The rapid growth of industrialization and the large-scale use of chemical fertilizers and pesticides have led to the continuous increase in heavy metal content in the soil. In plants, copper (Cu), zinc (Zn), iron (Fe), and manganese (Mn) are trace elements necessary for plant development and growth, but excessive accumulation can cause cell damage [1]. Some other metals such as cadmium (Cd), chromium (Cr), lead (Pb), aluminum (Al), arsenic (As), and mercury (Hg), as non-essential elements, are toxic even at low concentrations [2]. Studies have reported that heavy metal stress can inhibit activity of antioxidant enzyme systems and differential expression of a large number of proteins in plants, weakening photosynthesis, accompanied by a series of phenotypes such as suppressed root development and leaf senescence or even necrosis [3]. In order to avoid the destructive consequences of heavy metal toxicity, plants have developed corresponding coping mechanisms to resist heavy metal stress [3]. Plants can reduce heavy metal concentrations in their bodies by limiting the uptake of heavy metal ions and stimulating metal efflux, as well as by complexation of metal ligands such as glutathione (GSH), metallothionein (MT), and phytochelatins (PCs) [4,5,6]. In addition, the antioxidant defense mechanism will also be activated to reduce elevated reactive oxygen species (ROS) levels, thereby reducing oxidative damage [7,8]. A large number of studies have shown that gene expression plays a very important role in regulating the tolerance of HMs or individually regulating various stress response genes to form a gene network. Some functional group genes encode metabolites such as amines, alcohols, and sugars, which also play a crucial role in heavy metal stress tolerance [9,10].

With the discovery of small RNAs, there is growing interest in the importance of post-transcriptional gene regulation of microRNAs (miRNAs) in plant development and responses to environmental stresses. miRNAs are an extensive class of small noncoding (19~24 nt) RNAs molecules [11]. In plants, mature miRNAs are produced through a multistep process including the transcription, precursor processing, methylation, and assembly of miRNA-induced silencing complex (miRISC) [12]. Then, the mature miRNAs lead the RISC to target the complementary mRNAs, which play a vital post-transcriptional regulatory role in gene expression by target mRNA cleavage or translational inhibition [13]. In recent years, a crescendo of miRNA studies has demonstrated that miRNAs play important roles in tissue development and differentiation, phytohormones signaling, secondary metabolite production, and biotic and abiotic stress [14,15]. miRNAs affect multiple processes of plant growth, development, and response to stress by up-regulating or down-regulating their expression [16,17]. miR165/166 and miR394 have been shown to be involved in shoot apical meristem (SAM) development, including the direct post-transcriptional regulation of key SAM-related genes, which in turn maintains SAM development [18,19]. In addition, studies have shown that miR319 has a conserved regulatory function in leaf development. For example, ectopic upregulation of miR319 resulted in dramatic changes in tomato leaf size and shape [20]. CUC1 and CUC2 mRNAs accumulate in the axil of leaf primordia and play a key role in the establishment of axillary bud meristems. The regulatory mechanism of miR164-CUC1/CUC2 may be related to LAS-mediated initiation of axillary bud meristems [21]. In addition, miRNAs play important roles in plant root development. miR160 acts as a key controller and cleaves ARF10 and ARF16 transcripts during plant root cap formation [22]. In contrast, miR167 plays an active role in adventitious root formation, while miR156 and miR172 are well-studied miRNAs involved in floral control [23,24]. Overexpression of miR172 can promote flowering time in both monocotyledonous and dicotyledonous plants. In contrast, the expression level of miR156 decreased gradually from sowing to flowering, while upregulation of miR156 resulted in delayed flowering transition. These findings suggest that miRNAs play regulatory roles in different developmental transitions by mediating specific signaling pathways. In different models and crop plants (such as Arabidopsis, wheat, rice, maize, and barley), miRNAs regulate gene expression during different stress responses (drought, heat, salinity, cold, nutrition, and pathogens). In addition to plant growth and development, the role of conserved miRNA target modules is also critical for conferring stress tolerance through integration into metabolic pathways [25]. Studies have confirmed that miR160-ARF, miR156-SPL, miR159-MYB33, miR164-NAC, miR172-AP2, miR394-LCR, miR396-GRF, and miR398-CSD modules play important regulatory roles in different stress environments to mitigate the effects of adverse reactions [12]. For example, in Arabidopsis, increased expression of miR398 enhances plant heat tolerance by negatively regulating the expression of its targets CSD1, CSD2, and the copper chaperone (CCD) of CSD [26]. The highly conserved miR394-LCR module is involved in plant responses to cold stress [27]. The expression of auxin-responsive factors ARF10, ARF16, and ARF17, mediated by miR160 and miR167, resulted in enhanced salinity tolerance of cotton under high salt stress. Overexpression of osa-miR319a exhibited higher tolerance to drought and salt stress by regulating the TCP transcription factor [28].

Increasing evidence has also revealed that miRNA-mediated gene regulation plays a significant role in heavy metal regulatory networks. In addition, high-throughput genome-wide expression profiling has greatly improved our current understanding of the key involvement of miRNA in the toxic response of plant HMs and its targets. A substantial number of heavy-metal-responsive miRNAs have been identified in the Oryza sativa L., Zea mays L., Medicago truncatula L., Hordeum vulgare L., Vitis vinifera L., Brassica juncea L., and many other plants (Table 1) [6,29,30]. In different plant species, many miRNAs have significant differential expression of different HM such as Cd, Hg, Al, As, and Cr. Through the analysis of metal-regulated miRNA target genes, it has also been identified that many miRNAs are involved in the response of plants to HM. The target genes for various processes, including metal absorption and transport, sulfate distribution and assimilation, protein folding, antioxidant systems, and plant hormone signal transduction processes [11]. We searched the Web of Science core collection database for articles related to miRNA and heavy metals such as Cu, Cd, Hg, Al, As, and Cr in the past 10 years, and a total of 568 papers were retrieved. The research fields are mainly distributed in three aspects: Life Sciences Biomedicine, Science Technology, and Physical Sciences. Then, bibliometric analysis was performed using VOS software [31], and four clusters were formed with miRNAs, stress, cadmium, and strategy as the research centers (Figure 1). These keywords are categorized by publication year to deepen the analysis. The VOSviewer software represents the year with the highest number of publications between 2016 and 2019. However, articles from 2011 to 2022 were included in this analysis. The blue circles represent topics that have been intensively studied in the past decade, such as stress response, miRNA, target gene, etc. The results of the two co-occurrence maps showed that miRNAs and stress were most closely related, suggesting that the involvement of miRNAs in stress response is the current research hotspot.

In recent years, research on miRNA and HM stress has been increasing, and, with the rapid development of advanced technologies such as next-generation sequencing (NGS), a number of known and unknown miRNAs in response to metal stress are discovered through whole genome sequencing and miRNA sequencing. However, studies on the roles of different miRNAs in HMs signal transduction in plants and their targets are still in their infancy.

2. Response of miRNAs to Cu Toxicity