Over the last decades, climate change substantially intensifies drought incidences as a result of warming and the increased evaporation. Drought is undoubtedly one of the most important environmental stressors that severely affects a wide range of major food crops, leading to considerable yield losses, especially in the arid and semi-arid regions. Considering these adverse effects of drought to both plant growth and productivity, breeding for drought tolerant varieties is generally considered as an effective and sustainable means of ensuring economic viability of crops under water deficit conditions. The mitigation of yield losses under drought conditions primarily relies on improving traits that enhance drought tolerance, such as water consumption and water use efficiency (WUE) (Hatfield and Dold, 2019). WUE refers to plant’s ability to cope with moderate or severe soil water deficit and is considered as an important indicator of plant survival under limited water availability (Schulz et al., 2021). The response of plants to water deficit conditions includes a series of morphological, physiological, and biochemical modifications, including changes in photosynthesis, respiration, transpiration processes as well as the accumulation of reactive oxygen species (ROS) (Chevilly et al., 2021; Giordano et al., 2021; Nalina et al., 2021; Zhou et al., 2021). The molecular responses to drought include the activation of antioxidant enzymes and osmolyte accumulation which serve towards the stabilization of membranes, enzymes and proteins, as well as to the protection against oxidative damage through scavenging of ROS (Hasanuzzaman et al., 2020; Begum et al., 2022). Regarding the plant organs level, leaf morphology and functions may promote water use efficiency, thus contributing to mitigation of stress effects, while root characteristics such as length, weight, volume, and density, play a pivotal role in drought tolerance (Kapoor et al., 2020).

In the search to identify eligible strategies to improve drought tolerance of crops, several studies highlight the complexity of plant responses to drought, involving numerous factors such as the growth stage, the duration and severity of stress (Golldack et al., 2014), as well as the polygenic nature of quantitative tolerance traits (Mahmood et al., 2020). Considering that most plant species are drought-sensitive at all stages of their lifecycle, seed germination is considered as the most critical stage since water deficit impairs germination by limiting water imbibition and further reduces seedling vigor, via repressing root emergence and shoot elongation, thus preventing the establishment of a uniform stand (Queiroz et al., 2019; Rosero et al., 2020). As such, the most common plant response to reduced osmotic potential refers to delayed and reduced germination rates(Saberali and Shirmohamadi-Aliakbarkhani, 2020). Such adverse effects have been reported for a plethora of crop species, including maize (Queiroz et al., 2019), sorghum (Cokkizgin et al., 2019), faba bean (Li et al., 2018), cucumber(Mombeini et al., 2021), and melon (Saroj and Choudhary, 2020). Therefore, it is suggested that seed germination and seedling growth potential under stress conditions may enable the early selection of tolerant genotypes, thus enhancing the efficiency of selection procedures for suitable germplasm material to be cultivated or exploited in relative breeding programs (Tarchoun et al., 2022).

In the context of improving complex traits, such as drought tolerance, the research interest is progressively focused on the search of functional markers to be employed in breeding procedures aimed at early selecting for tolerant genotypes. Well known examples of stress indicators are certain amino acids of the group of quaternary amines with osmoprotective functions under water stress conditions, such as proline and glycine betaine(Bankaji et al., 2019). In the same line, plant protection against oxidative damage involves the induction of the activity of antioxidant enzymes(Gill and Tuteja, 2010). Carotenoids serve in multiple functions in plants, including photosynthesis and regulation of redox status(Gill and Tuteja, 2010), while phenolic compounds contribute to plant defense mechanisms against abiotic stress factors (Cheynier et al., 2013).

Given that photosynthesis is one of the most critical metabolic processes, its repression under drought stress adversely affects plant growth and development (Al Hassan et al., 2015). Upon severe drought stress, both in terms of duration and intensity, inhibition of photosynthesis is manifested by changes in chlorophyll content, mainly due to ROS-induced damage of chloroplasts, as well as damages in the whole photosynthetic apparatus (Porcar-Castell et al., 2014). Accordingly, ROS production is mainly driven by excess energy absorption in the photosynthetic apparatus, that might be reversed by degrading the absorbing pigments. Therefore, chlorophyll fluorescence is considered a fast, accurate and non-invasive tool to monitor photosystem II (PSII) (Baker, 2008), while photosynthetic activity can also be assessed since chlorophyll fluorescence can describe and investigate the photosynthetic light processes and quantum conversion by chlorophyll and accessory pigments of chlorophyll-protein complexes (Porcar-Castell et al., 2014).

Among the cultivated Cucurbita species, e.g. C. argyrosperma,C. ficifolia, C. maxima, C. moschata and C. pepo, the latter three are the most important in terms of worldwide production. In Tunisia, the squash (C. maximaDuch.) germplasm used for commercial cultivation is essentially derived from local landraces, produced by open pollination or farmer mass selection. Given the importance of local squash production, an increasing number of studies focuses on collection, ex-situ and in-situ conservation, characterization and maintenanceof local germplasm accessions (Hamdi et al., 2017; Hamdi et al., 2019; Tarchoun et al., 2022). Local landraces are often characterized by particularly broad leaves, thus leading to high evapotranspiration and high crop water requirements, since cultivation usually takes place during the warmer months of the year (Hamdi et al., 2017).

In this context, breeding efforts are focused on the selection of drought tolerant landraces so as to ensure economic viability of squash cropping in arid and semi-arid regions, such as Tunisia. Although several studies aimed at understanding the drought stress response in Cucurbita species, mainly C. pepo and C. moschata, so far (Biareh et al., 2022), there is a gap in relevant research for C. maxima. Therefore, this study aimed at (i) investigating the response of four squash local landraces of Tunisia to drought stress at germination stage, (ii) examining the possibilities of early selecting drought tolerant genotypes and (iii) determining the effects of drought stress at later plant growth stages. For this purpose, drought stress was imposed by different water potential (0, -0.24, -0.47 and -0.73 MPa). During germination, the evaluation of drought tolerance was based on traits related to germination and seedling growth (germination percentage, seedling vigor index, root and shoot length, root and shoot fresh weight, root volume and seed water absorbance). At later growth stages, the response of plants to stress was assessed using physiological (chlorophyll fluorescence, chlorophylls and carotenoid content, evapotranspiration, photosynthesis activity) and biochemical parameters (malondialdehyde (MDA), free proline, total phenolic compounds and total flavonoid content) and DPPH radical scavenging activity of roots and leaves.

The results of the present study underline the drastic effects of drought stress depending on the landrace, the stress level as well as their interaction. During germination, all traits related to both germination ability and rate (GP, WU, SVI), as well as seedling growth (RL, SL, RFW, SFW, RV) were negatively affected in all the studied landraces, with the effects being proportional to the stress level applied, thus providing evidence for their relative sensitivity or tolerance to drought stress. Such findings are further supportive of previous reports related to the severe effects of drought stress during germination in a plethora of crop species, including maize (Queiroz et al., 2019), sorghum (Cokkizgin et al., 2019), faba bean (Mombeini et al., 2021), cucumber (Rahmani et al., 2019), as well as pumpkin (Cucurbita pepo L.; Biareh et al., 2022). The drought stress effects during germination are widely attributed to the fact that this stage is considered as the most sensitive throughout the plant life cycle since water uptake, referred to as imbibition, is a prerequisite to initiate germination. Accordingly, the amount of the absorbed water depends both on the level of the initial seed moisture content, as well as on its chemical composition (McDonald et al., 1988). In fact, protein and pectin are hydrophilic colloids, thus requiring more water than starch to promote imbibition and germination processes (Queiroz et al., 2019). In addition to germination potential, our findings further pointed to the drastic effects of drought stress on seedling growth, with the increasing concentration of D-mannitol leading to gradually decreased elongation in both roots and shoots. Such seedling growth inhibitory effects have been also reported in a wide range of plant species and are mainly attributed to osmotic stress effects (Queiroz et al., 2019). In our study, the findings which suggest that roots were more drastically affected than shoots are in agreement with previous reports which also suggest that the aboveground parts are more affected than the aerial parts in several species when the plant is subjected to drought stress, broad been (Cokkizgin et al., 2019) and tomato plants (Al Hassan et al., 2015), but also in squash germplasm under salt stress conditions (Tarchoun et al., 2022). Despite the fact that drought stress posed a limiting factor in germination and growth potential of squash landraces, it is worth noting that the tested landraces showed a differential response to varying stress levels. Among landraces, “751” and”746”exhibited a higher germination potential at all stress levels, thus indicating their superiority in terms of drought tolerance. Given that these particular landraces were capable of germination and seedling growth even under severe stress conditions (-0.47 and - 0.73 MPa), it could be assumed that they consist a promising genetic material to be further exploited for breeding purposes.

Furthermore, our data underlined the significant effect of drought stress in all the studied physiological and biochemical traits, since chlorophyll b, F_m, PA and free proline content were the most variable traits, thus providing evidence for their suitability as screening criteria for drought tolerance among the tested landraces. Chlorophyll fluorescence analysis, which is routinely employed to assess the status of plant photosynthesis, revealed significant variations not only for the landrace and the drought stress level but also for their interactions. Changes in the minimum fluorescence yield (F_O) values suggest differential response to the transfer of excitation energy between pigments molecules (Liang et al., 2020). In our study, maximum fluorescence (F_m), whose values are maximum in healthy non-stressed leaves that have been dark-adapted (Huang et al., 2019), showed a decreasing trend as D-mannitol concentration increased. Furthermore, the F_v/F_m ratio, which refers to the maximum quantum yield of PSII photosynthetic apparatus and is commonly used as indicator of stress sensitivity (Shin et al., 2021), presented the lowest value at - 0.47 MPa (0.78), thus reflecting the existence of stress which lead to inactivation damage of PSII, referred to as photoinhibition. However, the F/Fm ratio showed higher values at - 0.73 MPa (0.83) which are indicative of unstressed leaves. Similar drought stress effects in photosynthesis activity were also reported in other plant species, including lettuce (Inoue et al., 2021), tomato and pepper (Parkash and Singh, 2020). According to previous studies, such disturbances due to abiotic stress, including water deficit, are attributed to changes in the activity of pigments in the course of photochemical and enzymatic reactions in thylakoids and the chloroplast, resulting in low amount of energy absorbed by PSII, as well as in changes in the membrane permeability of chloroplasts (Wang et al., 2018). Apart from the obvious stress level effects, our data pointed to differences in chlorophyll fluorescence attributed to the effect of landrace. Interestingly, landraces “747” and “746” showed a notable decrease at - 0.47 MPa, which is indicative of a stress state as a result of the initial plant damage closely related to PSII, whereas “751” and “748”exhibited an increased F_v/F_m ratio at both - 0.47 and - 0.73 MPa.

Moreover, our results suggested the drastic effect of drought stress on the content of chlorophylls a, b and carotenoids as well as on real evapotranspiration (RET) and photosynthetic activity (PA), while it was evidenced that the stress effects were analogous to its intensity. Our findings also confirm that the decreased content of chlorophylls and carotenoids, coupled with the decreased values for RET and PA, reflect the first defense reaction of leaves, involving stomatal closure as a means to reduce transpiration and photosynthesis as well as to limit CO₂ exchange, as previously reported in other vegetable crops (Parkash and Singh, 2020). Although the observed reduction in RET and PA values under D-mannitol stress conditions is in accordance with previous reports in other plant species, such as s, cucumber (Li et al., 2008) and watermelon (Mo et al., 2016), it is worth noting that landrace “746”, despite of its high RET values, also exhibited the highest PA values (1790.84 µmol photons m^-² s^-1) at - 0.73 MPa, thus reflecting an ability to maintain its photosynthetic activity even under severe drought stress. In contrast, landrace “751” was characterized by the lowest values for both RET and PA, probably as a result of adjustments in stomatal closure. Such regulation of stomatal control under drought stress conditions consists the main physiological factor in the context of optimizing water use and preventing excessive water loss, as it has already been proven in soybean (Liu et al., 2005). In the same line, it has been evidenced that wheat’s response to drought stress involved a decreased rate of photosynthesis and transpiration as well as decreased stomatal conductance (decrease by 78.4%, 85.4% and 92%, respectively), the latter being more correlated with the transpiration rate than the photosynthetic rate (Farooq et al., 2014).

It is well evidenced that plants’ response to abiotic stress involves the enhanced accumulation of osmolytes, such as free proline, MDA, total phenols and flavonoids, as well as increased radical-scavenging activity to mitigate oxidative stress (Kapoor et al., 2020; Parkash and Singh, 2020; Yadav et al., 2021). Among compounds with osmoprotective function, free proline is one of the most well studied indicators for drought tolerance as it plays a pivotal role in regulating osmotic pressure in stressed plants (Yadav et al., 2021). Similarly, MDA generated by the peroxidation of polyunsaturated fatty acids of the cell membranes as a response to ROS generation (Zhou et al., 2017), is routinely employed as an indicator to evaluate the degree of plasma membrane damage and the ability of plants to cope with drought stress. In relation to both free proline and MDA, our findings indicate a differential response of roots and shoots, with the former showing an increased content thus suggesting more intense stress conditions. Such observations are in agreement with the fact that under drought stress conditions roots play a fundamental role both in the perception of water deficit status and hormone signal transduction, mediated by abscisic acid (ABA), the latter triggering signals to aerial organs and tissues through vascular bundles, causing morphological/anatomical alterations, such as the root to shoot ratio (Takahashi et al., 2020). As the first organ to drought perception, roots respond with a series of morphological changes in the growth and architecture, including their length, weight and volume, while, at the same time, constituting the primary site of osmolyte accumulation (Fang and Xiong, 2015). Such drought adaption mechanisms in roots regulate water loss and promote water use efficiency, which are considered as key functional traits that contribute to drought tolerance (Takahashi et al., 2020). In accordance with the drought responses in typical roots, our findings suggest a more pronounced increase in total phenolic compounds and total flavonoids content as well as in DPPH activity in roots than in shoots, especially at high stress levels, which is indicative of the essential role of these parameters in mitigating the water deficit effects.

Conclusively, our findings contribute towards understanding the response of squash landraces to drought stress and further provide evidence regarding the tolerance ability of the landraces under study. Among landraces, “751” and”746”were classified as better performing in terms of drought tolerance both at germination and later plant growth stages. Considering the small number of landraces under study, it is concluded that squash germplasm exhibits considerable variation for drought tolerance traits, thus providing ground both for selecting appropriate landraces for cultivation under water deficit conditions and applying breeding approaches targeted at improving crop’s drought tolerance. However, further studies are needed with a broader group of germplasm accessions of squash germplasm that will help to valorize the existing variability through the selection of elite genotypes in targeted breeding programs.

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

WA: methodology; formal analysis; investigation; data curation; NT: conceptualization; writing-original draft preparation; writing-review and editing; visualization; supervision; project administration; IM and RA: methodology; formal analysis; investigation; data curation; OP: writing-original draft preparation; HF: methodology; formal analysis; investigation; data curation; CA: methodology; formal analysis; investigation; data curation; RK: writing-review and editing; visualization; supervision; project administration; SAP: conceptualization; writing-review and editing; visualization; supervision; project administration; funding acquisition. All authors contributed to the article and approved the submitted version.