Cytoplasmic assembly and selective nuclear import of Arabidopsis Argonaute4/siRNA complexes

In plants, DNA methylation can be mediated by a class of Argonaute4 (AGO4)-associated heterochromatic siRNAs (hc-siRNAs), through a pathway termed RNA-directed DNA methylation (RdDM). It has been thought that RdDM is solely a nuclear process, as both the biogenesis and functioning of hc-siRNAs take place in the nucleus. In this study, we unexpectedly found that hc-siRNAs are predominantly present in the cytoplasm. We demonstrated that AGO4 is loaded with hc-siRNAs in the cytoplasm and the formation of mature AGO4/siRNA complexes requires HSP90 and the cleavage activity of AGO4. Intriguingly, siRNA binding facilitates the redistribution of AGO4 into the nucleus, likely through inducing conformational change that leads to the exposure of the nuclear localization signal (NLS). Our findings reveal an unsuspected cytoplasmic step in the RdDM pathway. We propose that selective nuclear import of mature AGO4/siRNA complexes is a key regulatory point prior to the effector stage of RdDM.     

Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA‐directed DNA methylation

Argonaute (AGO) family proteins are conserved key components of small RNA‐induced silencing pathways. In the RNA‐directed DNA methylation (RdDM) pathway in Arabidopsis, AGO6 is generally considered to be redundant with AGO4. In this report, our comprehensive, genomewide analyses of AGO4‐ and AGO6‐dependent DNA methylation revealed that redundancy is unexpectedly negligible in the genetic interactions between AGO4 and AGO6. Immunofluorescence revealed that AGO4 and AGO6 differ in their subnuclear co‐localization with RNA polymerases required for RdDM. Pol II and AGO6 are absent from perinucleolar foci, where Pol V and AGO4 are co‐localized. In the nucleoplasm, AGO4 displays a strong co‐localization with Pol II, whereas AGO6 co‐localizes with Pol V. These patterns suggest that RdDM is mediated by distinct, spatially regulated combinations of AGO proteins and RNA polymerases. Consistently, Pol II physically interacts with AGO4 but not AGO6, and the levels of Pol V‐dependent scaffold RNAs and Pol V chromatin occupancy are strongly correlated with AGO6 but not AGO4. Our results suggest that AGO4 and AGO6 mainly act sequentially in mediating small RNA‐directed DNA methylation.     

Identification of genes required for de novo DNA methylation in Arabidopsis

De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1, and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.     

The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes

Key message

MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33.

Abstract

Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1–5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

     

Multi‐omics analyses reveal epigenomics basis for cotton somatic embryogenesis through successive regeneration acclimation process

Plant regeneration via somatic embryogenesis is time‐consuming and highly genotype‐dependent. The plant somatic embryogenesis process provokes many epigenetics changes including DNA methylation and histone modification. Recently, an elite cotton Jin668, with an extremely high regeneration ability, was developed from its maternal inbred Y668 cultivar using a Successive Regeneration Acclimation (SRA) strategy. To reveal the underlying mechanism of SRA, we carried out a genome‐wide single‐base resolution methylation analysis for nonembryogenic calluses (NECs), ECs, somatic embryos (SEs) during the somatic embryogenesis procedure and the leaves of regenerated offspring plants. Jin668 (R4) regenerated plants were CHH hypomethylated compared with the R0 regenerated plants of SRA process. The increase in CHH methylation from NEC to EC was demonstrated to be associated with the RNA‐dependent DNA methylation (RdDM) and the H3K9me2‐dependent pathway. Intriguingly, the hypomethylated CHH differentially methylated regions (DMRs) of promoter activated some hormone‐related and WUSCHEL‐related homeobox genes during the somatic embryogenesis process. Inhibiting DNA methylation using zebularine treatment in NEC increased the number of embryos. Our multi‐omics data provide new insights into the dynamics of DNA methylation during the plant tissue culture and regenerated offspring plants. This study also reveals that induced hypomethylation (SRA) may facilitate the higher plant regeneration ability and optimize maternal genetic cultivar.