**1. Introduction**

Human activity, natural pollution such as volcanic ash, climate change and the use of recycled water, among other reasons, have resulted in an increase in the area of cadmium (Cd)-contaminated soil [1–3]. The presence of Cd in soil is a serious environmental hazard, as the metal is not only toxic to plants but also transmissible through the food chain, threatening ecosystems and human health [4,5]. Compared with physicochemical treatment, phytoremediation is an economical, natural and effective method for managing Cd soil contamination [6,7]. Therefore, it is important to find suitable Cd-tolerant plants and elucidate their relevant molecular mechanisms.

Cd toxicity can inhibit carbon fixation and reduce chlorophyll content and photosynthetic activity [8]. When plants are exposed to Cd-contaminated soil, osmotic stress occurs, reducing the relative moisture content, stomatal conductance and transpiration [9]. Meanwhile, an excessive occurrence of reactive oxygen species (ROS) can damage plant membranes and destroy cell biomolecules and organelles [10]. Cd also interferes with the transport and uptake of mineral elements [9]. To avoid the harm caused by Cd stress, plants have undergone a series of morphological, physiological and biochemical evolutionary changes [11]. One major mechanism is external exclusion, which prevents plant cells from absorbing excessive Cd and hinders Cd transport within the plant [12,13]. Another mechanism is tolerance to Cd accumulation, whereby Cd exists in a non-biologically active conjugated form within the plant through chelation and compartmentalization [14]. Studies have reported that when Cd is absorbed by a plant, proteins such as metallothioneins (MTs) can form stable chelates with the Cd ion, thus reducing its toxicity [4,15]. The regulation of transcription factors is a vital mechanism in plant cells that protects them from heavy metal

**Citation:** Wang, Z.; Ni, L.; Liu, L.; Yuan, H.; Gu, C. *IlAP2*, an AP2/ERF Superfamily Gene, Mediates Cadmium Tolerance by Interacting with *IlMT2a* in *Iris lactea* var. *chinensis*. *Plants* **2023**, *12*, 823. https://doi.org/ 10.3390/plants12040823

Academic Editor: Andrzej Bajguz

Received: 25 December 2022 Revised: 10 February 2023 Accepted: 10 February 2023 Published: 12 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

exposure [16]. For example, previous studies showed that metal-responsive transcription factor 1 (MTF1) is involved in cellular protection against Cd stress signals [17]. Under Cd stress conditions, MTF1 can translocate into the nucleus and bind to metal-responsive elements (MREs), mediating the transcription of a series of downstream genes such as MTs, ZIP10, ferroportin 1 (FPN 1), selenoprotein H (Sel H) and selenoprotein W (Sel W) [16].

*Iris lactea* var. *chinensis*, an ornamental perennial groundcover plant that grows fast and forms a large biomass, is resistant to several stresses [18,19]. In particular, it has a very strong tolerance to Cd and the ability to accumulate Cd, which makes it suitable for improving Cd-contaminated soil [20]. We preliminarily analyzed the regulatory mechanism of *I. lactea* in response to Cd stress through transcriptome sequencing and mined an MT gene, *IlMT2a*, that may play a key role in Cd tolerance [21]. When constitutively expressed in *Arabidopsis thaliana*, *IlMT2a* leads to greater root length under Cd stress compared to the wild type (WT) [22]. In turn, we identified several proteins interacting with IlMT2a using a yeast two-hybrid assay (Y2H) with a yeast library constructed from cadmium-treated *I. lactea* seedlings and found that IlAP2, an apetala2/ethylene responsive factor, may be an interacting partner of IlMT2a [3].

Many transcription factors can be regulated by Cd stress and induce MTs to confer Cd tolerance [23–25], and *AP2* genes have been specifically induced by Cd in many plants, including *A. thaliana*, rice and kenaf [26–28]. Many reports have documented that AP2s are important regulators involved in plant growth and development, hormonal regulation, plant metabolite biosynthesis and the conferring of stress tolerance to plants [29,30]. Recently, potato *StAP2/ERF*s were found to be indispensable for Cd uptake and tolerance and may be useful in designing gene-modified plants with improved Cd tolerance [31]. To study the role of *IlAP2* in regulating the Cd tolerance of *I. lactea*, we cloned the open reading frame of *IlAP2* and confirmed the interaction between IlMT2a and IlAP2 using Y2H and a bimolecular fluorescence complementation test (BiFC). Following that, the regulation function and mechanism were explored through genetic transformation and transcriptome sequencing. This study provides information on the molecular mechanism of Cd tolerance and a gene resource for improving plant tolerance to Cd.

#### **2. Results**
