Ca addition alleviated Cd physiological toxicity
Cd stress induces alterations in photosynthetic rates, photosynthetic pigments, chlorophyll fluorescence, and nutrient homeostasis [39, 40]. Photosynthesis is especially sensitive to Cd. The chlorosis of leaves is one of the first visible symptoms of Cd toxicity, which is due to decreased rates of chlorophyll biosynthesis and chlorophyll contents caused by damage to thylakoid membranes . In this study, chlorosis symptoms in S. matsudana leaves exposed to different Cd concentrations were observed. This phenomenon was more obvious as Cd concentrations increased. The addition of Ca effectively alleviated Cd toxicity in Cd-treated plants. A previous study indicated a internal mechanism for depressing the Cd toxicity as Ca concentrations increased in plant roots exposed to Cd, that when both Cd and Ca exist in the soil system, Ca and Cd exhibit similar chemical properties, Ca competes with Cd at adsorption sites in the soil, as a result, Cd uptake is reduced by Ca, then reducing Cd toxic effects in plants [7, 13, 20, 23].
Chlorophyll fluorescence is an effective measure of photosynthesis in light reactions. Fv/Fm and Y(II) are representative fluorescence parameters that are widely used to evaluate the effects of environmental stress on plants . Chlorophyll fluorescence depends, to a great extent, on pigment contents and the capability of leaves to photosynthesize. In this study, the effects of Ca on Cd-induced damage in S. matsudana were investigated. The chlorophyll fluorescence parameters, F0, Fm, Fv/Fm, Y(II), and qP, significantly decreased and qN increased in S. matsudana after treatment with 50 µM Cd. Moreover, changes in these parameters revealed the damage of Cd toxicity to the photosynthetic apparatus. Cd disturbed the photosynthesis reaction center and restrained this process . Ge et al.  also indicated that Cd reduced Fe contents, which resulted in decreased chlorophyll contents in Populus leaves. This study demonstrated that the addition of exogenous Ca inhibited the decrease of F0, Fm, Fv/Fm, YII, and qP and the increase of qN, indicating that Ca played a positive role in this process. He et al.  found that exogenous Ca enhanced the electron transport capacity of cucumbers and reduced stress-induced damage. Moreover, exogenous Ca application increased the net photosynthesis rate, stomatal conductance, intercellular CO2 concentration, and maximum quantum efficiency of photosystem II photochemistry, YII, and qP .
Ca addition changed Cd uptake and translocation
EDXA is an analytical technique used for analyzing the elemental subcellular localization in biological specimens[31, 44]. The results by scanning electron microscope (SEM) and EDXA revealed that the Cd contents of root epidermis, cortex, and vascular cylinder cells in Cd-treated roots were lower than those in Cd + Ca-treated roots. Moreover, Cd absorbed in the roots passed through the root epidermis, cortex, and endodermis and was transported to the shoots in Cd and Cd + Ca-treated S. matsudana (Fig. 3). The Cd adsorption through the symplastic pathway was depressed in Cd + Ca-treated plants but enhanced in Ca-treated plants. However, the exact role that Ca addition plays in Cd uptake and transportation still remains unclear as no specific Cd transporter was ascertained in previous studies. Thus, Cd may be absorbed by metal transporters or through a similar mechanism, such as ZIP, NRAMP, or HMA. It was reported that the AtZIP2 and AtZIP4 expression levels correlated with Cd concentrations positively in Cd-treated plants, indicating that the Cd absorption by AtZIP2 and AtZIP4 depended on Cd concentrations, but after Ca application, their expression levels decreased obviously . Similar results in the expression levels of NRAMP1 and ZIP8 in S. matsudana were observed in this study (Fig. 2A). SmNRAMP1 and SmZIP8, which are involved in Cd absorption and transportation, exhibited higher expression levels in Cd-treated plants than Cd + Ca-treated plants. The investigations of Nakanishi et al. may explained the subtle changes in the expression level of IRT1 . It was concluded that Ca reduced Cd adsorption because adding Ca changed Cd transport process. Notably, the gene expression results uncovered an important fact that may lead to Cd uptake differences in various treatments of this study.
In previous studies, the addition of Ca decreased the amount of Cd in whole plants, including Arabidopsis thaliana , Zea mays , Trifolium repens , Brassica napus , Lens culinaris , Matricaria chamomilla , and Boehmeria nivea . Similar results were obtained in this study. The Cd contents in Cd + Ca-treated plants were significantly lower when compared to treatments with Cd alone, regardless of the dose (Table 2). Moreover, the TF of Cd in Cd + Ca-treated plants was higher than that in Cd- treated alone. Additionally, the changes in SmHMA2 and SmHMA4 expression levels in this study were induced by the Cd and Cd + Ca treatments (Fig. 2B), which confirmed that HMA2 and HMA4 participated in the upward-translocation of Cd. Similarly, the effluxion of Zn and Cd was resulted in by overexpression of HMA4 in yeast and E. coli, indicating that HMA4 played an important role in Cd xylem uploading [53, 54]. It was further noted that adding Ca promoted the xylem-loading and upward transportation of Cd based on the expression levels of related gene.
The radial translocation of Ca from the roots to shoots was promoted by the B. juncea plant cadmium resistance 1 (BjPCR1) protein, the transportation of Ca to the shoots was disturbed and the plant growth was inhibited by knock-out of BjPCR1[55, 56]. The expression levels of SmPCR1 was up-regulated in this study suggested that it play a major role in Ca adsorption and transportation. It was reported that Ca accumulate mainly in the epidermal cells and trichomes, which played an important role in some accumulative plant species, which was dependent on the plant species .
Ca addition induced Cd subcellular redistribution
The results of Fig. 4 revealed that Cd accumulated mainly in leaf epidermal and mesophyll cells, which was greater than in leaf main veins or guard cells. These results suggested that the leaf epidermal and mesophyll cells were the main site of Cd. Huguet et al.  also found that in leaves Cd accumulated mainly in leaf edges, and less concentrated in regions around leaf vascular bundles. Leitenmaier and Kupper  also reported that the Cd uptake rate in epidermal storage cells was greater than in epidermal or mesophyll cells. Shi et al.  observed high Cd levels in the epidermis, veins, and stomata near necrotic spots on leaves exposed to Cd. Cd accumulation and cell death have obvious relevance with the leaves exposed to Cd. After the addition of Ca, the Cd contents in the 3 leaf regions all decreased, and the proportion of Cd in leaf epidermal cells significantly increased, indicating that Ca application led to the redistribution of Cd in S. matsudana leaves. Based on these findings, it was assumed that the reduction of Cd toxicity in the leaves may have resulted from the Ca regulation of Cd contents in leaf epidermis cells. However, further investigations are required to verify these results.
The Cd contents of different subcellular fractions have extremely important influences on Cd uptake, translocation, distribution and detoxification in different plant species [60–62]. The data of this study demonstrated that Cd (86.7%–97.7%) in S. matsudana leaf and root cells was stored in the cell wall and soluble fractions. Cd (66%–77%) in S. matsudana roots was stored in the cell wall, while a small quantity of Cd was located in organelle fractions. However, in S. matsudana leaves, Cd (45%–55%) was found in soluble fractions. These results are the same as the findings of Huang et al.  and Lu et al. . Moreover, Wu et al.  also indicated that Cd was located in the cell wall of S. matsudana roots after treatment with 50 µM Cd. The cell wall is the first protective barrier that protects the protoplast from Cd toxicity and mainly comprised polyose and proteins, which can bind Cd ions on their surfaces and limit Cd migration across the cell membrane [21, 23, 60]. When Cd ions enter the cytosol, it is chelated by phytochelatin to form metal complexes in order to minimize Cd stress [25, 63, 64]. Wang et al.  found that most Cd in the cytosol was transformed into less toxic chemical forms and translocated into the vacuole, thereby minimizing Cd toxicity. Ma et al.  also reported that the internal detoxification of Cd and Zn in Thlaspi caerulescens leaves was achieved by vacuolar compartmentalization.
In this investigation, the application of Ca altered Cd subcellular distribution in S. matsudana, uncovering an important process of Ca alleviating Cd stress in plants (Fig. 5). After the addition of Ca, the percentage of Cd in the cell walls of S. matsudana roots and leaves decreased, while the percentage of Cd in soluble fractions increased. These results suggested that S. matsudana avoided Cd stress by chelating the free Cd in the protoplasm to form metal complexes, thereby fixing Cd in the vacuole through compartmentalization. Similarly, Choi et al.  found that seedlings treated with Cd in the presence of Ca exhibited increased tolerance, which was proportional to increases in Ca concentrations.
Ca addition modified Cd chemical forms
The chemical form of Cd in plants is very important and reflects the degree of Cd migration and toxicity. Different Cd chemical forms are connected with various Cd biological activities in plants . Cd in inorganic forms (CdW) and organic forms (CdE) are more mobile than other chemical forms, and more toxic to plant cells. Pectate- and protein-integrated Cd (CdNaCl), insoluble Cd phosphate (CdHAc), and Cd oxalate (CdHCl), are less mobile and less toxic to plant cells [60, 68–70]. In this study, Cd concentrations of different chemical forms in S. matsudana roots and leaves increased as Cd concentrations increased (Fig. 6). Cd extracted with 1 M NaCl and 2% HAc was the predominant chemical form in S. matsudana roots and leaves, while Cd extracted with other extracting solutions was rather low (Fig. 6). These results are consistent with earlier findings [25, 64, 71, 72, 73]. Wu et al.  demonstrated that NaCl extractants combine to pectic acids and proteins to which Cd was fixed. In S. matsudana roots and leaves, the proportion 1 M NaCl-extracted Cd decreased, while the proportion of 0.6 M HCl- and 2% HAc-extracted Cd increased as Cd concentrations increased from 10 to 50 μM (Fig. 6). The results suggested that, as Cd concentrations increased, Cd may have transformed into inactive metal complexes to protect the cells. Li et al.  also demonstrated that converting Cd into non-toxic pectate- and protein-bound forms could minimize the Cd toxicity. Qiu et al. drew the same conclusion that the Cd in pectate- and protein-chelated forms was correlated with Cd bound to cell wall fractions in B. parachinensis , then limited Cd translocation from roots to shoots .
The addition of Ca reduced the contents of different Cd chemical forms in S. matsudana (Fig. 6). The addition of Ca increased the percentage of 1 M NaCl-extracted Cd, but reduced the proportion of 0.6 M HCl- and 2% HAc-extracted Cd in S. matsudana leaves and roots compared to treatments with Cd alone, indicating that a larger proportion of Cd existed in the form of pectate- and protein-integrated Cd and non- or low-toxic complexes. A recent study also reported that the application of Ca may stimulate production of more peptides and proteins that can easily combine with Cd to alleviate Cd toxicity in plants . However, more research needs to be conducted in this area.
Cd and Ca interactions
In this study, the addition of Ca decreased the uptake of Cd in S. matsudana roots by altering its adsorption mode. Ca application also promoted Cd transportation from roots to shoots and modified Cd subcellular localization and its chemical forms in both roots and leaves to alleviate Cd toxicity (Fig. 7). Based on the collective findings, it is likely that Ca and Cd compete at Cd adsorption sites in the soil as they possess similar chemical properties. When both Ca and Cd exist in the soil system, Ca reduces Cd uptake, thus reducing Cd toxicity in plants [19, 45]. Apart from reducing Cd uptake, exogenous Ca alleviated Cd toxicity in S. matsudana by decreasing the percentage of Cd in the cell wall of roots and leaves and increasing the percentage of Cd content in soluble fractions, suggesting that plants minimize Cd toxicity by combining the free Cd in the protoplasm to form metal complexes, which further fixed Cd in the vacuoles through vacuolar compartmentalization. Exogenous Ca alleviated the toxicity of Cd in S. matsudana as well by changing the percentage of Cd chemical forms of 2% HAc- and 0.6 M HCl-extracted Cd to 1 M NaCl-extracted Cd .
Additionally, Toyota et al.  found that glutamic acid, a stress and mechanical damage signaling substance, transformed the signal due to the increased Ca2+ concentrations in the cytoplasm, thus spreading the signal to distal organs and inducing defense responses. In this study, the addition of Ca increased Ca concentrations in the cytoplasm. Therefore, it is proposed that similar processes, also referred to by Toyota et al. , were initiated in S. matsudana and that Ca coordinated with Cd, which led to Cd subcellular redistribution and modified Cd chemical forms by changing the expression levels of transporters involved in Cd xylem loading and upward-translocation in both the roots and leaves.