Abstract
Water deficit, salinity, and cadmium (Cd) contamination have generated an environmental problem worldwide, leading to damages to plant growth due to alteration in their metabolism. This study aimed to classify enzymatic and non-enzymatic antioxidant systems in Micro-Tom (MT) plants when subjected to two intensities (moderate and severe) of water deficit, salinity, and Cd exposure. The experimental design was a completely randomized 3 × 2 factorial, with the first factor representing the stress agents (water deficit, salinity, and Cd) and the second factor indicating stress intensities (moderate and severe), along with a control group. After an acclimation period, plants were exposed to 10 days of stress. Water deficit treatments were imposed using solutions adjusted to osmotic potentials of − 0.40 MPa and − 1.00 MPa; salinity stress was established with nutrient solutions containing 40 mM or 120 mM NaCl; and Cd stress was induced using nutrient solutions with 0.25 mM or 0.5 mM CdCl₂. Laboratory analyses included lipid peroxidation, hydrogen peroxide content, proline accumulation, protein quantification, and enzyme extraction. Descriptive analyses, and a Spearman’s correlation, identified the behavior of enzymatic and non-enzymatic systems for each stress agents and intensities, enabling the selection of key influencing factors. A factorial analysis of variance was performed to assess the mean differences among the treatments (α = 0.05) for enzymatic, non-enzymatic systems, MDA and, H₂O₂. Using this data, a decision tree model classified the stresses into four levels: low, low-medium, medium-high, and high. Variations in antioxidant response and stress biomarkers were detailed, with proline and superoxide dismutase identified as the primary variables of significance across stress indicators. Furthermore, the model achieved robust classification performance with Matthew’s correlation coefficients exceeding 0.80 in the extreme classes; however, it encountered limitations in distinguishing between classes with closely proximate values. The findings indicate the capability of the decision tree to classify stress levels in plants.
Data availability
All data are available in the manuscript. The raw data that support the findings of this study are available from the corresponding author upon reasonable request.
References
Contreras-Cornejo, H. A. et al. Abiotic plant stress mitigation by trichoderma species. Soil. Ecol. Lett. 6, 240240 (2024).
Chiconato, D. A., da Silveira Sousa Junior, G., dos Santos, D. M. M. & Munns, R. Adaptation of sugarcane plants to saline soil. Environ. Exp. Bot. 162, 201–211 (2019).
Alves, L. R. et al. Mechanisms of cadmium-stress avoidance by selenium in tomato plants. Ecotoxicology 29, 594–606 (2020).
Seleiman, M. F. et al. Drought stress impacts on plants and different approaches to alleviate its adverse effects. Plants 10, 259 (2021).
Akbari, M., Sabouri, H., Sajadi, S. J., Yarahmadi, S. & Ahangar, L. Classification and prediction of drought and salinity stress tolerance in barley using GenPhenML. Sci. Rep. 14, 17420 (2024).
Yang, X. et al. Response mechanism of plants to drought stress. Horticulturae 7, 50 (2021).
Montesinos, C. et al. Avaliação e caracterização Em Campo de Um Novo bioestimulante Para o cultivo de brócolis (Brassica Oleracea var. italica) Sob estresse hídrico e Salino Que aumenta o conteúdo de antioxidantes, Glucosinolatos e fitohormônios. Sci. Hort. 338, 113584 (2024).
Gisbert, C., Timoneda, A., Porcel, R., Ros, R. & Mulet, J. M. Overexpression of BvHb2, a class 2 non-symbiotic hemoglobin from sugar beet, confers drought-induced withering resistance and alters iron content in tomato. Agronomy 10, 1754 (2020).
Patel, M., Fatnani, D. & Parida, A. K. Mitigação do estresse hídrico induzida por silício em genótipos de amendoim (Arachis hypogaea L.) por meio da homeostase iônica, modulações do sistema de defesa antioxidante e regulações metabólicas. Plant Physiol. Biochem. 166, 290–313 (2021).
Behtash, F., Amini, T., Mousavi, S. B., Hajizadeh, S. & Kaya, O. Efficiency of zinc in alleviating cadmium toxicity in hydroponically grown lettuce (Lactuca sativa L. cv. Ferdos). BMC Plant Biol. 24, 648 (2024).
Rezaie, N., Razzaghi, F. & Sepaskhah, A. R. Different levels of irrigation water salinity and biochar influence on faba bean yield, water productivity, and ions uptake. Commun. Soil Sci. Plant Anal. 50, 611–626 (2019).
Saddiq, M. S. et al. Effect of salinity stress on physiological changes in winter and spring wheat. Agronomy 11, 1193 (2021).
Spormann, S., Soares, C., Azenha, M., Martins, V. & Fidalgo, F. A look into osmotic, ionic, and redox adjustments in wild tomato species under combined salt and water stress. Plant. Stress. 13, 100510 (2024).
Riaz, M. et al. O fornecimento de boro alivia a toxicidade do cádmio no arroz (Oryza sativa L.) aumentando a adsorção de cádmio na parede celular e ativando o sistema de defesa antioxidante nas raízes. Chemosphere 266, 128938 (2021).
Anwar, T., Qureshi, H., Jabeen, M., Zaman, W. & Ali, H. M. Mitigation of cadmium-induced stress in maize via synergistic application of biochar and gibberellic acid to enhance morpho-physiological and biochemical traits. BMC Plant Biol. 24, 192 (2024).
Zhang, Q. et al. Defense guard: strategies of plants in the fight against cadmium stress. Adv. Biotechnol. 2, 44 (2024).
Zulfiqar, U. et al. Cadmium phytotoxicity, tolerance, and advanced remediation approaches in agricultural soils: A comprehensive review. Front Plant. Sci 13 (2022).
Zhang, Y. et al. SlPHL1, a MYB-CC transcription factor identified from tomato, positively regulates the phosphate starvation response. Physiol. Plant. 173, 1063–1077 (2021).
Çelik, Ö., Ayan, A. & Atak, Ç. Enzymatic and non-enzymatic comparison of two different industrial tomato (Solanum lycopersicum) varieties against drought stress. Bot. Stud. 58, 32 (2017).
Caverzan, A. et al. Plant responses to stresses: role of ascorbate peroxidase in the antioxidant protection. Genet. Mol. Biol. 35, 1011–1019 (2012).
Soares, C., Carvalho, M. E. A., Azevedo, R. A. & Fidalgo, F. Plants facing oxidative challenges: a little help from the antioxidant networks. Environ. Exp. Bot. 161, 4–25 (2019).
Gratão, P. L. et al. Cadmium stress antioxidant responses and root-to-shoot communication in grafted tomato plants. Biometals 28, 803–816 (2015).
Todaka, D. et al. Application of ethanol alleviates heat damage to leaf growth and yield in tomato. Front Plant. Sci 15 (2024).
Sun, H. J., Uchii, S., Watanabe, S. & Ezura, H. A. Highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant Cell Physiol. 47, 426–431 (2006).
Yan, M. et al. The involvement of abscisic acid in hydrogen gas-enhanced drought resistance in tomato seedlings. Sci. Hort. 292, 110631 (2022).
Yang, R. et al. Strigolactones are involved in hydrogen sulfide-enhanced salt tolerance in tomato seedlings. Plant. Stress. 11, 100316 (2024).
Machado, J. et al. Enzymatic and Non-Enzymatic antioxidant responses of young tomato plants (cv. Micro-Tom) to single and combined mild nitrogen and water deficit: not the sum of the parts. Antioxidants 12, 375 (2023).
Chu, Z. et al. Linking phytohormones with growth, transport activity and metabolic responses to cadmium in tomato. Plant. Growth Regul. 90, 557–569 (2020).
Sandhu, K., Patil, S. S., Pumphrey, M. & Carter, A. Multitrait machine- and deep-learning models for genomic selection using spectral information in a wheat breeding program. Plant. Genome. 14, e20119 (2021).
Madhumitaa, P. S., Ragavi, C., Kiranmayi, C. & Prabhavathy, P. M, V. Drought and Salinity Stress Classification in Soyabean Crops: Comparative Analysis of Machine Learning Models. In International Conference on Signal Processing, Computation, Electronics, Power and Telecommunication (IConSCEPT), 1–5 (2024). https://doi.org/10.1109/IConSCEPT61884.2024.10627854
Oshternian, S. R., Loipfinger, S., Bhattacharya, A. & Fehrmann, R. S. N. Exploring combinations of dimensionality reduction, transfer learning, and regularization methods for predicting binary phenotypes with transcriptomic data. BMC Bioinform. 25, 167 (2024).
Sarker, I. H. Machine learning: Algorithms, real-world applications and research directions. SN Comput. Sci. 2, 160 (2021).
Houetohossou, S. C. A., Houndji, V. R., Hounmenou, C. G., Sikirou, R. & Kakaï, R. L. G. Deep learning methods for biotic and abiotic stresses detection and classification in fruits and vegetables: state of the art and perspectives. Artif. Intell. Agric. 9, 46–60 (2023).
Xu, L., Liu, H., Mittler, R. & Shabala, S. Useful or merely convenient: can enzymatic antioxidant activity be used as a proxy for abiotic stress tolerance? J. Exp. Bot. 76, 1524–1533 (2025).
Patel, J. et al. UV-C-induced reactive carbonyl species are better detoxified in the halophytic plants Salicornia brachiata and Arthrocnemum macrostachyum than in the halophytic Sarcocornia fruticosa plants. Plant J. 122, e70239 (2025).
Sana, S. et al. Differential responses of Chili varieties grown under cadmium stress. BMC Plant Biol. 24, 7 (2024).
Sultana, M. S., Sakurai, C., Biswas, M. S., Szabados, L. & Mano, J. Accumulation of reactive carbonyl species in roots as the primary cause of salt stress-induced growth retardation of Arabidopsis thaliana. https://doi.org/10.1111/ppl.14198.
Rabêlo, F. H. S. et al. Adequate S supply reduces the damage of high cd exposure in roots and increases N, S and Mn uptake by Massai grass grown in hydroponics. Environ. Exp. Bot. 148, 35–46 (2018).
Chen, L. et al. Gene identification and transcriptome analysis of cadmium stress in tomato. Front. Sustain. Food Syst. 7, 1–11 (2023).
Dar, M. I., Naikoo, M. I., Rehman, F., Naushin, F. & Khan, F. A. Proline Accumulation in Plants: Roles in Stress Tolerance and Plant Development. In Osmolytes and Plants Acclimation to Changing Environment: Emerging Omics Technologies (eds Iqbal, N., Nazar, R. & A. Khan, N.) 155–166 (Springer India, New Delhi, 2016). https://doi.org/10.1007/978-81-322-2616-1_9
Hayat, S. et al. Role of proline under changing environments: Uma revisão. Plant Signal. Behav. 7, 1456–1466 (2012).
López-Gómez, M., Hidalgo-Castellanos, J., Iribarne, C. & Lluch, C. Proline accumulation has prevalence over polyamines in nodules of medicago sativa in symbiosis with Sinorhizobium meliloti during the initial response to salinity. Plant. Soil. 374, 149–159 (2014).
Hosseinifard, M. et al. Contribution of exogenous proline to abiotic stresses tolerance in plants: A review. Int. J. Mol. Sci. 23, 5186 (2022).
Guan, C. et al. Proline biosynthesis enzyme genes confer salt tolerance to Switchgrass (Panicum virgatum L.) in Cooperation with polyamines metabolism. Front. Plant. Sci. 11, 1–14 (2020).
Souri, Z., Khanna, K., Karimi, N. & Ahmad, P. Silicon and plants: current knowledge and future prospects. J. Plant. Growth Regul. 40, 906–925 (2021).
Peña Calzada, K. et al. Regulatory role of silicon on growth, potassium uptake, ionic homeostasis, proline accumulation, and antioxidant capacity of soybean plants under salt stress. J. Plant. Growth Regul. 42, 4528–4540 (2023).
Chicco, D. & Jurman, G. The advantages of the Matthews correlation coefficient (MCC) over F1 score and accuracy in binary classification evaluation. BMC Genom. 21, 6 (2020).
de Barros, M. M., Silva, F. M., da Costa, A. G., Ferraz, G. A. & da Silva, F. C. S. Use of classifier to determine coffee harvest time by detachment force. Rev. bras. Eng. Agríc. Ambient. 22, 366–370 (2018).
Amoah, J. & Berko, D. Impact of salinity stress on membrane status, phytohormones, antioxidant defense system and transcript expression pattern of two contrasting sorghum genotypes. Egypt. J. Agron. 42, 123–136 (2020).
Gharsallah, C., Fakhfakh, H., Grubb, D. & Gorsane, F. Effect of salt stress on ion concentration, proline content, antioxidant enzyme activities and gene expression in tomato cultivars. AoB PLANTS. 8, plw055 (2016).
Jakovljević, D. Z., Topuzović, M. D., Stanković, M. S. & Bojović, B. M. Changes in antioxidant enzyme activity in response to salinity-induced oxidative stress during early growth of sweet Basil. Hortic. Environ. Biotechnol. 58, 240–246 (2017).
Mura, A. et al. Catalytic pathways of Euphorbia characias peroxidase reacting with hydrogen peroxide. 387, 559–567 (2006).
Manai, J., Gouia, H. & Corpas, F. J. A homeostase redox e do óxido nítrico é afetada em raízes de tomateiro (Solanum lycopersicum) sob estresse oxidativo induzido pela salinidade. J. Plant Physiol. 171, 1028–1035 (2014).
Murshed, R., Lopez-Lauri, F. & Sallanon, H. Effect of water stress on antioxidant systems and oxidative parameters in fruits of tomato (Solanum lycopersicon L, cv. Micro-tom). Physiol. Mol. Biol. Plants 19, 363–378 (2013).
El-Beltagi, H. S., Mohamed, H. I. & Sofy, M. R. Role of ascorbic acid, glutathione and proline applied as singly or in sequence combination in improving chickpea plant through physiological change and antioxidant defense under different levels of irrigation intervals. Molecules 25, 1702 (2020).
Gratão, P. l., Monteiro, C. c., Antunes, A. m., Peres, L. e. & Azevedo, R. a. Acquired tolerance of tomato (Lycopersicon esculentum cv. Micro-Tom) plants to cadmium-induced stress. Ann. Appl. Biol. 153, 321–333 (2008).
Hoagland, D. R. & Arnon, D. J. The Water-Culture Method for Growing Plants without Soil. vol. 347 (Arnon, D. I. & California Agricultural Experiment Station, California Agricultural Experiment Station- Berkeley, 1950).
Pino-Nunes, L. Obtenção e uso de mutantes com alterações no balanço auxina/citocinina no estudo da competência organogênica em micro-tomateiro (Lycopersicon esculentum cv Micro-Tom) (Universidade de São Paulo, 2005).
Heath, R. L. & Packer, L. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–198 (1968).
Gratão, P. L. et al. Dissecação bioquímica de mutantes de tomate Diageotropica e Never mature em condições estressantes de cd. Plant Physiol. Biochem. 56, 79–96 (2012).
Alexieva, V., Sergiev, I., Mapelli, S. & Karanov, E. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant. Cell. Environ. 24, 1337–1344 (2001).
Bates, L. S., Waldren, R. P. & Teare, I. D. Rapid determination of free proline for water-stress studies. Plant. Soil. 39, 205–207 (1973).
Azevedo, R. A., Alas, R. M., Smith, R. J. & Lea, P. J. Response of antioxidant enzymes to transfer from elevated carbon dioxide to air and Ozone fumigation, in the leaves and roots of wild-type and a catalase-deficient mutant of barley. Physiol. Plant. 104, 280–292 (1998).
Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).
Giannopolitis, C. N. & Ries, S. K. Superoxide dismutases: I. Occurrence in higher plants. Plant Physiol. 59, 309–314 (1977).
Kraus, T. E., McKersie, B. D. & Fletcher, R. A. Paclobutrazol-induced Tolerance of Wheat Leaves to Paraquat May Involve Increased Antioxidant Enzyme Activity. J. Plant Physiol. 145, 570–576 (1995).
Flohé, L. & Günzler, W. A. [12] Assays of glutathione peroxidase. In Methods in Enzymology Vol. 105 114–120 (Academic, 1984).
Nagalakshmi, N. & Prasad, M. N. V. Respostas das enzimas do ciclo da glutationa e do metabolismo da glutationa ao estresse de cobre em Scenedesmus bijugatus. Plant Sci. 160, 291–299 (2001).
Ozarda, Y. Reference intervals: current status, recent developments and future considerations. Biochem. Med. 26, 5–11 (2016).
Krzywinski, M. & Altman, N. Classification and regression trees. Nat. Methods. 14, 757–758 (2017).
Seliya, N., Khoshgoftaar, T. M. & Van Hulse, J. A study on the relationships of classifier performance metrics. In 2009 21st IEEE International Conference on Tools with Artificial Intelligence, 59-66 https://doi.org/10.1109/ICTAI.2009.25 (2009).
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To the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Finance Code 001 for financial support through the PhD scholarship, and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the postdoctoral scholarship, funding code 150129/2022-0.
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LMR, GSSJ and EPP designed the experiments. LMR, GSSJ and EPP performed the experiments. LMR, MDM and GSR analyzed the data. LMR, GSSJ and MDM wrote the manuscript (original draft, review and editing). GSR, RAA, PLG reviewed the manuscript, and all authors accepted the final version of the manuscript.
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Ribera, L.M., da Silveira Sousa Junior, G., Meneses, M.D. et al. Machine learning model provides stress biomarkers for the classification of abiotic stress in Micro-Tom. Sci Rep (2026). https://doi.org/10.1038/s41598-026-39117-y
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DOI: https://doi.org/10.1038/s41598-026-39117-y