Plastid signalling under multiple conditions is accompanied by a common defect in RNA editing in plastids

Abstract

Retrograde signalling from the plastid to the nucleus, also known as plastid signalling, plays a key role in coordinating nuclear gene expression with the functional state of plastids. Inhibitors that cause plastid dysfunction have been suggested to generate specific plastid signals related to their modes of action. However, the molecules involved in plastid signalling remain to be identified. Genetic studies indicate that the plastid-localized pentatricopeptide repeat protein GUN1 mediates signalling under several plastid signalling-related conditions. To elucidate further the nature of plastid signals, investigations were carried out to determine whether different plastid signal-inducing treatments had similar effects on plastids and on nuclear gene expression. It is demonstrated that norflurazon and lincomycin treatments and the plastid protein import2-2 (ppi2-2) mutation, which causes a defect in plastid protein import, all resulted in similar changes at the gene expression level. Furthermore, it was observed that these three treatments resulted in defective RNA editing in plastids. This defect in RNA editing was not a secondary effect of down-regulation of pentatricopeptide repeat protein gene expression in the nucleus. The results indicate that these three treatments, which are known to induce plastid signals, affect RNA editing in plastids, suggesting an unprecedented link between plastid signalling and RNA editing.

Fig. 1.

Effects on Arabidopsis seedlings of the ppi2-2 mutation and of treatment with norflurazon (NF) or lincomycin (Lin). (A) Phenotype of wild-type and ppi2-2 seedlings grown on either 1 μM NF, 500 μM Lin, or DMSO for 5 d. (B and C) Expression profiles of genes encoding photosynthesis-related (B) and non-photosynthetic (C) proteins in ppi2-2 mutant and wild-type plants treated with either 1 μM NF or 500 μM Lin. Wild-type plants treated with DMSO were analysed as a control. Plants were grown under continuous light (∼125 μmol m⁻² s⁻¹) for 5 d. Transcript levels were analysed by real-time PCR and normalized to the levels of ACTIN2. Each bar is plotted on a log scale and represents the mean of at least three independent samples. Error bars represent the standard deviations of the mean. AGI codes are listed in Supplementary Table S1 at JXB online.

Fig. 2.

Expression profiles of transcription factor-encoding genes in ppi2-2 mutant and wild-type plants treated with either 1 μM norflurazon (NF) or 500 μM lincomycin (Lin). Transcript levels were analysed by real-time PCR and normalized to the level of ACTIN2. Each bar is plotted on a log scale and represents the mean of at least three independent samples. Error bars represent the standard deviations of the mean. Transcription factors that are down-regulated in true leaves of ppi2-1 mutant plants (Kakizaki et al., 2009) were analysed. AGI codes are listed in Supplementary Table S1 at JXB online.

Fig. 3.

Expression profiles of several other transcription factors in ppi2-2 and wild-type plants treated with either 1 μM norflurazon (NF) or 500 μM lincomycin (Lin). Transcription factors were selected based on comparative analysis of SuperSAGE results and available microarray data sets (Supplementary Fig. S1 at JXB online). Transcript levels were analysed by real-time PCR and normalized to the level of ACTIN2. Each bar is plotted on a log scale and represents the mean of at least three independent samples. Error bars represent standard deviations of the mean. AGI codes are listed in Supplementary Table S1 at JXB online.

Fig. 4.

Analysis of chloroplast RNA editing. (A) Comparison of the RNA editing efficiencies of accD and rps14 in ppi2-2 and gun1-101 ppi2-2 mutant plants. RT-PCR products were sequenced directly to determine their editing efficiencies. (B) Quantitative analysis of the RNA editing efficiencies of rps14 and accD transcripts. RT-PCR products were subcloned into the pZErO-2 vector and their sequences analysed. At least 99 independent clones were sequenced for each treatment. WT, wild-type treated with DMSO; ppi2-2, ppi2-2 mutant treated with DMSO; NF, wild-type treated with norflurazon; Lin, wild-type treated with lincomycin.

Fig. 5.

Expression profiles of genes encoding pentatricopeptide repeat proteins in ppi2-2 mutant and wild-type plants treated with either 1 μM norflurazon (NF) or 500 μM lincomycin (Lin). Transcript levels were analysed by real-time PCR and normalized to the levels of ACTIN2. Each bar is plotted on a log scale and represents the mean of at least three independent samples. Error bars represent standard deviations of the mean. AGI codes are listed in Supplementary Table S1 at JXB online.