Chloroplast ribonucleoproteins - stabilizing chloroplast RNA pools during acclimation responses
Chloroplast ribonucleoproteins are unique in their abundance, ability to bind multiple RNAs and in their responsiveness to environmental signals like cold and light. We have demonstrated that mem-bers of this family are key modulators of RNA stability and are required for plant acclimation and survival at low temperatures (8°C). We propose to study the mechanism how cpRNPs mediate cold resistance in Arabidopsis by a combination of genetic, biochemical and systems approaches. In a second line of experiments, we will contribute to a joint effort to model tobacco chloroplast gene expression by quantifying cpRNP RNA ligands and by quantifying chloroplast RNA processing.
Analyses of the role of chloroplast ribonucleoproteins for plant acclimation
Left: Immunofluorescence microscopy localizes cpRNPs into discrete suborganellar domains.
Top: cpRNP mutants do not display phenotypes under normal growth conditions, but show chlorophyll deficiencies during cold acclimation (Kupsch et al. Plant Cell 2012).
Bottom: We are interested in understanding, how chloroplast gene expression changes are mediated by cpRNPs during acclimation to temperature and light changes. We focus on RNA maturation, storage and turn-over as a function of the presence of cpRNPs.
CP33A associates with mRNAs released after ribosome knock-off.
A) RIP-Chip of CP33A was carried out either with whole-plant extracts treated with puromycin or with mock-treated extracts. Plots with and without treatment were normalized to the median of all enrichment values of all probes on the microarray representing 16S and 23S rRNA. Note the increase of specific sections of the transcriptome in precipitates from puromycin-treated chloroplasts.
B) Dot-blot analysis of psbD mRNA co-precipitated with CP33A. Immunoprecipitations were carried out using either the CP33A antibody or non-specific IgGs as a negative control. RNA isolated from pellet (P) and supernatant (S) fraction was dot-blotted onto a nylon membrane, visualized with methylene blue (left) and hybridized with a psbD-specific, radiolabelled probe (phosphoimager scan shown on right). Please note that the ratio of pellet signal to supernatant signal shifts towards bound (pellet) RNAs after puromycin treatment (+Puro).