Melon (Cucumis melo L.) is a major crop with great agronomic and economic interest, considered a gourmet food in several markets and cultures. One of the main problems for melon farming is that its cultivation demands a lot of water (Cabello et al., 2009). In the current context of anthropogenic global warming and the subsequent climate change, aridity is increasing in traditional cultivation areas, and thus, melon culture is subjected to increasing abiotic stress, which compromises the yield. Specifically, drought stress is increasing, and salt stress is directly related to this water scarcity, given that excessive irrigation increases the salt deposition in the soil and diminishes the phreatic level, thus enabling the infiltration of sea water. It is estimated that 20% of all arable land and almost half of the land with water availability are affected by salts, significantly reducing yield below the genetic potential of most crops (Botella et al., 2007; Chandna et al., 2014). As a result of salinization, crop yields are declining while arable land is being irreversibly lost (Nawaz et al., 2010). High salinity levels also increase soil pH. In addition, saline stress leads to deterioration of soil structure and prevents the air-water balance, essential for biological processes occurring in the roots (Galvan-Ampudia et al., 2013). Saline soils reduce the biomass production of crops affecting important biochemical and physiological processes in the plant (Serrano et al., 1999).

We have generated considerable knowledge at the biochemical level and physiological level regarding how abiotic stress affects basic physiological processes, the cellular function and even the biochemical targets, but there are still large gaps in our knowledge about the limiting factors for stress responses. More specifically, we are lacking knowledge regarding which traits could be improved by breeding or genetic engineering that would have a major impact on plant growth and development under stress conditions. This explains the low success in breeding novel crops that are adapted to saline soils or are able to maintain yield under drought stress conditions (Ashraf et al., 2009). Proof of this scarcity of results is that there are only two GMO cultivars on the market whose trait is drought tolerance: the Droughtgard maize from BASF and the HB4 soy from Agroceres (Wang et al., 2015; Ribichich et al., 2020). To date, there are no marketed biotechnological crops with enhanced yield under saline conditions.

Several strategies have been developed to identify the limiting factors for stress tolerance. Evaluating the physiological and biochemical response of stress tolerant and stress sensitive plants is a well-established strategy to discover differential traits for abiotic stress tolerance (Taibi et al., 2017, 2018; Chevilly et al., 2021). All these analyses have been performed testing different cultivars from the same variety and a single stress. We have further developed this concept by evaluating, in the same analysis, different stresses and different cultivars of two different varieties to find limiting factors which are not particular to a specific variety or stress. In this report, we have applied this strategy to a pivotal horticultural crop for the economy in the Mediterranean area. There are several reports evaluating Galia melon performance under salt stress in field conditions (Akrami and Arzani, 2018; Akrami et al., 2019), but so far, there are no studies evaluating the plant response at the initial stages of development under controlled conditions. This work has been designed to determine the differences at the physiological and biochemical levels between different melon genotypes under two different abiotic stresses. These varieties had previously been characterized as sensitive or tolerant to abiotic stress. We have subjected these varieties to controlled drought or salinity stress, and have monitored different physiological or biochemical parameters, in order to find changes that are relevant among varieties or treatments. This will allow us to identify the limiting factors in abiotic stress tolerance and will help to define novel breeding strategies.

The main objective of this study is to compare physiological and biochemical responses of two cultivars of two different varieties, to both salt and drought stress, in order to find common patterns among different varieties. We included stress tolerant and sensitive cultivars as well to gain further insight into differential responses within varieties. Through the relativization of data (i.e., the ratio of the value under stress with respect to the value under control conditions) we have found that, irrespectively of the variety, tolerance to salt stress correlates with higher ratios (stress/control) for His (3.4 and 1.42 for tolerant vs. 0.92 and 1.18 for sensitive) and Na⁺/K⁺ (11, 0.37, and 91.3 for tolerant vs. 31.4 and 47.6 for sensitive) and lower Phe (0.93 and 1.06 for tolerant vs. 1.45 and 2.0 for sensitive) and Pro ratios (0.71 and 0.75 for tolerant vs. 1.41 and 0.83 for sensitive). In the case of drought stress, tolerance correlates with increased ratios of Ile (1.87 and 1.1 for tolerant vs. 0.78 and 0.5 for sensitive), Gly (5.68 and 1.12 for tolerant vs. 0.85 and 0.5 for sensitive), Ser (2.72 and 1.17 for tolerant vs. 0.75 and 0.46 for sensitive) and Asn (2.12 and 1.38 for tolerant vs. 0.79 and 0.32 for sensitive), and decrease ratios of Hydric potential (1.63 and 1.33 for tolerant vs. 2.67 and 1.84 for sensitive) and Phe (1.26 and 1.03 for tolerant vs. 2.0 and 1.69 for sensitive) and Trp (1.08 and 1.79 for tolerant vs. 2.0 and 7.61 for sensitive) (Figure 7A, Supplementary Table 1). All the results are summarized in the form of a heat map in Figure 7B. The numerical data of the ratios of the Stress/control for all values are presented in Supplementary Table 1.

One interesting aspect of our results is that, among varieties, physiological parameters are not a distinctive trait for abiotic stress tolerance. Several previous studies have determined the effect of stress on melon physiology (Zhang et al., 2021). In a recent study on muskmelon genotypes under drought stress, the net photosynthetic rate, stomatal conductance (Gs), and the transpiration (E) rate decreased (Ansari et al., 2019). Other reports described similar decreases in stomatal conductance in genotypes different from the ones that we have used in this study (Kusvuran, 2012; Wang et al., 2016). There are reports indicating that during drought stress in melon, there was a significant increase in water use efficiency in drought tolerant genotypes (Akhoundnejad and Dasgan, 2019). In our case, under drought conditions, we did not observe any significant differences with the control values, that is, efficiency is maintained, although in this report plants were grown in field conditions and measures were taken in older plants.

We have also calculated the differential traits between Galia and Piel de Sapo irrespectively of their stress tolerance (Supplementary Figure 1). In this case, the Piel de Sapo variety showed higher Stress/control ratio for E, A, WUEintr and WUEinst under salt stress (Supplementary Figure 1). In agreement with our results, it has been previously described that under salt stress in muskmelon there is a significant increase in WUEintr and WUEinst with respect to control conditions (Ansari et al., 2018). Our data under saline stress conditions showed no changes for Galia plants, but we confirmed the increase in our conditions for Piel de Sapo cultivars. The fact that most of the differences observed in the physiological traits are variety dependent and not stress dependent may be explained by the differences in the leaf morphology.

There is no description available in the literature regarding the behavior of the free amino acid pools under salt and drought stress in Cucumis melo comparing stress and different varieties, so here we have investigated the complete free amino acid profile in our plants under the studied conditions. Salt tolerance correlated with higher levels of His in tolerant plants. His has been related to tolerance against heavy metals as it can chelate them, but its role in salt stress tolerance has not been described. Proline it is known to act as and osmolyte, but we have found that tolerant plants have less stress/control ratio than the sensitive ones, pointing out that the increases observed upon salt stress are not determinant for salt stress tolerance. In the case of drought stress, Ile, Gly, Ser and Asn were higher in tolerant varieties. Glycine can act as an osmolyte and also is a component of the tripeptide glutathione. Serine is also a precursor of cysteine and other stress-related molecules. On the other hand, high stress/control ratios of Phe correlated with sensitivity to stress, and it is the only molecule that decreased under conditions of both drought and salt stress. Phe is a precursor of several molecules, among them lignin, a pivotal molecule for cell wall biosynthesis. The accumulation of Phe in sensitive cultivars, irrespectively of the varieties, may be a symptom that basic plant processes like cell wall biosynthesis are more affected by stress than in tolerant cultivars.

Potassium is the major ion in the cytoplasm and thus is largely responsible for the intracellular ionic environment. Sodium is toxic for melon plants and must be extruded from the cell, or accumulated in the vacuole. Regarding ion accumulation, our results suggest that the limiting factor for stress response is the ability to accumulate sodium (Serrano et al., 1999; Rodríguez-Navarro, 2000). Under salt stress, plants can extrude sodium from the root, or take it up, transport it to the aerial part and accumulate it in the vacuoles (Arzani and Ashraf, 2016). The higher Na⁺ and Na⁺/K⁺ ratio of a salt-tolerant variety in our study may be explained by the vacuolar accumulation of sodium in the leaf tissues. Similar results regarding the Na⁺/K⁺ ratio and water use efficiency were observed in a field trial with Cucumis melo cv. Huanghemi (Tedeschi et al., 2017) so the trend is the same, even when we compare field/greenhouse conditions and early development/late develompment. Here we demonstrate that the ability to accumulate sodium and maintain a high Na⁺/K⁺ ratio is a distinctive trait for tolerant cultivars. We did not find any distinctive pattern with the potassium levels under drought stress. Therefore, the role of potassium as an osmolyte is not a limiting factor for drought tolerance in these melon cultivars.

Melon is a climacteric fruit, so its hormonal levels are subjected to drastic changes (Dunlap et al., 1996). There are several descriptions in the literature of the hormonal levels in cucurbit plants under abiotic stress. For instance, exogenous application of SA increases drought tolerance in muskmelon (Korkmaz et al., 2007), similar to what is observed in other cultivated plants (Souana et al., 2020). In addition, SA and JA levels increase upon spermidine addition and increase tolerance to salt stress (Radhakrishnan and Lee, 2013). Also, JA levels tend to increase in cucumber plants subjected to drought stress (Llanes et al., 2016). It has also been described that under mild or moderate water stress, IAA concentrations tend to increase (Huang et al., 2018). ABA is the main player in the abiotic stress response in plants, and it has also been described to increase upon abiotic stress in melon (Sun et al., 2013). When we studied the phytohormone levels, we did not find any common pattern among the sensitive or tolerant varieties and cultivars studied, although the concentrations of SA and IAA were higher in Piel de Sapo cultivars (between 3- and 7-fold for SA and 4- to 17-fold for IAA).

Taken together, we have performed a complete study of two different melon varieties comparing sensitive and tolerant cultivars and applied statistical tools to the results to find common patterns in salt or drought stress responses that could be useful to predict the behavior of uncharacterized varieties and cultivars and to design novel classical or biotechnological breeding strategies. Varieties or cultivars with increased His content and/or the ability to accumulate sodium (likely in the vacuoles) may display improved tolerance to salt stress, while novel varieties with enhanced levels of Ile, Gly, Ser, and Asn could show better performance under drought stress conditions. High levels of Phe seem correlate with diminished tolerance to abiotic stress. Thus, our results have provided a useful framework for future studies which will examine the ability of these parameters to predict stress tolerance performance in additional melon varieties and cultivars.

The raw data supporting the conclusions of this article will be made available by the authors, witho undue reservation.

SC, LD-E, and GM-S cultivated the plants, obtained the samples, and performed the experiments. LM and AV did all the physiological determinations and the calculations. JL-N did the amino acid measurements. LY and JM analyzed the results and designed the figures. JB did the statistical analysis and analyzed the results. GM-S, LY, and JM wrote the manuscript. JM designed the study. All authors contributed to the article and approved the submitted version.

SC was a recipient of grant FPU19/01977 from the Spanish Ministerio de Universidades. LM and AV activities were funded by the Prometeu program (IMAGINA project, PROMETEU/2019/110). LM was also supported by the Spanish MICINN (PTA2019-018094). The CEAM foundation was funded by the Generalitat Valenciana.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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