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Plant Cell. Jan 2004; 16(1): 201–214.
PMCID: PMC301405

Maize Mutants Lacking Chloroplast FtsY Exhibit Pleiotropic Defects in the Biogenesis of Thylakoid MembranesW in Box

Abstract

A chloroplast signal recognition particle (SRP) that is related to the SRP involved in secretion in bacteria and eukaryotic cells is used for the insertion of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membranes. A conserved component of the SRP mechanism is a membrane-bound SRP receptor, denoted FtsY in bacteria. Plant genomes encode FtsY homologs that are targeted to the chloroplast (cpFtsY). To investigate the in vivo roles of cpFtsY, we characterized maize cpFtsY and maize mutants having a Mu transposon insertion in the corresponding gene (chloroplast SRP receptor1, or csr1). Maize cpFtsY accumulates to much higher levels in leaf tissue than in roots and stems. Interestingly, it is present at similar levels in etiolated and green leaf tissue and was found to bind the prolamellar bodies of etioplasts. A null cpFtsY mutant, csr1-1, showed a substantial loss of leaf chlorophyll, whereas a “leaky” allele, csr1-3, conditioned a more moderate chlorophyll deficiency. Both alleles caused the loss of various LHCPs and the thylakoid-bound photosynthetic enzyme complexes and were seedling lethal. By contrast, levels of the membrane-bound components of the thylakoid protein transport machineries were not altered. The thylakoid membranes in csr1-1 chloroplasts were unstacked and reduced in abundance, but the prolamellar bodies in mutant etioplasts appeared normal. These results demonstrate the essentiality of cpFtsY for the biogenesis not only of the LHCPs but also for the assembly of the other membrane-bound components of the photosynthetic apparatus.

INTRODUCTION

The biogenesis of chloroplast thylakoid membranes requires the coordinated expression and assembly of proteins encoded by both the nuclear and chloroplast genomes. Nucleus-encoded components are synthesized in the cytosol, imported into the chloroplast stroma, and subsequently targeted by one of four mechanistically distinct pathways to the thylakoid membrane. The four pathways engage different substrates and can be distinguished by their protein factor and energy requirements as follows. (1) The Sec pathway requires ATP and cpSecA and has been shown to be involved in the transport of plastocyanin, the 33-kD subunit of the oxygen-evolving complex (OE33), PSI-F (photosystem I-F), and the plastid-encoded cytochrome f. (2) A signal recognition particle (cpSRP) pathway requires GTP and cpSRP54 and cpSRP43 and is involved in the targeting of the abundant light-harvesting chlorophyll a/b binding proteins (LHCPs) that make up the antennae of photosystems I and II. (3) A ΔpH-dependent, or Tat, pathway uses a transthylakoidal pH gradient as its sole energy source and does not require the participation of stromal protein factors; this pathway transports OE23, OE17, PSII-T, and PSI-N. (4) A “spontaneous” pathway that seems not to require soluble factors or energy is responsible for the insertion of CFoII, PSII-W, PSII-X, and ELIP. In general, proteins destined for the thylakoid lumen are transported by either the Sec or the ΔpH pathway, whereas integral membrane proteins are targeted by the SRP or the spontaneous pathway. Components of the Sec, ΔpH, and cpSRP pathways all have homologs in extant bacteria and presumably were derived from the cyanobacteria-like ancestor of the chloroplast (for reviews, see Robinson et al., 1998; Keegstra and Cline, 1999).

The Escherichia coli SRP consists of Ffh (54 homolog) and 4.5S RNA and plays a central role by interacting with signal sequences as membrane-destined proteins emerge from the ribosome (for reviews, see Walter and Johnson, 1994; Rapoport et al., 1996; Fekkes and Driessen, 1999). The cpSRP in higher plant chloroplasts consists of the SRP54/Ffh homolog cpSRP54 and cpSRP43, which is unique for the chloroplast SRP system, but contains no RNA components (Schuenemann et al., 1998; Klimyuk et al., 1999; Tu et al., 1999; Groves et al., 2001). The LHCPs are the best characterized substrates of the cpSRP pathway. They are the most abundant thylakoid membrane proteins and bind almost half of the chlorophyll (for review, see Wollman et al., 1999). LHCPs form a superfamily of related nucleus-encoded proteins with three or four transmembrane helices. After their import into the chloroplast stroma, they are bound by cpSRP to form a soluble transit complex, which prevents the aggregation of these hydrophobic proteins and maintains their competence for thylakoid insertion (Payan and Cline, 1991; Li et al., 1995; Schuenemann et al., 1998). The details of the binding interactions between LHCP, cpSRP43, and cpSRP54 were elucidated recently (DeLille et al., 2000; Tu et al., 2000; Jonas-Straube et al., 2001). In addition to soluble cpSRP in the stroma, targeting of LHCP to the thylakoid membrane also requires a membrane-bound protein called cpFtsY, which is a homolog of the bacterial SRP receptor FtsY (Kogata et al., 1999; Tu et al., 1999). Finally, integration of LHCP into the thylakoid membrane is mediated by the integral membrane protein ALB3, which is a homolog of the mitochondrial Oxa1p and bacterial YidC proteins (Moore et al., 2000; Woolhead et al., 2001). Both cpSRP54 and cpFtsY contain a GTPase domain, and LHCP integration into the thylakoid membrane was shown to require GTP hydrolysis (Hoffman and Franklin, 1994; Kogata et al., 1999; Tu et al., 1999).

Several Arabidopsis mutants related to the cpSRP pathway have been isolated and characterized. Interestingly, these mutants seemed to vary in their phenotypes. A null mutant of cpSRP43, chaos, had pale green leaves throughout its growth, a high chlorophyll a/b ratio, and a specific reduction in 7 of the 11 different LHCPs tested (Amin et al., 1999; Klimyuk et al., 1999). A null mutant of SRP54, ffc, had yellow first true leaves that subsequently became pale green, a reduced level of half of the 11 LHCPs tested (Amin et al., 1999), and a reduction in many other thylakoid proteins (Pilgrim et al., 1998). The double mutant ffc/chaos had pale yellow leaves at all stages of growth and drastically reduced levels of all of the LHCPs except Lhcb4 (Hutin et al., 2002), whereas thylakoid proteins that use other targeting pathways seemed to accumulate normally. ffc, chaos, and ffc/chaos were not seedling lethal, indicating that they retained some photosynthetic competence. alb3 mutants showed the most severe phenotype: albino leaves and seedling lethality (Long et al., 1993; Sundberg et al., 1997).

To date, mutants lacking cpFtsY have not been described, and our knowledge of cpFtsY function is derived solely from in vitro assays. To elucidate the in vivo roles of cpFtsY, we obtained and characterized cpFtsY mutants in maize. As anticipated, null cpFtsY mutants showed reduced amounts of various LHCPs. However, they also lacked other thylakoid-bound components of the photosynthetic apparatus and exhibited severe defects in the architecture of the thylakoid membranes. These findings indicate that cpFtsY plays broad roles in thylakoid biogenesis that extend beyond the integration of the LHCPs.

RESULTS

cpFtsY Accumulates to High Levels in Both Green and Etiolated Leaf Tissue

A maize cDNA encoding cpFtsY was cloned by PCR amplification out of a maize leaf cDNA library, using primers designed to a maize EST whose product has a high level of identity with Arabidopsis cpFtsY. An alignment of the predicted amino acid sequence of maize cpFtsY with those of representative homologs in Arabidopsis (Kogata et al., 1999; Tu et al., 1999), rice, and bacteria is shown in Figure 1. cpFtsYs from Arabidopsis and maize exhibited 72.4% overall identity and 78.8% identity in their presumed mature sequences. Like the Arabidopsis cpFtsY, the maize homolog lacks the acidic N-terminal domain found in some bacterial FtsY proteins, which has been suggested to be essential for their membrane association. On the other hand, both the Arabidopsis and maize cpFtsYs preserve the NG domain, which includes three consensus motifs for GTP binding (boxed in Figure 1A) and is common to all SRP-type GTPases (Montoya et al., 1997). Completed genome sequences from rice and Arabidopsis revealed just one cpFtsY gene. The available maize sequence data also provide evidence for just one cpFtsY gene.

Figure 1.
Deduced Amino Acid Sequence of Maize cpFtsY and Corresponding Positions of Mu Insertions in csr1 Mutants.

The organ specificity of maize cpFtsY was examined by immunoblot analysis of root, stem, and leaf extracts with anti-maize cpFtsY antibody. The antibody detected a 34-kD protein (the size predicted for mature maize cpFtsY; molecular weight = 35,324) in leaf tissue. The protein detected is the product of the cloned cDNA, because it is missing in the cpFtsY mutants described below (see Figure 3C). cpFtsY accumulates to the highest level in leaves, to an intermediate level in stems, and was barely detectable in roots (Figure 2A). This expression pattern correlates with its predicted role in the biogenesis of the photosynthetic apparatus. cpFtsY was found not only in green leaf tissue but also in etiolated leaf tissue, and no obvious light induction was observed after light exposure (Figure 2B). By contrast, protochlorophyllide oxidoreductase showed light-depressed expression and Lhcb2 showed light-induced expression, as reported previously (Oosawa et al., 2000; Aronsson et al., 2003). The expression of other cpSRP components, such as cpSRP54, cpSRP43, and ALB3, and of cpHsp70 during the greening of etiolated seedlings was similar to that of cpFtsY.

Figure 2.
Immunoblot Analyses of Maize cpFtsY: Tissue and Light Dependence, and Suborganellar Localization.
Figure 3.
Pigment Deficiency of csr1 Mutant Seedlings.

Figure 2C demonstrates the distribution of cpFtsY within chloroplasts (left) and within etioplasts (right). Most of the cpFtsY was associated with thylakoid membranes, as we demonstrated previously for Arabidopsis cpFtsY (Kogata et al., 1999). Upon trypsin treatment, a luminal thylakoid protein, OE33, was unaffected, and membrane-embedded portions of the membrane proteins Lhcb2 and ALB3 were protected and detected as smaller fragments, as shown previously (Cline, 1986; Woolhead et al., 2001). By contrast, the thylakoid-bound cpFtsY was digested completely by the treatment, suggesting that cpFtsY is bound peripherally to the stromal face of the membrane, as shown previously for Arabidopsis cpFtsY (Kogata et al., 1999). In etioplasts, cpFtsY was localized mainly on the surface of prolamellar bodies (Figure 2C).

csr1 Mutants Harboring a Mu Transposon Insertion in the cpFtsY Gene Were Seedling Lethal

We named the gene that encodes the isolated cpFtsY cDNA csr1 (chloroplast SRP receptor1). To study the in vivo function of cpFtsY, we sought mutations in the csr1 gene using a reverse genetics approach involving the Photosynthetic Mutant Library (PML; http://pml.uoregon.edu/). PML is a collection of ~2000 transposon Mu-induced nonphotosynthetic maize mutants. Mutants contributed to PML exhibit either a seedling chlorophyll deficiency (e.g., pale green, yellow, virescent, albino, or striated leaves) or a high chlorophyll fluorescent (hcf) phenotype. Pooled mutant DNA samples were used in PCR with a Mu primer and a csr1 primer to identify mutants with Mu insertions in the csr1 gene. Three mutant alleles were recovered: csr1-1, csr1-2, and csr1-3. csr1-1 and csr1-2 contain a Mu insertion that disrupts the first exon in sequences that encode the middle of the transit peptide. csr1-3 has a Mu insertion in the second intron (Figures 1A and 1B). Crosses between plants heterozygous for the csr1-1, csr1-2, and csr1-3 alleles all yielded approximately one-quarter chlorophyll-deficient seedlings, indicating that the chlorophyll-deficient phenotype results from mutations in the csr1 gene. In this study, csr1-1 and csr1-3 were analyzed because they represent different degrees of the loss of function.

Like typical nonphotosynthetic maize mutants, homozygous csr1-1 and csr1-3 mutant plants were seedling lethal and died ~2 weeks after germination. Heterozygous plants appeared normal, indicating that these mutations are recessive. Before the depletion of seed reserves, the homozygous mutants were reduced only slightly in size and had normal morphology (Figure 3A). However, both mutant alleles were associated with a chlorophyll-deficient phenotype: leaves of csr1-1 mutants were affected more severely and had a pale yellow-green phenotype; csr1-3 mutant leaves were slightly pale green. Quantitative measurements of their chlorophyll content were consistent with their appearance: chlorophyll a and b in csr1-1 leaves were reduced to 10 and 14% of wild-type levels, respectively, whereas those in csr1-3 were 27 and 29% of normal (Figure 3B). The chlorophyll a/b ratio (3.49 ± 0.04 for the wild type) was moderately affected in csr1-1 (2.55 ± 0.08) but was nearly normal in csr1-3 (3.27 ± 0.05).

Immunoblots of the mutant leaves showed a deficiency for cpFtsY (Figure 3C). cpFtsY was not detectable in csr1-1 leaf extracts and was detected at very low levels in csr1-3 leaf extracts. These results suggest that csr1-1 might be a null allele, whereas csr1-3 clearly is a “leaky” allele. This leakiness is consistent with the fact that the Mu insertion maps within an intron in csr1-3.

Reduced Thylakoid Membrane Content in csr1 Mutants

In the Arabidopsis ffc and chaos mutant plants, which contained mutations in the cpSRP54 and cpSRP43 genes, respectively, chloroplast ultrastructure was not altered significantly. However, in the ffc/chaos double mutant and in alb3 mutants, thylakoid membrane development was affected severely (Sundberg et al., 1997; Pilgrim et al., 1998; Klimyuk et al., 1999; Hutin et al., 2002). To study the effects of cpFtsY deficiency on the ultrastructure of chloroplasts, leaf sections were analyzed by transmission electron microscopy as shown in Figure 4A. Both bundle sheath and mesophyll chloroplasts in csr1-1 mutants had large reductions in their thylakoid membrane contents, and the residual membranes were poorly organized. Furthermore, starch grains were lacking in the mutant bundle sheath chloroplasts, and grana stacking was lacking in the mutant mesophyll chloroplasts. In the weaker allele, csr1-3, the loss of thylakoid membrane was more moderate but still significant. The size of the chloroplasts in both cases was similar to that in wild-type plants.

Figure 4.
Ultrastructure of Chloroplasts and Etioplasts in csr1 Mutants.

As shown in Figure 2, cpFtsY is present in etioplasts of dark-grown seedlings. To determine whether cpFtsY is involved in the formation of prolamellar bodies, we visualized etioplasts in csr1 mutants by transmission electron microscopy. As shown in Figure 4B, etioplasts in csr1-1 mutants had rather normal crystalline prolamellar bodies, but the prothylakoid membranes extending from the prolamellar bodies were less abundant and shorter than normal (Figure 4B). Thus, although cpFtsY is located on the prolamellar bodies in etioplasts, it seems to be nonessential for prolamellar body formation. These results suggest that csr1 is required for light-dependent thylakoid membrane development and plays little or no role in etioplasts.

LHCP Integration Was Affected in csr1 Mutants

The participation of cpFtsY in the targeting of LHCPs to the thylakoid membrane has been shown through in vitro targeting assays (Kogata et al., 1999; Tu et al., 1999). To examine the role of cpFtsY in LHCP targeting in vivo, the steady state levels of various LHCPs were monitored by immunoblot analysis of csr1 mutant leaf extracts (Figure 5). The antisera used were generated against peptides that are conserved among various plant species and that are specific for particular members of the Lhc family (Andersson et al., 2001); however, we cannot be certain that the protein detected in maize is truly orthologous to the anticipated member of the family. Despite this ambiguity, it is clear that in csr1-1 mutants, most LHCPs tested were reduced dramatically in abundance, whereas in csr1-3 mutants, their levels were affected more moderately.

Figure 5.
LHCP Immunoblot Analyses of csr1 Mutants.

To test whether the reductions in LHCPs in the mutants are the consequence of inefficient LHCP integration into the thylakoid membranes, we performed in vitro import and thylakoid integration assays. Radiolabeled precursor LHCP (pLHCP, which corresponds to a pea Lhcb1 precursor) was synthesized in vitro and imported into chloroplasts isolated from wild-type and mutant seedlings. After a 15-min incubation, mature LHCPs appeared in both wild-type and csr1-3 chloroplasts (Figure 6A). The mature LHCPs were resistant to externally added thermolysin, suggesting that translocation of pLHCP across the envelope membrane and maturation by the stromal processing peptidase were not affected seriously in the csr1-3 chloroplasts. After suborganellar fractionation (Figure 6B), the mature LHCP in wild-type chloroplasts was mostly recovered with membranes containing the thylakoids.

Figure 6.
In Organello Import and Membrane Insertion of LHCP in csr1 Mutant Chloroplasts.

To test whether membrane-bound LHCP was integrated correctly into the membrane, the membranes were treated with thermolysin. Correctly integrated LHCP is digested by thermolysin only at its stroma-exposed N-terminal fragment (~2 kD); the remainder of the protein, which is integrated into the membrane, has been shown to be resistant to thermolysin digestion and should be observed as an LHCP fragment of 25 kD called LHCP-DP (Cline, 1986). LHCP-DP was observed after import into wild-type chloroplasts but was barely detectable after import into csr1-3 chloroplasts (Figures 6B and 6C). Furthermore, the mature LHCP recovered with thylakoids appeared to be mostly digested by thermolysin, suggesting that it has not inserted properly into the membrane. Interestingly, an increased fraction of the mature LHCP was found in the stroma in csr1-3 chloroplasts compared with wild-type stroma. Similar results were obtained for csr1-1 chloroplasts (see supplemental data online). These findings indicate that csr1 mutant chloroplasts can import and process LHCP normally but are defective in their ability to insert mature LHCP into the thylakoid membranes (Figure 6C). This is consistent with our previous findings that antibodies to Arabidopsis cpFtsY inhibited LHCP insertion into isolated thylakoid membranes in vitro (Kogata et al., 1999).

After import into the chloroplast, stromal LHCP has been shown to form a transit complex with the soluble cpSRP components cpSRP54 and cpSRP43 (Payan and Cline, 1991; Li et al., 1995; Schuenemann et al., 1998). To determine whether the small amount of stromal LHCP that accumulated in csr1-3 chloroplasts after in vitro import formed a complex with the cpSRP, the stromal fraction was subjected to immunoprecipitation using anti-cpSRP54 and anti-cpSRP43 antibodies (Figure 6D). Both antibodies coimmunoprecipitated the radiolabeled LHCP from the stroma of csr1-3 chloroplasts, whereas preimmune serum did not.

All Thylakoid-Bound Photosystem Complexes Are Reduced in the csr1 Mutants

The best established role for the cpSRP pathway is in the integration of the LHCPs. However, it also has been suggested to participate in the integration of chloroplast-encoded integral membrane proteins such as D1, the psbA gene product (Nilsson et al., 1999). To determine whether cpFtsY is important for the accumulation of thylakoid membrane proteins other than the LHCPs, the abundance of representative subunits of each thylakoid membrane complex was examined by immunoblot analysis (Figure 7A). Included in this analysis were proteins known to be targeted to the thylakoid membrane via different pathways: OE33 of PSII, cytochrome f of the cytochrome b6/f complex, and plastocyanin are known to be targeted to the thylakoids via the Sec pathway; OE23 uses the ΔpH pathway; and targeting and insertion of D1 (PsbA) was shown in vitro to be mediated by cpSRP54 and cpSecY (Nilsson et al., 1999; Zhang et al., 2001). All of these proteins are reduced in abundance in csr1-1 mutants, with csr1-3 mutants showing a more moderate decrease. Other thylakoid proteins whose targeting pathways are not yet known (PsaC and PsaD of PSI, CF1α and CFoI of the ATPase) also were reduced significantly in the mutants. These results indicate that the accumulation of all of the thylakoid-bound photosynthetic enzyme complexes is affected by the csr1 mutations. It is unlikely that cpFtsY is involved directly in the membrane targeting of all of these proteins. Possible interpretations of these observations are discussed below.

Figure 7.
Immunoblot Analyses of Photosynthetic Thylakoid Membrane Proteins, Stromal Chaperones, and Components of the Thylakoid Protein Translocation Machineries in csr1 Mutants.

Klimyuk et al. (1999) reported that the chaos mutation (which disrupts the gene for the SRP43 subunit of the SRP complex) caused a strong reduction of the oligomeric form of LHCPs, as detected by partially denaturing gel electrophoresis. To monitor the assembly of LHCP-containing complexes and other photosynthetic complexes, we used blue native gel electrophoresis (Schägger et al., 1994; see also http://www.hos.ufl.edu/clineweb/BNgel.htm) with the wild-type and mutant chloroplasts solubilized with 1% n-dodecyl-β-d-maltoside (Figure 8; see also the supplemental data online). Dramatic differences between wild-type and csr1-1 chloroplasts were observed. Although the mutants had a normal level of assembled ribulose-1,5-bisphosphate carboxylase/oxygenase, several thylakoid membrane complexes (PSII/LHCII supercomplexes, PSI/LHCI complex, ATPase, cytochrome b6/f complex, and LHCII trimer) were detected only faintly. By contrast, in the csr1-3 chloroplasts, corresponding complexes were detected, but at reduced levels. From these results, we conclude that the loss of cpFtsY causes a severe defect in the assembly and accumulation of photosynthetic complexes in the thylakoid membranes.

Figure 8.
Photosynthetic Complexes Were Decreased Dramatically in the csr1 Mutants.

Components Involved in the SRP-, Sec-, and ΔpH-Dependent Protein Translocation Pathways Were Not Decreased in the csr1 Mutants

One possible explanation for the global loss of thylakoid membrane proteins in csr1 mutants (Figure 7A) is that the polytopic membrane proteins cpSecY, Alb3, and cpTatC, which are the central components of the Sec, cpSRP, and ΔpH protein translocation machineries, respectively, might require cpFtsY to integrate into the thylakoid membrane. However, these proteins accumulated to normal or, in some cases, increased levels in the csr1-1 and csr1-3 mutants. The increased abundance of several of these proteins suggests that there may be compensatory mechanisms induced in the absence of cpFtsY (Figure 7B). Furthermore, membrane insertion of cpSecY and Alb3 in the csr1-1 mutant appeared to be normal, as judged by trypsin sensitivity and by solubilization with chaotropic agents (see supplemental data online) (Cline, 1986; Woolhead et al., 2001). The small reduction in cpSecA and cpSecY in csr1-3 mutants is unlikely to be a primary cause of the pleiotropic protein losses, because these two proteins accumulated to increased levels in the csr1-1 mutant (Figure 7B).

Stromal chaperone proteins also were increased significantly in both the csr1-1 and csr1-3 mutants (Figure 7B). A particularly striking increase was observed for the stromal cpHsp70. Yokthongwattana et al. (2001) reported that photodamage to PSII induced the nuclear gene that encodes the stromal Hsp70. Therefore, increased accumulation of stromal chaperones might occur in response to the disruption of photosystem assembly. The thylakoid-bound protease cpFtsH seemed to be increased slightly in csr1-1 but was not affected in csr1-3 (Figure 7B).

The Synthesis and Targeting of the Chloroplast-Encoded D1 Protein Are Not Disrupted Detectably in the Absence of cpFtsY

The PSII reaction center protein D1 is damaged during PSII photochemistry and then replaced by newly synthesized D1 protein (Rintamaki et al., 1996). This process involves both rapid D1 synthesis and efficient membrane targeting of the nascent protein. D1 is encoded by the chloroplast DNA and synthesized on thylakoid-bound polysomes (Kim et al., 1991; Zhang and Aro, 2002). cpSRP54 was shown to interact with nascent D1 protein during its in vitro translation in chloroplast lysate (Nilsson et al., 1999). By analogy with the SRP-mediated cotranslational translocation of proteins across the endoplasmic reticulum or bacterial cytoplasmic membranes, it seemed likely that cpFtsY would be required for the proper targeting of nascent D1 to the thylakoid membrane and for its cotranslational insertion into the membrane. Indeed, as shown in Figure 7, the steady state level of D1 protein in the csr1 mutants was reduced to some extent in csr1 mutants. To learn more about the cpFtsY dependence of D1 insertion, we examined the expression of D1 in csr1 mutants in detail.

As shown in Figure 9A, psbA mRNA accumulates to normal levels in csr1 mutants. The partitioning of the psbA transcripts between thylakoid and stromal fractions in csr1-1 was redistributed only slightly to the stromal fraction. This finding suggests that binding of the psbA transcript to the thylakoid surface does not strictly require cpFtsY (Figure 9B). To determine whether the psbA transcripts in the mutant are translated normally, we analyzed their association with polysomes by sedimenting leaf extract through sucrose gradients (Figure 9C). The sedimentation rate of an mRNA in this assay increases with the number of bound translating ribosomes. By this method, a dramatic decrease in the average number of ribosomes associated with psbA transcripts was detected in maize mutants lacking cpSecY (Roy and Barkan, 1998). By contrast, the psbA polysome profile was similar between the csr1-1 and wild-type samples. These results suggest that the psbA transcripts are translated normally in the absence of cpFtsY. Similar results were obtained for other chloroplast transcripts of rbcL (Figures 9A to 9C) and of atpF and petA (data not shown).

Figure 9.
Abundance, Membrane Association, and Polysome Association of the psbA mRNA Are Not Affected Significantly in the csr1-1 Mutant.

To determine whether the D1 protein that accumulates is inserted properly into the thylakoid membrane in the absence of cpFtsY, we examined its resistance to trypsin and solubilization by chaotropic regents using thylakoid membranes isolated from wild-type and csr1-1 chloroplasts. D1 responded similarly to these treatments in the csr1-1 and wild-type thylakoid membranes (see supplemental data online). This finding suggests that D1 was integrated firmly into the lipid phase of the membrane and probably folded correctly in the absence of cpFtsY.

DISCUSSION

This study provides an extensive analysis of the role of cpFtsY in vivo. We showed that maize cpFtsY accumulates mainly in the photosynthetic tissues, where it is localized exclusively on the thylakoid membranes (Figure 2), as found previously in Arabidopsis and pea (Kogata et al., 1999). cpFtsY accumulates to similar levels in light-grown and dark-grown etiolated leaves (Figure 2B). Although cpFtsY is expressed in etioplasts and bound on the surface of the prolamellar body, the functional importance of cpFtsY in the etioplast is unclear, because the crystalline structure of the prolamellar bodies appeared to be normal even in the absence of cpFtsY (Figure 4B). It is possible that the presence of cpFtsY in etioplasts facilitates the rapid biogenesis of the thylakoid membrane in response to light.

Tu et al. (1999) reported previously that cpFtsY was found partitioned almost equally between the stroma and the thylakoid membrane in pea chloroplasts. However, we found that cpFtsY was associated almost entirely with the thylakoid membranes in pea, Arabidopsis (Kogata et al., 1999), and maize (this study). Although the reason for this discrepancy is not clear, an interesting question raised by this work concerns the mechanism by which cpFtsY associates with the membrane. cpFtsY proteins lack the N-terminal acidic domain that has been shown to be essential for the membrane association of E. coli FtsY (Luirink et al., 1994; de Leeuw et al., 1997). Interestingly, FtsY homologs from some bacteria, including Thermus aquaticus, also lack the N-terminal acidic domain (Shepotinovskaya and Freymann, 2002). The mammalian FtsY homolog SRα, which participates in protein translocation across the endoplasmic reticulum membrane, is recruited to the membrane through interaction with the integral membrane protein SRβ (Tajima et al., 1986). However, no SRβ homolog has been found in chloroplasts (or in bacteria). Therefore, cpFtsY may associate with the membrane in a novel manner.

cpFtsY Is Important for Efficient Thylakoid Targeting of LHCPs but Is Not Absolutely Required

We showed here that cpFtsY deficiency disrupted the insertion of LHCP (Lhcb1 precursor) into the thylakoid membranes. Presumably as a result of this membrane integration defect, steady state levels of various LHCPs were reduced severely in the csr1-1 mutant. Some members of the LHCP family (e.g., Lhcb6) were less affected than others by the absence of cpFtsY, suggesting that certain LHCPs might be inserted into the thylakoid membrane without cpFtsY but with reduced efficiency.

Previous studies demonstrated that Arabidopsis null mutants lacking cpSRP54, cpSRP43, cpSRP43/cpSRP54, or ALB3 showed different phenotypes. The loss of ALB3 resulted in a white or light yellow seedling-lethal mutant (Sundberg et al., 1997). The chaos (ΔSRP43) mutant was slightly chlorotic (Amin et al., 1999; Klimyuk et al., 1999), whereas the ffc (ΔSRP54) mutant had yellow first true leaves that subsequently became green (Pilgrim et al., 1998; Amin et al., 1999). The double mutant ffc/chaos had pale yellow leaves at all stages (Hutin et al., 2002). Although it is difficult to compare mutant phenotypes across species, the appearance of maize csr1-1 seedlings resembles that of the Arabidopsis ffc/chaos and alb3 mutants. Different degrees of dependence of distinct LHCPs on cpSRP for their integration into the thylakoid also were reported previously in the ffc/chaos double mutant (Hutin et al., 2002). Interestingly, in both csr1-1 and ffc/chaos, Lhca3 and Lhcb3 were affected most severely, whereas Lhca2, Lhca4, and Lhcb6 were less affected, suggesting that these different degrees of dependence of distinct LHCP polypeptides on the cpSRP machinery are conserved between maize and Arabidopsis.

It is clear that certain LHCPs can be inserted into the thylakoid membranes even in the absence of cpSRP54/cpSRP43 or cpFtsY. Stromal chaperone proteins may promote such bypass targeting of LHCP to the thylakoid membranes. It is noteworthy that various stromal chaperones accumulate to increased levels in the csr1-1 chloroplasts. Upregulation of stromal chaperones may serve as a compensatory mechanism in chloroplasts in which thylakoid membrane assembly is disrupted. This is consistent with the previous observation that cpHsp70 expression is increased in response to photodamage to PSII (Yokthongwattana et al., 2001).

csr1 Mutants Showed a Pleiotropic Defect in the Accumulation of Thylakoid Membrane Complexes

We demonstrated that the absence of cpFtsY affects not only the LHCPs, which are known to use the cpSRP-dependent pathway, but also thylakoid proteins that are targeted to the membrane via the Sec- or ΔpH-dependent pathways as well as proteins whose integration mechanisms are uncharacterized. On this point, the csr1-1 mutant clearly differs from the Arabidopsis ffc chaos double mutant, which contains normal levels of thylakoid proteins whose targeting is known to be independent of cpSRP (e.g., OE23 and PC) (Hutin et al., 2002). Why might cpFtsY deficiency cause such a pleiotropic loss of thylakoid proteins? This does not seem to result from a general defect in chloroplast gene expression, because various chloroplast transcripts accumulated properly and chloroplast polysomes assembled normally (Figure 9). Moreover, subunits of the Sec, cpSRP, and ΔpH thylakoid-targeting machineries accumulated normally in the absence of cpFtsY, suggesting that these protein translocation components can be inserted into the membranes by a cpSRP-independent mechanism and that a defect in the assembly of these translocation machineries is not the cause of the pleiotropic protein losses in csr1 mutants. One possible explanation for the broad thylakoid protein defects in csr1 mutants is that at least one integral membrane subunit of each of the membrane-bound photosynthetic enzyme complexes depends on the cpSRP for its integration. Indeed, in csr1-1, peripheral components such as PsaC, PsaD, and CF1α, which bind on the stromal side of the thylakoid membrane and thus do not require membrane translocation or integration, also were affected severely. It is well established that a defect in the synthesis or assembly of a single subunit of these complexes causes a reduction in the accumulation of other closely associated subunits.

It also is plausible that the pleiotropic defects in csr1 mutants result from the severe disruption of the biogenesis of the LHCP complexes, which are the most abundant protein components in the thylakoid membranes and which have been proposed to govern grana stacking (Allen and Forsberg, 2001). Another possibility is that unidentified proteins may play critical roles in thylakoid biogenesis and are targeted via a cpFtsY-dependent mechanism. Further biochemical study would be needed to elucidate how many other thylakoid proteins actually require cpFtsY for their thylakoid targeting.

Recently, we found that an Arabidopsis mutant lacking cpFtsY exhibited pleiotropic defects in thylakoid biogenesis that are as severe as those in the maize csr1-1 mutant described here (our unpublished results). The Arabidopsis mutant has a pigmentation that is similar to that reported for the Arabidopsis alb3 mutant (Sundberg et al., 1997), suggesting that both ALB3 and cpFtsY contribute almost equally to thylakoid biogenesis. Therefore, cpFtsY and ALB3 may be specialized partner proteins for the efficient insertion and assembly of large amounts of membrane proteins during light-induced rapid thylakoid development in photosynthetic tissues. Together, these genetic data suggest either that the soluble cpSRP54/cpSRP43 complex is less important for membrane targeting than the membrane-bound cpFtsY/ALB3 components or that some proteins engage the membrane-bound cpFtsY/ALB3 machinery without previously engaging cpSRP54/cpSRP43. It is noteworthy that E. coli FtsY, which is essential for the biogenesis of several integral membrane proteins, also is required for the association of ribosomes with the inner membrane even in the absence of Ffh, the E. coli SRP54 homolog (Herskovits and Bibi, 1997, 2000). This finding suggests that FtsY homologs may have functions that do not involve their interaction with SRP.

Insertion of the Chloroplast DNA–Encoded D1 Protein (PsbA) into Thylakoid Membranes Can Occur Independently of cpFtsY

The chloroplast-encoded D1 protein is one of two core subunits of the PSII reaction center. Rapid light-dependent turnover of the D1 protein maintains and regulates PSII function. Although cpSRP54 was demonstrated to be cross-linked efficiently to nascent D1 polypeptides in vitro and thereby thought to be a targeting factor for D1 protein (Nilsson et al., 1999), interaction with cpSRP54 seems to be not essential for D1 targeting to the thylakoid membranes in vivo, because the ffc mutant lacking cpSRP54 contains nearly normal levels of inserted D1 protein (Amin et al., 1999). The results shown here suggest that cpFtsY also does not play an important role in the targeting of D1 protein to the membrane: in csr1-1, psbA transcripts were attached normally to the thylakoid membranes and D1 protein accumulated to only slightly reduced levels and appeared to be integrated normally into the membrane. Thus, we propose that the targeting of D1 protein to the thylakoid membrane occurs independently of cpFtsY or can bypass cpFtsY.

In conclusion, the results presented here provide information about the in vivo role of cpFtsY in the biogenesis of thylakoid membranes. The results suggest that cpFtsY plays a more essential and/or more general role than the soluble cpSRP54/cpSRP43 complex and that it is similar in this way to ALB3. Further biochemical analyses will be required to dissect the functional link between cpFtsY and ALB3 and should help to elucidate their precise roles in thylakoid membrane biogenesis.

METHODS

Plant Material and Growth Conditions

The csr1-1 and csr1-3 mutants of maize (Zea mays) were identified from the Photosynthetic Mutant Library (http://pml.uoregon.edu/) using a primer complementary to the Mu terminal inverted repeat (Bensen et al., 1995) and a primer specific for the csr1 gene. Homozygous mutants were seedling lethal, so the mutations were propagated as heterozygotes. Self-pollination of heterozygous plants was performed to recover homozygous mutant seedlings. Seedlings were grown on vermiculite at 26 to 28°C in a growth chamber (14-h-light/10-h-dark cycles, light intensity of ~700 μE·m−2·s−1) for 9 to 11 days. Etiolated plants were grown in the dark for 9 to 10 days at the same temperature.

Cloning the Maize cpFtsY (csr1) cDNA

An EST sequence potentially encoding maize cpFtsY was kindly provided by T. Helentjaris at Pioneer Hi-Bred International (Johnston, IA). 5′ rapid amplification of cDNA ends was performed to recover the 5′ end of the cDNA, as described by Hirohashi and Nakai (2000). A full-length cDNA then was isolated from a cDNA library prepared from greening leaves. The nucleotide sequence of the cDNA and the deduced amino acid sequence have been deposited in the EMBL/GenBank data libraries.

Electron Microscopy

Sample preparation was performed as described (Parthasarathy, 1994; Yano and Terashima, 2001). In brief, leaf tissue was fixed overnight at 4°C in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 6.9. After three washes in 0.1 M cacodylate buffer, the tissue was fixed with 2% osmium tetroxide for 3 h and washed with pure water three times on ice. Fixed sample were dehydrated in ethanol and then embedded in Spurr's resin. Ultra-thin sections (40 nm) were cut with a diamond knife on the ultramicrotome (Reichert Ultracut S; Leica, Vienna, Austria) and stained for 10 min in saturated uranium acetate and for 15 min in lead citrate. Micrographs were taken with a JEM-1200EX electron microscope (JEOL, Tokyo, Japan).

Chlorophyll Extraction and Quantification

Leaves of 10-day-old seedlings were frozen in liquid nitrogen and ground to a powder with a mortar and pestle. After extraction with 2 mL of 80% (v/v) acetone, the debris were removed by centrifugation at 18,800g for 1 min. Absorbances of the supernatant at 663.2 and 646.8 nm were measured, and the chlorophyll content was calculated using the following formulas: μg chlorophyll a/mL = 12.25 × A663.2 − 2.79 × A646.8; μg chlorophyll b/mL = 21.50 × A646.8 − 5.10 × A663.2 (Lichtenthaler, 1987).

Production of Antisera

Maize cpFtsY (the presumed mature protein of 327 amino acids), ALB3 (the C-terminal 310 amino acids), and cpSecY (the C-terminal 202 amino acids) were expressed in Escherichia coli as His-tagged fusion proteins after expression from a pET21d vector in BL21(DE3) cells (Novagen, Madison, WI). Expression was induced by the addition of 1 mM isopropyl-1-thio-β-d-galactopyranoside for 4 h. Cells were broken by sonication in buffer containing 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride. Recombinant cpFtsY was recovered in the soluble fraction, whereas ALB3 and cpSecY were recovered in inclusion bodies and membranes, respectively. ALB3 was solubilized with 7 M urea, 50 mM Tris-HCl, pH 7.5, and 300 mM NaCl. cpSecY was solubilized with 1% SDS, 50 mM Tris-HCl, pH 7.5, and 300 mM NaCl. The solubilized proteins were purified with a Talon metal affinity column (Clontech, Palo Alto, CA) according to the instruction manual. Purified proteins were desalted with NAP-10 columns (Amersham Biosciences) equilibrated with PBS (cpFtsY), PBS containing 6 M urea (ALB3), or PBS containing 0.01% Triton X-100 (cpSecY) and used for the generation of antibodies in rabbits.

Protein Extraction and Immunoblot Analysis

Proteins were extracted as described (Martinez-Garcia et al., 1999). Samples were denatured in 2× Laemmli buffer (Laemmli, 1970) and heated at 95°C for 5 min. For immunoblot analysis of cpSecY and cpTatC, samples were denatured in 2× urea buffer (8 M urea, 100 mM Tris-HCl, pH 8.8, 200 mM DTT, 4% SDS, 20% glycerol, and 0.2% bromphenol blue) and heated at 37°C for 1 h. Proteins were resolved by SDS-PAGE followed by electroblotting to polyvinylidene difluoride membranes (Millipore, Bedford, MA) using blotting buffer (99 mM Tris, 192 mM Gly, and 20% methanol).

Antisera to maize cpFtsY, ALB3, and cpSecY and pea cpSRP54 were affinity-purified with antigen-bound membranes (Nakai et al., 1994). The antisera against maize CF1α, CFoI, OE23, Tha4, and Hcf106 were described previously (Voelker and Barkan, 1995; Walker et al., 1999). Antisera against pea Cpn60α and Cpn60β were described by Nishio et al. (1999). The cpHsp70 and cpClpC antibodies were raised against the purified spinach stromal cpHsp70 and the recombinant pea cpClpC, respectively. The antiserum against pea cpTatC was kindly provided by K. Cline (University of Florida, Gainesville). IgGs used for the immunological detection of various light-harvesting chlorophyll proteins (LHCPs) were purchased from AgriSera (Stockholm, Sweden). Although these anti-LHCP antibodies were raised against distinct short peptides designed to be specific for conserved sequences characteristic of each Lhc protein (Andersson et al., 2001), it remains uncertain whether they recognize the corresponding maize ortholog. Protein gel blots were developed by enhanced chemiluminescence (Amersham Biosciences).

Intact Chloroplast Isolation

Intact chloroplasts were isolated as described previously (Nakai et al., 1994; Nohara et al., 1995; Hirohashi et al., 2001) with minor modifications. In brief, leaves of 40 to 80 seedlings were cut to ~5 mm in length and homogenized with a blender in ice-cold blending buffer (50 mM Hepes-KOH, pH 7.8, 0.33 M sorbitol, 2 mM EDTA, 1 mM MgCl2, 1 mM MnCl2, 25 mM Na-ascorbate, and 1 mM DTT) with several bursts of grinding for 3 to 5 s each. This homogenization was repeated three times with fresh blending buffer. Combined homogenates were filtered through two layers of Miracloth (Calbiochem). The filtrate was centrifuged at 4070g for 3 min (GSA rotor; Sorvall, Newtown, CT), and the pellet was suspended in 5 mL of blending buffer, overlaid onto 30% Percoll solution (30% [w/v] Percoll, 50 mM Hepes-KOH, pH 7.8, 0.33 M sorbitol, pH 7.8, 5 mM DTT, and 1% [v/v] Na-ascorbate), and centrifuged in a swinging-bucket rotor at 1350g for 15 min at 4°C. The broken chloroplasts in the upper band were removed with an aspirator. The pellet was washed with HS buffer (0.33 M sorbitol and 50 mM Hepes-KOH, pH 7.8), centrifuged again at 1200g for 2 min, and resuspended in the same buffer to give a final concentration of 1 mg/mL chlorophyll (for protein gel blot analysis) or 1.5 mg/mL protein (for import analysis). The chlorophyll concentration was determined as described (Arnon, 1949), and the protein concentration was determined with the Bio-Rad Protein Assay.

Suborganellar Fractionation

Isolated chloroplasts or etioplasts were suspended in hypotonic buffer (10 mM Hepes-KOH, pH 7.0) for 10 min on ice. Lysed chloroplast suspensions were centrifuged at 1100g for 10 min, and lysed etioplast suspensions were centrifuged at 15,000g for 10 min. The resulting pellets, containing thylakoids or prolamellar bodies, respectively, were washed once and resuspended with the same buffer. Supernatants were centrifuged again at 100,000g and 4°C for 1 h to obtain soluble stroma (supernatant) and envelope membranes (pellet). Protease treatment of thylakoids or prolamellar bodies was performed with trypsin (60 μg/mL) for 5 min on ice followed by quenching with the addition of trypsin inhibitor (type II-S soybean [Sigma], 600 μg/mL). Treatment of thylakoids with chaotropic regents was performed with 100 mM Na2CO3 or 4 M urea for 30 min on ice followed by centrifugation at 200,000g for 1 h to recover both the solubilized (supernatant) and membrane-bound (pellet) proteins.

Import of Precursor LHCP into Isolated Chloroplasts

A plasmid used for the in vitro expression of precursor LHCP was described previously (Kogata et al., 1999). In vitro transcription and translation were performed with the TNT coupled reticulocyte lysate systems (Promega) in the presence of Pro-mix l-35S in vitro cell-labeling mix (Amersham Biosciences) at 30°C for 90 min.

For in vitro import, 200-μL reaction mixtures were assembled in glass tubes and contained intact chloroplasts (0.3 mg of protein), 0.3 M sorbitol, 50 mM Hepes-KOH, pH 7.8, 5 mM MgCl2, 5 mM DTT, 5 mM ATP, 10 μL of translation product, and 2 mg/mL Met and Cys. Import reactions were performed under light illumination at 25°C for 7.5 or 15 min. Reactions were terminated on ice, and intact chloroplasts were recovered by centrifugation and washed twice with HS buffer. For protease treatment, import reaction mixtures were divided into two aliquots, one of which was treated with thermolysin (100 μg/mL) for 20 min (for the import assay) or 40 min (for the integration assay) on ice followed by quenching with the addition of 10 mM EDTA. Reisolated chloroplasts were denatured with 2× Laemmli buffer by heating to 95°C for 5 min and applied to 15% SDS-PAGE. Translation products that were equivalent to 10% of those added to each import reaction were loaded on the gel for comparison. The gels were treated with EN3HANCE (DuPont–New England Nuclear, Boston, MA) for 30 min, washed with pure water for 30 min, and subjected to fluorography.

Immunoprecipitation

Fifty microliters of the stromal fraction after protein import was diluted 10-fold in IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM KCl, and 0.1% Triton X-100). Twenty microliters each of Arabidopsis anti-cpSRP43 antibody, pea anti-cpSRP54 antibody, and Arabidopsis cpSRP43 preimmune antiserum was added and incubated for 2 h at 4°C. Protein A–Sepharose 4 Fast Flow beads (Amersham Biosciences) were prewashed three times with water and three times with IP buffer. After resuspension in a twofold volume of IP buffer, the beads were added to the immunoprecipitation reaction. These samples were incubated on a rotator for 30 min at 4°C. Protein A–Sepharose/antibody/antigen complexes were recovered by centrifugation at 7000g for 15 s and washed with IP buffer three times. The pellets were resusupended in 40 μL of 2× Laemmli buffer and heated at 95°C for 5 min. The supernatants were analyzed by SDS-PAGE, treated with EN3HANCE, and subjected to fluorography.

Blue Native Gel Electrophoresis

Blue native gel electrophoresis was performed as described (Schägger et al., 1994) with the following modifications. Chloroplasts (100 μg of protein) were solubilized in 100 μL of 50 mM BisTris-HCl, pH 7.0, 0.5 M epsilon-aminocaproic acid, and 10% (w/v) glycerol containing 1% (w/v) n-dodecyl-β-d-maltoside (Dojindo, Kumamoto, Japan) and incubated for 10 min on ice. Samples were centrifuged at 100,000g for 10 min at 4°C. Eighty microliters of the supernatant was combined with 2 μL of 5% Coomassie dye stock solution (5% [w/v] Serva Blue G [Serva, Heidelberg, Germany], 50 mM BisTris-HCl, pH 7.0, and 0.5 M epsilon-aminocaproic acid) to give a detergent:Coomassie ratio of 8:1 (w/w). Fifty microliters of sample (corresponding to 48.7 μg of protein) was loaded onto a blue native 5 to 14% polyacrylamide gradient gel. Electrophoresis was performed at 100 V and 4°C. The cathode buffer initially contained 0.02% Coomassie dye and was replaced by buffer lacking dye after approximately half of the electrophoresis run.

Isolation and Analyses of RNAs

Total leaf RNA was extracted and analyzed by RNA gel blot hybridization as described by Roy and Barkan (1998). The psbA and rbcL transcripts were detected with the spinach psbA fragment or the maize rbcL fragment after radiolabeling by random hexamer priming. Polysome analyses were performed as described by Roy and Barkan (1998).

Upon request, materials integral to the findings presented in this publication will be made available in a timely manner to all investigators on similar terms for noncommercial research purposes. To obtain materials, please contact Masato Nakai, pj.ca.u-akaso.nietorp@iakan.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank K. Cline (University of Florida, Gainesville) for anti-cpTatC antibodies, H. Oh-oka (Osaka University, Japan) for anti-PsaC antibody, W. Sakamoto (Okayama University, Japan) for anti-cpFtsH antibody, K. Takamiya (Tokyo Institute of Technology, Japan) for anti-LHCP antibody, F. Sato (Kyoto University, Japan) for anti-RbcL antibody, and H. Aronsson (Leicester University, United Kingdom) for anti-protochlorophyllide oxidoreductase antibody. We also thank K. Nishio and N. Kogata for the preparation of antibodies against cpHsp70, Cpn60α, Cpn60β, and cpSRP43, T. Helentjaris (Pioneer Hi-Bred International) for providing various EST clones, and T. Hase and Y. Kimata-Ariga for valuable discussion. We thank R. Monde and P. Williams (University of Oregon) for their helpful advice and C. Uchida and R. Nakanishi for technical and secretarial assistance, respectively. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas (14014225 and 14037236) and a Grant-in-Aid for the Encouragement of Young Scientists (14580626) to M.N. and by grants from the National Institutes of Health (R01 GM48179) and the National Science Foundation (DBI 0077756) to A.B.

Notes

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.014787.

Footnotes

W in BoxOnline version contains Web-only data.

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