The balance between protein synthesis and degradation in chloroplasts determines leaf variegation in Arabidopsis yellow variegated mutants

Abstract

An Arabidopsis thaliana leaf-variegated mutant yellow variegated2 (var2) results from loss of FtsH2, a major component of the chloroplast FtsH complex. FtsH is an ATP-dependent metalloprotease in thylakoid membranes and degrades several chloroplastic proteins. To understand the role of proteolysis by FtsH and mechanisms leading to leaf variegation, we characterized the second-site recessive mutation fu-gaeri1 (fug1) that suppressed leaf variegation of var2. Map-based cloning and subsequent characterization of the FUG1 locus demonstrated that it encodes a protein homologous to prokaryotic translation initiation factor 2 (cpIF2) located in chloroplasts. We show evidence that cpIF2 indeed functions in chloroplast protein synthesis in vivo. Suppression of leaf variegation by fug1 is observed not only in var2 but also in var1 (lacking FtsH5) and var1 var2. Thus, suppression of leaf variegation caused by loss of FtsHs is most likely attributed to reduced protein synthesis in chloroplasts. This hypothesis was further supported by the observation that another viable mutation in chloroplast translation elongation factor G also suppresses leaf variegation in var2. We propose that the balance between protein synthesis and degradation is one of the determining factors leading to the variegated phenotype in Arabidopsis leaves.

Figure 1.

Leaf Phenotypes of the Variegated Mutant var2 and Its Suppressor Line fug1. (A) Photographs of 5-week-old wild-type Col, var2-1, var2-1 fug1-1, fug1-3, and fug1-1 grown under normal conditions. (B) Photographs of detached rosette leaves from Col (5 weeks old) and var2-6 fug1-1 (6 weeks old) and a second true leaf from Col, var2-6 fug1-1, and var2-6. (C) Toluidine Blue–stained thin sections of a 3-week-old third true leaf from Col, var2-1, var2-1 fug1-1, and fug1-1. (D) to (G) Electron micrographs of chloroplasts from 3-week-old leaves of var2-6 fug1-1 (D), Col (E), var2-6 fug1-1 (F), and var2-1 fug1-1 (G). Bars = 833 nm in (D), 1.4 μm in (E), 5.0 μm in (F), and 1.3 μm in (G).

Figure 2.

Identification of the fug1 Alleles and RNAi Suppression Analysis. (A) Schematic representation of the gene At1g17220, which corresponds to FUG1 (also shown as cpIF2). Locations of T-DNA insertions in two lines (fug1-2 and fug1-3) as determined by sequence analysis. Insertion sites are indicated by white arrowheads with the nucleotide positions (+2517 and −367, respectively; +1 corresponds to A of the initiation codon ATG in the genomic sequence). An amino acid substitution found in fug1-1 (+4437, L963F) is indicated by an asterisk. The region corresponding to a DNA fragment that was used for the RNAi suppression experiment is indicated by a red bar. (B) Dissected young siliques from fug1-2 showing embryo lethality. Defective seeds detected in young siliques of fug1-2 are indicated by asterisks. Bars = 0.5 mm. (C) Suppression of the var2 leaf variegation by fug1-3. Photographs of 5-week-old var2-1 (left) and var2-1 fug1-3 (right) are shown. The genotype of the var2-1 fug1-3 plants was determined by PCR with gene-specific primers (see Methods). Bars = 5 mm. (D) Accumulation of FUG1 mRNA examined by RT-PCR. Primers for FtsH2(VAR2)+FtsH8, FUG1, and ACTIN2 were prepared, and RT-PCR was performed with total RNAs from 3-week-old plants as indicated above the gel. For each gene, two gels with different amplification cycles (shown on the right) are indicated. RT-PCR was repeated at least three times, and the representative result is shown. (E) Accumulation of cpIF2 protein examined by immunoblot analysis. Total protein extracts from cotyledons of Col, var2-1, var2-1 fug1-1, fug1-1, and fug1-3 were separated by 7.5% SDS-PAGE and probed with anti-FUG1 (top), anti-FtsH2 (middle), and Lhcb1 (bottom). Samples were equally loaded based on total chlorophyll contents. We repeated immunoblot analysis at least three times, and the representative result is shown. The band of 105 kD corresponding to the mature size of cpIF2 is indicated by an open arrowhead. An E. coli extract from the strain carrying pTrc99AcpIF2Δ64 (see Figure 4) also detected the band of 105 kD (right). Additional bands detected by the E. coli extract (indicated by closed arrowheads) likely represent degradation products of cpIF2, since none of the signals were detected by cell extracts without expressing cpIF2 (data not shown). (F) RNAi suppression of cpIF2 restores the formation of green sectors in true leaves. Schematic representation of the gene cassette used in RNAi (cpIF2-RNAi) is indicated on the top, consisting of the CaMV 35S promoter (Pro_35S), part of β-glucuronidase gene (ΔGUS), and the nopaline synthase terminator (NOS). The DNA fragment specific for FUG1 inserted in the gene cassette (shown in [A]) is highlighted by two red arrows (Antisense and Sense). Photographs of a parental var2-1 (var2-1) and two cpIF2-RNAi transgenics (transformants) displaying enhanced green sectors are indicated on the bottom. Bars = 1 mm.

Figure 3.

GFP Transient Assay and Phylogenetic Analysis. (A) Cellular localization of cpIF2 as revealed by a GFP transient assay. Protoplasts prepared from leaf tissues were transformed with N-terminal GFP fusion constructs that contained a putative targeting sequence from either cpIF2 (cpIF2TP-GFP) or from mtIF2 (mtIF2TP-GFP). A GFP construct that lacked the targeting signals was also transformed as a negative control (GFP). Images of bright-field (right), GFP fluorescence (middle), and chlorophyll autofluorescence (left) are shown. To ascertain mitochondrial localization, protoplasts expressing mtIF2-GFP were stained by a 50 nM Mito-Tracker Orange dye, and the signals were detected simultaneously with chlorophyll autofluorescence. Bars = 20 μm. (B) A phylogenetic tree of prokaryotic IF2 homologs. Bootstrap values (percentage) are shown in red, and expected distances as counts of amino acid substitutions per site are shown in black at the nodes.

Figure 4.

Complementation of the E. coli infB Null Mutation by Arabidopsis cpIF2. (A) The E. coli SL598R mutant strain, containing a functional infB in a thermosensitive lambda lysogen, was transformed with the four plasmids (indicated on the left). An empty pTrc99A vector was used as a negative control. The positive control plasmid (p18-1) is a pBR322 derivative and carries a wild-type infB. Each cell line, before and after heat-curing, was spotted on ampicillin plates, and photographs were taken after overnight incubation at 30°C. Rows 1 to 3 represent a series of cell dilutions (rows 2 and 3 are 10-fold and 50-fold dilutions of row 1, respectively) that were plated after heat-curing at 42°C for 1 h. The two truncated cpIF2 proteins that were used in this experiment (cpIF2Δ64 and cpIF2Δ421) are described in Results. We considered that the low recovery of the strain carrying p18-1 compared with others is due to the difference of vectors used. (B) PCR was conducted to verify the loss of infB due to the heat-curing treatment. Genomic DNA was isolated from cell lines before or after heat-curing, and samples were subjected to PCR analysis using primers specific for infB (top panel) and cpIF2 (bottom panel). The bands corresponding to an active form of infB (infB), an inactive form of infB [ΔinfB (cat)], cpIF2Δ64, and cpIF2Δ421 are indicated on the left. The bands in the p18-1 lanes shown by white arrowheads derive from infB cloned in p18-1.

Figure 5.

Protein Synthesis in Col and fug1-3 Studied by in Vivo Protein Labeling. (A) In vivo labeling of thylakoid membrane proteins in Col and fug1-3. Fully expanded mature leaves (∼7 weeks old under 12-h daylength) from Col and fug1-3 were thoroughly infiltrated with solution containing [³⁵S]Met and were transferred under a fluorescent bulb (150 μmol/m²/s) for a period of incubation (0, 5, 10, 15, 20, 30, and 60 min). Thylakoid membranes were separated by urea SDS-PAGE and stained by Coomassie blue (CBB). The bands corresponding to D1 and LHCII, confirmed by immunoblot analysis, are indicated by arrowheads in each panel. We repeated this experiment more than three times, and the representative result is shown. (B) Quantification of the LHCII and D1 protein labeling. Ratios of the LHCII radiolabel over the amount of LHCII detected by Coomassie blue (left) or over the LHCII radiolabel (right) was plotted at each time point (bars indicate sd; n = 3). To normalize values from three independent experiments, the ratio of Col at 60 min was adjusted as 1, and the relative ratios are indicated.

Figure 6.

Steady State Accumulation of Chloroplast Proteins. (A) Chloroplasts were purified by a Percoll step gradient from each plant line as indicated below the immunoblot. Leaves at the similar age in Figure 5 were subjected to chloroplast purification experiments. Proteins from the chloroplasts were equalized by total chlorophyll content and separated on SDS-PAGE. Results of immunoblots using anti-FtsH2 (VAR2), anti-D1 (PsbA), anti-PsbO, anti-PsaF, and anti-Lhcb1 antibodies are indicated. (B) Relative amounts of proteins estimated by immunoblot analysis. Signals of immunoblots were quantified by NIH Image, normalized by the signals of Lhcb1, and calibrated based upon the protein amount in Col (bars indicate sd; n = 3).

Figure 7.

Effects of fug1 and var2 fug1 on the Sensitivity to High Light. (A) The ratio of variable to maximum fluorescence (Fv/Fm) measured in detached leaves of Col, var2-1, and var2-1 fug1-1 using the FluorCam 700MF (bars indicate sd; n = 5 from different plants). Leaves were exposed to high light (800 μmol/m²/s for 240 min), and Fv/Fm was calculated. Results from leaves that were harvested from plants grown under normal light (70 μmol/m²/s) in MS plates or soil are shown in left and right panels, respectively. (B) Digital images of Fv/Fm. The values were measured by the FluorCam 700MF at time 0 and 240 min in detached leaves and were visualized by pseudocolor index as indicated on the bottom. Typical images from 3-week-old leaves (left) and from 5-week-old cauline leaves (right) are shown. Bars = 5 mm.

Figure 8.

Suppression of Leaf Variegation by var1 var2 fug1 and var2 sco1. (A) Leaf variegation of var1 and var1 var2 was suppressed by fug1. Photographs of a parental var1-1 (top left, 4 weeks) and var1-1 var2-1 double mutant (bottom left, 5 weeks) are shown, together with var1-1 fug1-1 double mutant (middle, 5 weeks; right, 10 weeks) and var1-1 var2-1 fug1-1 triple mutant (middle, 5 weeks; right, 10 weeks). Bars = 1 mm in left panel and 5 mm in right panels. (B) Another mutation, sco1, which is defective in translation elongation in chloroplasts, suppressed the leaf variegation of var2. Photographs of 4-week-old sco1 (left) and 5-week-old var2-1 sco1 double mutant (right) are shown. Bars = 1 mm in left panel and 5 mm in right panel.