With the global scarcity of water resources and increased of salinity in water and soil, these abiotic stresses constitute major limiting factors in plant growth and, consequently, agriculture productivity is decreasing (Bray et al. , 2000).

Plant responses to water and salinity stresses are complex and involve adaptive changes and/or deleterious effects (De Oliveira et al. , 2013). The outcomes of both stress types on plant performance are diverse, but have some points in common. The main effect when plants start becoming stressed is the reduced water content in their tissue, and therefore the closure of leaf stomatal complexes takes place. Consequently, transpiration (Bray et al. , 2000) and/or photosynthesis may decrease through reduced osmotic potential in the soil solution, which involves reduced water potential (Bojórquez-Quintal et al. , 2014; Penella et al. , 2014a, 2015, 2016). If salinity stress occurs, a specific ionic effect appears, mediated by the accumulation of toxic ions in cellular tissues (De Pascale et al. , 2003) with imbalances between nutrients (Hasanuzzaman et al. , 2013). All these factors have adverse effects on both plant growth and development at physiological and biochemical levels (Munns & James, 2003).

Plants have evolved mechanisms to overcome salinity and water deficit that allow them to perceive incoming stresses and to regulate their metabolic functions. In general, one of the important pathways to enhance water stress and salt tolerance is through osmotic adjustment (OA), in which leaf turgor remains necessary for stomatal opening and, thus, sustains photosynthesis and growth (Huang et al. , 2010; Nio et al. , 2011). Besides, various types of compatible solutes accumulate, such as sugars, proline, gycinebetaine or potassium, among others (Munns et al. , 1979; Morgan, 1992; Nio et al. , 2011), and can increase. These compounds can be added to the list of the non-enzymatic antioxidants that plants need to counteract the inhibitory metabolic effects of reactive oxygen species (ROS) provoked by stress (Gill & Tuteja, 2010; Penella et al. , 2014a, 2016). They also play a role in both the stabilisation of enzymes and proteins and the protection of membrane integrity (Patade et al. , 2012). Salt tolerance may arise from the ability to tolerate osmotic stress, from mechanisms of salt exclusion or from intercellular ion compartmentalisation (Munns & Tester, 2008). These mechanisms are not normally exclusive, so plants can combine some of these strategies at the same time (Chaves et al. , 2003). Furthermore, tolerance levels may vary between species (Munns, 2002) and within cultivars of the same species (Chartzoulakis & Klapaki, 2000).

Understanding the tolerance mechanisms that occur at the whole plant level has implications for screening and distinguishes plants that are tolerant to salinity and water stress (Munns, 2002). In the climate change scenario, new sources of salt and water stress tolerance are needed for the crops grown in areas with salinity and scarcity water problems. This available genotypic variability in terms of tolerance to abiotic stresses can provide plant species with a breeding opportunity to obtain better yields and production, and good fruit quality.

Pepper is a member of the family Solanaceae and is considered one of the most important crops in the Mediterranean area, where water shortage and salinity are major problems that limit productivity (Penella et al. , 2013, 2014b). Many crops belong to the genus Capsicum spp., and display wide genetic variability (Aktas et al. , 2006). Pepper has been classified from moderately sensitive to sensitive under salinity and water stress conditions (Tanji & Kielen, 2002; Penella et al. , 2015). In fact, some studies have reported reduced seedling growth with 50 mM concentration of NaCl (Chartzoulakis & Klapaki, 2000; De Pascale et al. , 2003). Sometimes pepper has been described as one of the most susceptible crops to water stress, mainly due to its large transpiring leaf surface and high stomatal conductance of water vapour (Alvino et al. , 1994; Delfine et al. , 2002). Consequently, pepper plants are particularly sensitive to water stress at flowering and fruit setting (Bosland & Votava, 2000). However, not all Capsicum genus cultivars have the same sensitivity to abiotic stresses (Penella et al. , 2013, 2014b; Aktas et al. , 2006). Therefore, the study and identification of the tolerance level and mechanisms of different pepper genotypes are immensely important to breed new cultivars that can overcome abiotic stresses, or be used as new crops in land with drought and salinity problems to help extend the cultivated property. For pepper, very rare information about genotype variability in terms of its behaviour under salinity and water stress is available.

Different physiological markers have been proposed as key traits to select salt and water stress tolerance. Our most recent works evaluated several pepper accessions. We selected some of them as a source of tolerance to salinity and water stress (Penella et al. , 2013, 2014b) using gas exchange as a useful technique to differentiate tolerance and susceptibility to these stresses.

In the present study, we tested new accessions of Capsicum annuum L. for them being the most economically important species from the Capsicum genus in the Mediterranean climate. Accessions selection was made according to previous results (Penella et al. , 2013; 2014b). To evaluate their behaviour under salinity and water stresses, we studied the physiological mechanisms that underlie tolerance strategies using efficient parameters to identify which pepper accessions are tolerant to salt and/or water stress to be used in marginal areas and/or in breeding programmes. Further, we describe the physiological parameters roles and discussing the possibility of using them as selection criteria for cast salt and water stress genotypes with tolerance. As predictive screening parameters to salinity and water stresses in these seven new pepper accessions, we measured photosynthesis (A _N ), stomatal conductance (g _s ), inner carbon (C _i ), water potential (ψ _w ), osmotic potential (ψ _s ), proline content, ion concentrations and biomass and their relationships.

The pepper accessions shown in this experiment exhibited physiological differences in response to drought and salinity stress. In particular, we obtained photosynthesis values connected to biomass, which indicated the ability to cope with these stresses. Therefore, these accessions would be suitable to be grown in semi-arid or salinity lands and/or to be used in breeding programmes as a source of tolerance.

It is well-known that water and salinity stress reduces plant growth and that there are differences among cultivars with peppers (Aktas et al. , 2006; Penella et al. , 2013, 2014b). According to our results, both stresses significantly suppressed the growth of pepper plants in dry weight terms, although their stress responses depended on the accession. It should be noted that among all accessions, A34 for salinity and A31 for water stress showed minor growth reduction; nevertheless, A31 under salinity experienced an important biomass reduction. However, the physiological, biochemical and genetics mechanisms involved in growth inhibition have not yet been well characterised (Misra et al. , 2002; Munns & Tester, 2008; Noreen et al. , 2010). Knowledge of plant certain capacities of cope with stress could be essential for characterising stress tolerance.

This differential growth of the seven pepper accessions under both stresses may have been due to a differential regulation of the distinct physiological attributes involved in growth processes. Previous studies have demonstrated a positive relationship between photosynthetic capacity and growth in the plants grown under both salinity (Praxedes et al. , 2010; Saleem et al. , 2011; Penella et al. , 2015; 2016) and water stress (Chaves et al. , 2002; Abbad et al. , 2004; Hassine et al. , 2008; Del Amor et al. , 2010). In spite of considerable reduction of carbon assimilation rate and biomass under both stresses for all accessions studied the genotype A31 under water stress showed the minor decrease for both physiological parameters showing not significant differences in A _N and a biomass reduction of 29% respect its control plants. Under salinity stress, among all accessions, the genotype A34 exhibited the higher photosynthesis rate and its biomass experienced the minor reduction respect to the rest of accessions.

Reduced photosynthesis can be caused by stomatal closure and/or non-stomatal inhibition, and the latter is associated with damage in photosynthetic machinery (Flexas et al. , 2004). In our experiment, a high correlation between A _N and g _s was observed for all data (A _N = 13.304 g _s + 6.63, r ² = 0.8579; p <0.05). This apparent linearity indicates that g _s and A _N reduced in a coordinated relation. This finding agrees with the interpretations under water and salinity stress conditions made by several researchers (Cowan & Farquhar, 1977; Delfine et al. , 2002; Filippou et al. , 2014; Penella et al. , 2015). The equation of the relationship between both parameters (A _N vs. g _s ) did not pass through the origin. This fact indicates that stomatal closure occurred earlier than CO ₂ fixation (Delfine et al. , 2002), although A _N reduction was due mainly to stomata closure, the partial inhibition of mesophyll conductance and/or photochemical efficiency cannot be ruled out.

Decrease in A _N /C _i indicates that both stresses affected the photosynthesis by metabolic limitations (Da Silva et al. , 2011; Penella et al. , 2015). The marked drop in A _N /C _i occurred more drastically under the salt-stressed pepper accessions. Excessive Na ⁺ and Cl ^- accumulation is harmful and may disrupt the integrity of chloroplasts and decrease photosynthetic capacity (Munns & Tester, 2008; Chaves et al. , 2009; Rouphael et al. , 2012; Penella et al. , 2015). We observed how the accumulation of mainly Na ⁺ , and of Cl ^- to a lesser extent, occurred in the roots and leaves of all the pepper accessions. However, A35 stood out with the greatest Na ⁺ accumulation in its leaves, affecting directivity to photosynthetic apparatus, which could cause the lowest A _N , g _s , E and FW (and DW) values. Under water stress, A _N /C _i decreased to a lesser extent, which supports the notion that the major photosynthesis inhibition was mainly resulted from stomatal closure, and that A36 was the most affected genotype with the lowest growth and CO ₂ fixation.

Under the osmotic stress provoked by water and salt stress in the root medium, plants lowered leaf ψ_s in an attempt to maintain water uptake (decrease ψ_w ) with a positive turgor, which is indispensable for cell growth and maintaining photosynthetic performance (Yadollahi et al. , 2011; Penella et al. , 2015, 2016). In order to face water loss, plants accumulate many compatible (organic) metabolites to increase tolerance against tissue dehydration (Yoshiba et al. , 1997; Patakas et al. , 2002). Proline accumulation is believed to be one of the most important metabolites that are implied in osmotic adjustment. Moreover, several studies have attributed multiple roles to proline, such as signalling molecule that influences defence pathways, complex metabolic regulation and development processes, and protective compounds (see Szabados & Savouré, 2010). In our experiment, proline content increased considerably under the salinity conditions (from 29% for A34 to 64% for A37 compared with their controls), but not under water stress. When proline was taken as an osmolyte, the role it played to contribute to lower ψ_s did not suffice under salinity (between 0.06 and 0.1 MPa) to generate osmotic pressure (Smirnoff & Cumbes, 1989; Penella et al. , 2015). Therefore, the increase in proline under salt stress was unable to explain the observed decrease in ψ_s , while the relationship between them was very weak ( r ² = 0.027). Nevertheless, the largest proline amount observed in all the pepper accessions, except A34, was related with the greater salt sensitivity of these genotypes. These findings are consistent with the research reported for pepper into higher leaf proline in salt-sensitive genotypes (Penella et al. , 2015), or for other species, such as wheat (Colmer et al. , 2005), barley (Chen et al. , 2007), or rice (Lutts & Guerrier, 1995). Moreover, proline or other compatible solutes may protect plants by scavenging the oxygen-free radicals caused by salt stress (Huang et al. , 2010; Penella et al. , 2016), a role as signalling molecule, implicated in regulation and developmental processes, and should be considered a protective compound (see Szabados & Savouré, 2010). Under water stress, no changes were observed in proline content respect to controls due to there were not differences in ψ_w and ψ_s was observed between treatments.

The adjustment of ψ_s through inorganic salt-ion uptake is a strategy that implies a much lower energy cost for cells compared with the organic molecules synthesised in cells (Munns, 2002). Pepper accessions showed a better correlation between osmotic potential and Na ⁺ levels in leaves ( ψ_s = -0.361 [Na ⁺ ] – 0.9288; r ² =0.666), but not for Cl ^- accumulation (data not shown). The decreased in ψ_s in the pepper accession leaves subjected to salt stress was largely the result of strong Na ⁺ accumulation. This result was also observed by Chen et al. (2007) in barley genotypes, Abideen et al. (2014) in Phragmite karka , or Navarro et al. (2003) and Penella et al. (2015) in pepper plants.

Overall, all the analysed physiological parameters, photosynthesis and stomatal conductance, can be reliable indicators of biomass under water and salinity stress. Both salinity and water stress lead to reduce photosynthesis, stomatal conductance and growth. The present study evidences a wide variability of the seven pepper accessions in response to both stresses, and in both drought and salinity treatments. Under our conditions, growth inhibition occurred under water stress, provoked mainly by stomatal closure, where A31 was the less affected, and A36 was the most sensitive one and also correlated with minor CO ₂ fixation and biomass. Under the salinity conditions applied in this experiment, our results showed that damage was greater compared with water stress. In this case, photosynthesis inhibition was due to the stomatal and non-stomatal effects caused by osmotic stress and toxic salt ion accumulation. Even with increased proline synthesis, the reduction in ψ_s and ψ_w was not enough to alleviate the salt effects. A34 was the most interesting accession due to its better tolerance to salinity with a major photosynthesis capacity, minor growth inhibition, but it had a lower proline concentration compared with A35, which suffered the worst adaptation. This genetic variation in response to both stresses can be exploited in pepper crops so they can be grown in marginal areas and/or in breeding programmes.