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First published online May 20, 2005; 10.1104/pp.105.063289 Plant Physiology 138:715-733 (2005) © 2005 American Society of Plant Biologists A Molecular-Genetic Study of the Arabidopsis Toc75 Gene Family1Department of Biology, University of Leicester, Leicester LE1 7RH, United Kingdom (A.B., A.W., R.P., P.D., S.K.P., D.T., P.J.); and Department of Plant Sciences, University of California, Davis, California 95616 (K.I.)
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.
The majority of plastid proteins are translated on cytosolic ribosomes and subsequently imported into plastids (Keegstra and Cline, 1999  ; Chen et al., 2000; Hiltbrunner et al., 2001a; Jarvis and Soll, 2001; Jarvis and Robinson, 2004). An amino-terminal transit peptide directs each of these proteins specifically to the plastid. Upon arrival in the stroma, the transit peptide is cleaved and the mature protein is either folded into its final conformation or targeted to another compartment of the plastid (Keegstra and Cline, 1999; Jarvis and Robinson, 2004). Preproteins are translocated through the plastid double membrane envelope by two membrane-bound protein complexes: the translocon at the outer envelope membrane of chloroplasts (Toc), and the translocon at the inner envelope membrane of chloroplasts (Tic).
Components of the Toc complex include Toc34, Toc75, and Toc159, which were first identified in pea (Pisum sativum) using biochemical approaches (Hirsch et al., 1994
Structural and electrophysiological studies on heterologously expressed pea Toc75 (psToc75) reconstituted into liposomes demonstrated that the protein forms a
Evidence that Toc75-related proteins are present in plant species other than pea was provided by Summer and Cline (1999)
The sequencing of the Arabidopsis (Arabidopsis thaliana) genome has revealed that this species contains multiple, homologous isoforms of many of the Toc and Tic components originally identified in pea (Jackson-Constan and Keegstra, 2001
Expression and Structure of the Different Arabidopsis Toc75 Gene Family Members
The sequencing of the Arabidopsis genome has enabled the identification, in silico, of putative homologs of proteins originally identified in other species. For example, it was previously reported that there are three psToc75-related sequences in the Arabidopsis genome: atTOC75-I, atTOC75-III, and atTOC75-IV (Jackson-Constan and Keegstra, 2001
To determine the reasons underlying the apparent inactivity of this putative gene, we conducted detailed studies of the surrounding genomic sequence. Our analysis revealed that homology with psToc75 extends outside of the predicted exons of At1g35860 (Fig. 1A) and even into the adjacent predicted gene, At1g35880, 5.8 kb upstream (Fig. 1A). These observations strongly suggested that the gene had been incorrectly annotated and led us to conduct a manual annotation of atTOC75-I (deposited in GenBank; accession no. BK005428). The corrected annotation revealed a 5.4-kb gypsy/Ty3-related retrotransposon inserted at the 5' end of the gene (Fig. 1A); this insertion incorporates 395-bp and 392-bp long terminal repeats that share 90% nucleotide sequence identity and is flanked by a perfect 5-bp target site duplication. A related element was previously identified at the waxy locus in maize (Zea mays; Purugganan and Wessler, 1994 ). Our analysis also revealed the presence of numerous nonsense, frame-shift, and splice-junction mutations, as well as a 75-bp insertion and a 48-bp direct repeat in exon 1 (Fig. 1A). This evidence, together with the absence of atTOC75-I ESTs and our inability to detect atTOC75-I expression by RT-PCR, led us to conclude that atTOC75-I is a pseudogene.
Next, we investigated the activity and structure of the atTOC75-IV gene (no. At4g09080). Expression analysis by RT-PCR provided clear evidence that the gene is active (Fig. 2A), and the nucleotide sequence of the PCR product obtained revealed that atTOC75-IV transcripts undergo accurate intron splicing (data not shown). Since no publicly available EST or cDNA clones were available for atTOC75-IV when we started these studies, we initiated experiments to identify our own atTOC75-IV cDNA clone. Attempts to identify such a clone by library screening were unsuccessful, presumably due to the low level of expression of the gene, so we instead generated a full-length clone using a combination of RACE-PCR and RT-PCR. The structure of the transcript at its 5' and 3' ends was determined by RACE-PCR, and this information was used to design primers for amplification of a full-length cDNA by RT-PCR. The RT-PCR product was cloned and sequenced (information deposited in GenBank; accession nos. AY585655 and AAT08975), and its structure is illustrated in Figure 1A. This experimentally determined structure differs slightly from the predicted structure given as part of the genome sequence annotation (accession no. NM_116977; Inoue and Potter, 2004
The structure of the atTOC75-III gene (no. At3g46740) was determined by sequencing a publicly available EST clone, APZL59c10R, and is shown in Figure 1A. The intron-exon boundaries were found to match exactly those predicted as part of the genome sequence annotation (accession no. AL096859), and the cDNA was essentially the same as a recently released full-length cDNA sequence (accession no. AY127014), except that the 5' and 3' untranslated regions were each slightly shorter.
The structures of the three Arabidopsis Toc75-related sequences, shown schematically in Figure 1A, are remarkably similar. All three genes have essentially the same intron-exon boundaries, with only two exceptions: exon 1 is absent from atTOC75-IV, and the penultimate intron is absent from the atTOC75-I pseudogene. The three Arabidopsis homologs are also very similar to each other, and to psToc75, at the amino acid level (Fig. 1B). Percentage sequence identities shared between the Arabidopsis proteins and the pea protein are as follows: 73% (atToc75-III), 60% (atToc75-IV), and 55% (atToc75-I). The 5' truncation of the atTOC75-IV gene means that the atToc75-IV protein includes only eight of the 16 transmembrane domains (nos. 7 and 10–16; Fig. 1B) predicted to be present in psToc75 (Sveshnikova et al., 2000a
To determine the evolutionary relationships between the different Arabidopsis Toc75 homologs, we conducted a phylogenetic analysis using amino acid sequences from several different species. In this analysis, we included all three Arabidopsis sequences, the psToc75 sequence, two Toc75 sequences from monocotyledonous species, as well as distantly related sequences from Synechocystis PCC 6803 (SynToc75) and Arabidopsis (atToc75-V/AtOEP80); SynToc75 is a cyanobacterial protein that shares only approximately 21% to 22% sequence identity with psToc75 (Bölter et al., 1998 The phylogeny suggests that the Arabidopsis Toc75 gene family formed after the pea and Arabidopsis species diverged and implies that pea and other species do not necessarily contain genes closely related to either atTOC75-IV or atTOC75-I. After speciation, an Arabidopsis TOC75 progenitor duplicated to form atTOC75-III and another TOC75 gene. Duplication of the latter then created progenitors of atTOC75-I and atTOC75-IV. Inactivation of atTOC75-I (due to the insertion of the gypsy/Ty3 retrotransposon or the accumulation of other mutations), and the truncation of atTOC75-IV, may have occurred after the final duplication event. Alternatively, retrotransposon insertion may have occurred prior to the duplication event that gave rise to atTOC75-I and atTOC75-IV.
Since the members of other Arabidopsis Toc component gene families have been shown to exhibit differential expression patterns and some degree of functional specialization (Bauer et al., 2000
The data shown in Figure 2C do not provide any information on the relative levels of expression of the two Toc75 genes. However, it is clear that atTOC75-III is expressed at much higher levels than atTOC75-IV for several reasons. First of all, amplification of comparable amounts of the atTOC75-III and atTOC75-IV transcripts by RT-PCR required 20 and 25 cycles, respectively (Fig. 2; data not shown). Assuming that each cycle of amplification produces a 2-fold increase in product yield, this difference in cycle number equates to a 32-fold difference in template concentration. These data are broadly consistent with recently reported microarray data (Vojta et al., 2004
The expression of atTOC75-III was found to be strong in young, rapidly dividing photosynthetic tissues and significantly weaker in mature or slow growing tissues, such as 28-d-old rosette leaves, and roots (Fig. 2C). Unexpectedly, atTOC75-III expression was found to be particularly low in 5-d-old etiolated plants. This suggests that atToc75-IV, the only other close homolog of psToc75 that is expressed in Arabidopsis, may play a relatively more important role in etioplast biogenesis. The expression of atTOC75-IV is more uniform than that of atTOC75-III; while the expression of atTOC75-III varied 10-fold across the samples tested, atTOC75-IV expression varied just 2-fold (Fig. 2C). Like atTOC75-III, the pea Toc75 gene is also expressed most strongly in young, rapidly dividing tissues (Tranel et al., 1995
Pea Toc75 is targeted to the chloroplast outer envelope membrane by a bipartite, amino-terminal transit peptide (Tranel et al., 1995
Common characteristics of outer envelope membrane proteins that lack a cleavable transit peptide are a small to moderate overall size and, in some cases, a well-defined hydrophobic region (Keegstra and Cline, 1999 ; Schleiff and Klösgen, 2001). Since atToc75-IV is relatively small (44 kD), we investigated the possibility that it is targeted by a similar mechanism. To this end, in vitro translated atToc75-IV was incubated with isolated pea chloroplasts under conditions favoring protein import. As expected, atToc75-IV efficiently associated with chloroplasts, and, in contrast with atToc75-III, was not processed to a lower molecular mass form during the experiment (Fig. 3A). Unlike the two Toc75 isoforms, a control protein that is normally targeted to peroxisomes (firefly luciferase) was not found to associate significantly with chloroplasts. Imported atToc75-IV was exclusively associated with the membrane fraction of chloroplasts and was resistant to extraction under high pH conditions, strongly suggesting that atToc75-IV is an integral membrane protein. By contrast, a peripheral protein of the inner envelope membrane, psTic22, was susceptible to extraction under alkaline conditions (Fig. 3A), as expected (Kouranov et al., 1998).
To further confirm the proper targeting of imported atToc75-IV, and to provide some clues about its suborganellar localization, we conducted postimport protease treatment experiments (Fig. 3, B–D). Four different precursors were imported into isolated pea chloroplasts, and then the chloroplasts were treated with three different concentrations of trypsin, or in the absence of trypsin; trypsin is a protease that is able to penetrate the outer envelope membrane and so gain access to proteins exposed in the intermembrane space, but not the inner envelope membrane (Cline et al., 1984
To determine if the targeting of atToc75-IV occurs in similar fashion in Arabidopsis, we conducted similar import experiments with isolated Arabidopsis chloroplasts. Like atToc75-IV imported into pea chloroplasts (Fig. 3A), atToc75-IV imported into Arabidopsis chloroplasts (as well as endogenous atToc75-III) was exclusively associated with the membranes and completely resistant to extraction under high salt and high pH conditions (data not shown). In an additional experiment, postimport treatment of the chloroplasts with thermolysin (a protease that cannot penetrate the outer envelope membrane; Cline et al., 1984
To verify that imported atToc75-IV was associated with the envelope membranes, we conducted fractionation experiments on Arabidopsis chloroplasts containing imported atToc75-IV protein (Fig. 4B). We expected to find that atToc75-IV is envelope-associated for two reasons. First, to date, only two proteins have been shown to gain access to the chloroplast interior without the aid of a transit peptide (Miras et al., 2002 ; Nada and Soll, 2004). Second, two proteins that are very closely related to atToc75-IV (atToc75-III and psToc75) have been shown to localize to the envelope (Schnell et al., 1994; Tranel et al., 1995; Tranel and Keegstra, 1996; Hiltbrunner et al., 2001b). Like endogenous atToc75-III and atTic110, imported atToc75-IV was detected in the whole chloroplast, total membrane, and envelope membrane preparations, but was completely absent from the thylakoid membrane preparation (Fig. 4B). By contrast, the control protein, light harvesting chlorophyll a/b-binding protein (LHCP), was detected in the whole chloroplast, total membrane, and thylakoid membrane preparations, but not in the envelope membrane preparation. We therefore conclude that imported atToc75-IV is an integral envelope membrane protein. To confirm that atToc75-IV is also localized to chloroplasts in vivo, we probed whole chloroplast protein samples with antibodies raised against an atToc75-IV-specific peptide. Two independent antisera recognized several different protein bands in wild-type and toc75-IV mutant chloroplasts, but no differences were observed between the genotypes in either case (Fig. 4C, lanes 1 and 3; data not shown). These data, together with the various lines of evidence indicating low atTOC75-IV transcript abundance in wild type (see earlier), suggested that atToc75-IV is a low abundance protein. To facilitate its detection, we also analyzed chloroplast samples from two transgenic lines overexpressing the atTOC75-IV cDNA (Fig. 2B). Chloroplasts from both overexpressor lines contained a protein of approximately 44 kD that was specifically detected by both atToc75-IV antisera, but not by the corresponding preimmune sera (Fig. 4C, lane 5; data not shown). The apparent molecular mass of the detected protein (44 kD) matched exactly the apparent molecular mass of the atToc75-IV in vitro translation product (data not shown) and the predicted molecular mass of the atToc75-IV protein (Fig. 1, A and B). The antibodies also detected an approximately 44-kD protein in envelope membranes isolated from the overexpressor line chloroplasts (Fig. 4C, lane 6; data not shown), which implies that the endogenous protein is localized in similar fashion to in vitro translated atToc75-IV (Fig. 4B). These data therefore support our conclusions drawn from the in vitro import experiments, i.e. that atToc75-IV is a chloroplast envelope membrane protein and that it is targeted without the aid of a cleavable transit peptide.
Most outer envelope membrane proteins are synthesized at their mature size without a cleavable transit peptide (Keegstra and Cline, 1999
Identification of atToc75-III and atToc75-IV Knockout Mutants To investigate the roles of the atToc75-III and atToc75-IV proteins in vivo, we identified T-DNA or transposon knockout lines for each gene. Two independent alleles of each knockout mutation were identified (Fig. 1A). In each case, the independent alleles behaved identically, indicating that the mutant phenotypes observed were due to the T-DNA or transposon insertions, rather than secondary, unlinked mutations. Unless stated otherwise, the toc75-III-1 and toc75-IV-1 alleles were used to generate the data presented below. All four knockout lines contained single-site insertions, based on the segregation of the insertion-associated marker genes (Table I). Families derived from heterozygous toc75-IV plants segregated three resistant plants for every one sensitive plant when plated on selective medium (Table I), indicating standard Mendelian inheritance. By contrast, families derived from heterozygous toc75-III-1 plants contained only two resistant plants for every one sensitive plant, and families derived from heterozygous toc75-III-2 plants segregated approximately two heterozygous plants for every one wild-type plant when tested by PCR (Table I). Furthermore, all toc75-III mutant plants tested by PCR (60 plants for toc75-III-1 and 35 plants for toc75-III-2) were found to be hemizygous for the corresponding T-DNA insertion. These data indicate that homozygous atToc75-III knockout mutations, unlike homozygous atToc75-IV mutations, are lethal during an early stage of development.
None of the single mutant seedlings (toc75-III heterozygotes and toc75-IV homozygotes) displayed obvious visible or chlorophyll accumulation phenotypes (Fig. 6). The absence of a phenotype in toc75-III heterozygotes indicates that the knockout mutation is recessive and that one atTOC75-III allele is sufficient to support normal growth under the conditions tested. The absence of a phenotype in toc75-IV homozygotes may reflect a degree of redundancy between atToc75-IV and other proteins, such as atToc75-III, or the nonessential nature of atToc75-IV under the conditions tested. In an attempt to reveal a phenotype for atToc75-IV, we generated double mutants by crossing toc75-IV-1 with toc75-III-1 and the atToc33 knockout mutant, ppi1, which displays protein import defects and a pronounced yellow-green phenotype (Jarvis et al., 1998 ; Kubis et al., 2003). Double mutants that were heterozygous for toc75-III and homozygous for toc75-IV (genotype, +/toc75-III; toc75-IV/toc75-IV) did not display any obvious visible phenotypes (Fig. 6). Furthermore, an extensive analysis of siblings among the progeny of plants that were homozygous for toc75-IV and heterozygous for toc75-III revealed no significant effect of a single toc75-III allele in the toc75-IV homozygous background (Fig. 6B). These data suggest that, if there is functional redundancy between the two proteins, a single atTOC75-III allele is sufficiently potent to compensate for the complete loss of atToc75-IV protein under normal circumstances. Similarly, even in the compromised, import-defective ppi1 background, the toc75-IV mutation did not give rise to any obvious visible phenotypes (Fig. 6).
Like the toc75-IV mutants, the previously identified atToc34 knockout mutants, ppi3-1 and ppi3-2, exhibit no obvious visible phenotypes in aerial tissues (Constan et al., 2004 ). However, the ppi3 mutants were found to display growth defects in the roots, a result that was interpreted to indicate an important role for atToc34 in the import of nonphotosynthetic proteins. To assess the possibility that the atToc75-IV protein plays a similar role in nonphotosynthetic protein import, we conducted root growth measurements much like those described previously (Constan et al., 2004). However, repeated measurements of plants grown under long-day conditions for 10 d (in total, approximately 100 plants of each genotype were measured) revealed no root length differences between the wild type and toc75-IV-1 or toc75-IV-2 (data not shown).
Since homozygous toc75-III seedlings could not be identified in the progeny of heterozygous toc75-III plants (Table I), we determined the stage of development at which the growth of toc75-III homozygotes terminates. In siliques of heterozygous toc75-III plants, aborted seeds were present at a frequency of approximately 25% (Table II; Fig. 7, K and L). This indicates that homozygous toc75-III mutations are embryo lethal, which is consistent with the notion that atToc75-III is the main Arabidopsis ortholog of psToc75 and with the fact that Toc75 plays a key, channel-forming role in the protein import mechanism.
To determine the exact stage at which development of homozygous toc75-III embryos terminates, we used Nomarski optics to observe developing seeds in heterozygous toc75-III siliques of different ages (Table III). Approximately 75% of embryos were observed to develop normally (Fig. 7, A–E). However, approximately 25% of the embryos in heterozygous toc75-III siliques did not develop beyond the two-cell stage (Fig. 7, F–I); i.e. when the normal embryos were at late globular stage (Fig. 7D), approximately 25% of the developing embryos had just two nuclei in the single apical embryo proper cell (Fig. 7I). When the normal embryos reached the heart stage (Fig. 7E), approximately 25% of the seed aborted (Fig. 7J). Additionally, the suspensor of homozygous toc75-III seed did not develop normally and was arrested after a single transverse division of the basal cell after the first zygotic division. Moreover, the free nuclear endosperm showed limited divisions and failed to cellularize, leading to nuclear fragmentation (Fig. 7I). Thus, we conclude that embryo development in toc75-III homozygotes arrests at the two-cell stage, and that this defect, together with endosperm failure, leads to seed abortion.
Etioplasts in the toc75-IV Mutants Have Structural Abnormalities
Since homozygous toc75-IV seedlings were found to have no obvious visible phenotypes (Fig. 6), we sought to identify a mutant phenotype by conducting more detailed studies. First, we compared the ability of isolated wild-type and toc75-IV mutant chloroplasts to import two different precursor proteins (preSSU and the 33-kD oxygen evolving complex precursor, preOE33), using published procedures (Aronsson and Jarvis, 2002
The expression data presented earlier (Fig. 2 and related discussion) suggest that atTOC75-IV may play a relatively more important role in etioplasts. We therefore analyzed the ultrastructure of etioplasts from 3-d-old, dark-grown wild-type and toc75-IV seedlings. While the prolamellar bodies and prothylakoids of toc75-IV etioplasts looked normal, we observed that some etioplasts contained enclosed bodies of cytosol, each apparently surrounded by normal envelope membrane (Fig. 8). We examined a relatively large number of etioplasts from wild-type, toc75-IV-1, and toc75-IV-2 seedlings, scored the number of etioplasts with cytosolic inclusions in each case, and then statistically analyzed the data (Table IV). Although approximately 20% of the wild-type etioplasts contained cytosolic inclusions, both toc75-IV alleles contained significantly more cytosolic inclusions than wild-type etioplasts (Table IV). The data were analyzed using the chi-squared test, and, because there are only two categories of data (one degree of freedom), we applied the Yates' correction, which makes the analysis more stringent. The null hypothesis that there was no significant difference between the wild-type and mutant data sets tested was rejected (critical
To investigate further the role of atToc75-IV, we transformed wild-type plants with an atTOC75-IV overexpression construct using the floral dip method (Clough and Bent, 1998 ). Twelve independent transformants were identified and shown to overexpress the atTOC75-IV gene by RT-PCR (data not shown). When grown in the light, these plants did not display any obvious phenotypes (data not shown). To assess the effect of atTOC75-IV overexpression on the cytosolic inclusions observed in the toc75-IV mutants (Table IV; Fig. 8), we examined the etioplasts of dark-grown seedlings in two of these transformed lines by electron microscopy. These two lines were estimated to display at least 30-fold increased levels of atTOC75-IV mRNA expression, relative to wild type, by RT-PCR (Fig. 2B). Remarkably, etioplasts from seedlings overexpressing atTOC75-IV contained significantly fewer cytosolic inclusions than wild-type etioplasts (Table IV). To assess whether or not the transgenic data deviated to a significant degree from the corresponding wild-type data, we again employed the chi-squared test with the Yates' correction. This analysis confirmed that the etioplasts of both transgenic lines contained significantly fewer cytosolic inclusions than wild-type etioplasts. We also found that there was no significant difference between the two wild-type samples (Table IV;  2-value = 1.68, P-value = 0.19), which were grown on separate occasions for comparison with the toc75-IV mutants and atTOC75-IV overexpression lines. Thus, these data strongly suggest that atToc75-IV has a role in etioplasts.
Expression (Fig. 2) and electron microscopy (Table IV; Fig. 8) data pointed toward a role for atToc75-IV in the etioplasts of dark-grown plants. To corroborate these findings, we carefully examined etiolated toc75-IV mutant plants for evidence of growth and developmental defects. First of all, we simply measured hypocotyl growth in dark-grown plants. However, repeated measurements of plants grown in the dark for either 5, 6, or 9 d (in total, approximately 800 plants of each genotype were measured) revealed no hypocotyl length differences between the wild type, toc75-IV-1, and toc75-IV-2 (data not shown). Since the most important function of etioplasts is the initiation of chloroplast development, following exposure to light, we next examined the ability of the toc75-IV mutants to undergo deetiolation. Wild-type and mutant plants were grown in the dark for 6 d and then transferred to continuous white light for a further period of 2 d. At the end of the 2-d light period, the plants were scored for visible evidence of deetiolation. Plants with green, expanded cotyledons were scored as having undergone deetiolation, whereas those with yellow, unexpanded cotyledons were scored as having failed to undergo deetiolation (Fig. 9A). This experiment was repeated 11 times, using multiple fresh batches of seed for each genotype (all of which were of the same age and produced by plants grown under identical conditions), including the same seed batches that were used to make the root and hypocotyl length measurements described earlier. Although the experiment was subject to substantial variability, presumably due to the stochastic nature of the deetiolation response and its acute sensitivity to environmental factors, the results revealed a significant difference between both toc75-IV mutants and the wild type (Fig. 9, B and C). In several individual experiments, a striking difference was observed between the mutants and wild type (e.g. Fig. 9B). In other experiments, this difference was less pronounced. Overall, however, the combined data of all 11 experiments (in total, >2,500 plants of each genotype were scored) revealed an approximately 50% reduction in deetiolation efficiency in the mutants by comparison with wild type (Fig. 9C). The combined toc75-IV-1 and toc75-IV-2 data sets were each statistically significantly different from the wild-type data set, as revealed by a Student's t test (P-values = 0.000016 and 0.0056, respectively); by contrast, the same test indicated that the two mutant data sets were not significantly different from each other (P-value = 0.073). These results demonstrate a physiological role for the atToc75-IV protein, most likely in etioplast biogenesis and/or the etioplast-chloroplast transition. They are also supportive of the ultrastructural data presented in Table IV and Figure 8, as well as of the data indicating that atToc75-IV is a plastidic protein shown in Figures 3 and 4.
The Toc75 protein forms the main protein import channel through the plastid outer envelope membrane (Schnell et al., 1994 ; Tranel et al., 1995; Hinnah et al., 2002). It works in concert with receptor proteins and is capable of recognizing, as well as translocating, chloroplast precursor proteins (Hinnah et al., 2002; Becker et al., 2004). Three Toc75-related sequences were previously identified in the Arabidopsis genome (Jackson-Constan and Keegstra, 2001). In this study, we used a range of techniques to characterize this small Arabidopsis gene family. Our data indicate that only two of the three family members, atTOC75-III and atTOC75-IV, are expressed genes. The third gene, atTOC75-I, has been inactivated due to a transposon insertion and the accumulation of numerous other mutations (Fig. 1, A and B). While atTOC75-III encodes a protein very similar to psToc75, atTOC75-IV encodes an amino-terminally truncated derivative (Fig. 1, A and B).
It is interesting to note that Schleiff et al. (2003b)
Based on the level and pattern of atTOC75-III expression, and on the high sequence identity shared between atToc75-III and psToc75, we conclude that atToc75-III is the main Arabidopsis ortholog of psToc75. The unique importance of atToc75-III was also demonstrated by the embryo lethality of the homozygous toc75-III mutations (Tables I–III; Fig. 7). As has been reported previously, plastids are essential for embryo development, presumably because they synthesize many important products that are utilized by the rest of the cell, including carbohydrates, fatty acids, amino acids, and terpenoids (Uwer et al., 1998
Various lines of evidence indicate that the atTOC75-III gene is expressed at much higher levels than atTOC75-IV (see "Results"). In Arabidopsis, the various homologous genes that encode other components of the Toc complex also display differing levels of expression. For example, atTOC159 is expressed at approximately 5- to 10-fold higher levels than its homologs, atTOC132 and atTOC120 (Bauer et al., 2000
It is interesting that the toc75-III mutation causes embryo lethality, especially since other individual components of the Toc complex (Toc34- and Toc159-related proteins, which are involved in preprotein recognition) are not essential for embryo development (Jarvis et al., 1998
Expression data suggested that the atToc75-IV protein might play a particularly important role during dark growth, or that a phenotype associated with the toc75-IV mutations might become more apparent in dark-grown seedlings (Fig. 2). Indeed, when the etioplasts of dark-grown plants were examined by electron microscopy, certain structural defects (an increased frequency of cytosolic inclusions) were observed in the mutants (Table IV; Fig. 8); as one might have expected, a diametrically opposed phenotype was observed in atTOC75-IV overexpressing plants (Table IV). Interestingly, elevated frequencies of very similar cytosolic inclusions were observed in the root plastids of atToc132/atToc120 double knockout mutants (Kubis et al., 2004
Since the envelope insertion of atToc75-IV is apparently not accompanied by proteolytic cleavage (Figs. 3 and 4), it is tempting to speculate that atToc75-IV gains access to the chloroplast using a different mechanism from its larger homolog, atToc75-III. This possibility is particularly interesting if one also assumes that atToc75-IV is a protein import channel with at least some functional similarity to atToc75-III. It may be significant that the targeting of atToc75-III is thought to be dependent upon a preexisting pool of correctly assembled atToc75-III (Tranel et al., 1995
Sequence Alignment and Phylogenetics
Amino acid sequences were aligned using ClustalW within the BioEdit program (Hall, 1999
All Arabidopsis (Arabidopsis thaliana) plants, both wild type and mutant, used in this study were of the Columbia-0 ecotype. For in vitro growth on Murashige and Skoog media, Arabidopsis seeds were surface-sterilized, and plants were grown at 20°C under a long-day cycle (16-h light/8-h dark) of 120 µmol photons m–2 s–1 white light, as described previously (Aronsson and Jarvis, 2002 Pea (Pisum sativum) plants were of the Little Marvel variety (SeedWay, Elizabethtown, PA). Pea seeds were soaked overnight with aeration, sown in vermiculite, and then allowed to grow at 20°C under a 12-h-light/12-h-dark cycle of approximately 30 µmol photons m–2 s–1 white light for 11 to 14 d.
RNA was isolated according to Kubis et al. (2003)
For RT-PCR analysis, first-strand cDNA was synthesized using 5 µg of total RNA, a primer with the sequence 5'-(T)17(A/G/C)N-3', and SuperScript II RNase H– reverse transcriptase (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. PCR amplifications were performed using the following primer pairs: atTOC75-I, forward (priF1 5'-TGG GCA TCG ATC GTG TAG CA-3', priF2 5'-AGT ATT GTT CCT GAT CCC-3', priF3 5'-ATA TCG AGA TCA TGC CAA T-3'), reverse (priR1 5'-AAG CTC AGC ACC AAC CTT TA-3', priR2 5'-TGT CCC TCT CTT CTG AAA GCC-3'); atTOC75-III, forward (5'-CGT ATC TGG ATG GTG TTT ACA ATC-3'), reverse (5'-GGA ATT CTT AAT ACC TCT CTC CAA ATC GGA AGA AC-3'); atTOC75-IV, forward (5'-CCA ATG TTT GTG GGT CGA GAT T-3'), reverse (5'-GGC TGC AGT TAG TAT CTC TCC CCG AAC C-3'); atTOC33, forward (5'-GGT CTC TCG TTC GTG AAT GG-3'), reverse (5'-CTG AGC GCC TAT GAT AAG AG-3'); eIF4E1, forward (5'-AAA CAA TGG CGG TAG AAG ACA CTC-3'), reverse (5'-AAG ATT TGA GAG GTT TCA AGC GGT GTA AG-3'). Each primer combination spanned an intron. For the expression profiles shown in Figure 2, PCRs used the minimum number of cycles necessary to observe a faint band following staining with ethidium bromide (atTOC75-III, 20 cycles; atTOC75-IV, 25 cycles; eIF4E1, 20 cycles). Products were resolved by agarose gel electrophoresis, blotted onto Hybond NX membrane (Amersham Pharmacia Biotech, Uppsala), and hybridized with gene-specific probes amplified using the primers listed above, according to the manufacturer's instructions (Amersham Biosciences, Little Chalfont, UK). Bands were visualized using a phosphorimager and quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). atTOC75-III and atTOC75-IV data were normalized using similar data for the uniformly expressed translation initiation factor gene, eIF4E1 (gene no. At4g18040; Rodriguez et al., 1998
The atTOC75-IV mRNA was characterized by RACE-PCR using the SMART RACE cDNA amplification kit (CLONTECH, Palo Alto, CA) according to the manufacturer's instructions. The gene-specific primers used were as follows: 5'RACE (5'-GCT GAA TGA AAC TTG TTA ATG ATA GT-3' and 5'-AAT CTC GAC CCA CAA ACA TTG G-3'); 3'RACE (5'-CCA ATG TTT GTG GGT CGA GAT T-3' and 5'-ACT ATC ATT AAC AAG TTT CAT TCA GC-3'). Sequences of the 5' and 3' RACE-PCR products were determined and then used to design primers for the amplification of a full-length cDNA clone by RT-PCR: forward, 5'-AAC TGC AGA ACA ATA TAG AGA GAA AGA AG-3'; reverse, 5'-CCA TCG ATG GAA AGA AAG CAG CAC AAA GTC-3'. The PCR product was cloned into pGEM-T Easy (Promega, Madison, WI), verified by sequencing, and then subcloned as a SacII/SpeI fragment into pBlueScript II KS– (Stratagene, La Jolla, CA). For overexpression in Arabidopsis plants, the atTOC75-IV cDNA was subcloned as a SacI/SalI fragment into the plant transformation/expression vector, pCHF2, which incorporates a double enhancer version of the cauliflower mosaic virus 35S promoter (Jarvis et al., 1998
For the pea import experiments, transcription/translation was performed in a coupled system containing rabbit reticulocyte lysate, [35S]Met, and T7 RNA polymerase, according to the manufacturer's instructions (TNT T7 Coupled Reticulocyte Lysate System; Promega). Plasmid DNA was used as template directly. The plasmids used have been described previously (Jackson et al., 1998
For the Arabidopsis import experiments, templates were amplified from cDNA clones by PCR using M13 primers. The atTOC75-III template was amplified from cDNA clone, APZL59c10R (the insert was subcloned into pBlueScript II KS- to enable use of the T7 promoter), and the atTOC75-IV template was amplified from the pBlueScript II KS– clone described above; the preSSU cDNA clone was described previously (Aronsson and Jarvis, 2002
Pea chloroplast isolations and in vitro import experiments, including high pH extractions and trypsin treatments, were carried out according to Inoue and Keegstra (2003)
Arabidopsis chloroplast isolations and in vitro import experiments were carried out essentially as described by Aronsson and Jarvis (2002)
For fractionation studies, chloroplasts were burst by incubation in 10 mM HEPES, 10 mM MgCl2, pH 8.0 (bursting buffer) for 10 min on ice. Total membrane samples were collected by centrifugation at 100,000g for 1 h. Thylakoid membranes were prepared according to Rawyler et al. (1992)
The atToc75-III protein was detected using an antibody raised against purified psToc75 (Tranel et al., 1995
The mutants used in this study were obtained from the Csaba Koncz Laboratory (Ríos et al., 2002
Gene-specific primers and insertion-specific primers (for the T-DNA left and right borders, LB and RB, or the 5' and 3' ends of the dSpm insertion) were used to follow the mutations by PCR. Plant DNA for PCR analysis was extracted as described previously (Edwards et al., 1991
Chlorophyll determinations and transmission electron microscopy were carried out as described previously (Porra et al., 1989
Siliques of different lengths were dissected on a microscope slide using syringe needle under a dissecting microscope (model Stemi-SV8, Zeiss, Germany). Embryos were cleared with a drop of clearing solution (240 g of chloral hydrate and 30 g of glycerol in 90 mL of water) for 30 min at room temperature. Preparations were examined with a microscope (model BHS, Olympus Optical, Tokyo) equipped for differential interference contrast (model BH2-NIC, Olympus). Images were captured and processed as previously described (Park et al., 1998 Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers N515928, At4g18040, AY127014, AL096859, At3g46740, NM_116977, AY585655, AAT08975, At4g09080, BK005428, and BX827624.
We thank Gabino Rios and Csaba Koncz for assisting in the identification of the toc75-III-1 mutant and Jonathan Jones for the toc75-IV-1 mutant. We are grateful to Natalie Allcock and Stefan Hyman for transmission electron microscopy, Jocelyn Bédard for assistance during the annotation of atTOC75-I, Colin Ferris for help with the phylogenetic analysis, and Henrik Aronsson, Jocelyn Bédard, Sabina Kovacheva, and Sybille Kubis for their comments on the manuscript. We thank Syngenta and SIGnAL for providing additional T-DNA lines. Funding for the SIGnAL indexed insertion mutant collection was provided by the National Science Foundation, and the seed were distributed by the Nottingham Arabidopsis Stock Centre. We are grateful to Neil Hoffman, Kenneth Keegstra, and Steven Theg for kindly providing antibodies and to the Kazusa DNA Research Institute for the atTOC75-III cDNA clone, APZL59c10R. Received March 23, 2005; returned for revision April 26, 2005; accepted April 27, 2005.
1 This work was supported by the University of Leicester (Ph.D. studentship to A.B.), by the United States Department of Agriculture Cooperative State Research, Education, and Extension Service (grant no. 2003–02860 to K.I.), by the Royal Society (Rosenheim Research Fellowship to P.J.), and by the Biotechnology and Biological Sciences Research Council (grant nos. 91/C12976, 91/P12928, and 91/C18638 to P.J.). 
2 These authors contributed equally to the paper.
3 Present address: Department of Plant Sciences, University of California, One Shields Avenue, Davis, CA 95616.
4 Present address: Division of Plant Bioscience, Kyungpook National University, Daegu 702–701, Korea. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.063289. * Corresponding author; e-mail rpj3{at}le.ac.uk; fax 44–116–252–3330.
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-barrel channel with a pore size of approximately 14 to 26 Å (Hinnah et al., 2002
gene. Plant Physiol 126: 717–730
pH pathways but not the chloroplast signal recognition particle pathway. Plant Physiol 119: 575–584
