**1. Introduction**

The rapid development of industrialization and urbanization has resulted in severe environmental pollution [1]. Sewage or waste produced by industries and garbage generated by anthropogenic activities lead to the release of heavy metals into the environment, causing contamination of agricultural soil and water [2,3]. Thus, heavy metal pollution affects both human health as well as plant health, particularly plant growth and development [4].

Cadmium (Cd), a toxic heavy metal, severely inhibits plant growth and crop production when it occurs in high concentrations in soils or growth media [5]. The absorption and translocation of Cd in plants include distinct phases, such as the absorption of Cd in roots, the transportation of Cd into xylem and phloem, and the transportation of Cd into aboveground tissues [6]. The absorption and transportation of Cd in plants mainly depend on transport proteins such as heavy-metal-associated P-type ATPase family protein (HMA),

**Citation:** Liu, C.-X.; Yang, T.; Zhou, H.; Ahammed, G.J.; Qi, Z.-Y.; Zhou, J. The E3 Ubiquitin Ligase Gene *Sl1* Is Critical for Cadmium Tolerance in *Solanum lycopersicum* L. *Antioxidants* **2022**, *11*, 456. https:// doi.org/10.3390/antiox11030456

Academic Editor: Nafees A. Khan

Received: 22 January 2022 Accepted: 23 February 2022 Published: 25 February 2022

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ATP-binding cassette transporters (ABC), natural resistance-associated macrophage protein (NRAMP), metal-tolerance protein (MTP), calcium exchanger protein (CAX), and zinc/iron-regulated transporter-like protein (ZRT/IRT) [6–12].

Owing to the interaction with the sulfhydryl group of proteins, Cd can affect the activity of multiple enzymes and the functions of proteins [13]. Hence, the accumulation of Cd in plant tissues disorders various growth, biochemical, and physiological processes, such as photosynthesis, antioxidant enzyme activity, cell structure, and plant morphology [13,14]. Specifically, Cd stress causes damage to chloroplast ultrastructure, inhibits pigment synthesis, and affects several photosynthesis-related protein complexes [15]. Cd stress also impairs the balance between reactive oxygen species (ROS) production and ROS scavenging [16]. In particular, Cd stress disturbs the electron transfer chain in mitochondria and chloroplasts and activates NADPH oxidases, which cause excessive ROS accumulation and associated oxidative stress in plants [17,18]. Moreover, Cd stress induces protein denaturation, which disturbs the balance of protein quality in plant cells [19].

Plants have also evolved very diverse and complex defensive mechanisms for resisting heavy metal stress [20]. The ascorbate–glutathione (AsA–GSH) cycle is an indispensable pathway for eliminating oxidative stress [21]. Moreover, antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), and glutathione reductase (GR) are vital for scavenging ROS [22]. Antioxidant GSH not only functions on relieving oxidative stress, but also participates in the synthesis of phytochelatins (PCs) in plants, which can bind with heavy metal ions [20,23,24]. Moreover, the protein quality control system needs chaperone proteins to refold the denatured proteins or operates a protein degradation system to clear misfolded proteins under Cd stress [24,25].

Ubiquitin proteasome system (UPS) has been recognized as a critical process to control the abundance, quality, and function of protein in cells [26,27]. Ubiquitin is an indispensable component of UPS, which acts as identifying target protein for degradation [28]. Ubiquitindependent protein degradation requires reactions of multiple ubiquitin-related enzymes, including ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3) [29,30]. The ubiquitin molecule is activated by E1 and then transferred to E2. The E3 interacts with E2-ubiquitin and targets proteins for labeling substrate protein with ubiquitin. Finally, the 26S proteasome degrades the target proteins [31–33]. E3 ubiquitin ligases play critical roles in recognizing substrate proteins [29]. Among the ubiquitinrelated enzymes, E3 ubiquitin ligases have the largest family in plants [19]. The E3 ligase has been divided into three types, as follows: really interesting new gene (RING)-type, homology to E6-associated carboxyl-terminus (HECT)-type, and U-box-type [31].

RING-type E3 ubiquitin ligases have a cysteine-rich domain that can bind with Zn ions [31]. Previous studies have revealed that RING-type E3 ubiquitin ligases are involved in mediating plant tolerance of heavy metal stress [19,25,34]. A RING-type E3 ubiquitin ligase HIR1 confers tolerance of arsenic (As) and Cd stress in rice [34]. Overexpression of *HIR1* increases root length in rice under As and Cd treatment. The E3 ubiquitin ligase protein HIR1 also interacts with tonoplast intrinsic protein TIP4;1 and regulates its abundance in rice, thereby alleviating heavy metal stress [34]. Tomato E3 ubiquitin ligase RING1 increases Cd tolerance by minimizing ROS levels due to enhanced antioxidant enzyme activities [19,25]. However, the roles of many tomato E3 ubiquitin ligases are largely unknown, particularly in Cd stress.

In a previous study, we found that the expression of the RING-type E3 ubiquitin ligase *Sl1* significantly increased in tomato roots when challenged with aluminum (Al) or Cd stress [25]. However, the precise role of *Sl1* remains elusive. We hypothesized that *Sl1* might play a crucial role in Cd tolerance in tomato plants. Here, we characterized the function of *Sl1* by generating *sl1* mutants and *Sl1* overexpressing lines in tomato. Our study unveils a novel role of *Sl1* in plant tolerance to Cd stress.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Treatments*

In this study, tomato (*Solanum lycopersicum* L., Tomato Genetics Resource Center, Davis, CA, USA, https://tgrc.ucdavis.edu (accessed on 4 March 2018)) cultivar "Ailsa Craig" was used to generate *sl1* mutants and *Sl1* overexpressing transgenic lines.

Tomato seedlings were raised in vermiculite and transferred to a hydroponic jar (40 cm × 25 cm × 15 cm) containing Hoagland nutrient solution, when two real leaves of seedlings unfolded. The growth conditions were temperature of 23/20 ◦C (day/night), 14 h photoperiod, 60% humidity, and photosynthetic photo flux density (PPFD) of 600 μmol m−<sup>2</sup> s<sup>−</sup>1. For Cd treatment, seedlings at the five-leaf stage were treated with 100 μM CdCl2 and the hydroponic nutrient solution was changed every 5 days (d).
