Deletion of the chloroplast-localized AtTerC gene product in Arabidopsis thaliana leads to loss of the thylakoid membrane and to seedling lethality

Authors


*(fax +82 2 312 5657; e-mail mhcho@yonsei.ac.kr).

Summary

Early seedling development in plants depends on the biogenesis of chloroplasts from proplastids, accompanied by the formation of thylakoid membranes. An Arabidopsis thaliana gene, AtTerC, whose gene product shares sequence similarity with bacterial tellurite resistance C (TerC), is shown to be involved in a critical step required for the normal organization of prothylakoids and transition into mature thylakoid stacks. The AtTerC gene encodes an integral membrane protein, which contains eight putative transmembrane helices, localized in the thylakoid of the chloroplast, as shown by localization of an AtTerC–GFP fusion product in protoplasts and by immunoblot analysis of subfractions of chloroplasts. T-DNA insertional mutation of AtTerC resulted in a pigment-deficient and seedling-lethal phenotype under normal light conditions. Transmission electron microscopic analysis revealed that mutant etioplasts had normal prolamellar bodies (PLBs), although the prothylakoids had ring-like shapes surrounding the PLBs. In addition, the ultrastructures of mutant chloroplasts lacked thylakoids, did not have grana stacks, and showed numerous globular structures of varying sizes. Also, the accumulation of thylakoid membrane proteins was severely defective in this mutant. These results suggest that the AtTerC protein plays a crucial role in prothylakoid membrane biogenesis and thylakoid formation in early chloroplast development.

Introduction

Proper development of plants during the early seedling phase is essential to the long-term health of the plant. During early seedling development, proplastids are converted into chloroplasts, whose purpose is to obtain energy. This conversion is not possible without thylakoid biogenesis. Recently, several studies have been carried out to elucidate which and how many genes are essential for seedling development and viability using mutant pools tagged with T-DNA and Ds transposons in Arabidopsis (Budziszewski et al., 2001; McElver et al., 2001).

According to Budziszewski et al. (2001), a high proportion of seedling-lethal mutants with pigmentation defects are likely to be affected in nuclear-encoded chloroplast proteins, because seedlings depend primarily on chloroplast function for energy supply. Many genes homologous to those mutated in the chloroplast-defective mutants are also found in prokaryotes (Adam et al., 2006; De Santis-Maciossek et al., 1999; Rodríguez-Concepción and Boronat, 2002; Settles and Martienssen, 1998; Wall et al., 2004). As all chloroplasts of photosynthetic eukaryotes are assumed to have arisen from photosynthetic bacterial endosymbionts that were engulfed by eukaryotic hosts (Douglas, 1998), and most endosymbiont genes have been transferred to the host genome (Martin and Herrmann, 1998; Martin et al., 1998), many processes and mechanisms occurring in this organelle have analogies to bacterial counterparts.

Although it is assumed that chloroplasts are the descendents of ancestors of present cyanobacteria, today’s higher plants have a more complex and elaborate internal membrane network of thylakoids than their presumed ancestors. Although many green algae and some cyanobacteria have lateral heterogeneity and areas of appressed thylakoid membrane, they do not possess such a highly structured multiple membrane (Mullineaux, 2005).

The formation of this sophisticated thylakoid membrane is initiated during the transition of proplastids into chloroplasts by light, followed by coordinated gene expression of nuclear and chloroplast genomes (Taylor, 1989). As the precise assembly of lipids and proteins into thylakoids is essential to chloroplast formation, mutations that affect this developmental process produce plastids that lack an internal membrane and show abnormal morphology. It has been suggested that the lipid component of the thylakoid membrane is derived from vesicles pinched off from the inner membrane (Hoober et al., 1994), and that thylakoid membrane formation is mediated by a eukaryotic-type vesicle transport system between the inner envelope and the thylakoid (Westphal et al., 2003). An Arabidopsis mutation, vipp1, is involved in vesicle formation at the inner membrane of the chloroplast. The vipp1 mutant shows a gradual disintegration of thylakoid membrane under normal light conditions (Kroll et al., 2001).

The photosynthetic multi-protein complexes, embedded in the thylakoid membrane, are mosaic structures, consisting of subunits encoded by both the nucleus and chloroplast genomes. In orchestrating coordinated gene expression, reciprocal signaling between both compartments is important in regulating balanced expression of chloroplast proteins (Strand, 2004; Surpin et al., 2002).

Mutations that interfere with thylakoid protein complex formation impair the structure of the plastids. Such mutants include hcf136, an essential protein for the stability of photosystem II (PSII) in Arabidopsis (Meurer et al., 1998), alb3, mutated in a subunit of the thylakoid Sec protein transport system in Arabidopsis (Klostermann et al., 2002; Sundberg et al., 1997), and apg2, mutated in a component of the Arabidopsis ΔpH-dependent thylakoid protein transport machinery (Motohashi et al., 2001). These mutants demonstrated abnormal ultrastructures of plastids, including reduction or loss of thylakoid membranes, and, in some cases, highly vacuolated structures.

Recent studies of the genes implicated in the complex biogenesis of the chloroplast have provided new insights into how some of these components work and how a functional complex is assembled in the thylakoid membrane along with numerous pigments, redox molecules and lipids. However, understanding of the mechanism regulating the transition from prothylakoid to thylakoid, as well as the spatial organization and stacking of the grana, is far from complete. This study isolated pigment-deficient and seedling-lethal mutants that possess a mutation in a gene that was previously annotated as the integral membrane protein tellurite resistance C (TerC) first described in prokaryotes and is involved in thylakoid membrane biogenesis in Arabidopsis.

Results

Phenotype analysis and molecular characterization of atterC mutants

A collection of Arabidopsis plants mutagenized with T-DNA (Alonso et al., 2003) was screened for plants with pigment-deficient and seedling-lethal phenotypes. The atterC-1 mutant (SALK_014736) was chosen for further characterization, as a second allele (atterC-2, GABI-Kat 844D10) of this mutation has been identified, which displayed identical phenotypes to atterC-1 in a different T-DNA mutant pool (Rosso et al., 2003).

As shown in Figure 1(a), both types of homozygous mutant seedling displayed identical visible phenotypes, including pigment deficiency and seedling lethality. Heterozygous plants of both mutants appeared identical to the wild-type. The progeny of self-pollinated heterozygous plants segregated as the wild-type and mutant phenotypes at a ratio of approximately 3:1 on non-selective medium. The wild-type siblings selected from self-pollinated heterozygotes showed wild-type phenotypes in successive generations. These results demonstrate that these mutations are recessive and that the mutant phenotype co-segregates with the T-DNA insertion.

Figure 1.

 Characterization of two AtTerC knockout mutants and Northern blot analyses.
(a) Pigment-defective and seedling-lethal phenotypes of atterC-1 and atterC-2. Plants were grown for 4 days (upper row) or 14 days (lower row) under continuous light. Scale bar = 1 mm.
(b) Disruption of AtTerC expression in two atterC mutants caused by T-DNA insertions. Northern blot analysis was performed using 15 μg of total RNA isolated from wild-type (WT) seedlings and two atterC mutants grown on agar medium for 14 days (16/8 h light/dark cycle). The 357 bp cDNA sequence corresponding to the 5′ region of the AtTerC gene (NB-P1) was used as a probe. A probe for 18S rRNA served as a loading control.
(c) Schematic representation of T-DNA insertions in the atterC-1 and atterC-2 mutants. Upper-case letters are the AtTerC sequence and lower-case letters are the T-DNA sequences in the T-DNA insertion positions. Three nucleotides of unknown origin in atterC-1 are shown in italics at the T-DNA border junction. Exons, black rectangles; introns, black solid lines; 5′ and 3′ untranslated regions, white rectangles; B, BamHI restriction site (the B indicated within the T-DNA is about 1 kb from the right LB border); NB-P1, the region corresponding to the probe used in Northern analysis in (b); SB-P, the region corresponding to the probe used in Southern analysis in (d); the arrows indicate the regions corresponding to the primers used in genotyping for each mutant. The T-DNAs are not drawn to scale.
(d) Genomic Southern analysis (BamHI digestion). The arrow indicates the atterC-1 mutant allele and the arrowhead indicates the AtTerC transgenic allele in a complemented (Comp.) line.
(e) Complementation of the atterC-1 mutant. The upper row shows 14-day-old wild-type, atterC-1, and complemented (Comp.) seedlings. The middle row shows Northern blot analysis for expression of the trans-AtTerC gene performed using a full-length cDNA probe of AtTerC. 18S rRNA was used as a loading control. Scale bar = 1 mm. (f) Northern blot analysis of the AtTerC transcript in various tissues. Fifteen micrograms of total RNA was purified from each tissue of a 4-week-old wild-type plant. The full-length AtTerC cDNA was used as a probe and 18S rRNA as a loading control.

To further characterize the mutant phenotype, the atterC-1 mutation was examined in more detail. Dark-grown atterC-1 mutants were indistinguishable from etiolated wild-type seedlings. However, after exposure to normal light conditions (approximately 150 μmol photons m−2 sec−1), the etiolated atterC-1 mutants failed to accumulate chlorophylls in the cotyledons. Mutants died within two weeks in soil. Under dim light conditions (approximately 5 μmol photons m−2 sec−1), atterC-1 mutants showed pale-green cotyledons and leaves (data not shown). These results indicate that although AtTerC is required for chlorophyll accumulation during early seedling development under normal growth conditions, AtTerC does not appear to be directly involved in chlorophyll biosynthesis.

As shown in Figure 1(b), Northern blot analysis indicated that the wild-type form of AtTerC mRNA was undetectable in both mutants when a probe corresponding to a fragment of 5′ region of AtTerC cDNA was used. However, multiple and aberrant transcripts were detected in atterC-2 when cDNA probes corresponding to another region of AtTerC mRNA were used (Figure S1). Similar results were shown previously in other studies (Matsuhara et al., 2000; Mengiste et al., 1999). As the transformation vector, pAC106, used for atterC-2 has two CaMV 35S promoters within the T-DNA delivered by the vector, these abnormal transcripts are probably initiated within the T-DNA insert and are not functional, considering the identical phenotype of both mutants.

PCR and sequence analysis revealed that the borders of the T-DNA were in the second intron of atterC-1 and in the third exon in atterC-2 (Figure 1c). For atterC-1, the inserted T-DNA was joined to the 67th nucleotide within the intron of the AtTerC gene. For atterC-2, the T-DNA was inserted between the 134th and 136th nucleotide of the third exon of AtTerC. The 135th guanosine nucleotide of the exon was deleted. These data indicate that the genomic DNA of AtTerC was rearranged in the mutant genome by T-DNA insertions.

Molecular complementation of the atterC mutation

To verify that the pigment-deficient and seedling-lethal phenotypes were associated with the T-DNA insertion into the AtTerC gene, molecular complementation was performed. A 4.2 kb, full-length AtTerC genomic copy was introduced into the heterozygous background (atterC/AtTerC) by in planta transformation. Twenty-six independent T1 transgenic seeds were selected, using the presence of a strong GFP fluorescent signal as a selection marker (see Experimental procedures). Four complemented lines were identified that have a homozygous mutant allele (atterC/atterC) background. The phenotype of one representative among the selected T2 complementation lines, which is indistinguishable from the wild-type plant, is shown in Figure 1(e). Genotypic characterization of the complemented line was performed by genomic DNA blot analysis to confirm that the transformant contained a homozygous atterC-1 mutant allele and a wild-type AtTerC transgene (Figure 1d). Northern blot analysis of the complemented line showed a wild-type band of AtTerC mRNA (Figure 1e). These analyses show that the ectopic expression of AtTerC prevented the pigment-deficient seedling growth and restored the seedling growth to that of wild-type plants. Therefore, the mutant phenotypes were conferred by the T-DNA insertional mutation in the AtTerC gene.

Identification of the AtTerC gene and phylogenetic analysis of the TerC homologues

DNA sequencing of the isolated regions flanking the two T-DNA insertions and subsequent database searches revealed that these sequences were contained in the bacterial artificial chromosome (BAC) clone MXC 9 (GenBank accession number AB007727). The MIPS (Munich Information Center for Protein Sequences) code for the gene is At5g12130. Although the gene was already annotated as the integral membrane protein TerC (tellurite resistance C) in Arabidopsis, and recently named as pigment-defective 149 (PDE149) in the database of the ‘Arabidopsis SeedGenes Project’ (Tzafrir et al., 2003), there are to date no published reports concerning its functions in plants.

The AtTerC gene encodes a polypeptide composed of 384 amino acids with an estimated molecular mass of 41.9 kDa. Analysis of the deduced amino acid sequence (Hofmann and Stoffel, 1993) of AtTerC indicates that it encodes an integral membrane protein containing eight possible transmembrane helices (Figure 2a). The ChloroP program (Emanuelsson et al., 1999) predicted localization of AtTerC to the chloroplast and that it contains a 48 amino acid cleavable transit peptide in the N-terminus (Figure 2a). A database search using the cDNA sequence of AtTerC revealed that the AtTerC gene is present as a singleton in the Arabidopsis genome.

Figure 2.

 Similarity of AtTerC to other homologous proteins and phylogenetic analysis.
(a) Alignment of the deduced amino acid sequences from Arabidopsis TerC and homologoues from other species. The sequences were aligned using clustal w (Thompson et al., 1994). Gaps have been introduced to maximize the alignment. Hatched lines represent the regions of the conserved TerC domain. The arrowhead denotes the putative cleavage site of the transit peptide in the AtTerC amino acid sequence. Lines above the sequences show the predicted transmembrane helix regions. Black boxes indicate amino acid residues that are >80% conserved, and gray boxes indicate amino acids that are >50% conserved. The amino acid sequences were GenBank NP_001054496 for Oryza sativa (O.s), CAL57657 for Ostreococcus tauri (O.t), ZP_01426499 for Herpetosiphon aurantiacus ATCC 23779 (H.a), NP_927355 for Gloeobacter violaceus PCC 7421 (G.v), YP_843886 for Methanosaeta thermophila PT (M.t) (all from Genbank), and TC332751 for Zea mays (Z.m), TC61933 for Saccharum officinarum (S.o), TC182062 for Lycopersicon esculentum (L.e) (all from the Institute for Genomic Research gene indices; http://tigrblast.tigr.org/tgi/), and aligned with the Arabidopsis thaliana protein (A.t) (GenBank accession number NP_568257). The deduced amino acid sequence of Saccharum officinarum is not full length.
(b) Phylogenetic analysis was performed with the alignment described in (a) using the mega 4 program (Tamura et al., 2007). The phylogenetic tree was constructed using the neighbor-joining method. The bootstrap values were determined from 1000 trials. Numbers along branches indicate the percentage of bootstrap support. The length of the branch lines indicates the extent of divergence according to the scale (relative units) at the bottom. Sequence identity (%) indicates amino acid sequence identity with the AtTerC protein.

A database search for AtTerC homologues in other species revealed that homologues to AtTerC are present in a variety of bacteria, archea, algae and other plant species. The common feature of these homologues was found to be a TerC domain. This conserved region comprises amino acids 145–356 for AtTerC (Figure 2a). However, there were no other sequence motives or domains other than the TerC domain that suggested the possible function of the AtTerC protein.

TerC was first cloned along with other components as a tellurite resistance (Ter) determinant encoded by the bacterial IncHI2 plasmid pMER610 (Jobling and Ritchie, 1987, 1988). However, the mechanisms responsible for this resistance remain elusive. As shown in Figure 2(a), there are very few similarities in the N-terminal regions among the TerC homologues. The N-termini of TerC homologues in higher plants are longer than those of homologues in other taxa. These N-terminal extensions may provide additional functions for higher-plant homologues. For example, they may serve to guide TerC proteins to intracellular organelles.

To investigate the relationship between TerC homologues in evolutionary history, a phylogenetic analysis was performed (Figure 2b). There is a close evolutionary relationship between AtTerC and homologues in the eukaryotic photosynthetic organisms. When comparing AtTerC with prokaryotic homologues, the TerC homologue of cyanobacteria, Gloeobacter violaceus PCC 7421, was found to have the highest similarity to AtTerC. Two TerC homologues are found in the strict anaerobic and methanogenic euryarchaeota archaea. In addition, there are TerC homologues in many other bacterial taxa, including proteobacteria, enterobacteria, eubacteria and high-GC Gram-positive bacteria. Previous studies on microbial molecular phylogeny have proposed that there have been extensive lateral gene transfers within bacteria and between bacteria and archaea in evolution (Doolittle, 1999). Therefore, the phylogenetic and sequence similarity analysis of TerC suggests that the TerC is an ancient gene, and that eukaryotic TerC genes possibly originated from bacteria.

To determine the spatial expression profiles of the AtTerC gene, RNA gel-blot analysis was carried out using total RNA extracted from various tissues. The AtTerC transcript was detected in all tissues analyzed from 6-week-old wild-type Arabidopsis plants (Figure 1f).

AtTerC is involved in the accumulation of photosynthetic pigments and in the expression of genes for photosynthetic proteins

To understand the nature of the pigment deficiency in mutant seedlings, the contents of several pigments were measured, including chlorophyll, carotenoid and anthocyanin. As shown in Figure 3(a), total chlorophyll content was the most severely reduced in atterC-1 compared to that of the wild-type, among all pigments analyzed. The total chlorophyll content of atterC-1 was approximately 10% of the wild-type level. In addition, it was shown that the carotenoid level of atterC-1 was also reduced to 34% of the wild-type level, whereas the atterC-1 anthocyanin content was similar to that of the wild-type.

Figure 3.

 Relative amounts of pigments and Pchlide in the wild-type (WT) and atterC-1 mutant.
(a) Content of pigments in wild-type and atterC-1. Seedlings were grown on MS agar medium for 4 days under continuous light. Mean values were obtained from three independent experiments (n = 30 or 40 each) are based on fresh weight (mg). Error bars represent SD.
(b) Relative fluorescence of Pchlide in wild-type and atterC-1. Seedlings were grown on MS agar medium in the dark for 4 days and then exposed to light for 20 h, and further incubated in the dark for 2 days. The fluorescence emission spectra represent the mean values for three independent experiments.
(c) Relative mRNA levels of light-inducible genes in the wild-type and atterC-1. Five micrograms of total RNA was purified from 4-day-old wild-type and atterC-1 seedlings grown under continuous light. A probe for 18S rRNA served as a loading control.

As the reduction of chlorophyll has been shown to be generally accompanied by a reduction in carotenoid content, it was necessary to determine whether the pigment-deficient phenotype is caused by the disruption in the pathway of chlorophyll biosynthesis. Thus, this study analyzed the relative amounts of protochlorophyllide (Pchlide) in wild-type and atterC-1 seedlings. Pchlide accumulates in the dark and is immediately converted to chlorophyllide a (Chlide a), a precursor of chlorophyll a, when plants are exposed to light (Reinbothe et al., 1996). Fluorescence spectroscopic data showed an approximately 1.4-fold higher Pchlide content in atterC-1 than in the wild-type, on the basis of fresh weight (Figure 3b). However, Pchlide levels were similar in the wild-type and atterC-1 when compared on a seedling basis (Figure 3b). When the fresh weights of the wild-type and atterC-1 were compared, the mutants always showed a reduced weight (about 70% of that of the wild-type). Considering that loss of the photosynthetic activity in atterC-1 led to an overall reduction of fresh weight, atterC-1 had higher relative amounts of Pchlide than the wild-type. Thus, these results suggest that the capability for chlorophyll biosynthesis is intact in atterC-1 mutant plants, as in the wild-type.

Plastid-emitted signals can effect the expression of nuclear-encoded photosynthesis proteins by means of a process known as retrograde signaling, depending on the functional state of the plastid (Koussevitzky et al., 2007; Mayfield and Taylor, 1984; Oelmüller et al., 1986; Strand et al., 2003). Therefore, the expression levels of the nuclear-encoded chloroplast proteins chlorophyll a/b binding protein (CAB) and ribulose-1,5-bisphophate carboxylase/oxygenase small subunit (rbcS) were examined. As shown in Figure 3(c), transcript levels of CAB and rbcS in atterC-1 were reduced to 42% and 58% of wild-type levels, respectively, when normalized to 18S rRNA. However, the expression levels of the nuclear-encoded non-photosynthetic proteins, chalcone synthase (CHS) and dihydroflavonol reductase (DFR) were similar in atterC-1 and the wild-type.

Taken together, these results strongly suggest that AtTerC is involved in chloroplast development rather than in the chlorophyll biosynthetic pathway.

AtTerC is localized into the thylakoid membrane of chloroplasts

To determine the precise intracellular localization of AtTerC, transgenic plants were produced, expressing AtTerC–GFP fusion constructs under the control of the CaMV 35S promoter. Confocal fluorescence microscopy of protoplasts isolated from 35S::AtTerC:GFP and 35S::GFP transgenic plants showed that the AtTerC–GFP fusion protein was localized to the chloroplast, as the green fluorescence of the AtTerC chimeric protein co-localized with the red autofluorescence of the chloroplast, while GFP fluorescence was detected only in the cytoplasm of protoplasts isolated from 35S::GFP transgenic plants (Figure 4a). This result indicates that AtTerC is translocated into the chloroplast.

Figure 4.

 Subcellular and sub-organellar localization of AtTerC–GFP.
(a) In vivo targeting of AtTerC:GFP in protoplasts. Protoplasts were isolated from 35S::GFP and 35S::AtTerC:GFP transgenic plants. Cells were observed under a confocal laser scanning microscope. Green fluorescence signals, chlorophyll red autofluorescence, an overlay of green and red signals, and bright-field images are shown. Scale bar = 5 μm.
(b) Immunoblot assay of chloroplast subfractions from a 35S::AtTerC:GFP transgenic plant. Tc, total chloroplast proteins; St, stroma proteins; En, envelope membrane proteins; Th, thylakoid proteins. Each fraction contained 7 μg of protein. Western blots were performed using polyclonal antibodies raised against GFP, rbcL (stroma marker), Tic 40 (envelope marker) and D1 (thylakoid marker).

To further investigate the sub-organellar localization of the AtTerC–GFP fusion protein, immunoblot analysis was performed using an anti-GFP antibody on chloroplast fractions isolated from AtTerC:GFP transgenic plants. The purity of each subfraction of chloroplast was assessed using antibodies against corresponding marker proteins including stromal rbcL (ribulose-1,5-bisphophate carboxylase/oxygenase large subunit), Tic 40 (translocon at the inner envelope membrane of chloroplasts), and the integral thylakoid membrane protein D1 (a core subunit of the PSII reaction center). As shown in Figure 4(b), AtTerC–GFP exhibited exactly the same fractionation pattern as D1. The molecular mass of the detected protein was approximately 8 kDa less than the predicted molecular mass of the fusion protein (approximately 69 kDa). This reduced size was expected as the N-terminal signal sequence of AtTerC is recognized by the stromal-processing peptidase and is removed after import into the chloroplast. Thus, these results indicate that AtTerC is targeted to the thylakoid membrane.

The product of the AtTerC gene is required for proplastid growth and thylakoid membrane formation

To investigate whether AtTerC is involved in the initial process of chloroplast development, this study examined the ultrastructure of plastids from atterC-1 plants and compared it to that of wild-type plants using transmission electron microscopy (Figure 5). Etioplasts from 4-day-old dark-grown atterC-1 plants had crystalline prolamellar bodies (PLBs) and double membrane envelopes similar to those of the wild-type (Figure 5b,d). However, the prothylakoids of atterC-1 were seen in the form of concentric rings surrounding the PLBs, and were observed in 95% of the 62 etioplasts examined for atterC-1 (Figure 5b,d). In addition, the intermembrane space of the prothylakoid lamellae of atterC-1 was shown to be about nine times as large as that of the wild-type: 0.13 versus 0.015 μm, respectively (Figure 5c,d).

Figure 5.

 Transmission electron microscopic analyses for etioplasts and chloroplasts of wild-type (WT) and atterC-1 plants.
(a, b) Four-day-old dark-grown cotyledons of WT and atterC-1. Black arrows indicate mitochondria.
(c, d) Enlarged images of etioplasts shown in (a) and (b), respectively. Triangles indicate prothylakoids and white arrows indicate PLBs.
(e, f) Twenty-four-day-old light-grown leaves of WT and atterC-1. St, starch granule. Plants were grown on sucrose-containing MS agar plates.

When the ultrastructures of chloroplasts from 24-day-old light-grown leaf sections of atterC-1 mutants were compared with those of wild-type plants grown on a sucrose-containing medium (Figure 5e,f), even more severely defective ultrastructures of chloroplasts were observed in atterC-1. As shown in Figure 5(f), the majority of the chloroplasts from atterC-1 did not have any lamellar structures, either stacked or unstacked thylakoids, but had many globular structures, which seemed to be formed from the fusion of internal membranes. These results suggest that AtTerC is involved in internal membrane formation or organization during chloroplast differentiation from etioplasts and/or proplastids.

Taken together, these results show that AtTerC is involved in normal prothylakoid lamellar growth in etioplasts before exposure to light, and in thylakoid biogenesis, including the stacking of grana and the growth of stromal lamellae, after light illumination.

Accumulation of thylakoid membrane proteins is defective in atterC-1

When plants are exposed to light, photosynthetic proteins encoded by the nuclear and chloroplast genomes accumulate in the chloroplasts. To investigate how the defects in thylakoid membrane biogenesis caused by the loss of AtTerC activity affect the accumulation of those proteins and their transcripts in the chloroplasts of the atterC-1 mutant, Western and Northern blot analyses were performed. For protein gel blotting, we used antibodies against distinct subunits of the photosynthetic membrane complexes and stromal proteins (Figure 6a). Of the four major protein complexes on the thylakoid, the subunits of D1, PsaA (PSI) and Cyt f (cytochrome b6f) were undetectable in the mutant, and the amount of AtpB (ATPase) detected was significantly reduced. The detection of AtpB is possibly due to the presence of intact mitochondria in atterC-1 (Figure 5b), and the fact that the anti-AtpB antibody used in the immunoblot assay can detect the AtpB subunit of both photosynthetic and respiratory ATP synthases. The levels of rbcL and CAB were greatly reduced or disappeared in the atterC-1 mutant compared to the wild-type over the period of light treatment, while the level of POR was similar in the mutant and wild-type. These results indicate that there are severe defects in the accumulation of photosynthetic complexes and the Rubisco complex in atterC-1. However, the failure of accumulation and the significant loss of photosynthetic proteins in the mutant does not result from transcriptional differences, because similar transcripts levels of relevant genes were observed in wild-type and in atterC-1 (Figure 6b).

Figure 6.

 Change in patterns of photosynthesis-related proteins and mRNAs in wild-type (WT) and atterC-1 seedlings.
(a) Immunoblot analysis for the accumulation of photosynthesis-related proteins during de-etiolation. Seedlings were grown for 4 days in the dark and then grown further in the light (approximately 100 μmol photons m−2 sec−1) for the indicated times. Fifteen micrograms of total protein were loaded per lane. An anti-actin antibody was used as a loading control.
(b) Northern blot analysis for the accumulation of photosynthesis-related transcripts during de-etiolation. Each lane contains 5 μg of total RNA isolated from seedlings grown as described in (a). A probe for 18S rRNA served as a loading control. Northern blot analysis was not performed for the POR gene because the anti-POR antibody is capable of recognizing all three Arabidopsis PORs (Frick et al., 2003).

To address whether the lack of accumulation of photosynthetic proteins in atterC-1 resulted from defects in translation, polysome profiling analysis was performed using probes against psbA, psaA, petA, atpB and rbcL (Barkan, 1998). As shown in Figure 7, there was no significant difference in the distribution of ribosomes between the mutant and the wild-type. Therefore, these results suggest that the impaired protein accumulation observed in the thylakoid of atterC-1 mutants is caused by accelerated degradation of proteins rather than defects in protein synthesis.

Figure 7.

 Association of photosynthesis-related mRNAs with polysomes in wild-type (WT) and atterC-1 seedlings. Ten fractions of equal volume were collected from the top to the bottom of the sucrose gradients, and equal proportions of RNA purified from each fraction were analyzed by gel-blot hybridization. Ribosomal RNAs were stained with ethidium bromide.

Taken together, these results demonstrate that AtTerC mutation causes the disruption of thylakoid membrane formation and results in severe defects in the accumulation of photosynthetic proteins, along with chlorophylls on thylakoids. As a result of these defects, plastids could not be normally converted into chloroplasts in atterC-1. Ultimately, the blockade of chloroplast biogenesis caused atterC-1 mutants to die at the early seedling stage.

Discussion

This study described the biological role of AtTerC, an Arabidopsis gene possibly involved in early thylakoid development, using T-DNA-tagged Arabidopsis mutants. The disrupted gene, which encodes the integral membrane protein AtTerC in Arabidopsis, appears to originate from bacterial ancestors. Analysis of the AtTerC amino acid sequence showed that the protein contains a conserved domain, previously annotated as tellurite resistance C (TerC) in bacteria (Figure 2a). Although there have been many efforts to identify the exact function of TerC, which serves as a key component of the tellurite resistance operon in prokaryotes, its function has remained undetermined until now. Nonetheless, it is thought that the TerC protein has a different function than the function of transmembrane transport postulated for TeO32- (Turner et al., 1995).

The loss of AtTerC activity caused a pigment-deficient and seedling-lethal phenotype, and this phenotype does not appear to be associated with perturbation of the chlorophyll biosynthetic pathway, as shown by the accumulation of comparable amounts of chlorophyll biosynthetic intermediates in the wild-type and the mutant (Figure 3b) and by a pale-green phenotype of atterC-1 in dim light conditions (data not shown).

The transmission electron microscopy analysis presented in Figure 5(f) clearly indicates blockage of chloroplast formation in the atterC-1 mutant. Thylakoid membrane structures do not exist in the mutant. Furthermore, one of the underlying causes of the defect in chloroplast biogenesis in atterC-1 may be compromised formation of prothylakoid membranes of etioplasts in atterC-1 (Figure 5b,d). As these mutant lines cannot be propagated in knockout homozygous seeds, all experiments with AtTerC homozygous knockout seedlings (atterC/atterC) must utilize heterozygous seeds. Then, pigment-deficient homozygous seedlings can be selected after light illumination. By detachment and storage of one cotyledon, and allowing growth to continue in the seedling bearing the remaining cotyledon (see Experimental procedures), it was possible to observe the ultrastructures of etioplasts of atterC-1 homozygous mutants that had not been exposed to light since germination.

In addition to the abnormal membrane structure of prothylakoids and thylakoids in mutants, a global loss of thylakoid membrane proteins was observed in the atterC-1 mutant after light illumination (Figure 6a). However, this does not appear to be a consequence of a global defect in chloroplast gene expression, because transcripts for these proteins accumulated properly, and chloroplast polysome profiles displayed similar patterns to those of the wild-type (Figure 7). The initial reduction and subsequent loss of CAB, a peripheral protein of photosystems, presumably resulted from the loss of core subunits caused by the rapid degradation of D1 and PsaA in atterC-1. In addition, a similar level of POR, another nuclear-encoded chloroplast protein, in the wild-type and the mutant, suggests that post-translational translocation of nuclear-encoded CAB into the chloroplast of the mutant is not affected, because proteins destined for plastids are generally imported using a single transport system (Keegstra and Cline, 1999). Taken together, this global loss of thylakoid membrane proteins is not the result of defects in protein synthesis or in the protein import system.

The severe reduction and steady-state level of stromal rbcL in atterC-1 appears to be the consequence of reduced expression of rbcS to about 50% of wild-type in the mutant (Figure 3c), because rbcS controls the rbcL level stoichiometrically without affecting the rbcL mRNA level (Khrebtukova and Spreitzer, 1996; Rodermel et al., 1988, 1996).

The biogenesis and assembly of both the plastid- and nuclear-encoded proteins into complexes on the thylakoid membrane require stoichiometric synthesis and sequential assembly of subunits, with a progressive increase in the stability of the complexes as various co-factors are incorporated. These are complicated and multi-step processes that are regulated by nuclear proteins to enforce stoichiometric assembly and to guarantee the coordinated development of all plastids in one cell (Choquet and Vallon, 2000; Vothknecht and Westhoff, 2001). Genetic and biochemical studies have provided insights into the control by nuclear-encoded factors of the biogenesis and assembly of thylakoid membrane protein complexes (Barkan et al., 1995; Choquet and Vallon, 2000; Leister, 2003). Each thylakoid complex assembles independently, and the lack of any core subunit of the complex results in specific loss of the affected complex (Barkan et al., 1995; Lezhneva and Meurer, 2004; Lezhneva et al., 2004).

It has also been reported that global loss of thylakoid membrane proteins is caused by mutations of upstream stages of the assembly of thylakoid proteins, such as defects in the targeting pathway to the thylakoid, and in chloroplast translation and mRNA metabolism (Asakura et al., 2004; Barkan, 1993; Roy and Barkan, 1998). Mutations of key components of the Sec-dependent thylakoid protein targeting pathway in maize, cpSecY (an ATP-driven translocation motor) and cpFtsY (a membrane-bound signal recognition particle receptor), resulted in broad thylakoid protein defects (Asakura et al., 2004; Roy and Barkan, 1998). For correct assembly of thylakoid protein complexes, the nuclear- and plastid-encoded proteins must be targeted to the correct position of the thylakoid with the help of thylakoid-targeting machineries. A similar pattern of global loss of thylakoid membrane proteins was also observed in atterC-1. The thylakoid membrane proteins D1, PasA and Cyt f were completely lost, and AtpB was severely reduced in atterC-1. Previous studies have proposed that translation of thylakoid membrane proteins, such as D1, PsaA and Cyt f, takes place on thylakoid-bound ribosomes, and that these proteins are co-translationally inserted into the thylakoid (Friemann and Hachtel, 1988; Klein et al., 1988; Zhang et al., 1999). In addition, Cyt f, which comprises a large lumenal domain and a single membrane anchor, has been proposed to require cpSecA for its integration into the thylakoid (Mould et al., 1997; Röhl and van Wijk, 2001; Voelker et al., 1997). Integration of D1 into the thylakoid has also been suggested to be assisted by the Sec pathway as well as the cpSRP (signal recognition pathway) pathway (Nilsson et al., 1999; Zhang et al., 2001). Therefore, we hypothesize that AtTerC may function as a component of the translocation machinery that assists the integration and translocation of thylakoid membrane proteins into the thylakoid and lumenal space.

Another example of global deficiencies in thylakoid membrane proteins is seen in maize mutants such as hcf7, cps1 and cps2 (Barkan, 1993). Despite the normal size and abundance of most plastid mRNAs, the levels of four distinct thylakoid membrane complexes were severely reduced in hcf7 and cps1, and to a minor extent in cps2. These mutants showed abnormal chloroplast mRNA polysome profiles in which a proportion of the non-polysomal region was substantially increased because of impaired translation initiation, probably caused by defects in rRNA processing. In contrast to the mutants mentioned above, the polysome profiles of chloroplast mRNAs in atterC-1 exhibited similar patterns to those of the wild-type (Figure 7). Furthermore, if the global defect is attributable to a reduction in the elongation step of translation in atterC-1, this would lead to an increase in the number of ribosomes associated with mRNAs; however, the polysomal regions (the bottom regions of sucrose gradients) displayed similar patterns between wild-type and atterC-1 (Figure 7), suggesting that the global defect in accumulation of thylakoid proteins is probably due to a post-translational problem. An alternative hypothetical function of AtTerC is that it might serve as a mediator to allow interaction between the polysomes and the thylakoid membranes for efficient integration of thylakoid proteins, synthesized within the chloroplast, into thylakoids.

The disordered ultrastructure of the prothylakoid membrane observed in etioplasts of atterC-1 suggests another putative role for AtTerC in thylakoid formation. The non-bilayer lipid monogalactosyldiacylglycerol (MGDG) constitutes up to 50% of the lipids of the prothylakoid and thylakoid membranes of green plants, and has a natural tendency not to be arranged into lamellar structures in aqueous media (Garab et al., 2000; Selstam and Sandelius, 1984). The transition from the non-bilayer lipid to bilayer lipid phase can be induced by integral membrane proteins as shown in previous studies (Batenburg and de Kruijff, 1988; Simidjiev et al., 1998, 2000). Thus, the failure to maintain prothylakoids and reconstitute lamellar thylakoids from non-bilayer lipids might result from loss of AtTerC in the mutant seedling during the very early de-etiolation period. To address these possibilities, further research is required to identify and functionally analyze interacting components.

In summary, the pre-existing disordered prothylakoid membranes and the impaired accumulation of thylakoid membrane complexes in atterC-1 are the direct cause of blockage of the reorganization of functional thylakoid membranes after light illumination. Ultimately, these defects may stop the stepwise processes for chloroplast biogenesis and subsequently cause death of the mutant seedlings at an early developmental stage.

Experimental procedures

Plant material and growth conditions

Seeds from wild-type Arabidopsis (Columbia) and atterC mutants were grown on half-strength MS medium (Duchefa, http://www.duchefa.com) supplemented with 1% w/v sucrose and 0.7% w/v phytoagar (Duchefa) in a growth room either in continuous light or in the dark at 22°C. Adult plants were grown in Sunshine-Mix 1 (SunGro, http://www.sungro.com) under a 16/8 h light/dark photoperiod at 22°C. All seeds were incubated at 4°C for 2 days to synchronize germination.

Oligonucleotide sequences and cDNA fragments for hybridization probes

Detailed information for all primer sequences and cDNA probe regions is given in Appendix S1.

Mutant identification

The AtTerC T-DNA insertion lines SALK_014736 (atterC-1) and GABI-Kat 844D10 (atterC-2) were obtained from the Arabidopsis Biological Resource Center (Ohio State University) and the GABI-Kat mutant collection at the Max-Planck-Institute for Plant Breeding Research, respectively. To determine the predicted positions of T-DNA insertions for AtTerC knockout homozygous mutants, genomic DNA was isolated from 10-day-old wild-type and mutant seedlings as described by Dellaporta et al. (1983), and then the flanking regions of the T-DNA insertions were amplified by PCR. The mutant-specific PCR products were then sequenced to confirm the predicted T-DNA insertions.

RNA gel-blot analysis

Total RNA was extracted from whole seedlings using an easy-BLUETM kit (iNtRON, http://www.intronbio.com) or was extracted from tissues of the whole plant grown in soil as described previously (Shi and Bressan, 2006). RNA gel-blot analyses were performed as described by Sambrook and Russell (2001). The hybridization signals were quantified using a phosphorimager (BAS-2500; Fujifilm, http://www.fujifilm.com).

Complementation analysis

For the complementation test of AtTerC−/− plants, a 4.2 kb genomic DNA fragment of the AtTerC gene, containing approximately 0.95 kb of a putative promoter sequence and approximately 0.75 kb of a 3′ sequence encompassing the 3′ UTR, was amplified by PCR using specific primers and cloned into a pFP101 binary vector (Bensmihen et al., 2004). The complementation vector was introduced into the Agrobacterium tumefaciens strain LBA4404. Plants heterozygous for the T-DNA disruption in AtTerC were transformed by the in planta method (Clough and Bent, 1998). Transformants were selected using a seed-expressed fluorescent marker (Bensmihen et al., 2004), then genotyped and analyzed for the introduced trans-AtTerC wild-type copy and AtTerC mRNA expression as described above. For the DNA gel blot, genomic DNA was isolated as described above. Ten micrograms of DNA were digested with BamHI overnight and run on a 1% agarose gel. DNA gel-blot analysis was performed out according to a standard method (Sambrook and Russell, 2001). A 1.1 kb fragment of AtTerC genomic DNA sequence was amplified from extracted wild-type genomic DNA and used as hybridization probe.

Pigment analysis

To measure the content of pigments, including chlorophyll, carotenoid and anthocyanin, seedlings were grown on MS agar medium for 4 days under continuous light. Further details are given in Appendix S1.

Subcellular localization of GFP fusion proteins in protoplasts

To construct the 35S::AtTerC:GFP gene, the AtTerC coding region was cloned into a pBI121 binary vector in which GUS was replaced by the GFP gene (U70495).

For protoplast preparation, 35S::AtTerC:GFP and 35S::GFP transgenic Arabidopsis plants were grown on MS medium for 2 weeks. The protoplasts were harvested as described previously (Jin et al., 2001). Expression of the fusion construct was monitored by confocal laser scanning microscopy, using an LSM Meta 510 (Carl Zeiss, http://www.zeiss.com).

Chloroplast isolation and subfraction

Leaves of 2-week-old 35S::AtTerC:GFP transgenic plants were used for sample preparation. Sample preparation was performed as described previously (Barneche et al., 2006; Miras et al., 2002; Robinson and Barnett, 1988) and detailed in Appendix S1.

Electron microscopy

For etioplast ultrastructural analysis, etiolated wild-type and atterC-1 seedlings were grown on MS agar plates in the dark for 4 days. Under dim green light, wild-type cotyledons were directly detached from seedlings using sharp forceps, and then immediately soaked in a brown-colored 1.5 ml tube containing fixation solution. For atterC-1, a batch of seeds from a heterozygous plant was sown on MS agar medium. One of the two cotyledons was carefully detached and numbered for a batch of seedlings in which wild-type, and heterozygous and homozygous atterC-1 seedlings were included. Plates containing the seedlings with one remaining cotyledon were transferred to light for 20 h. Next, atterC-1 homozygous background seedlings that did not show a greening cotyledon were identified, and the detached cotyledons in the corresponding tubes were used for transmission electron microscopic analysis.

For analysis of chloroplast ultrastructure, wild-type and atterC-1 seedlings were grown on MS agar medium under continuous light for 24 days. Small pieces of cut rosette leaves from the wild-type and the atterC-1 mutant were collected in fixation solution. Transmission electron micrographs were obtained as described by Lee et al. (2003).

Protein and polysome analysis

Total proteins were extracted as described previously (Frick et al., 2003), and immunoblot assay was performed by a standard method (Sambrook and Russell, 2001). Polysome analysis was performed as described by Barkan (1998). Detailed information is given in Appendix S1.

Acknowledgements

We thank G. Armstrong (Ohio State University) for kindly providing the POR antiserum. This work was supported by the Crop Functional Genomics Center funded by the Ministry of Science and Technology (MOST) of the Korean government (grant no. CG-2152) and the Second Phase of the BK21 Program (YBRI) of Korea.

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