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provided by Elsevier - Publisher Connector Current Biology, Vol. 14, 776–781, May 4, 2004, 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2004.04.031 GIANT 1 Is Essential for Correct Division in Arabidopsis

Jodi Maple,1 Makoto T. Fujiwara,1,2 ing frames. Although no functional information exists, Nobutaka Kitahata,2 Tracy Lawson,3 All2390 and Slr1223 were initially annotated as divi- Neil R. Baker,3 Shigeo Yoshida,2 sion-inhibitor SulA proteins. Alignment of the GC1 amino and Simon Geir Møller1,* acid sequence (347 amino acids) with the Anabaena sp. ;similarity %65ف 1Department of Biology PCC 7120 All2390 sequence revealed University of Leicester however, GC1 contained a 45 amino acid N-terminal Leicester LE1 7RH extension absent in Anabaena sp. predicted to harbor United Kingdom a 37 amino acid plastid-targeting transit peptide (Figure 2 Functions Laboratory 1A). Phylogenetic analysis using eleven GC1 homologs RIKEN from , mammals, Drosophila, and Arabidopsis Hirosawa 2-1, Wako, Saitama 351-0198 demonstrated a close relationship between GC1 and Japan GC1-like proteins from cyanobacteria, indicating a cya- 3 Department of Biological Sciences nobacterial origin of GC1 (Figure 1B). -overall identity to prokaryotic nucleo %40ف University of Essex GC1 has Colchester CO4 3SQ tide-sugar epimerases, and secondary structure predic- structural (%90–%80ف) United Kingdom tions showed that GC1 has high similarity to epimerases in the active site region. Epi- merases control and change the stereochemistry of car- Summary bohydrate-hydroxyl substitutions, often modifying pro- tein activity or surface recognition, and epimerases are vital plant organelles involved in many contain two crucial active site residues (S and Y) vital essential biological processes [1, 2]. Plastids are not for substrate binding [18, 19]. GC1 contains these two created de novo but divide by binary fission mediated residues (Figure 1A, S161 and Y168) in the correct sub- by nuclear-encoded proteins of both prokaryotic and protein environment, and although we have no evidence eukaryotic origin [3–7]. Although several plastid divi- to date, GC1 may have epimerase activity. sion proteins have been identified in [8–17], lim- As an initial characterization of GC1, we analyzed ex- ited information exists regarding possible division pression patterns in Arabidopsis. We cloned and se- control mechanisms. Here, we describe the identifica- quenced a full-length GC1 cDNA (1044 base pairs) and tion of GIANT CHLOROPLAST 1 (GC1), a new nuclear- performed semiquantitative RT-PCR analysis. These ex- encoded protein essential for correct plastid division periments demonstrated that GC1 is primarily ex- in Arabidopsis. GC1 is plastid-localized and is an- pressed in photosynthetic tissues, such as leaves and chored to the stromal surface of the chloroplast inner the stem, with negligible expression in roots (Figure 1C), envelope by a C-terminal amphipathic helix. In Arabi- indicating that GC1 mode of action is most probably dopsis, GC1 deficiency results in mesophyll cells har- restricted to photosynthetic tissues. bouring one to two giant , whilst GC1 overexpression has no effect on division. GC1 can GC1 Is Anchored to the Stromal Side of the Chloroplast form homodimers but does not show any interac- Inner Envelope by a C-Terminal Amphipathic Helix tion with the Arabidopsis plastid division proteins To test whether the GC1 N-terminal extension repre- AtFtsZ1-1, AtFtsZ2-1, AtMinD1, or AtMinE1. Analysis sented a bona fide chloroplast-targeting sequence, reveals that GC1-deficient giant chloroplasts contain we transiently expressed a CaMV35S promoter-driven densely packed wild-type-like thylakoid membranes GC1/YFP fusion protein (GC1/YFP) in onion epidermal and that GC1-deficient leaves exhibit lower rates of cells. Epifluorescence microscopy revealed that GC1 is CO assimilation compared to wild-type. Although GC1 2 exclusively targeted to (colorless plastids; shows similarity to a putative cyanobacterial SulA cell data not shown), demonstrating that GC1 contains a division inhibitor, our findings suggest that GC1 does functional transit peptide. To further analyze the in- not act as a plastid division inhibitor but, rather, as a traplastidic localization patterns of GC1, we generated positive factor at an early stage of the division process. transgenic Arabidopsis plants expressing the GC1/YFP fusion protein. Although overexpression of plastid divi- Results and Discussion sion components result in abnormal division [20], ele- vated GC1/YFP levels had no effect on chloroplast repli- Identification and Initial Characterization cation in Arabidopsis (data not shown). Epifluorescence of GIANT CHLOROPLAST 1 analysis and optical Z sectioning (0.5 ␮m sections) We identified GC1 in Arabidopsis (accession AF326895) through chloroplasts in these transgenic plants revealed based on its similarity to the Anabaena sp. PCC 7120 that GC1 localizes uniformly to the entire chloroplast all2390 (accession NP486430) and the Synechocystis envelope (Figure 2A). To further analyze the competency sp. PCC 6803 slr1223 (accession D90906) open read- of the chloroplast envelope in recruiting GC1 we trans- formed the CaMV35S/GC1/YFP cassette into three *Correspondence: [email protected] Arabidopsis accumulation and replication of chloroplast GC1 Is Essential for Plastid Division 777

Figure 1. Amino Acid Sequence of GC1, Phylogenetic Analysis, and Tissue-Specific GC1 Expression (A) Alignment of the deduced GC1 amino acid sequence with the Anabaena sp. PCC 7120 All2390 amino acid sequence. The black boxes represent identical amino acid residues and the gray boxes represent conservative substitutions. The predicted transit peptide cleavage site is indicated by an arrowhead. The putative active site epimerase serine (S) and tyrosine (Y) residues are indicated with asterisks. (B) Phylogenetic analysis of eleven GC1 homologs obtained from NCBI and CyanoBase aligned by using CLUSTAL W 1.81 [34]. The highly conserved 217 amino acid stretch was used to calculate the sequence distance matrix, and the phylogenetic tree was constructed by the neighbor-joining method and bootstrap analyses for 1000 replicates. (C) Semiquantitative RT-PCR analysis of GC1 transcript levels in leaf tissue, stem tissue, and in roots. UBQ10 was used as a control.

(arc) mutants. In arc10 [21] and arc11 [22, 23], which GC1 Deficiency, but Not Accumulation, Causes harbor expanded chloroplasts, and in arc6 [17, 24], Chloroplast Division Defects in Arabidopsis which contains one or two large chloroplasts per meso- To investigate the role of GC1 in chloroplast division, phyll cells, GC1 localization was identical to that in wild- we transformed Arabidopsis with a construct harboring type plants (data not shown). This suggested that the a CaMV35S-driven 600 bp GC1 cDNA fragment in the entire Arabidopsis chloroplast envelope is competent antisense orientation. Semiquantitative RT-PCR analy- for GC1 recruitment and, moreover, that GC1 mode of sis identified nine independent transgenic T2 seedlings action is probably envelope associated. with a 50%–70% reduction in endogenous GC1 tran- Because GC1 contains no obvious transmembrane script, and four representative lines are shown in Figure domains, envelope localization is either mediated by S1A (Supplemental Data). Mesophyll and hypocotyl cells interaction with a membrane-associated protein(s) or from all nine lines were analyzed with Nomarski differen- by direct interaction with the phospholipid bilayer. To tial interference contrast (DIC) microscopy [26], reveal- initially examine which domains of GC1 confer envelope ing that reduced levels (up to 70%) of endogenous GC1 localization, we transiently expressed a truncated ver- transcript have no effect on chloroplast division (Figure sion of GC1, lacking 88 amino acids at the C-terminal S1B). We then generated transgenic Arabidopsis plants containing a CaMV35S-driven, full-length GC1 cDNA in region (Figure 2B, GC1BsaA1, nucleotides 747–1011), fused to yellow fluorescent protein (YFP) in tobacco the sense orientation and identified 24 independent T2 cells. Epifluorescence analysis revealed punctate localiza- transgenic lines, eight of which showed greatly enlarged chloroplasts in mesophyll cells. Northern blot analysis tion in chloroplasts (Figure 2C), implying that the C-ter- of five independent lines revealed highly reduced levels minal region of GC1 is required for correct envelope of GC1 transcript, and we selected two lines (lines 4 localization. Moreover, the stromal punctuate localiza- and 5) for further characterization. Repeated Northern tion is indicative of fusion protein aggregation due to incor- blot analysis showed a near to complete absence (Ͻ5%) rect protein folding. Secondary structure predictions of both the transgene-derived and endogenous GC1 showed that GC1 contains a nine amino acid C-terminal transcripts, indicative of cosuppression (Figure 3A). We amphipathic helix (Figure 2E); because proteins can in- further analyzed a transgenic line showing normal chlo- teract with lipid bilayers through amphipathic helices roplast division (Figure 3B, line 13) and found elevated [25], we tested whether GC1 is anchored to the chloro- GC1 transcript levels compared to wild-type (Figure 3A, plast envelope through this helical region. We transiently line 13). These results demonstrated that GC1 deficiency expressed a second truncated version of GC1 lacking in Arabidopsis results in chloroplast division inhibition, this amphipathic helix (Figure 2D, GC1⌬H, nucleotides while increased levels of GC1 have no effect on the 1012–1044) fused to YFP in tobacco cells and found division process. ARTEMIS [14] and ARC6 [17] are the that in contrast to full-length GC1 envelope localization only other bona fide envelope-associated plastid divi- (Figures 2A and 2B), the GC1⌬H/YFP fusion protein sion proteins, and together with the fact that the arc6 showed uniform stromal localization in chloroplasts (Fig- phenotype is almost indistinguishable from the GC1- ure 2D). This indicated that the C-terminal amphipathic deficient phenotype, this may suggest that GC1 and helix is responsible for membrane localization, possibly ARC6 act in concert during plastid division. through direct interaction with the lipid bilayer, and, The effect of GC1 cosuppression was analyzed in moreover, that GC1 is localized to the stromal side of more detail, and although most mesophyll cells showed the inner chloroplast envelope. the presence of only one giant chloroplast (Figure 3B, Current Biology 778

more likely that the role of GC1 is less predominant in hypocotyl cells toward the hypocotyl/root transition zone. No plastid division defects were observed in root amyloplasts (data not shown) as expected from the GC1 transcript analysis (Figure 1C), underlining that GC1 mode of action is probably restricted to photosynthetic tissues. Transmission electron microscopy of GC1-deficient mesophyll cells revealed that the giant chloroplasts were folded around the central vacuole (Figure 4C) and 3D optical image analysis showed that the central vacu- ole was in most cases almost entirely enclosed (Movie 1). GC1-deficient giant chloroplasts contained wild- type-like thylakoid networks with normally stacked membranes (Figure 4D); however, the grana stacks were more densely packed (Figures 4C and 4D) than in wild- type (Figures 4A and 4B). This may reflect a compensa- tory mechanism due to a decrease in the total chloro- plast number or volume per cell. GC1-deficient chloro- plasts also contained less starch grains, but the significance of this is unclear.

GC1 Deficiency Results in Decreased

Photosynthetic CO2 Assimilation Although GC1-deficient plants grow and develop nor- mally, we investigated whether photosynthetic parame- ters were altered. Photosynthetic gas exchange mea- surements demonstrated that GC1-deficient leaves Figure 2. Localization of a GC1/YFP Fusion Protein in Arabidopsis exhibit lower rates of CO2 assimilation compared to wild- and Tobacco Chloroplasts type leaves over a range of light intensities (Figure 4E): (A) Schematic diagram of the GC1/YFP fusion protein used for the a 50% decrease in the light-saturated rate and a 27% localization studies together with a single 0.5 ␮m optical Z section decrease in the apparent maximum quantum yield of through the middle of an Arabidopsis chloroplasts harboring a GC1/ YFP fusion protein. CO2 assimilation. The response of CO2 assimilation (A) (B) Localization of a full-length GC1/YFP fusion protein (pWEN18/ to leaf intercellular CO2 concentration (ci) allows separa- GC1) to the envelope region of tobacco chloroplasts. tion of the relative limitations imposed on photosynthe- (C) Nonenvelope, punctate localization of a GC1/YFP fusion protein sis by the maximum carboxylation rate of ribulose 1,5- (pWEN18/GC1BsaA1) lacking an 88 amino acid C-terminal region of bisphosphate carboxylase/oxygenase (Rubisco) and GC1 but retaining the extreme C-terminal amphipathic helix. the capacity for regeneration of ribulose 1,5-bisphos- (D) Uniform stromal localization of a GC1/YFP fusion protein phate (RubP) via the Calvin cycle [28]. GC1-deficient (pWEN18/GC1⌬H) lacking the nine amino acid amphipathic helix. Arrow indicates small, nonfluorescent regions observed in some leaves exhibited a 23% decrease in the initial slope of chloroplasts [35]. (E) Schematic diagram showing the amino acid the A/ci curve (Figure 4F), indicating a large decrease sequence of the GC1 C-terminal helical region and a helical wheel in the maximum carboxylation efficiency of Rubisco. ϭ ␮ representation of these nine amino acids. Scale bar 2 min(A) Similarly the light- and CO2-saturated rate of CO2 assimi- and 5 ␮m in (B), (C), and (D). lation decreased by approximately 23% in GC1-defi- cient leaves (Figure 4F), demonstrating reduced RubP regeneration by the Calvin cycle. This could be attribut- line 4 and Figure 3C), a small proportion also contained able to either a decrease in the rate of supply of reduc- two large chloroplasts (Figure 3B, line 5 and Figure 3C). tants and ATP from photosynthetic electron transport Optical sectioning and 3D image analysis of the two- or an inactivation or loss of Calvin cycle enzymes other chloroplast-containing mesophyll cells detected no sig- than Rubisco. The reductions in the maximum apparent nificant chloroplast size heterogeneity within individual quantum yield and light- and CO2-saturated rates of CO2 cells (data not shown), which is in contrast to mesophyll assimilation together with the decreased maximum rate cells with altered levels of the plastid division proteins of carboxylation by Rubisco could result from decreased

AtMinD1 or AtMinE1 [11–13, 27]. Inhibition of chloroplast CO2 diffusion to Rubisco carboxylation sites. This is an division was also observed in GC1-deficient hypocotyl attractive possibility, since GC1-deficient chloroplasts cells (Figure 3B), but in some cases chloroplast size are both larger and thicker than wild-type chloroplasts heterogeneity was detected (Figure 3B, line 5). The de- (Movie 1), which may result in a greater average path gree of size heterogeneity was more pronounced in cells length for CO2 diffusion from the cytoplasm to Rubisco toward the hypocotyl base, showing the presence of carboxylation sites. both enlarged and wild-type-like chloroplasts (Figure 3B, line 5), while cells in the upper region of the hypocotyl GC1 Can Dimerize but Does Not Interact with FtsZ showed uniformly enlarged chloroplasts (Figure 3B, line Proteins, AtMinD1, or AtMinE1 from Arabidopsis 4). Although the smaller chloroplasts may represent divi- Several plastid division components can form protein sion-arrested organelles in early expansion stages, it is complexes [23] (J.M. and S.G.M., unpublished data), GC1 Is Essential for Plastid Division 779

and we tested whether GC1 forms homodimers or inter- acts with other plastid division proteins. Full-length GC1fused to the Gal4 activation domain (AD-GC1) and to the Gal4 DNA binding domain (BD-GC1) was coex- pressed in HF7c yeast cells. We found that His auxotro- phy was restored in AD-GC1/BD-GC1 yeast cells, dem- onstrating that GC1 can form homodimers (Figure 5). We further tested whether GC1 interacts with the Arabi- dopsis plastid division proteins AtFtsZ1-1, AtFtsZ2-1, AtMinD1, and AtMinE1 [10–13]. We generated BD con- structs containing AtFtsZ1-1, AtFtsZ2-1, AtMinD1, and AtMinE1 and transformed these into AD-GC1-containing HF7c yeast cells. Analysis on low-stringency growth me- dia (no 3-AT) detected no restoration of His auxotrophy, indicating that GC1 does not physically interact with AtFtsZ1-1, AtFtsZ2-1, AtMinD1, or AtMinE1 (data not shown). This suggested that GC1 is probably not directly involved in the FtsZ-mediated plastid division pathway in Arabidopsis. Recently, GC1 was annotated as being a SulA protein (Accession CAD56855) based on sequence similarity. In bacteria, SulA is a potent cell-division inhibitor inter- acting with FtsZ in a 1:1 ratio in response to DNA damage [29–33], and we would therefore expect a bona fide SulA protein in Arabidopsis to interact with either AtFtsZ1-1 or AtFtsZ2-1. However, we find no evidence for any inter- acting between GC1 and either AtFtsZ1-1 or AtFtsZ2-1. Together with the fact that GC1 overexpression does not result in plastid division arrest, we believe that GC1 does not represent an Arabidopsis SulA protein.

Conclusions GC1 represents a new nuclear-encoded plastid division component essential for correct plastid division in Arabi- dopsis. The fact that overexpression of GC1 in trans- genic plants has no effect on the division process sug- gests that GC1 most probably acts as a positive plastid division factor. The envelope localization of GC1, AR- TEMIS [14] and ARC6 [17] suggests that chloroplasts have retained an involvement of the envelope in the division process and, moreover, that GC1 and ARC6 may act together during division. The identification of GC1 has shed light on an unexplored aspect of chloro- plast division in higher plants and has undoubtedly added a new level of complexity to this fundamental biological process.

Supplemental Data Supplemental Data including Experimental Procedures, an addi- tional figure, and a QuickTime movie are available with this article online at http://www.current-biology.com/cgi/content/full/14/9/ 776/DC1/.

while transgenic line 13 shows highly elevated GC1 levels. Transcript level quantification is shown and wild-type GC1 transcript levels are taken as 100% expression. Figure 3. GC1 Deficiency Results in Near-to-Complete Inhibition of (B) Nomarski differential interference contrast (DIC) microscopy of Chloroplast Division mesophyll and hypocotyl cells from wild-type (wt) Arabidopsis and (A) Northern blot showing GC1 transcript levels in wild-type (wt) from the three transgenic Arabidopsis lines (line 4, 5, and 13) ana- Arabidopsis seedlings and in three transgenic Arabidopsis lines (4, lyzed in (A). Scale bar ϭ 25 ␮m. 5, and 13) harboring a CaMV35/GC1 transgene. Note that transgenic (C) Quantitative analysis of chloroplast numbers in 100 mesophyll lines 4 and 5 show a near to complete absence of GC1 transcript cells from transgenic lines 4 and 5. Current Biology 780

Figure 4. Ultrastructure and Photosynthetic Responses of Chloroplasts in GC1-Deficient Arabidopsis Leaves (A) and (B) Wild-type (wt) mesophyll chloro- plasts. (C) Mesophyll cell from a GC1-deficient seed- ling (GC1-co line 4) showing the presence of one large chloroplast. (D) Enlarged region of a giant chloroplast from a GC1-deficient mesophyll cell showing nor- mal but closely packed thylakoid stacks.

(E) Response of net CO2 assimilation (A )of GC1-deficient (filled squares) and wild-type (Ͻ) leaves to increasing incident light (PPFD).

(F) Response of net CO2 assimilation (A )of GC1-deficient (filled squares) and wild-type

(Ͻ) leaves to increasing leaf intercellular CO2

concentration (ci). Data are the means of three independent replicates and standard errors are shown when larger than the symbol. Scale bar ϭ 1 ␮m.

Acknowledgments laboratory is supported by grants from the Biotechnology and Bio- logical Sciences Research Council (91/C17189 and 91/REI18421), We thank Natalie Allcock and Stefan Hyman for electron microscopy The Royal Society (574006.G503/23280/SM), and Higher Education and Nottingham Arabidopsis Stock Centre for accumulation and Funding Council for England (HIRF/046) to S.G.M. replication of chloroplast mutants. J.M. was supported by The John Oldacre Foundation and by the Biotechnology and Biological Sci- Received: February 9, 2004 ences Research Council (A2/A1/C/08178). M.T.F. was supported in Revised: March 4, 2004 part by Grants-in-Aid from the Ministry of Education, Science, and Accepted: March 4, 2004 Culture of Japan, number 14760068. Plastid division work in our Published: May 4, 2004

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