Results here presented show that from the physiological, nutritional, and biochemical point of view the 4 native grass species considered of the Pampa biome responded differently to the excess of Zn availability. Considering their capability to survive and complete the vital cycle even when exposed to very high levels of Zn availability (accumulating as well very high metal levels in their tissues), it can be speculated that these plant species possess traits that make them interesting candidates for phytoremediation programs. This hypothesis is further corroborated by the evidence that some of these plant species are naturally living and replicating in soils of southern Brazil characterized by an excessive Cu availability (e.g. A. affinis, P. notatum e P. plicatulum) (Silva et al. 2022; Schwalbert et al. 2022).
In all the four plant species, a close positive relationship between the availability of Zn in the growth medium and the levels of metal accumulated in the plant tissues was highlighted (Fig. 2). This feature is of particular interest when a recovery of Zn contaminated soil is considered. However, even if united by the marked tolerance to the elevated Zn concentrations, the four native plant species seem to adopt different resistance strategies or diverse combinations of them. In fact, while in P. plicatulum and P. notatum plant species, the higher was the Zn accumulation in the tissues, the more limited was their size, the development and the biomass accumulation of A. affinis plants were completely unaffected by the Zn availability, regardless of its concentration (i.e. 2 or 300 µMol L-1) (Fig. 1A). In this context it is interesting to note that in A. lateralis plant species, there was not only an increase in the metal content in the tissues but also an increased accumulation of biomass even when the plants were fed with 300 µMol L-1 Zn. Taken together, these results demonstrate that in addition to the metal availability level, exposure time to the metal, and the phenological stage of the plant development at the moment of the treatment, there is a significant genetic component at the basis of the tolerance to different levels of Zn availability in these four native grasses (Balafrej et al. 2020).
It is well known that Zn concentration levels in leaves above 150 mg Zn kg-1 can normally induce the onset of toxicity symptoms in several plant species (Kabata-Pendias 2011) impacting, therefore, their development and reproduction capacity. It is worth to mention that in our work values ranging from 400 to 431 mg kg-1 have been measured in shoots of the four plant species when fed at 150 and 300 µMol L-1 (Fig. 1A). However, while in P. plicatulum and P. notatum plant species this metal accumulation process was accompanied by a marked inhibition of the plant development (about − 15% biomass accumulation) maybe due to the malfunction of different metabolic pathways as already described (Ghori et al. 2019), the plants of A. lateralis did not show any symptom of toxicity or stunted development. In this latter case, the activation of some specific physiological and biochemical processes linked to the tolerance to Zn excess (Sarwar et al. 2017) appears evident. The metal compartmentation within the cell (Jogawat et al. 2021) as well as the restraint of the metal free form (Krippner and Schubert 2021) are well described strategies acting directly on the acquired metal. On the contrary, the enhancement of the antioxidant systems can be counted among the biochemical strategies implemented to counter the effects of Zn excess like the ROS formation (Kaur and Garg 2021). In this respect, it should be noted that, except for P. plicatulum plant species, the levels of SOD activity were markedly increased in all the other three species when the plants were exposed to the highest level of Zn availability (Table 3).
From the nutritional and nutrients’ interactions point of view, results here reported show that a high availability of Zn can hinder a balanced acquisition of Mn (Table 1). All the four plants species are affected by this problem, albeit at different levels. The similarities in the ionic radii of the two divalent cations can be, at least in part, at the base of this competitive phenomenon of Mn and Zn for the transmembrane transport (Tewari et al. 2008). Similarly, high levels of Zn also hinder the acquisition of Mg. In this case, the lower Mg content at the leaf level can certainly have consequences not only on the chlorophyll functionality, but also on the RUBISCO activation levels (Küpper and Andresen 2016). In this latter case, a competition phenomenon between Mg and Zn for the functional sites of these molecules/proteins cannot be excluded. On the contrary, a nutrient whose acquisition is strongly stimulated by high availability of Zn in the growth medium is P, reaching in particular in A. lateralis values 1.75 times higher than those measured when the plants were fed with 2 µmol L-1 Zn (Table 1). Considering the role played by P at the cell wall level in terms of pectin and hemicellulose contents and pectin methylesterases activity (Wei et al. 2021), the increase in the P content could be considered a contribution to the plant's ability to counteract the excess of Zn by limiting its toxic effects.
With respect to the shoot and its analyzed parameters, it is interesting to highlight that the net CO2 assimilation rate (A) measured increased when the plants of the four native species were grown at the highest availability of Zn (Fig. 3A). Moreover, in this same growing conditions, P. notatum, P. plicatulum and A. lateralis plants also showed an enhancement in the instantaneous carboxylation efficiency of Rubisco (A/Ci) which is also associated, limited to P. notatum and P. plicatulum plant species, with an increased stomatal conductance, a higher internal CO2 concentration and a more pronounced transpiration rate (Fig. 3). These phenomena are in agreement with the substantial integrity (even at the very high availability of Zn) of the photosynthetic system evaluated by analyzing the pigments’ contents (Schwalbert et al. 2022). Moreover, the rather stable levels of H2O2 (with the only exception of A. lateralis plants) and malondialdehyde (MDA) concentration further corroborate the idea that not even the highest available Zn-dose is able to induce a significant level of stress in these plants. It is interesting to note that also at the root level the content of H2O2 and the levels of lipid peroxidation resulted not significantly affected by the Zn availability. Overall, it is clear that the plants of the four native plant species weren't suffering from significant oxidative stress due to Zn toxicity, at least from the type canonically described.
Considering the stress responses of each plant species analyzed individually, it is interesting to note that the plants of A. affinis are those not affected by the Zn excess in terms of biomass accumulation. Moreover, considering the Zn concentration at the root and shoot level (Fig. 2A and 2B) a mechanism of Zn exclusion at the rhizosphere (Taylor et al. 2008) appears to be adopted in this plant species. Furthermore, it is interesting to note that the values of the gas exchange and the oxidative stress parameters measured in A. affinis did not reflect a severe stress for these plant (Thakur et al. 2022), thus suggesting that there was still room of further plant's response expression to the high Zn availability, even if it was already at very high levels (i.e., 300 µMol L-1).
Differently, plants of A. lateralis, although the smallest, when exposed to high availability of Zn, stimulated the biomass accumulation, enhanced the Zn content at the root level, augmented the H2O2 and TBARS production in the roots. The plant showed the ability to maintain the metal in its roots, immobilizing so Zn thanks to the negative charges of the cell wall components or via a complexing process based on other elements such as P (Marta et. al. 2019). It should be noted that limiting the metal content at the leaf level means preserving the photosynthetic system from the damage while maintaining the plant's ability to produce carbohydrates (Yang et al. 2011). Moreover, it is interesting to note that in these plants a very high content of Fe has been detected at the root level, particularly when the treatment with 300 µM Zn is considered. In this respect it should be mentioned that Wu et al. (2022) have shown a marked inhibition of Zn allocation at the shoot level when Zn competes with Fe for the binding sites at the root level.
With respect to P. notatum and P. plicatulum plants, data here reported show a clear toxic effect of Zn in the biomass accumulation process with a positive trend of inhibition linked to the levels of metal availability. However, the values of root volume and root length as well as those of several gas exchange parameters (A, WUE and A/Ci) were clearly increased by higher Zn availability. With respect to the root length, it is interesting to note that its increase has been proposed as a positive sign of Zn stress tolerance in plants (Michael and Krishnaswamy 2011). In addition, the recognized capacity of P. plicatulum roots to modify the rhizosphere pH (De Conti et al. 2020) can surely and markedly affect the metal availability fraction in the soil near the roots. Moreover, the enhanced POD and SOD activity detected in these plants is also counted among the tolerance mechanisms to counter Zn excess (Wei et al. 2022). Furthermore, the proneness to accumulate significant quantities of Zn at the shoot level makes P. plicatulum plants particularly promising for recovery plans of contaminated soils through phytoremediation programs (De Conti et al. 2020).