The Whole Is Not Always the Sum of the Parts: Synergistic Plant Responses to Combined Environmental Stresses

Abstract

Numerous studies examining plant-environment stress interactions cultivate plants under optimal, non-stressful conditions before introducing individual stress factors to analyse response mechanisms, which clearly is an oversimplification. In nature, plants thrive under suboptimal conditions, encountering multiple environmental cues such as day-night temperature variation, abiotic stressors, soil heterogeneity, pathogens, herbivores and so forth. 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 herbivore pest in maize, causing significant agricultural losses worldwide (Block et al. 2020; Gorman et al. 2025; Tay et al. 2023).

A new report by 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 defence 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 analysed 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 defence 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. Interestingly, the amount of jasmonate-isoleucine, the active form of jasmonate (JA), a common hormone involved in insect resistance (Gao et al. 2025), was induced by S. frugiperda infestation at similar levels in control and after flooding in both, WT and the idf mutant. This data supports 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 analysed. 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 analysed 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 defence (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,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 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.

The authors declare no conflicts of interest.