In this study, we investigated the interaction between Si and Cd during the in vitro culture of A. tenella. The addition of Si to the culture medium resulted in plants that were more tolerant of the stress caused by Cd.
Alterations in the leaf anatomy play a fundamental role in plants' adjustment to environmental conditions. In this study, the first stress-regulation mechanisms were related to the investment in the number of stomata (stomatal density and stomatal index) in the function of Cd concentrations. These adjustments in anatomy were reflected in a decrease of the transpiration rate and mass flow, diminishing the uptake and translocation of mineral elements present in the soil or culture medium to the aerial part (Pereira et al. 2017; Martins et al. 2020; Pires-Lira et al. 2020). These alterations were in line with the number of vessel elements (xylem). A smaller number of vessels tends to reduce the translocation of water and nutrients, as well as of Cd, to the aerial part of the A. tenella plants. This observation is in accordance with the Hagen-Poiseuille law, which indicates that a reduction in the vessel elements' number and/or diameter has a negative exponential effect on the water conductivity (Scholz et al. 2013). Therefore, these combined alterations of the stomatal architecture and vessel elements might have been preponderant in controlling the translocation of Cd in the A. tenella plants.
The smaller flow of nutrients and water from the culture medium influenced the morphogenesis of the parenchyma tissues. The palisade parenchyma thickness declined, mainly in the plants grown with 100 and 200 µM of Cd. The reduced thickness of this tissue can be related to the smaller translocation of water, which interferes with cell expansion (Silva-Cunha et al. 2021). This process is fundamental for the growth of cell size and thus of the tissue as a whole. Likewise, the exposure to Cd led to thinner spongy parenchyma, correlated with the reduced size of the cells. Reducing this size is a mechanism to maintain functionality under stressful conditions because smaller cells tend to have a greater ability to maintain their turgor when exposed to water shortage than larger cells (Corso et al. 2020). Nevertheless, the plants cultivated with 200 µM Cd, regardless of the presence of Si, had thicker but statistically similar spongy parenchyma than the plants cultivated without Cd. This increase in the spongy parenchyma thickness of the plants exposed to 200 µM Cd was correlated with an increase in the intercellular spaces but not with the size of the cells. It makes sense because the intercellular spaces of the spongy parenchyma have the function of maximizing the surface area available for light capture and gas exchange for photosynthesis (Zhang et al. 2021). Besides this, the formation of larger intercellular spaces in spongy parenchyma can optimize the accumulation of CO2 and the accessibility of carboxylation sites on the chloroplasts inside the leaf cells (Acosta-Motos et al. 2015; Paradiso et al. 2017), in turn requiring less frequent stomatal opening for gas exchange. This morphological response of the spongy parenchyma is often related to water deficit (Acosta-Motos et al. 2015; Rouphael et al. 2017; Mott and Peak 2018). In this study, the A. tenella plants cultivated with 200 µM Cd presented an accentuated reduction in leaf size (leaf area) besides thicker spongy parenchyma. This suggests a compensatory mechanism (tradeoff) to maximize the accumulation of CO2 and minimize the evapotranspiration, preventing an increase in the mass flow and translocation of Cd.
One of the ways to determine physiological alterations and damages to the photosynthetic apparatus is by quantifying the contents of photosynthetic pigments in the leaves since plants under stress tend to have lower contents and modifications of their proportions to balance the levels and/or reduce the damages to the photosystem (Janečková et al. 2019). In the present study, we observed reductions in the contents of Chl a, Chl b, Car, and ChlTotal with rising concentrations of Cd in the culture medium, independently of the presence or absence of Si. Cd can cause modifications in the structure of the photosynthetic pigments due to competition for the binding sites of Mg2+ in the pheophytin ring and/or inhibition of the synthesis of the enzyme 5-aminolevulinic acid, which plays an important role in the biosynthesis of chlorophylls (Grajek et al. 2020); besides overloading of the antioxidant system, causing misshapen chloroplasts, dilation of thylakoid membranes and instability of chlorophyll (Pereira et al. 2017; Rodrigues et al. 2017). However, in the presence of Si, this decrease was less severe. That response might have been associated with the improvement of the antioxidant system, and therefore lesser oxidative damages of the structure and function of the thylakoid membranes (Yanhui et al. 2020). That hypothesis is corroborated by the values of WL obtained.
The Chl a/b ratio is a good indicator of how plants respond to the influence of metals on their photosynthetic apparatus (Ranjbarfordoei et al. 2006; Martins et al. 2020a, b; Houri et al. 2020). In this study, we observed a coordinated reduction of Chl a and b, but no significant change in the ratio, particularly in the treatments with 50 and 100 µM of Cd, indicating the efficacy of the plants' metabolism in minimizing the harmful effect of the excess of this metal. In contrast, the reduction of Chl a/b ratio in the treatment with 200 µM Cd indicated an imbalance between the contents of Chl a and Chl b, besides a high degradation rate of Chl a. In the presence of Si, the Chl a/b ratio of plants exposed to 200 µM Cd did not decline as much as in the plant cultivated with the same concentration of Cd but without Si. This response denotes lesser degradation of Chl a, indicating less damage to the active reaction centers (RCs) and hence in the performance of photosystem II (PSII) (Martins et al. 2021).
Alterations in the performance of the photosynthetic apparatus also demonstrated the effects of co-exposure to Si and Cd. A point-by-point analysis of the OJIP curve revealed the influence of this co-exposure and the response in the apparatus. At points VL and VK, we observed an increase in the plants grown with 200 µM Cd, irrespective of the presence of Si. This alteration at the highest Cd concentration indicated less energy connectivity of the PSII units and less efficiency of the oxygen-evolving complex (OEC) (Brestic et al. 2012; Begovic et al. 2020; Martins et al. 2020b). According to Zhang et al. (2018), an increased value of VK can be considered a specific marker of damage to the activity of the OEC from the electron donor side of PSII. In turn, alterations of the values of VJ and VI indicate the functioning of electron transport between the quinones (QA and QB) on the receptor side of PSII and changes in the efficiency/probability of movement of electrons between the photosystems, respectively (Santos et al. 2020; Martins et al. 2021). In this study, we observed that the presence of Si was associated with better electron transfer between the quinones and between PSII and PSI.
Another indicator to identify physiological disorders before visual manifestations of damage is the appearance of L- and K-bands. Positive L-bands indicate disturbances in the thylakoid membranes, reducing the connectivity between the CRs and PSII and causing less energy clustering among the photosystem units (Strasser et al. 2004). In other words, the behavior was denoted by an inverse function, in which a higher positive amplitude of band-L is associated with lesser connectivity. A positive band-K is closely related to an inactivation of the OEC (Xiang et al. 2013). We observed that the connectivity between CRs was maintained in the treatment with the highest concentration of Cd and the addition of Si. This maintenance may be attributed to the improvement of the antioxidant system, causing the values of WL to decline. According to Zhang et al. (2018), lower values of this parameter denote better functional and structural integrity of the thylakoid membranes. In counterpart, we observed an increase of the amplitude of band-K and the values of WK in the treatments with 200 µM Cd regardless of the presence of Si. This elevation might have been due to water restriction caused by the reduction of water connectivity (a characteristic determined by the stomatal and vessel elements), in turn causing an imbalance between the transfer of electrons from the OEC to P680+ and the electron acceptors of QA, along with possible partial inhibition of the water-splitting system.
The association among FV/F0, φP0, and φE0 allows analyzing the status of the electron transport system and the efficiency of the trapping, conversion, and transport of energy between the two photosystems (Strasser et al. 2004; Guo et al. 2020). In this respect, the presence of Cd caused a reduction of the values of φP0, FV/F0, and φE0 in the treatment with 200 µM Cd, resulting in lower photosynthetic apparatus efficiency. In contrast, when the plants were co-exposed to Cd and Si, the decrease in the values of those parameters were not as great (in relation to the control treatment), serving as an indicator of maintenance of the system for trapping, conversion, and transport of energy. This could have caused lesser dissipation of the energy (φD0).
The exposure to Cd also generated an increase in the values of ABS/RC, TR0/RC, and DI0/RC, indicating the susceptibility of the plants to photoinhibition caused by the down-regulation of the mechanism for dissipation of the energy absorbed by the reaction center (RC) (Franić et al. 2018). However, the presence of Si generated an adjustment that ameliorated the damages caused by the Cd. This improvement triggered an increase of ET0/RC and reduced energy loss by dissipation. At the highest Cd concentration, the Si also caused a less pronounced reduction of RC/ABS, corroborating the results found for the Chl a/b ratio.
The performance indices PI(ABS) and PI(Total) indicate how the stress factors affect the performance and integrity of the photosynthetic apparatus of plants (Kalaji et al. 2016). In particular, because PI(ABS) represents the combination of three factors (total number of active reaction centers for absorption of light; trapping of excitation energy; and conversion of that energy into the transport of electrons in PSII), and this parameter is sensitive to the stress caused by the metal (Martins et al. 2020; Guo et al. 2020). Therefore, the presence of Si reduced the damage to the system for trapping of excitation energy, resulting in a better performance of PSII, mainly at the highest Cd concentration. The Si also acted to reduce the photodamage beyond the intersystem, mainly at the highest Cd concentration, confirmed by the increased values of PI(Total). According to Guo et al. (2020), the increase of this parameter when plants are facing high stress is an indicator of improved ability to achieve photochemical reactions, i.e., their efficiency in using the energy absorbed by the antennas for conversion into energy in the form of ATP and NADPH, resulting in better physiological conditions for development and survival.
In summary, the presence of Si in the culture medium caused a reduction of the damages caused by Cd in the physiology of the plants, resulting in greater in vitro growth. In plants cultured with 40 µM Si, there was a greater biomass accumulation, mainly at the Cd concentrations of 100 and 200 µM, compared to those cultivated without Si. That response had a direct impact on the tolerance index (TI). According to Lux et al. (2004), the TI is classified as low when the values are below 35%; intermediate with values between 35% and 60%; and high when above 60%. In this work, the A. tenella plants exposed to 100 and 200 µM of Cd alone showed the lowest tolerance index values (53.31% and 28.06%, respectively). However, when the medium was supplemented with Si, the plants exposed to Cd were more tolerant (≥61.65%). Therefore, we can state that Si can mitigate the deleterious effects of Cd in A. tenella plants because these plants were more physiologically tolerant.