Arabidopsis OBG-Like GTPase (AtOBGL) Is Localized - Cell Press

Molecular Plant

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Volume 2

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Number 6

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Pages 1373–1383

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November 2009

RESEARCH ARTICLE

Arabidopsis OBG-Like GTPase (AtOBGL) Is Localized in Chloroplasts and Has an Essential Function in Embryo Development Fatima Chigria, Claudia Sippelb, Manuela Kolbb and Ute C. Vothknechta,b,1 a Center for Integrated Protein Science (Munich), Department of Biology, LMU Munich, D-81377 Munich, Germany b Department of Biology I, Botany, LMU Munich, D-82152 Planegg-Martinsried, Germany

ABSTRACT OBG-like GTPases, a subfamily of P-loop GTPases, have divers and important functions in bacteria, including initiation of sporulation, DNA replication, and protein translation. Homologs of the Bacillus subtilis spo0B GTP-binding protein (OBG) can be found in plants and algae but their specific function in these organisms has not yet been elucidated. Here, it is shown that AT5G18570 encodes an Arabidopsis thaliana OBG-like protein (AtOBGL) that is localized in chloroplasts. In contrast to the bacterial members of this protein family, AtOBGL and other OBG-like proteins from green algae and plants possess an additional N-terminal domain, indicating functional adaptation. Disruption of the gene locus of ATOBGL by TDNA insertion resulted in an embryo-lethal phenotype and light microscopy using Normarski optics revealed that embryo maturation in the atobgl mutant is arrested at the late globular stage before development of a green embryo. Expression of 35S::ATOBGL within the atobgl mutant background could rescue the mutant phenotype, confirming that embryo-lethality is caused by the loss of AtOBGL. Together, the data show that the bacterial-derived OBG-like GTPases have retained an essential role in chloroplasts of plants and algae. They furthermore corroborate the significance of chloroplast functions for embryo development — an important stage within the Arabidopsis lifecycle. Key words:

GTPase; OBG; embryo development; chloroplast.

INTRODUCTION GTPases are a large family of enzymes that hydrolyze guanosine triphosphate (GTP). GTP binding and hydrolysis take place in a highly conserved domain and specificity for GTP is often imparted by a specific base-recognition motif. GTPases are found ubiquitously in all kingdoms of life and they play a crucial role in many cellular processes (Bourne et al., 1990). P-loop GTPases, a subdivision of the P-loop NTPases, represent a large group of phylogenetically related proteins that can be divided into two major classes, called TRAFAC and SIMIBI, comprising 20 distinct families with more than 50 subfamilies (Bourne et al., 1991; Leipe et al., 2002; Saraste et al., 1990; Schweins and Wittinghofer, 1994). They appear to have originated from an ancestral GTPase with a possible function in protein translation by subsequent diversification. To date, involvement of P-loop GTPases has been shown for processes as diverse as signal transduction, intracellular trafficking, transcription/translation, protein translocation, and cell division (reviewed in Leipe et al., 2002). The translation factor-related class (TRAFAC) of P-loop GTPases can be further subdivided in several superfamilies, families, and subfamilies by characteristic motifs or structural features (Leipe et al., 2002). The spo0B

stage-related GTPase (OBG)-HflX-like superfamily includes the OBG family and within the subfamily of OBG-like proteins, whose members have been implicated in stress response, chromosome partitioning, replication initiation, mycelium development, and sporulation. The first member of this subfamily to be identified was the spo0B GTP-binding protein (OBG) from Bacillus subtilis, which is part of the stage 0 sporulation operon (Trach and Hoch, 1989). The TRAFAC class of GTPases also includes the extended Ras-like family, a predominantly eukaryotic branch of P-loop GTPases (Leipe et al., 2002; Vernoud et al., 2003), whose members play important roles in signal transduction and intracellular trafficking. Similarity searches in fully sequenced genomes, including those of plants and algae, have identified many P-loop

1 To whom correspondence should be addressed. E-mail vothknecht@bio. lmu.de, fax +49 89 2180 74661, tel. +49 89 2180 74660.

ª The Author 2009. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssp073, Advance Access publication 2 September 2009 Received 3 June 2009; accepted 3 August 2009

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AtOBGL Is a Chloroplast-Localized GTPase of the OBG Family

Figure 1. AT5G18570 Encodes a Canonical OBG-Like GTPase. (A) Sequence alignment of the deduced amino acid sequence of AtOBGL (AT) with proteins from Vitis vinifera (VV), Ricinus communis (RC), Ostreococcus tauri (OT), Nostoc punctiforme PCC 73102 (NP), and Bacillus subtilis (BS). Black boxes indicate identical amino acid residues in at least three out of six sequences, while gray boxes indicate a conserved amino acid substitution. The proposed cleavage site of the

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GTPases. While for most of these proteins, a concrete role has not yet been determined, the correct classification into their proper subfamily can assist in the elucidation of their potential cellular function. In a bioinformatic approach, Andersson and Sandelius tentatively identified At5g18570 as a potential chloroplast-targeted homolog of the Arf GTPase Sar1p, a member of the extended Ras-like family (Andersson and Sandelius, 2004), but experimental evidence has not yet verified the organellar localization or the specific function of this protein. Here, it is shown that the gene product of AT5G18570 is a bona fide member of the OBG subfamily of P-loop GTPases. The protein is localized in chloroplasts and its loss causes seed lethality due to an abortion of early embryo development. The protein therefore has an essential function in seed maturation, providing evidence for the importance of bacterialderived OBG-like GTPases in plants. The data furthermore support the significance of chloroplast functions for embryo development — an important stage within the Arabidopsis lifecycle.

RESULTS AT5G18570 Encodes a Plant Member of the OBG-Like GTPase Family Elucidation of the deduced amino acid sequence of AT5G18570 shows that the protein contains the secondary structure and sequence motifs common to P-loop GTPases (Figure 1A and 1B), such as the DxxG motif within Walker B and the GTP-binding motifs NKxD and SAV (Figure 1A, marked by asterisks). At5g18570 further possesses motifs characteristic of certain families within the P-loop GTPase superclass. These include a short FTT motif (Figure 1A, marked by #) within switch 1 region that is characteristic for the OBG-HflX-like superfamily within the TRAFAC class of P-loop GTPases. A motif in the switch 2 region (GAxxGxGxGxxxL, dotted line) localized just C-terminal of Walker B is found exclusively in subfamilies of the OBG family (Leipe et al., 2002). These sequence characteristics would place At5g18570 into the OBG subfamily of TRAFAC class GTPases. This is further supported by blast sequence alignments that revealed highest similarity of At5g18570 to other members of the OBG subfamily, including the OBG protein of Bacillus subtilis (BsOBG), the original namesake of this family (Figure 1A). Proteins similar to At5g18570 are also found in other plants, algae, and cyanobacteria as well as in many bacteria. Phylogenetic analysis by Neighbor-Joining shows that the OBG-like

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proteins of plants form a distinct cluster within a branch shared with the OBG-like proteins from bacteria (Figure 2). In contrast, other GTPases of the TRAFAC class, such as the Sar1-like proteins of yeast, animals, and plants, form a separate cluster distinct from the OBG-like proteins. Together, these features place At5g18570 clearly into the OBG subfamily of P-loop GTPases and we thus named the protein Arabidopsis thaliana OBG-like protein (AtOBGL). The sequence similarity of AtOBGL to BsOBG is especially high within the GTP-binding domain but extends further towards a domain between amino acids 209 and 360 of the AtOBGL sequence (Figure 1A). This region is called the Nterminal glycine rich domain or the OBG-fold (Buglino et al., 2002) and is common to all OBG-like proteins (Figure 1A, domain start marked by an arrowhead). Significant similarities are also found in the most C-terminal part, the TGS domain (Figure 1A, marked by a gray bar below the sequence). This domain of unknown function is not widely conserved in OBG-like proteins, but is found in proteins such as threonyltRNA synthetase and SpoT (Mittenhuber, 2001; Wolf et al., 1999). AtOBGL contains a further N-terminal extension of about 200 amino acids that is not present in bacterial OBG-like proteins. It includes a predicted presequence of about 40 amino acids (Figure 1A, marked by a triangle), which would allow for the transport of the protein into chloroplasts. A similar N-terminal extension is found in OBG-like proteins from seed plants and green algae, indicating that it might have a common function for the plant homologs. The sequence analysis makes it unlikely that AtOBGL is a phylogenetic ortholog to Sar1p from yeast, as suggested by Andersson and Sandelius (2004). The protein rather represents a plant member of the OBG subfamily. The additional N-terminal domain found exclusively in OBG-like proteins of plants but not bacteria and cyanobacteria might indicate a functional adaptation of these proteins in plants.

AtOBGL Is Localized in Chloroplasts and Associates with Chloroplast Membranes Bioinformatic prediction of a presequence suggested a chloroplast localization of AtOBGL. To elucidate the intracellular localization oftheprotein,analysisbyGFPfusion,immunodecoration, and in vitroimport assays was performed. Initially, a constructwas prepared encoding the first 65 amino acid residues of At5g18570, including the alleged presequence, fused to GFP and was used for transient expression in tobacco leaf protoplasts (Figure 3A, upper panel,pre-AtOBGL–GFP). GFP fluorescencewas foundexclusively

chloroplast transit peptide for AtOBGL is indicated by a triangle, while Walker A and B motifs are indicated by black bars above the sequence. The GTP-specific motifs NKxD and SAV are labeled by asterisks. Two motifs (switch 1 and 2) that are specific for the OBG-HflX-like superfamily and the OBG subfamily, respectively, are indicated by dotted lines. A FTT motif within switch 1 region (marked by #) is found exclusively in the OBG subfamily. The gray bar below the sequence marks the TGS domain as defined for the sequence from Bacillus subtilis. (B) Graphical display of the secondary structure of AtOBGL with a special focus on the GTPase domain. Gray arrows indicate beta-sheets conserved in P-loop GTPases of the TRAFAC class. Black boxes indicate alpha-helices within the GTPase domain. Gray boxes indicate domains outside the GTPase domain, including a domain conserved in all OBG-like proteins (glycine-rich domain or OBG-fold) and the predicted transit peptide (TP). Dotted lines indicate that this part of the scheme is not drawn to scale in correlation to the GTPase domain.

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Figure 2. Phylogenetic Relationship of AtOBGL to OBG-Like Proteins from Bacteria and Plants. Phylogenetic tree construction of different GTPases of the OBG family and the Arf subfamily was done by maximum likelihood using treepuzzle. Included in the analyses were homologs of BsOBG from bacteria, algae and plants, and homologs of yeast Sar1p from animal, plants, and algae. The alignment on which the phylogenetic analysis was based is provided in the supplements (Supplemental Text 1). The dotted line indicates that this part of the tree is not drawn to scale.

within chloroplasts and the overlay of chlorophyll fluorescence and GFP fluorescence further shows that the GFP protein is localized exclusively in areas free of red chlorophyll fluorescence. This distribution is typical for proteins that are localized in the chloroplast stroma and indicates that the N-terminal part of AtOBGL is sufficient to translocate GFP into the organelle. Fusion of the full-length protein to GFP revealed a slight but distinctive change in fluorescence distribution (Figure 3A, lower panel, AtOBGL– GFP). GFP fluorescence could now be observed evenly distributed within the chloroplast, indicating that the full-length GTPase might be associated with chloroplasts membranes.

To further address this question, in vitro import assays were preformed using isolated chloroplasts from pea (Figure 3B). Radioactively labeled AtOBGL, including its presequence (pAtOBGL), was obtained by in vitro transcription and translation from full-length cDNA using 35S-methionine (Figure 3B, pAtOBGL, lane 1). The dominant radioactive signal was observed at a molecular size slightly higher than expected for AtOBGL, even including its N-terminal targeting sequence. Correctness of the construct used for transcription/translation had been verified by DNA sequencing. Furthermore, a similar size was observed for recombinant AtOBGL when analyzed by

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Figure 3. AtOBGL Is Localized in Chloroplasts and Associates with Membranes. (A) Transient expression of GFP fused to the predicted transit sequence (upper panel, pre-AtOBGL–GFP) or to the full-length coding region (lower panel, AtOBGL–GFP) of At5g18570 in protoplasts from tobacco. Left panel: GFP fluorescence; middle panel: chlorophyll fluorescence; right panel: overlay of GFP and chlorophyll fluorescence. (B) 35S-labeled precursor proteins of AtOBGL (pAtOBGL) and Vipp1 (pVipp1) were imported into isolated pea chloroplasts and the import reaction was analyzed after zero and 20 min. Chloroplasts corresponding to one half of each import reaction were post-treated with thermolysin to remove proteins not imported into the organelle. Successful import is evidenced by the appearance of a proteolysisprotected mature protein of slightly smaller size (mAtOBGL and mVipp1). Pr, proteolysis after import; SW, chloroplast disrupted by hypertonic treatment prior to proteolysis; TL, 1/10 of the translation product used in the assay. (C) Determination of the localization of AtOBGL by Western blot analysis. Isolated chloroplasts (Chl) from Arabidopsis thaliana were separated into a fraction containing both outer and inner envelope membranes (Env), stroma (Str), and thylakoid membranes (Thy). The presence of AtOBGL was determined by immunodecoration with an antibody raised against the full-length protein (a-AtOBGL). Cross-contamination of the fractions was tested with antisera against different chloroplast proteins localized in the envelope membranes (a-Toc75, OEP16 and a-Tic40), the stroma (a-FBPase; Fructose-bis-phosphatase), and the thylakoid membrane (a-Alb4; Albino 4).

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SDS–PAGE (data not shown). Thus, it has to be concluded that the proteins displays an abnormal SDS–PAGE migration, as has been observed before for other proteins (Rath et al., 2009 and references within). Protein import assays showed a binding of the precursor protein (pAtOBGL) to the chloroplasts that increased with time of incubation (Figure 3B, upper panel, lanes 2 and 4). Also visible is the appearance of a slightly smaller, radioactively labeled protein that was not present in the translation reaction and was protected from proteolytic degradation (Figure 3B, upper panel, lanes 2–5, marked by asterisk). The protein could be degraded when envelope membranes were disrupted after the import reaction but prior to protease treatment (Figure 3B, upper panel, lane 6, SW), confirming its localization within the chloroplast. Thus, it represents the mature AtOBGL (mAtOBGL) after import and removal of the transit sequence. The import of pAtOBGL resembled that of the precursor form of the vesicle inducing protein in plastids 1 (pVipp1), a nuclear encoded chloroplast protein that was used as a control (Figure 3B, lower panel). Three radioactively labeled protein bands of lower molecular mass that can be observed in the import reaction of pAtOBGL are most likely caused by degradation of mAtOBGL within the chloroplast (Figure 3A, upper panel). Whereas the predicted transit peptide length was 40 amino acids (see Figure 1), the results of the import assay suggest a transit peptide of only about 1–2 kDa (;10–20 amino acids). The intra-organellar distribution of AtOBGL was further elucidated by immunodecoration with an antibody raised against recombinant AtOBGL protein (Figure 3C). The antibody clearly recognized the recombinant protein and reacted with a protein of about 85 kDa in whole plants (data not shown) as well as in isolated chloroplasts from Arabidopsis (Figure 3C, upper panel). Again, this is larger than expected from the amino acid sequence of the mature AtOBGL but corresponds well to the size of the recombinant protein as well as mAtOBGL observed in the import assays (see above). Upon subsequent fractionation of isolated chloroplasts into stroma, thylakoid membrane, and envelope membranes, the reaction with the antibody became more prominent and was most strongly visible in the envelope and the thylakoid membrane. A small amount of the protein could also be identified in the stromal fraction (Figure 3C, upper panel). Tests with antisera against different chloroplast proteins revealed only minor cross-contaminations within the different fractions (Figure 3C, lower panels). Thus, the immunoblot results not only corroborated that AtOBGL is a chloroplast protein, but also confirmed the association of the protein with chloroplast membranes.

AtOBGL Is Essential for Embryo Development In order to elucidate the function of AtOBGL, a mutant line (GABI_387B03) containing a TDNA insertion in the first exon of AT5G18570 was analyzed (Figure 4A). The localization of the TDNA insertion was verified by PCR using gene and TDNA specific primer (Figure 4B, right panel, line 6–1–4). Plants were

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Figure 4. Analysis of a TDNA Insertion Line in ATOBGL from A. thaliana. (A) Graphical display of the gene structure of ATOBGL including the positions of the TDNA insertion in exon one in the mutant line GABI_387B03 (Max-Planck Institute, Cologne, Germany). (B) Analysis of wild-type (wt) and heterozygous atobgl mutant (line 6–1–4) plants by PCR using primer to amplify the wild-type allel and the TDNA insertion. (C) Comparison of wild-type (wt) and heterozygous mutant plants (6–1–4). Wild-type and mutant plants showed not differences in phenotype when grown on soil (panel 1). Micrographs of siliques from heterozygous mutant plants showing aborted seeds looking white in younger siliques (panel 2, black arrowheads, and panel 3) and brown and shriveled in older siliques (panel 4 and 5, black arrowheads).

then analyzed for homozygous offspring. Even when seeds were grown heterotrophically, homozygous mutants of this line could not be obtained, indicating that the function of AtOBGL is essential for plant viability. Offspring from heterozygous plants after self-pollination segregated into one-third back-crossed wild-type and two-thirds heterozygous plants (data not shown) and no obvious difference in phenotype was observed between heterozygous mutants and wild-type under normal growth conditions (Figure 4C, panel 1). A closer examination of siliques from several different heterozygous plants revealed that about 25% of all seeds are aborted during development in contrast to ,2% in wild-type plants (Table 1 and Figure 4C, panels 2–5). At an early stage of development, in the yet green immature siliques of heterozygous mutant plants, ;25% of the seeds appear white, in contrast to the wild-type, where all seeds are green at this stage (Figure 4C, panels 2 and 3). No germination of the white seeds could be observed even if they were planted prematurely onto agar-plates supplemented with sucrose (data not shown). In mature siliques, ;25% of the seeds are aborted, indicating that the seeds that initially remained white are not able to develop into mature seeds (Figure 4C, panels 4 and 5). These findings suggest that the homozygous atobgl mutation causes embryo lethality. To identify the stage at which the embryo development is aborted, seeds from heterozygous plants were analyzed by light microscopy using Normarski optics (Figure 5). To ensure that the observed phenotype is representative, seeds were analyzed from different siliques originating from several different heterozygous mutant plants. Comparison of embryo development revealed that green seeds displayed a normal embryo development from globular to heart to torpedo stage (Figure 5A–5E). In contrast, in the white seeds, embryogenesis was arrested at the late globular stage (Figure 5F), since no heart or torpedo stage could be observed. Thus, it appears that loss of ATOBGL causes a seed-lethal phenotype due to abortion of embryo development at an early stage, indicating that the function of AtOBGL is required already early during seed maturation before the development of a green embryo. Subsequently, it was investigated whether the loss of AtOBGL is indeed responsible for the embryo-lethal phenotype observed in the homozygous atobgl mutant plants. First, evidence was provided by the fact that over several generations of self-pollination, the seed-lethal phenotype segregated with the TDNA insertion into AT5G18570 (data not shown). To provide more direct evidence, the coding sequence of AT5G18570 was cloned into a vector for stable plant transformation under the 35S promoter (Figure 6A). The construct 35S::ATOBGL was then transformed into heterozygous atobgl mutant plants by Agrobacterium tumefaciens-mediated flower transformation (Clough and Bent, 1998) and plants carrying the 35S::ATOBGL insert were initially selected by resistance to hygromycin. The F2 generation was subsequently screened for plants homozygous for the original TDNA

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Table 1. Comparison of Seeds in Siliques of Heterozygous atobgl Mutant Plants and Wild-Type.

Siliques counted

Number of viable seeds/silique

Number of aborted seeds/silique

Percentage of viable seeds/silique

atobgl 1

10

32.8 6 2.5

10.0 6 2.5

76.9 6 6.0

atobgl 2

10

37.3 6 3.6

9.7 6 2.5

73.9 6 4.6

atobgl 3

10

29.0 6 3.7

8.7 6 2.4

76.9 6 6.2

atobgl 4

10

30.7 6 3.3

11.1 6 2.3

73.6 6 4.5

atobgl 5

10

28.5 6 4.2

10.3 6 4.2

73.5 6 10.1

atobgl 6

8

27.5 6 2.1

7.5 6 1.9

76.7 6 5.3

28.8 6 3.2

9.6 6 2.7

75.3 6 5.8 99.3 6 1.2

Average mutant Wild type 1

10

39.8 6 5.3

0.3 6 0.5

Wild type 2

10

39.7 6 3.9

0.1 6 0.3

99.8 6 0.7

Wild type 3

10

37.7 6 2.5

0.0 6 0.0

100.0 6 0.0

Wild type 4

10

31.6 6 4.4

0.3 6 0.5

98.9 6 1.8

Wild type 5

10

35.4 6 5.4

0.2 6 0.4

99.5 6 1.1

Wild type 6

8

32.4 6 3.0

0.8 6 2.1

97.8 6 6.3

36.1 6 4.6

0.3 6 0.6

99.2 6 1.8

Average wild type

To determine the percentage of seeds that are aborted during development, seeds were removed from mature siliques of plants representing different lines of heterozygous mutants (atobgl) or wildtype. The number of viable and aborted seeds per silique was determined and the standard deviation was calculated.

Figure 6. Expression of 35S::ATOBGL in a Homozygous atobgl Mutant Background Can Rescue the Embryo Lethal Phenotype. (A) Graphical display of the 35S::ATOBGL construct for transformation into the atobgl mutant line. (B) Analysis of wt, heterozygous atobgl mutant (line 6–1), and atobgl-35S::ATOBGL rescue (line 6–1–5–19) plants. PCR analysis shows that the rescue line has a homozygous mutant background and contains the insertion of the 35S::ATOBGL construct. (C) Comparison of wild-type (left panel) and a homozygous atobgl plant containing the 35S::ATOBGL insertion (right panel).

insertion and carrying the 35S::ATOBGL insertion (Figure 6B, plant 6–1–5–19). Several such plants were identified in two different lineages. This result indicates that expression of ATOBGL from the inserted cDNA is able to rescue the embryo-lethal phenotype (Figure 6C, plant 6–1–5–19). Indeed, homozygous mutant plants carrying the 35S::ATOBGL insertion were able to grow on soil and showed no outward phenotype when compared to wild-type plants (Figure 6C).

DISCUSSION Figure 5. Loss of AtOBGL Causes Abortion of Embryo Development at the Globular Stage. Representative micrographs of embryos from green and white mutant seeds obtained by light microscopy using Normarski optics reveal that green seeds show normal embryo development from globular (A,B) to heart (C,D), and torpedo stage (E). Embryos in white seeds show no development further than the globular stage (F).

GTPases play an important role in many cellular processes, including signal transduction, intracellular trafficking, transcription/translation, protein translocation, and cell division (reviewed in Leipe et al., 2002). They are found in both prokaryotes as well as eukaryotes (Bourne et al., 1990) and due to a number of conserved motifs, members of this large superclass are readily identified in fully sequenced genomes. While, for most of these proteins, a concrete role has not yet been

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determined, the correct classification into their proper subfamily can assist in the elucidation of their potential cellular function. Sequence analysis shows that AtOBGL, the gene product of AT5G18570, clearly contains the secondary structure and sequence motifs found in members of the OBG subfamily of TRAFAC-class GTPases, including all the motifs required for GTP-binding and hydrolysis (Leipe et al., 2002). This placement is further supported by phylogenetic analysis showing AtOBGL in a cluster of OBG-like proteins of plants and algae that shares a common branch with the OBG-like proteins from bacteria. In contrast, GTPases of the RAS/RAB subfamily, which includes Sar1p from yeast, are also found in plants but are clearly localized on a separate branch. Together, these data show that AtOBGL is a bona fide member of the OBG subfamily of GTPases. The namesake of the OBG subfamily is the spo0B GTPbinding protein from Bacillus subtilis (BsOBG), which is part of the stage 0 sporulation operon (Trach and Hoch, 1989) and which was found to be essential for vegetative growth and efficient sporulation (Vidwans et al., 1995; Welsh et al., 1994). OBG-like proteins have been studied extensively in bacteria that undergo complex transitions, such as sporulation and differentiation, indicating that the protein might be important for these processes. In E. coli, ObgE is an essential protein required for cellular growth by inhibiting chromosome partition (Kobayashi et al., 2001). In Bacillus, BsOBG has later been shown to co-fractionate with ribosomes, probably as part of the sigma(B) stress response (Scott et al., 2000), but the exact function of the protein remains elusive. The wide distribution of OBG-like proteins in plants and algae indicates that the protein has retained an important function in these organisms. It can be speculated that the plant OBG-like proteins fulfill a role similar to their bacterial counterparts due to the conservation of important domains, including the OBG-fold located N-terminal of the GTPase domain. They also share significant similarities in the most C-terminal part of the protein, which, in BsOBG, comprises the so-called TGS domain. The specific function of the TGS domain is not known but it can also be found in threonyl-tRNA synthetases and SpoT (Mittenhuber, 2001; Wolf et al., 1999). While all these specific characteristics indicate a high conservation between the OBG-like proteins of bacteria and plants, the latter appear to contain an additional N-terminal extension that is not found in the bacterial proteins (see Figure 1). The N-terminal extension was found in OBG-like proteins from several plants and the alga Ostreococcus tauri and could be an indication for functional adaptation. It is not found in the only annotated OBG-like protein in the Physcomitrella impatiens genome, but further studies will have to show whether its lack in the moss sequence is due to a miss-annotation of the coding region. The sequence of AtOBGL furthermore includes a predicted transit peptide for chloroplast targeting and its localization within this organelle could be confirmed in this study. Potential transit peptides at the N-terminus of OBG-like proteins from plants and Ostreococcus indicate that chloro-

plast localization is common in the plant kingdom. In contrast to the plant proteins, the potential transit peptide of the Ostreococcus protein shows some different sequence features with regard to amino acid composition. Chloroplast localization of this protein is nevertheless quite likely since it has been shown before that chloroplast targeting sequences in algae differ from other chloroplast targeting sequences. Even though they exclusively promote import into chloroplasts, the transit peptides from green algae share features with plant transit peptides for both chloroplast and mitochondria (Franzen et al., 1990). Due to its chloroplast localization, it would seem likely that the plant OBG-like proteins originated from the endosymbiotic ancestor of chloroplasts. This is supported by the fact that OBG-like proteins are found in many cyanobacterial genomes, including those of Synechocystis, Synechococcus, and Nostoc. Nevertheless, it is noteworthy that the phylogenetic analysis performed in this study did not resolve the relationship of plant OBG-like proteins to their bacterial orthologs and that the plant and bacterial OBG-like proteins share a C-terminal TGS domain that is lacking in the cyanobacterial proteins. Further studies will have to show whether this domain was lost in the cyanobacterial lineage or whether the plant OBG-like proteins have an altogether different ancestry. BsOBG has been shown to co-fractionate with ribosomes probably as part of the sigma(B) stress response (Scott et al., 2000) but no indication for a co-localization of AtOBGL with ribosomes could be found in Arabidopsis (data not shown). Instead, the protein displays a strong association with chloroplast membranes. While Western Blot analysis showed a predominating presence of the protein in the envelope membrane, it can also be detected in thylakoids. The latter is supported by GFP-fusion experiments and suggests an additional association with thylakoid membranes. Since the amino acid sequence of the protein provides no indications for any transmembrane spanning domain or hydrophobic stretches, and small amounts of the protein were also detected in the stroma, AtOBGL most likely is a soluble protein that easily associates with the surface of both thylakoid and envelope membranes. Membrane association of AtOBGL might require the interaction with other proteins and could well be correlated to its cellular function in chloroplasts. There is one important correlation between OBG from bacteria and Arabidopsis that indicates functional conservation. Similar to the bacterial protein, this study suggests that AtOBGL is an essential protein in plants during a stage of complex transitions, more specifically for embryo development. The fact that the protein is localized in chloroplasts and therefore fulfills its function inside this organelle provides further evidence for the important role that chloroplast biogenesis plays in embryo maturation. AtOBGL was suggested as a functional homolog of Sar1p (Andersson and Sandelius, 2004) and would thus play a role in chloroplast vesicle transport (Morre et al., 1991; Westphal et al., 2001), implying that the ‘eukaryotic-type’

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Ras-like GTPase Sar1p was replaced in chloroplasts by the ‘prokaryotic-type’ OBG-like GTPase AtOBGL. Nevertheless, the severe phenotype of the atobgl mutant rather suggests a role of the protein in a chloroplast function that goes well beyond vesicle transport. OBG-like proteins in bacteria have been implicated in processes such as DNA partitioning and ribosome stability (Slominska et al., 2002; Tan et al., 2002) and in general terms to regulate the cell cycle. A similar function of AtOBGL in chloroplasts would certainly account for the lethal phenotype observed in homozygous mutant plants. Further studies, preferably by use of RNAi-induced gene silencing, will be required to shed light on the specific function of AtOBGL in plants.

METHODS Plant Material, Growth Conditions, and Mutant Selection For most experiments, seedlings of Arabidopsis thaliana wildtype (var. Columbia Col-0) or TDNA mutant line (GABI_387B03) were grown at 22C under a 16 h/8 h photoperiod at 80 lmol photons/m2 s 1 either on soil or on Murashige and Skoog medium supplemented with 1% (w/v) sucrose as described before (Kroll et al., 2001). All comparisons between mutant and wildtype plants were carried out with material grown under the same conditions and of the same developmental stage. For PCR analysis of the GABI_387B03 mutant plants, the following primers were used: the two gene specific primers At5g18570-for1 (5#-TGAGGAAGAAGAGAAAGAAAAGGA-3’) and At5g18570-rev1 (5#-AGCTTGTAACCATCATCAACACACT-3’) for the wild-type AT5G18570 gene and At5g18570-for1 and the TDNA specific primer tDNA-rev (5#-CCCATTTGGACGTGAATGTAGAGCAC-3’) for the at5g18570 TDNA insertion. Since no homozygous DAt5g18570 plant could be obtained, the line was propagated via heterozygous plants.

Preparation of Rescue Plants The rescue plants were obtained by cloning of the entire AT5G18570 reading frame into the plant expression vector pH2GW7 (Karimi et al., 2002) under control of the Cauliflower Mosaic Virus 35S (CaMV-35S) promoter. This construct, called 35S::ATOBGL, was transformed into heterozygous TDNA mutant plants by Agrobacterium tumefaciens-mediated flower transformation (Clough and Bent, 1998). Plants were grown on soil until they reached the flowering stage and were subsequently infected with Agrobacteria carrying the pH2GW7AT5G18570 plasmid. Offspring of these plants were initially selected for successful transformation by antibiotic resistance carried on the plasmid. Subsequent PCR analysis identified plants that were homozygous for the at5g18570 gene disruption (see above). Presence of the 35S::AT518570 insertion was then verified by PCR analysis using the following primers: pH2GW7-fw (5#-GCCGCCACTAGTGATAT-3’) and At5g18570rev4 (5#-CTTCAAACTCAAGTTCAGG-3’).

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In Vitro Transcription/Translation of Various Constructs For in vitro transcription/translation, the full-length coding sequence was cloned into pSP65. Radio-labeled protein was produced using the TNT SP6 Coupled Reticulocyte Lysate System from Promega-Deutschland (Mannheim, Germany) for 90 min at 30C in the presence of 35S-labeled methionine (1175 Ci mmol 1). The translation mixture was subsequently centrifuged for 20 min at 50 000 g to remove aggregated proteins and the supernatant was used in the import reaction.

Protein Import into Chloroplasts Import competent chloroplasts were isolated from leaves of 10–12-day-old peas (Pisum sativum, cultivar Arvika) as described previously (Waegemann and Soll, 1991). Plants were grown under a 16 h/8 h photoperiod at 250 lmol photons m2 s 1. Temperatures were 21C during the day and 16C in the night. Isolated chloroplasts were re-suspended in 330 mM sorbitol, 3 mM MgCl2, 50 mM Hepes-KOH pH 7.6 to a chlorophyll concentration of 2 mg ml 1 and kept on ice in the dark until use. A standard import reaction contained chloroplasts equivalent to 20 lg chlorophyll in 100 ll of import buffer (330 mM sorbitol, 3 mM MgCl2, 3 mM ATP, 10 mM methionine, 10 mM cysteine, 1 mM DTT, 20 mM potassium gluconate, 10 mM NaHCO3, 0.2% (w/v) bovine serum albumin) and 1–5% (v/v) translation product. Import assays were carried out for 20 min at 25C and chloroplasts were recovered from the import reaction by centrifugation through a 40% (v/v) Percoll cushion as previously described (Waegemann and Soll, 1991). One half of the reaction was treated with thermolysin while the other half of the reaction was kept on ice. For thermolysin treatment, the chloroplasts were re-suspended in 330 mM sorbitol, 50 mM Hepes pH 7.6 and 0.5 mM CaCl2 and thermolysin was added to a concentration of 100 lg ml 1. Proteolysis was carried out for 20 min on ice. For swelling reactions, the chloroplasts were re-suspended after a 20-min import reaction in 10 mM Hepes-KOH, pH 7.6 to disrupt the envelope membranes and were subsequently treated with thermolysin to digest all accessible proteins. All import reactions were analyzed by SDS–gel electrophoresis and phospho-imaging on a FUJI FLA-3000.

Localization of AtOBGL by Western Blot Analysis Purified recombinant AtOBGL protein was used to raise antibodies in rabbit (Biogenes, Berlin, Germany). Intact chloroplasts were isolated from Arabidopsis as previously described (Kunst, 1998). Isolated chloroplasts were subsequently lysed for 30 min in 50 mM Hepes/KOH, pH 7.6 on ice and stromal proteins and thylakoid membranes were separated by centrifugation at 3000 g for 10 min. Purified envelope membranes from Arabidopsis were a kind gift from J. Soll (LMU Munich). Equal amounts of protein were used for the Western Blot analysis. Immunoreactive proteins were detected by secondary antibodies coupled to alkaline phosphatase with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl

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phosphate as substrates. Purity of the fractions was determined with antisera against chloroplast proteins localized in the envelope (a-Toc75, OEP16 and a-Tic40), the stroma (a-FBPase; Fructose-bis-phosphatase), and the thylakoid membrane (a-Alb4; Albino 4).

Cloning of GFP Fusion Constructs Full-length and C-terminally truncated AT5G18570 including the predicted N-terminal presequence from Arabidopsis thaliana were amplified by polymerase chain reaction. Primers At5g18570-for2 (5#-GGACTAGTATGGCTTCCATATCCATTAACTGTTTCTTC-3’) and At5g18570-rev2 (5#-GGGGTACCCTTTCCATTGAGGCCATCTGACTG-3’) were used to obtain the full-length PCR product; primers At5g18570-for2 and At5g18570-rev3 (5#-GGGGTACCCACACCTCATCACAGCCGG-3’) were used to amplify the N-terminal part of AT5G18570 (1–195 bp). The PCR products were cloned into the pOL-LP vector, N-terminal to the coding sequence for GFP, thereby creating the plasmids pre-AtOBGL–GFP and AtOBGL–GFP (Mollier et al., 2002).

(Combet et al., 2000). Results of the NPS@ prediction were combined with the analysis provided by Leipe and coworkers for the classification of P-loop GTPases (Leipe et al., 2002) and by Buglino and coworkers for OBG-like GTPases (Buglino et al., 2002) to identify specific features of AtOBGL. Sequence alignments were obtained by ClustalX 1.8 (Thompson et al., 1997) and phylogenetic tree construction was performed by maximum likelihood using tree-puzzle-5.2 (Strimmer and von Haeseler, 1996). The alignment used as a base for tree construction is shown in the supplements (Supplemental Text 1).

SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online.

FUNDING This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB-TR1) to U.C.V.

Transient Expression in Tobacco Protoplasts Leafs of 3–4-week-old seedlings of Nicotiana tabacum cv. petite Havana, grown on B5-modified medium (Gamborg et al., 1976), were used in all experiments for protoplast isolation and transient transformation as described previously (Koop et al., 1996). All constructs were used at 50 lg DNA per 5 3 105 cells. Fluorescence images were obtained using a confocal laser scanning microscope (TCS-SP5, Leica SL, Wetzlar, Germany) using a 488-nm laser. GFP was detected by emission between 550 and 570 nm and chlorophyll fluorescence was recorded at 670 nm. Leica LAS AF software was used for 3-D reconstruction and image visualization.

Accession Numbers Sequence data from this article can be found in the EMBL/GenBank data libraries under the following accession number(s): NP_197358 (Arabidopsis thaliana, At5g18570), XP_002276482 (Vitis vinifera), EEF45504 (Ricinus communis), NP_001060586 (Oryza sativa), XP_002315565 (Populus trichocarpa), XP_001751788 (Physcomitrella impatiens), XP_001702482 (Chlamydomonas reinhardtii), CAL52961 (Ostreococcus tauri), YP_478554 (Synechococcus sp. JA-2–3B’a), NP_440268 (Synechocystis sp. PCC 6803), YP_001866647 (Nostoc punctiforme), ZP_04494139 (Sphaerobacter thermophilus), YP_002462987 (Chloroflexus aggregans), NP_390670 (Bacillus subtilis, BsOBG), NP_470908 (Listeria innocua), YP_002505657 (Clostridium cellulolyticum), NP_015106 (Saccharomyces cerevisiae, Sar1p), P0C583 (Neurospora crassa), NP_732717 (Drosophila melanogaster), NP_192117 (Arabidopsis thaliana, AT4G02080), EAY73867 (Oryza sativa), XP_001753154 (Physcomitrella impatiens), CAL57025 (Ostreococcus tauri).

ACKNOWLEDGMENTS The authors would kindly like to thank Dr J. Soll (LMU Munich) for the gift of purified envelope membranes from Arabidopsis as well as for the antibodies against Toc75, OEP16, Tic40, Albino 4, and Fructose-bis-phosphatase. The authors would further like to thank Dr E. Schroeder-Reiter (LMU Munich) for her assistance with light microscopy using Normarski optics. No conflict of interest declared.

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