• Punica granatum
• ozone stress
• salt tolerance

Climate change (CC) has become a reality much faster than all scenarios predicted. It may cause very large global-scale alterations that have a strong and decisive impact on human health, and welfare and plant life. Due to its location and diversity of ecosystems, the Mediterranean region has been identified as one of the most prominent “hot-spots” and an especially vulnerable region in future CC projections. In the context of CC, two major abiotic stresses with a strong impact on plants life are: fresh water salinization and increasing background tropospheric ozone (O3) concentrations. On the one hand, salinity is a progressive soil and water degradation process, affecting aquifers and the most productive agro-ecosystems, representing an increasing environmental concern. Salt stress disrupts homeostasis in water potential and ion distribution causing ion imbalance and toxicity, and hyperosmotic stress in plants. On the other hand, O3 is the most damaging air pollutant affecting plant production, severely affecting crops and natural vegetation both directly, due to its toxicity, and indirectly, through its role as a greenhouse gas contributing to global warming. The current background O3 levels are high enough to inhibit yields of sensitive species in many areas and to affect growth and development of natural plants. Studies on the combined effects of salt and O3 documented different responses from those observed when each stressor was applied independently. Results were sometimes contradictory: salt stress either counteracted O3 impact by reducing stomata opening, or further exacerbated negative O3-triggered effects.
Pomegranate (Punica granatum L.) is a deciduous shrub and its fruits have gained widespread popularity as a functional food and nutraceutical source, due to their antioxidant properties. Although it is still considered as a minor crop, nowadays it is widely cultivated throughout the world, indicating its flexibility and adaptability to different environmental conditions. Indeed, it is considered to be moderately tolerant to salinity but this feature depends on several factors, such as cultivar and stage of development. By contrast there are no works about O3 stress on pomegranate focused on macroscopic, physiological, biochemical and molecular traits.
Firstly, one-year-old saplings of an Italian cultivar (Dente di cavallo) were exposed to two levels of O3 concentrations (ambient and elevated, i.e. 21.51 and 58.74 ppm h, respectively) and salinity [salt and no salt, i.e. 50 and 0 mM of sodium chloride (NaCl), respectively] for five consecutive months, to evaluate the physiological and biochemical adjustments adopted under realistic field conditions. No evidence of visible injury due to salt was found. The maintenance of leaf water status, twinned with a significant increase of osmolytes and stomatal closure were reported as adaptive mechanisms against salt stress. By contrast, O3-treated plants were unable to protect cells against the negative impact of oxidative stress, as confirmed by the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) by-product, although plants tried to preserve themselves from further oxidative damage by adopting some biochemical adjustments. The interaction of the two stressors induced responses considerably different to those observed when each stressor was applied independently. An analysis of the antioxidant pool revealed that the biochemical adjustments adopted by P. granatum cv. Dente di Cavallo under both stressors were not sufficient to ameliorate the O3-induced oxidative stress.
Following the results that came out from this first experiment, other two sets of experiments related to salt stress were performed. The first one was focused on the strategies adopted by two commercial cultivars (Wonderful and Parfianka) in terms of sodium (Na+)/chloride (Cl-) translocation and tissue/organ compartmentalization, as well as of antioxidant and osmoprotectant mechanisms. They were subjected for 47 days to four salt concentrations (0, 100, 150 and 200 mM NaCl). Regardless of salt concentrations, mature leaves (ML) of both cultivars accumulated high concentrations of Na+ and Cl-, showed strong decreases in photosystem II (PSII) photochemical efficiency and finally died, suggesting that a salt-induced ion-specific toxicity occurred. Conversely, these detrimental effects occurred later and at a lower magnitude in young leaves (YL), likely because of a consequence of a Na+ and Cl- compartmentalization in ML and roots. Differential responses between cultivars were, instead, observed in terms of biochemical pathways, with Parfianka plants more activating osmolyte accumulation at root and leaf level, antioxidant system and macronutrient distribution than Wonderful plants. However, both cultivars resulted salt-tolerant, as confirmed by their ability to safeguard biomass production.
Considered the different tolerance between the two cultivars to moderate levels of chronic salinity, an another study was performed to investigate the molecular mechanisms adopted during the first hours of salt irrigation (i.e., episodic treatment of 100 mM NaCl). In particular, it was focused on transcriptional regulation of genes encoding for Na+ key transporters (NHX1, HKT1 and SOS1) to understand their role in mediating Na+ transport, translocation and intracellular compartmentalization. The obtained results suggested that Wonderful plants preferred to redirect Na+ to roots and ML, maintaining low concentrations of this cation in YL in order to prevent Na+ accumulation. Similarly, Parfianka plants seemed to be able to avoid Na+ accumulation, especially maintaining low Na+ concentration in YL, although there were not substantial differences in ML and roots Na+ distribution. These results reinforced the idea that difference in ion homeostasis and salt tolerance between cultivars is associated with transcriptional regulation of these genes.
Considering again the negative impact of chronic O3 exposure to pomegranate, a third experiment was performed in order to elucidate the sensitivity of the same two cultivars under O3 episodes at a gradient of concentrations (50, 100 and 200 ppb for 5 h), investigating the capability of hyperspectral reflectance (350-2500 nm) to characterize the cultivar-specific responses to O3-induced oxidative stress. Analyzing spectral signatures collected rapidly and non-destructively from asymptomatic leaves, the two cultivars have been accurately discriminated, as well as controls from plants exposed to O3, in particular those under the higher oxidative stress (i.e., 200 ppb). These discriminations were especially accurate in Wonderful at the end of the exposure and at 24 h from the beginning of exposure (FBE). Furthermore, using a partial least squares regression (PLSR) approach, it was possible to construct predictive spectral models to estimate from spectra an array of commonly used physiological and biochemical leaf traits related to plant/oxidative stress interaction (photosynthesis, lipid peroxidation, enzymatic and non-enzymatic antioxidant compounds). Most functional traits were relatively well predicted by spectroscopic models. Finally, variations of spectra-derived vegetation indices and leaf traits derived from spectra confirmed the lower O3-tolerance of the Wonderful cultivar, when exposed to 200 ppb, than Parfianka. Overall, this final study showed that the proposed spectroscopic approach can rapidly and non-destructively assess early oxidative stress conditions in plants, and consequently it can help in increasing plant yield and quality.
In conclusion in relation to salt stress, pomegranate plants seem to be tolerant to moderate levels of salinity. Anyway, this salt tolerance is a feature cultivar-dependent: Parfianka cultivar seems to be more tolerant than Wonderful, in terms of osmolytes production and Na+ distribution/compartmentalization, as confirmed by different molecular mechanisms activated. About O3 stress, it was observed that O3 chronic treatment has a negative impact on pomegranate plants, inducing oxidative stress. Nevertheless, both cultivars can tolerate O3 episodes showing a differential response, in particular Parfianka cultivar seems to be more tolerant than Wonderful due to a high production of antioxidant compounds.
Looking at the combination of both stressors, it was seen that salt did not counteract the negative impact of O3, but it exacerbated its deleterious effects.