A molecular-genetic study of the Arabidopsis Toc75 gene family

Abstract

Toc75 (translocon at the outer envelope membrane of chloroplasts, 75 kD) is the protein translocation channel at the outer envelope membrane of plastids and was first identified in pea (Pisum sativum) using biochemical approaches. The Arabidopsis (Arabidopsis thaliana) genome contains three Toc75-related sequences, termed atTOC75-I, atTOC75-III, and atTOC75-IV, which we studied using a range of molecular, genetic, and biochemical techniques. Expression of atTOC75-III is strongly regulated and at its highest level in young, rapidly expanding tissues. By contrast, atTOC75-IV is expressed uniformly throughout development and at a much lower level than atTOC75-III. The third sequence, atTOC75-I, is a pseudogene that is not expressed due to a gypsy/Ty3 transposon insertion in exon 1, and numerous nonsense, frame-shift, and splice-junction mutations. The expressed genes, atTOC75-III and atTOC75-IV, both encode integral envelope membrane proteins. Unlike atToc75-III, the smaller atToc75-IV protein is not processed upon targeting to the envelope, and its insertion does not require ATP at high concentrations. The atTOC75-III gene is essential for viability, since homozygous atToc75-III knockout mutants (termed toc75-III) could not be identified, and aborted seeds were observed at a frequency of approximately 25% in the siliques of self-pollinated toc75-III heterozygotes. Homozygous toc75-III embryos were found to abort at the two-cell stage. Homozygous atToc75-IV knockout plants (termed toc75-IV) displayed no obvious visible phenotypes. However, structural abnormalities were observed in the etioplasts of toc75-IV seedlings and atTOC75-IV overexpressing lines, and toc75-IV plants were less efficient at deetiolation than wild type. These results suggest some role for atToc75-IV during growth in the dark.

Figure 1.

Structural characteristics of the Arabidopsis Toc75 gene family. A, Schematic diagrams depicting the three Arabidopsis Toc75-related sequences. 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. Mutations in the atTOC75-I pseudogene are indicated as follows: F, frame-shift; S, nonsense; J, splice junction; I, missing intron. A 75-bp insertion and 48-bp direct repeat, both in exon 1, are represented by a black bar and two black arrows, respectively. Locations of forward (priF1, priF2, and priF3) and reverse (priR1 and priR2) primers used for RT-PCR analysis of atTOC75-I are shown. The gray diagram underneath atTOC75-I illustrates how the pseudogene is currently annotated as two separate genes (At1g35880 and At1g35860) in the GenBank database; vertical, dotted lines indicate four putative splice junctions that are identical in the two gene models. The locations of T-DNA and transposon insertions 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. B, Amino acid sequence alignment of pea Toc75 (ps) with the three Arabidopsis proteins (at-III, at-I, and at-IV). The atToc75-I sequence was derived by conceptual translation of a partially corrected version of the open reading frame illustrated in A. The locations of frame-shift (F) and nonsense (S) mutations in the atToc75-I sequence, a 25-residue insertion (IN), and a 16-residue direct repeat (DR) are indicated. Residues identical in at least three sequences are highlighted in black, whereas similar residues are highlighted in gray. The location of the first Asp residue of mature psToc75 is shown (Mat), as are the 16 predicted transmembrane domains of psToc75 (Tranel et al., 1995; Sveshnikova et al., 2000a). C, Phylogenetic analysis of Toc75-related proteins from Arabidopsis and other species. Full length pea, Arabidopsis, and cyanobacterial amino acid sequences were aligned, together with sequences from maize and rice and used to produce a phylogenetic tree. Numbers of mutations are given above the clades with bootstrap values below. Gene and accession numbers for the sequences used are as follows: psToc75 (L36858, S55344); atToc75-III (At3g46740, AAM83239); atToc75-I (BK005428); atToc75-IV (AY585655, AAT08975); atToc75-V/AtOEP80 (At5g19620, NP_568378); zmToc75 (AY106148); osToc75 (AK070010); SynToc75 (slr1227, NP_440832). Species of origin is indicated as follows: pea, ps; Arabidopsis, at; maize, zm; rice, os; Synechocystis PCC 6803, Syn. The cyanobacterial protein, SynToc75, was used as the outgroup.

Figure 2.

Expression studies on the Arabidopsis Toc75 genes. A and B, Analysis of atTOC75-IV expression in mutant and transgenic lines by RT-PCR. Total RNA samples isolated from the toc75-IV-1 mutant (A) and from two independent 35S-atTOC75-IV transgenic lines (B) were analyzed by RT-PCR, along with corresponding wild-type samples, using primers specific for the indicated genes (atTOC75-IV, atTOC33, and eIF4E1). In each case, the rosette leaves of a single mature plant were analyzed; the 35S-atTOC75-IV plants used were both homozygous for the transgene. For the experiment shown in A, PCR amplification was conducted over a total of 40 cycles. For the experiment shown in B, amplification was conducted using only 25 cycles. PCR products were resolved by agarose gel electrophoresis and stained with ethidium bromide. C, Expression profiles of atTOC75-III and atTOC75-IV during seedling development and in different tissues of Arabidopsis. Total RNA samples isolated from Arabidopsis tissues were analyzed by semiquantitative RT-PCR. RNA was isolated from wild-type seedlings grown in vitro for 5 or 10 d in the light (5 d L and 10 d L, respectively), or 5 d in the dark (5 d D), and from three different tissues of 28-d-old wild-type plants grown on soil (flower buds, rosette leaves, and roots). Amplifications were conducted under nonsaturating conditions using gene-specific atTOC75-III, atTOC75-IV, and eIF4E1 primers, and the products were quantified by hybridization with corresponding ³²P-labeled cDNA probes. The data for atTOC75-III and atTOC75-IV were normalized for eIF4E1 and then expressed as a percentage of the maximum level observed for each gene. Values shown are means (±sd) of four independent measurements.

Figure 3.

Import and localization studies on atToc75-IV using pea chloroplasts. A, High pH washes of imported proteins. In vitro translated, ³⁵S-labeled precursor proteins (atToc75-IV, atToc75-III, psTic22, and Luciferase, Luc) were incubated with isolated pea chloroplasts for 30 min under import conditions. Intact chloroplasts were reisolated and then treated with either 10 mm HEPES, 10 mm MgCl₂, pH 8.0 (bursting buffer), or 0.1 m Na₂CO₃ (high pH), before separation into pellet (P) and soluble (S) fractions by centrifugation. Samples were resolved by SDS-PAGE and visualized by fluorography. In each case, the first lane contains translation product equivalent to 10% of the amount added to each import reaction. The approximately 36-kD band below the main atToc75-IV translation product is presumably the result of translation initiation at Met-72, since this product was repeatedly observed in atToc75-IV translation mixtures. The precursor (p) and putative intermediate (i) and mature (m) forms of atToc75-III are indicated. B, Trypsin treatment of imported proteins. Import of four different precursors (atToc75-IV, atToc75-III, tp110-110N, and psTic22) was conducted as described in A. Reisolated chloroplasts (Imp) were then treated with three different concentrations of trypsin (1×, 2×, and 5× Trp indicates 0.1, 0.2, and 0.5 g trypsin/g chlorophyll, respectively, equivalent to 12.5, 25, and 62.5 ng trypsin/μL), or in the absence of trypsin (−), for 30 min on ice in the dark. Precursor (p), intermediate (i), and mature (m) forms of the various proteins, and a putative trypsin degradation of product of the psTic22 (*), are indicated. Bands corresponding to the imported proteins, in trypsin-treated and control samples, were quantified using ImageJ version 1.32j (http://rsb.info.nih.gov/ij/), and the data were used to produce the graph shown. Plotted values are means (±sd) derived from two independent experiments. The atToc75-III data were derived from the i and m forms, and the psTic22 data were derived from the p and m forms; it was previously demonstrated that cleavage of the psTic22 transit peptide is not essential for import of psTic22 to the intermembrane space (Kouranov et al., 1999). In each case, both bands exhibited essentially the same degree of trypsin resistance. C and D, Control trypsin treatment experiments. C, The in vitro translated atToc75-IV precursor (atToc75-IV IVT) utilized in A and B was treated in the absence (−) and presence (+) of 1× trypsin (Trp), as described in B, in the absence of isolated chloroplasts. D, Import of atToc75-IV was conducted as described in A and B. Samples were then treated with 2× trypsin, as described in B, in the presence or absence of the 1% (v/v) Triton X-100 (TX100), as indicated.

Figure 4.

Import and localization studies on atToc75-IV using Arabidopsis chloroplasts. A, Postimport thermolysin treatment experiment. In vitro translated, ³⁵S-labeled atToc75-IV was imported into chloroplasts isolated from 10-d-old Arabidopsis plants grown in vitro. The first lane contains translation product equivalent to 10% of the amount added to each import reaction, and the second lane contains a standard, 20-min import reaction (Imp). The remaining four lanes show an associated thermolysin treatment experiment: ³⁵S-labeled atToc75-IV was incubated for 20 min under import conditions in the presence/absence of chloroplasts (Chl), as indicated, before further treatment in the presence/absence of 60 μg/mL thermolysin (Th), as indicated. In each case, chloroplasts were recovered and the constituent proteins were resolved by SDS-PAGE and visualized by fluorography. B, Suborganellar fractionation experiment. In vitro translated,³⁵S-labeled atToc75-IV protein was imported into isolated chloroplasts as described in A. Recovered chloroplasts (Chl) were then fractionated by centrifugation to generate thylakoid (Thy), total membrane (Env/Thy), and envelope (Env) fractions. Following resolution by SDS-PAGE, atToc75-IV was visualized by fluorography, and atToc75-III, atTic110, and LHCP were detected by immunoblotting. Each lane contains protein equivalent to one import reaction (i.e. 10 million chloroplasts). C, Immunoblot experiment. Chloroplasts were isolated from 20-d-old wild-type, toc75-IV-1, and transgenic 35S-atTOC75-IV, line 13 (35S-75-IV, 13) plants and then used to prepare envelope membranes as in B. Whole chloroplast (Chl) and envelope membrane (Env) samples (equivalent to 4 μg chlorophyll for the atToc75-IV blots and 2 μg chlorophyll for the control blots) were then analyzed by immunoblotting. Identical blots were probed with antisera derived from two independent rabbits inoculated with an atToc75-IV-specific peptide, and the corresponding preimmune sera (the different rabbits gave similar results and so data are shown for one rabbit only). The antisera specifically detected a protein of 44 kD (the predicted size of atToc75-IV and the apparent molecular mass of in vitro translated atToc75-IV) in Chl and Env samples of the 35S-atTOC75-IV line, as indicated by the arrowhead and label at right. Also indicated are bands corresponding to Rubisco large subunit (RbcL), two prominent nonspecific proteins in the Chl samples (indicated with asterisks), and three prominent nonspecific bands in the Env samples (indicated with bullet points); all of these nonspecific bands were also detected by the preimmune sera. Positions of molecular mass standards (kD) are shown at left. Antibodies that recognize atToc75-III, atTic40, and two thylakoid proteins, the 33-kD and 23-kD subunits of the oxygen evolving complex (OE33 and OE23, respectively), were used as controls.

Figure 5.

Energetics of atToc75-IV membrane insertion. Chloroplasts isolated from 10-d-old Arabidopsis plants grown in vitro were incubated in the dark in the presence of 6 μm nigericin for 10 min to deplete endogenous ATP. In vitro translated, ³⁵S-labeled atToc75-IV and preSSU proteins were passed through Sephadex G-25 to remove small molecules and then incubated with the chloroplasts under import conditions for 20 min in the absence (Imp −ATP) or presence (Imp +ATP) of 5 mm ATP. Chloroplasts recovered from separate import assays were treated for 1 h with 0.1 m Na₂CO₃ (high pH) or 10 mm HEPES, 10 mm MgCl₂, pH 8.0 (bursting buffer), before separation into pellet (P) and soluble (S) fractions by centrifugation. Following resolution by SDS-PAGE, atToc75-IV and preSSU/SSU were visualized by fluorography. Bands corresponding to preSSU (p) and mature SSU (m) are indicated.

Figure 6.

Phenotypic analysis of the toc75-III and toc75-IV mutants. A, Plants grown under long-day conditions in vitro for 6- or 12-d postgermination are shown. The presented genotypes are, from left to right: wild type (WT), toc75-III heterozygote (+/toc75-III), toc75-III toc75-IV double mutant (+/toc75-III; toc75-IV/toc75-IV), toc75-IV homozygote, toc75-IV ppi1 double homozygote, and ppi1 homozygote. B, The visible phenotypes of 12-d-old plants of the indicated genotypes were quantified by making chlorophyll measurements. Sibling analysis was conducted on families segregating for the toc75-III mutation, which were either wild-type (bars 1 and 2) or homozygous mutant (bars 3 and 4) at the atTOC75-IV locus. For bars 1 to 4, measurements were of samples containing two primary leaves of the same plant; the remainder of each plant was used for genotyping by PCR. For bars 5 to 7 (toc75-IV, toc75-IV ppi1, and ppi1), measurements were of samples containing eight primary leaves from different plants. Values shown are means (±sd) derived from multiple independent measurements: wild type (WT; n = 11), +/toc75-III (n = 19), +/toc75-III; toc75-IV/toc75-IV (n = 45), toc75-IV (n = 15), toc75-IV (n = 20), toc75-IV ppi1 (n = 6), ppi1 (n = 6). The data are expressed as a percentage of the wild-type chlorophyll concentration.

Figure 7.

Embryo lethality of the toc75-III mutation. A to J, Morphology of normal (A–E) and mutant (F–J) embryos from the siliques of toc75-III heterozygous plants. The normal developmental stage in each corresponding pair of images is as follows: A and F, four-cell embryo stage; B and G, eight-cell embryo stage; C and H, 16-cell embryo stage; D and I, 32-cell embryo stage. Embryo cell stages refer to the number of cells in the embryo proper. A normal, heart-stage embryo (E) and an aborted seed (J) are also shown. Bars = 10 μm (A–D and F–I) and 50 μm (E and J). K, The appearance of part of a typical silique from a mature toc75-III heterozygous plant. Arrows indicate aborted seeds. Bar = 0.5 mm. L, Higher magnification image of the two central aborted seeds shown in K. Bar = 0.1 mm.

Figure 8.

Ultrastructure of etioplasts in the toc75-IV mutants. Etioplasts from wild-type (A) and toc75-IV-2 mutant (B) plants grown in vitro in the dark for 3 d; the toc75-IV plastid contains a large inclusion (i) of cytosolic material. In C, an enlargement of a typical cytosolic inclusion within a toc75-IV-1 mutant etioplast is shown. Bars = 0.5 μm.

Figure 9.

Deetiolation in the toc75-IV mutants. Wild-type and mutant Arabidopsis plants were grown in vitro in the dark, from germination, for a period of 6 d and then transferred to continuous white light of standard intensity for a further period of 2 d. At the end of the 2-d light period, plants were scored for evidence of deetiolation. This experiment was repeated 11 times. A, Typical plants from an individual experiment that revealed a striking difference in deetiolation efficiency between the toc75-IV mutants and wild type (data from this experiment are shown in B). The insets at right show the cotyledons of representative plants from the main image, at 2-fold higher magnification, and serve to illustrate the presence/absence of a deetiolation response. B, An individual experiment that revealed a striking difference in deetiolation efficiency between the toc75-IV mutants and wild type (representative plants from this experiment are shown in A). The data were derived from four separate petri plates per genotype, each one containing approximately 80 plants. Deetiolation frequencies were calculated on a per plate basis and then used to produce the mean values shown (±sd; n = 4). C, The combined data of all 11 experiments, each of which comprised three or four separate petri plates per genotype. Deetiolation frequencies were calculated on a per experiment basis and then used to produce the mean values shown (±sd; n = 11).