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The Plant Journal (2006) 48, 895–906 doi: 10.1111/j.1365-313X.2006.02922.x AKRP and EMB506 are two ankyrin repeat proteins essential for plastid differentiation and plant development in Arabidopsis C. Garcion1,†, J. Guilleminot1, T. Kroj2,3, F. Parcy2,4, J. Giraudat2 and M. Devic1,* Laboratoire Génome et Développement des Plantes, 52 Avenue Paul Alduy, 66860 Perpignan, France, 2 Institut des Sciences du Végétal, UPR 2355 CNRS,1. avenue de la terrasse, 91198 Gif-sur-Yvette cedex, France, 3 Laboratoire des interactions plantes micro-organismes UMR2594 CNRS-INRA, Chemin de Borde-Rouge BP 52627, 31326 Castanet Tolosan cedex, France, and 4 Laboratoire de physiologie cellulaire végétale UMR5168 17, Rue des martyrs 38054, Grenoble cedex 9, France 1 Received 6 June 2006; revised 9 August 2006; accepted 11 August 2006. *For correspondence (fax þ33 468 66 8499; e-mail [email protected]). † Present address: Department of Plant Biology, Rue Albert Gockel 3, Fribourg, Switzerland. Summary EMB506 is a chloroplast protein essential for embryo development, the function of which is unknown. A twohybrid interaction screen was performed to provide insight into the role of EMB506. A single interacting partner, AKRP, was identified among a cDNA library from immature siliques. The AKR gene (Zhang et al., 1992, Plant Cell 4, 1575–1588) encodes a protein containing five ankyrin repeats, very similar to EMB506. Protein truncation series demonstrated that both proteins interact through their ankyrin domains. Using reverse genetics, we showed that loss of akr function resulted in an embryo-defective (emb) phenotype indistinguishable from the emb506 phenotype. Transient expression of the signal peptide of AKRP fused to green fluorescent protein demonstrated the chloroplast localization of AKRP. The ABI3 promoter was used to express AKR in a seed-specific manner in order to analyse the post-embryonic effect of AKR loss of function in akr/akr seedlings. Homozygous fertile and viable akr/akr plants were obtained. These plants exhibited mild to severe defects in chloroplast and leaf cellular organization. We conclude that EMB506 and AKRP are involved in crucial and tightly controlled events in plastid differentiation linked to cell differentiation, morphogenesis and organogenesis during the plant life cycle. Keywords: embryogenesis, chloroplast, ankyrin repeat, Streptophytes, interaction, cell morphology. Introduction In higher plants, plastids differentiate from proplastids that are present in young meristematic cells to acquire a cellspecific morphology and function (Kirk and Tilney-Bassett, 1978). These essential functions encompass photosynthesis in leaves, metabolic processes (e.g. pigments, amino acids, lipid and starch biosynthesis) and a substantial part of hormone-biosynthesis pathways. The temporal and spatial differentiation of the different plastid types is tightly coordinated with plant development. The first step in proplastid differentiation occurs during embryogenesis at the globular-to-heart transition phase. This transition is rich in developmental and metabolic events: the embryo will acquire a bilateral symmetry by emergence of the cotyledons, ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd a relative metabolic autonomy and a photosynthetic capacity. It is therefore not surprising that arrest of embryo development at the globular-transition stage represents approximately 40% of the embryo-defective mutants (McElver et al., 2001). Using electron microscopy images of embryo cells, Mansfield and Briarty (1991) have illustrated that the globular-to-heart transition is indeed concomitant with chloroplast biogenesis. In addition, direct observations of seeds have established that embryos start to accumulate chlorophyll at the heart stage. However, the deficiency in the photosynthetic capacity of embryo plastids does not seem to be the cause for most of the embryonic lethal phenotypes. Albinos and depigmented mutants produce homozygous 895 896 C. Garcion et al. seeds that are morphologically normal, able to germinate and grow to variable extent on sugar rich medium (McElver et al., 2001). More specifically, the majority of mutations in genes directly involved in or related to photosynthesis result not in embryonic lethality, but in the production of pale green to yellow or variegated seedlings (Aluru et al., 2006) or seedlings with chlorophyll fluorescence defects (Leister, 2003). Equally, the phenotype of mutations in genes coding for enzymes of the plastid non-mevalonate isoprenoid biosynthetic pathway is not embryo-lethal (Estevez et al., 2000). An important set of vital functions of the plastid, which are required at globular-heart transition, is involved in the transcriptional and translational machineries of the plastid (Tzafrir et al., 2003). This is not surprising as it has been demonstrated that some genes encoded by the chloroplast DNA are essential for cell viability (Drescher et al., 2000). What is less expected is the relatively late requirement of the plant cell for a functional plastid. As plastids and mitochondria are both essential organelles, maternally inherited, one could expect that mutations in similar housekeeping functions in each organelle will result in similar phenotypes. Systematic analysis of knock-out mutants in aminoacyltRNA synthetases required for translation in the chloroplast and mitochondria have established that disruption of translation in chloroplasts often results in seed abortion at the transition stage of embryogenesis, while in mitochondria this deficiency is responsible for ovule abortion (Berg et al., 2005). The conclusion that disruption of an essential chloroplast function often results in embryo lethality, yet rarely in gametophyte lethality, is further supported by analysis of mutants in metabolism (Yu et al., 2004), protein import and protein assembly (Hust and Gutensohn, 2006). There are also several reports of proteins with as-yet unknown function playing essential roles in chloroplast biogenesis and development (Albert et al., 1999; Apuya et al., 2002). EMB506 is an example of such a protein, containing five ankyrin (ANK) repeats organized in tandem, and has been shown to be essential for embryogenesis and proplastid differentiation (Despres et al., 2001). Ankyrin repeat domains consist of a motif of 33 amino acids repeated in tandem of at least two repeats, and forming specific secondary and tertiary structures (Bork, 1993). A recent survey of the Arabidopsis genome has determined the presence of 105 genes encoding proteins containing ANK repeats, classified into 16 groups (Becerra et al., 2004). EMB506 belongs to the class B proteins, containing only ANK repeats as known motif and comprising 18 members. Proteins in the other classes possess additional motifs such as transmembrane domains, kinase signatures, zing or ring fingers (Becerra et al., 2004). At present, the major role of plant ANK repeat proteins is related to signalling in defence mechanisms (Cao et al., 1997) and in development (Hemsley et al., 2005; Li and Chye, 2004). Concerning chloroplast development, only one ANK repeat protein has been char- acterized in addition to EMB506, a member of the specific signal-recognition particle for light-harvesting chlorophyllbinding protein (LHCP) (Klimyuk et al., 1999). As ANK repeats are present in such a diversity of proteins, it is thought that, as in animals, the role of ANK repeats is to mediate protein–protein interactions. In this study we took advantage of the ANK repeats of EMB506 to obtain insight into its biological role. A single protein was identified as interacting with EMB506 in a yeast two-hybrid assay. Functional studies and intracellular localization of the EMB506 interacting protein support the involvement of these two proteins in plastid differentiation connected to embryogenesis, and later in plant development. Results Searching for partners of EMB506 in a yeast two-hybrid screen We previously described the essential role of the EMB506 protein in embryo and chloroplast development (Albert et al., 1999; Despres et al., 2001). Homozygous emb506 embryos are arrested at the globular stage. Analysis of the EMB506 protein sequence revealed a prominent C-terminal ankyrin domain composed of five ANK repeats. ANK are known to function as protein–protein interaction domains (Bork, 1993). To obtain further insight into the function of EMB506, we used the yeast two-hybrid screen to search for proteins interacting with EMB506. The EMB506 protein lacking the chloroplast targeting signal peptide was fused to the Gal4 activation domain to serve as bait. Nineteen cDNA clones were identified among a cDNA library constructed with RNA from immature siliques (Kroj et al., 2003). These positive clones corresponded to transcription products of the same gene. BLAST searches in databases identified the gene as At5g66055, which codes for AKRP (ankyrin repeat protein), the protein most similar to EMB506 (Albert et al., 1999). Series of truncations of each protein were performed to define the interacting domains of AKRP and EMB506. The results presented in Figure 1 demonstrate that the two proteins bind through their ANK domains, and that binding is independent of the type of Gal4 domains used for the fusion. However, binding was more efficient when the amino acids of exon II upstream of the ANK domain were present. When the ANK domain of AKR was reduced to three repeats, the interaction with EMB506 was abolished. As the ANK domains of EMB506 and AKRP are the most similar among the family of 105 Arabidopsis proteins containing ANK repeats (Becerra et al., 2004), we tested whether EMB506 or AKRP could form homodimers. No homodimerizations of EMB506 or AKRP were observed (data not shown). The latter result indicates that these two proteins possess specific amino ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 895–906 AKRP, EMB506 and plastid differentiation 897 Figure 1. Study of the interacting domains of EMB506 and AKRP in the yeast two-hybrid system. Upper panel, the two proteins schematized to scale: solid rectangle with hatched boxes, ANK repeats; EMB506 in black and AKRP in grey. On the protein schemes, numbers refer to the position of the amino acids that limit the portion of the protein used for fusion with the activation (AD) or DNA-binding (BD) domain of Gal4. Results of the interaction are presented on the right. On the histidine (HIS)-free medium supplemented with 20 mM 3-aminotriazole (3AT), yeast requires interaction to grow (selective medium). The control plate contains minimum medium containing HIS such that interaction is not required for growth (non-selective). acids that distinguish their ANK domain. Searching in databases for homologues of AKRP and EMB506 in other species may identify these key amino acid residues. ARKP and EMB506 putative orthologues are detected only in higher plants Using the AKRP sequence, at least 90 ESTs belonging to 30 different plant species could be recovered. When the conceptual translation of these ESTs contained an ankyrin domain, in two-thirds of cases it was associated with a partial N-terminal sequence similar to the AKRP N-terminus, ruling out the possibility of being an orthologue of EMB506 or of an ANK protein with no relationship to AKRP. Putative AKR orthologues could be identified only in higher plants, and none was detected in animals or microorganisms. Even among photosynthetic species including cyanobacteria, eukaryotic unicellular algae or mosses, no similar sequence was found. The AKR gene therefore appears to be restricted to the angiosperm and gymnosperm lineages. Studies with the EMB506 sequence revealed a similar higher plant-specific distribution. In organisms where sufficient sequence data were available, both AKRP and EMB506 were detected. The alignment of EMB506 and AKRP and their putative orthologues is shown in Figure 2. Although no similarity could be detected between AKRP and EMB506 in their Nterminal part, conservation of some residues in the immediate vicinity of the ankyrin domain encoded by exon II does exist, and these amino acids were included in the alignment in Figure 2. Residues that were similar between AtAKRP and AtEMB506 are also conserved between their putative orthologues (highlighted in grey scale) and belong mostly to the consensus of the ankyrin domain. Some residues were conserved only among the EMB506 proteins (highlighted in blue), others in the AKRP proteins (green). Despite the fact that EMB506 and AKRP share a high degree of similarity and are clustered together in a single clade (Becerra et al., 2004), the specific residues together with the N-terminal regions could confer properties and functions unique to the EMB506 or AKRP protein. This idea is consolidated further by the interaction of EMB506 and AKRP, the absence of homodimerization, and the lethal phenotype of EMB506 loss of function (Albert et al., 1999). ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 895–906 898 C. Garcion et al. Figure 2. Alignment of AKRP and EMB506 of Arabidopsis and their putative orthologues. The amino acid sequences encoded by exon II and the five ANK repeats of the Arabidopsis AKRP and EMB506 are aligned and divided as EMB506 orthologues (top) or AKRP orthologues (below). Genomic AKR and EMB506 sequences are also available for Oryza sativa var. japonica. Positions of the introns are the same as in Arabidopsis, and are indicated. Frequency of amino acid conservation shown as a grey-scale background from white to black for total conservation. Blue or green backgrounds indicate conservation among the EMB506 or AKRP proteins, respectively. The consensus for the ANK repeat is taken from Sedgwick and Smerdon (1999). Arrow, position after which the sequence of the AKRP splice variant starts to diverge. AKRP is directed to the plastidic compartment As EMB506 is localized in the chloroplast, we wanted to test whether this is also the case for its interacting partner, AKRP. Constructions were designed to fuse the AKR sequence to green fluorescent protein (GFP). However, further characterization of the AKR transcript was necessary before its utilization. Zhang et al. (1992) reported the first sequence of the AKR mRNA in the Landsberg ecotype (M82883). These authors translated the longest ORF of the messenger RNA into a protein of 439 amino acids, and suggested that AKRP was located in the nucleus, based on sequence similarities. However, the putative ATG-initiation codon in the AKR sequence in Landsberg was substituted by GTG in the Columbia genomic sequence and was not included in the full-length AKR mRNA sequence in Columbia (AY052363). Thus the AKR gene in Col0 encodes a protein of 435 amino acids, which lacked the first 29 amino acids of the original Landsberg sequence, but gained 25 amino acids at the Cterminus due to the position of the stop codon at the end of the last ANK repeat. With this new protein sequence, the bioinformatic tools predicted a putative signal peptide of 36 amino acids directing AKRP to plastids. In addition, two different gene products for AKR are predicted at The Arabidopsis Information Resource website (http://www.arabidopsis.org). At5g66055.1 produces a protein of 435 amino acids containing five ANK repeats; At5g66055.2, a splice variant, would code for a protein of 359 amino acids with an ANK domain lacking the last two repeats (arrow in Figure 2). As the alternative splicing occurs at the 3¢ end extremity of the mRNA, both gene products are presumably targeted to the chloroplast. We tested this hypothesis by fusing GFP to the C-terminus of either the signal peptide or the two AtAKRP proteins. These constructs were introduced into tobacco leaf cells by particle bombardment, and expression of the fusion proteins was determined in the guard cells and epidermal cells, which contain photosynthetically active chloroplasts (Dupree et al., 1991). EMB506 was used as a positive control for chloroplast localization. A typical bombarded guard cell showed a co-localization of the EMB506-GFP and the chlorophyll fluorescence signals (Figure 3j–l, n ¼ 49). Similar results were obtained when the 50 first amino acids of AKRP were fused to GFP (Figure 3a–c, n ¼ 113). This result confirmed the existence of a signal peptide for targeting AKRP to the chloroplasts. However, when the entire AKRP protein (n ¼ 27) or the variant protein vAKRP (n ¼ 34) was fused to GFP, the expression of either fusion protein was deleterious to the chloroplasts. Guard cells expressing AKRP-GFP contained several small structures emitting both the red chlorophyll fluorescence and the GFP signal (compared with the non-transformed guard cell of the same ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 895–906 AKRP, EMB506 and plastid differentiation 899 (a) (b) (c) (d) (e) (f) At1g45230) in Figure 3q. The GFP pattern of AKRP was clearly different from the labelling of the GFP fused to the signal peptide only or to EMB506 and AtDCL, markers of chloroplasts. Comparative expression of AKR and EMB506 during the plant life cycle (g) (h) (i) (j) (k) (l) (m) (n) (o) (p) (q) Figure 3. Transient expression of AKRP-GFP and EMB506-GFP fusions in tobacco leaf. Confocal sections of stomatal guard cells expressing the green fluorescent protein (GFP) fused to the first 50 amino acids of AKRP (a); to the entire AKRP sequence (d, g) and to EMB506 (j); (b, e, h, k) the same cells recorded for chlorophyll emission at 640 nm. (c, f, i, l) Merged images of GFP and chlorophyll fluorescence in the same cell. (g–i) Close-up of images (d–f). Arrows point to small structures emitting both chlorophyll and GFP fluorescence. Epidermal cells were examined under epifluorescence illumination using a photonic microscope: (m) SP-(AKRP)-GFP; (n) AKRP-GFP; (o) EMB506GFP. Projection of confocal images of epidermal cells expressing the fulllength AKRP protein fused to GFP (p) or the product of At1g45230, the homologue of DCL, the tomato chloroplast protein (Keddie et al., 1996) (q). Bar ¼ 20 lm (a–f, m–q); 5 lm (g–i). stomata), and are shown in Figure 3d–f. Figure 3g–i shows a close-up of these structures, which were absent in SP(AKRP)-GFP bombarded cells. A similar effect of overexpression of the full-length AKRP protein on chloroplasts was observed in epidermal cells (for AKRP-GFP, n ¼ 231; vAKRPGFP, n ¼ 170; SP-(AKRP)-GFP, n ¼ 209). Typical images of epidermal cells of tobacco leaves expressing the AKRP-GFP protein are shown in Figure 3n,p and compared with SP(AKRP)-GFP in Figure 3m, EMB506-GFP in Figure 3o and AtDEFECTIVE CHLOROPLASTS AND LEAVES-GFP (AtDCL, We analysed the expression of AKR in the major plant organs by RT-PCR in comparison with the expression of EMB506 in the same samples (Figure 4, upper panel). Transcripts of both genes were detected in all tissues. In contrast to EMB506, AKR was expressed at higher levels in roots than in leaves. Two bands were obtained for AKR and sequenced. The variant mRNA contained the unspliced fifth intron as predicted. Western blot experiments were performed to analyse the correlation between transcription and translation of the AKR gene. Antibodies were raised against the specific portion of AKRP protein, the N-terminal moiety from aa 36–247, and used to determine the occurrence and abundance of the protein in different plant tissues (Figure 4, upper panel). Three bands were detected with the AKRP antibodies. The band at 49 kDa may represent the precursor of AKRP before removal of the signal peptide. The lower band, the size of which corresponded to the truncated AKRP, was found predominantly in flower buds and siliques, although we cannot rule out the possibility of its being a proteolytic degradation of the full-length AKRP. The band at 45 kDa corresponded to the mature form of AKRP with five ANK repeats. This protein accumulated predominantly in leaves and inflorescence stems, and this result correlated with the RT-PCR data. akr mutant embryos are defective If EMB506 and AKRP interaction has a functional significance, we expect akr and emb506 mutants to display some mutant features in common. The function of the AKR gene was investigated by reverse genetics. Two independent TDNA akr alleles (emb2036-1 and emb2036-2) were identified in the SeedGenes database (http://www.seedgenes.org, Tzafrir et al., 2003). Allelic tests were performed by Meinke’s group, and demonstrated that loss of AKR function was responsible for the embryo defective phenotype. emb2036-1 was used further in this study and hereafter named the akr allele. akr mutant embryos are characterized by a developmental arrest at the globular stage. At late globular stage, akr embryos already showed slightly swollen cells compared with the wild type (Figure 4a,b), although a clear difference between wild-type and mutant embryos appeared at the transition from radial to bilateral symmetry (Figure 4c,d). When wild-type embryos reached the mature stage, akr mutants never developed cotyledons but did grow radially (Figure 4e–h). Cytological observations revealed that there ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 895–906 900 C. Garcion et al. (a) (b) (c) (e) (f) (g) (d) (h) akr mutant embryos were still blocked at the globular stage (Figure 4j). We examined the ultrastructure of plastids in akr embryos. Wild-type cells of the globular embryo contain undifferentiated proplastids (Figure 5a), while plastids of the endosperm tissue appeared more differentiated with a few thylakoids (Figure 5b). At wild-type torpedo stage, coincident with the greening of the seed, plastids of both the embryo proper and the endosperm exhibited grana and two to four starch granules. The starch accumulation was more intense in the endosperm than in the embryo (Figure 5c,d). In the akr seed at wild-type torpedo stage (as defined by the wild-type siblings of the same silique), plastids were poorly differentiated, with few internal membranes (Figure 5e,f). By these criteria, akr plastids were similar to plastids of wildtype embryos at the globular stage. However, plastids of akr mutants accumulated starch similarly to wild-type torpedo seeds. The plastids in emb506 embryo and endosperm cells (Figure 5g,h) were indistinguishable from those in akr cells. These observations suggest that AKRP and EMB506 are required for normal plastid differentiation, but not for starch accumulation. Complementation of the akr mutation using a seed-specific promoter (i) (j) (k) (l) To assess AKRP function during vegetative growth, a complementation experiment was undertaken using the (a) Figure 4. Study of AKR expression and loss of function during plant development. Upper panel, expression of ARK and EMB506 during plant development. Gene expression was assessed by RT-PCR in roots (R), rosette leaves (L), inflorescence stems (St), flower buds (FB), developing siliques (S) and dry seeds (DS). Co, negative control without DNA; EF1a, positive control. The AKRP protein was detected by Western blot on similar samples with the exception of dry seeds. PI, control blot with rosette leaf sample incubated with pre-immune serum. Lower panel: phenotype of akr mutant embryos during seed development. (a–h) Cleared seeds observed under Nomarski optics: (a, c, e, g) wild-type embryo development at globular, heart, torpedo and cotyledonary stages; (b, d, f, h) images of mutant seeds present in the same silique as seeds in (a, c, e, g), respectively. Arrows (b, f) point to cellular defects such as swollen cells (b) or apparent duplication of the protoderm layer (f). (i–l) Cytological observation of semi-fine section of wild-type (i, k) and akr (j, l) seeds: (i) wild-type seed at globular embryo stage containing a syncytial endosperm; (j) akr seed at wild-type heart-torpedo stage, when endosperm starts to cellularize. The arrow in (l) indicates a line of cells with abnormal patterns of cell division. Emb, embryo; Sus, suspensor. Bar ¼ 20 lM. was no easily recognizable tissue organization in late akr mutants (Figure 4i–l). In contrast, endosperm cellularization proceeded normally with the same developmental timing as in the wild type (beginning at the heart stage), although the (e) (b) (f) (c) (g) (d) (h) Figure 5. Ultrastructural study of plastids from wild-type, akr and emb506 cells. (a–d) Images of wild-type cells from seeds at globular stage (a, b) or torpedo stage (c, d). (a) Proplastid of a globular embryo cell; (b) plastid of endosperm cell; (c) plastid in torpedo embryo cell; (d) plastid of endosperm cell. (e, f) Images of plastids in embryo cell (e) and endosperm cell (f) of akr mutant seeds when wild-type seeds are at torpedo stage. (g, h) Images of plastids in embryo cell (g) and endosperm cell (h) of emb506 mutant seeds when wild-type seeds are at torpedo stage. Bar ¼ 350 nm. ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 895–906 AKRP, EMB506 and plastid differentiation 901 Table 1 Characterization of ABI3::AKR lines in the akr background Phenotype of T2 seedlings Transgenic lines AA17 AA19 AA21 AA22 AA24 AA25 Genotype of T1 plants a Class (%) Phenotype of T2 seeds akr mutation ABI3::AKR (no. copies) akr/akr Wild type Ratio (v2*) WT (%) A B C akr/þ akr/þ akr/þ akr/þ akr/þ akr/þ 2 2 1 1 2 2 13 9 21 20 3 5 162 236 239 228 82 116 0.08 (28.8) 0.04 (59.4) 0.09 (39.7) 0.09 (38.0) 0.04 (20.9) 0.04 (28.1) 81 86 84 88 80 76 9 3 16 12 8 11 0 4 0 0 12 12 10 8 0 0 2 1 a Estimated by antibiotic selection and PCR. *v2 based on the non-complementation ratio of 0.25. Ratios are significantly different when v2 > 3.84. Class: A, first rosette leaves small and white; B, rosette leaves display both white and green areas; C, rosette leaves present only very small white areas. seed-specific ABI3 promoter driving the expression of the AKR cDNA, encoding a protein of 435 amino acids (Despres et al., 2001). Six independent primary transformants (a) (b) (c) Figure 6. Molecular characterisation of complemented akr seedlings. (a) Schematic representation of intron–exon structure of the AKR gene and of the T-DNA insertion. Black line, intron; white box, coding part of the exon; grey box, non-coding part of the exon. The T-DNA is inserted as an inverted tandem and is not drawn to scale. Arrows show positions of primers used in the various PCR experiments. (b) Genotyping of T2 seedlings transformed with the ABI3:AKR construct was performed using a PCR strategy that allowed simultaneous amplification of wild-type and disrupted AKR alleles. DNA was extracted from phenotypically wild-type or malformed seedlings. Two controls were included: C1, nontransformed genomic DNA; C2, genomic DNA of akr/þ plants. (c) Gene expression was assessed by RT-PCR in wild-type seedlings (WT) and malformed seedlings of different severity class (A–C, see Figure 7). Co, PCR mix without DNA. EF1a is the constitutive control of the experiment. were studied during three to four successive generations. The ABI3::AKR transgene was able to complement the akr emb phenotype as the siliques contained significantly less than 25% emb seeds (Table 1). The T2 seeds, when sown on germinating medium without antibiotic, produced wildtype seedlings and 12–25% seedlings with abnormal morphology. Abnormal seedlings, classified A–C according to the severity of defects, were shown to be homozygous for the akr mutation (Figure 6a,b). RT-PCR with RNA extracted from class A–C seedlings confirmed the absence of AKR transcripts (Figure 6c), while the EMB506 transcripts were present at a level similar to the wild type (Figure 6c). We concluded that lack of AKR gene expression does not affect the transcription of the gene encoding its EMB506 partner. Class A akr seedlings were the most affected. On germination, cotyledons were green and phenotypically wild type, but the first two leaves were small, misshapen and white with small green patches (Figure 7a,d). Class B seedlings displayed rosette leaves that contained white and green patches (Figure 7b). Class C seedlings were able to form rosette leaves with limbs composed of green and yellow patches (Figure 7c). Growth and development of the seedlings correlated with the extent of green areas. Class A seedlings achieved their development with the first two leaves. Class B and C seedlings produced a rosette. In some cases for class B and for all cases of class C, plants were able to produce flowers (Figure 7g,h). Floral defects were observed in class B plants and included depigmentation of sepals, malformation of organs (Figure 7e,f) and reduced fertility (short siliques in Figure 7g). Plants from class C produced siliques of normal size and seed number. For classes B and C, the lines could be maintained as homozygous akr/akr. Some arrests at globular-torpedo stages of seed development were observed in siliques from class B plants. In class C, most of the seeds were green and apparently fully developed. When the seeds were examined more thoroughly, the majority presented morphological ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 895–906 902 C. Garcion et al. (b) (a) (d) (g) (e) Leaf cellular organization is perturbed in partially complemented akr/akr seedlings (c) (h) (f) (j) (i) (l) (m) (k) (n) Figure 7. Phenotypes of akr/akr plants partially complemented with the ABI3::AKR transgene. Based on their phenotypes, plants are grouped in three classes. Class A: an example of T2 seedlings of the AA22 line is shown in (a), with a close-up of a malformed leaf (d). Seedlings of class A do not develop further. Class B: development of plants of line AA24 belonging to class B: T2 seedlings (b); close-up of flowers exhibiting depigmented sepals and malformation of floral organs (e, f); plant at maturity (g). Class C: rosette of a T2 plant from line AA19 (c) and inflorescence at maturity (h). Phenotypes of embryos in self-pollinated flowers of class C (line AA17, i–k). Bar ¼ 20 lm. The principal defect resides in the formation of cotyledons while the formation of the hypocotyl and the root meristem are affected only marginally. In addition to defects in producing cotyledons with a wild-type shape, cells with irregular shape are often visible (k). Phenotype of 2-week-old CaMV35S:AKR seedlings ranging from mild chlorosis (l) to severe chlorosis and leaf morphology (m, n). defects (Figure 7i–k). Cotyledons were often misshapen, while the hypocotyl and root meristem were mostly normal. The surface of the cotyledons was irregular and wavy, indicating defects in tissue and cell organization (Figure 7k). By complementation using the CaMV35S promoter, homozygous akr/akr plants were also obtained, though the complementation was not complete. Mild to severe leaf defects were observed in akr/þ and akr/akr seedlings (Figure 7l–n). The phenotype ranged from reduction of chlorophyll content, usually in a patchy pattern reminiscent of partially complemented akr plants, to abnormal leaf morphology. As these defects were observed in the progeny of the three independent lines, this phenotype was probably due to ectopic and/or overexpression of AKR rather than a co-suppression event. As some depigmented Arabidopsis mutants exhibit defects in leaf tissue organization (Estevez et al., 2000; Reiter et al., 1994), we examined the leaf sections of akr/akr ABI3:AKR. A typical leaf of each class is represented in Figure 8(a–c); Figure 8(d) represents a wild-type leaf. In each mutant class, the thickness of the leaf (Figure 8e–g) was increased significantly in comparison with the wild type (Figure 8h). White areas of class A (Figure 8e,i) and class B (Figure 8f,j) leaves were severely disorganized with large intercellular spaces and a reduced number of mesophyll cells, while the epidermis, palisade and spongy mesophyll layers were still recognizable in class C plants. However, the greater blade thickness in class C plants was also due to an increase in cell diameter and a decrease in cell contact (Figure 8g,k). The number of plastids in the large cells of mutant leaves did not appear to be reduced significantly, indicating that plastids were not defective in division. We concluded that the lack of AKRP affects cell division, cell shape, cell adhesion and differentiation. The ultrastructures of plastids from wild-type leaves and white sectors of class A plants are presented in Figure 8m–p. Plastids in white sectors, although they did not contain chlorophyll like the proplastids found in globular embryo cells (Figure 5a) or akr/ akr embryos (Figure 5e), were larger than proplastids (Figure 8o–p). Lamellae were clearly visible and similar to those found in chloroplasts, but no grana stacks were present (Figure 8m,n). (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (m) (n) (o) (l) (p) Figure 8. Cytological observations of leaves from wild-type Col0 and akr/akr ABI3::AKR plants of class A–C. (a–d) Typical leaves of class A (a); class B (b); class C (c); and wild type (d). White arrows, bases of trichomes. (e–l) Toluidine blue-stained sections of leaf blades of class A (white leaf, e, i); class B (white and green sectors, f, j); class C (pale green and dark green sectors, g, k); and wild type (h, l). Bar ¼ 20 lm. (m–p) Ultrastructure of plastids in wild-type leaf (n, o) and white sector of akr/ akr ABI3::AKR plants of class A (m,p). Bar ¼ 500 nm (m, o); 100 nm (n, p). ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 895–906 AKRP, EMB506 and plastid differentiation 903 Discussion Interaction of AKRP and EMB506 is mediated via their ankyrin repeats AKRP was identified as the unique partner of EMB506 in a yeast two-hybrid screen. Surprisingly, AKRP is the protein most similar to EMB506, both possessing an ANK domain of five repeats sharing up to 50% identity. A series of truncations in each protein have demonstrated that the two partners interact through their ANK repeats. In the literature, these repeats have been shown to be involved in binding other proteins as well as in mediating homodimerization. Usually, the interacting proteins do not contain ANK repeats and the interaction involves another type of motif in the protein partner. In the rare case of homodimerization, protein interaction occurs through the ANK repeats (JonasStraube et al., 2001). The mode of interaction of ARKP and EMB506 is therefore peculiar as it involves their ANK repeats uniquely for heterodimer formation. Functional similarities of ARKP and EMB506 Both proteins are located in the chloroplast to permit their function in the plant cell. We have demonstrated the essential role of EMB506 (Albert et al., 1999) and of AKRP (this study) in the initial differentiation of the proplastid at the globular-heart embryo transition. The phenotype of akr embryos was undistinguishable from emb506 embryos. The inability to mutually complement each other’s function despite the similarities of their proteins is in agreement with the existence of an in vivo AKRP-EMB506 complex. Using the strategy of partial complementation (Despres et al., 2001), we provided further evidence for their similarities of function. By providing AKRP during seed development only, viable and fertile homozygous akr/akr plants were obtained. During vegetative development, loss of AKRP was partly compensated. The partially complemented akr plants essentially resemble the ABI3:EMB506 emb506/emb506 plants. This approach is complementary to the antisense strategy employed by Zhang et al. (1992) for AKR. In both studies, loss or severe reduction of AKR function produced pigmentation defects. In our experiments, in the depigmented white leaf area of akr/akr plants, the plastids did not resemble proplastids as they were larger and produced a few lamellae, but nor did they resemble chloroplasts. In contrast to embryogenesis, AKRP does not affect the onset of proplastid differentiation in meristematic cells. However, we cannot rule out the possibility that there might be a low level of AKRP due to residual ABI3 promoter activity in the shoot apical meristem, even though plants were grown under constant illumination to prevent the quiescent state of the cells (Rodhe et al., 1999). Alternatively, the severity of the seedling phenotype may be a consequence of the amount of AKRP produced during embryogenesis. AKRP is required for the control of both chloroplast and leaf development, as the leaf tissue showed abnormal cell morphology. Cellular abnormalities resulted in very large cells with less adhesion, leading to an increase in air space, principally in the mesophyll tissue. Alterations in leaf anatomy are not common in chloroplast-development mutants, but have been described in Arabidopsis (Estevez et al., 2000; Reiter et al., 1994) and other species (Chatterjee et al., 1996; Keddie et al., 1996). Such alterations have been interpreted as an impairment of plastid-to-nucleus signalling pathways. Although analysis of the embryonic and vegetative loss-of-function phenotypes supports the existence of an EMB506/AKRP complex in vivo, the variegation patterns in the leaves of akr plants that were not observed in emb506 plants represent a significant difference. This indicates that a compensatory mechanism for an ARKP function not involving EMB506 is switched on in a cell-autonomous manner. Transcriptional and post-transcriptional regulation of AKR and EMB506 gene expression We know that there is no strict correlation between the presence of the EMB506 transcript and the accumulation of EMB506 protein (Albert et al., 1999). The EMB506 protein accumulated in flowers and siliques, but not in rosette leaves, while the transcript was ubiquitously present. When the EMB506 cDNA was cloned downstream of the constitutive CaMV35S promoter and introduced into Arabidopsis plants, the EMB506 protein was not detected in the leaf extract by Western blot (data not shown), despite the ability of this construction to complement the emb506 mutation. Taken together, these observations point to the existence of a post-transcriptional regulation of the EMB506 gene. The level of this regulation has not been established to date. The control of AKR gene expression is also complex, although different. Expression of AKR is light-dependent and developmentally regulated (Zhang et al., 1992). This transcriptional regulation of AKR is apparently important, as complementation was not complete when the CaMV35S promoter was fused to the AKR cDNA. Similarly, AKR sense plants in Zhang et al.’s (1992) study, expressing only the ANK domain, presented a chlorotic phenotype, which was probably caused not by co-suppression but by ectopic or overexpression of the AKRP ANK repeats. Mechanistically, a molar ratio between EMB506 and AKRP may be necessary for regulation of the complex by association–dissociation. Furthermore, we have shown that expression of AKRP-GFP fusion is detrimental for the chloroplast. These results suggest that, in contrast to the control of EMB506, there is no mechanism to prevent the overaccumulation of AKRP in the cell, and therefore that transcription is an important regulatory point for this gene. Remarkably, two transcripts can be produced from transcription of the AKR gene, producing ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 895–906 904 C. Garcion et al. proteins of different sizes. This feature is not unique to AKR: it has been reported for other nuclear genes, encoding chloroplast proteins. For example, three transcripts have been detected for the VDL (variegated and distorted leaf) gene of tobacco (Wang et al., 2000). Although the three transcripts are produced in wild-type plants, the phenotype of the vdl mutant is due only to a lack of VDL-1 transcript, encoding the largest protein. The existence of alternative splicing adds another level to the regulation of AKR gene expression and function. At this time, we have not attempted to decipher the role of each AKRP protein. Our preliminary results in the yeast two-hybrid system demonstrated that the truncated form of AKRP could not interact with EMB506. Production of the short AKRP may represent a way to regulate the interaction with EMB506 by competition with the full-length AKRP or to allow binding with other partners. The presence of the AKRP proteins in most chlorophytic tissues is compatible with a more general role for AKRP. We propose that transcriptional and post-transcriptional regulatory mechanisms act in combination in order to produce the appropriate amount of AKRP and EMB506 proteins necessary for normal plastid differentiation/development in different organs. Possible function for the AKRP/EMB506 complex The AKR and EMB506 genes have been found only in plant genomes, more precisely in Streptophytes. We can deduce that these genes did not originate in the chloroplast genome, and that their function is associated to plastids and plant development rather than photosynthesis. This hypothesis is also supported by the unaltered expression of nuclear and chloroplastic photosynthesis-related genes in AKR antisense plants (Zhang et al., 1994). Relatively few genes have been described encoding chloroplast proteins that are present only in higher plants. The PEND gene encodes a DNAbinding protein in the inner envelope membrane of the chloroplast, and related sequences have not been found in non-flowering plants and algae (Terasawa and Sato, 2005). This indicates that these proteins are required for a higher plant-specific function of the chloroplast, such as hormone biosynthesis or conversion to specialized plastids. While the evolution of chloroplasts from an endosymbiotic cyanobacterium is readily accepted, it is more difficult to understand the evolutionary processes that have created the multiple forms of plastids. There is no indication that the structures found in proplastids, chromoplasts or leucoplasts have been part of the genetic developmental plan that the endosymbiont transferred to the host cell. It may be assumed, therefore, that this development took place after the endosymbiotic event and was imposed on the plastid by the host cell (Vothknecht and Westhoff, 2001). In conclusion, we propose that AKRP and EMB506 act together to play an essential role in the first differentiation of the proplastid during embryo formation, and a less vital task later in plant development. Their different modes of regulation of gene expression (transcript and protein levels) and the minor differences in their vegetative phenotype of loss of function indicate that AKRP may also have additional partners. When acting together or separately, these two proteins are involved in crucial and tightly controlled events in plastid differentiation linked to cell differentiation, morphogenesis and organogenesis during the plant life cycle. Experimental procedures Plant material and growth conditions Arabidopsis plants were grown in soil or on Petri dishes under constant illumination at 22C. The akr mutation used in this work is the emb2036-1 allele of At5g66055 in Columbia ecotype (Tzafrir et al., 2003). Plants were selected in vitro on germination medium containing 10 mg l)1 glufosinate-ammonium (Cluzeau Info Lab, Sainte Foy-La-Grande, France). Yeast two-hybrid experiments The experiments were performed using the Gal4 system vectors of Clontech (Takara Bio Inc., Shiga, Japan) and according to Vignols et al. (2003). The cDNA library from immature siliques was cloned in fusion with the activation domain of Gal4 in the Stratagene Hybridzap 2.1 system (Stratagene, La Jolla, CA, USA; Kroj et al., 2003). The cDNA corresponding to the EMB506 protein (without the signal peptide) was amplified by PCR with primers 506-2 (5¢-AAGTCGACGAGGAAGCTCCGTCAAG-3¢) and 506-4 (5¢-CTCTGCAGCTCTTTCTATATATCCC-3¢), cloned into the SalI and PstI sites of the pBD vector and used as a bait. For the study of the interacting domains of EMB506 and AKRP, series of primers, including the SalI or PstI sites for directional cloning in pAD or pBD vectors, were designed for amplification of the chosen sequences. Analysis of embryo development Siliques were partially opened under the binocular and fixed in ethanol/acetic acid (3/1 vol) for 20 min and rehydrated by several passages in ethanol series. The samples were mounted in Hoyer’s solution on microscopy slides (Albert et al., 1999). Preparations were observed using Nomarski optics with a Zeiss Axioplan microscope equipped with a digital Leica camera (DC 300FX; LeicaMicrosystems GmbH, Wetzlar, Germany). Light and electron microscopy protocols Histological sections were produced as described by Albert et al. (1999). For electron microscopy, leaves of wild-type or partially complemented plants were cut into small pieces of a few mm. Wildtype or akr seeds were isolated from the same silique. Seeds were punctured with a needle to ensure efficient penetration of the fixative. The material was incubated for 6–12 h in 6% glutaraldehyde in 200 mM phosphate buffer pH 6.8 at 4C and post-fixed overnight in 2% OsO4. Samples were dehydrated in ethanol series and embedded in Araldite (Agar Scientific, Stansted, UK). Ultrathin sections of 100 nm were stained for 15 min in uranyl acetate solution followed ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 895–906 AKRP, EMB506 and plastid differentiation 905 by 5 min in lead citrate. The sections were examined at 80 kV in a Hitachi 7500 microscope (Hitachi High-Technologies Corp., Tokyo, Japan). Partial complementation of the akr mutation A 5-kb ABI3-promoter containing a KpnI restriction site near the initiation codon (Despres et al., 2001) was used to build the translational ABI3:AKR fusion. The AKR coding sequence was amplified using primers akr20 (5¢-TTGGTACCAATGCAGTCACTCTCCACCCCACA-3¢) and akr16 (5¢-TTCTGCAGGGATCCCTATTCAATATCTTCATCTGTTGT-3¢). The ABI3:AKR fusion was inserted into pC23, a pCAMBIA 2300 derivative containing a kanamycin-resistance cassette. The 35S:AKR fusion was created by introducing an NcoI site at the translation initiation site of the AKR coding sequence, using primers akr9 (5¢-AACCATGGCATCACTCTCCACCCCACACACC-3¢) and akr16, and ligating the fragment to the duplicated CaMV35S promoter of the pRTL2-GUS plasmid (Kay et al., 1987). The 35S::AKR fusion was inserted into pC23. Heterozygous akr plants were selected on Basta prior to transformation. T1 seeds were selected in vitro on Basta (10 mg l)1) and kanamycin (50 mg l)1, Duchefa Biochemie B.V., Haarlem, The Netherlands). Genotyping of the individual T1 seedling was performed (Despres et al., 2001). Primers akr5M (5¢-AGGAGGAGAGGAGAAAGCTCCGTTATTCA-3¢) and akr14 (5¢-TTGTCCTTCTTCGTCAGATTCA-3¢) were used to amplify the wild-type allele of AKR, and primers akr5M and LB3 (5¢TAGCATCTGAATTTCATAACCAATCTCGATACAC-3¢) to verify the presence of the T-DNA in AKR. Transient expression of AKR-GFP fusions in tobacco leaves The AKR cDNA was modified by introducing an NcoI site at the translation initiation site using the akr9 primer. Amplification of the DNA sequence coding for the first 50 amino acids was performed with akr9 and akr15 (5¢-GCCATGGCACCTGCTCCGAGGATCGACGTTTGAGAAGAA-3¢). For fusion of the AKR protein of 435 amino acids, the amplification was performed with akr9 and akr12 (5¢-TCTAGACTCGAGGTACCGAGCTCACCATGGCAGCTGCTCCTTCAATATCTTCATCTGTTGTTACC-3¢). In addition, a small linker of one glycine and four alanines were added between the AKRP and GFP proteins. For amplification of the splice variant, the primer akr22 (5¢-TTCCCATGGATCCTACCCTGTCCTGAGCGT-3¢) was designed. The fragments were inserted into the pPK100 vector (kindly provided by R. Blanvillain, Plant Gene Expression Centre, Albany, CA, USA) in frame with the EGFP sequence and downstream of a duplicated CaMV35S promoter. The EMB506-GFP fusion was described by Albert et al., 1999). The plasmids were introduced into plant cells by biolistic using the Biorad gene-transformation apparatus (PDS-1000/He, Bio-Rad, Hercules, CA, USA). Gold beads of 1 lm diameter were coated with plasmid DNA and bombarded onto the lower face of tobacco leaves with a helium pressure of 1300 psi. Leaves were observed 12–24 h after bombardment using a confocal microscope (Leica TCS SP). The sample was excited with an argon ion laser at 488 nm. For visualization of EGFP fluorescence, the emission window was set at 500–540 nm with a passband filter and at 640 nm for chlorophyll emission. Optical slices were approximately 1 lm thick. RT-PCR analysis RNA was extracted from two to three seedlings (cotyledons were removed) using an Invitrogen kit (Invitrogen, Carlsbad, CA, USA). Total RNA (250 ng) was used for synthesis of the first-strand cDNA following the protocol of the Stratagene Prostar RT-PCR kit. Two sets of primers were designed to test the expression of AKR. The first set, akr5M/akr14, flanked the T-DNA insertion site; the second set, akr14/akr7 (5¢-TCGTCGACGGTCTCTTTCCTATTCTTC-3¢), was positioned downstream of the T-DNA insertion. Specific amplification of the EMB506 cDNA sequence was obtained with the primers EMB5 (5¢-TCTGCAAGGCTCCAACTTGAAC-3¢) and EMB3 (5¢-AGTGATTGGGAAGATGACTC-3¢). EF1a (elongation factor1 a, At1g07940) was used as a marker for constitutive expression and amplified with primers EF5 (5¢-CTGCTAACTTCACCTCCCAG-3¢) and EF3 (5¢-TGGTGGGTACTCAGAGAAGG-3¢). To test the existence of the splice variant, RT-PCR was performed with akr13 (5¢-TACATATGGGTCGACGGAATCCTGACCTAGCCGTT-3¢ and ak3¢ (5¢GGAAACTCTTTCAACAGCTTC-3¢) primers. Production of polyclonal antibodies and Western blot analysis The cDNA portion encoding the first 247 amino acids of AKRP and lacking its ANK domain was amplified by PCR using primers akr10 (5¢-AACATATGGGTCGACTTTCCTATTCTTCTCAAACGTC-3¢) and akr11 (5¢-TTCTGCAGCTAGGATCCATTGAGCATAAACTTCTCTTCTT3¢). The fragment was cloned in the pGEM-T vector (Promega, La Charbonnières, France). The insert was transferred to the pET16b expression vector (Novagen, EMDbiosciences, Darmstadt, Germany). The recombinant protein was expressed in the BL21 strain of Escherichia coli and purified on His-bind resin. Polyclonal antibodies from rabbits immunized with the recombinant AKRP were produced by Eurogentec (Liège, Belgium). Western blots and protein extraction were performed as described by Albert et al. (1999). Acknowledgements We are grateful to Nicole Lautrédou-Audouy at IURC Montpellier and Genevieve Conejero at IFR127 for technical expertise in confocal imaging. Electron microscopy was performed in Laboratoire Arago at Banyuls. We thank Thomas Roscoe for critical reading of the manuscript and help in improving the English. C.G. would like to thank J.P. Metraux for his constant encouragement and enthusiasm. 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