**3. Discussion**

Heavy metal pollution of soil and water is a serious environmental problem. Among heavy metal pollutants, Cd is considered to be one of the most toxic ones [24]. Cd can be quickly absorbed by plants due to its high solubility in water, which is the main way it enters the food chain [33]. Plant roots can absorb and transport Cd to nutritional and reproductive organs even at low Cd concentrations, which has a negative impact on the regulation of the plant's nutrition and its mineral balance during growth [34].

Several genes in plants, such as those encoding an ATP-binding transporter, ZRT/IRTlike proteins and glutathione, have been reported to participate in reducing Cd ion toxicity by inhibiting Cd ion absorption and transport, improving Cd ion chelation ability and other processes [35,36]. Gene *BcMT2* could enhance Cd tolerance and reduce the production of

reactive oxygen species (ROS) in *A. thaliana* [37]. Similarly, we previously discovered that *IlMT2a* in *I. lactea* increased plant tolerance to Cd by potentially reducing the accumulation of H2O2 and O2 •− [22]. However, *IlMT3*, another MT, was identified as an interacting protein of *IlMT2a* but did not provide protection against Cd toxicity in transgenic *A. thaliana* [3]. In this study, an AP2/ERF superfamily gene that encodes an interacting protein of IlMT2a, further validated its role in *I. lactea*'s response to Cd stress.

The transformation of transcriptomics under Cd stress has been studied in several plant species, which has helped to identify many Cd-responding transcription factors [24]. Different transcription factors, such as ERF (ethylene-responsive factor), WRKY, bZIP (basic leucine zipper) and MYB (myeloblastosis protein), control the expression of specific stressrelated genes under Cd stress and play important roles [24]. Based on the ORF sequence amplified by PCR, the gene was named *IlAP2* following the phylogenetic tree and amino acid sequence analyses. To further understand the cytological function of the IlAP2 protein, we studied its expression and found that it was localized in the nucleus. In addition, the BiFC assays showed that the site of interaction between *IlAP2* and *IlMT2a* was also in the nucleus. The expression pattern analysis indicated that *IlAP2* was a stress-responsive transcription factor that could be regulated by Cd stress and maintained a steady high level of expression under Cd stress treatment. These findings indicate that *IlAP2* may be involved in the response to Cd stress and has a positive effect on Cd-stress tolerance, similar to some *StAP2/ERF* genes in kenaf [23].

Plant-specific AP2/ERF transcription factors can regulate plant responses to environmental stimuli or plant growth and development, depending on the presence of a highly conserved 60-amino-acid AP2 domain in the protein [38,39]. For example, rice *OsEREBP1* attenuates disease caused by *Xanthomonas* and confers drought and submergence tolerance by activating the jasmonate and abscisic acid signaling pathways, thereby priming the rice plants for enhanced survival under abiotic or biotic stress conditions [40]. *A. thaliana RAP2.6* participates in abiotic stress, including abscisic acid (ABA), salt and osmotic stress, possibly through the ABA-dependent pathway [41]. A previous study found that *StAP2/ERF*s are indispensable in Cd uptake and tolerance and may be useful in designing gene-modified potato plants with improved Cd tolerance [31]. To further demonstrate that *IlAP2* can improve plant Cd tolerance, we performed phenotype analysis on overexpressing *A. thaliana* plants. Under normal conditions, the phenotypic difference between the transgenic and WT *A. thaliana* plants was not significant. After Cd treatment, the growth of all *A. thaliana* plants was inhibited, but the root length and growth of the WT were inhibited to a greater extent, indicating that *IlAP2* influences the response of *A. thaliana* to Cd stress.

The overexpression of *TdSHN1*, a wheat ERF transcription factor, in transgenic tobacco conferred Cd tolerance, produced less ROS under excess Cd compared to WT plants and had higher activities of ROS-scavenging enzymes, which might contribute to Cd tolerance [42]. IlAP2 was confirmed to interact with IlMT2a and improve Cd tolerance. Although there is no report on the overexpression of *AtAP2* in *Arabidopsis*, the expression of potato *StAP2/ERF075/077/126* has been shown to increase tolerance to Cd [31]. Therefore, *AP2* may play an important role in the regulation of cadmium tolerance. Based on these results, we used transgenic *Arabidopsis* to preliminarily explore the regulatory mechanism through transcriptome sequencing, laying the foundation for subsequent research. A total of 1,340 and 870 genes were identified as up- and down-regulated, respectively. Enrichment analysis showed that *IlAP2* could alter ion signal transduction, plant hormones and other transcription factors at the transcriptional level, playing an important role in the plant's response to Cd stress. Previous studies have preliminarily analyzed the regulatory mechanism of *I. lactea* in response to Cd stress through proteome data and found that Cd regulatory proteins were mainly involved in signal transduction, ion transport, redox reactions, ion binding and other functions [43]. These findings indicate that *IlAP2* interacts with *IlMT2a* in the nucleus and may cooperate with other transcription factors to regulate genes related to signal transduction and plant hormones, thereby reducing Cd toxicity.
