Plant polymerase IV sensitizes chromatin through histone modifications to preclude spread of silencing into protein-coding domains.

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.

Degradome comparison between wild and cultivated rice identifies differential targeting by miRNAs.

BACKGROUND: Small non-coding (s)RNAs are involved in the negative regulation of gene expression, playing critical roles in genome integrity, development and metabolic pathways. Targeting of RNAs by ribonucleoprotein complexes of sRNAs bound to Argonaute (AGO) proteins results in cleaved RNAs having precise and predictable 5` ends. While tools to study sliced bits of RNAs to confirm the efficiency of sRNA-mediated regulation are available, they are sub-optimal. In this study, we provide an improvised version of a tool with better efficiency to accurately validate sRNA targets. RESULTS: Here, we improvised the CleaveLand tool to identify additional micro (mi)RNA targets that belong to the same family and also other targets within a specified free energy cut-off. These additional targets were otherwise excluded during the default run. We employed these tools to understand the sRNA targeting efficiency in wild and cultivated rice, sequenced degradome from two rice lines, O. nivara and O. sativa indica Pusa Basmati-1 and analyzed variations in sRNA targeting. Our results indicate the existence of multiple miRNA-mediated targeting differences between domesticated and wild species. For example, Os5NG4 was targeted only in wild rice that might be responsible for the poor secondary wall formation when compared to cultivated rice. We also identified differential mRNA targets of secondary sRNAs that were generated after miRNA-mediated cleavage of primary targets. CONCLUSIONS: We identified many differentially targeted mRNAs between wild and domesticated rice lines. In addition to providing a step-wise guide to generate and analyze degradome datasets, we showed how domestication altered sRNA-mediated cascade silencing during the evolution of indica rice.

Cultivar-specific miRNA-mediated RNA silencing in grapes.

MAIN CONCLUSION: In-depth comparative degradome analysis of two domesticated grape cultivars with diverse secondary metabolite accumulation reveals differential miRNA-mediated targeting. Small (s)RNAs such as micro(mi)RNAs and secondary small interfering (si) often work as negative switches of gene expression. In plants, it is well known that miRNAs target and cleave mRNAs that have high sequence complementarity. However, it is not known if there are variations in miRNA-mediated targeting between subspecies and cultivars that have been subjected to vast genetic modifications through breeding and other selections. Here, we have used PAREsnip2 tool for analysis of degradome datasets derived from two contrasting domesticated grape cultivars having varied fruit color, habit and leaf shape. We identified several interesting variations in sRNA targeting using degradome and 5'RACE analysis between two contrasting grape cultivars that was further correlated using RNA-seq analysis. Several of the differences we identified are associated with secondary metabolic pathways. We propose possible means by which sRNAs might contribute to diversity in secondary metabolites and other development pathways between two domesticated cultivars of grapes.

Double DJ-1 domain containing Arabidopsis DJ-1D is a robust macromolecule deglycase.

Plants, being sessile, are prone to genotoxin-induced macromolecule damage. Among the inevitable damaging agents are reactive carbonyls that induce glycation of DNA, RNA and proteins to result in the build-up of advanced glycated end-products. However, it is unclear how plants repair glycated macromolecules. DJ-1/PARK7 members are a highly conserved family of moonlighting proteins having double domains in higher plants and single domains in other phyla. Here we show that Arabidopsis DJ-1D offers robust tolerance to endogenous and exogenous stresses through its ability to repair glycated DNA, RNA and proteins. DJ-1D also reduced the formation of reactive carbonyls through its efficient methylglyoxalase activity. Strikingly, full-length double domain-containing DJ-1D suppressed the formation of advanced glycated end-products in yeast and plants. DJ-1D also efficiently repaired glycated nucleic acids and nucleotides in vitro and mitochondrial DNA in vivo under stress, indicating the existence of a new DNA repair pathway in plants. We propose that multi-stress responding plant DJ-1 members, often present in multiple copies among plants, probably contributed to the adaptation to a variety of endogenous and exogenous stresses.

Rice-specific Argonaute 17 controls reproductive growth and yield-associated phenotypes.

KEY MESSAGE: This manuscript describes the functions of an Argonaute protein named AGO17 in rice. AGO17 is required for the development of rice reproductive tissues. Argonaute (AGO) proteins are a well-conserved multigene family of regulators mediating gene silencing across eukaryotes. Monocot plants have additional members of AGO, the functions of which are poorly understood. Among the non-dicot AGO1 clade members in monocots, AGO17 expresses highly in reproductive tissues. Here we show that overexpression of Oryza sativa indica AGO17 in rice resulted in robust growth and increased yield, whereas its silencing resulted in reduced panicle length, less fertility, and poor growth. Small (s)RNA transcriptome analysis revealed misregulation of several miRNAs and other categories of sRNAs in silenced and overexpression lines, in agreement with its likely competition with other AGO1 clade members. Targets of differentially expressed miRNAs included previously unreported target RNAs coding for proteins involved in development, phase transition, and transport. Our results indicate a distinctive role for OsAGO17 in rice reproductive development that could be harnessed to improve yield.

Major Domestication-Related Phenotypes in Indica Rice Are Due to Loss of miRNA-Mediated Laccase Silencing.

Domestication of rice (Oryza sativa) included conversion of perennial wild species with few seeds to short plants that produced abundant seeds. Most domestication-associated changes were due to variations in transcription factors and other key proteins such as enzymes. Here, we show that multiple yield-related traits associated with indica rice domestication are linked to micro (mi) RNA-mediated regulation. Analysis of small (s) RNA data sets from cultivated indica rice lines, a few landraces, and two wild relatives of rice revealed the presence of abundant 22-nucleotide (nt) reads in wild relatives that mapped to miR397 precursors. miR397 was expressed at very high levels in wild relatives and at negligible levels in high-yielding cultivated lines. In its genera-specific form of 22-nt, miR397 targeted mRNAs encoding laccases that decayed and induced robust secondary cascade silencing in wild species that required RNA-dependent RNA polymerase 6. In wild species of rice, reduced expression of laccases resulted in low lignification. As expected, overexpression of miR397 induced de-domestication phenotypes. At least 26 uncharacterized QTLs previously implicated in rice yield overlapped with laccases and miR397 genes. These results suggest that miRNAs contribute to rice domestication-associated phenotypes.

miR828 and miR858 regulate VvMYB114 to promote anthocyanin and flavonol accumulation in grapes.

MicroRNAs are a class of non-coding small RNAs involved in the negative regulation of gene expression, which play critical roles in developmental and metabolic pathways. Studies in several plants have identified a few microRNAs and other small RNAs that target regulators of the phenylpropanoid metabolic pathway called the MYB transcription factors. However, it is not well understood how sRNA-mediated regulation of MYBs influences the accumulation of specific secondary metabolites. Using sRNA sequencing, degradome analysis, mRNA sequencing, and proteomic analysis, we establish that grape lines with high anthocyanin content express two MYB-targeting microRNAs abundantly, resulting in the differential expression of specific MYB proteins. miR828 and miR858 target coding sequences of specific helix motifs in the mRNA sequences of MYB proteins. Targeting by miR828 caused MYB RNA decay and the production of a cascade of secondary siRNAs that depend on RNA-dependent RNA polymerase 6. MYB suppression and cascade silencing was more robust in grape lines with high anthocyanin content than in a flavonol-rich grape line. We establish that microRNA-mediated silencing targeted the repressor class of MYBs to promote anthocyanin biosynthesis in grape lines with high anthocyanins. We propose that this process regulates the expression of appropriate MYBs in grape lines to produce specific secondary metabolites.

Loss of function of Oryza sativa Argonaute 18 induces male sterility and reduction in phased small RNAs.

KEY MESSAGE: In this manuscript, we show that Oryza sativa indica Argonaute protein AGO18 is required for male gametophyte development likely to through a small RNA-mediated mechanism. Monocots have evolved unique gene silencing pathways due to the presence of unique members of Dicer-like and Argonaute (AGO) family members. Among the monocot AGO homologs, AGO18 occupies a unique position. Previous reports have implicated this protein in viral resistance as well as in gametogenesis, likely through its competition with AGO1 clade members for micro(mi)RNAs and other small (s)RNAs. Although expression of rice AGO18 in specific stages of male gametogenesis has been documented, its major functions in plant development remain poorly understood. Here, we show that Oryza sativa indica AGO18 is involved in male gametophyte development. Knockdown (KD) of AGO18 in transgenic rice lines resulted in stunted plants that are male sterile, whereas their carpels were functional. Transcriptome analysis revealed downregulation of several pollen development-associated genes in KD lines. sRNA sequencing in vegetative and reproductive tissues of KD lines indicated reduction of miRNAs and phased secondary sRNAs implicated in male gametophyte development. Our results indicate a distinct role for rice AGO18 in male fertility.

Extraction and Purification of Laccases from Rice Stems.

Laccases are found in cell walls of plants in very low amounts. This protocol provides an efficient method to purify laccases from rice stems. The method involves three steps: 1) Isolation of total protein from rice stems using buffers with high salt concentration to extract protein from cell walls; 2) Purification of laccases using concanavalin-A beads; and, 3) In-gel staining of laccases with 4-hydroxyindole. Concanavalin-A specifically binds to internal or non-reducing terminal α-D-mannosyl and α-D-glucosyl groups found in glycoproteins and glycolipids. Laccases being glycoproteins binds to concanavalin-A during purification process and eluted with mannose.