Davis, G. E. (2000). Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines. Proceedings of the National Academy of Sciences, 97(24), 13009–13014.

Mayer, A. M. (2006). Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry, 67(21), 2318–2331.

Nawaz, M., Sun, J., Shabbir, S., Khattak, W. A., Ren, G., Nie, X., Bo, Y., Javed, Q., Du, D., & Sonne, C. (2023). A review of plants strategies to resist biotic and abiotic environmental stressors. Science of the Total Environment, 900, 165832.

Park, S., Choi, K. S., Kim, S., Gwon, Y., & Kim, J. (2020). Graphene oxide-assisted promotion of plant growth and stability. Nanomaterials, 10(4), 758.

Rashid, A. N., Janda, M., Mahmud, M. Z., Valentová, O., Burketová, L., & Alekber, Q. A. (2021). Influence of salt stress on the Flg22-induced ROS production in Arabidopsis thaliana leaves. Pakistan Journal of Botany, 53(5), 1605–1610.

Ryan, A., Kaplan, E., Nebel, J. C., Polycarpou, E., Crescente, V., Lowe, E., Preston, G. M., & Sim, E. (2014). Identification of NAD(P)H quinone oxidoreductase activity in azoreductases from P. aeruginosa: Azoreductases and NAD(P)H quinone oxidoreductases belong to the same FMN-dependent superfamily of enzymes. PloS One, 9(6), e98551.

Taghiyeva, N., Hasanova, U., Millet, M., Gardiennet, C., Gakhramanova, Z., Mirzayev, M. H., Gahramanli, L., Pham-Huu, C., Aliyeva, S., Aliyeva, G., Rzayev, F., Gasimov, E., Boulogne, C., & Akhundzada, H. V. (2024). Synthesis and characterization of novel adsorbents based on functionalization of graphene oxide with Schiff base and reduced Schiff base for pesticide removal. Materials, 17(16), 4096.

Trapet, P., Kulik, A., Lamotte, O., Jeandroz, S., Bourque, S., Nicolas-Francès, V., Rosnoblet, C., Besson-Bard, A., & Wendehenne, D. (2015). NO signaling in plant immunity: a tale of messengers. Phytochemistry, 112, 72–79.

Wellburn, A. R., & Lichtenthaler, H. (1984). Formulae and program to determine total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Advances in Photosynthesis Research, 2, 9–12.

Yadeta, K. A., & J. Thomma, B. P. (2013). The xylem as battleground for plant hosts and vascular wilt pathogens. Frontiers in Plant Science, 4, 97.

Yusupova, Z. R., Khairullin, R. M., & Maksimov, I. V. (2006). The activity of peroxidase in various cell fractions of wheat plants infected with Septoria nodorum berk. Russian Journal of Plant Physiology, 53(6), 807–813.

Zhang, T., Jin, Y., Zhao, J. H., Gao, F., Zhou, B. J., Fang, Y. Y., & Guo, H. S. (2016). Host-induced gene silencing of the target gene in fungal cells confers effective resistance to the cotton wilt disease pathogen Verticillium dahliae. Molecular Plant, 9(6), 939–942.

Zhang, Y., Zhu, H., Ye, Y., & Tang, C. (2021). Antifungal activity of chaetoviridin A from Chaetomium globosum CEF-082 metabolites against Verticillium dahliae in cotton. Molecular Plant-Microbe Interactions, 34(7), 758–769.

Zhou, B., Guo, Z., Xing, J., & Huang, B. (2005). Nitric oxide is involved in abscisic acid-induced antioxidant activities in Stylosanthes guianensis. Journal of Experimental Botany, 56(422), 3223–3228.

Zhu, Y., Zhao, M., Li, T., Wang, L., Liao, C., Liu, D., Zhang, H., Zhao, Y., Liu, L., Ge, X., & Li, B. (2023). Interactions between Verticillium dahliae and cotton: Pathogenic mechanism and cotton resistance mechanism to Verticillium wilt. Frontiers in Plant Science, 14, 1174281.