In plants, 24 nucleotide long heterochromatic si
All eukaryotes possess three DNA‐dependent RNA polymerases, Pols I–III, while land plants possess two additional polymerases, Pol IV and Pol V. Derived through duplication of Pol II subunits, Pol IV produces 24‐nt short interfering RNAs that interact with Pol V transcripts to target
- Award ID(s):
- 1929678
- NSF-PAR ID:
- 10374785
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 111
- Issue:
- 3
- ISSN:
- 0960-7412
- Page Range / eLocation ID:
- p. 748-755
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Summary RNA s (het‐siRNA s) transcriptionally regulate gene expression byRNA ‐directedDNA methylation (RdDM ). The biogenesis of most het‐siRNA s depends on the plant‐specificRNA polymeraseIV (PolIV ), andARGONAUTE 4 (AGO 4) is a major het‐siRNA effector protein. Through genome‐wide analysis ofsRNA ‐seq data sets, we found thatAGO 4RNA s. The accumulation ofAGO 4RNA s also requires several factors known to participate in the effector portion of the RdDM pathway, includingRNA POLYMERASE V (POL V),DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM 2) andSAWADEE HOMEODOMAIN HOMOLOGUE 1 (SHH 1). Like manyAGO proteins,AGO 4 is an endonuclease that can ‘slice’RNA s. We found that a slicing‐defectiveAGO 4 was unable to fully recoverAGO 4‐RNA accumulation fromago4 mutant plants. Collectively, our data suggest thatAGO 4RNA s are secondary siRNA s dependent on the prior activity of the RdDM pathway at certain loci. -
more » « less
Summary Small RNAs trigger repressive DNA methylation at thousands of transposable elements in a process called RNA‐directed DNA methylation (RdDM). The molecular mechanism of RdDM is well characterized in Arabidopsis, yet the biological function remains unclear, as loss of RdDM in Arabidopsis causes no overt defects, even after generations of inbreeding. It is known that 24 nucleotide Pol IV‐dependent siRNAs, the hallmark of RdDM, are abundant in flowers and developing seeds, indicating that RdDM might be important during reproduction. Here we show that, unlike Arabidopsis, mutations in the Pol IV‐dependent small RNA pathway cause severe and specific reproductive defects in
Brassica rapa . High rates of abortion occur when seeds have RdDM mutant mothers, but not when they have mutant fathers. Although abortion occurs after fertilization, RdDM function is required in maternal somatic tissue, not in the female gametophyte or the developing zygote, suggesting that siRNAs from the maternal soma might function in filial tissues. We propose that recently outbreeding species such asB. rapa are key to understanding the role of RdDM during plant reproduction. -
more » « less
Summary As rapid changes in climate threaten global crop yields, an understanding of plant heat stress tolerance is increasingly relevant. Heat stress tolerance involves the coordinated action of many cellular processes and is particularly energy demanding. We acquired a knockout mutant and generated knockdown lines in
Arabidopsis thaliana of the d subunit of mitochondrial ATP synthase (gene name:ATPQ , AT3G52300, referred to hereafter asATPd ), a subunit of the peripheral stalk, and used these to investigate the phenotypic significance of this subunit in normal growth and heat stress tolerance. Homozygous knockout mutants forATPd could not be obtained due to gametophytic defects, while heterozygotes possess no visible phenotype. Therefore, we used RNA interference to create knockdown plant lines for further studies. Proteomic analysis and blue native gels revealed thatATPd downregulation impairs only subunits of the mitochondrial ATP synthase (complex V). Knockdown plants were more sensitive to heat stress, had abnormal leaf morphology, and were severely slow growing compared to wild type. These results indicate that ATPd plays a crucial role in proper function of the mitochondrial ATP synthase holoenzyme, which, when reduced, leads to wide‐ranging defects in energy‐demanding cellular processes. In knockdown plants, more hydrogen peroxide accumulated and mitochondrial dysfunction stimulon (MDS) genes were activated. These data establish the essential structural role of ATPd and support the importance of complex V in normal plant growth, and provide new information about its requirement for heat stress tolerance. -
more » « less
Across eukaryotes, gene regulation is manifested via chromatin states roughly distinguished as heterochromatin and euchromatin. The establishment, maintenance, and modulation of the chromatin states is mediated using several factors including chromatin modifiers. However, factors that avoid the intrusion of silencing signals into protein-coding genes are poorly understood. Here we show that a plant specific paralog of RNA polymerase (Pol) II, named Pol IV, is involved in avoidance of facultative heterochromatic marks in protein-coding genes, in addition to its well-established functions in silencing repeats and transposons. In its absence, H3K27 trimethylation (me3) mark intruded the protein-coding genes, more profoundly in genes embedded with repeats. In a subset of genes, spurious transcriptional activity resulted in small(s) RNA production, leading to post-transcriptional gene silencing. We show that such effects are significantly pronounced in rice, a plant with a larger genome with distributed heterochromatin compared with
Arabidopsis . Our results indicate the division of labor among plant-specific polymerases, not just in establishing effective silencing via sRNAs and DNA methylation but also in influencing chromatin boundaries. -
more » « less
Abstract In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without pre-existing DNA methylation. We find that Pol V follows and is dependent on the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of pre-existing DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.
