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.