*3.2. E3 Ubiquitin Ligase Activity and Subcellular Localization of Sl1*

To verify the E3 ubiquitin ligase activity of Sl1 protein, we successfully purified the maltose-binding protein-Sl1 (MBP-Sl1) fusion protein and maltose-binding proteinempty vector (MBP-EV) fusion protein following the manufacturer instructions (Figure 2A). We performed the in vitro ubiquitination assay and showed that Sl1 protein had selfubiquitination ability when present with E1, E2, and ubiquitin. However, lacking any of E1, E2, or ubiquitin in the reaction system undermined the self-ubiquitination ability of Sl1. Meanwhile, MBP-EV also did not show self-ubiquitination ability (Figure 2B).

**Figure 2.** E3 ligase activity and subcellular localization of Sl1. (**A**) In vitro expression of MBP-Sl1 and MBP-EV. T: total protein; S: soluble protein; B: before expression; A: after expression. (**B**) In vitro E3 ligase activity of Sl1 protein. The reaction system included E1, E2, MBP-Sl1, and ubiquitin-His, the replacement of MBP-Sl1 with MBP-EV and the absence of E1, E2, and His-Ub as control. The Western blot was detected with anti-MBP and anti-His. (**C**) Subcellular localization of GFP-Sl1 and GFP-EV. The GFP-Sl1 was transiently expressed in *Nicotiana benthamiana* (tobacco with nucleuslocated mCherry). Images were pictured by confocal microscope after 48 h infiltration. Bar = 25 μm.

To examine the subcellular localization of Sl1 protein, we transiently expressed GFP-Sl1 in transgenic tobacco (with nucleus signal). Images captured by a confocal microscope revealed that the Sl1 protein is located in plasma membranes rather than the nucleus (Figure 2C).

#### *3.3. Sl1 Positively Regulates Cd Tolerance in Tomato*

Since the expression of the *Sl1* gene was induced by Cd stress, we investigated the tolerance of *sl1* mutant lines, wild-type, and *Sl1* overexpressing lines to Cd stress. We generated the *sl1* mutant lines and *Sl1* overexpressing lines as described in the Methods and Materials section (Figure S1). Two *sl1* mutant lines were mutated at different sites that both induced early termination of translation. The *sl1-1* mutant line was found 311 bp deletion between sgRNA1 and sgRNA2 and the translation was terminated after 98 amino acids (Figure S1B). The *sl1-2* mutant line was deleted 1 bp after the protospacer adjacent motif (PAM) of sgRNA1 and the translation was terminated after 89 amino acids (Figure S1B). Two overexpressing lines of *Sl1* were checked with Western blot that both had bright bands near the predictive molecular weight (Figure S1C). The phenotypes of *sl1* mutants were similar to wild-type plants; however, *Sl1* overexpressing lines grew more slowly and showed smaller leaves compared with wild-type plants when they were grown under optimal (nonstress) environments (Figure 3A).

**Figure 3.** Sl1 positively regulates tomato Cd tolerance. (**A**) The phenotype of *Sl1* mutant lines (*sl1-1/2*), wild-type (WT), and *Sl1* overexpressing lines (*Sl1*-OE-1/2) under control and Cd stress after 15 d treatment. Bar = 10 cm. (**B**) The content of hydrogen peroxide (H2O2) in the roots of *Sl1* mutant lines (*sl1-1/2*), WT, and *Sl1* overexpressing lines (*Sl1*-OE-1/2) under control and Cd stress after 3 d treatment. (**C**,**D**) the image and level of actual quantum efficiency of PSII photochemistry (ΦPSII) of *sl1-1/2*, WT, and *Sl1*-OE-1/2 plants with and without Cd treatment for 15 d. Bar = 1 cm. The data presented here are the average of three biological replicates (±SD). Different letters indicate a significant difference (*p* < 0.05, Tukey's test).

After 15 d of Cd treatment, *sl1* mutants showed sensitivity to Cd stress, while *Sl1*-OE showed enhanced tolerance to Cd stress (Figure 3). Compared to control conditions, Cd-stress-induced changes in leaf size, leaf color, level of ΦPSII, and content of hydrogen peroxide (H2O2) in *sl1* mutants and wild-type plants. In particular, the leaves of *sl1* were

more etiolated than those of wild-type, and the leaf sizes of mutants were smaller than that of wild-type. However, the leaf size and color in *Sl1* overexpressing plants were just slightly affected by Cd stress (Figure 3). Since H2O2 is a major ROS generated under stress conditions, we then detected the accumulation of H2O2 in roots under Cd stress. Importantly, the accumulation of H2O2 increased by 43.1% and 30.5% in *sl1-1* and *sl1-2* mutants, while it decreased by 31.7% and 26.5% in two lines of *Sl1* overexpressing plants compared with wildtype plants after Cd treatment, respectively (Figure 3B). Moreover, we detected the value of actual quantum efficiency of PSII photochemistry, ΦPSII, which reflects the state of photosystem II as a reliable marker of plant health status. As shown in Figure 3C,D, the ΦPSII value of *sl1* mutants, wild-type, and *Sl1* overexpressing lines exhibited no significant difference under control conditions. However, the ΦPSII levels of *sl1-1* and *sl1-2* mutants decreased by 12.1% and 12.7% respectively, compared with wild-type under Cd stress (Figure 3D). The ΦPSII levels of two lines of *Sl1* overexpressing plants were significantly greater than that in wild-type under Cd stress (Figure 3C,D). These results indicate that Sl1 is critical for alleviating Cd-induced H2O2 accumulation and damage to the photosynthetic system.

#### *3.4. Sl1 Promotes Antioxidant Enzyme Activity*

To understand whether *Sl1* influenced antioxidant enzyme activities in tomato under Cd stress, we examined the enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). The results showed that Cd stress increased antioxidant enzyme activities in wild-type and overexpressing lines. However, in *sl1* mutant lines, there were no significant differences in antioxidant enzyme activities between control and Cd treatment (Figure 4). The activities of SOD, CAT, APX, and GR in two lines of *Sl1* overexpressing plants were all induced compared with wild-type plants under Cd stress (Figure 4). These results suggest that *Sl1* promotes the activities of antioxidant enzymes under Cd stress.

**Figure 4.** Sl1 promotes antioxidant enzyme activities in tomato plants under Cd stress. The activities of SOD, CAT, APX, and GR in the roots of *Sl1* mutant lines (*sl1-1/2*), wild-type (WT), and *Sl1* overexpressing lines (*Sl1*-OE-1/2) under Cd stress for 3 d. The data presented here are the average of three biological replicates (±SD). Different letters indicate a significant difference (*p* < 0.05, Tukey's test).

#### *3.5. Sl1 Reduces Cd Accumulation and Transportation*

To investigate whether *Sl1* is involved in Cd accumulation in tomato plants, we detected the content of Cd in shoots and roots under Cd stress. Results revealed that the Cd content in the roots was higher than that in the shoots (Figure 5A,B). Obviously, overexpression of *Sl1* decreased Cd content in both shoots and roots, while Cd content in *sl1* mutants significantly increased compared with that in wild-type (Figure 5A,B). The content of Cd in roots of two *Sl1* overexpressing lines both decreased by 25.6% compared with wild-type, while the Cd content increased by 34.7% and 41.6% in roots of *sl1-1* and *sl1-2* mutants compared with wild-type, respectively. Similarly, Cd accumulation was also higher in the shoots of *sl1* mutants than wild-type plants, while it was lower in the shoots of *Sl1* overexpressing lines.

**Figure 5.** Sl1 decreases Cd content in tomato plants under Cd stress. The Cd content in the shoot (**A**) and root (**B**) of *Sl1* mutant lines (*sl1-1/2*), wild-type (WT), and *Sl1* overexpressing lines (*Sl1*-OE-1/2) under Cd stress for 10 d. (**C**) Cd accumulation in tomato root tips stained by the Cd-specific probe LeadmiumTM Green AM. Bar = 25 μm. (**D**) Relative fluorescence intensity of Cd staining over tomato root tips of *sl1* mutants, wild-type, and *Sl1* overexpressing lines after 10 d Cd treatment. The relative fluorescence intensity is normalized to the intensity of wild-type in (**C**). The data presented here are the average of three biological replicates (±SD). Different letters indicate a significant difference (*p* < 0.05, Tukey's test).

To further investigate whether *Sl1* decreased Cd accumulation by altering Cd delivery, we used a Cd-specific probe to study the Cd distribution in the root tips. The Cd-specific probe stained signals were not detected in the root tips of all plants without Cd treatment (Figure S2E). However, Cd treatment induced the accumulation of Cd in the root tips as reflected by the increased fluorescence intensity. The relative fluorescence intensity of *sl1-1* and *sl1-2* mutants were 1.40-fold and 1.41-fold of that in wild-type plants, respectively, while the relative fluorescence intensity of *Sl1*-OE-1 and *Sl1*-OE-2 plants were only 49.2% and 54.7% of that in wild-type plants (Figure 5C,D).

To investigate how *Sl1* regulated Cd transportation, we examined the expression of heavy metal transportation-related genes (*CAX3*, *HMA-A*, *HMA-B*, and *IRT1*). There was no significant difference in the expression of these four genes between *sl1* mutants, wild-type, and *Sl1* overexpressing lines under control conditions. Although these four genes were highly expressed in *sl1* mutants under Cd stress, no significant difference was found between control and Cd treatment in two lines of *Sl1* overexpressing plants. We found that Cd stress dramatically increased the transcript level of *CAX3* in *sl1-1* and *sl1-2* mutants under Cd stress, which were 3.2-fold and 3.4-fold of that in wild-type, respectively (Figure 6). The heavy metal transport gene *HMA-A/B* was also upregulated by Cd stress in *sl1-1* and *sl1-2* plants by 76.5%/73.6% and 86.5%/79.9% compared with wild-type, respectively (Figure 6). Similarly, the expression of *IRT1*, which plays a prominent role in heavy metal transportation, was also increased by Cd stress in *sl1* mutants compared with wild-type plants (Figure 6). These results suggest *Sl1* potentially functions in resisting heavy metal transportation through repressing the transcription of heavy metal transportation-related genes.

**Figure 6.** Sl1 negatively regulates the transcripts of genes related to heavy metal transportation. The relative expression of *CAX3*, *HMA-A*, *HMA-B,* and *IRT1* in the roots of *Sl1* mutant lines (*sl1-1/2*), wild-type (WT), and *Sl1* overexpressing lines (*Sl1*-OE-1/2) under Cd stress for 3 d. The data presented here are the average of three biological replicates (±SD). Different letters indicate a significant difference (*p* < 0.05, Tukey's test).

#### **4. Discussion**

As a significant component of the food chain, plants play a crucial role in the transportation and accumulation of toxic elements such as Cd in humans [44]. Nonetheless, plants also suffer from the stress induced by heavy metals and they address the stress by multiple pathways, including eliminating ROS, resisting heavy metal transportation, and maintaining protein quality [19,20,24,25]. The E3 ubiquitin ligase-mediated protein degradation plays an important role in plant stress tolerance [25,34,36,45,46]. Here, we characterized a RING-type E3 ubiquitin ligase Sl1, which conferred Cd tolerance in tomato. Our study advances the understanding of the mechanism of UPS-mediated heavy metal tolerance in plants.

The RING-type E3 ligase is closely associated with plant tolerance to various stress [47]. For example, a C3H2C3-type E3 ubiquitin ligase AtAIRP1 positively regulates ABA-dependent drought tolerance by mediating AtAIRP1 degradation [48,49]. The roles of E3 ubiquitin ligases in the positive regulation of heavy metal stress tolerance have been reported recently in different plant species [19,25,34,46]. Overexpression of *HIR1* increases the tolerance of rice to As and Cd stress [34]. *HIR1* that encodes an E3 ubiquitin ligase interacts with TIP4;1 for resisting heavy metal absorption in rice [34]. Moreover, a U-box type E3 ubiquitin ligase, *SlUPS*, is highly expressed under Cd stress in tomato. Heterologous expression of *SlUPS* in yeast increases the concentration of yeast bacterial fluid exposed to Cd and overexpression of *SlUPS* in *Arabidopsis* enhances Cd tolerance [46]. The RING-type E3 ligase AtIDF1 degrades IRT1 to modulate iron homeostasis in *Arabidopsis* [50]. Heterologous expression of a soybean RING-type E3 ubiquitin ligase gene *GmARI1* in *Arabidopsis* enhances Al tolerance [51]. Furthermore, tomato E3 ubiquitin ligase SlRING1 positively mediates Cd tolerance by enhancing antioxidant enzyme activities and inhibiting Cd accumulation [19,25]. Similar to *SlRING1*, *Sl1*, which is highly expressed under Cd stress in tomato roots, plays a pivotal role in Cd tolerance (Figures 1 and 3). We also found that Sl1 protein possessed the E3 ligase activity and overexpression of *Sl1* inhibited Cd accumulation in tomato. These results are consistent with previous studies [19,34], suggesting that RING-type E3 ubiquitin ligases play crucial roles in regulating metal ion transport.

Cadmium has a broad variety of negative impacts on plants, including oxidative stress, nutrient absorption disruption, and even plant mortality. Antioxidant enzymes such as SOD, POD, CAT, APX, and GR function in collaboration with nonenzymatic antioxidants such as AsA and GSH to prevent Cd-induced oxidative damage [52,53]. Furthermore, GSH directly participates in the synthesis of PCs [54,55]. PCs form complexes with Cd that can be compartmentalized into the vacuoles; thus, PCs and other thiols play an important role in determining the sensitivity or tolerance in contrasting genotypes of a plant species [56]. Moreover, studies on the semihalophytic plant *Mesembryanthemum crystallinum* L. and different Cd hyperaccumulators such as *Arabidopsis halleri*, *Thlaspi caerulescens*, *Solanum nigrum*, and *Sedum alfredii* species indicate that both antioxidative enzymes and nonenzymatic antioxidants play a vital role in Cd tolerance [57–59]. Previously, we found that an E3 ligase gene *SlRING1* positively regulates relative expression levels of *CAT*, *MDHAR*, *GSH1*, and *PCS*, while it decreases H2O2 content in tomato under Cd stress [19,25]. Consistent with those studies, *Sl1* overexpression increased the activities of SOD, CAT, APX, and GR, and decreased the content of H2O2 under Cd stress in *Sl1* overexpressing lines compared with those in wild-type and mutant lines (Figures 3 and 4).

Metal absorption and transportation are critical for plant tolerance to heavy metal stress [44]. The rice E3 ubiquitin ligase OsHIR1 targets TIP4;1 that functions as a heavy metal absorption protein, and thus OsHIR1-induced degradation of TIP4;1 increases rice tolerance to As and Cd stress [34]. Moreover, another RING-type E3 ubiquitin ligase in rice, OsAIR3, regulates protein degradation of molybdate transporter (OsMOT1;3) in rice to increase plant tolerance to arsenate stress [60]. In agreement with these studies, we found that overexpression of *Sl1* significantly attenuated the relative expression level of several genes related to heavy metal transportation, such as *CAX3*, *HMA-A*, *HMA-B,* and *IRT1*, along with decreased Cd content in *Sl1* overexpressing lines compared with the wild-type and mutant lines (Figures 5 and 6). *CAX* gene family is an important plant gene family involved in heavy metal transportation [11]. It is plausible that Sl1 functions as an E3 ubiquitin ligase for the degradation of proteins involved in heavy metal transporters or regulating the abundance of transcription factors upstream of those transporters. Interestingly, heavy-metal-induced stress also results in protein denaturation that aggravates the oxidative stress in plants [24]. Thus, further studies are essentially needed to investigate whether *Sl1* could degrade denatured proteins to relieve cell oxidative stress. Moreover, it will also be interesting to study whether *Sl1* can coordinate with autophagy to clear denatured proteins [61].

Plants do not have a Cd-selective transporter, therefore, Cd absorption happens through plasma membrane transporters that also take up other divalent cations [62]. Thus, it is indeed difficult to reduce plant Cd accumulation without compromising plant growth since the majority of well-known Cd transporters also transport various essential micronutrients such as Zn, Fe, Mn, and Cu. Despite the fact that CAXs are mostly Ca2+ specific transporters, AtCAX2 and AtCAX4 have been demonstrated to transport various other metals, such as Cd, Zn, and Mn in *Arabidopsis* [58]. Similarly, Fe-specific transporter OsIRT1 has been found to participate in Cd uptake in rice [63]. Thus, suppression of the metal transporter may lead to essential nutrient deficiency and compromised plant growth. In our study, overexpression of *Sl1* in tomato not only decreased Cd accumulation but also suppressed plant growth. Reduced Cd accumulation was associated with decreased expression of several metal transporters such as *CAX3*, *HMA-A*, *HMA-B*, and *IRT1*. Thus, it is possible that decreased plant growth in *Sl1* overexpressing lines could be a consequence of the suppression of the metal transporters under Cd stress. However, reduced growth of the *Sl1* overexpressing lines under control conditions could be attributed to some other reasons such as impaired hormone homeostasis since the expression levels of *CAX3*, *HMA-A*, *HMA-B*, and *IRT1* were not significantly different among *sl1* mutants, wild-type, and *Sl1* overexpressing lines under control conditions. Thus, it would be interesting to explore hormonal involvement in *Sl1*-regulated plant growth and stress tolerance in future studies.

#### **5. Conclusions**

In the present study, we characterized an E3 ubiquitin ligase Sl1 in tomato, which is located in plasma membranes and highly expressed in roots under Cd stress. For functional characterization of Sl1, we generated knockout lines and overexpressing lines of *Sl1* in tomato. The parameters of chlorophyll fluorescence and content of H2O2 demonstrated that *Sl1* overexpressing lines suffered less photosystem damage and oxidative stress compared with wild-type and mutants, suggesting that *Sl1* overexpressing lines are resistant to Cd stress, while mutant lines are sensitive to Cd stress. Moreover, Sl1 positively regulated antioxidant enzyme activities and negatively mediated gene expression associated with heavy metal transportation. Thus, the current study unveils a novel role of an E3 ubiquitin ligase Sl1 in tomato that may have potential implications in enhancing heavy metal tolerance in plants. However, identification of the substrate protein of Sl1 needs further study to precisely verify its association with heavy metal transportation.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11030456/s1, Figure S1: Generation of *Sl1* mutant lines and overexpression lines; Figure S2: Structure analysis of Sl1 protein and histochemical staining of Cd accumulation of tomato root tips; Table S1: The primers used for qRT-PCR. Supplementary material related to this article can be found in the online version.

**Author Contributions:** J.Z. and G.J.A. designed the research; C.-X.L., T.Y., H.Z. and Z.-Y.Q. performed the experiments; C.-X.L. and J.Z. analyzed the data; J.Z., C.-X.L. and G.J.A. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Key Research and Development Program of China (2019YFD1000300), the National Natural Science Foundation of China (31922078 and 31872089), the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study (SN-ZJU-SIAS-0011), the National Natural Science Foundation of China (31950410555), and the Ministry of Science and Technology of the People's Republic of China (QNJ20200226001, QNJ2021026001).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and Supplementary Materials.

**Conflicts of Interest:** The authors declare no conflict of interest.
