Chloroplast development and genomes uncoupled signaling are independent of the RNA-directed DNA methylation pathway

Abstract

The Arabidopsis genome is methylated in CG and non-CG (CHG, and CHH in which H stands for A, T, or C) sequence contexts. DNA methylation has been suggested to be critical for seed development, and CHH methylation patterns change during stratification and germination. In plants, CHH methylation occurs mainly through the RNA-directed DNA methylation (RdDM) pathway. To test for an involvement of the RdDM pathway in chloroplast development, we analyzed seedling greening and the maximum quantum yield of photosystem II (Fv/Fm) in Arabidopsis thaliana seedlings perturbed in components of that pathway. Neither seedling greening nor Fv/Fm in seedlings and adult plants were affected in this comprehensive set of mutants, indicating that alterations in the RdDM pathway do not affect chloroplast development. Application of inhibitors like lincomycin or norflurazon inhibits greening of seedlings and represses the expression of photosynthesis-related genes including LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN1.2 (LHCB1.2) in the nucleus. Our results indicate that the LHCB1.2 promoter is poorly methylated under both control conditions and after inhibitor treatment. Therefore no correlation between LHCB1.2 mRNA transcription and methylation changes of the LHCB1.2 promoter could be established. Moreover, we conclude that perturbations in the RdDM pathway do not interfere with gun signaling.

The RNA‐binding protein RBP45D of Arabidopsis promotes transgene silencing and flowering time

“…The function of RBP45D in silencing could be indirect; for example, it might bind to or alter expression of mRNAs for proteins involved in silencing. We therefore assessed the degree of overlap between the ‘up’ and ‘down’ gene lists and a list of genes whose products are involved in epigenetic processes (Wang et al., 2020b). No overlap was found with the ‘down’ gene set, while 11 genes from the ‘up’ gene set were identified, including genes for DCL3, DCL4, RDR6 and NRPB1 (the largest subunit of RNA polymerase II), but increased activity of these in the rls6 mutant would not explain its silencing phenotype.…”

“…The methylation status of selected DNA sites was determined using Chop‐PCR essentially as described before (Wang et al., 2020b). To discriminate between the endogenous and transgenic LHCB1.2 promoter, primer positions were chosen as depicted in Figure S6.…”

Viroids as a Tool to Study RNA-Directed DNA Methylation in Plants

“…Finally, another interesting topic that has not been investigated yet, is whether members of the Avsunviroidae can also trigger RdDM. Of course, these viroids replicate in the chloroplast, where no RNAi/RdDM occurs [ 75 ]. However, when they enter the plant cell, it is thought that they are first transported into the nucleus before being targeted to the chloroplasts [ 76 ].…”

The RNA-binding protein RBP45D of Arabidopis plays a role in epigenetic control of flowering time and DCL3-independent RNA-directed DNA methylation

“…Hence, the function of RBP45D in silencing could be indirect; e.g., it might bind to or alter expression of mRNAs for proteins involved in silencing. We therefore assessed the degree of overlap between the "up" and "down" gene lists and a list of genes whose products are involved in epigenetic processes (Wang et al, 2020b). No overlap was found with the "down" gene set, while eleven genes from the "up" gene set were identified (Figure S3C), including genes for DCL3, DCL4, RDR6 and NRPB1 (the largest subunit of RNA polymerase II), but increased activity of these in the rls6 mutant would not explain its silencing phenotype.…”

“…Homozygous lines were identified using gene-specific primers and T-DNA border primers designed with the aid of the website http://signal.salk.edu/isectprimers.html. Details of T-DNA insertion sites in lines associated with RNA-directed DNA methylation can be found in (Wang et al, 2020b). Seeds were surfacesterilized with 0.6% (v/v) sodium hypochlorite solution containing 0.01% (v/v) Triton X-100 for 10 min, and then washed four times with ddH2O.…”