In the context of global climate change, more frequent and severe drought episodes are expected in the coming years (Wood et al., 2023). These events will negatively affect crop growth and therefore food production. Over 20% of the world’s agricultural areas suffer from drought problems (Mansoor et al., 2022), and Tunisia is one of the countries with the lowest availability of water in the world (Hachani et al., 2022). Since many rivers and dams have already dried up, Tunisia is using up to 80% of the available water resources in agriculture (Douh et al., 2022). Wheat is consumed as a fundamental food grain all over the world (Hussein et al., 2023), with Tunisia being an important consumer. Thus, the country is facing a great challenge to preserve national food security under serious drought threats. Plant responses in arid conditions involve complex functional and structural adaptations. For wheat crops, different levels of drought-tolerance mechanisms, involving changes in physiological processes of plant leaves, have been observed. Metabolic disruptions and great yield loss result from the activation of different mechanisms, including water and minerals uptake, CO₂ assimilation, transpiration, stomatal conductance, chlorophyll concentrations, stomatal density and cell wall integrity (Hachani et al., 2022; Srinatha et al., 2023). Various study have shown differences in the capacity of plant species to perform stomatal regulation, osmotic adjustment and to change the leaf tissues elastic properties (Martínez et al., 2007; Hessini et al., 2008; Sampaio Filho et al., 2018; Leuschner et al., 2019; Wood et al., 2023). The determination of water relations and leaf gas exchange parameters are fundamental to analyze and quantify the effects of drought on the cell and tissue physiology of wheat plants and to understand how they impact on their survival, growth, and productivity. Leaf turgor loss point (Ψ_π ⁰) was identified as a key parameter to evaluate the response of many plants to drought stress (Wood et al., 2023). During water deficits, isohydric species maintained a constant Ψ_π ⁰ through sensitive stomatal regulation, reduced stomatal conductance (gs) and transpiration, and was more depended on stored carbohydrates to meet continued carbon demands. On the other hand, anisohydric plants showed marked drops in Ψ_π ⁰, high gs levels, and increased evaporative demand, as stomata close later and reached more negative xylem water potentials. Plants often regulate the use of water by balancing water supply and demand to safely maintain leaf osmotic potential above Ψ_π ⁰ (Wood et al., 2023). As solutes accumulate, the turgor pressure increases until the cell reaches Ψ_π equilibrium in the immediate surroundings (Juenger and Verslues, 2023). This effect can derive from either simple passive solute concentration resulting from dehydration, or from net solute accumulation, considered as active osmotic adjustment (OA) (Patakas et al., 2002). The compounds involved in OA differ widely in plants species and appear to be organic within wheat plants (Hussein et al., 2023). Besides differences in stomatal regulation, anisohydric and isohydric plants differ in the elastic properties of leaf tissues. Cell wall elasticity is considered one of the most important physiological mechanisms of adaptation to water stress (Martínez et al., 2007). At the cellular level, plants with lower Ψ_π ⁰ tended to cope with drought stress through changes in the elasticity of cell walls to maintain turgor (Leuschner et al., 2019). Rigid cell walls help maintaining lower water potential at any given volume compared to elastic ones. This effect can lead to increased water potential gradient between the soil and the plant, thereby promoting more effective water uptake from drying soils and/or accelerating recovery after re-watering (Patakas et al., 2002; Hessini et al., 2008).

The physiological mechanisms underlying stomatal response most likely involve the accumulation of the drought hormone abscisic acid (ABA) (Munemasa et al., 2015; Ali et al., 2020; Aslam et al., 2022). Recent studies on the isohydric specie Arabidopsis thaliana have shown that ABA reduces leaf hydraulic conductance through the downregulation of aquaporin activity (Shatil-Cohen et al., 2011; Coupel-Ledru et al., 2017). These results suggest that ABA promotes stomatal closure either through a local biochemical mechanism on the guard cells, or via a remote hydraulic impact with a decrease in water permeability within the bundle sheath. It has been hypothesized that the hydraulic effect of ABA could underlie the apparent interaction between hydraulics and ABA on the stomatal control of isohydric species and therefore could give rise to the genetic differences between isohydric and anisohydric behaviors. It has also been reported that the molecular mechanism determining the conversion of plants from isohydric behavior to a more tolerant anisohydric one may involve the expression of aquaporins (AQPs) (Sade et al., 2009). AQPs are considered as the main channels for the transport of water, as well as small neutral solutes and CO₂, through the plant cell membrane (He et al., 2023).

A better comprehension of the morphoanatomical and physiological basis of drought stress in wheat crops could facilitate the selection or creation of new germplasm resources to increase productivity in drought- prone areas. This study presents the findings of a comparative study on drought responses strategies of six wheat genotypes and highlighted the features that can be used for the selection of genotypes showing a water stress resistance. The objectives were: (i) to analyze water relation parameters obtained from pressure-volume (P-V) curves of plants subjected to optimal watering and drought conditions; (ii) to identify the compounds involved in leaf osmotic adjustment; (iii) to characterize the physiological processes and response strategies of the six wheat genotypes under drought stress conditions; and iv) to evaluate the expression level of key genes involved in drought stress response.

Preliminary screening focused on the measurement of dry matter (DM) and grain yield (GY) of 28 Tunisian wheat genotypes grown under control and drought stress conditions revealed that the tested varieties showed very contrasting phenotypes ( Table 1 ). Starting from these data we selected a total of 6 genotypes (4 durum wheat and 2 soft wheat) for further analyzes of drought stress resistance.

Drought stress poses a serious threat to crop productivity. Reduced soil water availability strongly affects several aspects of plant physiology leading plants to adjust their water balance. Water related parameters are among the first physiological traits that are affected by osmotic stress during the vegetative phase in Damask rose (Al-Yasi et al., 2020) and Spartina alterniflora (Hessini et al., 2009). Our findings indicated that the selected wheat genotypes responded to drought stress by activating an adaptive mechanism that involved the maintenance of cell turgor through osmotic adjustment (OA) and cellular elasticity (Ɛmax) ( Table 2 ). At the end of the drought treatment, stressed plants of the tolerant genotypes D117 and Syndiouk exhibited a significant higher apoplasmic water content (AWC) compared to control plants. This adaptation could contribute to the accumulation of solutes and thereby could help maintaining turgor in stressed leaves (Hessini et al., 2008). Accordingly, D117 and Syndiouk displayed a decrease in leaf osmotic potential at full turgor (Ψπ¹⁰⁰) and at turgor loss point (Ψπ⁰), while leaf relative water content (RWC₀) did not change significantly.

Based on the findings of Martínez et al. (2007), we suggest that the increase in apoplastic water reserves and the decrease in Ψπ were sufficient to prevent significant water loss. In addition to the decrease of the Ψπ, drought stress induced a reduction in cell walls elasticity. Both increase and decrease in the bulk modulus of elasticity (Ɛmax) have been described as adaptive responses to drought stress (Leuschner et al., 2019). The increase in Ɛmax results in stiffer cell walls (Juenger and Verslues, 2023). In almost all genotypes ( Table 2 ), higher Ɛmax facilitated water extraction creating a greater gradient in water potential from the soil to the leaves (Hessini et al., 2008). This gradient promoted a more efficient water uptake during period of high transpirational demand ( Figure 2 ). Our findings agree with previous study on plants subjected to drought stress (Patakas et al., 2002; Martínez et al., 2007; Hessini et al., 2008). Syndiouk, D117, Td7, and Utique genotypes exhibited a significant increase in Ɛmax, with the highest increase observed for Syndiouk. On the other hand, AG3 and BT did not show statistically significant changes in cell wall extensibility. These results indicate that the alterations in the mechanical properties of cell walls in AG3 and BT were mainly influenced by their distinct behavior, which differed significantly from the other genotypes. An increase in hemi-cellulose content of the cell membrane has been reported in wheat plants exposed to drought stress (Wakabayashi et al., 1997). In addition, genotypes exhibiting active osmotic adjustments and accumulating high levels of solutes after drought stress maintained cell integrity through inelastic cell walls (Patakas et al., 2002).

Our results showed that plants ability to adjust the cell osmolarity and membrane elasticity under drought stress was related to higher cell membrane stability. The significant increase in Ɛmax and the higher osmotic adjustment values recorded for the genotypes D117, Syndiouk, Td7, and Utique ( Table 2 ) were associated with lower levels of malondialdehyde (MDA) ( Figure 5A ) and lower increase in electrolyte leakage ( Figure 5B ). The osmotic changes (ΔΨπ) observed for the selected genotypes after drought stress indicated an active accumulation of solutes in the leaves. Soluble sugars play a key role in osmotic adjustment (Abeed and Dawood, 2020), and drought stress alters carbon assimilation in response to a photosynthetic rate inhibition (Patakas et al., 2002). However, in our study, leaf carbohydrate levels did not decrease in stressed plants. On the contrary, we observed an accumulation of these organic solutes in drought-stressed wheat plant leaves. This accumulation resulted from the inhibition of assimilate production, not strong enough to compensate for its consumption (Quick et al., 1992). It has been shown that carbohydrate and/or sugars deriving from starch degradation accumulated in the leaves of stressed plants and could be transferred from mature to growing leaves, thus contributing to osmotic adjustment (Patakas et al., 2002).

Compared to soluble sugars, the contribution of proline to the osmotic adjustment was not significant (2.9%) ( Table 4 ). In addition, there was no clear correlation between proline accumulation and specific drought response strategy, indicating that it cannot be considered as a reliable selection criterion for drought tolerance in the studied wheat genotypes. Sanchez et al. (2004) reported that, in response to drought stress, proline mainly accumulates in the symplast and therefore, in addition to its contribution to osmotic adjustment it may have a more complex role in conferring osmotic resistance. Furthermore, the accumulation of different ions during drought is of great interest. In our study, potassium (K⁺) and calcium (Ca²⁺) content significantly increased only in Syndiouk stressed plants ( Table 3 ). These ions can be used as economic and efficient osmotic regulators to improve water status and increase drought resistance. Ca²⁺ ions stimulate photosynthesis, enhance nutrient uptake and control water use efficiency (Patakas et al., 2002; Cui et al., 2019; Huihui et al., 2021). Furthermore, calcium is generally believed to enhance cell wall rigidity and its protection against oxidative damage (Patakas et al., 2002). These effects could explain the significant increase in bulk modulus of elasticity (Ɛmax) ( Table 2 ), the reduced levels of malondialdehyde (MDA) and the lower electrolyte leakage (EL) rates observed for Syndiouk under water stress ( Figure 5 ).

Since the content of inorganic ions in the leaves of other wheat varieties did not increase under drought stress ( Table 3 ), it is possible that other solutes, such as organic acids, glycine betaine and asparagine, could be involved in the osmotic adjustment of these two genotypes (Hessini et al., 2008). Our data showed that organic solutes, especially carbohydrates, played a significant role in osmotic adjustment of wheat genotypes ( Table 4 ). These findings are consistent with the results of Bhutto et al. (2023) who reported that under drought stress, the increase in soluble carbohydrate content enabled wheat plants to lower their water potential and to protect cell membranes, soluble proteins and phospholipids.

In our study, the consistent accumulation of soluble sugars under water deficit conditions could be considered one of the key mechanisms adopted by wheat plants to tolerate drought stress. This strategy enhanced the capacity for osmotic adjustment, improved water preservation, regulated stomatal behavior, facilitated CO₂ fixation, and stabilized macromolecules involved in photosynthetic efficiency and stress tolerance (Abeed and Dawood, 2020).

Regarding changes in water relations and osmolyte accumulation, our results indicate that the six selected wheat genotypes employed distinct strategies to cope with stress. The genotypes AG3 and BT, exhibited an isohydric response, as evidenced by their smaller differences in Ψπ¹⁰⁰ and Ψπ⁰ between the control and stressed conditions compared to the other genotypes, and a decrease in transpiration rate (E), suggesting an effective stomatal control to maintain internal water balance (Onyemaobi et al., 2021).

Due to the crucial role of stomata in gas exchange, their closure during drought stress can have a negative impact on CO₂ diffusion, and subsequently on photosynthetic rate (Pitaloka et al., 2022). Under drought stress, the repression of photosynthetic activity observed for the genotypes AG3 and BT was not only due to the reduced stomatal conductance but also to a more pronounced decrease in leaf area ( Figure 4A ) and chlorophyll content ( Figure 4B ). This decline was associated with a decrease in stomatal density on both the lower and upper leaf surfaces ( Figure 4C ), indicating that under such drought stress, photosynthesis was constrained by both stomatal and non-stomatal limitation in AG3 and BT genotypes (Flexas et al., 2006). The inhibition of chloroplast activity (Hasanuzzaman et al., 2023) led to an excessive amount of excitation energy in chloroplasts, which destroyed the equilibrium of electron transfer reactions, leading to the accumulation of highly reactive oxygen species (Liu et al., 2022) and the over-production of malondialdehyde (MDA). In absence of osmoprotectants, the high level of MDA content and the increase in electrolyte leakage observed for AG3 and BT stressed plants led to dysfunction of the cell membranes (Abeed and Dawood, 2020). In drought stress conditions, the preservation of water is the main strategy employed by the genotypes AG3 and BT to minimize water loss. However, this approach resulted in a premature shut down of physiological activity, which could explain the relatively high sensitivity of yield components to drought observed for these two genotypes as compared to other ones.

Anisohydric varieties adopt a different approach. They exhibit higher ΔΨπ⁰ values and accumulate osmotic compounds, which results in a greater adjustment of membrane elasticity and cell osmolarity. For the genotypes D117, Syndiouk, Td7 and Utique, a more pronounced decrease in Ψπ⁰ was observed. This characteristic enabled these plants to absorb water from drying soil even under limited water availability conditions (Leuschner et al., 2019). Consequently, in these genotypes cells could maintain turgor for a longer time before reaching the point of turgor loss (Hachani et al., 2022).

This finding was in line with the increase in cell membrane rigidity observed for these plants ( Table 2 ). The thicker cell walls also play a compensatory role in counteracting the effect of reduced Ψπ (Patakas et al., 2002). The enhanced rigidity of leaf tissue and the ΔΨπ values measured in the selected genotypes allowed the maintenance of open stomata, thereby facilitating dynamic gas exchange. This phenomenon contributed to the preservation of stomatal conductance (gs) and photosynthetic assimilation (A), as illustrated in Figure 3 (Hachani et al., 2022). Among the selected genotypes studied, Syndiouk and Utique exhibited the highest A rate maintenance under drought conditions, while D117 showed the highest gs. This behavior of anisohydric plants highlighted their acclimatization ability during periods of stress, as evidenced by the growth parameters measured (Gallé et al., 2013).

Stomatal functioning can be influenced by hormonal or morphological behavior resulting in higher gs values. To further analyse this aspect, qRT-PCR analysis was carried out to measure the expression levels of the gene AAO involved in the last step of ABA biosynthesis and the gene PIP2:1, encoding for a plasma membrane intrinsic protein (PIP), which is part of the aquaporin family. We showed that the genotypes exhibiting higher gs values (D117 and Utique) were those in which AAO gene for ABA biosynthesis was only slightly induced after drought stress, allowing stomata to remain open for a longer period. The lack of correlation between stomatal conductance (gs) and photosynthesis (A) observed for the genotype D117 could arise from the low induction of gene AAO involved in ABA biosynthesis, which allowed stomata to remain open for a longer period. However, even if an adequate supply of CO₂ was ensured by the increase in stomatal conductance, the efficiency of photosynthesis could still be hampered by other limiting factors (such as slow activation of electron transport, key enzymes such as Rubisco in the Calvin-Benson, and the synthesis of sucrose) (Stitt and Schreiber, 1988; Carmo-Silva and Salvucci, 2013; Yamori, 2016; Kaiser et al., 2016). Consistent with our data, results reported in von Caemmerer et al. (2004) revealed that stomatal conductance was not strictly correlated with the photosynthetic capacity of guard cells or leaf mesophyll.

Moreover, drought stress triggered a strong up-regulation of the gene PIP2:1 in the same genotypes. This confirmed that the overexpression of PIP2:1 could enhance not only water movement across the cell membrane, but also gas exchange, as PIP2:1 is a CO₂-permeable aquaporin (Wang et al., 2016). Our results are consistent with the findings of Sade et al. (2009), who reported that SlTIP2:2 overexpression limited the reduction in transpiration under drought stress, which ensured continuous CO₂ uptake, promoting plant growth and yield production. Based on these findings, we suggest that: i) an elevated ABA content may have led to a transcriptional down-regulation of aquaporins, and ii) the variations in aquaporin transcript levels in response to drought could be linked to a divergent root hydraulic conductivity between isohydric and anisohydric wheat genotypes (Coupel-Ledru et al., 2017).

Furthermore, D117, Syndiouk, Td7, and Utique displayed elevated MDA contents in leaves ( Figure 5A ) and maintained stable yield levels following the stress period ( Table 5 ). The accumulation of MDA under drought stress could indicate an adaptive mechanism. It has been reported that to maintain gs in the absence of ABA hormone repression, MDA might act as a signal for the expression of genes involved in the synthesis of enzymes and antioxidant molecules (Gallé et al., 2013; Onyemaobi et al., 2021). This suggestion is consistent with recent studies demonstrating that leaves are the primary site for ABA biosynthesis (Cardoso et al., 2020). Moreover, D117 and Utique were able to adjust their stomatal densities to optimize the functioning of the photosynthetic machinery. They reduced the stomatal density on the lower leaf surface (SDlower), thereby limiting transpiration, and compensated this reduction by increasing the number of stomata on the upper leaf surface (SDupper) ( Figure 4C ).

Our study showed that, among the yield components, Grain yield (GY) was the most adversely affected by the drought treatment ( Table 5 ). The reduction was particularly pronounced in the genotypes AG3 and BT, which showed a decrease of 69% and 61%, respectively.

In contrast, Syndiouk was the least affected by water stress, as its GY, seed number, and the weight of 1000 seed were unchanged. On the other hand, the AG3 and BT genotypes produced significantly fewer and lighter seeds following drought treatment, as indicated by the decrease in 1000 seed weight, suggesting a decline in seed quality ( Table 5 ).

The analysis of drought tolerance indices allows the classification of wheat varieties based on their productivity under normal and stressful conditions. The stress susceptibility index (SSI) and stress tolerance (TOL) are used to evaluate drought-tolerant genotypes able to achieve high yields under both normal and stress conditions (Mardeh et al., 2006). High values of these indices mean high susceptibility to drought stress (Fischer and Maurer, 1978; Rosielle and Hamblin, 1981). The genotypes AG3 and BT exhibited the highest TOL and SSI values. In addition, the high mean productivity (MP) values indicated a high productivity even in extreme environments. In the principal components analysis ( Figure 6 ), the Utique and Td7 genotypes were close to Yp, Ys, and MP, demonstrating their high performance in terms of grain yield production under both control and drought conditions. The drought resistance index (DI) and yield stability index (YSI) are suitable parameters for selecting the most drought-tolerant genotypes (Khare et al., 2022). The D117 and Syndouk genotypes displayed the highest DI and YSI values, and the lowest TOL and SSI indices, suggesting a stable yield production due to their higher drought resistance. Based our results, the genotypes Syndiouk, D117, Td7 and Utique genotypes were classified as drought-tolerant, while AG3 and BT were categorized as sensitive.

The findings of the present study highlighted the relationship between drought tolerance and specific physio-molecular adaptations in the selected wheat genotypes. These adaptations included the adjustment of osmotic potential, the enhancement of cell wall rigidity and the maintenance of photosynthetic activity through the regulation of stomatal behavior. We suggest that measures such as ΔΨπ, Ɛmax and gs could be used as primary selection criteria for the robust screening of numerous genotypes in wheat breeding programs. Further exploration of the molecular mechanisms underlying the proposed differentiation between isohydric and anishydric genotypes showed that the overexpression of the PIP2:1 gene and the absence of induction of AAO gene expression under water-stressed conditions represent potential indicators for preliminary selection, even at a very early stage, of drought-tolerant genotypes.

Based on the overall responses of the six studied genotypes, Syndiouk, D117, Utique, and Td7 could be promising for future wheat breeding programs aimed at developing high-yielding and drought-tolerant varieties.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

AG, CB, HA, and FG designed the project and the strategy. HA, MA, contributed to plant sample collection and processing. AG, CB, FG, HA wrote and revised the manuscript. NR and MF-S were involved in the revision of the manuscript. NR and MF-S performed the molecular analysis. EB and RD helped with a critical discussion on the work. All authors contributed to the article and approved the submitted version