Rapid shift in global climatic conditions due to industrialization and urbanization has raised concerns like food and freshwater availability for a growing population (Islam and Karim, 2019). This situation is notably worse in the arid and semi-arid regions of the world, for instance, the food demand in Africa and Asia has exceeded the capacity of the food supply chain (Hussain et al., 2019; Fukase and Martin, 2020). Salinity and drought have affected 7% and 40% of the total land area, respectively, which makes these abiotic stressors as primary factors for agro-economic losses (Trenberth et al., 2014). The sustainable development goals were designed to overcome this distressing situation, which has united researchers across the globe to improve productivity under stress (Morton et al., 2019). Natural habitat often exhibited multiple stress factors, therefore, research needed to identify responses of plants under multiple stress conditions (Cominelli et al., 2013) to establish stress resilience of a plant.

Halophytes are the native flora of saline landscapes, of which many species also exhibit tolerance to drought conditions (Weber, 2009). Their utilization for phytoremediation, medicines, and biofuel has far surpassed their traditional uses as feed and fodder (Hamed and Custódio, 2019). The concurrence of stress factors, i.e., drought and soil salinity, is ubiquitous in natural habitats of the halophytes. Since many of the physiological and biochemical mechanisms adapted in response to these stressors are common, the exposure of halophytes to combined stressors may act additively, synergistically, or antagonistically to escalate their tolerance to stress environments (Hamed et al., 2013).

Excessive concentrations of Na⁺ and Cl^- limit the availability of other beneficial ions and disturb cellular osmotic potential. Conversely, plants under drought stress defend against water shortage by utilizing mainly K⁺ and Cl^- at a lesser extent for stomatal closure (Ruzin, 1999). The accumulation of Cl^-, a common anion in response to both salt and drought stressors, benefits plants by improving the size of leaf cells (i.e., more water storage capacity) and by increasing the mesophyll conductance to atmospheric carbon (Colmenero-Flores et al., 2019). In addition, the oxidative stress generated either due to an enhanced ROS production or inhibited antioxidant activity is a typical response to salinity and water-deficit conditions (Miller et al., 2010).

Physiological water-deficit, common to salt and drought stress, restricts the cellular turgor. Reduced leaf area coupled with stomatal closure limits the availability of CO₂ to chloroplasts and, therefore, the photosynthetic performance (Flexas et al., 2009; Osakabe et al., 2013). Although plants employ various anatomical adaptations, such as an increase in vascular tissues and thickness of cell walls, to overcome water shortage (Guerfel et al., 2009), this phenotypic plasticity has not been resolved concerning combined stresses.

Panicum antidotale Retz. is a cash crop halophyte that can survive up to seawater salinity (500 mM NaCl) concentration (Koyro et al., 2013; Hussain et al., 2015). Extensively distributed along with the marginal lands of Pakistan, this C4 perennial grass is utilized as a typical fodder. Detailed investigations have been conducted to characterize the physiological, molecular, and biochemical markers of salt tolerance in this fodder grass (Hussain et al., 2015; Hussain et al., 2021). Our previous work distinguished single and combined effects of drought and salinity stress on the gas exchange properties of this grass (Hussain et al., 2020). To our surprise, a combination of salinity and drought treatment ameliorated the plant’s performance by assisting water-retaining and carbon-fixing properties.

Salt-tolerant grasses are generally salt excluders (Munns, 2005). P. antidotale retains toxic ions within roots and hence, a lower concentration of Na⁺ and Cl^- in shoots (Hussain et al., 2015). However, an understanding of the management of toxic ions in response to combined stresses has not been developed for this grass. The current study was, therefore, designed to explore the ion homeostasis management in this species to a combination of stresses. We presume that better resilience of this grass to salinity + drought stress will be due to either restricted accumulation of toxic ions or anatomical adaptations that favor water and carbon supply. Following hypotheses were specifically tested: i) the exposure of plants to combined stressors will enhance the selective transport of toxic ion to shoots. ii) Plants will exhibit a more activated antioxidative machinery to tolerate combined stress. iii) Plants treated with combined stressors will develop a larger area of transport tissues.

The present study investigated the effects of salinity (low and high), drought stress, and their combination on the management of ion homeostasis, antioxidant activity, and anatomical features of P. antidotale. These findings indicated that a combination of low salt and drought stress mostly improved plant performance by relieving plants from damaging effects of drought treatment. In contrast, the interaction between high salt and drought was mostly synergistic and escalated the harm caused by individual stressors. Thus, this halophytic grass can be naturally cultivated in low saline-arid areas to meet the increasing pressure on fodder and ecological needs.

Stimulated growth at low salinity (LS) and inhibited growth at high salinity (HS) of test species ( Figure 1 ) are consistent with the response of other halophytic grasses (Flowers and Colmer, 2008). In addition, the equally reduced plant biomass at D and HS supports the similar effects of these conditions (Askari-Khorasgani et al., 2021). While a combination of low salinity and drought (LSD) alleviated the adverse effects of D on biomass accumulation (dry weight), the synergistic effects of high salinity and drought combination (HSD) mainly inhibited leaf biomass (fresh weight). The negative effects of HSD on plant growth and development were also evident with fewer upper and lower epidermal cells. However, the comparable leaf dry weight at HS, D, and HSD suggests similar influence of individual and combined stresses in impeding plants water acquisition. Although t reduction in epidermal cells was more pronounced at HS; the maximum area of these cells suggested a rigid outer leaf coat associated with epidermal cell wall-loosening proteins and is consistent with the protective and water-conserving function of the epidermis under stress (Zörb et al., 2015). The similar epidermal area in D and control plants highlights the regulatory adaptions that P. antidotale exhibits to overcome a water-deficit environment. It is worth mentioning that the drastic effects of D on roots’ fresh weight were not alleviated even with LSD. Since roots are the primary organs to perceive and respond to environmental stimuli, the altered expression of various genes may influence the development of the root system under stress (Cominelli et al., 2013). The compromised root growth, however, may be beneficial as it allows plants to redistribute the limited resources and fulfill the energy requirements of stress tolerance mechanisms (Munns and Gilliham, 2015).

Stress environments often cause a carbon/nitrogen imbalance shown with an increased C: N ratio suggesting the poor health (Rong et al., 2015) and discrepancies in the biophysical properties of plants (Xu et al., 2011). Significantly low C in the root of HS, D, and HSD-treated plants may reflect a disturbance in the transport of photosynthate from leaf or green stem to root. It also suggests a lower accumulation of C-based secondary metabolites, including soluble sugars, which function as osmolytes. Osmolytes have a promising role in maintaining the water influx while protecting the cells against structural and functional damage during stress (Jogawat, 2019). Interestingly, the unchanged nitrogen (N) content in root of HS, D, and HSD, reflected a non-disturbed nitrogen metabolism. P. antidotale treated with LSD showed the lowest C: N ratio in the leaves owing to the highest N and C content which implies abundant chlorophyll (Xu et al., 2018) and maximum carboxylation rates of Rubisco to sustain the photosynthetic performance of plants (Evans and Clarke, 2019). However, these plants could not mitigate the negative effects of D on the mesophyll region. Besides, the diffusional resistance of CO₂ to bundle sheath reduces the carbon fixation in C4 plants (Ellsworth and Cousins, 2016). Therefore, determining the contribution of these factors behind non-optimal photosynthetic rates is equally important.

The limited plant growth under stress is mainly due to disturbed water relations, ionic toxicity, and nutritional imbalance. As expected, plants exposed to D did not experience ionic stress (i.e., comparable levels of Na⁺ and Cl^- in control and D plants). Moreover, the maintained K⁺ levels in the root and stem to that of control plants ( Table 1 ) indicated a high resilience of our test species against the water deficit conditions. The accumulation of Na⁺ increased in all the organs with increasing NaCl, but that of Cl^- was limited to root, at least in LS-treated plants, which indicates the amplified susceptibility of below-ground plant parts to ionic stress. Combining drought with low salinity (i.e., LSD) enabled plants to retain ion homeostasis (Na⁺/K⁺, Na⁺/K⁺ + Ca²⁺ + Mg²⁺) at least in the root. The non-selectively transported ions (in low concentrations) may act as cheap osmoticum in the above-ground plant parts to develop a water potential gradient and improve the carbon assimilation properties of P. antidotale (Hussain et al., 2020). The combination of high salinity and drought (HSD) acted additively, and excessive accumulation of Na⁺ and Cl^- restricted the availability of essential ions (K⁺, Ca²⁺, and Mg²⁺) even in the root. The elevated Na⁺ and Cl^- damage cellular machinery and biomolecules, and, therefore, must either be excluded, extruded, or compartmentalized (Roy and Chakraborty, 2014). This is why the cytosolic ratio of beneficial ions, such as K⁺, to Na⁺, rather than the actual content of Na⁺, is considered more relevant to achieve stress tolerance (Anschütz et al., 2014). Salt-resistant species successfully maintain ion homeostasis due to the coordinated functioning of various ion transporters and channels (Huang et al., 2020). For instance, the upregulated expression of SOS1 has been shown to restrict Na⁺ loading in the transpiration stream while enhancing K⁺ uptake under NaCl stress (El Mahi et al., 2019). Halophytic grasses express a supreme resiliency against salt stress by effectively managing toxic ions between different organs (Munns, 2011; Shabala, 2013). The reduced vascular bundle at HS proposed disturbed ions transportation efficiency, in agreement with the previous reports (Arafa et al., 2009; Nassar et al., 2020). The onset of drought caused a further reduction in the vascular bundle area, indicating the severe effects of water-deficit environments on the anatomical features of this halophyte. However, combining salt with drought, i.e., LSD and HSD, alleviated the negative impacts of drought on the area of vascular tissues, but the area was still low if compared to salinity stressor alone ( Table 3 ). Our results, disapprove the hypothesis, “Plants treated with combined stressors will develop a larger area of transport tissues.”

Based on the analyses of selective absorption and transport of beneficial ions, we, for the first time, demonstrated differences in the ion regulatory capability of P. antidotale under saline-drought conditions. Combined stress improved the plant’s selective absorption of K⁺, Ca²⁺, and Mg²⁺ over Na⁺; however, the selective transport of the absorbed ions did not occur ( Figure 3 ). Our data rejected the hypothesis that “the exposure of plants to combined stressors will enhance the selective transport of toxic ions to shoots”. In contrast, relatively less selective absorption under salinity (LS and HS) was followed by an active selective transport of beneficial nutrients. This enabled salt-treated plants to transport K⁺ from roots to stems and leaves while retaining most of the Na⁺ in the roots. This observation is surprising as an increased allocation of potassium in the below-ground parts of Desmostachya bipinnata – a halophytic grass with C4 photosynthesis, disturbed ionic homeostasis in the above-ground parts (Adnan et al., 2021). In our study, the LS-treated plants also selectively transported other ions such as Ca²⁺ from root to leaf and N O 3   − from root to stem. Organ-specific nutrient allocation is related to the involvement of specific nutrients in cellular processes such as membrane stabilization, stomatal opening, regulation of enzyme activity, etc. (Wu and Wang, 2012). Better nutrient utilization of plant at LSD ( Table 2 ) allowed us to claim an economical and environmentally sustainable solution to cultivate saline arid lands.

Plants under environmental stress experience an imbalance in photochemical reactions and electron flow from PSII, which affects their carbon assimilation properties. The energized electrons in such a case generate reactive oxygen species (ROS) (Foyer, 2018). The lowest ratio of ΦPSII/ΦCO₂ in LSD plants suggested less availability of free electrons and an optimal photosynthetic performance as previously reported (Hussain et al., 2020). Interestingly, the high values of ΦPSII/ΦCO₂ obtained in response to individual treatments, i.e., LS, HS, and D, decreased under combined treatments, i.e., LSD and HSD. The improved mesophyll region in HSD-treated plants ( Table 3 ), however, does not guarantee an enhanced carbon fixation as the contribution of non-stomatal factors in limiting carbon fixation in high salinity + drought-treated plants was acknowledged (Hussain et al., 2020). Similarly, the enhanced ROS generation at HSD may also be reasoned to fewer stomata and a small stomatal aperture (Hussain et al., 2020).

SOD, the first cellular enzyme for ROS detoxification, was active in high salinity and drought-stressed plants, agreeing with the report on salt tolerant plants (Al Hassan et al., 2017). A combination of these conditions, i.e., HSD, interacted additively to produce more superoxide radicals and therefore, an enhanced SOD activity ( Figure 5 ) as noted for other halophytes (Lu et al., 2021). The activity of subsequent H₂O₂-detoxifying enzymes, however, either remained unchanged (catalase) or decreased (APx) at HSD. While H₂O₂ in low concentrations modulate many physiological processes, high concentrations may damage metabolic machinery (Petrov and Van Breusegem, 2012). These findings allowed us to postulate a H₂O₂-derived damage to cellular biopolymers such as proteins and lipids in HSD-treated plants. The HSD-induced oxidative stress was also perceived with low activities of other antioxidants such as GR and GPx and the total antioxidant capacity. The relatively low (APx, GR) or comparable (GPOx, CAT) activities at LSD, to that of control plants, agreed with the posit that ROS production was low in these plants. The findings of the study reject the hypothesis that “plants will have more activated antioxidants machinery to tolerate combined stress”. In contrast, high ROS-detoxification efficiency under the combination of abiotic stresses has been reported for other plants (Ramu et al., 2016; Lopez-Delacalle et al., 2021).

Panicum antidotale is a suitable candidate to grow on low saline-arid areas, as shown by dry weight accumulation ( Figure 6 ). It was mainly due to the selective absorption of beneficial ions to fulfill the nutrient requirements and to utilize the accumulated ions as cheap osmoticum. In the absence of NaCl, drought-treated plants were able to accumulate high content of potassium, and this ability persisted after the addition of low salinity, i.e., LSD. Consequently, nutrient utilization efficiencies in these plants were improved compared with any of these two stressors individually (LS and D). Similarly, the production of ROS was minimal (i.e., ΦPSII/ΦCO2), and the T-AOC of test species was sufficient to detoxify the generated ROS. Nevertheless, the reduced area of the vascular bundle and mesophyll region explained many physiological strains in the current study (such as low photosynthesis and water content). Combining high salinity with drought (HSD) escalated the adverse effects caused by individual stressors on the antioxidant capacity and ion homeostasis. The non-selectively transported ions caused an ionic imbalance, and a high ratio of Na⁺/K⁺ was evident in all the organs. A reduction in epidermal tissues (number and area) predicted that these plants were prone to mechanical injury and water loss. Given all this, we would suggest highly antagonistic interactions between low salt and drought while synergistic/additive interactions between high salt and drought on most plant physiological mechanisms. Future studies must efficiently explore the molecular basis of combined tolerance to drought and salt stress to transfer tolerance traits in related crops.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

TH and XL perceived the idea. TH and WZ performed the experiments, TH evaluated data, TH and HA wrote the manuscript, WZ and XL corrected the manuscript. All authors contributed to the article and approved the submitted version.