RNA-directed DNA methylation (RdDM) in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV)1,2 and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA3-7. The SU(VAR)3-9 homologs SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown8,9. Here we show that genome-wide Pol V association with chromatin redundantly requires, SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-adenosyl methionine (SAM) binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins8. SUVH2 and SUVH9 both contain SET- and RING-ASSOCIATED (SRA) domains capable of binding methylated DNA8, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH2 with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results suggest that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing.

SUMMARY RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nucleotide (nt) small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30-to-40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5’-adenine preference. P4RNAs exhibit transcription-start-sites similar to Pol II products, and are featured with 5’-monophosphates and 3’-misincorporated nucleotides. The 3’-misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a “one-precursor, one-siRNA” model for the biogenesis of 24-nt siRNAs in Arabidopsis.

Polycomb group (PcG) proteins form essential epigenetic memory systems for controlling gene expression during development in plants and animals. However, the mechanism of plant PcG protein functions remains poorly understood. Here, we probed the composition and function of plant Polycomb repressive complex 2 (PRC2). This work established the fact that all known plant PRC2 complexes contain MSI1, a homologue of Drosophila p55. While p55 is not essential for the in vitro enzymatic activity of PRC2, plant MSI1 was required for the functions of the EMBRYONIC FLOWER and the VERNALIZATION PRC2 complexes including trimethylation of histone H3 Lys27 (H3K27) at the target chromatin, as well as gene repression and establishment of competence to flower. We found that MSI1 serves to link PRC2 to LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), a protein that binds H3K27me3 in vitro and in vivo and is required for a functional plant PcG system. The LHP1-MSI1 interaction forms a positive feedback loop to recruit PRC2 to chromatin that carries H3K27me3. Consequently, this can provide a mechanism for the faithful inheritance of local epigenetic information through replication.

Starch contains phosphate covalently bound to the C6-position (70 to 80% of total bound phosphate) and the C3-position (20 to 30%) of the glucosyl residues of the amylopectin fraction. In plants, the transient phosphorylation of starch renders the granule surface more accessible to glucan hydrolyzing enzymes and is required for proper starch degradation. Phosphate also confers desired properties to starch-derived pastes for industrial applications. In Arabidopsis thaliana, the removal of phosphate by the glucan phosphatase Starch Excess4 (SEX4) is essential for starch breakdown. We identified a homolog of SEX4, LSF2 (Like Sex Four2), as a novel enzyme involved in starch metabolism in Arabidopsis chloroplasts. Unlike SEX4, LSF2 does not have a carbohydrate binding module. Nevertheless, it binds to starch and specifically hydrolyzes phosphate from the C3-position. As a consequence, lsf2 mutant starch has elevated levels of C3-bound phosphate. SEX4 can release phosphate from both the C6-and the C3-positions, resulting in partial functional overlap with LSF2. However, compared with sex4 single mutants, the lsf2 sex4 double mutants have a more severe starch-excess phenotype, impaired growth, and a further change in the proportion of C3-and C6-bound phosphate. These findings significantly advance our understanding of the metabolism of phosphate in starch and provide innovative options for tailoring novel starches with improved functionality for industry.

Import of nuclear-encoded precursor proteins from the cytosol is an essential step in chloroplast biogenesis that is mediated by protein translocon complexes at the inner and outer envelope membrane (TOC). Toc159 is thought to be the main receptor for photosynthetic proteins, but lacking a large-scale systems approach, this hypothesis has only been tested for a handful of photosynthetic and nonphotosynthetic proteins. To assess Toc159 precursor specificity, we quantitatively analyzed the accumulation of plastid proteins in two mutant lines deficient in this receptor. Parallel genomewide transcript profiling allowed us to discern the consequences of impaired protein import from systemic transcriptional responses that contribute to the loss of photosynthetic capacity. On this basis, we defined putative Toc159-independent and Toc159-dependent precursor proteins. Many photosynthetic proteins accumulate in Toc159-deficient plastids, and, surprisingly, several distinct metabolic pathways are negatively affected by Toc159 depletion. Lack of Toc159 furthermore affects several proteins that accumulate as unprocessed N-acetylated precursor proteins outside of plastids. Together, our data show an unexpected client protein promiscuity of Toc159 that requires a far more differentiated view of Toc159 receptor function and regulation of plastid protein import, in which cytosolic Met removal followed by N-terminal acetylation of precursors emerges as an additional regulatory step.

(A.M.S.) To uncover components of the mechanism that adjusts the rate of leaf starch degradation to the length of the night, we devised a screen for mutant Arabidopsis thaliana plants in which starch reserves are prematurely exhausted. The mutation in one such mutant, named early starvation1 (esv1), eliminates a previously uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a nonlinear fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a similar uncharacterized Arabidopsis protein (named Like ESV1 [LESV]) are located in the chloroplast stroma and are also bound into starch granules. The region of highest similarity between the two proteins contains a series of near-repeated motifs rich in tryptophan. Both proteins are conserved throughout starch-synthesizing organisms, from angiosperms and monocots to green algae. Analysis of transgenic plants lacking or overexpressing ESV1 or LESV, and of double mutants lacking ESV1 and another protein necessary for starch degradation, leads us to propose that these proteins function in the organization of the starch granule matrix. We argue that their misexpression affects starch degradation indirectly, by altering matrix organization and, thus, accessibility of starch polymers to starch-degrading enzymes.

Edited by Ulf-Ingo FlüggeKeywords: Protochlorophyllide FLU Glutamyl-tRNA reductase Arabidopsis a b s t r a c t Regulation of tetrapyrrole biosynthesis in higher plants has been attributed to negative feedback control. Two effectors of feedback inhibition have been identified, heme and the FLU protein. Inhibition by heme implicates the Fe-branch via regulation of the initial step of tetrapyrrole synthesis. In the present work a FLU-containing chloroplast membrane complex was identified, that besides FLU comprises the four enzymes catalyzing the final steps of chlorophyll synthesis. The results support the notion that FLU links chlorophyll synthesis and the target of feedback control, glutamyl-tRNA reductase, thereby allowing also the Mg-branch to control the initial step of tetrapyrrole synthesis. Structured summary of protein interactions:FLU, CHL27, PORB, PORC and CHLP physically interact by blue native page (View interaction) FLU physically interacts with CHL27, PORB, PORC, CHLP and GluTR by anti tag co-immunoprecipitation (View interaction) FLU physically interacts with CHL27 by anti bait co-immunoprecipitation (View Interaction: 1, 2) FLU physically interacts with CHL27 by anti tag co-immunoprecipitation (View interaction) FLU physically interacts with CHL27, PORB, PORC and CHLP by anti tag co-immunoprecipitation (View interaction)

Epigenetic gene silencing is of central importance to maintain genome integrity and is mediated by an elaborate interplay between DNA methylation, histone posttranslational modifications, and chromatin remodeling complexes. DNA methylation and repressive histone marks usually correlate with transcriptionally silent heterochromatin, however there are exceptions to this relationship. In Arabidopsis, mutation of Morpheus Molecule 1 (MOM1) causes transcriptional derepression of heterochromatin independently of changes in DNA methylation. More recently, two Arabidopsis homologues of mouse microrchidia (MORC) genes have also been implicated in gene silencing and heterochromatin condensation without altering genome-wide DNA methylation patterns. In this study, we show that Arabidopsis microrchidia (AtMORC6) physically interacts with AtMORC1 and with its close homologue, AtMORC2, in two mutually exclusive protein complexes. RNA-sequencing analyses of high-order mutants indicate that AtMORC1 and AtMORC2 act redundantly to repress a common set of loci. We also examined genetic interactions between AtMORC6 and MOM1 pathways. Although AtMORC6 and MOM1 control the silencing of a very similar set of genomic loci, we observed synergistic transcriptional regulation in the mom1/atmorc6 double mutant, suggesting that these epigenetic regulators act mainly by different silencing mechanisms.epigenetics | plant biology