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An Arabidopsis Mutant Defective in the Plastid General Protein Import Apparatus

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Science  02 Oct 1998:
Vol. 282, Issue 5386, pp. 100-103
DOI: 10.1126/science.282.5386.100

Abstract

Elaborate mechanisms have evolved for the translocation of nucleus-encoded proteins across the plastid envelope membrane. Although putative components of the import apparatus have been identified biochemically, their role in import remains to be proven in vivo. AnArabidopsis mutant lacking a new component of the import machinery [translocon at the outer envelope membrane of chloroplasts (Toc33), a 33-kilodalton protein] has been isolated. The functional similarity of Toc33 to another translocon component (Toc34) implies that multiple different translocon complexes are present in plastids. Processes that are mediated by Toc33 operate during the early stages of plastid and leaf development. The data demonstrate the in vivo role of a translocon component in plastid protein import.

The routing of newly synthesized proteins to appropriate subcellular compartments is a fundamental process that is common to all organisms. In plants, chloroplasts (the photosynthetic plastids of green tissues) are the major target of such protein trafficking because they account for >50% of the total soluble protein in leaves and because >80% of the proteins required for their formation are encoded in the nucleus (1). The translocation of proteins across the envelope membrane is especially important early on in chloroplast and leaf development, when the photosynthetic apparatus is being assembled for the establishment of photoautotrophic growth (1). The capacity of plastids to import proteins is regulated developmentally and is maximal during these early stages of organ expansion (2).

Genetic screens for loci affecting the expression of nucleus-encoded photosynthetic proteins have identified several mutants with defects in plastid biogenesis (3, 4). Here, we describe a new mutant, initially referred to by the number 127-4, which belongs in this category. The 127-4 mutant was identified as having a recessive, pale phenotype in a population of transferred DNA (T-DNA)–mutagenized plants. Although the mutant appeared uniformly pale during the first 2 weeks of its life cycle (Fig. 1, A and B), the oldest leaves of mature plants frequently had an appearance closer to that of the wild type (Fig. 1C) (5).

Figure 1

The visible phenotype of the 127-4 (ppi1) mutant. Plants were grown under long-day conditions (16 hours of light and 8 hours of dark) for (A) 5 days, (B) 13 days, and (C) 25 days (5). Wild-type plants are shown on the left, andppi1 plants are shown on the right. Scale bars, 2 mm.

The gene disrupted by the T-DNA insertion (Fig. 2A) encodes a 33-kD protein that is very similar to Toc34 from Arabidopsis (61% amino acid identity) and pea plants (59% amino acid identity) (Fig. 2B);Arabidopsis Toc34 and pea Toc34 share 64% amino acid identity (6–8). Toc34 is a guanosine triphosphate (GTP)–binding protein of the plastid outer envelope membrane. Toc34, Toc75, and Toc86 are components of the outer envelope segment of the general import apparatus through which the majority of proteins destined for the plastid are believed to pass (9). The involvement of Toc34 in protein import is inferred from its interaction with preproteins during import and from its association with Toc75 and Toc86 (10). However, its precise role in the import process is not known. The 127-4 mutant was named ppi1(for plastid protein import), but we will refer to the protein encoded by the PPI1 gene as Toc33, in accordance with biochemical nomenclature (11). The size (an estimated 13 kb) and location (in intron 2) of the T-DNA insertion, combined with our failure to detect any mRNA in mutant plants (12), indicated that the ppi1 mutation was most likely null.

Figure 2

Identity of the 127-4 (PPI1) gene. (A) Schematic representation of the gene disrupted in the 127-4 (ppi1) mutant (8). The depicted gene encodes the Toc33 protein. ATG, translation initiation codon; LB, left border of T-DNA insertion (not to scale); stop indicates translation termination codon; and poly(A), polyadenylation site. (B) Alignment of the Toc33, Arabidopsis thaliana Toc34 (atToc34), and pea (Pisum sativum) Toc34 (psToc34) amino acid sequences (8,19). Dashes indicate gaps introduced to maximize alignment; residues conserved in at least two of the three sequences are shaded. (C) Complementation of theppi1 mutant. Mutant plants that were transformed with a wild-type (WT) copy of the PPI1 gene (Toc33 genomic), with a Toc33 overexpression construct (35S-Toc33), or with a Toc34 overexpression construct (35S-Toc34) were assayed for their chlorophyll content after 13 days of growth in continuous white light (14, 15). The presented data are average values from at least six transformants in each case. Error bars indicate 1 SE.

Given their strong sequence similarity, we tested the hypothesis that Toc33 and Toc34 might correspond to functionally equivalent translocon components. In vitro transcribed and translated Toc33 protein was found to insert itself into the outer envelope membrane of isolated pea chloroplasts in a similar manner to ArabidopsisToc34 (12, 13), which suggests that the two proteins are similarly localized in vivo. We subsequently overexpressed Toc33 and Toc34 cDNA clones in ppi1 plants and found that either protein could complement the chlorophyll deficiency of ppi1, which confirms the functional similarity of the two proteins (Fig. 2C) (14, 15). The existence of two functionally similar Toc proteins suggests that at least two distinct translocon complexes exist in Arabidopsis. This observation is of particular interest because biochemical studies of pea chloroplasts have revealed the presence of only one Toc complex (9).

Analyses of plastids from pea and wheat plants have demonstrated that protein import capabilities can vary substantially through development (2). The abundance of Toc75 mRNA was shown in pea plants to be greater in young tissues than in mature leaves (2). To assess the role of Toc33 in the regulation of plastid import in Arabidopsis, we examined the expression patterns of Toc33 and Toc34 and characterized the ppi1phenotype with respect to chlorophyll accumulation, plastid ultrastructure, and plastid protein import. Steady-state levels of Toc33 and Toc34 mRNAs were examined over the course of theArabidopsis life cycle (Fig. 3A). Toc33 was expressed at high levels in very young plants, but its expression declined rapidly as the age of the plant increased. Although the expression of Toc34 appeared to follow a similar trend, Toc34 mRNA remained at a relatively low level throughout development. The expression of both genes was about three times higher in the young, expanding tissues of 25-day-old plants than in the mature, fully expanded leaves of the same plants; this suggests that the expression of Toc33 in very young plants (Fig. 3A) is elevated because these plants have more young, expanding tissues.

Figure 3

Developmental time-course analyses of gene expression and chlorophyll accumulation. (A) Northern (RNA) blot analyses of Toc33 and Toc34 expression at different stages in the development of wild-typeArabidopsis. RNA was extracted from whole plants or from the young and old aerial tissues of 25-day-old plants (d, day). Probes of similar specific activities were prepared with full-length cDNA clones of each gene; no cross-hybridization between Toc33 and Toc34 was observed. Quantitation was performed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and was normalized for 18S ribosomal RNA. (B) and (C) show chlorophyll accumulation in wild-type and ppi1 plants throughout development. (B) Total chlorophyll per plant was determined for each genotype at different stages of development (15). Error bars indicate 1 SE. (C) Alternative presentation of the data in (B);ppi1 data are expressed as a percentage of the wild-type data. All plants were grown in continuous white light.

Mutant plants contained reduced levels of chlorophyll at all developmental stages (Fig. 3B) (15). However, they were extremely pale at the earliest measured time (day 2), and except for a period between days 5 and 10, chlorophyll levels improved steadily throughout development (Fig. 3C). The first observation indicates that Toc33 functions very early in development (before day 2) in the expanding cotyledons. The second observation suggests that Toc33 has a role during the emergence of the primary leaves (between days 5 and 10) and not during other periods; it is likely that the effect ofppi1 on chlorophyll accumulation during the emergence of later leaves is masked in this assay by the presence of older leaves. Together, these data imply an involvement of Toc33 in chloroplast development that occurs concomitantly with the expansion of newly formed tissues.

The ultrastructure of mutant plastids from different stages of development was examined [electron microscopy was carried out according to described procedures (3)]. The most pronounced defect of etioplasts was the markedly reduced size of the prolamellar body (an estimated 50% reduction in cross-sectional area) (Fig. 4, A and B). The nucleus-encoded protochlorophyllide oxidoreductase (POR) enzyme is the primary proteinaceous component of the prolamellar body (16). The mutant chloroplasts were smaller than those in the wild type and contained fewer thylakoid membranes with smaller granal stacks. Chloroplast defects appeared far more pronounced in 5-day-old plants (Fig. 4, C and D) than in 4-week-old plants (Fig. 4, E and F), and thus, the defects parallel the visible phenotype of the mutant (Fig. 1, A and C). These data again indicate that Toc33 functions during the early stages of plastid development. At no stage do ppi1plastids appear aberrant to a degree that is indicative of a complete block in their development; this observation can be accounted for by the observed basal expression of Toc34 (Fig. 3A).

Figure 4

Ultrastructure ofppi1 plastids at different stages of development. Representative plastids from (A) 5- day-old etiolated wild-type cotyledons; (B) 5-day-old etiolatedppi1 cotyledons; (C) 5-day-old light-grown wild-type cotyledons; (D) 5-day-old light-grownppi1 cotyledons; (E) 4-week-old light-grown wild-type leaves; and (F) 4-week-old light-grownppi1 leaves [electron microscopy was carried out according to described procedures (3)]. (A) and (B) are at higher magnification than (C) through (F). Scale bars, 1 μm.

To determine the precise nature of the defect in ppi1plants, we assayed the import of several proteins into mutant chloroplasts isolated from plants grown for either 10 or 28 days (13). Chloroplasts isolated from 28-day-old plants were largely derived from mature, fully expanded leaves, whereas a substantial proportion of chloroplasts isolated from 10-day-old plants were derived from newly expanding primary leaves. We found that eight tested proteins (RBCS, PORA, PORB, CAB, OEP14, Toc33, Toc34, and Toc75) were imported into the plastids of mature ppi1 plants with wild-type efficiencies (12). However, the translocation efficiencies of all four tested proteins (RBCS, PORA, PORB, and CAB) into the plastids of 10-day-old mutant plants were reduced (Fig. 5A); the amounts of each protein imported into mutant plastids, which were expressed as percentages of the protein amounts imported into wild-type plastids, were 59% (RBCS), 68% (PORA), 61% (PORB), and 75% (CAB). To corroborate the findings of these studies in organelles, we conducted immunoblot experiments that were designed to assess plastid protein import defects in vivo. Five-day-old etiolated mutant plants contained reduced levels of the mature POR form (POR; 36 kD) and a pronounced accumulation of nonprocessed POR preprotein (pPOR; 44 kD) (Fig. 5B). The accumulated pPOR was stable for at least 48 hours after the transfer of etiolated plants to continuous light, which implied that the pPOR had not been imported because a chloroplast-specific protease degrades both POR and pPOR (12, 17).

Figure 5

Analyses of plastid protein import in the ppi1 mutant. (A) In-organelle analysis of protein import into wild-type and mutant plastids isolated from 10-day-old plants. The import of four different protein precursors was investigated: RBCS, PORA, PORB, and CAB. Chloroplasts were repurified after the import reactions, and equal numbers of chloroplasts were loaded in each lane (13). (B) In vivo immunoblot analysis of POR protein in 5-day-old etiolated wild-type and mutant plants. Precursor proteins are indicated with open triangles; mature proteins are indicated with solid circles.

The data suggest that the changing import capabilities of maturing plastids result from variations in the number of translocon complexes, which are brought about in turn through changes in translocon gene expression. Given the functional similarity between Toc33 and Toc34, the reason for the presence of both proteins is unclear. One possibility is that two separate genes, whose transcription can be regulated independently, provide the simplest means of achieving the necessary pattern of expression. The fact that the translocation efficiencies of some preproteins vary between different plastid types suggests that the import machinery possesses a certain degree of specificity (18). Although our transgenic data indicate functional similarity between the two proteins (Fig. 2C), it is possible that, under normal conditions, subtle functional differences exist.

  • * To whom correspondence should be addressed. E-mail: chory{at}salk.edu

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