The A. blanchetiana plants cultivated under the in vitro conditions imposed showed different anatomical and physiological responses due to the presence or absence of Si and the variation in concentrations of NaCl. The morphophysiological responses induced by Si had an attenuating effect on salt stress, through anatomical alterations, increased content of photosynthetic pigments, and greater activity of the enzymes of the antioxidant system, besides their contribution to enhance the performance of the photosynthetic apparatus.
The root and leaf anatomy of the plants was in accordance with the previous description of Martins et al. (2018). The reduction of the diameter of the root cross-sections under salt stress conditions found in this study might have resulted from reductions in the size and number of cells, especially in the internal cortex. The alterations of the cell size can be related to resistance to salt stress since smaller cells can indicate an essential response to increase the water potential, possibly contributing to more effective maintenance of turgor under water deficit (Munns and Tester 2008; Terletskaya et al. 2019). Reduced root diameters can be a sign of adaptation to the high pressure of the water column on the conductor system (Rewald et al. 2013; Terletskaya et al. 2019).
The thickening of the cell wall of the exodermis of the roots was modulated by the concentrations of NaCl. This thickening occurs naturally by deposition of lignin and/or suberin, and the degree of thickening can moderate the uptake and translocation of mineral nutrients to the entire plant (Martins et al. 2019). The exposure to excessive NaCl in the shoots induced a thinner exodermis in the roots, which could have been the key to the tolerance of excess NaCl in the shoots. The thinner exodermis permitted a greater flow of nutrients from the culture medium to the shoots, which in turn improved the nutritional balance. Furthermore, in the presence of Si, the thickness of the exodermis was smaller compared to the control plants, showing that Si can act as a modulator and contribute positively to the absorption of nutrients, as previously described by Martins et al. (2019) in A. blanchetiana plants grown in vitro with Si.
In the leaves, the direct exposure to NaCl at the leaf base reduced the stomatal density. Besides this, the epidermis was thicker in the plants exposed to salt. These responses together suggest a morphological adjustment to control the entry of NaCl through the symplastic and transcellular veins (Morton et al. 2019). Considering that plants can also absorb nutrients through the leaves, an increase in the thickness of the epidermis can function as a mechanism to control the absorption of excessive NaCl (Mahmood et al. 2019). It has been suggested that the movement of nutrients to the interior of plants can involve diffusion through the cuticle and absorbed by leaf cells. Absorption through the stomatal pore can also occur since the stomata act as potential pathways for the movement of nutrients applied to the leaves (Li et al. 2019).
In the middle region of the leaves, the stomatal density was greater than in the base region, as previously observed by Santos et al. (2020). However, a comparison of the treatments revealed that Si could influence the morphology of the stomata of other leaf regions. The morphophysiological modulations in the middle leaf region in plants grown with Si, such as smaller stomatal density and size, might have occurred to reduce the osmotic stress (Mahmoudi et al. 2019; Morton et al. 2019). This reduction resulting from the action of Si might be a mechanism to maintain the prompt functioning of the stomata for osmotic control. The size of the stomata is related to their functionality because smaller guard cells respond (open/close) faster than larger ones, and consequently maintain the stomatal conductance (Rouphael et al. 2017). Another alteration observed in this study was an increase in the thickness of the chlorenchyma, apparently related to a trade-off mechanism in which the smaller leaf area is offset by the greater thickness of this tissue (Pereira et al. 2016). This capacity for the protection of the photosynthetic tissues permits maintenance of the plant's biomass production (Pereira et al. 2016; Ribeiro et al. 2019).
The excess of NaCl altered the content of mineral nutrients in A. blanchetiana, namely reduction of the contents of the macronutrients S and Ca and the micronutrients Fe, Zn, and Mn. The excessive accumulation of Na+ competitively inhibits the absorption of other cations, including K+, Ca2 +, and Fe2 +, leading to an imbalance in cell homeostasis, oxidative stress, and interference in the functions of Ca2 + and K+ (Kim et al. 2021). Besides this, the limited availability of Ca can reduce the tolerance of plants to salt stress since this is involved in the gene induction of tolerance to salt stress and regulation of the antioxidant defense (Liu et al. 2019). K plays a fundamental role in synthesizing proteins, photosynthesis, and the activity of glycolytic enzymes in plants (Liu et al. 2019). Therefore, we suggest that reducing the contents of S, Ca, Fe, Zn, and Mn reduced the stress tolerance of the plants, generating oxidative stress and affecting the functioning of the photosynthetic apparatus.
The modulations of the contents of mineral nutrients in A. blanchetiana promoted by Si contributed to improve the nutritional balance and mitigated the damages caused by the toxicity of NaCl in the leaf cells. The increase promoted by Si in the contents of the nutrients Ca, B, Zn, Mn, N, and Mg was probably due to the thinner exodermis in the roots, modulated by Si, which allowed greater absorption of these nutrients. The resulting better nutritional balance contributed to increase the content of photosynthetic pigments and the activity of the enzymes of the antioxidant system (SOD, APX, and CAT). This promoted the protection of the plants' tissues against oxidative damages to the membrane under salt stress, thus alleviating the toxicity of salt and increasing the growth of the A. blanchetiana plants. These nutrients are structural components of the chlorophyll molecule and play a role in forming the amino acids necessary for the processes of the antioxidant defense system, acting as enzymatic cofactors, for example (Rahman et al. 2016; Tewari et al. 2019; Santos et al. 2021). Besides this, the greater activity of the antioxidant system enzymes leads to lower degradation of chlorophyll (Gong et al. 2018). Higher B content contributes to increasing the defense of the antioxidant system and diminishing oxidative stress (Rahman et al. 2021). The increase in the activity of antioxidant enzymes is also responsible for reducing oxidative stress and eliminating the ROS produced during salt stress (Tewari et al. 2019; Zhang et al. 2019; Chung et al. 2020; Kim et al. 2021). In this study, the activity of the antioxidant enzymes was greater in the shoots than in the roots of the plants cultivated in the medium supplemented with Si. This result indicates that the direct exposure to NaCl on the leaves had an impact, generating oxidative stress.
Even though the presence of Na caused stress, as indicated by the biochemical alterations described, this element also appears to play a fundamental role in the metabolism of A. blanchetiana plants, and its absorption in minimum quantities seems to have occurred. Plants grown in the 0 µM NaCl + 14 µM Si condition had greater content of Na than in the control plants (Na added only in the form of Na-EDTA in the MS medium). Other studies have shown that A. blanchetiana has crassulacean acid metabolism (CAM) for the fixation of carbon under adverse conditions (Chaves et al. 2014; Krause et al. 2016). We suggest that even in in vitro conditions, A. blanchetiana plants can have some CAM behavior level, such as reducing leaf area making the leaves more compact. Plants that use CAM metabolism can require sodium ions (Na+) (Scholl et al. 2020). In this species, Na+ seems to be fundamental for the regeneration of phosphoenolpyruvate, the substrate for initial carboxylation in plants with C4 and CAM metabolism (Scholl et al. 2020). CAM metabolism is a mechanism that protects against increased salinity, but the most critical tolerance mechanism can be the accumulation of ions in leaf vacuoles for osmotic adjustment (Montero et al. 2018). In halophytes, the accumulation of Na and its compartmentalization in vacuoles modulate the osmotic potential and help guarantee water absorption under salt stress conditions (Zeng et al. 2015). The Na ions stimulate growth by promoting cell expansion and partially substitute K ions as an osmotically active solute (Hussain et al. 2010). This modulation of the content of Na+ can partly explain the salt tolerance and thus the existence of A. blanchetiana in the sandbank (Restinga) region studied here. Furthermore, this can also explain the increase in the Na/ K ratio with higher salt concentration and the presence of Si observed in this study, which has been confirmed to be one of the main determinants of resistance to salts (Liu et al. 2020). Despite this increase in the Na/ K ratio, the use of Si was responsible for modulating the competitive absorption between Na and K and maintaining the balance in the intercellular distribution of K in the A. blanchetiana plants since the content of K was not different between the treatments.
The morphophysiological modulations promoted by Si, such as the greater activity of the enzymes SOD, APX, and CAT, reduced the stress on the photosynthetic apparatus, as demonstrated by the analysis of the chlorophyll a fluorescence. The plants grown in the medium supplemented with Si had the highest values of qP and qL, implying a more remarkable ability for photochemical conversion and transfer of electrons from PSII (Wang et al. 2018). This suggests that even though the plants grown with high NaCl suffered photodamage, the Si was able to ameliorate this damage by maintaining a proper balance of nutrients, as well as enhancing the activity of the antioxidant system, impeding oxidative damages to the photosystems (Liu et al. 2020). The Si also contributed to maintain the electron transport, as evidenced by the higher FV/FM ratio, indicating greater potential photochemical activity of PSII (Lotfi et al. 2018). Factors for the photosynthetic apparatus's functioning were also evidenced by the lower values of the parameters of non-photochemical quenching, such as qN, ΦNPQ, and NPQ, in comparison with the plants grown without Si. These responses helped to reduce the damages to the plants caused by the stress, which in turn helped to maintain the plants' growth since they had greater fresh weight when cultivated with higher concentrations of NaCl. The excess of NaCl in plants grown without Si caused increases of qN, ΦNPQ, and ΦNO, leading to over-reduction of the photosynthetic electron transport chain, excess excitation energy, and consequently reduction of the photochemical step and biochemical processes. The increase of ΦNPQ indicates that the regulation of the excitation energy loss occurred by dissipation of the heat involving the mechanisms dependent on ΔpH and zeaxanthin and by means of the xanthophyll cycle (Wang et al. 2018; Shu et al. 2019). Furthermore, the increase of ΦNO indicates that this energy loss did not involve the action of trans-thylakoid ΔpH and zeaxanthin, meaning the excess flow of energy was out of control (Yao et al. 2018; Wang et al. 2018).
The increase of the stress level caused by NaCl affected the functioning of the photosynthetic apparatus by reducing the values of ΦPSII and ETR. The decrease might have partly inhibited the transport of electrons and effective photochemical activity of PSII and increased the formation of ROS since the activity of the antioxidant system was affected. This reduction indicates a smaller density of the flow of photons absorbed by PSII (Wang et al. 2018). These responses induced by excess of NaCl in the absence of Si caused a reduction of the plants' growth. The decline of the fresh and dry weights of the leaves and roots, and thus the reduction in the plants' growth, are symptoms commonly observed in plants under salt stress (Dias et al. 2017). This result can be attributed to the osmotic effect of the salt solution beyond the roots, as well as an imbalance in the absorption and assimilation of nutrients (Dias et al. 2017; Rezende et al. 2018).