Abstract
In higher plant chloroplasts the accumulation of plastid-encoded mRNAs during leaf maturation is regulated via gene-specific mRNA stabilization. The half-lives of chloroplast RNAs are specifically affected by magnesium ions. psbA mRNA (D1 protein of photosystem II), rbcL mRNA (large subunit of ribulose-1,5-bisphosphate carboxylase), 16 S rRNA, and tRNA(His) gain stability at specific magnesium concentrations in an in vitro degradation system from spinach chloroplasts. Each RNA exhibits a typical magnesium concentration-dependent stabilization profile. It shows a cooperative response of the stability-regulated psbA mRNA and a saturation curve for the other RNAs. The concentration of free Mg(2+) rises during chloroplast development within a range sufficient to mediate gene-specific mRNA stabilization in vivo as observed in vitro. We suggest that magnesium ions are a trans-acting factor mediating differential mRNA stability.
Highlights
The efficiency of gene expression depends on the stability of mRNAs by determining the pool of templates available for synthesis of the respective gene products
We suggest that magnesium ions are a trans-acting factor mediating differential mRNA stability
Chloroplast transformation with constructs of the psbD leader fused to a reporter gene showed a destabilized chimeric transcript in the mutant background and normal accumulation in the wild type, indicating that the 74-nucleotide leader of the mRNA includes a determinant for psbD mRNA degradation [13, 14]
Summary
Plant Material—Spinach plants (Spinacea oleracea L. cv. Monnopa) were grown on soil in the greenhouse with additional illumination during wintertime to result in 12 h of light per day. Northern Analysis—For Northern analysis 2 g of chloroplast RNA per lane were separated on 1.2% agarose-formaldehyde gels according to Ref. 32. High Resolution Northern Analysis—For high resolution Northern analysis 2 g of chloroplast RNA per lane were separated on denaturating 5% sequencing polyacrylamide gels containing 8 M urea, 0.5ϫ TBE (10ϫ: 89 mM Tris, 89 mM boric acid, 1 mM EDTA). Relative RNA stability for magnesium-dependent stability profiles was determined by normalizing the data points obtained after 180 min of incubation to the “0” time point. To plot the relative RNA stability against concentrations of free Mg2ϩ, the data were fitted using a modification of the Hill algorithm for binding of small ligands to a macromolecule [36]. UV Cross-linking Analysis—For analysis of protein binding, label transfer experiments were performed according to Ref. 38. The gels were stained with silver nitrate [40], dried, and exposed to Kodak XAR x-ray films
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