Ca alleviated Cd-induced toxicity in Salix matsudana by affecting Cd absorption, translocation, subcellular distribution, and chemical forms

Background: Cadmium (Cd), a ubiquitous and highly toxic heavy metal pollutant, is toxic to animals and plants. Calcium (Ca) is an essential component for plant growth and reduces plant Cd adsorption by competing with Cd. To gain deeper insight into the effects of Ca on Cd absorption, translocation, subcellular distribution, and chemical forms in S. matsudana seedlings under Cd stress, an investigation was conducted on these properties. Results: Adding Ca alleviated Cd physiological toxicity in S. matsudana, reduced Cd adsorption, increased the translocation from roots to shoots, lead to subcellular redistribution of Cd by increasing the proportion of Cd in soluble fractions but decreasing Cd in the cell wall and changed the chemical forms of Cd from 0.6 M HCl- and 2% HAc-extracted Cd to 1 M NaCl-extracted Cd. The energy dispersive X-ray analyses (EDXA) results revealed that after adding Ca, Cd was transferred through the root epidermis, cortex, endodermis, and vascular cylinder, transported to the shoots, and was highly accumulated in leaf epidermal and mesophyll cells, but less in leaf vein and guard cells. The genes involved in Cd uptake and xylem loading included NRAMP1, ZIP8, HMA2, and HMA4, which were up-regulated significantly (p < 0.05) in the Cd and Cd + Ca treatments compared to the control. Conclusions: The findings of this study provide new insight into the mechanism that Ca alleviates Cd toxicity in woody tree species, as well as propose an important prospect of Ca addition for improving the phytoremediation of Cd contamination.

The EDXA data also uncovered the subcellular localization of Cd in the leaves exposed to 50 µM Cd and 50 µM Cd + 5 mM Ca after 28 d (Fig. 4). The Cd contents in the three zones of the leaves exposed to Cd differed and were ordered as follows: leaf epidermal cells (1.05 wt%) > leaf main vein (0.69 wt%) > leaf epidermal stomata (0.40 wt%). After the addition of 5 mM Ca, the Cd contents of the three leaf zones were ordered as follows: leaf epidermal cells (0.88 wt%) > leaf main veins (0.58 wt%) > leaf epidermal stomata (0.34 wt%). Collectively, these results suggested that Cd was mainly

Cd chemical forms
The diverse chemical forms of Cd in S. matsudana roots and leaves exposed to all Cd and Cd + Ca treatments after 28 d were extracted and determined with different extracting solutions. The Cd contents and percentages of the chemical forms are presented (Table 4, Fig. 6). The Cd contents with different chemical forms raised obviously (p < 0.05) as Cd concentrations increased. The percentage of different chemical forms varied with Cd and Ca concentrations (Fig. 6). Results revealed that 1 M NaCl and 2% HAc extracted the highest Cd content in all the Cd treatments. The proportion of Cd chemical forms in the leaves and roots were ordered as follows: Cd NaCl > Cd HAc > Cd HCl > Cd W > Cd E > Cd R . Moreover, in the roots and leaves, the proportion of Cd extracted with 1 M NaCl decreased as the Cd concentration increased from 10 to 50 μM, while the proportion of Cd extracted using 2% HAc and 0.6 M HCl increased (Fig. 6).
The results also revealed that the addition of Ca decreased the contents of distrinct Cd chemical forms in both roots and leaves when contrasted with treatments with Cd alone (Fig. 6). The addition of Ca altered the proportion of different Cd chemical forms in S. matsudana by means of elevating the percentage of 1 M NaCl-extracted Cd and lowering the proportion of 0.6 M HCl-and 2% HAc-extracted Cd in the leaves and roots.

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 [33].
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. F v /F m and Y(II) are representative fluorescence parameters that are widely used to evaluate the effects of environmental stress on plants [41]. 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 Cdinduced damage in S. matsudana were investigated. The chlorophyll fluorescence parameters, F 0 , F m , F v /F m , 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 [33]. Ge et al. [33] 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 F 0 , F m , F v /F m , YII, and qP and the increase of qN, indicating that Ca played a positive role in this process. He et al. [42] 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 CO 2 concentration, and maximum quantum efficiency of photosystem II photochemistry, YII, and qP [43].

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 + Catreated 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 [45]. 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 [46]. 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 (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 (

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. [58] also found that in leaves Cd accumulated mainly in leaf edges, and less concentrated in regions around leaf vascular bundles.
Leitenmaier and Kupper [59] also reported that the Cd uptake rate in epidermal storage cells was greater than in epidermal or mesophyll cells. Shi et al. [30] 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 [67] 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 [64].
Cd in inorganic forms (Cd W ) and organic forms (Cd E ) are more mobile than other chemical forms, and more toxic to plant cells. Pectate-and protein-integrated Cd (Cd NaCl ), insoluble Cd phosphate (Cd HAc ), and Cd oxalate (Cd HCl ), are less mobile and less toxic to plant cells [60,[68][69][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. [25] 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. [74] 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 [75], then limited Cd translocation from roots to shoots [26].
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 [23]. 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, Additionally, Toyota et al. [76] found that glutamic acid, a stress and mechanical damage signaling substance, transformed the signal due to the increased Ca 2+ 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. [76], were initiated in S. matsudana and that Ca coordinated with Cd,

Determination of Cd contents and TF calculation
Roots, old stem, new stem and leaves from each treatment were gathered separately after 28 d. The samples were washed thoroughly with running tap water and subsequently with deionized water. All the samples were dried in an oven at 45°C for 3 d, at 80°C for 1 d, and at 105°C for 12 h. After that, the samples were digested using wet-digestion methods. Cd contents were determined using inductively coupled plasma atomic emission spectrometry (ICP-AES) (Leeman Labs Inc., Mason, OH, USA). TF was calculated as Cd content in the aerial parts of plants ÷ total Cd content in the plant.

Measurement of chlorophyll fluorescence parameters
The chlorophyll fluorescence parameters quenching analysis was conducted at room temperature with a Dual-PAM/F portable fluorometer (Walz, Effeltrich, Germany). The plants of each treatment were darkened for 20 min prior to measurements. The fifth fully expanded leaf from the top shoot after 28 d was collected for measuring the chlorophyll fluorescence parameters with five replicates. F 0 , F m , F v /F m , Y(II), qP, qN, and other data were provided by the fluorometer.

Subcellular tissue separation by differential centrifugation
The 28-d-treated roots and leaves were cut and rinsed with deionized water. After absorbing the surface moisture, 3 g treated roots and leaves were accurately weighed, frozen, and homogenized in pre-cooled (4°C) extraction buffer (50 mM Tris-HCl, 250 mM sucrose, 1.0 mM C 4 H 10 O 2 S 2 , pH 7.5) with a chilled mortar and pestle [77]. Samples were divided into three parts (i.e., cell wall, cell organelles, and soluble fractions) following the methods reported by Xin et al. [78] and Wu et al. [25]. Cd concentrations in the three fractions were measured by an Analyst 400 atomic absorption spectrometer (PerkinElmer, Waltham, MA, USA).

Extraction of different Cd chemical forms
Different Cd chemical forms were successively extracted by designated solutions in the following order [25,78]: (1) 80% ethanol, Cd E ; (2) deionized water, Cd W ; (3) 1 M NaCl, Cd NaCl ; (4) 2% HAc, Cd HAc ; (5) 0.6 M HCl, Cd HCl ; (6) Cd in residues, Cd R . The extracted Cd by the above extracting solutions and residues were named Cd E , Cd W , Cd NaCl , Cd HAc , Cd HCl , Cd R respectively.
S. matsudana roots and leaves exposed to the five treatments after 28 d were collected, then rinsed with 20 mM EDTA and deionized water successively. Two-gram samples were accurately weighed, and 20 mL of the above extractants was added and homogenized for extraction. The homogenate was shaken at 25°C for 22 h, then centrifuged at 5000 rpm for 10 min. The supernatant liquid was poured off and stored, then 10 mL buffer solution was added and subsequently shaken at 25°C for 2 h, centrifuged at 5000 rpm for 10 min, and the supernatant liquid was combined and dried in an oven.

Scanning electron microscope observation and EDXA
The elemental composition and subcellular localization in freeze-dried leaf and root samples were  Table S1).

Statistical analyses
Data were visualized using SigmaPlot v12.5 (Systat Software Inc., San Jose, CA). Data are presented as the mean ± standard error (SE). Analyses were performed using SPSS v24.0 for Windows (SPSS Inc., Illinois, USA). For the equality of averages, Student's t test was conducted.

Availability of data and materials
All data generated or analysed during this study are included in this published article [and its supplementary information files].

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