The whole is not always the sum of the parts: Synergistic plant responses to combined environmental stresses

The whole is not always the sum of the parts: Synergistic plant responses to combined environmental stresses | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Plant, Cell & Environment This is a preprint and has not been peer reviewed. Data may be preliminary. 15 April 2025 V1 Latest version Share on The whole is not always the sum of the parts: Synergistic plant responses to combined environmental stresses Authors : Vitor Amorim-Silva 0000-0002-3978-7205 and Miguel A. Botella 0000-0002-8867-1831 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174471890.05910119/v1 Published Plant, Cell & Environment Version of record Peer review timeline 328 views 205 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Since this is a commentary paper, it does not have an abstract. The whole is not always the sum of the parts: Synergistic plant responses to combined environmental stresses Vítor Amorim-Silva 1 (orcid: 0000-0002-3978-7205) Miguel A. Botella 1 (orcid: 0000-0002-8867-1831) Correspondance: Vítor Amorim‐Silva ( [email protected] ) | Miguel A. Botella ( [email protected] ) 1 Área de Mejora y Fisiología de Plantas, Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga‐Consejo Superior de Investigaciones Científicas (IHSM‐UMA‐CSIC), Universidad de Málaga, 29010, Málaga, Spain. Numerous studies examining plant-environment stress interactions cultivate plants under optimal, non-stressful conditions before introducing individual stress factors to analyze response mechanisms, which clearly is an over-simplification. In nature, plants thrive under sub-optimal conditions, encountering multiple environmental cues such as day-night temperature variation, abiotic stressors, soil heterogeneity, pathogens, etc. While individual stresses often have minor negative effects on growth and survival, the cumulative impact of multifactorial stress combinations can be highly detrimental (Zandalinas et al. 2021). However, sometimes these stress combinations lead to unexpected outcomes. A good example of this is that maize plants exposed to flooding, but not under normal conditions, exhibited increased salicylic acid (SA)-dependent resistance to the fall armyworm Spodoptera frugiperda (Figure 1a), a major pathogen in maize, causing significant agricultural losses worldwide (Block et al. 2020, Gorman et al. 2025, Tay et al. 2023). A new report by Gorman and colleagues (Gorman et al. 2025) provides further insights into the mechanisms behind this induced resistance using transcriptomic, metabolomic, and genetic approaches. RNA-seq analysis of maize plants exposed to flooding, herbivory, or their combination revealed that the combined stress resulted in a greater number of differentially expressed genes (DEGs) than individual stresses. Importantly, many DEGs were uniquely expressed under the combined stress (Gorman et al. 2025). Gene Ontology enrichment showed that flooding alone enriched DEGs related to water transport, herbivory enriched DEGs related to wounding and biosynthesis of defense compounds, while their combination enriched DEGs specifically involved in phenylpropanoid metabolism, particularly those associated with SA and ROS responses (Gorman et al. 2025). Due to the complexity of the phenylpropanoid pathway, the authors used targeted LC-MS/MS to determine which specific pathway branches were relevant for flood-induced S. frugiperda resistance. They analyzed phenolic responses under flooding, S. frugiperda infestation, and their combination (Gorman et al. 2025). Combined stress induced the amount of benzoates, lignin breakdown products, chlorogenic acid, and flavonoids. Flavones, especially monohydroxy-B-ring flavonoids (MHBFs) apigenin and luteolin, derived from naringenin and eriodictyol via flavone synthases (FNS), were the most induced (Figure 1b). This correlated with strong induction after stress-combination of type-I FNS transcripts, particularly ZmFNSI-2 , highlighting a role of the flavonoid pathway in maize defense against S. frugiperda (Figure 1b). To test flavonoid function in resistance, mutants of key enzymes in flavonoid biosynthesis were studied. The idf mutant, with impaired chalcone synthase (CHS), the first enzyme in flavonoid biosynthesis, showed reduced resistance and lower SA accumulation under combined stress (Gorman et al. 2025), suggesting flavonoids are required for both SA production and resistance. Interentingly, the amount of jasmonate-isoleucine, the active form of jasmonate (JA), a common hormone involved in insect resistance (Gao et al. 2024), was induced by S. frugiperda infestation at similar levels in control and after flooding in both, WT and the idf mutant. This data support that JA are not involved in flooding-induced resistance or flavonols accumulation. To investigate which flavonols could be involved in the resistance phenotype, mutants lacking specific flavonoid branches, such as anthocyaninless1 ( a1 , deficient in ZmDFR) and pr1 (defective in ZmPR1) were analyzed. These mutants showed no significant differences in larval growth under combined stress, suggesting that anthocyanidins and phlobaphenes are not essential for flood-induced resistance (Gorman et al. 2025). Next, the a1 (anthocyanins via ZmDFR), and pr1 (flavonoid 3’-hydroxylase via ZmPR1) were analyzed to further explore the link between specific flavonoids, S. frugiperda resistance and SA accumulation. Neither mutant showed reduced resistance, suggesting that anthocyanidins and phlobaphenes are not essential in this context. However, pr1 mutants accumulated more SA and apigenin but lacked eriodictyol, pointing to a role for monohydroxy B-ring flavonoids (MHBFs), such as apigenin, in SA induction. SA in plants is synthesized via the isochorismate synthase (ICS) and phenylalanine ammonia-lyase (PAL) pathways (Ullah et al. 2023). Of the nine putative PAL isoforms exist, RT-qPCR showed that ZmPAL5 , ZmPAL7 , ZmPAL8 , and ZmPAL9 (from the same phylogenetic cluster) were induced by combined stress, with ZmPAL7 and ZmPAL8 showing the strongest induction (Gorman et al. 2025). Except for ZmPAL9 , these genes were also upregulated by herbivory alone, though to a lesser extent. Notably, idf mutants showed strongly reduced expression of ZmPAL5 , ZmPAL7 , and ZmPAL8 , suggesting that flavonoids may regulate PAL gene expression (Figure 1b). While the ICS pathway is well characterized in Arabidopsis (Torrens-Spence et al., 2019), its role in maize remains unclear. The maize ICS gene showed no change in expression under stress combination, strongly supporting that the PAL pathway is the dominant SA synthesis route in maize (Gorman et al. 2025). Flavonoids are a diverse class of plant metabolites with protective roles. Known for their antioxidant properties, they scavenge harmful ROS that accumulate during stress (Shoaib et al. 2024). They also have insecticidal and antimicrobial effects, contributing to plant defense (Chatterjee et al. 2023). Using phenotyping, global transcriptome profiling, directed metabolomics, and mutant analysis, this study identifies a specific group of flavonoids involved in SA production, which underlies flood-induced S. frugiperda resistance (Gorman et al. 2025), adding complexity to the functional roles of these molecules (Figure 1a and b). Despite the wealth of information provided in this study, important questions remain open. Since the idf mutant does not fully replicate the enhanced resistance observed in wild-type plants, additional mechanisms beyond flavonols are likely contributing to this resistance. Another key question is to identify the mechanism by which flavonols induce the expression of PAL , leading to increased salicylic acid (SA) content. Once this mechanism is elucidated, it will be important to determine whether it is specific to maize or conserved across species. Finally, from an applied perspective, it is also crucial to explore how this knowledge can be leveraged to enhance resistance in maize production. A key takeaway from this work is that, despite methodological challenges, the study of combined stresses can uncover novel resistance mechanisms that would otherwise remain hidden, thereby advancing our understanding of plant stress responses under natural conditions. Figure Legend FIGURE 1 (a) Maize ( Zea mays ) plants in flooding or nonflooding conditions were infested with S. frugiperda neonates. Measurement of larval growth on flooded versus control plants revealed that S. frugiperda had a significantly slower growth rate on flooded maize than on non-flooded controls. This combined stress leads to elevated production of the defense hormone salicylic acid, which does not occur under individual stresses, resulting in a salicylic acid-dependent increase in S. frugiperda resistance. (b) Simplified overview of salicylic acid-dependent flood-induced S. frugiperda resistance at the plant subcellular level. Two flavone synthase (FNS) genes ( ZmFNSI-1 and ZmFNSI-2 ) were uniquely upregulated in response to the combined flooding and herbivory stress, leading to the accumulation of flavonoids, with the flavones apigenin and luteolin being the most prominently induced. Since several flavonoids possess insecticidal activity, they could play a direct role in resistance to herbivory stress. In addition, the accumulation of specific flavonoids leads to transcriptional induction of phenylalanine ammonia lyase (PAL) genes 5, 7 and 8 ( ZmPAL5/7/8 ) and elevated levels of salicylic acid (SA), leading to SA binding to the non-expressor of pathogenesis-related (PR) proteins (ZmNPR). This, in turn, promote its nuclear localization and transcriptional activation of defense responses, leading to enhanced resistance to S. frugiperda . This figure was created in BioRender. Botella, M. (2025) https://BioRender.com/fmbhr9u References Block A.K., Hunter C.T., Sattler S.E., Rering C., McDonald S., Basset G.J., Christensen S.A., 2020, Fighting on two fronts: Elevated insect resistance in flooded maize, Plant, Cell & Environ , 43: 223–234. https://doi.org/10.1111/pce.13642 Chatterjee D., Lesko T., Peiffer M., Elango D., Beuzelin J., Felton G.W., Chopra S., 2023, Sorghum and maize flavonoids are detrimental to growth and survival of fall armyworm Spodoptera frugiperda , Journal of Pest Science, 96(4):1551–1567. https://doi.org/10.1007/s10340-022-01535-y Gao, Q., Jin, N., Shen, Z., Guo, J., Lu, H., Han, S., Xiao, W., Lu, J. and Lou, Y., 2025, Both Jasmonic Acid- and Abscisic Acid-Mediated Signalling Pathways Regulate the Ovicidal Defence of Plants Against Phloem-Feeding Insects. Plant, Cell & Environment. https://doi.org/10.1111/pce.15445 Gorman, Z., Liu, H., Sorg, A., Grissett, K.S., Yactayo-Chang, J.P., Li, Q.-B., Rivers, A.R., Basset, G.J., Rering, C.C., Beck, J.J., Hunter, C.T. and Block, A.K. (2025), Flood-Induced Insect Resistance in Maize Involves Flavonoid-Dependent Salicylic Acid Induction. Plant, Cell & Environment . https://doi.org/10.1111/pce.15496 Tay W.T., Meagher Jr R.L., Czepak C., Groot A.T., 2023, Spodoptera frugiperda: ecology, evolution, and management options of an invasive species. Annual Review of Entomology , 68(1):299–317. https://doi.org/10.1146/annurev-ento-120220-102548 Torrens-Spence M.P., Bobokalonova A., Carballo V., Glinkerman C.M., Pluskal T., Shen A., Weng J.K., 2019, PBS3 and EPS1 complete salicylic acid biosynthesis Arabidopsis, Molecular plant, 12(12):1577-1586. https://doi.org/10.1016/j.molp.2019.11.005 Shoaib, N., Pan, K., Mughal, N., Raza, A., Liu, L., Zhang, J. et al. (2024) Potential of UV-B radiation in drought stress resilience: A multidimensional approach to plant adaptation and future implications. Plant, Cell & Environment , 47, 387–407. https://doi.org/10.1111/pce.14774 Ullah, C., Chen, Y.H., Ortega, M.A., Tsai, C.T (2023), The diversity of salicylic acid biosynthesis and defense signaling in plants: Knowledge gaps and future opportunities. Current Opinion in Plant Biology . 72: 102349. https://doi.org/10.1016/j.pbi.2023.102349 Zandalinas, S.I., Sengupta, S., Fritschi, F.B., Azad, R.K., Nechushtai, R. and Mittler, R. (2021), The impact of multifactorial stress combination on plant growth and survival. New Phytologist , 230: 1034-1048. https://doi.org/10.1111/nph.17232 Acknowledgements M.A.B was funded by the Ministerio de Ciencia e Innovación (grant no. PID2023‐147983OB‐I00) and by the Junta de Andalucia (PAIDI 2020‐PY20_00084). V.A.‐S. was funded by a grant (“Programa Emergia 2023”, DGP_EMEC_2023_00375) by “Consejería de Universidad, Investigación e Innovación de la Junta de Andalucía” and the European Union: European Social Fund Plus (ESF+). Funding for open access charge: Universidad de Málaga/CBUA. Conflicts of Interest The authors declare no conflicts of interest. Data Availability Statement Data sharing not applicable to this article as no datasets were generated or analysed during the current study. Information & Authors Information Version history V1 Version 1 15 April 2025 Peer review timeline Published Plant, Cell & Environment Version of Record 11 May 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Plant, Cell & Environment Keywords flavonoid flooding hormones maize resistance to s. frugiperda salicylic acid secondary metabolism Authors Affiliations Vitor Amorim-Silva 0000-0002-3978-7205 Instituto de Hortofruticultura Subtropical y Mediterranea View all articles by this author Miguel A. Botella 0000-0002-8867-1831 [email protected] Instituto de Hortofruticultura Subtropical y Mediterranea View all articles by this author Metrics & Citations Metrics Article Usage 328 views 205 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Vitor Amorim-Silva, Miguel A. Botella. The whole is not always the sum of the parts: Synergistic plant responses to combined environmental stresses. Authorea . 15 April 2025. DOI: https://doi.org/10.22541/au.174471890.05910119/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. 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