Crop productivity and yield are seriously affected by soil salinity, which can result from natural phenomena or human activities Isayenkov and Maathuis, 2019). Contextually, since climate change is worsening environmental cues including salinity stress (Ahuja et al., 2010), the research for solutions to mitigate such events is a priority for modern agriculture (Chaudhry and Sidhu, 2022). It has been demonstrated that an important role in the coordination of plant responses to different biotic and abiotic stresses is played by peptides or protein fragments enclosed in larger precursors (Chen et al., 2020). For example, Arabidopsis Pep1, a 23-amino-acid peptide derived from a precursor protein of 92 amino-acid, enhances resistance to both root (filamentous fungi) and leaves parasites (hemibiotrophic bacteria and necrotrophic fungi) (Yamaguchi et al., 2010; Liu et al., 2013), while Arabidopsis Pep3, a 30-amino-acid peptide derived from a 96 amino-acid precursor, activates components of the innate immune response and enhances tolerance to high salinity (Nakaminami et al., 2018). The former also triggers the immune signals Huffaker et al., 2006), while the latter is highly induced by salt stress, within minutes to hours (Nakaminami). In addition, precursor proteins involved in defence responses, may undergo different modification at molecular level beside releasing protein fragments that play significant functions [26]. This group includes tomato ProSys, that is involved in the release of the Sys peptide and serves as a key hub for multiple plant stress-related activities (Natale et al., 2023). Indeed, its interaction plasticity, favoured by its disordered feature (Buonanno et al., 2018), makes ProSys to cope with plant response to both biotic and abiotic stresses. Here we explored, for the first time, the potential of three ProSys derived protein fragments to promote resilience against salt stress in tomato.
Other than the biostimulant effect on plant shoot fresh weight (Table 1), a single irrigation with EFs markedly reduced the salt damages making the plants more tolerant, since they showed a significant improvement in root area under salt stress conditions. This result is in line with what Orsini et al., 2010 observed in plants overexpressing ProSys. In response to salt stress, ProSys transgenic plants maintained a higher stomatal conductance compared with the wild-type control. Leaf concentrations of abscisic acid and proline were lower in stressed transgenic plants than in wild-type control, suggesting that either the former perceived a less stressful environment or they adapted more efficiently to it. In addition, under salt stress, transgenic plants produced a higher biomass. However, salt tolerance cannot be attributed exclusively to an overproduction of Sys, but also to the ability of other regions of the precursor, such as those tested in the present work that appear to be able to reduce the damages of salt stress likely by integrating multiple signals. Indeed, it has been demonstrated that stress-related pathways are not necessarily independent, but rather characterized by an intricate crosstalk (Ludwing et al. 2004; Fujita et al., 2006). Salt and wounding stress, for example, appear to be interconnected by the oxidative burst involving calcium ions and by the production of JA and Sys (Dombrowski, 2003); Orsini et al., 2010). The key elements that may mediate this crosstalk are calmodulins (CMs) and calcium-dependent protein kinases (CDPKs) (Ludwing et al., 2004; Capiati et al., 2006), which interestingly appear to take part of ProSys interaction network (Natale et al., 2023).
Abiotic stress, like salt stress, reduces water uptake disrupting plant physiological processes and produce not only ionic and osmotic stresses, but also oxidative damages as a consequence of a plethora of stress-induced ROS. High level of ROS alters cell structure and degrades proteins and nucleic acids (Umeno et al., 2017). Thus, reducing stress-induced ROS over-accumulation is important to protect plants under adverse environments. Our results showed that EFs increased transcripts associated with antioxidative activity both in absence and in presence of salt suggesting a role in shaping plant oxidative status with a reduction of ROS accumulation. These results correlate with the improved performance of salinized plants showing an increased value in their shoot and in their stomatal density. It was demonstrated that a salt tolerant pepper genotype showed a much higher level of transcripts of CAT2, APX2 and other antioxidant genes in respect to susceptible genotype when subjected to salt stress (Zhang et al., 2022) suggesting a correlation between ROS scavenger and plant performance in salt stress conditions. Moreover, increased root biomass is often associated with an enhanced tolerance to salinity and drought stresses and in this context an important role is played by HSP90 (Yadav et al., 2021), also upregulated following plant treatments with specific EFs. For example, tobacco and tomato plants overexpressing HSP90 were more tolerant to salt stress conditions and their roots grew faster than the wild types (Yadav et al., 2021; Liu and Zao, 2006). It was proposed that HSP90 plays a vital role in improving the salinity tolerance by way of enhancing the root biomass and architecture (Yadav et al., 2021). Although salt stress leads to a reduction in stomatal density (Prakash and Ramachandran, 2000), plants treated with PS1-70, PS1-120 and G do not show such a reduction (Fig. 2). Reduced stomatal density, which is determined by multiple and interconnected morpho-physiological and metabolic factors, increases salinity tolerance and water-use efficiency under salt stress, but reduces the rate of photosynthetic efficiency and thus plant biomass production (Barbieri et al., 2012; Parihar et al., 2015). The application of the EFs, under salt stress, results in a stomatal density similar to that of controls without salt. The increased stomatal density may allow tomato plants to restore gas exchange, avoiding the accumulation of ROS (Campobenedetto et al., 2021; Goñi et al., 2018). It is worth noting that an increased stomatal density is associated with increased salinity tolerance in barley (Hasanuzzaman et al., 2018).
Salt stress induces not only antioxidants but also proline (Fu et al., 2018). Proline plays several roles in plants under stress conditions, such as stabilization of membranes and subcellular structures, and also acts as ROS scavenger and as compatible osmolyte that contributes to the conservation of the osmotic gradient in salt stress circumstances (Acosta-Motos et al., 2017; Arif et al., 2020). In our experimental conditions, upon salt stress, proline content highly increased in control plants while a more modest increase was observed in EFs-treated plants. This may be the consequence of the redox status of these plants, already ameliorated by the upregulation of CAT2, APX, as well as due to a priming effect exerted by fragment treatments, similarly to that induced by proline treatments.
Recently, it was demonstrated that plant biostimulants are very effective to ameliorate the sustainability and resilience of production systems also under stress conditions (Rouphael et al., 2022). As a matter of fact, since biostimulants are based on molecules or microorganisms that regulate plant physiology and metabolism they may promote plant growth as well as resilience against abiotic stress (Thakur et al., 2023; Di Mola et al., 2021).
Several research articles have demonstrated the positive role of protein hydrolysate biostimulants in enhancing tolerance to abiotic stresses, such as heat, drought and salinity (Ikuyinminu et al., 2022). Protein hydrolysates exhibit a molecular mechanism that is not fully understood, but the activity appears to be closely related to the presence of peptide fragments that interact with membrane receptors to act as hormonal signaling by triggering the response at the gene level and changing the secondary metabolism in favor of growth and development.
The biostimulant activity of EFs in promoting biomass growth, even under salinity condition, is a very interesting characteristics of the EFs, that highlights novel tools in agricultural biotechnology.
In recent past, multiple lines and levels of evidence revealed a prominent role for peptides not only in the antiherbivore and antipathogen responses but also in the modulation of high salinity and drought stress (Chen et al., 2020). Three plant peptides, characterized in Arabidopsis, are known to regulate high salinity and drought stress responses. CLE25, a 12-amino-acid peptide derived from the C-terminal region of the 69-amino-acid CLE25 precursor protein (Takahashi et al., 2018), controls stomatal closure under dehydration to prevent water loss by transpiration. Cle25 knockout mutants are more sensitive to dehydration than wild type. CAPE1, derived from the C-terminus of the 172-amino-acid PROCAPE1, negatively regulates salt tolerance under high salinity (Chien et al., 2015). AtPep3, released form a member of the PROPEP gene family, was recently found to enhance the tolerance to high salinity (Nakaminami et al., 2018). Both overexpression of AtPROPEP3 and exogenous treatment of synthetic AtPep3 peptide induces salt stress tolerance. Conversely, AtPROPEP3-RNAi lines are hypersensitive under salinity stress, which is recovered by AtPep3 peptide application.