The AT5G08670 mutant is a genomes uncoupled (gun)mutant
Retrograde signaling is triggered when plastid gene expression (PGE) is disrupted at the transcriptional or translational levels (Woodson & Chory, 2008). Inhibition of PGE causes changes in the expression ofPhANG and the LIGHT-HARVESTING CHLOROPHYLL A/B-BINDING(LHCB ) genes (Zhang et al., 2011). When WT seedlings are exposed to the plastid development inhibitors norflurazon (NF) and lincomycin (LIN), plastid retrograde signaling is induced (Strand et al., 2003). However, in plastid-to-nucleus signaling mutants, retrograde signaling is impaired and LIN or NF treatment no longer represses the expression of LHCB genes to the same extent as in WT (Strand et al., 2003). To determine whetherAT5G08670 is involved in plastid retrograde signaling, T-DNA insertion mutants ofAT5G08670 (SALK_047877, SALK_083115 ) were obtained (Figure 2a), and the homozygous T-DNA insertion was confirmed (Figure S1). To further check the impairment of retrograde signaling, the linesSALK_047877 , SALK_083115 , and genomes uncoupled1 (gun1 ) (as an experimental control) were treated with LIN and NF. Expression of LHCB1.2 was found by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) to be significantly higher in gun1 , SALK_047877 and SALK_083115 than in WT seedlings treated with LIN and NF treatment (Figure 2b, c), thus revealing a conspicuous gun phenotype (Figure 2b, c). These results suggest that AT5G08670 may play a role in plastid retrograde signaling.
AT5G08670 protein is localized in mitochondria
Fluorescence microscope analysis of protoplasts of Arabidopsis from a transgenic line expressing a35S::AT5G08670-GFP fusion protein indicated thatAT5G08670-GFP is targeted to mitochondria (Figure 3) thus confirming its mitochondrial localization, In public databases (The Arabidopsis Information Resource, TAIR; www.arabidopsis.org),AT5G08670 is also annotated as a mitochondrial protein.
Loss of expression of AT5G08670 leads to a decrease in mitochondrial ATP synthase level and activity
Because AT5G08670 and AT5G08690 encode an identical mature ATP synthase b -subunit, it was important to determine whether the level of mitochondrial ATP synthase was diminished and whether its activity was affected in the AT5G08670 mutants. Immunoblotting with ATPB antibody revealed that the level of ATP b -subunit dropped to 25% of WT level in the AT5G08670 mutants, and total ATP synthase activity was found to be significantly lower than in WT (Figure 4a-b). Taken together, these results indicate that changes in mitochondrial ATP synthase activity affect plastid retrograde signaling.
Detection of differentially expressed genes (DEGs ) ofATG08670 mutants by transcriptome analysis.
RNA-seq analysis was performed to study the effects of the T-DNA insertions in AT5G08670 on the transcriptome. Principal component analysis (PCA) was performed using the expression of genes to examine the distribution of samples and explore relationships between samples. Samples in the same group were more concentrated in spatial distribution. In the control group, the samples clustered in the same region. After LIN treatment, the distributions of mutant and WT differed (Figure 5a). The number of counts for each gene was normalized using DESeq software. The negative binomial distribution test was performed to determine the multiplicity of differences in data, estimate expression, and evaluate the significance of reading differences using the base mean values. The results of the difference in ploidy and significance tests were used to screen for differential expression of protein-coding genes. These differences are shown in the volcano plot, with non-significant differences in gene expression in grey, and significantly down- and up-regulated genes in red and green, respectively (Figure 5b). In WT,gun1 , SALK_047877 , and SALK_083115 , DEGs were found to be, respectively, 11,192 (42 percent up-regulated, 58 percent down-regulated), 6842 (35 percent up-regulated, 65 percent down-regulated), 6242 (35 percent up-regulated, 65 percent down-regulated), and 5837 (38 percent up-regulated, 62 percent down-regulated) (Figure 4c). Unsupervised hierarchical clustering of DEGs was performed. The results show that after LIN treatment, WT, the two SALK lines, and thegun1 mutant cluster each in separate branches (Figure 5d). These results indicate that the transcriptional patterns of the mutants and WT differ considerably after LIN treatment.