MicroRNAs (miRNAs) are a class of short noncoding RNAs (20–24nt) that mediate gene expression by complementarily binding to their targeted transcripts for mRNA cleavage or protein translation inhibition (Sanei and Chen, 2015). Mature miRNAs are generated from precursor stem-loop structures (pre-miRNAs), which are the intermediates processed from primary miRNA transcripts (pri-miRNAs) transcribed by RNA polymerase II from MIR genes (Yu et al., 2019). Substantial evidence has shown that miRNAs play crucial roles in diverse biological processes, including plant development and biotic and abiotic stress responses. A growing number of yield-related agronomic traits have also been found to be associated with miRNAs (Zheng and Qu, 2015; Peng et al., 2019), which makes miRNAs promising targets for crop improvement.

Since the first set of plant miRNAs was identified in Arabidopsis in 2002 (Llave et al., 2002; Park et al., 2002; Reinhart et al., 2002), an increasing number of miRNAs have been discovered and annotated due to high-throughput sequencing. However, the biological functions of most miRNAs have not been thoroughly explored. To decipher the function of miRNAs, multiple molecular-based approaches have been applied, such as the overexpression of MIR genes (Aukerman and Sakai, 2003), anti-microRNA oligonucleotides (AMOs; Hutvágner et al., 2004), RNA interference (RNAi; Vaistij et al., 2010), artificial miRNA (amiRNA; Eamens et al., 2011), endogenous and artificial target mimicry (Ebert et al., 2007; Franco-Zorrilla et al., 2007; Yan et al., 2012), transcription activator-like effector nucleases (TALEN; Hu et al., 2013), and clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9; Jacobs et al., 2015; Figure 1). Functional analyses of miRNAs have been achieved through overexpression for gain-of-function or knockdown/knockout for loss-of-function. Among these techniques, RNAi, amiRNA, TALEN, and CRISPR/Cas9, which were initially applied for the functional analysis of protein-encoding genes, have proven to be useful for the subsequent regulation of miRNAs (Fire et al., 1998; Schwab et al., 2006; Morbitzer et al., 2010; Shan et al., 2013). AMOs and sponges (SPs) were initially developed to inhibit miRNA action in animal systems and subsequently adopted in plants (Xian et al., 2014; He et al., 2016). MiRNA decoys/mimics such as target mimic (TMs) and short tandem target mimics (STTMs) were developed based on the discovery of miRNA sequestration by endogenous target mimics in plants (Franco-Zorrilla et al., 2007; Todesco et al., 2010; Yan et al., 2012).

These techniques have greatly expanded our abilities in plant miRNA research; more importantly, these methods are constantly being improved for broader applications in research and plant breeding. Thus, there is a strong need for a comprehensive understanding of the key features of each technique and effective controls on these methods for various research purposes. Here, we reviewed the major approaches used for determining the function of miRNAs, with a focus on the STTM technique and its applications in the functional characterization of miRNAs involved in various rice agronomic traits, and illustrated transgene-free breeding practices based on transgenic outcomes via CRISPR/Cas9. We also discussed the challenges and potential future trends of STTM applications in the functional analysis of miRNAs and in crop breeding.

A gain-of-function system that constitutively overexpresses a miRNA may alter its localization and dose. Therefore, the non-authentic phenotypes may not reflect the normal function of the miRNA. Thus, various loss-of-function strategies have been extensively exploited as alternative and effective approaches to evaluate the roles of many miRNAs. Of these approaches, miRNA decoy techniques such as TM and STTM, and the genome-editing system CRISPR/Cas9 are applied much more widely than others (Figure 3). As the first miRNA decoy technique created for plants, the TM approach has been applied in many studies, and the functions of many miRNAs have been successfully uncovered (Todesco et al., 2010). In recent years, STTM and CRISPR/Cas9 techniques have been developed rapidly and have become the two most widely adopted approaches in functional analysis of miRNAs in plants.

The STTM technique is widely accepted in miRNA functional studies in plants because it can be applied in a variety of ways. First, the STTM structure has two miRNA-binding sites, and it can efficiently silence some highly abundant miRNAs (e.g., miR156/157, miR165/166, and miR398) and generate visible phenotypes (Yan et al., 2012; Reichel et al., 2015; Zhang et al., 2017). Second, STTMs can be used to study the interactions between two miRNAs by inserting two different miRNAs in the same STTM construct (Peng et al., 2018). Moreover, it has been reported that STTMs can silence multiple distinct miRNAs simultaneously with an increased number of tandem miRNA-binding sites (Fei et al., 2015). Finally, the STTM approach can be adopted as an important complement to CRISPR/Cas9 in certain situations, such as the knockout of miRNAs resulting in drastic or lethal developmental defects (Teotia et al., 2016) or the locations of miRNAs overlapping with other genes in the chromosome.

The STTM approach can be applied in a constitutive (Tang et al., 2012), inducible (Peng et al., 2018) or tissue-specific (Zhao et al., 2019) manner driven by the corresponding promoter. This makes it possible to study miRNA functions spatiotemporally and to precisely modify the crop traits of interest. Since its development, the STTM approach has been extensively adopted for the functional analysis of numerous miRNAs in multiple species, including rice (Xia et al., 2015; Zhang et al., 2017; Zhao et al., 2019), tomato (Damodharan et al., 2016; Jiang et al., 2018; Yang et al., 2020), soybean (Wang et al., 2014; Nizampatnam et al., 2015), cotton (Wang et al., 2017; Liu et al., 2019), maize (Zhang et al., 2019), wheat (Guo et al., 2018), and tobacco (Diao et al., 2019; Figure 3). Rice is the most important staple crop worldwide, and several of its agriculturally important traits are controlled by miRNAs (Tang and Chu, 2017; Peng et al., 2019). Given the extensive application of STTMs in rice, we reviewed STTM-based functional studies of miRNAs in rice and focused specially on agronomic traits.

Higher yield and enhanced stress resistance are important breeding goals of crop breeders. Yield is a sophisticated agronomic trait in rice that is generally determined by four decisive factors: the number of tillers per plant, number of grains per panicle, grain size/weight, and the ratio of filled grains (Sakamoto and Matsuoka, 2008; Xing and Zhang, 2010). In addition, tiller angle and plant height are important agronomic traits that determine the ideal architecture and the leaf size, shape, and inclination determine the leaf architecture, all of which eventually affect the grain yield (Zuo and Li, 2014). Extensive studies have supported the vital roles of miRNAs in regulating diverse important agronomic traits and stress responses in crops (Zheng and Qu, 2015; Tang and Chu, 2017; Peng et al., 2019). To date, dozens of miRNA families have obtained STTM transgenic lines in rice, and several of them show obvious phenotypic alterations associated with yield-related agronomic traits and stress responses (Figure 4; Table 1). Hereafter, we provide an overview of the application of STTM in rice classified by different agronomic traits.

Although many agronomically improved STTM transgenic lines have been generated, these achievements have not yet been commercialized. Currently, the commercial development of genetically modified organisms (GMOs) is considered a threat to human health and the environment and is thus under stringent governmental regulations (Prado et al., 2014). Transgene-free edited crops created via CRISPR/Cas9 receive the regulatory waivers in many countries (Chen and Gao, 2020). Transgene-free edited crops can be generated through conventional and transient expression methods. In conventional transformation methods, transgene-free edited progenies can be obtained by eliminating CRISPR transgenes via genetic segregation through selfing or crossing (Gao, 2021). In the transient expression methods, transgene-free genome editing can be generated through the transient expression of CRISPR/Cas9 DNA, RNA, or RNP (ribonucleoprotein) delivered into plant cells (Woo et al., 2015; Zhang et al., 2016b; Liang et al., 2017). Recently, transgene-free gene-edited non-browning white button mushrooms (Waltz, 2016) and high-GABA tomato (EUROFRUIT, 2021) have been commercialized outside of GMO regulations. Furthermore, due to the precise genome modifications, time savings, and transgene-free nature of these crops (Zhu et al., 2020), some practices for translating the outcomes of transgenic into transgene-free breeding by CRISPR/Cas9 in crops have been developed.

Great progress has been made by using a transgene-free edited system on protein-coding genes. A series of transgene-free edited crops with increased yield (Li et al., 2016), improved quality with low amylose content (Zhang et al., 2018a), high amylose content (Sun et al., 2017; Tuncel et al., 2019; Li et al., 2021), higher grain fragrance (Usman et al., 2020), enriched γ-aminobutyric acid (Akama et al., 2020), and enhanced biotic stress resistance (Zhou et al., 2020) have been generated. Currently, several studies are also trying to convert the accumulating knowledge of miRNA functions into a transgene-free edited system. In rice, the overexpression of OsmiR535 resulted in reduced tolerance to drought and salinity, while the knockdown of OsmiR535 by STTM conferred enhanced tolerance to drought and salinity (Yue et al., 2020). A transgene-free, drought-tolerant osmir535 mutant was generated by CRISPR/Cas9 mediated knockout, which provides a successful example for abiotic stress-resistant breeding (Yue et al., 2020). In soybean (Glycine max L.), gma-miR398c functions negatively in drought tolerance by targeting multiple peroxisome-related genes, GmCSDs and GmCCS (Zhou et al., 2020). Arabidopsis and soybean overexpressing gma-miR398c both showed decreased drought tolerance, while the gma-miR398c mutant generated by CRISPR/Cas9 showed increased drought resistance. Gma-miR398c was demonstrated to be a valuable locus for crop improvement by creating drought resistance-enhanced soybean via CRISPR/Cas9 (Zhou et al., 2020). In tomato, one of the most important horticultural crops, late blight caused by Phytophthora infestans (P. infestans) is a great threat to production (Fry et al., 2015). MiR482 was identified as a negative regulator of the resistance to P. infestans by targeting NBS–LRR disease-resistance genes in tomato (Jiang et al., 2018; Canto-Pastor et al., 2019). The overexpression of miR482b caused more severe disease symptoms in plants infected by P. infestans, while the silencing of miR482b by STTM resulted in enhanced resistance to P. infestans in tomato (Jiang et al., 2018). Based on these transgenic outcomes, mir482b and mir482b/c were generated by CRISPR/Cas9, and both showed enhanced resistance to P. infestans. In addition, mir482b/c was more resistant to P. infestans than mir482b in tomato, demonstrating that miR482 is an important target for cultivating pathogen-resistant tomatoes via CRISPR/Cas9 (Hong et al., 2020). The ever-expanding genetic resources created by these miRNA techniques provide us with a promising prospect for crop improvement and breeding.

Multiple tools have been developed for the functional analysis of miRNAs, thus contributing to the crop improvement and future agriculture. Given the advantages and drawbacks of each approach, it is important to design proper strategies for each specific study and the miRNAs of interest. STTM is one of the most widely adopted miRNA techniques, and a large collection of STTM lines have been generated in crops, especially in rice. STTM and related miRNA techniques help to reveal the complex molecular mechanisms involved in the regulation of agronomic traits, and also provide potential breeding materials for crop improvement and breeding. These transgenic achievements, together with the CRISPR/Cas gene-editing system and many other breeding techniques, will allow the generation of transgene-free plants and greatly facilitate precision crop breeding.

To date, as the majority of functional studies focus on conserved miRNAs, our knowledge about species and tissue specific miRNAs is extremely limited, which offers a broad space for the application of STTM and other approaches. Further identification and validation of these miRNAs will lead to a great leap for the application of miRNAs in crop improvement. Due to the multiple roles of miRNAs in plant developmental processes, STTM lines of these miRNAs driven by the 35S promoter show pleiotropic effects that alter more than one trait (Table 1). Tissue-specific or inducible promoters may be an appropriate alternative to facilitate STTM as a useful tool to study the functions of miRNAs in specific tissues and increase yield without penalty on other agronomic traits. Not all STTM rice lines showed apparently altered morphologies (Zhang et al., 2017), and other approaches mentioned previously are advocated to be adopted for the desired loss-of-function outcome. Multiple genetics-based approaches are imperative and may work complementarily in fully mining and using vital miRNAs associated with superior agronomic traits. MiRNAs are important regulators in biotic and abiotic stress responses. Fine-tuning the accumulation of stress-related miRNAs by STTM techniques may provide valuable resources for the assessment of varying stress responses. Additionally, due to public safety concerns over transgenic crops, the commercialization of improved crops bred by STTM-based techniques is restricted. Some successful breeding practices on translation of transgenic outcomes into transgene-free outcomes via CRISPR/Cas9 in rice have been adopted. Useful targets for crop improvement confirmed by STTM have huge potential for application in breeding through non-transgenic translation, which may accelerate the creation of new varieties and strengthen food security.

GT supervised the project. JC and TL wrote this paper. ST edited and gave suggestions for the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

This work was supported by funding from the Guangdong Innovation Research Team Fund (grant no. 2014ZT05S078), the National Natural Science Foundation of China (grant no. 32070852) and the open fund of the Guangdong Key Laboratory of Plant Epigenetics.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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