3.1. Physiological and Biochemical Parameters of Pepper Plants
In the present study, the responses of the ungrafted and grafted pepper plants to the salt stress induced by these salinity treatments were evaluated by analyzing some of the physiological and biochemical parameters in the leaves and fruit over increased treatment duration.
The lower accumulation of plant biomass and the reduced fruit yield associated with these treatments will probably have arisen as a consequence of the negative effects of salt stress, which is often a result of the inhibition of photosynthesis [
35]. In general, a Pn reduction might be due to lower intercellular CO
2 concentrations in the leaves as a result of stomatal closure, or because of nonstomatal factors, including conversion and dissipation of the photon energy into heat through the xanthophyll cycle, or degradation of photosynthetic pigments due to severe stress [
36]. In the present study, with the longer duration of salt stress, Pn in the ungrafted plants always showed a decrease with increased salt stress, whereas Pn in the grafted plants decreased only under the higher salt stress. At the same time, no differences between ungrafted and grafted plants were seen for the increasing salt stress and its duration in terms of the reduction in stomatal conductance (g
s) and transpiration (
E). This indicated that the stomatal restriction of photosynthesis [
37] was similar in the ungrafted and grafted plants. As the stomatal function reflects the water status of the plants, we can assume that the grafting did not reduce the disturbance in the water balance under the increased salt stress. These data do not support previous studies that have shown that grafting can improve the water status of pepper plants [
38,
39,
40]. The rootstock used in this study failed to obtain scions that showed better physiological performance and consequently higher yield, although it underwent a rigorous screening program for salt tolerance [
41]. It has been reported that some of the salt-tolerant rootstocks were tested for many years under real salinity field conditions and showed higher yields than ungrafted plants or other commercial rootstocks tested [
16]. Therefore, further studies with different salt-tolerant rootstocks are needed in the future to confirm the tolerance of grafted plants in prolonged salt stress conditions.
The analyses of the photosynthetic pigments and fluorescence measurements indicated that Pn was also affected by nonstomatal limitations. There was a decrease in Chl
a+b levels with increasing duration of the salt stress. The other effect that can reduce Pn that was also monitored here was photoinhibition [
12,
39,
42]; in the ungrafted plants, Fv′/Fm′ decreased significantly with increased salt stress, whereas in grafted plants, it decreased only under the higher salt stress (40 mM NaCl). This decrease can mainly be attributed to the efficiency of the excitation energy capture by the open PSII reaction centers [
43], which suggests that in the grafted plants, the photoinhibition of PSII was triggered with a delay. The de-epoxidation state ratio (EOS) did not indicate the involvement of the xanthophyll cycle pigments in this Pn reduction.
Comparisons of the data for the measurements carried out for the different treatment durations through the season suggest that the mitigating effects of grafting were seen most strongly when these pepper plants were exposed to unfavorable growth conditions, which will be combined with the increased duration of the primary stressor, salinity. The reduction in Pn and Fv′/Fm′ in August was most likely also due to the high temperatures and low humidity, which will affect leaf physiology. From this, it can be concluded that grafted plants appear to maintain higher stomatal conductivity and photochemical efficiency under these conditions.
The underlying mechanism here will not be related to the mineral contents of the leaves because no significant differences between the ungrafted and grafted plants were seen for the K
+ and Na
+ levels in the salt-exposed pepper plants. This indicates that in the present study, the grafting did not reduce the risk of ionic and osmotic imbalance. Studies with grafted plants that have reported this effect have proposed salt exclusion from the shoots and its retention in the roots [
38], or compartmentalization of the salt ions in the cell vacuole [
26]. The ability of some rootstocks to retain Na
+ ions in their roots is genotype-specific, as has been reported for rootstock of peppers, cucumbers, and tomatoes [
4,
20,
44,
45]. However, this was not confirmed in the present study because no measurements of Na
+ concentration were carried out for the roots of these pepper plants.
Better maintenance of K
+ homeostasis in plant tissue is another salt tolerance mechanism that grafting can affect [
26]. The K
+ content in leaves of pepper plants in our study is in agreement with a previous study [
20] where no significant effect of salt treatment on K+ content in leaves of pepper plants was found when salt stress was induced by adding NaCl to the common nutrient solution. K
+ assists in the cation–anion balance, osmoregulation, and water movement and is essential for plant acclimation to biotic and abiotic stress [
46]. The retention of K
+ ions in the cells of the roots and leaves has been shown to be a selection criterion between salt-tolerant and salt-sensitive varieties. Although the “Rocal” rootstock used in this study was selected as salt-tolerant, there were no significant effects of grafting on the K
+ and Na
+ levels in the leaves of these pepper plants under moderate salt exposure during the summer growing season. These results are in agreement with previous findings reported by Aktas et al. [
5], who also observed no differences in leaf K
+ content between control and NaCl-stressed pepper plants of genotypes classified as "tolerant." The salt stress in the present study, induced by the addition of NaCl to the common nutrient solution presumably resulted in competition between Na
+ and the cations already present in the nutrient solution, thereby attenuating the salt stress. Indeed, it is known that the addition of external Ca
2+ and K
+ can significantly mitigate salinity stress symptoms in many species [
47]. In our study, cation competition probably reduced salt stress to the extent that tolerance of grafted plants to salinity could not be expressed. Therefore, in future studies, modified solutions with lower cation concentrations of macronutrients should be used in salt stress treatments in addition to the usual nutrient solution to investigate whether the reduction of salt stress due to competitive cation effects has an influence on the growth and yield of grafted and ungrafted pepper plants.
Although the K
+ levels in the leaves of these ungrafted and grafted plants indicated similar salt stress responses, the Cl
– levels showed the opposite. Here, the Cl
– levels significantly increased under the higher salt stress, by about 27.8-fold in ungrafted plants and 37.4-fold in grafted plants. This resulted in significant differences in Cl
– concentration in these treated plants and reflected the inability of the plant/ rootstock to limit the transport of toxic Cl
– ions to the shoot. Similar data on the lack of rootstock retention of Cl
– ions have been obtained in other studies on salt-tolerant grafted pepper plants [
16,
17] and salt-tolerant grafted tomato plants [
24]. Navarro et al. [
2] also reported negative effects of salinity on pepper growth, and they concluded that the yield reduction induced by salt stress can be linked to the toxic effects of Cl
– accumulation in the plant tissues. Indeed, this might be one of the reasons for the yield reduction under the salt stress in the present study. It is known that the Cl
− concentration in the shoot of non-halophyte plants varies greatly, ranging from 1 mg·g
−1 to 20 mg·g
−1 DW [
48,
49], which means that the Cl
− concentrations in this study, which is expressed in DW amounted to 35 mg·g
−1 and 41 mg·g
−1, indeed reached toxic amounts. In the future, whole-plant Cl
− content analysis may be included in studies to provide data for a better understanding of the salt stress mechanism that will enable grafted pepper plants to overcome salt stress problems during the growing season. Proline is one of the compounds that can accumulate in plant tissues, most frequently as an osmolyte or protective substance under unfavorable environmental conditions, such as drought and salt stress [
6,
50]. Salt-induced proline accumulation was also evident in the present study. Under these prolonged moderate salt stress conditions, the ungrafted plants showed higher leaf proline levels than the grafted plants, but at the same time, they had lower effective quantum efficiency (Fv′/Fm′), which indicated higher stress intensity. However, as the rootstock did not contribute to the exclusion of salt from the shoots, a similar level of osmotic disturbance would be expected in the leaves of both of these plant groups. Differences in leaf proline levels might be due to different involvement of this compatible organic solute in osmotic adjustment, which is also favored by salt ions deposited in the vacuole [
14]. Here, the high Cl
− levels in the leaves of the grafted plants might have an important role. As the ungrafted and grafted plants showed similar water status throughout the experimental period and had similar responses of the stomata under different conditions, it can be excluded that the differences in the proline levels are a result of a specific response of plants to water deficit (e.g., high vapor pressure deficit; see Grossiord et al. [
51]).
3.2. Fruit Quality
In addition to providing resistance to soil-borne pathogens [
52], the main objective of vegetable grafting is an improvement of tolerance to abiotic stress [
15] thus promoting increased yields, although often at the expense of fruit quality [
33]. It is therefore of utmost importance to understand the effects of grafting on the fruit quality parameters and to understand and define the mechanisms involved [
23].
To the best of our knowledge, there are no reports in the literature of changes in the biochemical profiles of pepper fruit induced by salinity and grafting, with treatment durations continuing across different harvest periods (i.e., July to September). In the present study, the data showed no influence of salt stress on the total sugars content in the pepper fruit, and no differences in the total sugars content between ungrafted and grafted plants, under both the short treatment duration for fruit harvested in July and the longer treatment to September. The total acidity showed similar results, although, for the fruit harvested in September, the total acidity of the grafted plants tended to be lower than that of the ungrafted plants, even if the differences did not show significance. The results of the fruit acidity might be associated with less severe salt stress in the grafted plants because total sugar content and titratable acidity in the pepper fruit increase as water loss of the plant increases, due to osmotic drought caused by salt stress [
53].
Among the functional compounds reported to be influenced by salinity and grafting [
15], ascorbic acid and polyphenols were analyzed in this study. In the literature, contradictory data have been reported regarding variations in the ascorbic acid content in response to grafting [
25,
29,
30,
31,
32]. The present study showed no significant impact of grafting on ascorbic acid content for both the short-term and the long-term treatments. This is consistent with data reported by Sánchez-Torres et al. [
32], who showed that grafting of two pepper varieties onto two different rootstocks did not alter the ascorbic acid contents in the pepper fruit. In the present study, the salt stress influenced the ascorbic acid content only with the long-term treatment in September, which resulted in ascorbic acid increases with increased salt stress under both of these grafting conditions. Effects of salt stress on ascorbic acid content have been shown in some studies, although the data are again contradictory. For example, in green pepper, osmotic stress induced by low irrigation frequency strongly increased the ascorbic acid content (by 23%), while an increase in salinity from 0 mM to 30 mM NaCl resulted in a decrease in ascorbic acid content [
53]. In contrast, in cherry tomato fruit, increased salt in the nutrient solution resulted in increased ascorbic acid. This suggested that the increase was a consequence of the increase in fruit dry matter due to the different salinity conditions and the activation of specific metabolic pathways in tomato plants under high salt stress conditions [
54].
Polyphenols are phytochemicals that contribute to antioxidant activities in plants, and they are present in pepper fruit at moderate to high levels [
55]. The synthesis of phenolics has been described as actively involved in neutralization of free radicals as a response to oxidative stress caused by abiotic factors [
15,
33]. The high pressure liquid chromatography HPLC–mass spectrometry analysis of the pepper fruit in the present study showed that the phenolic profile mainly consisted of hydroxycinnamic acid and two types of flavonoids—flavones (for the most part) and flavonols. Here, the phenolic content in pepper fruit decreased with the duration of the salt stress. This higher total phenolics in fruit from the short-term treatments compared to those from the long-term treatments might be a further indicator of the stress caused by environmental factors compared to the fruit from the long-term treatments because stress conditions such as high solar radiation and temperature induce an accumulation of phenolics [
29,
56]. We obtained similar data in our previous study, in which the symptoms of salinity were differently expressed in tomato plants that were stressed in mid-summer or late summer [
57].
Data on variations in the phenolics in grafted vegetable fruit are contradictory. Most previous studies have investigated biochemical responses of plants to salt treatment, while few have investigated instead the biochemical responses of fruit. For instance, Koleška et al. [
58] reported that moderate salinity led to their greatest reductions in flavonoids in ungrafted tomato plants, while at the same time, it caused the highest increase in flavonoids in the fruit. This suggested that the phenolics are transported via the phloem from the leaves to the fruit, where their antioxidant protection occurs. Similarly, López-Marín et al. [
30] reported a decrease in total phenolics in “Herminio” pepper plants, but only when “Creonte” rootstock was used. In contrast, López-Serrano et al [
34] reported significant increases in total phenolics in grafted pepper plants under salinity treatment, which coincided with stimulation of antioxidant capacity because phenolic compounds are reported to help prevent the formation of reactive oxygen species and protect the photosynthetic apparatus [
59]. In the present study, for the short-term treatments in July, the grafting led to significant increases in total phenolics content in these pepper fruit under the higher salt stress (40 mM NaCl), compared to the ungrafted plants. This might indicate that higher salt stress in July, accompanied by harsh environmental conditions, triggers a defense mechanism in grafted plants that includes an increase in phenolics and has a protective role against ion-induced oxidative stress [
34]. With the longer-term treatments in September, however, there were no significant differences in the total phenolic content between the fruit of the ungrafted and grafted plants.