Multifaceted roles of RNA polymerase IV in plant growth and development

We discuss the latest findings on RNA polymerase IV (Pol IV) in plant growth and development, providing new insights and expanding on new ideas for further, more in-depth research on Pol IV.

RNA-directed DNA methylation (RdDM) is a small RNA-mediated epigenetic process in plants. The biogenesis of small RNAs and initiation of RdDM rely on complex transcriptional machineries, including two plant-specific RNA polymerases (Pol IV and Pol V) and other auxiliary proteins. Pol IV is known to play a critical role in generating 24-nt siRNAs in the RdDM pathway, and is involved in Capsella pollen development, rice tillering, and rice resistance to viruses. Here, we discuss the most recent findings on the functions of Pol IV in plant growth and development and consider other possible functions that need further investigation.
In plants, RNA-directed DNA methylation (RdDM) is a conserved epigenetic process that mediates the silencing of DNA with repetitive sequences and transposable elements (TEs). Thus, RdDM is considered to be an important mechanism for the maintenance of genome stability (Tsukahara et al., 2009;Law et al., 2010). In the canonical RdDM pathway, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) converts RNA polymerase IV (Pol IV)-generated transcripts into double-stranded RNAs (dsRNAs), while in the noncanonical RdDM pathway, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) converts RNA polymerase II (Pol II)-generated RNA transcripts into dsRNAs. The canonical RdDM pathway includes the following steps. (i) SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1) protein recognizes histone H3K9me2 and then recruits Pol IV to recognize the heterochromatic regions to transcribe precursor RNAs, which are in the order of 25 to 50 nucleotides (nt) in length (Yang et al., 2016). (ii) RDR2 physically interacts with Pol IV  and converts the Pol IV transcripts into dsRNAs. Among them, the chromatin remodeler proteins CLASSYs (CLSYs; CLSY1-4) function as components of the Pol IV complex; their main function is to act as locus-specific regulators of both 24-nt small interfering RNA (siRNA) production and DNA methylation (Yang et al., 2018;Zhou et al., 2018). (iii) dsRNAs are processed by DICER-LIKE 3 (DCL3) into 24-nt siRNAs, which are loaded into ARGONAUTE4 (AGO4) and then processed through Pol V-mediated de novo DNA methylation (Wierzbicki et al., 2008;Zhong et al., 2014;Gallego-Bartolomé et al., 2019;Singh et al., 2019).
Apart from DCL3, other DCLs are also capable of generating distinct small RNA species. Among them, DCL1 is known to be responsible for the maturation of 21-nt microRNAs (miRNAs) or siRNAs processed from hairpin-structured precursors. DCL2 acts mainly in the biogenesis of 22-nt viral siRNAs (vsiRNAs), while DCL4 generates mainly 21-nt trans-acting siRNAs (ta-siRNAs). Furthermore, DCL2, DCL3, and DCL4 are known to function partially redundantly in the establishment and maintenance of DNA methylation as well as the biogenesis of Pol IV-generated RNA transcripts. In addition, there is a unique and DCL-independent class of siRNAs (sidRNAs) of the order of 20 to 60 nt in length (Yang et al., 2016;Ye et al., 2016). The precursor RNA transcripts of sidRNAs are associated with AGO4 and are subsequently trimmed by 3′-5′ exonuclease to produce mature sidRNAs to initiate de novo DNA methylation (Ye et al., 2016). Because the RdDM pathway has been found in both vegetative and reproductive organs of plants, it is likely to have prominent roles in the whole life cycle.

Multifaceted roles of Pol IV in plant growth and development
Very recently, two articles have shed new light on the functions of Pol IV in rice (Oryza sativa). Zhang et al. (2020) have reported that the stable expression of rice grassy stunt virus (RGSV)-encoded P3 protein in rice plants can cause a dwarfing and excessive tillering phenotype similar to the disease symptoms caused by RGSV infection. The authors conclude that stable expression of P3 protein or RGSV infection in rice plants can lead to an enhancement of ubiquitination and the ubiquitin proteasome system (UPS)-dependent degradation of rice NUCLEAR RNA POLYMERASE D1a (OsNRPD1a), one of the two orthologs of the largest subunit of plant-specific Pol IV holoenzyme. This degradation mechanism is accomplished mainly by recruiting P3IP1, a P3-inducible U-box type E3 ubiquitin ligase, to ubiquitinate and degrade OsNRPD1a protein by the UPSdependent pathway. This report also revealed that RGSV can target host Pol IV for UPS-dependent degradation and RdDM core protein can serve as a potential target for the UPS, a novel virulence mechanism underlying plant-virus interactions .
The other study, by Xu et al. (2020), revealed that RdDM inhibits rice tillering by regulating the expression of three agriculturally important genes, OsMIR156d, OsMIR156j, and DWARF14 (D14). Reduced expression of rice OsNRPD1a and OsNRPD1b results in a pronounced loss of genome-wide 24-nt siRNAs, a remarkable reduction of DNA methylation in the miniature inverted-repeat transposable element (MITE) regions, especially CHH methylation, and the subsequent control of the expression of key genes associated with rice tillering. Mechanistically, RdDM targets two MITEs in the promoter regions of OsMIR156d and OsMIR156j and significantly inhibits the transcription of these two miRNAs, which controls the expression of key genes related to rice tillering. Rice tillering determines the plant structure and grain yield, and Ideal Plant Architecture 1 (IPA1) is an important factor that has been identified to regulate rice tillering. Three MITEs were found in the promoter of IPA1. However, the degree of methylation of these MITEs was not significantly different between wild-type plants and osnrpd1-1 mutants. To a certain extent, the possibility of RdDM involvement in the regulation of rice tillering by directly controlling the transcription of IPA1 was ruled out. Studies have found that the expression of IPA1 can be inhibited by OsmiR156 at the shoot tip (Jiao et al., 2010;Miura et al., 2010). OsmiR156a-j transcripts accumulated excessively in osnrpd1-1/2 and osnrpd1ab double knockout lines, and the expression of the target IPA1 was down-regulated, highlighting that RdDM regulates rice tillering through the OsmiR156-IPA1 module. In contrast, the expression of D14, which encodes a strigolactone receptor and can repress the outgrowth of rice tillers, is activated by CHH methylation in a MITE region located at its downstream. In the osnrpd1-1/2 mutant, MITE#1 in the downstream region of D14 was hypomethylated, resulting in the down-regulation of D14 and enhanced protein stability of D53. Furthermore, D53 inhibits the transcriptional activation ability of IPA1 (Song et al., 2017), leading to an increase in rice tillering, indicating that RdDM also controls rice tillering through the strigolactone signaling pathway. This finding indicates an important RdDM-dependent mechanism controlling rice tillering and provides potential targets for the improvement of agronomic traits through epigenome editing.
In addition to its above-mentioned roles, Pol IV is also critical for basal heat tolerance in Arabidopsis. Transient heat stress can affect the epigenetic program in plants as well as the long-term thermal responses triggered by the depletion of loci silencing within constitutive heterochromatin. Recent findings have indicated that mutant plants defective in NRPD2, which encodes a common (and the second largest) subunit of the Pol IV and Pol V complexes, are hypersensitive to heat exposure. All the dysregulated genes in nrpd2 mutants recovering from heat stress are located near the transposon residues or the siRNA-producing clusters, suggesting that these dysregulated thermal-responsive genes are modulated by defective epigenetic regulation near the transposons in plants lacking a functional NRPD2. These results also point toward a certain signal-controlled correlation between the RdDM pathway and plant tolerance to heat stress (Popova et al., 2013).
Recently, Pol IV has been shown to play an important role in pollen development in Arabidopsis. The formation of pollen is strongly affected by the reprogramming of CHH methylation. During meiosis, the global level of CHH methylation is greatly reduced and the accumulation of meiosis-specific small RNAs is dependent on Pol IV (Walker et al., 2018). Although many functions of Pol IV have been documented, its loss of function does not cause an obvious pollen-deficient phenotype in Arabidopsis. Based on the obvious difference in TE contents between Arabidopsis thaliana and Capsella rubella, the loss of function of Pol IV has a greater impact on the latter species, resembling the defects in Brassica rapa (Grover et al., 2018). Recent studies have also demonstrated that the loss of Pol IV function in Capsella can lead to an arrest of microspore development. Small RNA profiling has shown that depletion of Pol IV can block the production of 21-and 22-nt siRNAs (Wang et al., 2020), suggesting that Pol IV is required for the synthesis of epigenetically activated 21-and 22-nt siRNAs (easiRNAs) in pollen. The biogenesis of easiRNAs is known to be triggered by certain miRNAs (e.g. miRNA845b) and requires the involvement of DCL2 and/or DCL4.
Pol IV-dependent paternal easiRNA can cause barriers to cross-breeding using plants of different ploidy (Martinez et al., 2018). Seed development is sensitive to parental genome doses, and excessive paternal genomes can cause defective phenotypes, including large endosperm reproduction without cellularization and seed abortion. Paternal loss of Pol IV function can inhibit easiRNA biogenesis, and depletion of easiRNA can overcome the triploid block to rescue triploid seed formation via the restoration of RdDM on TEs. This restoration will increase paternal ploidy in Arabidopsis. It is noteworthy that easiRNA is not only a quantitative signal for paternal chromosomes, but also a balanced dose required for post-fertilization genome stabilization as well as seed vigor. How easiRNA is generated, and the nature of its downstream reaction mechanisms, are still not fully understood and thus need more in-depth research.
Coinciding with Arabidopsis, the maize (Zea mays) Pol IV-mediated RdDM pathway also plays an extensive role in At5G66750 Snf2 chromatin remodeler acting in siRNA-independent DNA methylation (Zemach et al.,

At3G42670
Putative Snf2 chromatin remodeling factor, involved in the Pol IV pathway (Smith et al., 2007) Protein components involved in the RdDM pathway and DNA methylation in Arabidopsis

Gene ID Description Reference
AtCHR34

2011) Protein components involved in the RdDM pathway and DNA methylation in rice OsNRPD1a
LOC_Os04g48370 One of two orthologs of the largest subunit of Pol IV  OsNRPD1b LOC_Os09g38268 One of two orthologs of the largest subunit of Pol IV (Xu et al., 2020) OsDCL1a LOC_Os03g02970 Responsible for the processing of 21/24-nt miRNAs (Liu et al., 2005) OsDCL2a LOC_Os03g38740 Responsible for the processing of rice miRNAs (Kapoor et al., 2008) OsDCL2b LOC_Os09g14610 Responsible for the processing of rice miRNAs (Kapoor et al., 2008) OsDCL3a LOC_Os01g68120 Required for the biogenesis of lmiRNAs (Kapoor et al., 2008) OsDCL3b LOC_Os10g34430 Responsible for the processing of 21/24-nt miRNAs (Song et al., 2012) OsDCL4 LOC_Os04g43050 Affects the production of 21nt siRNA in the panicle (Song et al., 2012) OsAGO1a LOC_Os02g45070 Has the ability to bind small RNA and has cleavage activity (Wu et al., 2009)

OsAGO1b
LOC_Os04g47870 Has the ability to bind small RNA and has cleavage activity (Wu et al., 2009)

OsAGO1c
LOC_Os02g58490 Has the ability to bind small RNA and has cleavage activity (Wu et al., 2009)

LOC_Os01g16870
Involved in the biogenesis of small RNAs (Kapoor et al., 2008) OsAGO4b LOC_Os04g06770 Involved in the biogenesis of small RNAs (Kapoor et al., 2008) OsMEL1 LOC_Os03g58600 Participates in the regulation of the division of germ cells before meiosis, the correct modification of meiotic chromosomes, and the accurate progress of meiosis through the RdDM pathway (Nonomura et al., 2007) OsAGO16

LOC_Os07g16224
Involved in transcriptional gene silencing by guiding DNA methylation (Wu et al., 2010)

LOC_Os01g34350
Participates in the plant defense responses to viruses, bacteria, and fungi (Wagh et al., 2016) OsRDR2

LOC_Os04g39160
Has roles in siRNA-mediated DNA methylation and histone modifications (Vrbsky et al., 2010) OsRDR4 LOC_Os01g10140 Specifically activated in response to dehydration stress (Kumar and Singh, 2016)

Future perspectives
A comprehensive list of components associated with Arabidopsis and rice RdDM pathways, including DCLs, AGOs, and RDRs, is given in Table 1. In addition, we have summarized the Fig. 1. The all-round role of RNA polymerase IV (Pol IV) in plants. In Arabidopsis, SHH1/DTF1 binds to the nucleosome through reading H3K9me2, and recruits Pol IV to transcribe the target region. RDR2 and RDR6 interact with Pol IV to convert and then process the Pol IV transcripts into 21/22/24-nt siRNAs and easiRNAs with the assistance of DCL proteins. Among these, DCL3 is the main enzyme for processing Pol IV-synthesized RNA transcripts, and other DCLs might be more important for easiRNA biosynthesis. As components of the Pol IV complex, the CLSYs (CLSY 1-4) regulate the Pol IVchromatin association and 24-nt siRNA production at thousands of distinct loci, but whether CLSYs directly bind chromatin is not known. Subsequently, the guide strand is incorporated into AGO4/6, and then enters de novo DNA methylation or builds the triploid block using excess 21/22-nt easiRNAs.
In rice, depletion of Pol IV (OsNRPD1a and OsNRPD1b) results in a remarkable loss of CHH-type DNA methylation in MITEs, thereby affecting the expression of key agronomically important genes (OsMIR156d/j and D14) to regulate rice tillering. By recruiting E3 ubiquitin ligase P3IP1, rice grassy stunt virus (RGSV) P3 protein enhances the ubiquitination and UPS-dependent degradation of rice OsNRPD1a. These findings highlight a new virulence mechanism underlying plant-virus interaction, and further integrate the crosstalk between the RdDM pathway and UPS-dependent degradation during virus infection. LOC_Os03g02010 Regulates rice vegetative and reproductive growth through DNA methylation (Dangwal et al., 2013) multifaceted role of Pol IV in plants in Fig. 1 Although many studies using Arabidopsis, rice, maize, and other plants have significantly advanced our knowledge on the functions of Pol IV, many fundamental questions are still unanswered. For example, Pol IV is an important component in RdDM, and rice nrpd1 mutant plants exhibit a dwarfed and excessive tillering phenotype, and maize rpd1 mutants are shorter, with delayed flowering, feminization of male tassels, depolarization of leaf tissue, and tissue outgrowths on their stems (Parkinson et al., 2007, Erhard et al., 2009. In contrast to these representative monocotyledonous species, Arabidopsis mutants in Pol IV function have no such developmental defects. Perhaps Pol IV controls different regulatory mechanisms in monocotyledonous and dicotyledonous plants. As far as monocotyledonous species are concerned, the loss of Pol IV activity also has different effects on plant development in rice and maize, and its underlying fine mechanisms still need to be urgently elucidated in future research. In addition, it remains unknown how RGSV can target host Pol IV to disrupt the UPS-dependent pathways but not the downstream regulatory networks involved in plant-pathogen interactions. Although OsNRPD1a and OsNRPD1b are the orthologs of the largest subunit in rice Pol IV, do they have functional divergence, especially in the regulation of plant responses to stresses? Can Pol IV play roles in other abiotic stress responses in addition to heat stress,?
Future biochemical, functional, and genetic studies are necessary to address these questions. As with other molecular biology studies, the studies on the functions of Pol IV have entered a new phase to explore much broader and more in-depth mechanisms in many other plant species. Understanding the mechanisms underlying the functions of Pol IV in other plant species, especially monocotyledonous species, will provide us with opportunities to identify the links between RdDM and other molecular pathways, such as the UPS-dependent pathway. Collectively, the information described above will uncover the multifaceted roles of Pol IV in plant development and reproduction.