Sly-miR398 Participates in Cadmium Stress Acclimation by Regulating Antioxidant System and Cadmium Transport in Tomato (Solanum lycopersicum)

Cadmium (Cd) pollution is one of the major threats in agricultural production, and can cause oxidative damage and growth limitation in plants. MicroRNA398 (miR398) is involved in plant resistance to different stresses, and the post-transcriptional regulation of miR398 on CSDs plays a key role. Here, we report that miR398 was down-regulated in tomato in response to Cd stress. Simultaneously, CSD1 and SOD were up-regulated, with CSD2 unchanged, suggesting CSD1 is involved in miR398-induced regulation under Cd stress. In addition, the role of miR398 in Cd tolerance in tomato was evaluated using a transgenic line overexpressing MIR398 (miR398#OE) in which the down-expression of miR398 was disrupted. The results showed that Cd stress induced more significant growth inhibition, oxidative damage, and antioxidant enzymes disorder in miR398#OE than that in wild type (WT). Moreover, higher Cd concentration in the shoot and xylem sap, and net Cd influx rate, were observed in miR398#OE, which could be due to the increased Cd uptake genes (IRT1, IRT2, and NRAMP2) and decreased Cd compartmentalization gene HMA3. Overall, our results indicate that down-regulated miR398 plays a protective role in tomato against Cd stress by modulating the activity of antioxidant enzymes and Cd uptake and translocation.


Introduction
Cadmium (Cd) is one of the major metal pollutants in arable soil, and adversely affects plant growth and food safety [1]. With an increase in chemical fertilizer application and sewage irrigation in agricultural practice, the area of arable lands under Cd pollution is growing larger, thereby threatening sustainable agricultural production severely [2,3]. Cd stress causes numerous toxic effects including membrane lipid peroxidation, photosynthesis system destruction, and growth limitation in plants, which is, at least partially, due to Cd-induced accumulation of reactive oxygen species (ROS) [4,5], since the ability of ROS scavenging in plants is disrupted by Cd stress [6,7]. Superoxide dismutase (SOD) plays an important role as the first line of antioxidant defense in plants to convert superoxide anion (O 2 − ) into H 2 O 2 , thereby participating in ROS scavenging and stress acclimation in plants [8,9]. Considering the role of SOD in antioxidant defense and Cd-induced oxidative toxicity, it is not surprising that the connection between SOD and Cd stress tolerance has been repeatedly documented in different kinds of plants species including rice [10], wheat [11], and tomato [12,13].
MicroRNAs (miRNAs) play diverse roles in plant development and stress response by regulating gene expression through directing mRNA cleavage and/or inhibiting translation [14]. As a key regulator of SOD in plants, microRNA398 (miR398) can negatively (F) in tomato shoot and root. Tomato seedlings (25 days old, cv. Microtom) were treated with control (CK) and 100 μM Cd(NO3)2 (Cd) for 3 or 6 h. The data are the mean values + SD of four individual replicates and asterisks indicate significant differences between CK and Cd treatment (p < 0.05). One unit (U) of activity of SOD is defined as 50% inhibition of the photochemical reduction of nitroblue tetrazolium. SOD, superoxide dismutase, Pro, protein. Along with the down-expression of miR398, the expression of CSD1 and the activity of SOD was promoted in both the shoot and root under Cd stress ( Figure 1B,D), while no obvious change in CSD2 expression was observed ( Figure 1C). As for reactive oxygen species (ROS) concentration, although no obvious change in O 2 − content was induced by Cd stress (Figure 1E), the content of H 2 O 2 was significantly increased in both the shoot and root of tomato under Cd exposure ( Figure 1F). In accordance with the time-dependent response of miR398 to Cd stress, an earlier increase in CSD1, SOD, and H 2 O 2 was also observed in the root (at 3 HAT) than that in the shoot (at 6 HAT) of tomato under 100 µM Cd treatment (Figure 1).

Cd Stress Induced More Significant Growth Inhibition and Oxidative Damage in MiR398#OE
Based on the results mentioned above, a transgenic line overexpressing MIR398 (miR398#OE) in which the down-regulation of miR398 was disrupted was used in this study, and growth parameters including shoot height, root length, biomass, and chlorophyll content were determined and compared in wild type (WT, cv. Microtom) and miR398#OE after 9 d of treatment. As shown in Figure 2, no significant difference in growth was found between WT and miR398#OE under control, while Cd stress caused severe growth inhibition in both WT and miR398#OE. However, more significant decline in shoot height, root length, shoot biomass, and chlorophyll content was observed in miR398#OE under Cd treatment in contrast with WT ( Figure 2). treatment in both WT and miR398#OE ( Figure 3). However, the results of NBT and DAB staining showed that 100 μM Cd stress induced more significant accumulation of O2 − and H2O2 in tomato leaf of miR398#OE than that in WT ( Figure 3A). Consistently, the contents of MDA, H2O2, and O2 − in the shoot were more significantly increased by Cd stress in miR398#OE in contrast with WT ( Figure 3). In tomato root, Cd stress induced more significant accumulation of H2O2 in miR398#OE, but no obvious change in MDA and O2 − was found between WT and miR398#OE under Cd exposure ( Figure 3).

Figure 2.
Effects of cadmium stress on tomato growth in wild type (WT) and transgenic line overexpressing MIR398 (miR398#OE). Shoot height (A), root length (B), fresh weight of the shoot (C) and root (D), and chlorophyll content (E). Tomato seedlings (25 d old) of WT and miR398 #OE were grown under control (CK) and 100 μM Cd(NO3)2 (Cd) for 9 d. The data are the mean values + SD of four individual replicates and different letters indicate significant differences (p < 0.05). FW, fresh weight.   Figure 3). In addition, severe oxidative damage was induced by Cd treatment in both WT and miR398#OE ( Figure 3). However, the results of NBT and DAB staining showed that 100 µM Cd stress induced more significant accumulation of O 2 − and H 2 O 2 in tomato leaf of miR398#OE than that in WT ( Figure 3A). Consistently, the contents of MDA, H 2 O 2 , and O 2 − in the shoot were more significantly increased by Cd stress in miR398#OE in contrast with WT ( Figure 3). In tomato root, Cd stress induced more significant accumulation of H 2 O 2 in miR398#OE, but no obvious change in MDA and O 2 − was found between WT and miR398#OE under Cd exposure ( Figure 3).

Overexpression of MIR398 Enhanced Cd-Induced Disorder of Antioxidant Enzymes in Tomato
Under control treatment, overexpression of MIR398 decreased the activity of SOD and APX, with CAT, GPOD, GR, and DHAR unchanged in tomato shoot. In the root, overexpression of MIR398 decreased the activity of SOD, CAT, and GR and increased the activity of APX, with no effect on GPOD and DHAR ( Table 1).
As for the effects of Cd stress on antioxidant enzymes, 100 µM Cd induced significant changes in the activity of antioxidant enzymes in both the shoot and root of WT (Table 1). In tomato shoot, Cd stress decreased the activity of SOD, CAT, and APX, and increased the activity of GPOD and DHAR in WT (Table 1). Under Cd exposure, more significant decrease in SOD, CAT, APX, and increase in GPOD was observed in the shoot of miR398#OE in comparison with that in WT (Table 1). However, the aggregative effects of MIR398 overexpression on Cd-induced antioxidant enzyme disorder in tomato shoot was not found in tomato root. Although Cd stress increased the activity of APX, GR, and DHAR and decreased the activity of SOD in the root of WT, no obvious difference in the activity of these enzymes was found between WT and miR398#OE under Cd exposure (Table 1). In addition, Cd stress increased the activity of GPOD in the root of WT with no effect on CAT, while overexpression of MIR398 decreased the activity of CAT and GPOD under Cd stress (Table 1).

Overexpression of MIR398 Enhanced Cd-Induced Disorder of Antioxidant Enzymes in Tomato
Under control treatment, overexpression of MIR398 decreased the activity of SOD and APX, with CAT, GPOD, GR, and DHAR unchanged in tomato shoot. In the root, overexpression of MIR398 decreased the activity of SOD, CAT, and GR and increased the activity of APX, with no effect on GPOD and DHAR ( Table 1).
As for the effects of Cd stress on antioxidant enzymes, 100 μM Cd induced significant changes in the activity of antioxidant enzymes in both the shoot and root of WT (Table 1). In tomato shoot, Cd stress decreased the activity of SOD, CAT, and APX, and increased the activity of GPOD and DHAR in WT (Table 1). Under Cd exposure, more significant decrease in SOD, CAT, APX, and increase in GPOD was observed in the shoot of miR398#OE in comparison with that in WT (Table 1). However, the aggregative effects of MIR398 overexpression on Cd-induced antioxidant enzyme disorder in tomato shoot was not found in tomato root. Although Cd stress increased the activity of APX, GR, and DHAR and decreased the activity of SOD in the root of WT, no obvious difference in the activity of these enzymes was found between WT and miR398#OE under Cd exposure (Table 1). In addition, Cd stress increased the activity of GPOD in the root of WT with no effect on CAT, while overexpression of MIR398 decreased the activity of CAT and GPOD under Cd stress (Table 1).

Overexpression of MIR398 Enhanced Cd Uptake and Accumulation in Tomato
The concentrations of Cd in the shoot, root, and xylem sap of WT and miR398#OE were determined in this study ( Figure 4). Although no obvious difference in root Cd concentration was observed between WT and miR398#OE, the concentration of Cd in the shoot and xylem sap was significantly higher in miR398#OE than that in WT by 25.3% and 90.5%, respectively ( Figure 4). ment for 9 d. The data are the mean values ± SD of four individual replicates and different letters show significant differences (p < 0.05). One unit (U) activity of SOD is defined as 50% inhibition of the photochemical reduction of nitroblue tetrazolium. SOD, superoxide dismutase, U mg −1 Pro; CAT, catalase, mM H2O2 mg −1 Pro min −1 ; APX, ascorbate peroxidase, mM ASA mg −1 Pro min −1 ; GPOD, guaiacol peroxidase, mM guaiacol mg −1 Pro min −1 ; GR, glutathione reductase, mM NADPH mg −1 Pro min −1 ; DHAR, dehydroascorbate reductase, mM DHA mg −1 Pro min −1 ; Pro, protein; ASA, ascorbate; DHA, dehydroascorbate.

Overexpression of MIR398 Enhanced Cd Uptake and Accumulation in Tomato
The concentrations of Cd in the shoot, root, and xylem sap of WT and miR398#OE were determined in this study ( Figure 4). Although no obvious difference in root Cd concentration was observed between WT and miR398#OE, the concentration of Cd in the shoot and xylem sap was significantly higher in miR398#OE than that in WT by 25.3% and 90.5%, respectively ( Figure 4). Following the assay of Cd concentration in tomato, root net Cd influx rate was further measured using the non-invasive scanning ion-selected electrode technique (SIET), and a 5 min of stable Cd flux rate was recorded ( Figure 5A). After calculating the net Cd influx rate based on time, the results showed overexpression of MIR398 significantly increased net Cd influx rate by 23.6% ( Figure 5B), which was in line with the enhanced Cd accumulation in tomato shoot of miR398#OE ( Figure 4).
In addition, the expression of genes concerning Cd uptake (IRT1, IRT2, NRAMP2) and compartmentalization (HMA3) in the root of WT and miR398#OE was evaluated (Figure 6). The results showed that the expression of IRT1, IRT2, and NRAMP2 was Following the assay of Cd concentration in tomato, root net Cd influx rate was further measured using the non-invasive scanning ion-selected electrode technique (SIET), and a 5 min of stable Cd flux rate was recorded ( Figure 5A). After calculating the net Cd influx rate based on time, the results showed overexpression of MIR398 significantly increased net Cd influx rate by 23.6% ( Figure 5B), which was in line with the enhanced Cd accumulation in tomato shoot of miR398#OE (Figure 4). significantly up-regulated in miR398#OE root in contrast with that in WT ( Figure 6A-C). Furthermore, the expression of HMA3 was significantly down-regulated in the root of miR398#OE in comparison with WT ( Figure 6D). The enhanced expression of Cd uptake genes and decreased expression of Cd compartmentalization genes could be responsible for the improved Cd concentration and net Cd influx rate in miR398#OE (Figures 4 and  5).  In addition, the expression of genes concerning Cd uptake (IRT1, IRT2, NRAMP2) and compartmentalization (HMA3) in the root of WT and miR398#OE was evaluated ( Figure 6). The results showed that the expression of IRT1, IRT2, and NRAMP2 was significantly up-regulated in miR398#OE root in contrast with that in WT ( Figure 6A-C). Furthermore, the expression of HMA3 was significantly down-regulated in the root of miR398#OE in comparison with WT ( Figure 6D). The enhanced expression of Cd uptake genes and decreased expression of Cd compartmentalization genes could be responsible for the improved Cd concentration and net Cd influx rate in miR398#OE (Figures 4 and 5).

Discussion
MicroRNAs (miRNAs) play key roles in regulating plant growth, development, and stress response [39,40]. As an important member of miRNAs in plants, microRNA398 (miR398) is involved in the acclimation to kinds of biotic and abiotic stresses, while the miR398-CSD module has been shown to play a central role in the regulatory network of miR398 in plants under stressful conditions [25].

Discussion
MicroRNAs (miRNAs) play key roles in regulating plant growth, development, and stress response [39,40]. As an important member of miRNAs in plants, microRNA398 (miR398) is involved in the acclimation to kinds of biotic and abiotic stresses, while the miR398-CSD module has been shown to play a central role in the regulatory network of miR398 in plants under stressful conditions [25].
In this study, our results showed the expression of miR398 was down-regulated in tomato under Cd exposure ( Figure 1A), which is in line with the results in Arabidopsis under Cu stress [25]. Along with the decrease in miR398 expression, an increase in CSD1 expression and SOD activity was observed in both the shoot and root in tomato under Cd stress ( Figure 1B,D). However, the expression of CSD2 was not changed by the downexpression of miR398 as CSD1 in tomato under Cd exposure ( Figure 1C). In plants, miR398 is able to modulate the activity of SOD by negatively regulating the expression of its target genes including CSD1 and/or CSD2 [21,25], while sequence match analysis showed the match between miR398 and CSD1 was tighter than that between miR398 and CSD2 [35]. In this study, our results indicated that CSD1 but not CSD2 is involved in the miR398-induced regulation of SOD in response to Cd toxicity in tomato. In addition, it should be noted that the down-regulated miR398 and up-regulated CSD1 and SOD were found at 3 h after Cd treatment in the root but at 6 h after treatment of Cd in the shoot (Figure 1). In plants, the toxicity of Cd in plants is mainly based on Cd uptake and accumulation. Therefore, it is not surprising that the root is earlier in suffering from Cd toxicity than the shoot under Cd exposure [41,42], which was also proved by the earlier accumulation of H 2 O 2 in the root (3 h) than the shoot (6 h) in this study. Overall, these results clearly indicate the down-expression of miR398 and its successive regulation on CSD1 and SOD was in response to Cd accumulation and toxicity in tomato.
In plants under stressful conditions, the response of miR398 varies among stress types and plant species [21], implying that the distinct roles of miR398 in stress adaption. Therefore, to investigate the function of miR398 in Cd stress tolerance in tomato, a transgenic line overexpressing MIR398 (miR398#OE) was further used in this study. Consistent with previous reports [43,44], Cd stress induced toxicity symptoms including growth inhibition, chlorophyll degradation, ROS accumulation, and membrane peroxidation in both wild type (WT, cv. Microtom) and miR398#OE. However, compared with WT, Cd stress induced more significant growth inhibition and oxidative damage in miR398#OE (Figures 2 and 3). The results are consistent with that in Arabidopsis under Cu stress and high light stress [25], and in tomato under salt stress [35], which confirmed that the down-regulation of miR398 played a protective role in tomato against Cd stress.
However, it should be noted although Cd stress caused severe oxidative damage in both the shoot and root of tomato, overexpression of MIR398 aggregated Cd-induced oxidative damage in the shoot but not in the root (Figure 3). Therefore, the activity of antioxidant enzymes in the shoot and root of miR398#OE and WT under Cd exposure was measured (Table 1). In the shoot of tomato, Cd stress induced significant decrease in SOD, CAT, and APX, and increase in GPOD and DHAR, while the Cd-induced disorder of antioxidant enzymes was further enhanced by MIR398 overexpression (except DHAR) ( Table 1). The results were in line with the enhanced oxidative damage in the shoot of miR398#OE (Figure 3). In the root, although Cd stress caused increase in APX, GPOD, GR, and DHAR, and decrease in SOD in WT, no aggregative effect of MIR398 overexpression on these enzymes and oxidative damage (except H 2 O 2 ) was found in this study. The regulation of miR398 on SOD under stress has been repeatedly documented in previous research [45]. In line with previous research concerning miR398 and the antioxidant system under biotic and abiotic stresses [35][36][37], our results indicate that, besides SOD, the miR398-CSD1 module could also modulate the activity of other antioxidant enzymes in tomato under Cd stress, which could be based on the systematic cooperation of different kinds of enzymes in plant antioxidant defense [46][47][48].
Given the fact that the toxicity of Cd is based on Cd accumulation in plant tissue, which is mediated by root uptake and translocation of Cd [4], the concentration of Cd in tomato and Cd uptake flux rate was determined in this study. The content of Cd in the shoot and xylem sap was significantly higher in miR398#OE under Cd stress than that in WT, while no significant difference in root Cd concentration between miR398#OE and WT was observed ( Figure 4). Moreover, the results of non-invasive scanning ion-selected electrode technique (SIET) showed that the net Cd influx rate was significantly higher in the root of miR398#OE than that in WT ( Figure 5). By comparing the activity of antioxidant enzyme and Cd concentration in WT and miR398#OE under Cd toxicity, it can be concluded that the protective regulation effects of miR398 on antioxidant enzymes and Cd concentration were mainly observed in the shoot but not root of tomato. When subject to Cd stress, the shoot is more sensitive to Cd toxicity than the root [43,49]. Considering the important roles of the shoot in plant growth (e.g., photosynthesis), the protective effects of miR398 in the shoot would be more efficient in promoting Cd tolerance in tomato.
In plants, different genes including IRT1, IRT2, and NRAMP2 have been shown to be involved in the process of root Cd uptake [41], while HMA3 is responsible for vacuole compartmentalization, thereby influencing Cd translocation [50]. In this study, our results showed that overexpression of MIR398 increased the expression of IRT1, IRT2, and NRAMP2 ( Figure 6A-C), and decreased the expression of HMA3 (Figure 6D), which could be responsible for the enhanced Cd uptake and translocation in miR398#OE under Cd stress in comparison with WT. In the initial hypothesis, we have assumed that miR398 could influence the ability of ROS scavenge in plants under Cd stress, and successively influence affect membrane peroxidation and Cd transport in tomato under Cd exposure. However, overexpression of MIR398 did not enhance Cd-induced oxidative damage in tomato root (Figure 3), implying that the regulation of miR398 on the activity of Cd transport and related genes is independent of membrane peroxidation. The mechanisms behind this still need further investigation.

Plant Growth and Treatment Application
Seeds of tomato (Solanum lycopersicum L.) were sterilized with distilled water at 55 • C for 10 min and then germinated on moist filter paper in dark at 28 • C for 2 d. After 20 d of growth on the substrate (Golden No.3, Jinhai, Hangzhou, China), each three uniform tomato seedlings were transplanted to a plastic bottle containing 1.2 L nutrient solution for another 3 d of growth. The nutrient solution (pH 6.2) was renewed each 3 d and contained 5 mM Ca(NO 3 ) 2 , 5 mM KNO 3 , 2.5 mM KH 2 PO 4 , 2 mM MgSO 4 , 29.6 µM H 3 BO 3 , 10 µM MnSO 4 , 50 µM Fe-EDTA, 1.0 µM ZnSO 4 , 0.05 µM H 2 MoO 4 , and 0.95 µM CuSO 4 . The experiment was conducted in a growth chamber with a controlled environment: 70% relative humidity, 16/8 h, and 28/20 • C for light/dark period, respectively. After growth under the condition mentioned above, short-and long-time treatment experiment was conducted.
Experiment I: Tomato seedlings (cv. Microtom) were treated with or without 100 µM Cd(NO 3 ) 2 addition for 6 h, the expression of miR398, CSD1, CSD2, the activity of SOD, and the contents of superoxide anion (O 2 − ) and H 2 O 2 in tomato shoot and root were measured. Experiment II: Tomato seedlings of Microtom (wild type, WT) and transgenic line overexpressing miR398 (miR398#OE) obtained from our previous work [35] were grown under control and 100 µM Cd(NO 3 ) 2 addition for 9 d. Then, tomato growth, oxidative damage, antioxidant enzyme activity, Cd concentration, net Cd flux rate, and the expression of genes concerning Cd uptake was evaluated.

Tomato Growth and Chlorophyll Concentration
Tomato seedlings were harvested with the root and shoot separated, and then fresh weight, shoot height, and root length were measured. For chlorophyll assay, leaf samples were homogenized with alcohol solution (95%, v/v), and the content of chlorophyll was determined by measuring the absorbance of the supernatant at 665, 649, and 470 nm with an UV/Vis spectrophotometer (UV-2600, Shimadzu, Kyoto, Japan) according to Porra et al. [51].

Oxidative Damage and ROS Accumulation
To evaluate the oxidative damage caused by Cd stress, nitroblue tetrazolium (NBT) and diaminobenzidine (DAB) staining were used for the visualization of superoxide anion (O 2 − ) and H 2 O 2 in tomato shoot samples according to the methods described by Jabs et al. [52] and Vanacker et al. [53], respectively. The contents of malonaldehyde (MDA), O 2 − , and H 2 O 2 in the shoot and root of tomato under different treatments were measured using the trichloroacetic acid-thiobarbituric acid (TCA-TBA) method [54], hydroxylamine oxidation method [55], and potassium iodide method [56], respectively.

Antioxidant Enzyme Activity
Fresh tomato samples were homogenized with 1.5 mL phosphate butter (50 mM, pH 7.8) containing 0.2 mM ethylenediamine tetraacetic acid and 2% (w/v) polyvinyl pyrrolidone, and 1 mL supernatant was collected for the detection of soluble protein and antioxidant enzyme activity after centrifugation at 10,000 rpm for 10 min at 4 • C [57]. Superoxide dismutase (SOD) activity was determined with the NBT method and one unit (U) activity of SOD was defined as 50% inhibition of the photochemical reduction of NBT [58]. Catalase (CAT) activity was determined based on the extinction rate of H 2 O 2 according to Cakmak and Maschner [59]. Ascorbate peroxidase (APX) activity was determined by ascorbate oxidation according to Nakano and Asada [60]. Guaiacol peroxidase (GPOD) activity assay was conducted with the method described by Egley et al. [61]. Dehydroascorbate reductase (DHAR) activity was calculated by the formation of ascorbate according to Mano et al. [62]. Glutathione reductase (GR) activity was defined by nicotinamide adenine dinucleotide phosphate oxidation according to Foyer and Halliwell [63].

Cd Concentration
Tomato shoot and root samples were oven-dried at 80 • C for 96 h, and then digested with 5 mL nitric acid and 1 mL hydrogen peroxide at 180 • C on a thermal digestion system (SH230N, Sineo, Shanghai, China). Xylem sap was collected according to Yan et al. [64] by cutting the stem off 2 cm above the root base. Cd concentrations were analyzed with inductively coupled plasma-mass spectrometry (ICP-MS, NexION 300X, Perkin Elmer, Waltham, MA, USA).

Net Cd Influx Rate
After 9 d of treatment, root samples (about 1.5 cm) were cut off above the root apex for Cd flux determination using non-invasive scanning ion-selected electrode technique (SIET, BIO-001A, Younger, Amherst, MA, USA) according to Yan et al. [65]. Briefly, root samples were placed in plastic dishes containing test buffer for about 1 h, and the electric potential was measured at about 4 µm above the root surface with a microelectrode for about 15 min. Then, the change in electric potential was converted into Cd flux rate according to Cd standard curves based on 50 and 500 µM Cd(NO 3

Gene Expression
Total RNA was extracted from frozen tomato samples (approximately 100 mg) using Trizol reagent (Invitrogen, Thermo Fisher, Waltham, MA, USA). After the measurement of A 260/280 and A 260/230 to check the purity and integrity, miRNA and RNA were converted to cDNA with a miRNA first-strand cDNA synthesis kit (by stem-loop) (Vazyme, Nanjing, China) and HiScript III first-strand cDNA synthesis kit (Vazyme, Nanjing, China), respectively. Quantitative real-time PCR was operated on a quantitative PCR system (qTower3, Analytik jena, Jena, Germany) using chamQ SYBR qPCR master mix (Vazyme, Nanjing, China) following the instruction. The relative expression rate was calculated using the ∆∆Ct method. U6 was for miR398, while Actin was used as the internal reference for CSD1, CSD2, IRT1, IRT2, NRAMP2, and HMA3. The sequence of primers used in this study were listed in supplementary Table S1.

Statistical Analysis
Statistical analysis was performed with Excel software (2019 version, Microsoft, Redmond, WA, USA) and data were compared using one-way or two-way analysis of variance (ANOVA). The significant difference was defined at the 0.05 probability level.

Conclusions
In conclusion, our results show the down-regulation of miR398 plays a protective role in tomato against Cd stress through modulating antioxidant system and Cd uptake and translocation, thereby alleviating oxidative damage and promoting plant growth under Cd contamination (Figure 7). The results of this study would expand our knowledge concerning miRNA and metal stress tolerance in plants, which would also provide new insight for breeding and Cd pollution remediation. China) following the instruction. The relative expression rate was calculated using the ΔΔCt method. U6 was for miR398, while Actin was used as the internal reference for CSD1, CSD2, IRT1, IRT2, NRAMP2, and HMA3. The sequence of primers used in this study were listed in supplementary Table S1.

Statistical Analysis
Statistical analysis was performed with Excel software (2019 version, Microsoft, Redmond, WA, USA) and data were compared using one-way or two-way analysis of variance (ANOVA). The significant difference was defined at the 0.05 probability level.

Conclusions
In conclusion, our results show the down-regulation of miR398 plays a protective role in tomato against Cd stress through modulating antioxidant system and Cd uptake and translocation, thereby alleviating oxidative damage and promoting plant growth under Cd contamination (Figure 7). The results of this study would expand our knowledge concerning miRNA and metal stress tolerance in plants, which would also provide new insight for breeding and Cd pollution remediation.