1. Introduction
Global food production should increase by 50% by 2050 to feed a population that will rise to 10 × 10
9 people [
1]. However, soil salinity affects over 109 × 10
6 ha of cropland worldwide, reducing yields in more than 50% of the surface of the most productive agricultural areas—those cultivated under irrigation in arid and semi-arid regions [
2,
3,
4,
5,
6]. The source of high salt concentration is primarily the presence of salt in irrigation water and the accumulation of Na
+ and Cl
− in soils [
2].
The effects of soil salinity on plants vary widely depending on multiple factors, but salt tolerance is mainly controlled by genotype [
7,
8]. An operational threshold has been established to classify species into salt-sensitive (glycophytes) and salt-tolerant (halophytes), as well as those able to complete their life cycle under soil salinities equivalent to more than 200 mM NaCl [
9,
10,
11]. Different species have developed protective mechanisms to cope with salt stress, from the physiological and biochemical to the molecular level, involving complex networks of genes, proteins and metabolites [
12].
Salinity induces detrimental effects in glycophytes, such as (i) reduced water availability, (ii) ion toxicity, (iii) oxidative stress and (iv) K
+ deficiency [
13,
14], which lead to a reduction in plant growth and biomass accumulation, and eventually, plant death [
15,
16].
Salt stress alters the normal metabolic processes of the plant: photosynthesis and energy production, lipid metabolism, nutrient acquisition, the integrity of cellular membranes and the activity of enzymes [
17]. Plant growth under such stressful conditions depends on the efficiency of the mechanisms of stress responses in each species. In this regard, tolerance mechanisms can be divided into two major groups: defence against osmotic stress and ion toxicity, which includes the control of ion transport and osmolyte biosynthesis and defence against oxidative stress, which includes the activation of antioxidant mechanisms.
To maintain intracellular osmotic balance, some plants accumulate toxic ions such as Na
+ and Cl
− in the vacuoles [
18,
19]. An excessive accumulation of Na
+ is generally accompanied by K
+ deficiency by competition between the two cations because of their similar physicochemical properties. Thus, maintaining Na
+/K
+ homeostasis is crucial to develop normal metabolic processes in the cytoplasm, such as enzymatic reactions and protein synthesis [
14]. In addition, different metabolites are involved in the responses to osmotic stress as osmolytes and osmoprotectants, including sugars, polyalcohols, amino acids, ammonium compounds, betaines and sulphonium compounds. Sugars are direct products of photosynthesis that play essential functions in the cell; their increase could be a response to stress or a signal for activating other cellular processes. During abiotic stress, their primary role is stress mitigation by osmoprotection, carbon storage and the scavenging of Reactive Oxygen Species (ROS). Amino acids also have some regulatory and signalling functions. In stress tolerance mechanisms, a significant role is played by the flagship compatible solute proline (Pro) [
18,
20,
21,
22].
Toxic ions absorbed by roots move into photosynthetic organs, causing harmful nutritional imbalances and the generation of oxidative stress by an increase in the production of ROS. Through the oxidation of fatty acids, amino acid residues in proteins and the DNA bases, ROS accumulation leads to membranes degradation, protein inactivation and DNA mutations, causing cellular damage, and eventually, cell death [
23,
24,
25]. To cope with oxidative stress, plant cells activate antioxidant systems, including the activation of redox regulatory enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) (and other peroxidases) or glutathione reductase (GR), and the synthesis of antioxidant metabolites, such as phenolic compounds [
26].
Eggplant (
Solanum melongena L.) is one of the most economically important crops worldwide, reaching 1.86 × 10
6 cultivated hectares and an annual production of over 54 × 10
6 tonnes [
1]. Eggplant is moderately sensitive to salinity [
27], which partially reduces growth and yield.
Solanum melongena can be crossed with many wild relatives from the primary, secondary and tertiary gene pools, adapted to a wide range of environments [
28]. Therefore, it should be possible to identify new sources of genetic variation in wild relatives adapted to saline areas. The responses of
Solanum insanum L. to salt stress have recently been studied, and the wild genotype seems more tolerant than the cultivated eggplant, as it can stabilise its growth and photosynthetic rate under saline conditions [
29]. In
S. insanum, toxic Na
+ and Cl
− ions are transported to the leaves at high external salinity, where they most likely accumulate in vacuoles, according to the ‘ion compartmentalisation hypothesis’ [
30,
31], whereas K
+ concentrations are maintained. In the presence of high salt concentrations, compared with cultivated eggplant, the wild species also shows a higher accumulation of proline, an excellent marker of a stress tolerance phenotype [
27].
Little is known about the inheritance of quantitative or complex traits such as stress tolerance. However, introgression can be used to introduce favourable characteristics in cultivated
S. melongena [
32,
33,
34] for future breeding prospects.
This study compares the response of cultivated eggplant (S. melongena L.), a wild relative (S. insanum L.) and their interspecific hybrid, under salt stress conditions in short- and long-term treatments by determining growth parameters and the levels of several biochemical stress markers—such as ions, osmolytes or antioxidant compounds—and the activity of antioxidant enzymes. This work represents the first step in assessing the possibilities of success of the introgression of salt tolerance traits from the wild relative into the cultivated species.
3. Discussion
Recent studies have revealed that, even though
Solanum melongena has been described as moderately susceptible—or tolerant—to salt stress, the response largely depends on the genotype [
35,
36,
37,
38]. The effects of salt stress on plant development are well established [
3,
16], although specific response mechanisms depend on the genotype and the experimental conditions tested [
29,
39]. The present work compared the behaviour of an eggplant cultivar (MEL), the wild relative
Solanum insanum (INS) and their hybrid (HYB), with the aim of establishing possible differences in salt tolerance between the tested genotypes and the inheritance of those tolerance mechanisms. Our general assumption was that both parents, closely related genetically, would not differ in the type of responses to salt stress but could differ in the magnitude or efficiency of those responses.
High salt concentrations cause growth inhibition in plants [
35,
36,
40], which was also observed in the present work for the three studied genotypes, especially at 400 mM NaCl. Some quantitative differences between both parents were detected in our experiments; for example, the wild relative seemed to grow more rapidly than the cultivated eggplant in the absence of salt, as shown by the larger increase in leaf number and plant height. However, considering most measured growth variables, MEL and INS plants did not differ substantially in terms of their relative growth inhibition under salt stress, indicating that the degree of salt tolerance was similar for the tested
S. melongena cultivar and
S. insanum. However, in a previous study, another
S. melongena cultivar was shown to be less salt-tolerant than the wild relative at high salinities [
29], confirming the genotype dependence of this trait in eggplant. On the other hand, our results pointed to a higher tolerance of the hybrid than either parent: a smaller relative reduction in the leaf surface and plant height and, most importantly, a relatively higher biomass accumulation (FW of stems and leaves) in the presence of 200 mM of NaCl. Moreover, while root FW did not change in response to the salt treatments in MEL and INS plants, it significantly increased in hybrid plants treated with 200 mM of NaCl. Since the primary root length actually decreased, the intermediate salt treatment most likely induced the growth of secondary roots to enhance water uptake from the soil, as has been observed in some other species [
41,
42,
43,
44], suggesting a specific adaptation to salt stress in the hybrid. The higher efficiency of the defence mechanisms against salt stress observed in the hybrid may relate to the well-known phenomenon of heterosis that hybrids show in comparison to their parents, leading to improving important agronomical quantitative traits, such as growth vigour, seed production, yield or even stress survival [
45,
46]. Heterosis has been observed and studied in many different plant species, including, for example, maize, rice or tomato [
47,
48,
49], as well as in eggplant [
50]. This hybrid vigour would be of great interest in future eggplant breeding prospects, but the genetic basis of heterosis is still under study.
Salinity is generally associated with the degradation of photosynthetic pigments, which is more pronounced in less tolerant species, reducing photosynthesis activity in the plant. Consequently, chlorophylls
a and
b (ChlA and ChlB), and carotenoid (Caro) contents, might be used as stress biomarkers [
51,
52]. In the present work, however, we did not observe a significant salt-induced reduction in ChlA, ChlB or Caro in any of the genotypes, thus, supporting the moderate salt tolerance of eggplant and its wild relative. Interestingly, the ChlA contents were higher in the hybrid than in both parents in the presence of 400 mM of NaCl, which was probably also due to an heterotic effect.
Na
+ and Cl
− contents accumulated in leaves and roots when salinity increased, with similar patterns for
S. melongena, S. insanum and the hybrid, as previously reported in different eggplant cultivars [
53,
54]. However, we did not observe significant differences between roots and leaves, except for
S. insanum, with a more significant Na
+ accumulation in leaves. This result supports the idea that in
S. insanum, Na
+ is transported from roots to leaves and is sequestered in leaf vacuoles with a somewhat higher efficiency than in the cultivated eggplant.
An increase in Na
+ contents in response to increasing salinity would generally promote a decrease in K
+ concentration, as both cations compete for the same transport proteins of the membrane [
36,
53,
54]. For this reason, the Na
+/K
+ ratio has been reported as a possible indicator of salt tolerance in plants [
14,
55]. Some studies revealed a limited Na
+ accumulation in eggplant leaves to maintain lower Na
+/K
+ ratios, reducing Na
+ uptake or compartmentalising it into root vacuoles [
56,
57]. In this study, we observed that the plants of the three genotypes maintained root K
+ levels under salt stress, which likely contributes to salt tolerance in eggplant, its wild relative and their hybrid. Changes in leaf K
+ concentrations were also not detected in salt-treated HYB plants, which in this respect showed an intermediate behaviour between its parent species. The Na
+ contents measurements indicated that ion transport in response to salt differs in both parental species. In INS, Na
+ levels were higher in leaves, whereas Na
+ predominantly accumulated in roots in MEL and the HYB, which in this respect was more similar to the cultivated eggplant than the wild relative.
The Ca
2+ contents increased in the presence of salt, with higher accumulation in roots than in leaves, supporting the notion that Ca
2+ is beneficial for maintaining Na
+ and K
+ homeostasis, at least partly via the SOS pathway [
58]. Moreover, due to its critical regulatory and signalling roles in plant growth and development [
59], increasing Ca
2+ in the leaves of salt-stressed plants is important to maintain essential metabolic and cellular processes.
Proline (Pro) is a reliable marker of abiotic stress in many plant species, increasing its concentration in stressed plants in relation to its background levels in non-stressed controls [
15,
60]. In addition to its role in osmotic adjustment, Pro is an osmoprotectant because of its function as a low-molecular-weight chaperon, maintaining protein structure and membrane integrity, as well as ROS detoxification under stress conditions [
61]. Several reports have shown a positive correlation between Pro accumulation and stress tolerance when comparing related taxa, suggesting the direct participation of Pro in the mechanisms of tolerance [
20,
62,
63]. In other cases, however, Pro contents simply indicate the relative degree of stress affecting the plants, accumulating at higher levels in the more-stressed, less-tolerant genotypes, indicating that Pro is not directly involved in relevant tolerance mechanisms [
37,
38,
64]. Our results showed higher Pro contents in the control plants of the hybrid than in
S. melongena or
S. insanum, suggesting a constitutive mechanism of stress response. The salt treatments led to significant, concentration-dependent increases in Pro accumulation in all three genotypes; however, the absolute concentrations reached were much higher, around 25-fold, in INS and HYB than in MEL. Therefore, regarding Pro biosynthesis and accumulation in response to salt stress, the hybrid was much closer to the wild relative than the cultivated eggplant.
Soluble sugars (TSS) play a role in osmoregulation under stress environments in some plant species, amongst other multiple functions unrelated to specific responses to stress [
65,
66]. This report does not show any significant variation in TSS contents in response to salt stress, indicating that sugar accumulation is not a relevant mechanism of salt tolerance in eggplant.
Malondialdehyde (MDA), a product of lipid peroxidation, is an excellent marker of oxidative stress generated as a secondary effect of high salinity and other abiotic stresses due to the increase in cellular ROS levels [
67]. Therefore, in general, it should be expected that MDA contents increase in plants in the presence of salt, as should the levels of antioxidant compounds and enzyme activities, in order to counteract oxidative stress [
68,
69,
70]. In this study, however, MDA contents did not vary significantly in response to the salt treatments. Similarly, no salt-induced increase in H
2O
2 contents was detected, in agreement with the maintenance of low ROS levels, but for a slight increase in INS at 400 mM of NaCl. These data suggest that salt stress responses based on the control of ion transport and Pro accumulation are efficient enough to avoid the generation of oxidative stress in salt-treated MEL, INS or HYB plants under our experimental conditions. In the absence of oxidative stress, the activation of antioxidant enzymes or the synthesis of antioxidant metabolites, such as phenolic compounds, was not detected, as should be expected.
The responses of adult plants to lower salt concentrations during longer treatment were qualitatively similar, in most cases, to those described above for younger plants, albeit generally weaker. This behaviour could be explained by the increase in stress tolerance with plant age, which has been observed in many plant species [
71,
72,
73]. The salt treatment also caused an inhibition of growth in adult plants, mainly reflected in a reduction in the FW of the aerial part, stem and leaves, but without significant differences between the three genotypes.
The Na+ contents significantly increased in salt-treated plants with respect to the non-treated controls, in roots and leaves. However, contrary to what was observed in younger plants, Na+ concentrations were much lower in leaves than in roots. It seems that adult plants can efficiently block the transport of the toxic ion to the aerial part of the plants. On the other hand, K+ and Ca2+ concentrations were slightly (but significantly) higher in leaves than in roots and did not vary in response to the salt treatment or between genotypes. Compared to young plants, where Ca2+ is accumulated primarily in roots, Ca2+ concentrations were slightly but significantly higher in leaves in adult plants. Moreover, Na+/K+ ratios were significantly lower in adult hybrid roots compared to young hybrid roots. The relatively low accumulation in the leaves of Na+ ions, and the higher contents of K+ and Ca2+, partly counteracting the deleterious effects of salt stress, most likely contribute to the higher tolerance of adult plants.
Finally, Pro contents increased in the plants treated with 80 mM of NaCl, although the relative increase over control levels and the maximum values reached were lower than in young plants. In this case, however, the lowest Pro concentrations were measured in INS plants and the highest in MEL, with the hybrid showing intermediate values. Since toxic Na+ accumulated in leaves at much lower levels than in young plants, it seems logical to assume that lower Pro concentrations are also required for osmotic adjustment.