The dicot homolog of maize PPR103 carries a C-terminal DYW domain and may have a role in C-to-U editing of some chloroplast RNA transcripts.

In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain. IPI1/emb175/PPR103 is a nuclear gene encoding a PLS-type PPR protein essential for survival in Arabidopsis thaliana and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and Nicotiana IPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing in N. benthamiana to gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in the Nicotiana and Arabidopsis IPI1 orthologs. Virus-induced gene silencing of NbIPI1 led to defects in chloroplast ribosomal RNA processing and changes to stability of rpl16 transcripts, revealing conserved function with its maize ortholog. NbIPI1-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing in N. benthamiana chloroplasts.

The dicot homolog of maize PPR103 carries a C-terminal DYW domain and is required for C-to-U editing of chloroplast RNA transcripts.

In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain. IPI1/emb175/PPR103 is a nuclear gene encoding a PLS-type PPR protein essential for survival in Arabidopsis thaliana and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and Nicotiana IPI1 homologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. We examined the function of ISE2 and IPI1 in chloroplast RNA processing in N. benthamiana. A combination of deep sequencing and Sanger sequencing revealed C-to-U editing at 41 sites in 18 transcripts, with 34 sites conserved in the closely related N. tabacum. Virus induced gene silencing of NbISE2 or NbIPI1 led to defective C-to-U revealed that they have overlapping roles at editing a site in the rpoB transcript but have distinct roles in editing other transcripts. This finding contrasts with maize ppr103 mutants that showed no defects in editing. The results indicate that NbISE2 and NbIPI1 are important for C-to-U editing in N. benthamiana chloroplasts, and they may function in a complex to edit specific sites while having antagonistic effects on editing others. That NbIPI1, carrying a DYW domain, is involved in organelle C-to-U RNA editing supports previous work showing that this domain catalyzes RNA editing.

Chloroplast-to-nucleus retrograde signalling controls intercellular trafficking via plasmodesmata formation.

The signalling pathways that regulate intercellular trafficking via plasmodesmata (PD) remain largely unknown. Analyses of mutants with defects in intercellular trafficking led to the hypothesis that chloroplasts are important for controlling PD, probably by retrograde signalling to the nucleus to regulate expression of genes that influence PD formation and function, an idea encapsulated in the organelle-nucleus-PD signalling (ONPS) hypothesis. ONPS is supported by findings that point to chloroplast redox state as also modulating PD. Here, we have attempted to further elucidate details of ONPS. Through reverse genetics, expression of select nucleus-encoded genes with known or predicted roles in chloroplast gene expression was knocked down, and the effects on intercellular trafficking were then assessed. Silencing most genes resulted in chlorosis, and the expression of several photosynthesis and tetrapyrrole biosynthesis associated nuclear genes was repressed in all silenced plants. PD-mediated intercellular trafficking was changed in the silenced plants, consistent with predictions of the ONPS hypothesis. One striking observation, best exemplified by silencing the PNPase homologues, was that the degree of chlorosis of silenced leaves was not correlated with the capacity for intercellular trafficking. Finally, we measured the distribution of PD in silenced leaves and found that intercellular trafficking was positively correlated with the numbers of PD. Together, these results not only provide further support for ONPS but also point to a genetic mechanism for PD formation, clarifying a longstanding question about PD and intercellular trafficking. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

RNA on the move: The plasmodesmata perspective.

It is now widely accepted that plant RNAs can have effects at sites far away from their sites of synthesis. Cellular mRNA transcripts, endogenous small RNAs and defense-related small RNAs all move from cell to cell via plasmodesmata (PD), and may even move long distances in the phloem. Despite their small size, PD have complicated substructures, and the area of the pore available for RNA trafficking can be remarkably small. The intent of this review is to bring into focus the role of PD in cell-to-cell and long distance communication in plants. We consider how cellular RNAs could move through the cell to the PD and thence through PD. The protein composition of PD and the possible roles of PD proteins in RNA trafficking are also discussed. Recent evidence for RNA metabolism in organelles acting as a factor in controlling PD flux is also presented, highlighting new aspects of plant intra- and intercellular communication. It is clear that while the phenomenon of RNA mobility is common and essential, many questions remain, and these have been highlighted throughout this review.

The chloroplast RNA helicase ISE2 is required for multiple chloroplast RNA processing steps in Arabidopsis thaliana.

INCREASED SIZE EXCLUSION LIMIT2 (ISE2) is a chloroplast-localized RNA helicase that is indispensable for proper plant development. Chloroplasts in leaves with reduced ISE2 expression have previously been shown to exhibit reduced thylakoid contents and increased stromal volume, indicative of defective development. It has recently been reported that ISE2 is required for the splicing of group II introns from chloroplast transcripts. The current study extends these findings, and presents evidence for ISE2's role in multiple aspects of chloroplast RNA processing beyond group II intron splicing. Loss of ISE2 from Arabidopsis thaliana leaves resulted in defects in C-to-U RNA editing, altered accumulation of chloroplast transcripts and chloroplast-encoded proteins, and defective processing of chloroplast ribosomal RNAs. Potential ISE2 substrates were identified by RNA immunoprecipitation followed by next-generation sequencing (RIP-seq), and the diversity of RNA species identified supports ISE2's involvement in multiple aspects of chloroplast RNA metabolism. Comprehensive phylogenetic analyses revealed that ISE2 is a non-canonical Ski2-like RNA helicase that represents a separate sub-clade unique to green photosynthetic organisms, consistent with its function as an essential protein. Thus ISE2's evolutionary conservation may be explained by its numerous roles in regulating chloroplast gene expression.