ENB1 encodes a cellulose synthase 5 that directs synthesis of cell wall ingrowths in maize basal endosperm transfer cells.

Development of the endosperm is strikingly different in monocots and dicots: it often manifests as a persistent tissue in the former and transient tissue in the latter. Little is known about the controlling mechanisms responsible for these different outcomes. Here we characterized a maize (Zea mays) mutant, endosperm breakdown1 (enb1), in which the typically persistent endosperm (PE) was drastically degraded during kernel development. ENB1 encodes a cellulose synthase 5 that is predominantly expressed in the basal endosperm transfer layer (BETL) of endosperm cells. Loss of ENB1 function caused a drastic reduction in formation of flange cell wall ingrowths (ingrowths) in BETL cells. Defective ingrowths impair nutrient uptake, leading to premature utilization of endosperm starch to nourish the embryo. Similarly, developing wild-type kernels cultured in vitro with a low level of sucrose manifested early endosperm breakdown. ENB1 expression is induced by sucrose via the BETL-specific Myb-Related Protein1 transcription factor. Overexpression of ENB1 enhanced development of flange ingrowths, facilitating sucrose transport into BETL cells and increasing kernel weight. The results demonstrated that ENB1 enhances sucrose supply to the endosperm and contributes to a PE in the kernel.

The biosynthesis of storage reserves and auxin is coordinated by a hierarchical regulatory network in maize endosperm.

Grain filling in maize (Zea mays) is intricately linked to cell development, involving the regulation of genes responsible for the biosynthesis of storage reserves (starch, proteins, and lipids) and phytohormones. However, the regulatory network coordinating these biological functions remains unclear. In this study, we identified 1744 high-confidence target genes co-regulated by the transcription factors (TFs) ZmNAC128 and ZmNAC130 (ZmNAC128/130) through chromatin immunoprecipitation sequencing coupled with RNA-seq analysis in the zmnac128/130 loss-of-function mutants. We further constructed a hierarchical regulatory network using DNA affinity purification sequencing analysis of downstream TFs regulated by ZmNAC128/130. In addition to target genes involved in the biosynthesis of starch and zeins, we discovered novel target genes of ZmNAC128/130 involved in the biosynthesis of lipids and indole-3-acetic acid (IAA). Consistently, the number of oil bodies, as well as the contents of triacylglycerol, and IAA were significantly reduced in zmnac128/130. The hierarchical regulatory network centered by ZmNAC128/130 revealed a significant overlap between the direct target genes of ZmNAC128/130 and their downstream TFs, particularly in regulating the biosynthesis of storage reserves and IAA. Our results indicated that the biosynthesis of storage reserves and IAA is coordinated by a multi-TFs hierarchical regulatory network in maize endosperm.

SWI2/SNF2 ATPase CHR2 remodels pri-miRNAs via Serrate to impede miRNA production.

Chromatin remodelling factors (CHRs) typically function to alter chromatin structure. CHRs also reside in ribonucleoprotein complexes, but little is known about their RNA-related functions. Here we show that CHR2 (also known as BRM), the ATPase subunit of the large switch/sucrose non-fermentable (SWI/SNF) complex, is a partner of the Microprocessor component Serrate (SE). CHR2 promotes the transcription of primary microRNA precursors (pri-miRNAs) while repressing miRNA accumulation in vivo. Direct interaction with SE is required for post-transcriptional inhibition of miRNA accumulation by CHR2 but not for its transcriptional activity. CHR2 can directly bind to and unwind pri-miRNAs and inhibit their processing, and this inhibition requires the remodelling and helicase activity of CHR2 in vitro and in vivo. Furthermore, the secondary structures of pri-miRNAs differed between wild-type Arabidopsis thaliana and chr2 mutants. We conclude that CHR2 accesses pri-miRNAs through SE and remodels their secondary structures, preventing downstream processing by DCL1 and HYL1. Our study uncovers pri-miRNAs as a substrate of CHR2, and an additional regulatory layer upstream of Microprocessor activity to control miRNA accumulation.

Expansion and improvement of ChinaMu by MuT-seq and chromosome-level assembly of the Mu-starter genome.

ChinaMu is the largest sequence-indexed Mutator (Mu) transposon insertional library in maize (Zea mays). In this study, we made significant improvements to the size and quality of the ChinaMu library. We developed a new Mu-tag isolation method Mu-Tn5-seq (MuT-seq). Compared to the previous method used by ChinaMu, MuT-seq recovered 1/3 more germinal insertions, while requiring only about 1/14 of the sequencing volume and 1/5 of the experimental time. Using MuT-seq, we identified 113,879 germinal insertions from 3,168 Mu-active F1 families. We also assembled a high-quality genome for the Mu-active line Mu-starter, which harbors the initial active MuDR element and was used as the pollen donor for the mutation population. Using the Mu-starter genome, we recovered 33,662 (15.6%) additional germinal insertions in 3,244 (7.4%) genes in the Mu-starter line. The Mu-starter genome also improved the assignment of 117,689 (54.5%) germinal insertions. The newly upgraded ChinaMu dataset currently contains 215,889 high-quality germinal insertions. These insertions cover 32,224 pan-genes in the Mu-starter and B73Ref5 genomes, including 23,006 (80.4%) core genes shared by the two genomes. As a test model, we investigated Mu insertions in the pentatricopeptide repeat (PPR) superfamily, discovering insertions for 92% (449/487) of PPR genes in ChinaMu, demonstrating the usefulness of ChinaMu as a functional genomics resource for maize.

A SnRK1-ZmRFWD3-Opaque2 Signaling Axis Regulates Diurnal Nitrogen Accumulation in Maize Seeds.

Zeins are the predominant storage proteins in maize (Zea mays) seeds, while Opaque2 (O2) is a master transcription factor for zein-encoding genes. How the activity of O2 is regulated and responds to external signals is yet largely unknown. Here, we show that the E3 ubiquitin ligase ZmRFWD3 interacts with O2 and positively regulates its activity by enhancing its nuclear localization. Ubiquitination of O2 enhances its interaction with maize importin1, the α-subunit of Importin-1 in maize, thus enhancing its nuclear localization ability. We further show that ZmRFWD3 can be phosphorylated by a Suc-responsive protein kinase, ZmSnRK1, which leads to its degradation. We demonstrated that the activity of O2 responds to Suc levels through the ZmSnRK1-ZmRFWD3-O2 signaling axis. Intriguingly, we found that Suc levels, as well as ZmRFWD3 levels and the cytonuclear distribution of O2, exhibit diurnal patterns in developing endosperm, leading to the diurnal transcription of O2-regulated zein genes. Loss of function in ZmRFWD3 disrupts the diurnal patterns of O2 cytonuclear distribution and zein biosynthesis, and consequently changes the C/N ratio in mature seeds. We therefore identify a SnRK1-ZmRFWD3-O2 signaling axis that transduces source-to-sink signals and coordinates C and N assimilation in developing maize seeds.

Multiple Quality Control Mechanisms in the ER and TGN Determine Subcellular Dynamics and Salt-Stress Tolerance Function of KORRIGAN1.

Among many glycoproteins within the plant secretory system, KORRIGAN1 (KOR1), a membrane-anchored endo-ß-1,4-glucanase involved in cellulose biosynthesis, provides a link between N-glycosylation, cell wall biosynthesis, and abiotic stress tolerance. After insertion into the endoplasmic reticulum, KOR1 cycles between the trans-Golgi network (TGN) and the plasma membrane (PM). From the TGN, the protein is targeted to growing cell plates during cell division. These processes are governed by multiple sequence motifs and also host genotypes. Here, we investigated the interaction and hierarchy of known and newly identified sorting signals in KOR1 and how they affect KOR1 transport at various stages in the secretory pathway. Conventional steady-state localization showed that structurally compromised KOR1 variants were directed to tonoplasts. In addition, a tandem fluorescent timer technology allowed for differential visualization of young versus aged KOR1 proteins, enabling the analysis of single-pass transport through the secretory pathway. Observations suggest the presence of multiple checkpoints/branches during KOR1 trafficking, where the destination is determined based on KOR1's sequence motifs and folding status. Moreover, growth analyses of dominant PM-confined KOR1-L48L49→A48A49 variants revealed the importance of active removal of KOR1 from the PM during salt stress, which otherwise interfered with stress acclimation.

Arabidopsis retains vertebrate-type telomerase accessory proteins via a plant-specific assembly.

The recent discovery of the bona-fide telomerase RNA (TR) from plants reveals conserved and unique secondary structure elements and the opportunity for new insight into the telomerase RNP. Here we examine how two highly conserved proteins previously implicated in Arabidopsis telomere maintenance, AtPOT1a and AtNAP57 (dyskerin), engage plant telomerase. We report that AtPOT1a associates with Arabidopsis telomerase via interaction with TERT. While loss of AtPOT1a does not impact AtTR stability, the templating domain is more accessible in pot1a mutants, supporting the conclusion that AtPOT1a stimulates telomerase activity but does not facilitate telomerase RNP assembly. We also show, that despite the absence of a canonical H/ACA binding motif within AtTR, dyskerin binds AtTR with high affinity and specificity in vitro via a plant specific three-way junction (TWJ). A core element of the TWJ is the P1a stem, which unites the 5' and 3' ends of AtTR. P1a is required for dyskerin-mediated stimulation of telomerase repeat addition processivity in vitro, and for AtTR accumulation and telomerase activity in vivo. The deployment of vertebrate-like accessory proteins and unique RNA structural elements by Arabidopsis telomerase provides a new platform for exploring telomerase biogenesis and evolution.

Accumulation of 22 kDa α-zein-mediated nonzein protein in protein body of maize endosperm.

Protein bodies (PBs), the major protein storage organelle in maize (Zea mays) endosperm, comprise zeins and numerous nonzein proteins (NZPs). Unlike zeins, how NZPs accumulate in PBs remains unclear. We characterized a maize miniature kernel mutant, mn*, that produces small kernels and is embryo-lethal. After cloning the Mn* locus, we determined that it encodes the mitochondrial 50S ribosomal protein L10 (mRPL10). MN* localized to mitochondria and PBs as an NZP; therefore, we renamed MN* Non-zein Protein 1 (NZP1). Like other mutations affecting mitochondrial proteins, mn* impaired mitochondrial function and morphology. To investigate its accumulation mechanism to PBs, we performed protein interaction assays between major zein proteins and NZP1, and found that NZP1 interacts with 22 kDa α-zein. Levels of NZP1 and 22 kDa α-zein in various opaque mutants were correlated. Furthermore, NZP1 accumulation in induced PBs depended on its interaction with 22 kDa α-zein. Comparative proteomic analysis of PBs between wild-type and opaque2 revealed additional NZPs. A new NZP with plastidial localization was also found to accumulate in induced PBs via interaction with 22 kDa α-zein. This study thus reveals a mechanism for accumulation of NZPs in PBs and suggests a potential application for the accumulation of foreign proteins in maize PBs.

The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans.

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3' long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

OsWUS promotes tiller bud growth by establishing weak apical dominance in rice.

Two branching strategies are exhibited in crops: enhanced apical dominance, as in maize; or weak apical dominance, as in rice. However, the underlying mechanism of weak apical dominance remains elusive. OsWUS, an ortholog of Arabidopsis WUSCHEL (WUS) in rice, is required for tiller development. In this study, we identified and functionally characterized a low-tillering mutant decreased culm number 1 (dc1) that resulted from loss-of-function of OsWUS. The dc1 tiller buds are viable but repressed by the main culm apex, leading to stronger apical dominance than that of the wild-type (WT). Auxin response is enhanced in the dc1 mutant, and knocking out the auxin action-associated gene ABERRANT SPIKELET AND PANICLE 1 (ASP1) de-repressed growth of the tiller buds in the dc1 mutant, suggesting that OsWUS and ASP1 are both involved in outgrowth of the rice tiller bud. Decapitation triggers higher contents of cytokinins in the shoot base of the dc1 mutant compared with those in the WT, and exogenous application of cytokinin is not sufficient for sustained growth of the dc1 tiller bud. Transcriptome analysis indicated that expression levels of transcription factors putatively bound by ORYZA SATIVA HOMEOBOX 1 (OSH1) are changed in response to decapitation and display a greater fold change in the dc1 mutant than that in the WT. Collectively, these findings reveal an important role of OsWUS in tiller bud growth by influencing apical dominance, and provide the basis for an improved understanding of tiller bud development in rice.

Maize kernel development.

Maize (Zea mays) is a leading cereal crop in the world. The maize kernel is the storage organ and the harvest portion of this crop and is closely related to its yield and quality. The development of maize kernel is initiated by the double fertilization event, leading to the formation of a diploid embryo and a triploid endosperm. The embryo and endosperm are then undergone independent developmental programs, resulting in a mature maize kernel which is comprised of a persistent endosperm, a large embryo, and a maternal pericarp. Due to the well-characterized morphogenesis and powerful genetics, maize kernel has long been an excellent model for the study of cereal kernel development. In recent years, with the release of the maize reference genome and the development of new genomic technologies, there has been an explosive expansion of new knowledge for maize kernel development. In this review, we overviewed recent progress in the study of maize kernel development, with an emphasis on genetic mapping of kernel traits, transcriptome analysis during kernel development, functional gene cloning of kernel mutants, and genetic engineering of kernel traits.

Maize endosperm development.

Recent breakthroughs in transcriptome analysis and gene characterization have provided valuable resources and information about the maize endosperm developmental program. The high temporal-resolution transcriptome analysis has yielded unprecedented access to information about the genetic control of seed development. Detailed spatial transcriptome analysis using laser-capture microdissection has revealed the expression patterns of specific populations of genes in the four major endosperm compartments: the basal endosperm transfer layer (BETL), aleurone layer (AL), starchy endosperm (SE), and embryo-surrounding region (ESR). Although the overall picture of the transcriptional regulatory network of endosperm development remains fragmentary, there have been some exciting advances, such as the identification of OPAQUE11 (O11) as a central hub of the maize endosperm regulatory network connecting endosperm development, nutrient metabolism, and stress responses, and the discovery that the endosperm adjacent to scutellum (EAS) serves as a dynamic interface for endosperm-embryo crosstalk. In addition, several genes that function in BETL development, AL differentiation, and the endosperm cell cycle have been identified, such as ZmSWEET4c, Thk1, and Dek15, respectively. Here, we focus on current advances in understanding the molecular factors involved in BETL, AL, SE, ESR, and EAS development, including the specific transcriptional regulatory networks that function in each compartment during endosperm development.

A Sequence-Indexed Mutator Insertional Library for Maize Functional Genomics Study.

Sequence-indexed insertional libraries are important resources for functional gene study in model plants. However, the maize (Zea mays) UniformMu library covers only 36% of the annotated maize genes. Here, we generated a new sequence-indexed maize Mutator insertional library named ChinaMu through high-throughput sequencing of enriched Mu-tagged sequences. A total of 2,581 Mu F2 lines were analyzed, and 311,924 nonredundant Mu insertion sites were obtained. Based on experimental validation, ChinaMu contains about 97,000 germinal Mu insertions, about twice as many as UniformMu. About two-thirds (66,565) of the insertions are high-quality germinal insertions (positive rate > 90%), 89.6% of which are located in genic regions. Furthermore, 45.7% (20,244) of the 44,300 annotated maize genes are effectively tagged and about two-thirds (13,425) of these genes harbor multiple insertions. We tested the utility of ChinaMu using pentatricopeptide repeat (PPR) genes. For published PPR genes with defective kernel phenotypes, 17 out of 20 were tagged, 11 of which had the previously reported mutant phenotype. For 16 unstudied PPR genes with both Mu insertions and defective kernel phenotypes, 6 contained insertions that cosegregated with the mutant phenotype. Our sequence-indexed Mu insertional library provides an important resource for functional genomics study in maize.

Maize pentatricopeptide repeat protein DEK53 is required for mitochondrial RNA editing at multiple sites and seed development.

Pentatricopeptide repeat (PPR) proteins were identified as site-specific recognition factors for RNA editing in plant mitochondria and plastids. In this study, we characterized maize (Zea mays) kernel mutant defective kernel 53 (dek53), which has an embryo lethal and collapsed endosperm phenotype. Dek53 encodes an E-subgroup PPR protein, which possesses a short PLS repeat region of only seven repeats. Subcellular localization analysis indicated that DEK53 is localized in the mitochondrion. Strand- and transcript-specific RNA-seq analysis showed that the dek53 mutation affected C-to-U RNA editing at more than 60 mitochondrial C targets. Biochemical analysis of mitochondrial protein complexes revealed a significant reduction in the assembly of mitochondrial complex III in dek53. Transmission electron microscopic examination showed severe morphological defects of mitochondria in dek53 endosperm cells. In addition, yeast two-hybrid and luciferase complementation imaging assays indicated that DEK53 can interact with the mitochondrion-targeted non-PPR RNA editing factor ZmMORF1, suggesting that DEK53 might be a functional component of the organellar RNA editosome.

KETCH1 imports HYL1 to nucleus for miRNA biogenesis in Arabidopsis.

MicroRNA (miRNA) is processed from primary transcripts with hairpin structures (pri-miRNAs) by microprocessors in the nucleus. How cytoplasmic-borne microprocessor components are transported into the nucleus to fulfill their functions remains poorly understood. Here, we report KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1) as a partner of hyponastic leaves 1 (HYL1) protein, a core component of microprocessor in Arabidopsis and functional counterpart of DGCR8/Pasha in animals. Null mutation of ketch1 is embryonic-lethal, whereas knockdown mutation of ketch1 caused morphological defects, reminiscent of mutants in the miRNA pathway. ketch1 knockdown mutation also substantially reduced miRNA accumulation, but did not alter nuclear-cytoplasmic shuttling of miRNAs. Rather, the mutation significantly reduced nuclear portion of HYL1 protein and correspondingly compromised the pri-miRNA processing in the nucleus. We propose that KETCH1 transports HYL1 from the cytoplasm to the nucleus to constitute functional microprocessor in Arabidopsis This study provides insight into the largely unknown nuclear-cytoplasmic trafficking process of miRNA biogenesis components through eukaryotes.

The splicing machinery promotes RNA-directed DNA methylation and transcriptional silencing in Arabidopsis.

DNA methylation in transposons and other DNA repeats is conserved in plants as well as in animals. In Arabidopsis thaliana, an RNA-directed DNA methylation (RdDM) pathway directs de novo DNA methylation. We performed a forward genetic screen for suppressors of the DNA demethylase mutant ros1 and identified a novel Zinc-finger and OCRE domain-containing Protein 1 (ZOP1) that promotes Pol IV-dependent siRNA accumulation, DNA methylation, and transcriptional silencing. Whole-genome methods disclosed the genome-wide effects of zop1 on Pol IV-dependent siRNA accumulation and DNA methylation, suggesting that ZOP1 has both RdDM-dependent and -independent roles in transcriptional silencing. We demonstrated that ZOP1 is a pre-mRNA splicing factor that associates with several typical components of the splicing machinery as well as with Pol II. Immunofluorescence assay revealed that ZOP1 overlaps with Cajal body and is partially colocalized with NRPE1 and DRM2. Moreover, we found that the other development-defective splicing mutants tested including mac3a3b, mos4, mos12 and mos14 show defects in RdDM and transcriptional silencing. We propose that the splicing machinery rather than specific splicing factors is involved in promoting RdDM and transcriptional silencing.

Two novel NAC transcription factors regulate gene expression and flowering time by associating with the histone demethylase JMJ14.

The histone demethylase JMJ14 catalyzes histone demethylation at lysine 4 of histone 3 and is involved in transcriptional repression and flowering time control in Arabidopsis. Here, we report that JMJ14 is physically associated with two previously uncharacterized NAC transcription factors, NAC050 and NAC052. The NAC050/052-RNAi plants and the CRISPR-CAS9-mediated nac050/052 double mutant plants show an early flowering phenotype, which is similar to the phenotype of jmj14, suggesting a functional association between JMJ14 and NAC050/052. RNA-seq data indicated that hundreds of common target genes are co-regulated by JMJ14 and NAC50/052. Our ChIP analysis demonstrated that JMJ14 and NAC050 directly bind to co-upregulated genes shared in jmj14 and NAC050/052-RNAi, thereby facilitating H3K4 demethylation and transcriptional repression. The NAC050/052 recognition DNA cis-element was identified by an electrophoretic mobility shift assay at the promoters of its target genes. Together, our study identifies two novel NAC transcription repressors and demonstrates that they are involved in transcriptional repression and flowering time control by associating with the histone demethylase JMJ14.

Folate polyglutamylation is involved in chromatin silencing by maintaining global DNA methylation and histone H3K9 dimethylation in Arabidopsis.

DNA methylation and repressive histone Histone3 Lysine9 (H3K9) dimethylation correlate with chromatin silencing in plants and mammals. To identify factors required for DNA methylation and H3K9 dimethylation, we screened for suppressors of the repressor of silencing1 (ros1) mutation, which causes silencing of the expression of the RD29A (RESPONSE TO DESSICATION 29A) promoter-driven luciferase transgene (RD29A-LUC) and the 35S promoter-driven NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) transgene (35S-NPTII). We identified the folylpolyglutamate synthetase FPGS1 and the known factor DECREASED DNA METHYLATION1 (DDM1). The fpgs1 and ddm1 mutations release the silencing of both RD29A-LUC and 35S-NPTII. Genome-wide analysis indicated that the fpgs1 mutation reduces DNA methylation and releases chromatin silencing at a genome-wide scale. The effect of fpgs1 on chromatin silencing is correlated with reduced levels of DNA methylation and H3K9 dimethylation. Supplementation of fpgs1 mutants with 5-formyltetrahydrofolate, a stable form of folate, rescues the defects in DNA methylation, histone H3K9 dimethylation, and chromatin silencing. The competitive inhibitor of methyltransferases, S-adenosylhomocysteine, is markedly upregulated in fpgs1, by which fpgs1 reduces S-adenosylmethionine accessibility to methyltransferases and accordingly affects DNA and histone methylation. These results suggest that FPGS1-mediated folate polyglutamylation is required for DNA methylation and H3K9 dimethylation through its function in one-carbon metabolism. Our study makes an important contribution to understanding the complex interplay among metabolism, development, and epigenetic regulation.

DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV.

DNA methylation is an important epigenetic mark in many eukaryotic organisms. De novo DNA methylation in plants can be achieved by the RNA-directed DNA methylation (RdDM) pathway, where the plant-specific DNA-dependent RNA polymerase IV (Pol IV) transcribes target sequences to initiate 24-nt siRNA production and action. The putative DNA binding protein DTF1/SHH1 of Arabidopsis has been shown to associate with Pol IV and is required for 24-nt siRNA accumulation and transcriptional silencing at several RdDM target loci. However, the extent and mechanism of DTF1 function in RdDM is unclear. We show here that DTF1 is necessary for the accumulation of the majority of Pol IV-dependent 24-nt siRNAs. It is also required for a large proportion of Pol IV-dependent de novo DNA methylation. Interestingly, there is a group of RdDM target loci where 24-nt siRNA accumulation but not DNA methylation is dependent on DTF1. DTF1 interacts directly with the chromatin remodeling protein CLASSY 1 (CLSY1), and both DTF1 and CLSY1 are associated in vivo with Pol IV but not Pol V, which functions downstream in the RdDM effector complex. DTF1 and DTF2 (a DTF1-like protein) contain a SAWADEE domain, which was found to bind specifically to histone H3 containing H3K9 methylation. Taken together, our results show that DTF1 is a core component of the RdDM pathway, and suggest that DTF1 interacts with CLSY1 to assist in the recruitment of Pol IV to RdDM target loci where H3K9 methylation may be an important feature. Our results also suggest the involvement of DTF1 in an important negative feedback mechanism for DNA methylation at some RdDM target loci.

A Pre-mRNA-splicing factor is required for RNA-directed DNA methylation in Arabidopsis.

Cytosine DNA methylation is a stable epigenetic mark that is frequently associated with the silencing of genes and transposable elements (TEs). In Arabidopsis, the establishment of DNA methylation is through the RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification and characterization of RDM16, a new factor in the RdDM pathway. Mutation of RDM16 reduced the DNA methylation levels and partially released the silencing of a reporter gene as well as some endogenous genomic loci in the DNA demethylase ros1-1 mutant background. The rdm16 mutant had morphological defects and was hypersensitive to salt stress and abscisic acid (ABA). Map-based cloning and complementation test led to the identification of RDM16, which encodes a pre-mRNA-splicing factor 3, a component of the U4/U6 snRNP. RNA-seq analysis showed that 308 intron retention events occurred in rdm16, confirming that RDM16 is involved in pre-mRNA splicing in planta. RNA-seq and mRNA expression analysis also revealed that the RDM16 mutation did not affect the pre-mRNA splicing of known RdDM genes, suggesting that RDM16 might be directly involved in RdDM. Small RNA expression analysis on loci showing RDM16-dependent DNA methylation suggested that unlike the previously reported putative splicing factor mutants, rdm16 did not affect small RNA levels; instead, the rdm16 mutation caused a decrease in the levels of Pol V transcripts. ChIP assays revealed that RDM16 was enriched at some Pol V target loci. Our results suggest that RDM16 regulates DNA methylation through influencing Pol V transcript levels. Finally, our genome-wide DNA methylation analysis indicated that RDM16 regulates the overall methylation of TEs and gene-surrounding regions, and preferentially targets Pol IV-dependent DNA methylation loci and the ROS1 target loci. Our work thus contributes to the understanding of RdDM and its interactions with active DNA demethylation.