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BIOENERGETICS AND PHOTOSYNTHESIS

Functional Redundancy of AtFtsH Metalloproteases in Thylakoid Membrane Complexes¹

Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50011

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
FtsH is an ATP-dependent metalloprotease found in bacteria, mitochondria, and plastids. Arabidopsis (Arabidopsis thaliana) contains 12 AtFtsH proteins, three in the mitochondrion and nine in the chloroplast. Four of the chloroplast FtsH proteins are encoded by paired members of closely related genes (AtFtsH1 and 5, and AtFtsH2 and 8). We have previously reported that AtFtsH2 and 8 are interchangeable components of AtFtsH complexes in the thylakoid membrane. In this article, we show that the var1 variegation mutant, which is defective in AtFtsH5, has a coordinate reduction in the AtFtsH2 and 8 pair, and that the levels of both pairs are restored to normal in var1 plants that overexpress AtFtsH1. Overexpression of AtFtsH1, but not AtFtsH2/VAR2, normalizes the pattern of var1 variegation, restoring a nonvariegated phenotype. We conclude that AtFtsH proteins within a pair, but not between pairs, are interchangeable and functionally redundant, at least in part. We further propose that the abundance of each pair is matched with that of the other pair, with excess subunits being turned over. The variegation phenotype of var1 (as well as var2, which is defective in AtFtsH2) suggests that a threshold concentration of subunits is required for normal chloroplast function. AtFtsH1, 2, 5, and 8 do not show evidence of tissue or developmental specific expression. Phylogenetic analyses revealed that rice (Oryza sativa) and Arabidopsis share a conserved core of seven FtsH subunit genes, including the AtFtsH1 and 5 and AtFtsH2 and 8 pairs, and that the structure of the present-day gene families can be explained by duplication events in each species following the monocot/dicot divergence.

Reverse and forward genetics experiments have revealed that most AtFtsH mutants do not have readily visible phenotypes (Sakamoto et al., 2003; F. Yu and S. Rodermel, unpublished data). Notable exceptions include the var1 and var2 variegation mutants of Arabidopsis (Martínez-Zapater, 1993; Chen et al., 1999, 2000; Takechi et al., 2000; Rodermel, 2001; Sakamoto et al., 2002). Whereas cells in the green leaf sectors of these mutants contain morphologically normal chloroplasts, cells in the white sectors contain vacuolated plastids lacking organized lamellae. At least in the case of var2, the white sectors are heteroplastidic and contain rare, normal-appearing chloroplasts in addition to the white plastids (Chen et al., 1999). This plastid autonomy suggests that plastids do not respond similarly in the mutant background. The distinctive phenotypes of var1 and var2 permitted the cloning of the responsible genes, namely, AtFtsH5 and AtFtsH2, respectively (Chen et al., 2000; Takechi et al., 2000; Sakamoto et al., 2002). Because var1 and var2 have uniform genetic constitutions, a major question is how cells in the green sectors are able to bypass the requirement for AtFtsH5 (in the case of var1) or AtFtsH2 (in the case of var2) during the process of chloroplast biogenesis.

One consequence of the dearth of AtFtsH mutants is that little is known about the functions of FtsH in higher plants. FtsH-like proteins have been implicated in the degradation of unassembled cytochrome b₆f Rieske FeS proteins in the thylakoid membrane (Ostersetzer and Adam, 1997). The first identified activity of FtsH came from in vitro studies in the Adam group showing that AtFtsH1 is involved in the D1 repair cycle of PSII, where it catalyzes the proteolytic degradation of photodamaged D1 proteins (Lindahl et al., 2000). AtFtsH2 and 5 also appear to be involved in photoprotection, inasmuch as var2 and var1 are more sensitive than wild type to PSII photoinhibition, and D1 degradation is slowed in var2 (Bailey et al., 2002; Sakamoto et al., 2002). AtFtsH2 might also be involved in membrane fusion and/or translocation events since it bears high similarity to the pepper Pftf (plastid fusion and/or translocation factor) protein (Hugueney et al., 1995; Chen et al., 2000).

AtFtsH is present in multimeric complexes in plastid membranes (Sakamoto et al., 2003; Yu et al., 2004). These appear to be heteromeric and homomeric in nature. To better characterize these complexes, we reported that two AtFtsH-containing bands migrate near one another on two-dimensional (2-D) green gels of Arabidopsis thylakoid membranes (Yu et al., 2004). These bands contain the products of two of the three plastid AtFtsH gene pairs—an upper band (containing AtFtsH1 and 5) and a lower band (containing AtFtsH2 and 8). We observed that both bands are decreased in amount in var2 and, further, that the levels of these bands are restored to normal in transgenic var2 that overexpresses AtFtsH8. Overexpression of AtFtsH8 also rescues the variegation phenotype of var2, indicating that AtFtsH2 and 8 are functionally redundant, at least in part. Consistent with this notion, both genes have similar expression patterns, being abundantly expressed in photosynthetic tissues (Yu et al., 2004).

Based on these data, we suggested a model in which AtFtsH2 and 8 are functionally interchangeable and are capable of stabilizing AtFtsH1 and 5 in the thylakoid membrane (Yu et al., 2004). In support of this model, Sakamoto et al. (2003) demonstrated via coimmunoprecipitation that members of each pair might interact, although the precise interactions could not be determined because the polyclonal antibodies that were used detect both members of each pair (Sakamoto et al., 2003). The purpose of this investigation was to test elements of our model. First, we wanted to assess the functional relatedness of AtFtsH1 and AtFtsH5. Are they interchangeable? Do they have similar expression patterns? Second, we wanted to test whether members of the AtFtsH2 and 8 pair are interchangeable with members of the AtFtsH1 and 5 pair. Finally, we wanted to take advantage of the nearly complete genome sequence of rice (Oryza sativa) to ask whether the two AtFtsH pairs are unique to Arabidopsis or whether they have a broader significance in FtsH complex formation.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Interchangeability of Subunits in the Upper AtFtsH-Containing Band

AtFtsH1 and AtFtsH5 are the most closely related members of the AtFtsH gene family in Arabidopsis (approximately 90% amino acid identity). Based on our previous results showing that members of another closely related AtFtsH gene pair—AtFtsH2 and AtFtsH8—have redundant functions, we wanted to examine whether the same is true for AtFtsH1 and AtFtsH5. To test this hypothesis, we overexpressed AtFtsH1 in the var1-1 variegation mutant, which defines the AtFtsH5 locus (Fig. 1A; Sakamoto et al., 2002). The var1-1 allele has a nucleotide insertion in its N terminus that is predicted to generate an early stop codon; if stable, a truncated translation product would be produced lacking most of the functional domains of the protein. Taken together with the finding that AtFtsH5 mRNAs are not detectable, this suggests that var1-1 is likely a null allele (Sakamoto et al., 2002).

View larger version (73K): [in this window] [in a new window]   Figure 1. Complementation of var1-1 by overexpression of AtFtsH1. A, Phenotypes of wild type, var1-1, and var1-1 transformed with an AtFtsH1 cDNA under the control of the CaMV 35S promoter (C5W1No2). A T2 generation C5W1No2 plant is shown. All the plants were 10 d old. B, Southern-blot of wild type and C5W1No2. OE, Overexpression. Genomic DNAs from wild type and C5W1No2 were digested with HindIII, and the Southern-blot was probed with 32P-labeled NPTII sequences. C, Total cell RNAs were isolated from the green sectors of var1-1 and from leaves of C5W1No2 (OE). RNA amounts were determined by semiquantitative RT-PCR as described in "Materials and Methods." The RT-PCR products were electrophoresed through 1.5% agarose gels and stained with ethidium bromide. Actin2 served as a control.

Whereas the data in Figure 1A are consistent with the idea that overexpression of AtFtsH1 rescues the variegation phenotype of var1-1, it was necessary to confirm this at the level of AtFtsH1 expression. Semiquantitative RT-PCR analyses (Fig. 1C) revealed that the transcript levels of AtFtsH1 are sharply elevated in C5W1No2 compared to var1-1. By contrast, AtFtsH2 and AtFtsH8 mRNA levels are normal in the overexpressor. Only trace amounts of the AtFtsH5 transcript are present in var1-1 (as anticipated from Sakamoto et al., 2002) and C5W1No2.

To examine protein accumulation in the overexpression plants, we performed 2-D green gel analyses of Arabidopsis thylakoid membrane proteins. Previous studies have shown that two AtFtsH-containing bands can be resolved on green gels of Arabidopsis thylakoids: upper and lower bands consisting, respectively, of AtFtsH1 and 5 and AtFtsH2 and 8 (Yu et al., 2004). Whereas both AtFtsH-containing bands are reduced in abundance in var1-1 (Fig. 2A), they are restored to normal in C5W1No2 (Fig. 2B). To confirm this finding, we performed western-immunoblot analyses on thylakoid membrane proteins fractionated by one-dimensional SDS-PAGE. To detect AtFtsH1 specifically, we generated an antibody against a peptide that is unique to this protein (see "Materials and Methods"). This antibody reacts with AtFtsH1 proteins that are expressed in E. coli, but not with E. coli-expressed AtFtsH5 or AtFtsH2 (data not shown). We detected similar levels of AtFtsH1 in wild type and var1-1, but a significant increase in C5W1No2 (Fig. 2C). Western immunoblots were also probed with an antibody that detects both AtFtsH2 and 8 (Yu et al., 2004). As illustrated in Figure 2D, the band containing AtFtsH2 and 8 is decreased in amount in var1-1, but is present at normal levels in C5W1No2. In summary, the data in Figure 2, A to C, are consistent with the idea that (1) wild-type AtFtsH complexes contain optimal levels of the 1-5 and 2-8 pairs; (2) var1-1 has a reduction in 5, but not 1, and this is matched by a reduction in 2 and 8; and (3) optimal complex levels (as found in the wild type) can form when 1 is overexpressed in var1-1; these complexes contain 1 and 2-8.

View larger version (38K): [in this window] [in a new window]   Figure 2. Two-dimensional green gel and western-immunoblot analyses. A and B, Comparison of the upper and lower AtFtsH-containing bands of var1-1 and wild type (A) and var1-1 and C5W1No2 (B). Thylakoid membranes corresponding to equal amounts of chlorophyll were loaded on the native gels. C, Western immunoblots of wild type, var1-1, and C5W1No2 thylakoid membranes. Thylakoid membranes corresponding to 2.5 µg of chlorophyll were loaded in each lane of a denaturing, 12% SDS-polyacrylamide gel. Membranes were incubated with polyclonal antibodies specific for AtFtsH1. D, Western immunoblots of wild type, var1-1, and C5W1No2 thylakoid membranes. Nitrocellulose filters were incubated with polyclonal antibodies to AtFtsH2/VAR2, the D1 protein of PSII, the PetC (Rieske) protein of the cytocrome b6f complex, and the -subunit of the proton-translocating ATP synthase. Protein loadings were the same as in Figure 2C. E, Coimmunoprecipitation of AtFtsH1 and AtFtsH2 and 8. Immunoprecipitates were resolved by SDS-PAGE and immunoblots were probed with AtFtsH1 and VAR2 antibodies. Shown are immunoprecipitated samples from VAR2 preimmune serum (lane 1), VAR2 antibody (lane 2), AtFtsH1 preimmune serum (lane 3), and AtFtsH1 antibody (lane 4). Each lane represents a thylakoid membrane corresponding to approximately 10 µg of chlorophyll. Lane 5 is a thylakoid membrane control corresponding to 5 µg of chlorophyll.

Using polyclonal AtFtsH5/VAR1 and AtFtsH2/VAR2 antibodies, it was reported that VAR1 and VAR2 might interact directly (Sakamoto et al., 2003). One factor complicating these studies was the lack of isoform-specific antibodies, i.e. their AtFtsH5/VAR1 antibody detected 1 and 5, and their AtFtsH2/VAR2 antibody detected 2 and 8. With the availability of AtFtsH1-specific antibodies, we wanted to test whether 1 interacts with the 2-8 pair, as suggested in our model of AtFtsH complex formation (Yu et al., 2004). In these experiments, we coimmunoprecipitated thylakoid membrane samples from wild-type Columbia using our AtFtsH1 or AtFtsH2/VAR2 antibodies; the latter antibodies, as mentioned earlier, detect 2 and 8. Figure 2E (lane 5) shows a western immunoblot of control thylakoid membranes: Bands corresponding to 2 and 8 (top) and 1 (bottom) can be seen. The relatively low abundance of AtFtsH1 in the membranes of wild type confirms the data in Figure 2C. In lane 4, the AtFtsH1 antibody was used to coimmunoprecitate thylakoid proteins. The immunoprecipitates were then electrophoresed on polyacrylamide gels and subjected to western analysis, using either the VAR2 or AtFtsH1 antibodies as probes. Bands containing 1 and 2 and 8 are visible, suggesting that 1 interacts with 2 and/or 8. In complementary experiments, the VAR2 antibody was used to coimmunoprecipitate thylakoid proteins (lane 2). Again, bands corresponding to 1 and 2 and 8 were observed on the western blots. This confirms the data in lane 4. As expected, coimmunoprecitation with preimmune sera did not give rise to bands on the western blots (lanes 1 and 3). We conclude that 1 interacts with 2 and/or 8 in AtFtsH complexes in the thylakoid.

Considered together, the data in Figures 1 and 2 show that AtFtsH1 overexpression rescues the var1-1 variegation phenotype. This suggests that AtFtsH1 and 5 are interchangeable in thylakoid membranes. The data also show that there are coordinate changes in the levels of the proteins in the upper and lower bands in wild type, var1-1, and the overexpression plants. Control of subunit abundance likely occurs posttranscriptionally, since the overexpression plants have normal levels of AtFtsH2 and 8 mRNAs, but reduced amounts of AtFtsH2 and 8 proteins. Perhaps the simplest hypothesis is that AtFtsH1 is interchangeable with AtFtsH5 in AtFtsH complexes, and that AtFtsH1 exerts its effect by stabilizing AtFtsH2 and 8 in the membrane.

One prediction of the above hypothesis is that down-regulation of AtFtsH1 protein levels in a var1-1 background should result in plants with a more severe phenotype than var1-1. A complete lack of AtFtsH1 and 5 might be lethal if these proteins are essential for viability. To test this, we transformed var1-1 with an antisense AtFtsH1 cDNA driven by the CaMV 35S promoter. A number of antibiotic-resistant T1 lines were selected and selfed. Figure 3 shows the T2 progeny from a representative T1 line. These plants have a variety of color phenotypes that range from variegations similar to var1-1 (Fig. 3A) to partially albino (Fig. 3, B–D) and completely albino (Fig. 3, E and F). PCR was used to confirm the presence of the antisense construct in the individual T2 plants (data not shown). However, we did not try to monitor changes in AtFtsH1 expression in these plants because the parental var1-1 mutants have extremely low levels of AtFtsH1 mRNAs and proteins (see Figs. 1C and 2C). Nonetheless, the phenotypes of the antisense mutants are consistent with the idea that down-regulation of AtFtsH1 in a background that lacks AtFtsH5 can be lethal.

View larger version (121K): [in this window] [in a new window]   Figure 3. Antisense down-regulation of AtFtsH1 expression in var1-1. A partial AtFtsH1 cDNA was cloned in reverse orientation behind the CaMV 35S promoter, and the construct was transformed into var1-1. Representative T2 plants from an antibiotic-resistant T1 plant are shown. These plants have a range of color phenotypes, from those that are similar to var1-1 (A) to those that are partially albino (B–D) or completely albino (E and F). This variability was observed in the T2 progeny of multiple independent T1 lines. Bars = 5 mm.

To examine AtFtsH1 and AtFtsH5 expression, we generated transgenic Columbia plants that express AtFtsH1 promoter-GUS and AtFtsH5 promoter-GUS fusions. Figure 4 shows that GUS activities are similar in seedlings (Fig. 4, A and C) and fully expanded older leaves in both sets of transformants (Fig. 4, B and D). Examination of other growth stages did not reveal obvious staining differences between the two constructs (data not shown). We conclude that AtFtsH1 and AtFtsH5 promoter activities are similar to one another, and that these genes are ubiquitously expressed, especially in green organs of the plants. These expression patterns resemble those of AtFtsH2 and AtFtsH8, as previously reported (Yu et al., 2004).

View larger version (85K): [in this window] [in a new window]   Figure 4. AtFtsH1 and AtFtsH5/VAR1 expression. Constructs containing promoter-GUS fusions were generated and transformed into wild type (Columbia). GUS activities were visualized by histochemical staining of fusions with the AtFtsH1 promoter (A and B) and the AtFtsH5/VAR1 promoter (C and D). Illustrated are 10-d-old green seedlings (A and C) and fully expanded leaves from 20-d-old plants (B and D).

Considered together with previous results (Sakamoto et al., 2003; Yu et al., 2004), the current data suggest that AtFtsH forms multimeric complexes in thylakoid membranes, and that these complexes contain at least two pairs of interchangeable subunits: AtFtsH2 and 8 (lower band) and AtFtsH1 and 5 (upper band). The question arises as to whether FtsH homologs in different pairs are interchangeable. To address this question, we tested whether overexpression of AtFtsH2 can rescue the var1 variegation phenotype, i.e. whether AtFtsH2 can substitute for AtFtsH5. For these experiments, we generated a construct in which the full-length AtFtsH2 cDNA was placed under the control of the CaMV 35S promoter, then transformed into var1-1. As a control, the construct was transformed into the var2-4 variegation mutant, which lacks AtFtsH2. Figure 5, B and D, shows that AtFtsH2 transcripts are significantly elevated in both sets of transformed plants. Yet, AtFtsH2 overexpression is only able to rescue the var2-4 variegation (Fig. 5A). A failure to rescue the var1-1 variegation was observed in multiple lines (Fig. 5C).

View larger version (33K): [in this window] [in a new window]   Figure 5. Overexpression of AtFtsH2/VAR2 in var2-4. A, A construct containing a full-length AtFtsH2/VAR2 cDNA driven by the CaMV 35S promoter was transformed into var2-4. C2W2 is a representative T2 plant. B, Total cell RNAs were isolated from var2-4 and C2W2, and northern-blot analysis was performed using 2 µg of RNA per gel lane; the blot was probed with 32P-labeled AtFtsH2/VAR2 cDNA. Ethidium bromide-staining of the 18S RNA band on the agarose gel is shown as a loading control. C, The same construct was transformed into var1-1. C5W2 is a representative T2 plant. D, Total cell RNAs were isolated from var1-1 and C5W2, and northern-blot analysis was performed as described in B. E, Steady levels of AtFtsH2/VAR2 and AtFtsH1 in var1-1 and C5W2 thylakoid membranes were compared by western-immunoblot analysis. Thylakoid membranes corresponding to 5 µg of chlorophyll were loaded in each lane.

OsFtsH Gene Family in Rice

At least three different efforts are under way to sequence the rice genome (about 420 megabases in size), and draft sequences have been published from the japonica cultivar (approximately 389 megabases; Goff et al., 2002) and from the indica cultivar (approximately 361 megabases; Yu et al., 2002). In addition, The Institute for Genomic Research (TIGR) has recently released its third version (December 30, 2004) of annotations of pseudomolecules (virtual contigs) from the International Rice Genome Sequencing Project (IRGSP; http://rgp.dna.affrc.go.jp/IRGSP/index.html), encompassing approximately 370 megabases of the genome. This resource is easy to access (http://www.tigr.org/tdb/e2k1/osa1). To determine the FtsH gene complement in rice (OsFtsH genes), we searched the annotated pseudomolecule database and identified a total of nine FtsH homologs, designated OsFtsH1 through OsFtsH9 (Table I). All nine appear to be expressed insofar as each is represented in the databases by cDNAs and expressed sequence tags (ESTs).

View larger version (23K): [in this window] [in a new window]   Figure 6. Conservation of FtsH genes in Arabidopsis and rice. Phylogenetic analysis of FtsH genes from Arabidopsis, rice, and Synechocystis. Full-length protein sequences were aligned using the ClustalW program, and the aligned sequences were analyzed by the Mega 2.1 program (Kumar et al., 2001; see also http://www.megasoftware.net). The neighbor-joining method was used to construct the phylogenetic tree.

View larger version (14K): [in this window] [in a new window]   Figure 7. Structures of rice and Arabidopsis FtsH genes. Gene structures were constructed based on annotation of the Arabidopsis and rice genomes. Intron-exon boundaries were confirmed by examination of cDNA and EST sequences. Boxes indicate exons and lines represent introns. Numbers above each box refer to size of the exon (bp). Untranslated regions were not included in the structures. Colored regions correspond to various FtsH domains. Blue represents transit peptide region, green represents ATP-binding region, and red represents zinc-binding site.

View larger version (43K): [in this window] [in a new window]   Figure 8. Comparative 2-D green gel analyses. A, 2-D green gels were prepared as described in Figure 2A using isolated thylakoid membranes from Arabidopsis, rice, and pea. The regions of each gel containing the upper and lower FtsH-containing bands are shown. B, Western immunoblots of 2-D gels probed with AtFtsH1 and VAR2 antibodies are shown.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Subunit Interchangeability: Gene Duplication and Redundant Functions

E. coli has one FtsH gene, cyanobacteria have four (Kaneko et al., 1996), rice has at least nine (Table I), and Arabidopsis has 12 (Fig. 6). Why are so many FtsH genes needed in higher plants? These data show that a core complement of 7 FtsH genes was in place by the time of the monocot/dicot divergence (see Fig. 7). Some of these core genes subsequently became duplicated in Arabidopsis or rice. These include the genes in the upper and lower bands of the 2-D gels. One hypothesis is that the various FtsH proteins became specialized in their activity. For instance, the chaperone activity of FtsH normally acts to promote the protease activity of the enzyme (Suzuki et al., 1997). However, it is possible the enzyme could act independently as a chaperone, as found in the Clp class of proteases (Nielsen et al., 1997). We found that members of the 2-8 and 1-5 duplicated pairs in Arabidopsis are interchangeable, while proteins of different pairs are not interchangeable. This lack of interchangeability does not necessarily mean that the members of the two pairs have different functions, e.g. it could simply signify an inability to assemble efficiently. Nevertheless, whether the two have similar, or different, functions is now under investigation.

Another hypothesis is that multicellularity required a subspecialization of function such that it became necessary for the various FtsH gene family members to differ in the timing and/or cell specificity of their expression. In this respect, it is interesting that all four AtFtsH genes (AtFtsH1, 2, 5, and 8) have similar expression profiles, with abundant expression in green organs of the plant. Although our experiments were not refined enough to allow us to detect cell-specific differences in expression, the absolute levels of expression of these genes vary: AtFtsH2 is the most highly expressed (at the mRNA and protein levels), followed by AtFtsH1 and AtFtsH5, with approximately 50% of the mRNA and protein expression levels of AtFtsH2, and finally AtFtsH8, whose mRNA and protein expression are about 20% that of AtFtsH2 (Sinvany-Villalobo et al., 2004). As Sinvany-Villalobo et al. (2004) have pointed out, these expression levels roughly correlate with the severity of phenotype observed in mutants that have lesions in these genes: Null mutants of AtFtsH2 and AtFtsH5 are variegated, whereas null (T-DNA insertion) mutants of AtFtsH1 and AtFtsH8 have no obvious phenotypic changes (Sakamoto et al., 2003; F. Yu and S. Rodermel, unpublished data).

Although we do not understand the reason for the quantitative differences in expression among the various AtFtsH genes, our data indicate that chloroplast biogenesis requires AtFtsH complexes with two types of subunits (AtFtsH2 and 8) and (AtFtsH1 and 5). As discussed below, the attainment of threshold concentrations of these subunit types might be critical. If so, processes that promote this might have been important during evolution (e.g. increased gene copy number, increased expression levels). In this context, our data are reminiscent of Rubisco, where the large size of the nuclear Rubisco small subunit gene family has been attributed, in part, to the need to obtain high Rubisco concentrations in the chloroplast, rather than to a need for individual gene specialization (for review, see Rodermel, 1999). Consistent with this hypothesis is the observation that AtFtsH2 can form homo-oligmers, which might constitute a fraction of the AtFtsH complex pool (Sakamoto et al., 2003). This might not be unexpected because AtFtsH2 is the most abundant FtsH subunit. Regardless, how the total optimum AtFtsH pool size is attained is not known.

Mechanism of Variegation

The data in this study confirm and extend a model of variegation in which chloroplast function requires a threshold concentration of AtFtsH multimers (Yu et al., 2004). Below this threshold, a white plastid forms, while above this threshold, a normal chloroplast is produced. According to our revised model, AtFtsH complexes contain two types of subunits—AtFtsH2 and 8 and AtFtsH1 and 5. Because alterations in AtFtsH protein abundance do not influence AtFtsH transcript abundance, we propose that levels of the AtFtsH1 and 5 pair are matched with those of the AtFtsH2 and 8 pair, and that excess subunits are turned over. In cells in which the number of AtFtsH complexes is below the threshold, chloroplast differentiation is impaired, leading to the generation of white plastids. Conversely, plastids with threshold or higher levels of AtFtsH complex formation develop normal chloroplasts. We speculate that these decisions occur during the conversion of undifferentiated proplastids in the leaf meristem into mature chloroplasts early in leaf development. The process of sorting out of dividing plastids would then result in the generation of clones of white plastids and cells (white sectors) or clones of chloroplast-containing cells (green sectors).

If our model is correct, there are at least two possibilities to explain the threshold phenomenon. One is that there is unequal partitioning of a nuclear gene product to the plastids (>100) within a cell. For instance, in the case of var1, there could be unequal partitioning of AtFtsH1, with some plastids receiving more of this protein than others. An alternative explanation is that all plastids receive similar amounts of AtFtsH1, but that there might be intrinsic differences among plastids within a developing leaf cell in a biochemical process that is required for normal chloroplast development. For instance, there might be variability in rates of D1 repair, such that not all plastids need the same amount of AtFtsH1 for this process to occur efficiently. Regardless of the precise mechanism, our model is in accord with early data showing that cells in the white sectors of var2 are heteroplastidic and contain some normal-appearing chloroplasts, as well as a large number of abnormal plastids (Chen et al., 1999). This plastid autonomy indicates that not all plastids in a given cell are equivalent in function.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material

Arabidopsis (Arabidopsis thaliana ecotype Columbia) and pea (Pisum sativum) were grown in growth rooms under continuous fluorescent lights (about 100 µmol m^–2 s^–1) at 22°C. Rice (Oryza sativa sp. japonica cv Nipponbare) was grown in growth chambers under 16-h-day/8-h-night cycles at 28°C.

2-D Green Gel Analysis

Leaf tissues used in this study were collected from 3-week-old Arabidopsis plants, 3-week-old rice plants, and 2-week-old pea plants. Chloroplast membranes were isolated as previously described (Yu et al., 2004) by grinding the tissue in 0.33 M sorbitol, 10 mM EDTA, 50 mM HEPES, pH 8.0, and 0.05% bovine serum albumin using a Waring blender. The homogenate was filtered through two layers of Miracloth and centrifuged at 2,600g for 3 min. Crude chloroplasts were washed with 10 mM MOPS, pH 8.0, followed by centrifugation at 10,000g for 10 min. The pellet, which contains thylakoid membranes, was resuspended in 0.33 M sorbitol, 5 mM MgCl₂, and 50 mM HEPES, pH 8.0. Chlorophyll concentrations were measured on the resuspended membranes using 95% ethanol (Lichtenthaler, 1987). All the steps were performed at 4°C under dim light.

First-dimension green gel analysis was carried out as previously described (Yu et al., 2004). In brief, thylakoid membranes were solubilized in a mixture of detergents (0.45% octyl glucoside, 0.45% decyl maltoside, 0.1% lithium dodecyl sulfate, 10% glycerol, 2 mM Tris maleate, pH 7.0), and samples corresponding to 45 µg of chlorophyll were loaded onto a 3-mm 8% polyacrylamide (native) gel (MiniProtein II; Bio-Rad, Hercules, CA). The gels were electrophoresed at 4°C at 10 mA for 90 min. For the second dimension, gel strips were cut out of the native gels and incubated in 2x SDS sample buffer (Laemmli, 1970) at 65°C for 2 h. SDS-PAGE was then performed as described using 14% SDS-polyacrylamide gels (Xu et al., 1994).

Antibody Production, Coimmunoprecipitation, and Western Immunoblotting

To generate a specific antibody for AtFtsH1, a 15-amino acid peptide PLFIQNEILKAPSPK that is specific for AtFtsH1 was synthesized, and polyclonal antibodies were generated in rabbits (ProSci, Poway, CA). The antibody was used at a 1:10,000 dilution.

Nucleotide fragments corresponding to amino acids P196-G353, A51-S229, and G367-V507 of the D1 protein of PSII, the cytochrome b₆f Rieske protein (PetC), and the ATP synthase -subunit, respectively, were amplified by PCR and subcloned into the pET15b vector (Novagen, Madison, WI). The resulting constructs were transferred and expressed in Escherichia coli BL21(DE3) (Novagen). All the expressed peptides formed inclusion bodies, which were purified and solubilized (by procedures suggested by the manufacturer) and injected into rabbits. After three injections, cleared sera were used as antibodies against each antigen. All three antibodies were used at a 1:1,000 dilution.

Coimmunoprecipitations were carried out essentially as described by Sakamoto et al. (2003). Briefly, thylakoid membranes were suspended in phosphate-buffered saline at a concentration of 0.8 mg chlorophyll/mL, then solubilized with 0.8% dodecylmaltoside. After 30-min incubation on ice, samples were diluted to 0.1 mg chlorophyll/mL and antibodies against AtFtsH1 or AtFtsH2/VAR2 were added. Sepharose-coupled protein A was added after 4 h and the samples were incubated overnight at 4°C. Immunoprecipitates were recovered by centrifugation at 12,000g for 15 s, then washed three times with phosphate-buffered saline containing 0.05% dodecylmaltoside. Finally, the samples were boiled for 5 min in 2x SDS buffer and subjected to standard SDS-PAGE.

For western immunoblotting, proteins were transferred from 12% SDS-polyacrylamide gels to nitrocellulose membranes (Immobilon-NC; Millipore, Billerica, MA) and probed with various antibodies. The AtFtsH2/VAR2 antibody was described in Chen et al. (2000); it detects both AtFtsH2 and 8. The SuperSignal West Pico chemiluminescence kit (Pierce, Rockford, IL) was used for signal detection. The nitrocellulose membranes were stained after transfer with Ponceau S to examine loading efficiency and transfer quality.

Plasmid Construction and Arabidopsis Transformation

To generate an AtFtsH1 overexpression construct, an AtFtsH1 cDNA was amplified using pfx DNA polymerase (Invitrogen, Carlsbad, CA) with forward primer 5'-AACCTCGAGACGAAGAAGAAGAAACAGAGCTGC-3' and reverse primer 5'-AACCTCGAGTGTAAGCACTAAGCAATGGCAACT-3' (XhoI sites underlined). The PCR product was digested with XhoI and cloned into the XhoI site of pBlueScript. The resulting plasmid was sequenced for errors. The AtFtsH1 cDNA fragment was isolated and cloned into the XhoI site of a binary vector derived from pBI121 (Yu et al., 2004). This vector lacks the GUS gene, but contains the CaMV 35S promoter and the NPTII (neomycin phosphotransferase) gene separated by a multiple cloning site. The resultant plasmid was named pBI111LF1C. To generate var1-1 plants that overexpress AtFtsH1, pBI111LF1C was mobilized into Agrobacterium tumefaciens and var1-1 plants were transformed by the A. tumefaciens-mediated floral-dip method (Clough and Bent, 1998). Kanamycin-resistant plants were selected at the T1 generation on plates containing 1x Murashige and Skoog salts, 1% Suc, 0.8% agar (pH 5.7) supplemented with 50 µg/mL kanamycin. PCR and Southern blotting were performed to verify that the plants were transformed. Segregation of phenotypes was scored in the T2 generation; RNA and protein analyses were also performed using T2 generation plants.

To generate an AtFtsH1 antisense construct, a 1,500-bp AtFtsH1 cDNA fragment from pBI111LF1C was cut out using XhoI and SstI and then inserted into a pBI111L plasmid digested with XhoI and SstI. In this way, this AtFtsH1 cDNA fragment is under 35S promoter control in antisense orientation.

To generate an AtFtsH2/VAR2 overexpression construct, forward primer 5'-ATAGGATCCAGATTCTCCTTCCCTATACAACTCT-3' and reverse primer 5'-ATAGGATCCGAGATAGTATCACATTTCTAGAGTG-3' (BamHI site underlined) were used to amplify AtFtsH2/VAR2 full-length cDNA. The cloning procedure is essentially the same as the AtFtsH1 construct above, except the restriction enzyme used is BamHI.

To generate AtFtsH1 and AtFtsH5/VAR1 promoter-GUS fusion constructs, a similar approach was taken as in Yu et al. (2004). The primers for AtFtsH1 are forward primer 5'-CATTCTAGAAGCTCTCACTGTTGTGTTACTCTT-3' and reverse primer 5'-CATTCTAGATACTTGCGACGAGAATGGCGAATT-3' (XbaI site underlined). The primers for AtFtsH5/VAR1 are forward primer 5'-CATGGATCCCAACATCTATGTACTAGAATCTAG-3' and reverse primer 5'-CATGGATCCGAACAACAGAGCTGCTAACGCAGC-3' (BamHI site underlined).

Manipulations of DNA and RNA

Genomic DNAs were isolated and Southern-blot analyses were performed as described in Wetzel et al. (1994). Total cell RNAs were isolated from plants using the Trizol RNA Reagent (Invitrogen). Semiquantitative RT-PCR was carried out essentially as described in Yu et al. (2004). In brief, the RNA was reverse transcribed using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen). The RT reaction products were diluted 10 times, and the PCR reactions were performed using 32 cycles in a total volume of 50 µL. This is within the linear range of amplification for each of the fragments examined. Gene-specific primers for AtFtsH1 were 5'-GTCTCAAGACGAAAATTCGC-3' and 5'-GCCTGAAAAGCAAGAAGAGT-3'. Gene-specific primers for AtFtsH2/VAR2 were 5'-ACTCTCATCTGTAATCGATA-3' and 5'-ACCAATCAAGTTGAATAACAC-3'. Gene-specific primers for AtFtsH5/VAR1 were 5'-CAACCACCAGCACCAACCAT-3' and 5'-CTCTAAAGAGATAAAACAACC-3'. Gene-specific primers for AtFtsH8 were 5'-TCTTCTGCCAAATTCCACAG-3' and 5'-CCCAATCAAGTTGAGTATTGG-3'. Gene-specific primers for the Actin 2 gene were 5'-TCAAAGACCAGCTCTTCCATCGAGA-3' and 5'-ACACACAAGTGCATCATAGAAACGA-3'.

	ACKNOWLEDGMENTS

 
We thank the ABRC for Arabidopsis seed stocks and the National Small Grains Collection (Aberdeen, Idaho) for rice seeds.

Received February 15, 2005; returned for revision April 28, 2005; accepted May 11, 2005.

	FOOTNOTES

 
¹ This work was supported by funding from the U.S. Department of Agriculture Competitive Research Grants Program (Biochemistry; grant no. 20013531810004 to S.R.R.) and from the U.S. Department of Energy, Energy Biosciences Panel (DE–FG02–94ER20147).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.061234.

^* Corresponding author; e-mail rodermel{at}iastate.edu; fax 515–294–1337.

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