Climate change is increasing the frequency and severity of drought and flooding events, hampering food security. Drought affects plants throughout all developmental stages by impairing key physiological processes including photosynthesis (Hossain et al., 2012; Kilic and Yagbasanlar, 2010). The loss of photosynthesis can lead to reduced growth and ultimately seed yield (Ahmad et al., 2022; Chen et al., 2012; Ahmad et al., 2018). Conversely, flooding affects approximately 10% of arable land (Yaduvanshi et al., 2012) and causes a reduction of oxygen levels in the soil with hypoxic conditions created. Flooding increased the accumulation of CO₂ and ethylene that disrupts the normal physiological and morphological development of plants (Kreuzwieser et al., 2004). The severity of flooding depends on factors such as duration, developmental stage and species or cultivar characteristics (Manghwar et al., 2024). Key effects include reductions of photosynthetic rate (P_n), transpiration (E) and water use efficiency (WUE), along with damage to the photosynthetic apparatus, particularly during early growth stages (Shao et al., 2013). Flooding leads to a loss of chlorophyll in any growth stage (Chen et al., 2005). Stomatal closure is a primary response to both drought and flooding that leads to decrease the rate of net photosynthesis and under severe water stress conditions, metabolic impairment maybe added to the stomatal limitation. At progressive stages of water stress, stomatal traits such as size, density and distribution can be altered (Pirasteh-Anosheh et al., 2016; Zhou et al., 2020).

Climate change forecasts predict increasing frequency of both drought and flooding events across cereal growing region (Borowski, 2020; Eckstein et al., 2020). Importantly, plants are increasingly exposed to these stresses successively at the same growth phase, and yet, their responses to such sequential stresses are complex and not simply additive. Evidence suggests that the physiological responses to alternating drought and flooding are distinct from single drought or flooding responses (Rivero et al., 2022; Walter et al., 2013; Biswas and Jiang, 2011). For example, drought followed by flooding (D-F) induced photosynthetic, growth and yield reductions in rice (Xiong et al., 2018; Zhu et al., 2020), and cabbage (Barber and Müller, 2021). Qian et al. (2020) showed that all yield indices of cotton were reduced by flooding followed by drought (F-D), but the reduction was lower than the additive effects of both stresses imposed separately. Winter flooding followed by summer drought reduced yield in wheat, with this effect being additive but not more than additive (Dickin and Wright, 2008). However, a research gap remains regarding species-specific responses of major cereals like wheat and barley to successive alternating water stresses, particularly at physiological and biochemical levels.

Wheat and barley are important cereal crops, directly or indirectly providing a significant part of the calories in human diets globally. With expanding global populations, food security is becoming increasingly difficult, especially with unpredictable severe weather patterns (Wheeler and Von Braun, 2013). Water stress is critical for wheat production and this is a serious problem, as wheat is the staple food of 35% of the world population (Ostrowska and Hura, 2022; Soares et al., 2019; Tadesse et al., 2019). Barley is the fourth most important cereal crop (Giraldo et al., 2019) and is regarded as being better adapted to drought stress (Dawson et al., 2015). The high amount of genetic variability towards stress tolerance has led to its use as model plant in studying water use efficiency (Ostrowska and Hura, 2022).

Despite many studies published on separate drought and flooding responses in wheat and barley, little is known about the physiological or biochemical traits resulting from cycles of drought and flooding in these important cereals. Such knowledge is necessary to identify novel traits conferring resilience and for cereal crop improvement to withstand sequential water stresses. The present study aims to investigate the physiological responses of wheat and barley to cycles of drought and flooding, with single drought or flooding and drought followed by flooding (D-F) or flooding followed by drought (F-D) treatments, as well as their recovery responses. We have focused on identifying physiological features that give rise to cyclical drought-flooding stress tolerance. To achieve this, we have focused on photosynthesis, chlorophyll content and growth parameters as photosynthesis is essential for plant growth and yield and its stability under water stress can indicate better physiological resilience. We hypothesize that successive applications of drought and flooding (D–F and F–D) will lead a greater inhibition of photosynthesis than either drought or flooding alone. Furthermore, we predict that barley will exhibit greater physiological resilience to these cyclical stress conditions than wheat, as evidenced by better photosynthetic performance.

Episodes of extreme weather are projected to increase and fluctuate between extreme dry and wet conditions. To enhance stress tolerance in crop plants, particularly cereals, we need to better understand the traits possessed by tolerant plants. In this study, a barley cultivar, Chariot- generally considered a relatively stress tolerant cereal species under different environmental conditions, and a wheat cultivar, Gleam- a high yielding adaptable variety that can adapt to different environmental stress, were examined, with the hypothesis that differences between these two plant species might exist in their responses to cycles of drought and flooding. Our results show clear contrasts between the two cultivars studied, indicating that successive stresses increase the sensitivity by the exposure to a first stress, and that barley is less sensitive than wheat. Inhibition in barley could mostly be explained by stomatal limitation of photosynthesis, whilst in wheat, non-stomatal limitations were important.

In this study, drought or flooding were applied as single stress (15 days) or as sequential stresses, in which one stress was followed by the other (15 days each, total 30 days). While this introduces a difference in total stress duration between single and sequential stresses, the experimental design was set up to reflect ecologically relevant scenarios, where plants encounter temporally separated but contrasting water stresses. To enable us to isolate the effects of stress sequence and transition (drought to flooding or flooding to drought) on photosynthetic responses, each individual stress duration was standardized to 15 days. Although the extended duration of the sequential stress treatments may contribute to cumulative effects, our primary focus was on how the transition between stress types influence physiological performance. This approach is supported by previous findings emphasizing the significance of both stress duration and sequence stimulating plant responses to sequential abiotic stress. For example, Ru et al. (2022) showed that prior exposure to drought can enhance tolerance to subsequent heat stress in maize through mechanisms such as improved photosynthetic efficiency and by activating stress memory in plants under water stress (Luis de la Fuente et al., 2023). By comparing responses in wheat and barley, we further examined whether prior exposure to one stress stimulates the response to a subsequent, contrasting stress- highlighting possible mechanism or recovery effects specific to species and stress order. These insights contribute to a broader understanding of how cereals cope with dynamic and fluctuating environmental conditions, particularly in the context of climate change.

Previously, only a few studies have examined responses to cycles of drought and flooding. Zhu et al. (2020) showed the cumulative effects of drought followed by flooding (D-F) reduced photosynthesis followed by recovery and compensation in rice. They also observed both synergistic or antagonistic effects on photosynthesis in previously droughted rice following flooding at different drought or flooding intensities. To be best of our knowledge, no work has been reported on photosynthetic responses in plants exposed to flooding followed by drought (F-D) and importantly, photosynthetic responses of relatively stress tolerant species like barley have not so far been examined under drought-flood water stress. Though some studies have focused on yield loss in wheat (Dickin and Wright, 2008), cotton (Qian et al., 2020) and cabbage (Barber and Müller, 2021) under drought-flood stress, photosynthetic responses, that may be important to find out tolerance traits under double water stress have not been described. In our experiments, two-week-old wheat and barley plants were studied under drought, flooding and D-F or F-D treatments. We chose in this study to measure photosynthetic parameters along with growth, as a previous study on barley photosynthesis showed a significant correlation between photosynthetic efficiency, especially maximum quantum yield of photosystem II, and yield related traits that suggests the improving crop yield through optimizing photosynthetic light use efficiency (Gao et al., 2024).

Wheat plants exposed to a single stress, either drought or flooding, experienced a decrease of photosynthetic capacity ( Figure 3A ), and growth of droughted wheat was reduced post-flooding ( Figures 2A, E , grey area). By contrast, neither drought nor flooding as a single stress inhibited the growth of barley ( Figures 2C, G ), with flooding increasing root growth post-stress, Day 30 ( Figure 2C grey area, Supplementary Figure S3 ). There was no decrease of maximum photosynthetic rate during stress in barley, though this was inhibited post-flooding ( Figure 3C ). Overall, results are consistent with this species generally being more stress tolerant than wheat. The increasing root:shoot ratio in barley following flooding, Day 30 ( Figure 2K , grey area) indicates that this had the greatest impact on plant development, which might be expected to affect subsequent stress responses. Yang et al. (2004) reported that extensive root systems can help maintain photosynthetic rate by supplying water, nutrients and plant hormones under stress conditions. The reduction of growth and photosynthesis by single- drought (Ahmad et al., 2018; Zhao et al., 2020) or flooding (Shao et al., 2013) stress has been reported previously in wheat and reduction in the photosynthetic capacity depends on the intensity of water stress (Chen et al., 2025). Also, an increase in photosynthetic capacity in tolerant barley varieties induced by single- drought has also been observed (Harb et al., 2020), and the tolerance of flooded barley measured by Fv/Fm (Langan et al., 2024) but the post-flooding inhibition of photosynthesis in this species has not to our knowledge been reported before. Here, we observed that barley photosynthetic capacity is less sensitive to single- drought or flooding stress than wheat. We assessed physiological reasons associated with these contrasting responses by investigating chlorophyll fluorescence, chlorophyll content and C-N content. ϕPSII was unaffected by drought and flooding treatments in both species ( Figures 5A, C ). This contrasts with P_n, which was inhibited in wheat in both stresses ( Figure 3A ). This implies that changes in P_n are a result mainly of stomatal closure limiting CO₂ supply, while ϕPSII is maintained either through acclimation of the photosynthetic apparatus (reducing PSII capacity) or simply through an increase in photorespiration. The loss of chlorophyll in wheat after flooding ( Figure 6A , grey area) may reflect an acclimation response in this case, correlating with a loss of leaf N, whilst C did not change ( Figures 7E, M , grey area). In contrast, in drought or flooded barley there was no reduction of photosynthesis or ϕPSII, and no change in chlorophyll or leaf N. There was an increase in shoot C in flooded barley, which continued after the stress, Day 30 ( Figure 7O ) and is suggested to reflect the accumulation of sugars in leaves (Martínez-Alcántara et al., 2012) which may help the osmotic adjustment of stressed barley.

Droughted wheat which was then exposed to flooding (D-F) showed reduced stomatal conductance ( Figure 3F ) which was similar to the findings of experiments on rice (Zhu et al., 2020). In our experiment, the inhibited stomatal conductance (g_s) in the early droughted wheat ( Figure 3E ) was followed by a greater inhibition of g_s following subsequent exposure to flooding ( Figure 3F ), which contributed to a decreased rate of photosynthesis ( Figure 3B ). At the same time, D-F treatment reduced ϕPSII, accompanied by a loss of chlorophyll, neither of which were affected by drought alone ( Figures 5B , 6B ). Alongside this loss of photosynthesis, wheat growth was highly retarded following D-F treatment, as compared to drought alone ( Figures 2B, F ). This suggests D-F stress induces some non-stomatal factors that limit photosynthesis and growth in wheat. C-N content ( Figure 7 ) reflects a loss of nitrogen, which may affect growth. The loss of N in shoots, without any change of carbon content, may contribute to the loss of photosynthesis in D-F stress in wheat. Therefore, we found that the effect of flooding on the previously droughted plants was continued and aggravated in wheat, as also previously observed in wheat (Schweiger et al., 2023). The prior inhibition of photosynthesis of single-droughted wheat could be explained by stomatal closure, but after subsequent flooding, non-stomatal factors were added, which were probably linked to the reduced PSII efficiency and chlorophyll content.

In contrast to wheat, when barley was flooded after drought, there was a recovery of stomatal conductance, whilst P_n was unaffected in either drought or D-F treatments ( Figure 3 ). In addition, there was no change of ϕPSII ( Figure 5D ) or chlorophyll content ( Figure 6D ) in either treatment. There was no effect of D-F treatment on root growth, but the shoot growth was retarded ( Figures 2D, H ). This effect on growth was also reflected in their C-N content, showing that there was no change of N-content in the shoots in droughted barley, but a loss of N, accompanied by an increase in C- content in the shoot of D-F barley ( Figures 7H, P ). Therefore, we conclude that barley can retain stomatal conductance and photosynthesis under D-F stress whilst decreasing shoot growth, increasing root:shoot ratio. The increased leaf C- content suggests an accumulation of sugar in shoots.

For the F-D treatment in wheat, stomatal closure was greater than seen for drought as a single stress ( Figures 3E, F ) although there was no greater inhibition of photosynthesis ( Figures 3A, B ). This is accompanied by a similar downregulation ϕPSII in the F-D treatment ( Figure 5B ). This suggests a downregulation of electron transport to match production of ATP and NADPH to the decreased CO₂ assimilation (Lu and Zhang, 1999). In addition, the loss of chlorophyll content under F-D stress may contribute to the low rate of photosynthesis ( Figure 6B ). Though there was no effect on their growth exposed to prior flooding, after F-D stress (Day 45) the growth was greatly retarded ( Figures 2B, F ). Therefore, the inhibition induced by flooding is continued when plants are exposed to subsequent drought. Like the D-F treatment, our observations showed the involvement of non-stomatal factors decreasing photosynthesis and growth under F-D stress conditions in wheat.

In barley, the F-D treatment, unlike the D-F treatment, induced a large inhibition of g_s, accompanied by an inhibition of the rate of photosynthesis ( Figures 3D, H ), together with the downregulation of ϕPSII followed by recovery ( Figure 5D ). There was no accompanying change of chlorophyll content ( Figure 6D ). However, they retained their root growth under F-D stress ( Figure 2D ), but shoot growth was retarded, giving a higher root/shoot ratio, similar to that in D-F stressed barley ( Figures 2H, L ). When previously flooded barley was droughted, the inhibition of g_s was greater than that caused by drought as a single stress ( Figure 3G ) which did not fully recover when plants returned to normal watering conditions ( Figure 3H ), although P_n recovered ( Figure 5D ). The response of g_s may be explained by developmental differences in leaves formed during flooding ( Figure 4 ). Leaves formed during the initial flooding showed a higher stomatal density, but these stomata were smaller than those formed under control conditions. A similar developmental effect has previously been seen in barley grown at a high CO₂ concentration (Haworth et al., 2023; Kreuzwieser et al., 2004). Moreover, F-D stressed barley had increased C-content in both roots and shoots ( Figures 7L, P ) possibly reflecting an accumulation of carbohydrates in their tissue. Overall, we conclude that previous flooding increases barley sensitivity to subsequent drought, exacerbating the post-flooding effect seen when plants are returned to normal watering. Further study is required to understand the stomatal limitation to photosynthesis of barley in F-D stress.

The present study has investigated the physiological responses of wheat and barley to cycles of drought and flooding and shown contrasting responses in the two species ( Figure 8 ). We observed that exposure to the first stress stimulated the inhibition of photosynthesis induced by further stress. Importantly, preceding exposure to flooding increased the inhibition of photosynthesis induced by subsequent drought in both wheat (36%) and barley (44%). Barley is relatively more tolerant to successive water stresses retaining photosynthetic capacity and PSII efficiency following single as well as double water stress, with only F-D double stress inhibiting photosynthesis and only via a stomatal limitation. The greater root growth (79% increase) of barley may be important in this tolerance. By contrast, wheat may increase its chances of survival by having the flexibility to reengineer the leaf, even if it is unable to maintain its growth and final yield. These findings have important implications for agricultural production in sequential water stressed environments. The relative successive drought and flood resilience of barley suggests it could be a more sustainable crop choice under climate change with frequent water stress. It could potentially ensure more stable yields leading to food security. Future research should focus on underlying photosynthetic mechanisms of barley under sequential flood-drought conditions and yield variations to optimize crop performance.