Cadmium effect on biomass yield and tillering of P. maximum used for phytoextraction in mildly polluted Oxisol along of two successive shoot growths
The exposure of P. maximum to the highest Cd concentrations decreased both leaves and stems biomass yield in the growth period (Figs. 2A-B), but there was no reduction on the number of tillers per plant in this growth period (Fig. 3A). Thereby, the reduction observed on shoot biomass yield can be attributed to a Cd-induced reduced number of leaves and shortening of stems and leaves (Supplementary Fig. 2). As gibberellins regulate the stem elongation rate in grasses (Zhang et al. 2016), Cd may have repressed gibberellins synthesis in P. maximum by affecting the KNOTTED1- like homeodomain (KNOX) proteins. KNOX proteins either activate or repress gibberellins synthesis genes, modifying levels of active gibberellins in the meristems and boundary regions of grasses (Pautler et al. 2013), which is the tillers initiation region (Chrysler 1906). Cadmium-induced changes on shoot meristematic region are also pointed out as a factor to reduce the number and length of leaves in plants of the family Poaceae. The decreased leaf length Cd-induced in maize (Zea mays) grown in a mildly polluted soil (46.5 mg Cd kg−1 soil) was attributed to the lower number of meristematic cells, longer cell cycle duration and inhibition of cell elongation rate (Bertels et al. 2020).
During the regrowth, leaf and stem biomass yields of P. maximum exposed to the highest Cd concentrations did not differ from those plants of control treatment (Figs. 2A-B). These data indicate that P. maximum was able to cope with Cd-induced stress under prolonged exposure by adapting its mechanisms of tolerance against Cd-induced stress (for a comprehensive review we suggest Rabêlo et al. 2021a). Such assumption is supported by the fact the number of tillers increased during plant regrowth compared to growth period (Fig. 3A), even the basal node (tiller initiation region) presenting high Cd concentrations (Fig. 4F). Moreover, there was not the trend of Cd accumulated in the basal node reduces the number of tillers during regrowth, differently from which was observed in the plant growth (Fig. 3B). It means that Cd probably is more harmful in the early stages of development of P. maximum grown in mildly polluted soils. Sunflower (Helianthus annuus) is also more susceptible to Cd-induced stress in the early stages of development because an uncontrolled Cd uptake that results in high Cd concentrations in its plant tissues (De Maria et al. 2013). However, as Cd concentrations in the leaf and stem tissues collected at the end of the growth and regrowth period were similar (Figs. 4A-E), other factors than Cd concentrations in the plant tissues limited the growth of P. maximum exposed to the highest Cd concentration in the first growth.
Interestingly, a decrease in Mg concentration in the leaves has been associated with plant protection against Cd-induced stress under prolonged exposure by improving the action of the antioxidant system (Chou et al. 2011; Hermans et al. 2011). Such mechanism possibly was employed by P. maximum, since lower Mg concentrations were observed in the leaves compared to stems, in the plants exposed to the highest Cd concentration compared to the other Cd concentrations, and in the regrowth compared to growth period (Table 2). This conferred higher tolerance to plants exposed to the highest Cd concentration in the regrowth period, but not in the growth period. Other nutritional adjustments occurred in P. maximum under Cd exposure (Table 2) and probably contributed for a higher Cd tolerance in the regrowth compared to growth period. Such nutritional adjustments are addressed in the next session.
As the number of tillers of P. maximum was higher in the regrowth compared to growth period (Fig. 3A), we can assume that plant density was higher in the regrowth period. Under such circumstance, plants show a clear increase in the stem fraction due to changes on carbohydrates allocation (Poorter et al. 2012). This explains why the stem biomass yield was higher and the leaf/stem ratio was lower in the regrowth than growth period (Figs. 2B and 3C). Furthermore, there was no reduction on basal node biomass under Cd exposure (Fig. 2B), which may have contributed for the higher tillering in the regrowth period, since the tillers grow up from the axillary buds located at the basal node of the plant (Chrysler 1906). As observed for basal node biomass, the root biomass of P. maximum did not decrease due to Cd exposure (Fig. 2D), even this structure presenting high Cd concentrations (Fig. 4G). Maybe such result is associated to the fact that P. maximum preferentially accumulates Cd bound to cell wall in the root apoplast (Rabêlo et al. 2021b). In this case, the deleterious effects of Cd are more noticeable on root length and root surface than root weight (Rabêlo et al. 2020b), since the thickening of the roots due to lignification and suberization (Lux et al. 2011) can compensate the root weight.
Distribution and accumulation of Cd within the plant tissues of P. maximum and its relationship with Cd phytoextraction efficiency and nutritional disorders
Cadmium concentration increased in all plant tissues of P. maximum as a consequence of Cd exposure, but our results suggest the existence of restrictive mechanisms on Cd translocation from lower to upper plant parts because Cd concentration followed a decreasing gradient in the sequence: basal node > stems > leaves (Fig. 4). During Cd translocation in the xylem, Cd2+ ions interact with the cell walls of xylem vessels and are partly adsorb on them (Sterckeman and Thomine 2020). Furthermore, Cd accumulated in the leaves can be redistributed to other plant organs via phloem or even to roots where Cd could be excreted (Sterckeman and Thomine 2020). Thus, with exception of hyperaccumulators plants, a restriction on Cd translocation upwards is expected, mainly under higher Cd exposure. Cadmium translocation from roots to shoot was higher when P. maximum was grown in the Oxisol presenting the available Cd concentration of 2.86 mg kg−1 soil, but from this point there was a reduction on Cd TF (Fig. 5F). Our results are similar to those reported for lettuce (Lactuca sativa), spinach (Spinacia oleracea), cauliflower (Brassica oleracea) and oat (Avena sativa), in which Cd concentrations were higher in the shoots than roots when plants were grown on low Cd-polluted soil, but Cd concentrations in the roots became higher than of the shoots when these plants were grown on more polluted soil (John 1973). Similarly to Cd TF, the higher Cd BCFs were observed when P. maximum was exposed to the available Cd concentration of 2.86 mg kg−1 soil, and from this point the Cd BCFs decreased (Figs. 5A-E). Although the two factors remained higher than 1 under Cd exposure, Cd TF and Cd BCFs decreased under the highest Cd concentrations in the Oxisol, indicating lowered phytoextraction efficiency in such conditions. The potential of phytoextraction tends to decrease when grass species are faced to more high Cd concentrations due to Cd-induced toxicity, such as nutritional disorders (Rabêlo et al., 2021a).
Nutritional disorders are common in grasses exposed to Cd (Rabêlo and Borgo 2016), which can decrease Cd phytoextraction efficiency (Rabêlo et al. 2020a), even the lower NUE of P, K, Ca, S, Cu, Fe, Mn and Zn observed in P. maximum under Cd exposure (Table 3) did not negatively correlating with Cd BCF (Supplementary Fig. 1). Changes on nutrients´ concentration and use are coupled to negative outcomes on the development of plants under Cd exposure, but there is evidence that nutritional adjustments are necessary for plants cope with Cd stress (for a review, see Carvalho et al. 2020). For instance, a reduction in leaves Mg concentration of plants exposed to Cd can improve the action of the antioxidant system (Chou et al. 2011; Hermans et al. 2011), making possible an increase on biomass yield (Carvalho et al. 2020), as observed in our study (Figs. 2A-B; Table 2). Another example is K, which is involved on biomass allocation due its role on carbohydrates loading into the phloem for long-distance transport. Under lower K concentration sucrose export into the phloem is reduced (Cakmak et al. 1994), but increased K+ may promote sugar unloading in sink tissues and speed the conversion of sucrose to synthetic metabolites (Conti and Geiger 1982), which allow biomass yield. Thus, the increase in the stem biomass yield induced by the higher plant density in the regrown P. maximum (Figs. 2B and 3A), especially in those plants exposed to the highest Cd concentrations, possibly is associated with the increased K concentrations verified in the stems, in the regrown plants and in the plants exposed to the highest Cd concentration (Table 2).
The concentrations of P, S and Cu of P. maximum increased after Cd exposure (Table 2). Under high P concentrations, more P is accumulated in the root and may form insoluble phosphate precipitates with Cd in the cell wall and vacuoles, which prevents the transport of Cd to the protoplasm and xylem and inhibits the transport of Cd to the shoot (Guo et al., 2018). Indeed, Cd TF was reduced in P. maximum exposed to the available Cd concentrations of 5.93 and 10.91 mg kg−1 soil (Fig. 5F), where higher shoot P concentrations were found (Table 2). Plants under Cd exposure tends to uptake more S due its role on glutathione (GSH, γ-Glu-Cys-Gly) and phytochelatins [PCs, (γ-Glu-Cys)n-Gly, with n = 2-11] synthesis, which are peptides involved in plant tolerance against to Cd-induced stress (for a comprehensive review we suggest Gill and Tuteja 2011). Sulfur concentration was higher in the growth than regrowth period (Table 2), which makes sense, since Cd is more stored as chelates (e.g., PC-Cd) in the vacuoles of plants in the early stages of development, whereas other detoxification mechanisms (e.g., Cd bound to cell walls) are more employed under prolonged Cd exposure (Rabêlo et al. 2018, 2021a; Sterckeman and Thomine 2020). As an increase on antioxidant activity of P. maximum was speculated due to decreased Mg and increased S concentrations, an increase on Cu concentration due to Cd exposure, especially in the growth period (Table 2), makes sense since Cu is a cofactor of the enzyme superoxide dismutase (SOD, EC 126.96.36.199) (Gratão et al. 2005). Superoxide dismutases, such as the isoenzyme Cu/Zn-SOD, act as the first line of defense against reactive oxygen species by dismutating superoxide (O2•−) in H2O2 (Gratão et al. 2005). In addition, the presence of Cu/Zn-SOD in the apoplast of spinach was positively correlated to sites of lignification (Ogawa et al. 1996). In this sense, is plausible to assume that the higher Cu concentration observed in the roots of P. maximum (Table 2) favored root lignification through the action of Cu/Zn-SOD in the root apoplast (main local of Cd storage in this species; Rabêlo et al. 2021b), which avoid a strong reduction on the root weight of plants exposed to the highest Cd concentrations in the Oxisol due to a root thickening (Fig. 2D).
The concentrations of B and Mn tended to decrease in the plant tissues of P. maximum exposed to the highest Cd concentrations, differently from which was observed for P, K, S and Cu (Table 2). In tomato (Solanum lycopersicum), Cd toxicity was related to B and Mn excess in leaves, in addition to the own Cd accumulation, since the symptoms of Cd toxicity in leaf tissues resembled those triggered by B and Mn excess (Carvalho et al. 2018). It is possible that P. maximum had decreased both B and Mn concentrations in its tissues as a strategy of adaptation to Cd-induced stress, as no visual symptoms similar to those triggered by B and Mn excess were observed in our study (Supplementary Fig. 2).
The most part of nutrients´ concentrations (Table 2), if not all, indicate that P. maximum cv. Massai poses strategies to cope with Cd-induced stress through nutritional adjustment (Carvalho et al., 2020). Even so, the NUE of P, K, Ca, S, Cu, Fe, Mn and Zn by P. maximum decreased under Cd exposure (Table 3). Lower NUEs in plants grown in polluted soils are expected due to phytotoxicity or internal adjustments which affect plant growth in such conditions (Baligar et al. 2001). Although there were no significant negative correlations between the NUEs and Cd BCFs (Supplementary Fig. 1), the data of NUE (Table 3) together with Cd TF and Cd BCFs (Fig. 5) support the statement of Rabêlo et al. (2021a), who described that the potential of phytoextraction of grass species faced to more high Cd concentrations tends to decrease due to Cd-induced toxicity.