Whirly1 in chloroplasts associates with intron containing RNAs and rarely co-localizes with nucleoids

Abstract

The nucleic acid binding protein Whirly1 of barley has been located to both chloroplasts and the nucleus of the same cell. Immunogold labelling furthermore showed that in vivo Whirly1 does not strictly co-localize with DNA in chloroplasts, while it is closely associated with DNA in the nucleus. High-resolution imaging of Whirly1-GFP and PEND-RFP fusion proteins revealed that only a minor part of Whirly1 co-localizes with nucleoids. The co-localization with nucleoids is in accordance with the detection of Whirly1 in a conventionally prepared fraction of the transcriptionally active chromosome (TAC). By further purification and enrichment of transcriptional activity Whirly1, however, was lost from the TAC fraction. Knockdown of Whirly1 in transgenic barley plants had neither impact on transcription of selected protein coding genes nor on genes coding for ribosomal RNAs or tRNAs. The results of RIP-chip experiments showed that barley Whirly1 as its maize orthologue associates with a set of intron containing plastid RNAs. Taken together, the results suggest that plastid-located Whirly1 functions primarily in RNA metabolism rather than as a DNA binding protein.

New insights into plastid nucleoid structure and functionality

“…In fact, sizes and positions of different nucleoid sub-populations could very well reflect the different requirements for enzymatic activities within the plastid nucleoids during plastid biogenesis. First support for this comes from co-localization studies of different plastid nucleoid-associated proteins (ptNAPs) (Melonek et al 2010). …”

“…Another protein possibly involved in anchoring of plastid nucleoids to thylakoid membranes is the tetratricopeptide-containing chloroplast protein of 34 kDa (TCP34), which was shown to stably associate with thylakoid membranes (Weber et al 2006). Immunological analysis revealed that the protein is a TAC component (Weber et al 2006;Melonek et al 2010). The recombinant TCP34 protein showed specific binding to chloroplast DNA isolated from spinach (Weber et al 2006).…”

“…Considering the tight association of nucleoids and thylakoids, a direct perception of redox changes in the photosynthetic apparatus by redox sensitive proteins in the nucleoids as also reported for a soluble RNA polymerase preparation from chloroplasts (Schröter et al 2010) must be expected. Sato and Ohta 2001) Unknown (Terasawa and Sato 2009) MFP1 At3g16000 Anchorage to thylakoid membranes (Jeong et al 2003) Chromatin attachment to the nuclear envelope (Meier et al 1996) Whirly1 At1g14410 SsDNA and RNA binding; recombination (Marechal and Brisson 2010;Melonek et al 2010;Pfalz et al 2006;Prikryl et al 2008) Transcription factor and telomere homeostasis (Desveaux et al 2000;Yoo et al 2007) SWIB-4 At3g03590 Unknown (Melonek et al 2012) Component of SWI/SNF chromatin remodeling complexes (Melonek et al 2012) pTAC12/ HEMERA/ PAP5 At2g34640 Component of the plastid transcriptional apparatus, subunit of plastid-encoded RNA-polymerase complex (Gao et al 2011;Pfalz et al 2006;Steiner et al 2011) Phytochrome signaling (Chen et al 2010) SIB1 ? 2 a At3g56710…”

The Single-Stranded DNA-Binding Protein WHIRLY1 Represses WRKY53 Expression and Delays Leaf Senescence in a Developmental Stage-Dependent Manner in Arabidopsis

“…In fact, it was proposed that plastidic WHY1 in maize (Zea mays) and barley both have functions in RNA processing and that the plastidic WHY proteins in Arabidopsis have a function as antirecombination proteins that could be involved in maintaining plastome DNA stability (Maréchal et al, 2008(Maréchal et al, , 2009Prikryl et al, 2008;Cappadocia et al, 2010;Maréchal and Brisson, 2010;Melonek et al, 2010).…”

The functions of WHIRLY1 and REDOX-RESPONSIVE TRANSCRIPTION FACTOR 1 in cross tolerance responses in plants: a hypothesis

“…Here, we propose that WHIRLY1 is involved in the perception of redox changes in the photosynthetic apparatus and in an SA-dependent transduction of this information to the nucleus. Biochemical studies have shown that chloroplast WHIRLY1 is present in nucleoid fractions [65,66]. Moreover, immunogold-labelling studies have shown that WHIRLY1 is closely associated with thylakoid membranes [59], as shown in figure 3.…”