The Biosynthesis Process of Small RNA and Its Pivotal Roles in Plant Development. (2024)

Author(s): Quan Li [†]; Yanan Wang [†]; Zhihui Sun; Haiyang Li (corresponding author) [*]; Huan Liu (corresponding author) [*]

1. Introduction

sRNAs are found across all domains of life, including in bacteria, archaea, and various eukaryotes. Their diverse compositions and functions have continued to astonish researchers over the past two decades [1]. Synthesized by the RNAIII enzyme, these sRNAs are excised from dsRNA or single-stranded RNA (ssRNA) harboring a stem–loop architecture [2,3]. As non-coding entities, sRNAs pervade plant cellular machinery, orchestrating the regulation of target gene mRNAs—a process central to gene expression control, genomic integrity, stress response, and a myriad of vital biological functions [4,5,6]. The unearthing of a vast spectrum of sRNAs in the plant has elucidated the complexities inherent in plant gene regulatory mechanisms. This discovery has significantly propelled the field of agricultural biotechnology forward, offering novel insights and applications in the modulation and enhancement of plant traits [7,8,9].

Amidst the vast constellation of non-coding RNAs, this review narrows its focus to plant sRNAs, elucidating their biosynthetic origins and biological mandates, specifically miRNAs and siRNAs [6]. The sRNA processing imparts distinct length characteristics to these molecules. Contrastingly, the preferential selection of the initial nucleotide at the sRNA’s 5'-terminus is pivotal in dictating the recruitment of Argonaute (AGO) proteins, thereby delineating the sRNA’s functional spectrum [7,8]. miRNAs are single-stranded, non-coding RNA molecules containing 20 to 24 nucleotides. They are widely found in eukaryotes and play a crucial role in regulating gene expression. Derived from RNA transcripts, miRNAs bind specifically to target mRNA, inhibiting post-transcriptional gene expression. Their functions extend to cell cycle regulation and developmental timing in organisms [9]. miRNAs are engendered through the precise processing of stem–loop structured precursor RNAs (pre-miRNAs), which are transcribed by the esteemed RNA polymerase II [9,10]. siRNA is a class of double-stranded RNA molecule, typically 21–24 base pairs long. Similar to miRNA, siRNA functions within the RNA interference (RNAi) pathway. It specifically targets and degrades mRNAs that express genes with complementary nucleotide sequences, thereby inhibiting translation [10]. Concurrently, the origin of siRNAs is ascribed to the cleavage of dsRNA substrates, underscoring the divergent pathways that shape the sRNA landscape [6,10]. To enhance our understanding of sRNAs and facilitate their application in agricultural production, we offer an overview of sRNA biogenesis, transport processes, and the underlying mechanisms of their functions.

2. sRNA

2.1. miRNA

miRNAs, a cadre of diminutive RNA entities, span approximately 20–24 nucleotides. In the verdant realm of plants, these miRNAs typically manifest at a length of 21 nucleotides and are pivotal in the orchestration of gene regulatory networks. Through their affinity for target gene mRNAs, miRNAs enact a process of RNA-induced silencing [11]. The molecular dialogue between miRNAs and their cognate target sequences is governed by the extent of nucleotide complementarity, which consequently determines the fate of both the miRNA and the target mRNA. In plants, high-precision alignments between miRNAs and their targets instigate the total disintegration of the target gene’s transcript. Conversely, miRNAs that exhibit partial complementarity to their targets predominantly attenuate the translational process of the corresponding gene products (Figure 1) [10,12].

Within the elaborate choreography of gene expression, miRNA genes are initially transcribed by RNA polymerase II into primary transcripts, known as pri-miRNAs, which consist of several hundred nucleotides. These nascent transcripts are then refined by the RNaseIII family’s Dicer-like enzymes, predominantly DCL1, into pre-miRNA. Subsequently, DCL1 meticulously carves the pre-miRNA into a double-stranded configuration, which is then elegantly modified by the addition of a methyl group at the 3'-terminus’s ultimate nucleotide, a reaction catalyzed by the miRNA methyltransferase HUA ENHANCER 1 (HEN1) [13,14]. In the nucleus, mature miRNA strands are integrated into ARGONAUTE 1 (AGO1). Within the nucleus, these mature miRNA strands are assimilated into ARGONAUTE 1 (AGO1), completing their journey to functionality. In this final act of the post-transcriptional gene regulation ballet, the mature miRNA, ensconced within the ARGONAUTE 1 (AGO1) complex, executes its role with precision. It either catalyzes the mRNA excision—a process akin to molecular surgery, excising the target mRNA with exactitude—or it imposes translational repression, subtly silencing the mRNA without cleavage. Through these mechanisms, miRNAs exert their influence, conducting the intricate symphony of gene silencing that fine-tunes the expression of the plant’s genetic repertoire [15,16,17,18].

Currently, it is acknowledged that a multitude of miRNAs with well-characterized functions exhibit a high degree of conservation; however, this is not a universal trait among plant miRNAs. While many miRNAs are conserved, a significant number are not [19]. A considerable proportion of plant miRNAs are endemic, flourishing solely within their native species and a select cadre of closely allied taxa. These miRNAs, termed species-specific miRNAs, often elude identification of their target genes or may not be associated with any targets at all. Moreover, the expression levels of these species-specific miRNAs are markedly subdued when contrasted with their conserved counterparts [20,21].

2.2. siRNA

In plants, siRNAs are critical modulators of gene silencing, comprising 21 to 24 nucleotides derived from dsRNA precursors. Predominantly, these endogenous siRNAs originate from genomic repetitive sequences and transposable element (TE) intergenic regions, with a characteristic length of 24 nucleotides. The biogenesis of these molecules involves their generation from dsRNA and subsequent processing via Dicer-like 3 (DCL3) enzyme cleavage. Intriguingly, the majority of 24-nucleotide miRNAs identified in both Arabidopsis and rice exhibit species-specific patterns. This specificity implies that these 24-nucleotide miRNAs may represent evolutionary novelties, having emerged recently and, thus, not having been subjected to extensive evolutionary selection, as suggested by recent studies (Figure 1) [22,23].

The synthesis of plant siRNAs is distinct from that of miRNAs, primarily because siRNAs are typically derived from long non-coding RNAs (lncRNA), regions proximal to heterochromatin, repetitive sequences, and TEs within the genome [24,25]. In the intricate regulatory networks of plant genomics, the plant-specific RNA polymerase IV (Pol IV) is of paramount importance. It is strategically recruited to the loci designated for RNA-directed DNA methylation (RdDM), where it initiates the synthesis of single-stranded siRNA precursors. This recruitment is not merely a procedural formality but a critical juncture in the pathway, pivotal for the subsequent steps that culminate in the nuanced orchestration of gene expression regulation [26]. In the context of plant molecular biology, the transformation of single-stranded siRNA precursors into dsRNA is facilitated by RNA-dependent RNA polymerase 2 (RDR2). Following this, the DCL3 enzyme meticulously processes the dsRNAs, culminating in the production of 24-nucleotide mature siRNAs [27,28]. These siRNA molecules are subsequently methylated by HEN1 and then translocated to the cytoplasm [29]. Upon integration with the RISC, the dsRNA is bifurcated into single strands. One strand assumes the role of the guide strand within the RISC, while its counterpart is relegated to the passenger strand [30]. Concurrently, the plant-specific Pol V engages in the transcription of non-coding RNAs at the RdDM loci. These non-coding RNAs are pivotal in magnetizing the siRNA–AGO4 complex, a process orchestrated by the nucleotide complementarity between the non-coding RNAs and the guide strand of the RISC. This molecular affinity facilitates the enlistment of DNA Remethylase 2 (DRM2), thereby initiating the process of DNA methylation at the RdDM sites [31].

Phased small interfering RNAs (phasiRNAs) represent a distinct subset of endogenous siRNAs that exert a pivotal regulatory influence in plants. A subset of phasiRNAs has been documented to orchestrate the degradation of complementary mRNAs, a critical aspect of gene regulation [32]. The genesis of phasiRNAs is predicated upon the meticulous cleavage of either target mRNA or lncRNA by a 22-nucleotide miRNA. This cleavage event triggers the conversion of the cleaved RNA into dsRNA through the enzymatic activity of RDR6. The dsRNA is subsequently subjected to systematic cleavage by Dicer-like proteins, notably DCL4, culminating in the formation of a double-stranded phasiRNA body. Analogous to the miRNA double-stranded bodies, the RISC laden with phasiRNA engages in hom*ologous base pairing with the target RNA, leading to its degradation [32].

LncRNAs are associated with sRNAs due to their roles as sRNA targets, sRNA precursors, or miRNA sponges. These interactions contribute to the regulation of plant immunity and growth. Experimental evidence has revealed specific connections between lncRNAs and sRNAs in plants [33]. In addition to miRNAs, lncRNAs can also be targeted by siRNAs. Notably, vsiRNA-mediated lncRNA cleavage seems to play an emerging regulatory role for siRNAs. Previous studies have demonstrated that virus-infected plants exhibit an accumulation of viral siRNAs. These viral siRNAs, which exhibit high sequence complementarity to host genes or host gene promoters, can induce the cleavage of host mRNAs or transcriptional inactivation of host gene promoters [33].

3. Key Protein Involved in the Biosynthesis of sRNAs

In the realm of plant biology, a diverse array of sRNAs are generated via distinct biogenesis pathways (Figure 2). A cadre of proteins, each playing an indispensable role in these pathways, have been identified. Within the intricate molecular machinery of plant cells, RNA polymerases and RNA-dependent RNA polymerases (RDRPs) are indispensable catalysts in the transcriptional genesis of sRNA precursors. These enzymes are central to the complex network of pathways that facilitate the synthesis and subsequent processing of sRNAs, thereby underpinning the sophisticated regulatory systems that modulate gene expression through sRNA-mediated interactions. Dicer/Dicer-like proteins, belonging to the ribonuclease class III, are responsible for the cleavage of these sRNA precursors. The methylesterase HEN1 contributes to the stabilization of sRNA, while the ARGONAUTE (AGO) protein is pivotal in the formation of the RISC with sRNA [34].

3.1. RNA Polymerases (Pols)

In the botanical realm, plants possess a quintet of RNA polymerases, each with distinct roles: Pol I–III, which are ubiquitous among eukaryotic organisms, and Pol IV and V, which are unique to the plant kingdom. Specifically, Pol I is tasked with the transcription of the 45S rRNA genes, a critical component of protein synthesis machinery. In contrast, Pol II oversees the transcription of the majority of genes, including those encoding proteins. Pol III transcribes shorter structural RNAs, such as tRNA and 5S rRNA [35,36]. Within the complex tapestry of plant genetic regulation, a cadre of RNA polymerases meticulously orchestrates the synthesis of small RNA (sRNA) precursor sequences. Pol II is primarily responsible for the transcription of microRNA (miRNA) precursors. Conversely, the transcriptional duties for small interfering RNA (siRNA) precursors are allocated to either Pol IV or Pol V [24,31]. Both Pol IV and V are critical to the RdDM pathway; Pol IV is indispensable for the biogenesis of siRNAs, while Pol V synthesizes non-coding transcripts that act as scaffolds for the genesis of 24-nucleotide siRNAs [31]. This intricate interplay of RNA polymerases underscores the complexity of sRNA biogenesis in plants.

The eukaryotic Pol I–III protein complex is composed of 12–17 proteins [37,38]. In Arabidopsis, the principal subunits of RNA polymerases I–III—specifically NRPA1, NRPB1, and NRPC1—exhibit evolutionary conservation with the ß’ subunit of prokaryotic RNA polymerase. The subunits of secondary magnitude, namely NRPA2, NRPB2, and NRPC2, display hom*ology with the ß subunit of the same enzyme [39]. The Pol I–III complex is indispensable for the transcription of life-sustaining genes. Mutations in the secondary largest subunits of RNA polymerases I–III in Arabidopsis, namely nrpa2, nrpb2, and nrpc2, all result in lethality [40]. In the specialized ensemble of plant RNA polymerases, Pol IV and V are distinguished by their composition of 12 subunits, which exhibit hom*ology with those of Pol II. Pol IV is characterized by four unique subunits that differentiate it from Pol II, whereas Pol V is defined by six distinct subunits. In contrast to Pol I–III, mutations in the largest and second-largest subunits of Pol IV and V do not result in lethality, indicating their non-essential nature for plant viability. Moreover, the functional roles of Pol IV and V are distinct and do not intersect with the fundamental processes governed by Pol I–III [41]. In the nrpd1 mutant of Arabidopsis, the expression level of 24 nt sRNA is significantly reduced, and the level of CHG methylation is decreased [42]. Transcription products generated by Pol IV are shorter in length (mostly 30–40 nt) compared to those produced by Pol II, suggesting that a single transcription product from Pol IV can yield only one mature siRNA [43]. In the context of Arabidopsis thaliana, mutants of RNA Pol IV, specifically nrpd1 and nrpd2a, do not manifest substantial phenotypic growth disparities when juxtaposed with the wild-type counterparts. However, in maize, the Pol IV mutant rmr6 exhibits discernible developmental aberrations at the internodes. Similarly, the tomato slnrpd1 mutant is characterized by marked dwarfism, highlighting the divergent phenotypic consequences of Pol IV mutations across different plant species [44,45]. Pol V, despite being less studied, has been implicated in the regulation of gene expression regulation and heterochromatin formation in a Pol IV-independent manner, although the exact mechanism remains elusive [46].

3.2. RNA-Dependent RNA Polymerase (RdRP)

RdRP is a distinct class of RNA polymerase protein and is found in organisms possessing a conserved catalytic domain capable of synthesizing complementary dsRNA molecules from ssRNA templates [47]. RdRP primarily orchestrates the synthesis of siRNA in plants. In the presence of RdRP, ssRNA and primary siRNA molecules can engender copious amounts of dsRNA, which are subsequently processed by Dicer-like proteins to yield siRNAs [22]. siRNAs can be amplified by RdRP, which uses mRNA as a template and siRNA as a primer to generate sufficient dsRNA for Dicer to produce additional siRNAs. These siRNAs can then form RISCs and continue to degrade mRNA, thereby amplifying the siRNA-mediated silencing cascade [48]. In Arabidopsis, the RdRP family encompasses six distinct members, designated as RDR1 through RDR6. RDR1 is principally involved in the amplification of exogenous siRNAs, playing a pivotal role in the plant’s defense mechanisms against viral pathogens. On the other hand, RDR2 is implicated in the process of transcriptional gene silencing (TGS), a critical epigenetic regulatory mechanism that modulates gene expression at the transcriptional level. The functional dichotomy between RDR1 and RDR2 highlights the specialized roles these enzymes play within the broader context of RNA-mediated gene regulation in plants. RDR6 participates in the biosynthesis of phased, secondary phasiRNAs in Arabidopsis. To date, no functions have been identified for Arabidopsis RDR3, RDR4, and RDR5 in the gene silencing pathway [49,50,51].

In eukaryotic organisms, RDRPs are classified into three primary subfamilies: RDRa, RDRß, and RDR?. Plants typically harbor multiple RDRs. The RDRa subfamily, which includes RDR1/2/6, is characterized by a conserved C-terminal catalytic motif, DLDGD. The RDR? subfamily, comprising RDR3/4/5, possesses an atypical catalytic motif, DFDGD [52]. Despite sharing the same catalytic motif, the functions of RDR1/2/6 are somewhat divergent. They participate in distinct endogenous silencing pathways in conjunction with specific Dicer-like proteins. RDR1/2/6 contribute to plant resistance against viruses and fungi. Mutations in RDR1/2/6 diminish plant resistance to DNA and RNA viruses [53]. Members of the RDRa family can indirectly influence plant resistance to fungi, bacteria, and nematodes by modulating endogenous sRNA levels [54]. In biotic stresses, the RDRa family impact abiotic stress responses in plants. Various abiotic stresses and hormones induce the expression level of Arabidopsis RDR1. Rice RDR1 mutant exhibit increased sensitivity to salt and enhanced tolerance to heavy metal ions, such as copper and mercury [55]. Rice RDR6 mutants display spikelet abnormalities at high temperatures [56]. The rdr1 rdr2 rdr6 triple mutant of Arabidopsis exhibits significantly inhibited root length under polyethylene glycol (PEG) treatment [57]. While numerous studies have found that the RDRa family can influence sRNA synthesis, mutants of Arabidopsis and rice RDRa members did not exhibit severe development defects. However, maize RDR6 mutants displayed pronounced developmental abnormalities [58,59]. Phylogenetic scrutiny has uncovered variations in the number of RDR 1/2/6 among different species [60]. The operational dynamics of the RDR? subfamily are yet to be fully deciphered, accentuating the imperative for augmented investigative efforts to demystify the contributions of the RDR contingent to the intricate regulatory networks within plants.

3.3. Dicer/Dicer-like (DCLs)

Dicer-like proteins, essential to the biosynthesis of sRNAs, are nucleic acid endonucleases that belong to the RNaseIII family. They exhibit specificity in recognizing and processing dsRNA, initiating sRNA production through the cleavage of dsRNA precursors [61]. In the verdant realm of plant biology, a minimum of four gene family members are known to encode the Dicer-like proteins. DCL1 is tasked with the generation of 21-nucleotide miRNAs, which are processed from primary transcripts exhibiting stem–loop configurations. DCL2 is responsible for the production of 22-nucleotide siRNAs, playing an integral role in certain facets, such as plant growth, development, antiviral defense, and stress resilience. DCL3 is involved in the synthesis of 24-nucleotide siRNAs, which are key components in RdDM. DCL4 produces 21 nt siRNAs that guide mRNA cleavage and also contribute to antiviral defense [61]. Interestingly, certain plant species encode a greater diversity of Dicer-like proteins; for example, the soybean genome harbors seven distinct DICER-LIKE protein genes [62].

A prototypical Dicer protein is distinguished by its composite structural domains, which include a decapping enzyme domain, a domain of unknown function (DUF283), a PAZ domain, dual RNase III domains, and a domain for double-stranded RNA binding [63]. These domains equip DCL with the ability to accurately recognize and process dsRNA substrates into sRNA. The PAZ and RNase III domains are integral to the dsRNA cleavage process. The PAZ domain specifically recognizes and binds the 2-nucleotide overhang at the 3' termini of the dsRNA precursor. Concurrently, the bifunctional RNase III domains orchestrate the cleavage of both strands of the dsRNA. This conformation acts as a molecular caliper, dictating the dimension of the resultant sRNA product. Structural analysis has revealed additional dsRBDs in the C-terminal region [64,65]. Expression of DCL in plant tissues is contingent upon the developmental stage and stress response. DCL3, the enzyme responsible for generating the most copious siRNAs in plants, occupies a critical position in TGS via RdDM pathway. Upon encountering virus infestation, DCL2 and DCL4 generate copious amounts of virus-derived siRNAs to mount a defense [66]. Recent scholarly investigations have elucidated that DCL2 exerts a regulatory effect on the biosynthesis of 22-nucleotide siRNAs, which are instrumental in targeting and impeding the translation of the genes NIA1 and NIA2. This ultimately curtails the efficiency of nitrogen assimilation, ensuring plant survival. Furthermore, mutations in the soybean genes GmDCL2a and GmDCL2b precipitate the absence of 22-nucleotide small interfering RNAs (siRNAs), culminating in a marked augmentation of ketone synthase within the seed coat. This biochemical change is concomitant with a phenotypic alteration, transmuting the seed coat color from yellow to brown [62]. These discoveries shed new light on the role of DCL proteins in plant biology.

3.4. HUA ENHANCER1 (HEN1)

HEN1, the inaugural sRNA methyltransferase identified in Arabidopsis, adds a methyl group to the 2'-OH at the 3'-termini of each strand of Dicer-like-cleaved sRNA precursors. This methylation prevents labelling by HESO1 and subsequent degradation by sRNA Degrading Nuclease1 (SDN1) [67]. Mutations in HEN1 lead to miRNA instability and heterogeneity at the 3'-termini. Sequencing analysis reveals a significant addition of uracil, a process known as uridylation, catalyzed by HESO1, the primary terminal uridylyltransferase in Arabidopsis [68]. The catalytic locus of HEN1 is uniquely differentiated from the ‘KDK’ motif that characterizes the active site within the RFM superfamily of 2'-O-methyltransferases (2'-O-MTases), as well as from the established active sites of 2'-O-MTases belonging to the SPOUT superfamily [69].

Arabidopsis HEN1, a pivotal player in sRNA biosynthesis, exhibits a quintet of structural domains. Among these, four domains engage directly with sRNA duplexes: a pair of double-stranded RNA-binding domains (dsRBDs), a conserved methyltransferase (MTase) domain, and a La-motif-containing domain (LCD) that discerns the 3' terminal hydroxyl (-OH) group. The quinary domain, a peptidyl-prolyl isomerase (PPIase)-like domain (PLD), remains unaffiliated with dsRNA binding [69]. In vitro co-crystallization analyses have demonstrated that HEN1 accommodates dsRNA substrates in a monomeric form. The dsRBDs annex to both flanks of the A-form sRNA substrate, while the LCD domain sheathes the 3' termini of the non-methylated strand. The MTase domain’s active site encases the protruding bi-nucleotide terminus of the methylated strand [70]. The determination of substrate length by HEN1 is potentially contingent upon the spatial interplay between the MTase and LCD domains, in addition to the cap site [30]. Methylation modification by HEN1 is indeed pivotal in sRNA biosynthesis and plant function. However, the mechanism of factor recruitment remains an open question. HEN1 expression is regulated by light, and it finetunes photomorphogenesis by regulating miR157d and miR319 expression [71,72]. Deletion mutants of HEN1 manifest a spectrum of developmental anomalies, including aberrant photomorphogenesis, diminutive plant stature, delayed flowering, and infertility [25]. Conversely, deletion mutants of HEN1 SUPPRESSOR 1 (HESO1) mitigate the phenotypic manifestations of HEN1. Overexpression of HESO1 within HEN1 mutants exacerbates the reduction in miRNA abundance and intensifies the developmental aberrations, thereby implicating uridylation as a precipitant of miRNA degradation [73]. This intricate interplay of molecular interactions underscores the complexity and sophistication of sRNA biogenesis in plants.

3.5. Argonaute (AGO)

Arabidopsis is endowed with ten AGO proteins, which are associated with RNA silencing and form RISCs [74]. Argonaute (AGO) proteins are categorized into three distinct clades: AGO1, 5, 8, and 10; AGO2, 3, and 7; and AGO4, 6, 8, and 9 [10]. AGO1, the preeminent member of the AGO family, orchestrates the assembly of miRNAs into RISCs and exerts regulatory control over gene expression at the post-transcriptional and translational echelons [75]. AGO10 positively regulates the expression levels of HD-ZIP-III-like transcription factors and mediates the translational repression of several genes, including AGO1 [76]. AGO5, expressed during macro sporogenesis, may be involved in gametophyte development or cell differentiation [77]. AGO2, which loads miRNAs with an A at the 5'-termini, plays a role in the plant immune system [78]. It can bind virally produced siRNAs (vsiRNAs) and dsDNA break-induced sRNAs (diRNAs) [79]. AGO3, which shares a highly similar protein sequence with AGO2, may have redundant functions [80]. AGO7 loads trans-acting siRNAs (ta-siRNAs) generated by TAS3 [81]. AGO4, 6, and 9 predominantly load 24 nt siRNAs with an A at the 5'-termini, processed by DCL3 [82]. AGO4 exerts a substantial effect on RdDM and the TGS of transposable elements [83]. Beyond its role in siRNA incorporation, AGO4 is capable of accommodating a minor subset of 24-nucleotide miRNAs and effectuating sequence-specific DNA methylation [84]. It can also replace AGO1 and AGO7 in the biosynthesis of trans-acting siRNAs (ta-siRNAs) produced by miR172 and miR390 [85]. High-throughput sequencing data analysis revealed that AGO4/6/9 loaded 24 nt siRNAs from different production sites [82]. Using the AGO4 promoter to initiate the expression of AGO6 and AGO9 reduced this difference, suggesting that AGO4/6/9 mediate the RdDM pathway in specific tissues [86]. AGO8, which exhibits low expression levels in all tissues and periods in Arabidopsis, is generally considered to be a pseudogene with no reported function [87].

The AGO protein, a cornerstone of RNA silencing, is characterized by four major structural domains: an adaptable N-terminal domain, a conserved PAZ domain, a conserved MID domain, and a conserved PIWI domain. The conformation of the folded AGO protein assumes a bilobate architecture, engendering a channel conducive to RNA engagement [88]. The MID domain is responsible for the recognition of the 5' termini of the small RNA (sRNA), whereas the PAZ domain secures the 3' termini. The PIWI domain is characterized by its intrinsic endonuclease activity. Different AGO proteins exhibit preferences for the length and 5'-termini of the sRNA [89]. AGO1 and AGO10 prefer U sRNAs at the 5'-termini, AGO5 prefers C sRNAs at the 5’-termini, and AGO2/4/6/7/9 prefer A sRNAs at the 5’-termini. This base preference is related to the structure of nucleotide-specific recognition in the MID domain [90,91]. Mutants of AGO1 in Arabidopsis display dwarf and sterile phenotypes [92]. Exclusive to the AGO7 mutant is the manifestation of lobed leaf margins and an increased intercalary distance of lateral organs, in contrast to other AGO mutants which do not exhibit phenotypic deviations of significance when compared to the wild type [93]. AGO6 serves as a TGS suppressor with partial functional redundancy with AGO4 [94]. Rice and maize possess more AGO protein family members (19 and 17, respectively) than Arabidopsis [51]. In addition to proteins hom*ologous to the three subfamilies in Arabidopsis, the grass family has a specific subfamily, AGO18. ZmAGO18b in maize, expressed in male ears, influences spikelet development [95]. Contemporary theoretical frameworks propose that AGO10 safeguards HD-ZIP-III-like transcription factors from proteolytic breakdown by engaging in competitive binding with AGO1 for the incorporation of miR165/166 [76].

4. The Regulatory Function of sRNAs in Plant Development

4.1. Impact of sRNAs on Shoot Morphogenesis

The ontogeny of shoot development is typified by the orchestrated emergence of lateral organs, which are spatially and temporally patterned in distinct configurations at the shoot apical meristem (SAM). This developmental ballet necessitates the interplay of intricate and resilient genetic networks, which synchronize the proliferation programs across the diverse cellular and tissue landscapes. Within this intricate network, sRNAs stand out as key players (Figure 3). They constitute the foundational regulatory mechanisms that delineate shoot morphogenesis, conferring a degree of control and exactitude indispensable for the plant’s appropriate ontogeny and maturation. This specific role of sRNAs in regulating shoot development has elucidated the importance of these tiny molecules in the grand scheme of shoot morphogenesis [96].

The orchestration of shoot growth is contingent upon the meticulous organization and preservation of the SAM, a process quintessential to plant morphogenesis. The SAM, delineated during the early stages of embryogenesis, encompasses a reservoir of pluripotent stem cells that give rise to all aerial organs, ensconced within a milieu of undifferentiated meristematic cells [97]. It has been discovered that PHABULOSA (PHB) and PHAVOLUTA (PHV) are repressed by miRNA165/166, with PHB playing a significant role in SAM development [98,99,100]. AGO10 prevents miRNA165/166 from binding to AGO1, thereby enabling its function. In AGO10 mutants, increased activity of miRNA165/166 precipitates a diminution in HD-ZIP III expression, concomitantly inhibiting the proliferation of the plant SAM [23,101]. Recent scholarly contributions have disclosed a contributory role for miR394 in this regulatory cascade. The SAM is architecturally partitioned into three distinct strata (L1–L3). miR394, expressed in the outermost L1 layer, migrates to the L3 layer to repress its target, the F-Box protein LCR, and the transcription factor WUSCHEL (WUS) in the innermost L3 cells, thereby mediating stem cell functional diversity [102]. WUS enhances cellular proliferation within a reciprocal regulatory circuit with the CLV genes [103]. Phenotypic analyses of miR394 mutants indicate that the repression of LCR by miR394 is imperative for stem cell proliferation, underscoring that the sustenance of the SAM necessitates the synergistic interaction of both WUS and miR394 [104,105]. Expressing the regulator miR171 under the WUS promoter reduces the activity of the hairy meristem family, leading to premature termination of nutritive meristem organization. This implies that a consortium of microRNAs (miRNAs) may orchestrate cellular proliferation within the stem cell ecotone [106,107].

4.2. Functional Dynamics of sRNAs in Leaf Morphogenesis

Leaf morphogenesis, an exquisitely orchestrated process unfolding in a sequential cascade, entails phases of initiation, specification, transition, proliferation, and maturation. An array of coding genes, augmented by a curated cohort of non-coding sRNAs (Figure 3), has been implicated in this complex ballet of foliar development [5,108].

sRNAs and their intricate interplays not only dictate gene expression and regulatory frameworks but also assume pivotal roles in leaf morphogenesis via their synergistic integration with diverse genetic networks and physiological pathways [109]. TAS3 siRNAs, originating from miR390, are synthesized on the proximal aspect of juvenile foliage through the cleavage mediated by the AGO1/AGO7 complex. These siRNAs function to inhibit the transcription of factors ARF3/4, which are pivotal in fostering cellular polarity along the distal axis [110]. The interaction of SPL proteins regulated by miR156 with CIN-TCP prevents CIN-TCP from interacting with NAC proteins to produce leaf serrations [111]. miR159 promotes leaf cell proliferation by suppressing MYB33 and MYB65 expression. miR160 selectively modulates members of the ARF family, specifically ARF10/16/17, thereby influencing leaf morphogenesis through the modulation of auxin signaling [112]. Concurrently, miR164 orchestrates the expression of NAC domain genes, facilitating the delineation of leaf margins in Arabidopsis by targeting the CUC genes CUC1/2, which are integral NAC transcription factors implicated in the embryonic formation of the SAM and demarcation of developmental boundaries [113]. miR165/166 represses HD-ZIP III family members on the distal side, causing proximal axialization of vascular bundles [101]. miR319 is expressed as a TCP transcription factor and targets CINCINNATA (CIN)-TCP to promote cell differentiation, mitotic arrest, and to control leaf shape in angiosperms [114]. CIN-TCP also promotes leaf senescence via the jasmonic acid pathway. miR393, through its interaction with the target loci TIR1 and AFB1/2/3, exerts a regulatory influence on foliar morphology and dimensions by modulating hormonal pathways [115]. The equilibrium between miR396 and GRFs is instrumental in dictating the cellular composition of leaves and the dimensionality of meristematic tissues [116]. Additionally, miR824 and its associated gene AGL16 play a critical role in the ontogeny of stomatal structures [23].

4.3. Regulatory Mechanisms of sRNAs in Flowering Development

Floral ontogenesis represents a critical event within the life cycle of higher plant taxa, characterized by a complex coordination of gene networks. An extensive array of these characterized sRNAs has been associated with the regulation of diverse biological frameworks (Figure 3), mechanisms, and signaling pathways, functioning in concert with developmental cues [4]. Nonetheless, the preponderance of sRNA research in the context of floral development has predominantly centered on miRNAs, with scant attention accorded to siRNAs.

miRNAs play a crucial role in the regulation of floral organ identity and the timing of flowering in Arabidopsis. A significant number of miRNAs, such as miR156/157, 159, 164, 165/166, 167, 169, 171, 172, 319, 390, 393, and 824, are involved in the intricate control mechanisms that govern flowering processes [117]. The modulation of miR156 expression, specifically its reduction, coupled with an elevation in the abundance of SPL proteins, instigates flowering. This is achieved through the activation of LFY, FUL, and AP1, thereby transitioning plant growth from vegetative to reproductive phases [118,119]. The influence of miR156 and SPL on the development of rice flowers has been documented [120]. During periods of short daylight, miR159 governs the floral transition by suppressing genes associated with floral meristem organization. Its target, MYB33, enhances the transcription of ABA INSENSITIVE 5 (ABI5), which functions proximal to miR156 in the regulatory hierarchy, orchestrating the transition from vegetative growth to the reproductive phase [121,122]. miR164 orchestrates organ boundary formation by targeting CUC genes, with overexpression resulting in sepal fusion and a reduction in petal number [123]. miR165/166 oversees floral morphogenesis and meristem activity [99]. miR167 specifically targets the ARF6 and ARF8 genes that are integral to the regulation of pistils and stamens. Additionally, miR167 is instrumental in directing the morphogenesis of both male and female reproductive structures in Arabidopsis [124]. Concurrently, miR169 plays a pivotal role in modulating stress-induced floral induction by repressing the AtNF-YA transcription factor, culminating in the downregulation of FLC expression. This suppression facilitates the activation of FLC downstream target genes, including FT and LFY, thereby promoting the floral transition [125]. miR171 targets LOM1, which regulates SPL transcription factor activity and delays flowering [118]. The overexpression of miR172 in Arabidopsis leads to the precocious onset of flowering and the perturbation of floral organ morphogenesis. miR172 suppresses APETALA2 (AP2) expression by activating SPL, thereby promoting flowering time and floral organ determination. During specific phases of the plant growth cycle, miR156 and miR172 exhibit interactive regulatory roles, as evidenced by miRNA-mediated modulation [126]. miR156 exerts an inhibitory effect on the expression of the SPL gene family, whereas certain SPL gene members reciprocally augment miR172 expression. Within foliar tissues, the SPL9–miR172b/c complex is responsible for the regulation of flowering time-associated genes through the modulation of FT expression. Conversely, in apical meristematic regions, the SPL15–miR172d complex facilitates the floral initiation by inducing the expression of MADS-box genes. The transcriptional activity of the miR172 gene is subject to regulation by environmental cues, such as ambient temperature and photoperiodicity [127,128]. miR319 exerts regulatory control over the TCP gene family, facilitating the induction of flowering through transcriptional activation. This is achieved by the binding of miR319 to the promoter region of the CONSTANS (CO) gene, thereby influencing its expression [129,130]. miR390-tasiRNA3-ARF4 regulates flowering through the AP1/FUL pathway [23]. The overexpression of miR393 has been demonstrated to impede the translation of AFB2 and TIR1, consequently influencing the temporal regulation of flowering [131,132]. AGL16 protein and its negative regulator, miR824, are pivotal in controlling the timing of flowering in Arabidopsis. AGL16 is capable of direct interaction with SVP and an indirect association with FLC, leading to the formation of a repressive complex that inhibits the expression of FT. miR824 and AGL16 are integral in the suppression of floral induction during extended photoperiods [119,133]. In Brachypodium distachyon, a species within the Pooideae subfamily, the species-specific miR5200 targets FT, thereby modulating the timing of flowering and influencing the floral transition [134].

4.4. sRNA-Mediated Regulation Mechanisms of Root Development

Recent scholarly focus has been directed towards the sRNA-mediated regulatory pathways in root development within plant biology research [135]. A comprehensive body of scientific literature has highlighted the critical functions of numerous miRNAs and ta-siRNAs in various aspects of root development (Figure 3). These facets encompass root morphogenesis, vascular tissue patterning, lateral root (LR) formation and extension, as well as adventitious root development [136].

An abundance of empirical evidence highlights the significant regulatory influence of miRNAs on the development of plant roots. miR156 in conjunction with its target SPL gene plays a crucial role in the modulation of root length [137]. The dynamism of root meristematic cells is directed by miR159 via its target gene MYB65 [138]. miR160 and its associated target genes ARF10/16 are pivotal in affecting root elongation, influencing cellular division and differentiation processes at the root apex [139]. Mutations in miR163 or overexpression of PXMT1 result in diminished primary root lengths, indicating that miR163 and its target gene PXMT1 are integral to the configuration of root architecture during the nascent stages of Arabidopsis seedlings development. This provides further evidence that HY5-miR163-PXMT1-mediated post-transcriptional regulation affects root development [136,140]. In Arabidopsis, miR164 is known to target quintuple mRNAs encoding NAC domain proteins. Mutants of miR164a and miR164b exhibit upregulated expression of NAC1, which correlates with an increased production of lateral roots [141]. Plant miR165/166 enhances cell division and meristematic tissue activity by negatively regulating HD-ZIPIIIs to promote root elongation [142]. It also fosters xylem differentiation by repressing PHABULOSA (PHB) expression. miR165 regulates Arabidopsis root differentiation [143]. The transcription factors SHR and SCR of the GRAS family play pivotal roles in root tip meristem formation [23,98]. miR169 targets the NF-YA10 gene and its target gene NF-YA2 through the ABA pathway to control root morphological structure. The suppression of NF-YA2 regulation by miR169 indirectly influences the development of lateral roots [144]. Furthermore, miR171 orchestrates the elongation of primary roots by inhibiting HAM genes, thereby modulating the activity of the quiescent center and overall root growth. Its target genes SCL6-II and SCL6-III are also involved [107,145]. miR319b and its target gene MYB33 are implicated in the ethylene regulation of CBP20 phosphorylation, leading to a reduction in root tip number and a significant alteration in root morphology [146]. The miR390-associated trans-acting short interfering RNAs (TAS3-tasiRNAs) and their target genes ARF2/3/4 provide quantifiable positive or negative feedback mechanisms that selectively modulate the length of lateral roots, without affecting their number or density [147]. miR393 negatively regulates TIR1 and AFB (AFB1, AFB2, and AFB3), critical genes of the growth hormone signaling pathway that promote primary and lateral root elongation in Arabidopsis seedlings. Under nitrate treatment, Arabidopsis miR393 expression levels are upregulated, and morphological changes in both primary and lateral roots are observed in AFB3 mutants, indicating that miR393/AFB3 is induced by nitrate to regulate root architecture. Enhanced expression of TIR1 augments the responsiveness to auxin treatment, culminating in the suppression of primary root elongation and a concomitant increase in the density of lateral roots. TIR1 promotes miR393 expression through a feedback pathway [115]. Arabidopsis miR396a targets seven GRF and bHLH74 genes. Overexpression of miR396a results in shorter roots, while overexpression of bHLH74 promotes root elongation. miR396 is also involved in root development through interaction with GRF. The overexpression of miR396 is correlated with a reduction in the cell cycle rate at the root apex and an enlargement of the root apical meristem. This suggests a significant role for miR396 in root development through the modulation of its target gene, GRF [148]. The accumulated evidence conclusively demonstrates that a vast array of miRNAs is essential in the regulation of plant root elongation and morphogenesis. These miRNAs function through their target genes, responding adaptively to the multifaceted environmental conditions within the root ecosystem.

Specific siRNAs have been elucidated as pivotal regulators in the modulation of root growth dynamics. For instance, in Arabidopsis, the dcl2/3/4 mutant markedly diminishes the accumulation of siRNAs that influence RdDM, leading to an increase in cellulose and callus accumulation in the root xylem, thereby impacting root growth [149]. Particular siRNAs are capable of orchestrating plant root growth and development through the modulation of endogenous hormonal concentrations, including jasmonic acid, gibberellin, and ethylene [150]. In Arabidopsis, certain siRNAs are associated with root development and can influence root development by regulating various pathways, including root hair formation and cell division.

4.5. sRNA-Mediated Regulatory Mechanisms in Nodule Morphogenesis

Given the considerable energetic demands, symbiotic nitrogen fixation in legume nodules requires substantial carbon inputs, predominantly sourced from sugars. Legumes utilize the Autoregulation of Nodulation (AON) pathway, which is centrally modulated by miRNAs, to preserve a balanced nodule count [151,152]. These miRNAs are also instrumental in the morphogenesis and development of nodules (Figure 3). The emergence of high-throughput small RNA sequencing (sRNA-seq) technologies has significantly accelerated the identification of miRNAs associated with nodule formation [153].

The majority of research on miRNA regulation in the symbiosis between soybeans and rhizobia has been centered on nodulation. A host of miRNAs, including miR160, 167, 171, 172, 393, and 2111, have been implicated in the developmental processes of rhizobia [153]. The miR156b–SPL9d complex, along with NINa, acts as a principal regulatory axis in the autoregulation of nodulation in soybeans [154]. Additionally, miR172c is known to inhibit the expression of NNC1, thereby facilitating the activation of the AON pathway [155]. MiR2111 suppresses the negative regulator of nodulation, Too Much Love (TML), thereby maintaining the default susceptible state of legumes. Suppression of miR2111 expression precipitates the accumulation of TML proteins in roots, concomitantly inhibiting the organogenesis of root nodules [156,157]. MiR393j-3p exerts an influence on nodulation by modulating the expression of the early nodulin gene ENOD93 [158]. Conversely, the overexpression of miR482, miR1512, and miR1515 is correlated with an increase in the number of nodules [159]. However, our understanding of how miRNAs, such as those involved in symbiotic nitrogen fixation (SNF) in mature nodules, regulate nodule functions remains limited. Future studies will elucidate the molecular mechanisms of miRNAs in root nodule formation and development and provide new strategies for regulating root nodule formation and development using miRNAs. In summary, the regulatory oversight of both the antecedent and subsequent elements within the AON pathway is attributed to the governance of sRNAs.

5. Conclusions and Future Perspectives

Recent accelerated progress in next-generation sequencing technologies has catalyzed significant breakthroughs in sRNA research within the plant sciences, particularly in crop species. Notably, many of these crops, such as oilseed rape and soybean, present challenges for genetic analysis owing to their protracted growth periods and intricate genomic structures. It is evident that agricultural crops exhibit both conserved and unique miRNA and siRNA pathways, analogous to those observed in other model plant species. These conserved pathways are integral to the regulation of vital growth and developmental processes, including, but not limited to, floral induction, root architecture formation, and pathogen resistance mechanisms. In contrast, lineage-specific or species-specific miRNAs and siRNAs hold significant biological interest due to their potential to bestow or modulate phenotypic characteristics that are absent or undeveloped in other plant taxa. Consequently, a comprehensive understanding of the regulatory dynamics between specific miRNAs/siRNAs and the manifestation of distinct phenotypic traits is imperative for the advancement of targeted breeding strategies.

Despite the considerable progress achieved in sRNA research within certain crop species, this progress remains disproportionately distributed, with a vast array of other crops yet to be thoroughly investigated. This disparity underscores the imperative for systematic sequencing and profiling of sRNAs, leveraging computational methodologies to discover new miRNAs and elucidate their intricate regulatory networks. It is paramount that these newly identified miRNAs undergo empirical validation within their respective host organisms, employing techniques, such as RNA interference (RNAi) or clustered regularly interspaced short palindromic repeats (CRISPR) for gene silencing, and various overexpression methods for gene activation. However, the execution of such analyses poses a significant challenge across numerous plant species, attributed to the necessity for robust translation mechanisms. To summarize, the preceding decade has witnessed only the nascent stages of sRNA exploration in agricultural crops. Prospective research endeavors must integrate comprehensive bioinformatic analysis and profound functional interpretation to fully demystify the roles and impacts of sRNAs.

Author Contributions

Q.L.: formal analysis, writing—original draft, funding acquisition. Y.W.: formal analysis, writing—original draft. Z.S.: supervision, writing, funding acquisition. H.L. (Haiyang Li) and H.L. (Huan Liu): conceptualization, supervision, project administration, funding acquisition, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Acknowledgments

We thank our group members at Guangzhou University for their helpful comments on the manuscript. We apologize for the inadvertent omission of any pertinent studies from this review due to space limitations.

References

1. J. Shi; T. Zhou; Q. Chen Exploring the expanding universe of small RNAs., 2022, 24,pp. 415-423. DOI: https://doi.org/10.1038/s41556-022-00880-5. PMID: https://www.ncbi.nlm.nih.gov/pubmed/35414016.

2. D.L. Court; J. Gan; Y.H. Liang; G.X. Shaw; J.E. Tropea; N. Costantino; D.S. Waugh; X. Ji RNase III: Genetics and function; structure and mechanism., 2013, 47,pp. 405-431. DOI: https://doi.org/10.1146/annurev-genet-110711-155618. PMID: https://www.ncbi.nlm.nih.gov/pubmed/24274754.

3. Y.G. Chen; S. Hur Cellular origins of dsRNA, their recognition and consequences., 2022, 23,pp. 286-301. DOI: https://doi.org/10.1038/s41580-021-00430-1. PMID: https://www.ncbi.nlm.nih.gov/pubmed/34815573.

4. J. Wang; X. Meng; O.B. Dobrovolskaya; Y.L. Orlov; M. Chen Non-coding RNAs and their roles in stress response in plants., 2017, 15,pp. 301-312. DOI: https://doi.org/10.1016/j.gpb.2017.01.007. PMID: https://www.ncbi.nlm.nih.gov/pubmed/29017967.

5. G. Reshetnyak; J.M. Jacobs; F. Auguy; C. Sciallano; L. Claude; C. Medina; A.L. Perez-Quintero; A. Comte; E. Thomas; A. Bogdanove et al. An atypical class of non-coding small RNAs is produced in rice leaves upon bacterial infection., 2021, 11,p. 24141. DOI: https://doi.org/10.1038/s41598-021-03391-9. PMID: https://www.ncbi.nlm.nih.gov/pubmed/34921170.

6. Y. Tang; X. Yan; C. Gu; X. Yuan Biogenesis, trafficking, and function of small RNAs in plants., 2022, 13, 825477. DOI: https://doi.org/10.3389/fpls.2022.825477. PMID: https://www.ncbi.nlm.nih.gov/pubmed/35251095.

7. L. He; G.J. Hannon MicroRNAs: Small RNAs with a big role in gene regulation., 2004, 5,pp. 522-531. DOI: https://doi.org/10.1038/nrg1379. PMID: https://www.ncbi.nlm.nih.gov/pubmed/15211354.

8. W. Islam; M. Adnan; Z. Huang; G.-D. Lu; H.Y.H. Chen Small RNAs from seed to mature plant., 2019, 38,pp. 117-139. DOI: https://doi.org/10.1080/07352689.2019.1608404.

9. X. Chen; O. Rechavi Plant and animal small RNA communications between cells and organisms., 2022, 23,pp. 185-203. DOI: https://doi.org/10.1038/s41580-021-00425-y.

10. H. Zhang; R. Xia; B.C. Meyers; V. Walbot Evolution, functions, and mysteries of plant ARGONAUTE proteins., 2015, 27,pp. 84-90. DOI: https://doi.org/10.1016/j.pbi.2015.06.011.

11. M.W. Jones-Rhoades; D.P. Bartel; B. Bartel MicroRNAS and their regulatory roles in plants., 2006, 57,pp. 19-53. DOI: https://doi.org/10.1146/annurev.arplant.57.032905.105218. PMID: https://www.ncbi.nlm.nih.gov/pubmed/16669754.

12. F.F. Salgado; L.R. Vieira; V.N.B. Silva; A.P. Leao; P. Grynberg; M.M. do Carmo Costa; R.C. Togawa; C.A.F. de Sousa; M.T.S. Junior Expression analysis of miRNAs and their putative target genes confirm a preponderant role of transcription factors in the early response of oil palm plants to salinity stress., 2021, 21, 518. DOI: https://doi.org/10.1186/s12870-021-03296-9. PMID: https://www.ncbi.nlm.nih.gov/pubmed/34749653.

13. L. Gonzalo; I. Tossolini; T. Gulanicz; D.A. Cambiagno; A. Kasprowicz-Maluski; D.J. Smolinski; M.F. Mammarella; F.D. Ariel; S. Marquardt; Z. Szweykowska-Kulinska et al. R-loops at microRNA encoding loci promote co-transcriptional processing of pri-miRNAs in plants., 2022, 8,pp. 402-418. DOI: https://doi.org/10.1038/s41477-022-01125-x. PMID: https://www.ncbi.nlm.nih.gov/pubmed/35449404.

14. X. Shi; H. Yang; C. Chen; J. Hou; T. Ji; J. Cheng; J.A. Birchler Dosage-sensitive miRNAs trigger modulation of gene expression during genomic imbalance in maize., 2022, 13,p. 3014. DOI: https://doi.org/10.1038/s41467-022-30704-x.

15. S. Diederichs; D.A. Haber Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression., 2007, 131,pp. 1097-1108. DOI: https://doi.org/10.1016/j.cell.2007.10.032. PMID: https://www.ncbi.nlm.nih.gov/pubmed/18083100.

16. X.A. Cambronne; R. Shen; P.L. Auer; R.H. Goodman Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach., 2012, 109,pp. 20473-20478. DOI: https://doi.org/10.1073/pnas.1218887109. PMID: https://www.ncbi.nlm.nih.gov/pubmed/23184980.

17. A.A. Sarshad; A.H. Juan; A.I.C. Muler; D.G. Anastasakis; X. Wang; P. Genzor; X. Feng; P.F. Tsai; H.W. Sun; A.D. Haase et al. Argonaute-miRNA complexes silence target mRNAs in the nucleus of mammalian stem cells., 2018, 71,pp. 1040-1050.e8. DOI: https://doi.org/10.1016/j.molcel.2018.07.020.

18. C.J. Stavast; S.J. Erkeland The non-canonical aspects of MicroRNAs: Many roads to gene regulation., 2019, 8, 1465. DOI: https://doi.org/10.3390/cells8111465.

19. X. Zhu; Y. Chen; Z. Zhang; S. Zhao; L. Xie; R. Zhang A species-specific miRNA participates in biomineralization by targeting CDS regions of Prisilkin-39 and ACCBP in Pinctada fucata., 2020, 10,p. 8971. DOI: https://doi.org/10.1038/s41598-020-65708-4.

20. C. Chen; Z. Zeng; Z. Liu; R. Xia Small RNAs, emerging regulators critical for the development of horticultural traits., 2018, 5,p. 63. DOI: https://doi.org/10.1038/s41438-018-0072-8.

21. H. Pietrykowska; I. Sierocka; A. Zielezinski; A. Alisha; J.C. Carrasco-Sanchez; A. Jarmolowski; W.M. Karlowski; Z. Szweykowska-Kulinska Biogenesis, conservation, and function of miRNA in liverworts., 2022, 73,pp. 4528-4545. DOI: https://doi.org/10.1093/jxb/erac098.

22. Q. Wang; Y. Xue; L. Zhang; Z. Zhong; S. Feng; C. Wang; L. Xiao; Z. Yang; C.J. Harris; Z. Wu et al. Mechanism of siRNA production by a plant Dicer-RNA complex in dicing-competent conformation., 2021, 374,pp. 1152-1157. DOI: https://doi.org/10.1126/science.abl4546. PMID: https://www.ncbi.nlm.nih.gov/pubmed/34648373.

23. Q. Dong; B. Hu; C. Zhang microRNAs and their roles in plant development., 2022, 13, 824240. DOI: https://doi.org/10.3389/fpls.2022.824240.

24. F. Borges; R.A. Martienssen The expanding world of small RNAs in plants., 2015, 16,pp. 727-741. DOI: https://doi.org/10.1038/nrm4085. PMID: https://www.ncbi.nlm.nih.gov/pubmed/26530390.

25. J. Wang; J. Mei; G. Ren Plant microRNAs: Biogenesis, homeostasis, and degradation., 2019, 10, 360. DOI: https://doi.org/10.3389/fpls.2019.00360. PMID: https://www.ncbi.nlm.nih.gov/pubmed/30972093.

26. M. Zhou; J.A. Law RNA Pol IV and V in gene silencing: Rebel polymerases evolving away from Pol II’s rules., 2015, 27,pp. 154-164. DOI: https://doi.org/10.1016/j.pbi.2015.07.005. PMID: https://www.ncbi.nlm.nih.gov/pubmed/26344361.

27. V. Mishra; J. Singh; F. Wang; Y. Zhang; A. f*ckudome; J.C. Trinidad; Y. Takagi; C.S. Pikaard Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV., 2021, 118,p. e2019276118. DOI: https://doi.org/10.1073/pnas.2019276118.

28. M. Yoshikawa; Y.W. Han; H. Fujii; S. Aizawa; T. Nishino; M. Ishikawa Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in Arabidopsis., 2021, 118,p. e2102885118. DOI: https://doi.org/10.1073/pnas.2102885118.

29. Z. Yang; Y.W. Ebright; B. Yu; X. Chen HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2' OH of the 3' terminal nucleotide., 2006, 34,pp. 667-675. DOI: https://doi.org/10.1093/nar/gkj474.

30. L. Ji; X. Chen Regulation of small RNA stability: Methylation and beyond., 2012, 22,pp. 624-636. DOI: https://doi.org/10.1038/cr.2012.36. PMID: https://www.ncbi.nlm.nih.gov/pubmed/22410795.

31. M.J. Sigman; K. Panda; R. Kirchner; L.L. McLain; H. Payne; J.R. Peasari; A.Y. Husbands; R.K. Slotkin; A.D. McCue An siRNA-guided ARGONAUTE protein directs RNA polymerase V to initiate DNA methylation., 2021, 7,pp. 1461-1474. DOI: https://doi.org/10.1038/s41477-021-01008-7. PMID: https://www.ncbi.nlm.nih.gov/pubmed/34750500.

32. R. Komiya Biogenesis of diverse plant phasiRNAs involves a miRNA-trigger and Dicer-processing., 2017, 130,pp. 17-23. DOI: https://doi.org/10.1007/s10265-016-0878-0.

33. M.C.D.O. Urquiaga; T. Flávia; A.S. Hemerly; P.C.G. Ferreira From trash to luxury: The potential role of plant LncRNA in DNA methylation during abiotic stress., 2021, 11, 603246. DOI: https://doi.org/10.3389/fpls.2020.603246. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33488652.

34. L. Morgado Chapter 3—Biogenesis of small RNA: Molecular pathways and regulatory mechanisms., Academic Press: Cambridge, MA, USA, 2020,pp. 49-64.

35. E. Lata; K. Choquet; F. Sagliocco; B. Brais; G. Bernard; M. Teichmann RNA polymerase III subunit mutations in genetic diseases., 2021, 8, 696438. DOI: https://doi.org/10.3389/fmolb.2021.696438. PMID: https://www.ncbi.nlm.nih.gov/pubmed/34395528.

36. M. Girbig; J. Xie; H. Grotsch; D. Libri; O. Porrua; C.W. Muller Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly(dT) termination signals., 2022, 40,p. 111316. DOI: https://doi.org/10.1016/j.celrep.2022.111316.

37. D.B. Nikolov; S.K. Burley RNA polymerase II transcription initiation: A structural view., 1997, 94,pp. 15-22. DOI: https://doi.org/10.1073/pnas.94.1.15.

38. H. Khatter; M.K. Vorlander; C.W. Muller RNA polymerase I and III: Similar yet unique., 2017, 47,pp. 88-94. DOI: https://doi.org/10.1016/j.sbi.2017.05.008.

39. E. Babiychuk; K.T. Hoang; K. Vandepoele; E. Van De Slijke; D. Geelen; G. De Jaeger; J. Obokata; S. Kushnir The mutation nrpb1-A325V in the largest subunit of RNA polymerase II suppresses compromised growth of Arabidopsis plants deficient in a function of the general transcription factor IIF., 2017, 89,pp. 730-745. DOI: https://doi.org/10.1111/tpj.13417.

40. H. Zhao; Y. Qin; Z. Xiao; K. Liang; D. Gong; Q. Sun; F. Qiu Knockdown NRPC2, 3, 8, NRPABC1 and NRPABC2 affects RNAPIII activity and disrupts seed development in Arabidopsis., 2021, 22, 11314. DOI: https://doi.org/10.3390/ijms222111314.

41. J.R. Haag; C.S. Pikaard Multisubunit RNA polymerases IV and V: Purveyors of non-coding RNA for plant gene silencing., 2011, 12,pp. 483-492. DOI: https://doi.org/10.1038/nrm3152.

42. Z. Wang; N. Butel; J. Santos-Gonzalez; F. Borges; J. Yi; R.A. Martienssen; G. Martinez; C. Kohler Polymerase IV plays a crucial role in pollen development in Capsella., 2020, 32,pp. 950-966. DOI: https://doi.org/10.1105/tpc.19.00938. PMID: https://www.ncbi.nlm.nih.gov/pubmed/31988265.

43. J. Zhai; S. Bischof; H. Wang; S. Feng; T.F. Lee; C. Teng; X. Chen; S.Y. Park; L. Liu; J. Gallego-Bartolome et al. A one precursor one siRNA model for Pol IV-dependent siRNA biogenesis., 2015, 163,pp. 445-455. DOI: https://doi.org/10.1016/j.cell.2015.09.032. PMID: https://www.ncbi.nlm.nih.gov/pubmed/26451488.

44. L. Ferrafiat; D. Pflieger; J. Singh; M. Thieme; M. Bohrer; C. Himber; A. Gerbaud; E. Bucher; C.S. Pikaard; T. Blevins The NRPD1 N-terminus contains a Pol IV-specific motif that is critical for genome surveillance in Arabidopsis., 2019, 47,pp. 9037-9052. DOI: https://doi.org/10.1093/nar/gkz618. PMID: https://www.ncbi.nlm.nih.gov/pubmed/31372633.

45. Z. Wang; N. Butel; J. Santos-Gonzalez; L. Simon; C. Wardig; C. Kohler Transgenerational effect of mutants in the RNA-directed DNA methylation pathway on the triploid block in Arabidopsis., 2021, 22, 141. DOI: https://doi.org/10.1186/s13059-021-02359-2. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33957942.

46. D. Holoch; D. Moazed RNA-mediated epigenetic regulation of gene expression., 2015, 16,pp. 71-84. DOI: https://doi.org/10.1038/nrg3863. PMID: https://www.ncbi.nlm.nih.gov/pubmed/25554358.

47. Y. Jiang; W. Yin; H.E. Xu RNA-dependent RNA polymerase: Structure, mechanism, and drug discovery for COVID-19., 2021, 538,pp. 47-53. DOI: https://doi.org/10.1016/j.bbrc.2020.08.116.

48. B. Hu; L. Zhong; Y. Weng; L. Peng; Y. Huang; Y. Zhao; X.J. Liang Therapeutic siRNA: State of the art., 2020, 5,p. 101. DOI: https://doi.org/10.1038/s41392-020-0207-x. PMID: https://www.ncbi.nlm.nih.gov/pubmed/32561705.

49. N. Pinzon; S. Bertrand; L. Subirana; I. Busseau; H. Escriva; H. Seitz Functional lability of RNA-dependent RNA polymerases in animals., 2019, 15, e1007915. DOI: https://doi.org/10.1371/journal.pgen.1007915. PMID: https://www.ncbi.nlm.nih.gov/pubmed/30779744.

50. Y.H. Hung; R.K. Slotkin The initiation of RNA interference (RNAi) in plants., 2021, 61, 102014. DOI: https://doi.org/10.1016/j.pbi.2021.102014.

51. D.B. Krishnatreya; P.M. Baruah; B. Dowarah; S. Chowrasia; T.K. Mondal; N. Agarwala Genome-wide identification, evolutionary relationship and expression analysis of AGO, DCL and RDR family genes in tea., 2021, 11,p. 8679. DOI: https://doi.org/10.1038/s41598-021-87991-5.

52. D. Diez; F. Sanchez-Jimenez; J.A. Ranea Evolutionary expansion of the Ras switch regulatory module in eukaryotes., 2011, 39,pp. 5526-5537. DOI: https://doi.org/10.1093/nar/gkr154.

53. S. Wang; H. Liang; Y. Xu; L. Li; H. Wang; D.N. Sahu; M. Petersen; M. Melkonian; S.K. Sahu; H. Liu Genome-wide analyses across Viridiplantae reveal the origin and diversification of small RNA pathway-related genes., 2021, 4, 412. DOI: https://doi.org/10.1038/s42003-021-01933-5.

54. D. Pires; C.S.L. Vicente; E. Menendez; J.M.S. Faria; L. Rusinque; M.J. Camacho; M.L. Inacio The fight against Plant-Parasitic Nematodes: Current status of bacterial and fungal biocontrol agents., 2022, 11, 1178. DOI: https://doi.org/10.3390/pathogens11101178.

55. H. Sun; H. Dai; X. Wang; G. Wang Physiological and proteomic analysis of selenium-mediated tolerance to Cd stress in cucumber (Cucumis sativus L.)., 2016, 133,pp. 114-126. DOI: https://doi.org/10.1016/j.ecoenv.2016.07.003. PMID: https://www.ncbi.nlm.nih.gov/pubmed/27434422.

56. X. Song; D. Wang; L. Ma; Z. Chen; P. Li; X. Cui; C. Liu; S. Cao; C. Chu; Y. Tao et al. Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development., 2012, 71,pp. 378-389. DOI: https://doi.org/10.1111/j.1365-313X.2012.05001.x.

57. V. Jha; A. Narjala; D. Basu; T.N. Sujith; K. Pachamuthu; S. Chenna; A. Nair; P.V. Shivaprasad Essential role of gamma-clade RNA-dependent RNA polymerases in rice development and yield-related traits is linked to their atypical polymerase activities regulating specific genomic regions., 2021, 232,pp. 1674-1691. DOI: https://doi.org/10.1111/nph.17700.

58. P. Bustos-Sanmamed; E. Hudik; C. Laffont; C. Reynes; E. Sallet; J. Wen; K.S. Mysore; A.C. Camproux; C. Hartmann; J. Gouzy et al. A Medicago truncatula rdr6 allele impairs transgene silencing and endogenous phased siRNA production but not development., 2014, 12,pp. 1308-1318. DOI: https://doi.org/10.1111/pbi.12230. PMID: https://www.ncbi.nlm.nih.gov/pubmed/25060922.

59. N. Jiang; A. Gutierrez-Diaz; E. Mukundi; Y.S. Lee; B.C. Meyers; M.S. Otegui; E. Grotewold Synergy between the anthocyanin and RDR6/SGS3/DCL4 siRNA pathways expose hidden features of Arabidopsis carbon metabolism., 2020, 11,p. 2456. DOI: https://doi.org/10.1038/s41467-020-16289-3.

60. S. Polydore; M.J. Axtell Analysis of RDR1/RDR2/RDR6-independent small RNA s in Arabidopsis thaliana improves MIRNA annotations and reveals unexplained types of short interfering RNA loci., 2018, 94,pp. 1051-1063. DOI: https://doi.org/10.1111/tpj.13919.

61. A. f*ckudome; T. f*ckuhara Plant dicer-like proteins: Double-stranded RNA-cleaving enzymes for small RNA biogenesis., 2017, 130,pp. 33-44. DOI: https://doi.org/10.1007/s10265-016-0877-1.

62. J. Jia; R. Ji; Z. Li; Y. Yu; M. Nakano; Y. Long; L. Feng; C. Qin; D. Lu; J. Zhan et al. Soybean DICER-LIKE2 regulates seed coat color via production of primary 22-nucleotide small interfering RNAs from long inverted repeats., 2020, 32,pp. 3662-3673. DOI: https://doi.org/10.1105/tpc.20.00562.

63. K. Ciechanowska; M. Pokornowska; A. Kurzynska-Kokorniak Genetic insight into the domain structure and functions of Dicer-type ribonucleases., 2021, 22, 616. DOI: https://doi.org/10.3390/ijms22020616. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33435485.

64. T. Montavon; Y. Kwon; A. Zimmermann; P. Hammann; T. Vincent; V. Cognat; M. Bergdoll; F. Michel; P. Dunoyer Characterization of DCL4 missense alleles provides insights into its ability to process distinct classes of dsRNA substrates., 2018, 95,pp. 204-218. DOI: https://doi.org/10.1111/tpj.13941. PMID: https://www.ncbi.nlm.nih.gov/pubmed/29682831.

65. S. Su; J. Wang; T. Deng; X. Yuan; J. He; N. Liu; X. Li; Y. Huang; H.W. Wang; J. Ma Structural insights into dsRNA processing by Drosophila Dicer-2-Loqs-PD., 2022, 607,pp. 399-406. DOI: https://doi.org/10.1038/s41586-022-04911-x.

66. D.L. Cui; J.Y. Meng; X.Y. Ren; J.J. Yue; H.Y. Fu; M.T. Huang; Q.Q. Zhang; S.J. Gao Genome-wide identification and characterization of DCL, AGO and RDR gene families in Saccharum spontaneum., 2020, 10,p. 13202. DOI: https://doi.org/10.1038/s41598-020-70061-7.

67. J. Chen; L. Liu; C. You; J. Gu; W. Ruan; L. Zhang; J. Gan; C. Cao; Y. Huang; X. Chen et al. Structural and biochemical insights into small RNA 3' end trimming by Arabidopsis SDN1., 2018, 9,p. 3585. DOI: https://doi.org/10.1038/s41467-018-05942-7. PMID: https://www.ncbi.nlm.nih.gov/pubmed/30181559.

68. X. Wang; Y. Wang; Y. Dou; L. Chen; J. Wang; N. Jiang; C. Guo; Q. Yao; C. Wang; L. Liu et al. Degradation of unmethylated miRNA/miRNA*s by a DEDDy-type 3' to 5' exoribonuclease Atrimmer 2 in Arabidopsis., 2018, 115,pp. E6659-E6667. DOI: https://doi.org/10.1073/pnas.1721917115.

69. K.L. Tkaczuk; A. Obarska; J.M. Bujnicki Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis., 2006, 6, 6. DOI: https://doi.org/10.1186/1471-2148-6-6. PMID: https://www.ncbi.nlm.nih.gov/pubmed/16433904.

70. Z. Deng; L. Ma; P. Zhang; H. Zhu Small RNAs participate in plant-virus interaction and their application in plant viral defense., 2022, 23, 696. DOI: https://doi.org/10.3390/ijms23020696.

71. H.L. Tsai; Y.H. Li; W.P. Hsieh; M.C. Lin; J.H. Ahn; S.H. Wu HUA ENHANCER1 is involved in posttranscriptional regulation of positive and negative regulators in Arabidopsis photomorphogenesis., 2014, 26,pp. 2858-2872. DOI: https://doi.org/10.1105/tpc.114.126722.

72. M.C. Lin; H.L. Tsai; S.L. Lim; S.T. Jeng; S.H. Wu Unraveling multifaceted contributions of small regulatory RNAs to photomorphogenic development in Arabidopsis., 2017, 18, 559. DOI: https://doi.org/10.1186/s12864-017-3937-6. PMID: https://www.ncbi.nlm.nih.gov/pubmed/28738828.

73. X. Wang; W. Kong; Y. Wang; J. Wang; L. Zhong; K. Lao; X. Dong; D. Zhang; H. Huang; B. Mo et al. Uridylation and the SKI complex orchestrate the Calvin cycle of photosynthesis through RNA surveillance of TKL1 in Arabidopsis., 2022, 119,p. e2205842119. DOI: https://doi.org/10.1073/pnas.2205842119. PMID: https://www.ncbi.nlm.nih.gov/pubmed/36095196.

74. Z. Zhang; X. Liu; X. Guo; X.J. Wang; X. Zhang Arabidopsis AGO3 predominantly recruits 24-nt small RNAs to regulate epigenetic silencing., 2016, 2,p. 16049. DOI: https://doi.org/10.1038/nplants.2016.49. PMID: https://www.ncbi.nlm.nih.gov/pubmed/27243648.

75. Y. Xu; Y. Zhang; Z. Li; A.K. Soloria; S. Potter; X. Chen The N-terminal extension of Arabidopsis ARGONAUTE 1 is essential for microRNA activities., 2023, 19, e1010450. DOI: https://doi.org/10.1371/journal.pgen.1010450.

76. H. Zhu; F. Hu; R. Wang; X. Zhou; S.H. Sze; L.W. Liou; A. Barefoot; M. Dickman; X. Zhang Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development., 2011, 145,pp. 242-256. DOI: https://doi.org/10.1016/j.cell.2011.03.024.

77. H. Vaucheret; O. Voinnet The plant siRNA landscape., 2024, 36,pp. 246-275. DOI: https://doi.org/10.1093/plcell/koad253.

78. X. Zhang; H. Zhao; S. Gao; W.C. Wang; S. Katiyar-Agarwal; H.D. Huang; N. Raikhel; H. Jin Arabidopsis Argonaute 2 regulates innate immunity via miRNA393(*)-mediated silencing of a Golgi-localized SNARE gene, MEMB12., 2011, 42,pp. 356-366. DOI: https://doi.org/10.1016/j.molcel.2011.04.010. PMID: https://www.ncbi.nlm.nih.gov/pubmed/21549312.

79. O. Voinnet Revisiting small RNA movement in plants., 2022, 23,pp. 163-164. DOI: https://doi.org/10.1038/s41580-022-00455-0.

80. Y. Chu; A. Kilikevicius; J. Liu; K.C. Johnson; S. Yokota; D.R. Corey Argonaute binding within 3'-untranslated regions poorly predicts gene repression., 2020, 8,pp. 7439-7453. DOI: https://doi.org/10.1093/nar/gkaa478.

81. N. Fahlgren; T.A. Montgomery; M.D. Howell; E. Allen; S.K. Dvorak; A.L. Alexander; J.C. Carrington Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis., 2006, 16,pp. 939-944. DOI: https://doi.org/10.1016/j.cub.2006.03.065.

82. W. Liu; S.H. Duttke; J. Hetzel; M. Groth; S. Feng; J. Gallego-Bartolome; Z. Zhong; H.Y. Kuo; Z. Wang; J. Zhai et al. RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis., 2018, 4,pp. 181-188. DOI: https://doi.org/10.1038/s41477-017-0100-y. PMID: https://www.ncbi.nlm.nih.gov/pubmed/29379150.

83. D. Zilberman; X. Cao; S.E. Jacobsen ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation., 2003, 299,pp. 716-719. DOI: https://doi.org/10.1126/science.1079695.

84. M.A. Matzke; R.A. Mosher RNA-directed DNA methylation: An epigenetic pathway of increasing complexity., 2014, 15,pp. 394-408. DOI: https://doi.org/10.1038/nrg3683.

85. C.G. Duan; H. Zhang; K. Tang; X. Zhu; W. Qian; Y.J. Hou; B. Wang; Z. Lang; Y. Zhao; X. Wang et al. Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA-directed DNA methylation., 2015, 34,pp. 581-592. DOI: https://doi.org/10.15252/embj.201489453. PMID: https://www.ncbi.nlm.nih.gov/pubmed/25527293.

86. G. Bradamante; V.H. Nguyen; M. Incarbone; Z. Meir; H. Bente; M. Donà; N. Lettner; O.M. Scheid; R. Gutzat Two ARGONAUTE proteins loaded with transposon-derived small RNAs are associated with the reproductive cell lineage in Arabidopsis., 2024, 36,pp. 863-880. DOI: https://doi.org/10.1093/plcell/koad295.

87. Z. Liao; K.P. Hoden; R.K. Singh; C. Dixelius Genome-wide identification of Argonautes in Solanaceae with emphasis on potato., 2020, 10,p. 20577. DOI: https://doi.org/10.1038/s41598-020-77593-y. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33239724.

88. S. Niaz The AGO proteins: An overview., 2018, 399,pp. 525-547. DOI: https://doi.org/10.1515/hsz-2017-0329. PMID: https://www.ncbi.nlm.nih.gov/pubmed/29447113.

89. G. Meister Argonaute proteins: Functional insights and emerging roles., 2013, 14,pp. 447-459. DOI: https://doi.org/10.1038/nrg3462.

90. S. Jin; J. Zhan; Y. Zhou Argonaute proteins: Structures and their endonuclease activity., 2021, 48,pp. 4837-4849. DOI: https://doi.org/10.1007/s11033-021-06476-w.

91. W. Liu; K. Shoji; M. Naganuma; Y. Tomari; H.O. Iwakawa The mechanisms of siRNA selection by plant Argonaute proteins triggering DNA methylation., 2022, 50,pp. 12997-13010. DOI: https://doi.org/10.1093/nar/gkac1135.

92. K. Bohmert; I. Camus; C. Bellini; D. Bouchez; M. Caboche; C. Benning AGO1 defines a novel locus of Arabidopsis controlling leaf development., 1998, 17,pp. 170-180. DOI: https://doi.org/10.1093/emboj/17.1.170. PMID: https://www.ncbi.nlm.nih.gov/pubmed/9427751.

93. C. Zhou; L. Han; C. Fu; J. Wen; X. Cheng; J. Nakashima; J. Ma; Y. Tang; Y. Tan; M. Tadege et al. The trans-acting short interfering RNA3 pathway and no apical meristem antagonistically regulate leaf margin development and lateral organ separation, as revealed by analysis of an argonaute7/lobed leaflet1 mutant in Medicago truncatula., 2013, 25,pp. 4845-4862. DOI: https://doi.org/10.1105/tpc.113.117788. PMID: https://www.ncbi.nlm.nih.gov/pubmed/24368797.

94. X. Zheng; J. Zhu; A. Kapoor; J.K. Zhu Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing., 2007, 26,pp. 1691-1701. DOI: https://doi.org/10.1038/sj.emboj.7601603. PMID: https://www.ncbi.nlm.nih.gov/pubmed/17332757.

95. W. Sun; X. Xiang; L. Zhai; D. Zhang; Z. Cao; L. Liu; Z. Zhang AGO18b negatively regulates determinacy of spikelet meristems on the tassel central spike in maize., 2018, 60,pp. 65-78. DOI: https://doi.org/10.1111/jipb.12596. PMID: https://www.ncbi.nlm.nih.gov/pubmed/28875539.

96. M. D’Ario; S. Griffiths-Jones; M. Kim Small RNAs: Big impact on plant development., 2017, 22,pp. 1056-1068. DOI: https://doi.org/10.1016/j.tplants.2017.09.009. PMID: https://www.ncbi.nlm.nih.gov/pubmed/29032035.

97. C.M. Ha; J.H. Jun; J.C. Fletcher Shoot apical meristem form and function., 2010, 91,pp. 103-140.

98. F. Du; W. Gong; S. Bosca; M. Tucker; H. Vaucheret; T. Laux Dose-dependent AGO1-mediated inhibition of the miRNA165/166 pathway modulates stem cell maintenance in Arabidopsis shoot apical meristem., 2020, 1,p. 100002. DOI: https://doi.org/10.1016/j.xplc.2019.100002. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33404539.

99. T. Yang; Y. Wang; S. Teotia; Z. Wang; C. Shi; H. Sun; Y. Gu; Z. Zhang; G. Tang The interaction between miR160 and miR165/166 in the control of leaf development and drought tolerance in Arabidopsis., 2019, 9,p. 2832. DOI: https://doi.org/10.1038/s41598-019-39397-7.

100. D. Jiang; L. Hua; C. Zhang; H. Li; Z. Wang; J. Li; G. Wang; R. Song; T. Shen; H. Li et al. Mutations in the miRNA165/166 binding site of the HB2 gene result in pleiotropic effects on morphological traits in wheat., 2023, 11,pp. 9-20. DOI: https://doi.org/10.1016/j.cj.2022.05.002.

101. Q. Du; H. Wang The role of HD-ZIP III transcription factors and miR165/166 in vascular development and secondary cell wall formation., 2015, 10,p. e1078955. DOI: https://doi.org/10.1080/15592324.2015.1078955.

102. L. Lu; A. Holt; X. Chen; Y. Liu; S. Knauer; E.J. Tucker; A.K. Sarkar; Z. Hao; F. Roodbarkelari; J. Shi et al. miR394 enhances WUSCHEL-induced somatic embryogenesis in Arabidopsis thaliana., 2023, 238,pp. 1059-1072. DOI: https://doi.org/10.1111/nph.18801.

103. M. Somssich; B.I. Je; R. Simon; D. Jackson CLAVATA-WUSCHEL signaling in the shoot meristem., 2016, 143,pp. 3238-3248. DOI: https://doi.org/10.1242/dev.133645.

104. C. Hu; Y. Zhu; Y. Cui; K. Cheng; W. Liang; Z. Wei; M. Zhu; H. Yin; L. Zeng; Y. Xiao et al. A group of receptor kinases are essential for CLAVATA signalling to maintain stem cell homeostasis., 2018, 4,pp. 205-211. DOI: https://doi.org/10.1038/s41477-018-0123-z. PMID: https://www.ncbi.nlm.nih.gov/pubmed/29581511.

105. L. Zhang; D. DeGennaro; G. Lin; J. Chai; E.D. Shpak ERECTA family signaling constrains CLAVATA3 and WUSCHEL to the center of the shoot apical meristem., 2021, 148,p. dev189753. DOI: https://doi.org/10.1242/dev.189753. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33593817.

106. H. Han; A. Yan; L. Li; Y. Zhu; B. Feng; X. Liu; Y. Zhou A signal cascade originated from epidermis defines apical-basal patterning of Arabidopsis shoot apical meristems., 2020, 11,p. 1214. DOI: https://doi.org/10.1038/s41467-020-14989-4.

107. H. Han; Y. Zhou Function and regulation of microRNA171 in plant stem cell homeostasis and developmental programing., 2022, 23, 2544. DOI: https://doi.org/10.3390/ijms23052544. PMID: https://www.ncbi.nlm.nih.gov/pubmed/35269685.

108. Z. Tu; H. Xia; L. Yang; X. Zhai; Y. Shen; H. Li The Roles of microRNA-long non-coding RNA-mRNA networks in the regulation of leaf and flower development in liriodendron chinense., 2022, 13, 816875. DOI: https://doi.org/10.3389/fpls.2022.816875. PMID: https://www.ncbi.nlm.nih.gov/pubmed/35154228.

109. H. Wang; F. Kong; C. Zhou From genes to networks: The genetic control of leaf development., 2021, 63,pp. 1181-1196. DOI: https://doi.org/10.1111/jipb.13084. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33615731.

110. R. Xia; J. Xu; B.C. Meyers The emergence, evolution, and diversification of the miR390-TAS3-ARF pathway in land plants., 2017, 29,pp. 1232-1247. DOI: https://doi.org/10.1105/tpc.17.00185.

111. R.K. Tripathi; P. Bregitzer; J. Singh Genome-wide analysis of the SPL/miR156 module and its interaction with the AP2/miR172 unit in barley., 2018, 8,p. 7085. DOI: https://doi.org/10.1038/s41598-018-25349-0.

112. Y. Zhao; S. Wang; W. Wu; L. Li; T. Jiang; B. Zheng Clearance of maternal barriers by paternal miR159 to initiate endosperm nuclear division in Arabidopsis., 2018, 9,p. 5011. DOI: https://doi.org/10.1038/s41467-018-07429-x. PMID: https://www.ncbi.nlm.nih.gov/pubmed/30479343.

113. J. Wang; J. Bao; B. Zhou; M. Li; X. Li; J. Jin The osa-miR164 target OsCUC1 functions redundantly with OsCUC3 in controlling rice meristem/organ boundary specification., 2021, 229,pp. 1566-1581. DOI: https://doi.org/10.1111/nph.16939. PMID: https://www.ncbi.nlm.nih.gov/pubmed/32964416.

114. Y. Mao; F. Wu; X. Yu; J. Bai; W. Zhong; Y. He MicroRNA319a-targeted Brassica rapa ssp. pekinensis TCP genes modulate head shape in chinese cabbage by differential cell division arrest in leaf regions., 2014, 164,pp. 710-720. DOI: https://doi.org/10.1104/pp.113.228007. PMID: https://www.ncbi.nlm.nih.gov/pubmed/24351684.

115. J. Jiang; H. Zhu; N. Li; J. Batley; Y. Wang The miR393-target module regulates plant development and responses to biotic and abiotic stresses., 2022, 23, 9477. DOI: https://doi.org/10.3390/ijms23169477. PMID: https://www.ncbi.nlm.nih.gov/pubmed/36012740.

116. R.E. Rodriguez; M.F. Ercoli; J.M. Debernardi; N.W. Breakfield; M.A. Mecchia; M. Sabatini; T. Cools; L. De Veylder; P.N. Benfey; J.F. Palatnik MicroRNA miR396 regulates the switch between stem cells and transit-amplifying cells in Arabidopsis roots., 2015, 27,pp. 3354-3366. DOI: https://doi.org/10.1105/tpc.15.00452. PMID: https://www.ncbi.nlm.nih.gov/pubmed/26645252.

117. S. Waheed; L. Zeng The critical role of miRNAs in regulation of flowering time and flower development., 2020, 11, 319. DOI: https://doi.org/10.3390/genes11030319. PMID: https://www.ncbi.nlm.nih.gov/pubmed/32192095.

118. M. Xu; T. Hu; J. Zhao; M.Y. Park; K.W. Earley; G. Wu; L. Yang; R.S. Poethig Developmental functions of miR156-Regulated SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes in Arabidopsis thaliana., 2016, 12, e1006263. DOI: https://doi.org/10.1371/journal.pgen.1006263. PMID: https://www.ncbi.nlm.nih.gov/pubmed/27541584.

119. J.Y. Hu; Y. Zhou; F. He; X. Dong; L.Y. Liu; G. Coupland; F. Turck; J. de Meaux miR824-regulated AGAMOUS-LIKE16 contributes to flowering time repression in Arabidopsis., 2014, 26,pp. 2024-2037. DOI: https://doi.org/10.1105/tpc.114.124685.

120. Y. Li; Y. Tong; X. He; Y. Zhu; T. Li; X. Lin; W. Mao; Z. Ghulam Nabi Gishkori; Z. Zhao; J. Zhang et al. The rice miR171b–SCL6-IIs module controls blast resistance, grain yield, and flowering., 2022, 10,pp. 117-127. DOI: https://doi.org/10.1016/j.cj.2021.05.004.

121. C. Guo; Y. Xu; M. Shi; Y. Lai; X. Wu; H. Wang; Z. Zhu; R.S. Poethig; G. Wu Repression of miR156 by miR159 regulates the timing of the juvenile-to-adult transition in Arabidopsis., 2017, 29,pp. 1293-1304. DOI: https://doi.org/10.1105/tpc.16.00975.

122. C. Guo; Y. Jiang; M. Shi; X. Wu; G. Wu ABI5 acts downstream of miR159 to delay vegetative phase change in Arabidopsis., 2021, 231,pp. 339-350. DOI: https://doi.org/10.1111/nph.17371.

123. Z. Chang; R. Xu; Q. Xun; J. Liu; T. Zhong; Y. Ding; C. Ding OsmiR164-targeted OsNAM, a boundary gene, plays important roles in rice leaf and panicle development., 2021, 106,pp. 41-55. DOI: https://doi.org/10.1111/tpj.15143. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33368800.

124. X. Yao; J. Chen; J. Zhou; H. Yu; C. Ge; M. Zhang; X. Gao; X. Dai; Z.N. Yang; Y. Zhao An essential role for miRNA167 in maternal control of embryonic and seed development., 2019, 180,pp. 453-464. DOI: https://doi.org/10.1104/pp.19.00127. PMID: https://www.ncbi.nlm.nih.gov/pubmed/30867333.

125. M.Y. Xu; L. Zhang; W.W. Li; X.L. Hu; M.B. Wang; Y.L. Fan; C.Y. Zhang; L. Wang Stress-induced early flowering is mediated by miR169 in Arabidopsis thaliana., 2014, 65,pp. 89-101. DOI: https://doi.org/10.1093/jxb/ert353.

126. B. Zhang; X. Chen Secrets of the MIR172 family in plant development and flowering unveiled., 2021, 19, e3001099. DOI: https://doi.org/10.1371/journal.pbio.3001099.

127. Y. Xie; Y. Liu; H. Wang; X. Ma; B. Wang; G. Wu; H. Wang Phytochrome-interacting factors directly suppress MIR156 expression to enhance shade-avoidance syndrome in Arabidopsis., 2017, 8,p. 348. DOI: https://doi.org/10.1038/s41467-017-00404-y.

128. H. Lian; L. Wang; N. Ma; C.M. Zhou; L. Han; T.Q. Zhang; J.W. Wang Redundant and specific roles of individual MIR172 genes in plant development., 2021, 19, e3001044. DOI: https://doi.org/10.1371/journal.pbio.3001044. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33529193.

129. J. Liu; X. Cheng; P. Liu; D. Li; T. Chen; X. Gu; J. Sun MicroRNA319-regulated TCPs interact with FBHs and PFT1 to activate CO transcription and control flowering time in Arabidopsis., 2017, 13, e1006833. DOI: https://doi.org/10.1371/journal.pgen.1006833.

130. Y. Zhou; A.A. Myat; C. Liang; Z. Meng; S. Guo; Y. Wei; G. Sun; Y. Wang; R. Zhang Insights into microRNA-mediated regulation of flowering time in cotton through small RNA sequencing., 2022, 13, 761244. DOI: https://doi.org/10.3389/fpls.2022.761244.

131. K. Xia; R. Wang; X. Ou; Z. Fang; C. Tian; J. Duan; Y. Wang; M. Zhang OsTIR1 and OsAFB2 downregulation via OsmiR393 overexpression leads to more tillers, early flowering and less tolerance to salt and drought in rice., 2012, 7, e30039. DOI: https://doi.org/10.1371/journal.pone.0030039.

132. Z. Chen; L. Hu; N. Han; J. Hu; Y. Yang; T. Xiang; X. Zhang; L. Wang Overexpression of a miR393-resistant form of transport inhibitor response protein 1 (mTIR1) enhances salt tolerance by increased osmoregulation and Na+ exclusion in Arabidopsis thaliana., 2015, 56,pp. 73-83. DOI: https://doi.org/10.1093/pcp/pcu149. PMID: https://www.ncbi.nlm.nih.gov/pubmed/25336111.

133. H.M. Szaker; E. Darko; A. Medzihradszky; T. Janda; H.C. Liu; Y.Y. Charng; T. Csorba miR824/AGAMOUS-LIKE16 module integrates recurring environmental heat stress changes to fine-tune poststress development., 2019, 10, 1454. DOI: https://doi.org/10.3389/fpls.2019.01454. PMID: https://www.ncbi.nlm.nih.gov/pubmed/31824525.

134. M. McKeown; M. Schubert; J.C. Preston; S. Fjellheim Evolution of the miR5200-FLOWERING LOCUS T flowering time regulon in the temperate grass subfamily Pooideae., 2017, 114,pp. 111-121. DOI: https://doi.org/10.1016/j.ympev.2017.06.005.

135. A. Singh; V. Gautam; S. Singh; S. Sarkar Das; S. Verma; V. Mishra; S. Mukherjee; A.K. Sarkar Plant small RNAs: Advancement in the understanding of biogenesis and role in plant development., 2018, 248,pp. 545-558. DOI: https://doi.org/10.1007/s00425-018-2927-5.

136. X.-X. Yan; X.-Y. Liu; H. Cui; M.-Q. Zhao The roles of microRNAs in regulating root formation and growth in plants., 2022, 21,pp. 901-916. DOI: https://doi.org/10.1016/S2095-3119(21)63818-2.

137. C. Zheng; M. Ye; M. Sang; R. Wu A regulatory network for miR156-SPL module in Arabidopsis thaliana., 2019, 20, 6166. DOI: https://doi.org/10.3390/ijms20246166. PMID: https://www.ncbi.nlm.nih.gov/pubmed/31817723.

138. T. Xue; Z. Liu; X. Dai; F. Xiang Primary root growth in Arabidopsis thaliana is inhibited by the miR159 mediated repression of MYB33, MYB65 and MYB101., 2017, 262,pp. 182-189. DOI: https://doi.org/10.1016/j.plantsci.2017.06.008.

139. X. Dai; Q. Lu; J. Wang; L. Wang; F. Xiang; Z. Liu MiR160 and its target genes ARF10, ARF16 and ARF17 modulate hypocotyl elongation in a light, BRZ, or PAC-dependent manner in Arabidopsis: miR160 promotes hypocotyl elongation., 2021, 303,p. 110686. DOI: https://doi.org/10.1016/j.plantsci.2020.110686. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33487334.

140. P.J. Chung; B.S. Park; H. Wang; J. Liu; I.C. Jang; N.H. Chua Light-inducible miR163 targets PXMT1 transcripts to promote seed germination and primary root elongation in Arabidopsis., 2016, 170,pp. 1772-1782. DOI: https://doi.org/10.1104/pp.15.01188.

141. H.S. Guo; Q. Xie; J.F. Fei; N.H. Chua MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development., 2005, 17,pp. 1376-1386.

142. X. Jia; N. Ding; W. Fan; J. Yan; Y. Gu; X. Tang; R. Li; G. Tang Functional plasticity of miR165/166 in plant development revealed by small tandem target mimic., 2015, 233,pp. 11-21. DOI: https://doi.org/10.1016/j.plantsci.2014.12.020. PMID: https://www.ncbi.nlm.nih.gov/pubmed/25711809.

143. S. Miyashima; S. Koi; T. Hashimoto; K. Nakajima Non-cell-autonomous microRNA165 acts in a dose-dependent manner to regulate multiple differentiation status in the Arabidopsis root., 2011, 138,pp. 2303-2313. DOI: https://doi.org/10.1242/dev.060491. PMID: https://www.ncbi.nlm.nih.gov/pubmed/21558378.

144. C. Sorin; M. Declerck; A. Christ; T. Blein; L. Ma; C. Lelandais-Briere; M.F. Njo; T. Beeckman; M. Crespi; C. Hartmann A miR169 isoform regulates specific NF-YA targets and root architecture in Arabidopsis., 2014, 202,pp. 1197-1211. DOI: https://doi.org/10.1111/nph.12735. PMID: https://www.ncbi.nlm.nih.gov/pubmed/24533947.

145. T. Um; J. Choi; T. Park; P.J. Chung; S.E. Jung; J.S. Shim; Y.S. Kim; I.Y. Choi; S.C. Park; S.J. Oh et al. Rice microRNA171f/SCL6 module enhances drought tolerance by regulation of flavonoid biosynthesis genes., 2022, 6,p. e374. DOI: https://doi.org/10.1002/pld3.374. PMID: https://www.ncbi.nlm.nih.gov/pubmed/35028494.

146. F. Zhang; L. Wang; J.Y. Lim; T. Kim; Y. Pyo; S. Sung; C. Shin; H. Qiao Phosphorylation of CBP20 links microRNA to root growth in the ethylene response., 2016, 12, e1006437. DOI: https://doi.org/10.1371/journal.pgen.1006437. PMID: https://www.ncbi.nlm.nih.gov/pubmed/27870849.

147. K.V. Hobecker; M.A. Reynoso; P. Bustos-Sanmamed; J. Wen; K.S. Mysore; M. Crespi; F.A. Blanco; M.E. Zanetti The microRNA390/TAS3 pathway mediates symbiotic nodulation and lateral root growth., 2017, 174,pp. 2469-2486. DOI: https://doi.org/10.1104/pp.17.00464. PMID: https://www.ncbi.nlm.nih.gov/pubmed/28663332.

148. M. Bao; H. Bian; Y. Zha; F. Li; Y. Sun; B. Bai; Z. Chen; J. Wang; M. Zhu; N. Han miR396a-Mediated basic helix-loop-helix transcription factor bHLH74 repression acts as a regulator for root growth in Arabidopsis seedlings., 2014, 55,pp. 1343-1353. DOI: https://doi.org/10.1093/pcp/pcu058. PMID: https://www.ncbi.nlm.nih.gov/pubmed/24793750.

149. R. Kaushal; L. Peng; S.K. Singh; M. Zhang; X. Zhang; J.I. Vilchez; Z. Wang; D. He; Y. Yang; S. Lv et al. Dicer-like proteins influence Arabidopsis root microbiota independent of RNA-directed DNA methylation., 2021, 9, 57. DOI: https://doi.org/10.1186/s40168-020-00966-y.

150. M. Li; Y. Zhu; S. Li; W. Zhang; C. Yin; Y. Lin Regulation of phytohormones on the growth and development of plant root hair., 2022, 13, 865302. DOI: https://doi.org/10.3389/fpls.2022.865302.

151. N.T. Hoang; K. Toth; G. Stacey The role of microRNAs in the legume-Rhizobium nitrogen-fixing symbiosis., 2020, 71,pp. 1668-1680. DOI: https://doi.org/10.1093/jxb/eraa018.

152. J. Yun; C. Wang; F. Zhang; L. Chen; Z. Sun; Y. Cai; Y. Luo; J. Liao; Y. Wang; Y. Cha et al. A nitrogen fixing symbiosis-specific pathway required for legume flowering., 2023, 9,p. eade1150. DOI: https://doi.org/10.1126/sciadv.ade1150. PMID: https://www.ncbi.nlm.nih.gov/pubmed/36638166.

153. K. Fan; J. Wong-Bajracharya; X. Lin; M. Ni; Y.S. Ku; M.W. Li; C.F. Tian; T.F. Chan; H.M. Lam Differentially expressed microRNAs that target functional genes in mature soybean nodules., 2021, 14,p. e20103. DOI: https://doi.org/10.1002/tpg2.20103. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33973410.

154. J. Yun; Z. Sun; Q. Jiang; Y. Wang; C. Wang; Y. Luo; F. Zhang; X. Li The miR156b-GmSPL9d module modulates nodulation by targeting multiple core nodulation genes in soybean., 2022, 233,pp. 1881-1899. DOI: https://doi.org/10.1111/nph.17899. PMID: https://www.ncbi.nlm.nih.gov/pubmed/34862970.

155. L. Wang; Z. Sun; C. Su; Y. Wang; Q. Yan; J. Chen; T. Ott; X. Li A GmNINa-miR172c-NNC1 regulatory network coordinates the nodulation and autoregulation of nodulation pathways in soybean., 2019, 12,pp. 1211-1226. DOI: https://doi.org/10.1016/j.molp.2019.06.002. PMID: https://www.ncbi.nlm.nih.gov/pubmed/31201867.

156. D. Tsikou; Z. Yan; D.B. Holt; N.B. Abel; D.E. Reid; L.H. Madsen; H. Bhasin; M. Sexauer; J. Stougaard; K. Markmann Systemic control of legume susceptibility to rhizobial infection by a mobile microRNA., 2018, 362,pp. 233-236. DOI: https://doi.org/10.1126/science.aat6907. PMID: https://www.ncbi.nlm.nih.gov/pubmed/30166437.

157. M. Zhang; H. Su; P.M. Gresshoff; B.J. Ferguson Shoot-derived miR2111 controls legume root and nodule development., 2021, 44,pp. 1627-1641. DOI: https://doi.org/10.1111/pce.13992. PMID: https://www.ncbi.nlm.nih.gov/pubmed/33386621.

158. Z. Yan; M.S. Hossain; S. Arikit; O. Valdés-López; J. Zhai; J. Wang; M. Libault; T. Ji; L. Qiu; B.C. Meyers et al. Identification of microRNAs and their mRNA targets during soybean nodule development: Functional analysis of the role of miR393j-3p in soybean nodulation., 2015, 207,pp. 748-759. DOI: https://doi.org/10.1111/nph.13365.

159. H. Li; Y. Deng; T. Wu; S. Subramanian; O. Yu Misexpression of miR482, miR1512, and miR1515 increases soybean nodulation., 2010, 153,pp. 1759-1770. DOI: https://doi.org/10.1104/pp.110.156950.

Figures

Figure 1: In plant cells, the biogenesis of miRNA and siRNA and their regulatory mechanisms are of paramount importance. The transcription of miRNAs is orchestrated by RNA polymerase II within the confines of the nucleus, culminating in the genesis of primary miRNAs (pri-miRNA). These pri-miRNAs are characterized by their distinctive hairpin structure. The transformation of double-stranded RNA into siRNA is expedited by the concerted efforts of DCL3, HEN, and RDR2. In the subsequent stages, the RISC–AGO complex assumes a pivotal role in guiding the specific strands of the siRNA duplex towards either post-transcription gene silencing (PTGS) or transcriptional gene silencing (TGS). [Please download the PDF to view the image]

Figure 2: Small RNAs guide their effector proteins to specific loci to initiate either post-transcriptional gene silencing (PTGS) through transcript cleavage, RNA decay (degradation) and translation repression, or transcriptional gene silencing (TGS), which involves histone H3 Lys9 (H3K9) methylation at DNA loci that are hom*ologous to the sRNA, often transposable element (TE) loci. Different mechanisms observed in different organisms are summarized in the same scheme. [Please download the PDF to view the image]

Figure 3: Functions of sRNAs in plant development and an overview of the current understanding of miRNA-mediated regulation in plants. [Please download the PDF to view the image]

Author Affiliation(s):

Guangdong Key Laboratory of Plant Adaptation and Molecular Design, Guangzhou Key Laboratory of Crop Gene Editing, Innovative Center of Molecular Genetics and Evolution, School of Life Sciences, Guangzhou University, Guangzhou 510006, China; [emailprotected] (Q.L.); [emailprotected] (Y.W.); [emailprotected] (Z.S.)

Author Note(s):

[*] Correspondence: [emailprotected] (H.L.); [emailprotected] (H.L.)

DOI: 10.3390/ijms25147680