Functional specialization amongst the Arabidopsis Toc159 family of chloroplast protein import receptors

Abstract

The initial stages of preprotein import into chloroplasts are mediated by the receptor GTPase Toc159. In Arabidopsis thaliana, Toc159 is encoded by a small gene family: atTOC159, atTOC132, atTOC120, and atTOC90. Phylogenetic analysis suggested that at least two distinct Toc159 subtypes, characterized by atToc159 and atToc132/atToc120, exist in plants. atTOC159 was strongly expressed in young, photosynthetic tissues, whereas atTOC132 and atTOC120 were expressed at a uniformly low level and so were relatively prominent in nonphotosynthetic tissues. Based on the albino phenotype of its knockout mutant, atToc159 was previously proposed to be a receptor with specificity for photosynthetic preproteins. To elucidate the roles of the other isoforms, we characterized Arabidopsis knockout mutants for each one. None of the single mutants had strong visible phenotypes, but toc132 toc120 double homozygotes appeared similar to toc159, indicating redundancy between atToc132 and atToc120. Transgenic complementation studies confirmed this redundancy but revealed little functional overlap between atToc132/atToc120 and atToc159 or atToc90. Unlike toc159, toc132 toc120 caused structural abnormalities in root plastids. Furthermore, when proteomics and transcriptomics were used to compare toc132 with ppi1 (a receptor mutant that is specifically defective in the expression, import, and accumulation of photosynthetic proteins), major differences were observed, suggesting that atToc132 (and atToc120) has specificity for nonphotosynthetic proteins. When both atToc159 and the major isoform of the other subtype, atToc132, were absent, an embryo-lethal phenotype resulted, demonstrating the essential role of Toc159 in the import mechanism.

Figure 1.

Phylogenetic Analysis of Toc-GTPases from Arabidopsis and Other Species. Amino acid sequences of the G-domains (equivalent to residues 828 to 1062 of psToc159) of Toc159 and Toc34 homologs from different species were aligned and used to produce a phylogenetic tree. Numbers of mutations are given above the clades, with bootstrap values below. Genes and accession numbers of the sequences used are as follows: atToc159, At4g02510; atToc132, At2g16640; atToc120, At3g16620; atToc90, At5g20300; atToc33, At1g02280; atToc34, At5g05000; psToc159, AAF75761; psToc34, Q41009; osToc86-like_1, AAG48839; osToc86-like_2, AAK43509; zmToc34-1, CAB65537; zmToc34-2, CAB77551; H-Ras p21, P01112. Species of origin of the sequences are indicated as follows: at, Arabidopsis thaliana; ps, Pisum sativum; os, Oryza sativa; zm, Zea mays. H-Ras p21 is from human.

Figure 2.

Expression Profiles of the Four Arabidopsis Toc159 Homologous Genes. (A) Fifteen-microgram samples of total RNA isolated from the indicated Arabidopsis tissues were analyzed by RNA gel blotting. RNA was isolated from wild-type seedlings grown in vitro for 10 d in the light (10d), from three different tissues of 28-d-old wild-type plants grown on soil (rosette leaves, young inflorescence tips, and roots), and from the four original Toc159 homolog knockout mutants (Figure 3A) grown in vitro for 10 d in the light. Filters were probed using ³²P-labeled gene-specific probes of similar lengths with identical specific activities. rRNA (28S) was used as a loading control. The images shown were obtained after 10-d or 22-d exposures, as indicated, but quantifications were performed using identical exposures. (B) Relative levels of expression of the atTOC159, atTOC132, atTOC120, and atTOC90 genes, normalized for 28S rRNA, are shown in the chart; y axis values between ∼55 and ∼95 have been removed to aid visualization.

Figure 3.

Visible Phenotypes of the Toc159 Homolog Knockout Mutants. (A) Schematic diagrams showing the structure of the four Arabidopsis Toc159-related genes and the location of each T-DNA insertion. Protein-coding exons are represented by black boxes, and untranslated regions are represented by white boxes; introns are represented by thin lines between the boxes. T-DNA insertion sites are indicated precisely, but the insertion sizes are not to scale. ATG, translation initiation codon; stop, translation termination codon; p(A), polyadenylation site; LB, T-DNA left border; RB, T-DNA right border. (B) Homozygous, single knockout mutant seedlings grown in vitro alongside the wild type. (C) Pale and bleached toc132 toc120 double mutant seedlings grown in vitro alongside the wild type. The double mutants shown were homozygous for toc132 and heterozygous for toc120 (toc132/toc132; +/toc120; pale) or homozygous for both mutations (toc132 toc120; bleached). (D) Mature (24-d-old) single and double mutants. Apart from the individual labeled toc132/toc132; +/toc120, all plants shown were homozygous for the indicated mutations. Plants were germinated in vitro and transferred to soil (or, in the case of toc132 toc120 and toc159, to medium containing 3% [w/v] sucrose) after 10 d of growth. (E) Rosette leaves of the toc132 single mutant have a reticulate appearance. (F) The toc132 toc120 double homozygote is able to survive to maturity on soil. The double mutant was allowed to establish itself on medium containing 3% (w/v) sucrose for 2 weeks before transfer to soil. Bars = 5 mm.

Figure 4.

Chlorophyll Accumulation in the Toc159 Homolog Knockout Mutants. The chlorophyll contents of 10-d-old (B) and 24-d-old ([A] and [C]) plants are shown. The charts in (B) and (C) have a dual y axis, such that the right side of each chart is at 10-fold higher magnification than the left side. The data shown in (C) are also shown in (A). All values shown are means (±sd) derived from five independent samples.

Figure 5.

Embryo Lethality of the toc159 toc132 Double Mutation. (A) Three to four siliques from three different individuals (at least nine siliques in total) of the indicated genotypes were scored for the presence of aborted seeds. The F3 plants scored were all heterozygous for the toc159 mutation (introgressed into the Col-0 ecotype) and homozygous for the second mutation (Table 3). Error bars indicate sd. (B) The appearance of aborted seeds within the silique of an F3 individual that was homozygous for toc132 and heterozygous for toc159.

Figure 6.

Transgenic Complementation of the toc159 Single Mutant and the toc132 toc120 Double Mutant. Plants carrying the toc159 (A) or toc132 and toc120 (B) knockout mutations were stably transformed with four different T-DNA constructs: 35S-atTOC159, 35S-atTOC132, 35S-atTOC120, and 35S-atTOC90. Mutant complementation was quantified in the T3 generation by making chlorophyll measurements on plants with appropriate mutant genotypes (toc159/toc159 [A] and toc132/toc132; +/toc120 [B] or, occasionally, amongst the 35S-atTOC132 and 35S-atTOC120 transformants toc132/toc132; toc120/toc120 [B]) and that were either heterozygous or homozygous for the relevant transgene. The chlorophyll values shown are means (±sd) derived from measurements on four to six independent transformants; for each transformant, five independent measurements were made. Values are expressed as a percentage of the wild-type chlorophyll concentration. The extent of transgene overexpression in each transformant was estimated by conducting RT-PCR experiments using T2 plants that were either heterozygous or homozygous for the relevant transgene. Mean fold changes in expression (±sd), relative to the wild type, for each transgene/mutant combination are given textually above the corresponding chlorophyll data bars.

Figure 7.

Ultrastructure of Plastids in the Toc159 Homolog Knockout Mutants at Different Developmental Stages. (A) Plastids from the cotyledons of young (10-d-old) wild type, pale toc132 toc120 double mutant (genotype: toc132/toc132; +/toc120; arrowheads indicate five cytoplasmic inclusions, one of which contains a plastid [p]), bleached toc132 toc120 double homozygous (arrowheads indicate two cytoplasmic inclusions, one of which contains a mitochondrion [m]), and toc159 (two mitochondria and one plastid are indicated) plants are shown. All images are at the same magnification. Bar = 1 μm. (B) Chloroplasts from the leaves of mature (28-d-old) wild type, toc132, and pale toc132 toc120 double mutant (genotype: toc132/toc132; +/toc120) plants are shown. The wild-type chloroplast is from an interveinal region; mutant chloroplasts are from veinal (v) and interveinal (iv) regions, as indicated. Arrowheads indicate five cytoplasmic inclusions. An enlargement of the indicated region (Z1) in the pale toc132 toc120 double mutant interveinal chloroplast is shown. All of the chloroplasts shown are at the same magnification; bar = 1 μm. The enlargement (Z1) is at eightfold higher magnification; bar = 0.1 μm. (C) Plastids from the roots of 10-d-old wild type, toc159, and toc132 toc120 double homozygous plants are shown. Cytoplasmic inclusions are indicated (i). An enlargement of the indicated region (Z2) in the toc132 toc120 double mutant plastid is shown. All of the plastids are at the same magnification; bar = 1 μm. The enlargement (Z2) is at 5.3-fold higher magnification; bar = 0.1 μm.

Figure 8.

Comparison of Import Rates into Isolated Wild-Type and Mutant (toc132/toc132; +/toc120) Chloroplasts for Different Preproteins. In vitro–translated, ³⁵S-Met–labeled preSSU (A) and preL11 (B) were imported into wild-type and mutant (toc132/toc132; +/toc120) chloroplasts for the times indicated in the graphs. The amount of protein imported into chloroplasts was expressed as a percentage of the amount imported into wild-type chloroplasts at the final time point in each case. The data shown are means (±sd) of four (A) or three (B) independent experiments.

Figure 9.

Effects of the toc132 and ppi1 Mutations on the Accumulation of Nuclear Transcripts Encoding Chloroplast Proteins. (A) Nylon filter DNA array technology was used to characterize the ppi1 (Kubis et al., 2003) and toc132 (see Supplemental Table 1 online) nuclear chloroplast transcriptome responses. Hierarchical clustering of the expression profiles of the 288 genes that show significant differential expression, relative to the wild type, in both mutants (ppi1 and toc132) is shown. Colors indicate downregulated (green) or upregulated (red) gene expression relative to the wild type. (B) The behavior of those genes that are downregulated in ppi1, relative to the wild type, in the toc132 mutant is shown in the top pie chart. Similarly, the behavior of those genes that are downregulated in toc132, relative to the wild type, in the ppi1 mutant is shown in the bottom pie chart. The top chart describes 161 genes, and the bottom chart describes 200 genes. Colors indicate downregulated (green), upregulated (red), or unchanged (gray) gene expression relative to the wild type.