EMB1211 is required for normal embryo development and influences ...

Copyright © Physiologia Plantarum 2010, ISSN 0031-9317

Physiologia Plantarum 2010

EMB1211 is required for normal embryo development and influences chloroplast biogenesis in Arabidopsis Qiuju Lianga,† , Xiaoduo Lub,† , Ling Jianga,c , Chongying Wangd , Yunliu Fana,c and Chunyi Zhanga,c,∗ a Department

of Plant Biotechnology, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China of Metabolism and Signal Transduction, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China c National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), Beijing 100081, China d School of Life Sciences, Lanzhou University, Lanzhou 730000, China b Center

Correspondence *Corresponding author, e-mail: [email protected] Received 22 June 2010; revised 23 July 2010 doi:10.1111/j.1399-3054.2010.01407.x

Chloroplast biogenesis is tightly linked with embryogenesis and seedling development. A growing body of work has been done on the molecular mechanisms underlying chloroplast development; however, the molecular components involved in chloroplast biogenesis during embryogenesis remain largely uncharacterized. In this paper, we show that an Arabidopsis mutant carrying a T-DNA insertion in a gene encoding a multiple membrane occupation and recognition nexus (MORN)-containing protein exhibits severe defects during embryogenesis, producing abnormal embryos and thereby leading to a lethality of young seedlings. Genetic and microscopic studies reveal that the mutation is allelic to a previously designated Arabidopsis embryo-defective 1211 mutant (emb1211). The emb1211 +/− mutant plants produce approximately 25% of white-colored ovules with abnormal embryos since late globular stage when primary chloroplast biogenesis takes place, while the wild-type plants produce all green ovules. Transmission electron microscopic analysis reveals the absence of normal chloroplast development, both in the mutant embryos and in the mutant seedlings, that contributes to the albinism. The EMB1211 gene is preferentially expressed in developing embryos as revealed in the EMB1211::GUS transgenic plants. Taken together, the data indicate that EMB1211 has an important role during embryogenesis and chloroplast biogenesis in Arabidopsis.

Introduction Formation of plant embryo body is a consecutive complex developmental process which can be divided conceptually into three overlapping phases (Goldberg et al. 1994, Jurgens ¨ and Mayer 1994, West and Harada 1993). During the first phase, the pattern of mature embryo is established throughout the embryo body

with apical-basal axis (Goldberg et al. 1994, Laux and Jugens ¨ 1997, Meinke 1995). Subsequent events include embryo growth and the accumulation of storage reserves (Goldberg et al. 1989). During the final phase, the embryo undergoes desiccation and dehydration, and eventually enters seed dormancy. An extensive investigation on plant embryogenesis has been performed during the past decades using light and

Abbreviations – bp, base pair; DAP, days after pollination; DMSO, dimethyl sulfoxide; GFP, green fluorescence protein; GUS, β-glucuronidase; MS, Murashige and Skoog media; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; TEM, transmission electron microscope; X-Gluc, 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid. † These

two authors contributed equally to this work.

Physiol. Plant. 2010

electron microscopy, and detailed descriptions about the morphological and anatomical changes that characterize the embryo development are presented; however, the cellular and molecular mechanisms governing the early events of embryogenesis remain poorly understood. One approach to address this question is the isolation and characterization of the mutants that display impaired embryo development. To date, a large number of embryo-defective mutants have been isolated in Arabidopsis, and some of the mutated genes have been cloned. These genes encode proteins that participate in a variety of biological processes such as metabolism, transport, morphogenesis and transcription etc. (Tzafrir et al. 2004). One of the cellular processes required for normal embryogenesis involves the formation of organelles such as peroxisome, mitochondria and plastid (Huang et al. 2009, Sparkes et al. 2003, Tzafrir et al. 2004, Yamaoka and Leaver 2008). In Arabidopsis embryos, chloroplast biogenesis starts from the late globular stage or early heart stage when the ovules get green in appearance (Mansfield and Briarty 1991, Ruppel and Hangarter 2007). The functional chloroplasts in green embryos are capable of photosynthesis to provide nourishment for the embryos and are responsible for the synthesis of many important products (carbohydrates, fatty acids, amino acids, etc.) (Mansfield and Briarty 1991, Ruuska et al. 2004). Efficient storage of assimilates by seeds is essential to provide metabolic precursors and chemical energy to power the young seedling until it can capture its own energy from the sun (Goffman et al. 2005, Alonso et al. 2007). Therefore, chloroplast biogenesis is closely associated with embryo development and seedling growth. Up to now, a great body of work has been done on chloroplast development during embryogenesis, demonstrating that the function and integrity of chloroplasts and plastids are important for the development of embryos. In Arabidopsis, the mutation in the nuclear gene EDD1 (Embryo-Defective Development), which encodes a plastid-localized Glycyl-tRNA synthetase, is lethal, arresting embryo growth between the globularto-heart transition stage. Furthermore, mutant embryos develop an abnormal embryo structure without normal cotyledon initials or chlorophyll accumulation and cultured embryos form small albino plantlets which fail to grow but turn into calli. EDD1 is predicted to be involved in the production of a very early signal that is required to initiate the first step in the differentiation of proplastids, which in turn triggers cotyledon morphogenesis (Uwer et al. 1998). slp (schlepperless) mutation in the chaperonin-60α gene causes a retardation of embryo development before the heart stage. The homozygous mutant embryos have no well-developed

plastids with unstacked or seemingly collapsed membrane structures, and they develop albino seedlings (Apuya et al. 2001). The plastid-targeted elongation factor G (SCO1) is essential for plastid development during embryogenesis. Two of its T-DNA insertion mutant alleles sco1-2 and sco1-3 are embryo-lethal and develop white ovules. The point mutation allele sco1-1 and the allele sco1-4 with a T-DNA insertion in the promoter display ovule, cotyledon and hypocotyl albinism because of improper chloroplast development, and normally die shortly after germination (Ruppel and Hangarter 2007). EMB506, an ankyin-repeat protein, together with its interacting partner AKRP, are involved in crucial and tightly controlled events in plastid differentiation linked to cell differentiation, morphogenesis and organogenesis. The homozygous mutant exhibits poorly differentiated plastids with few internal membranes in torpedo-stage embryos (Albert et al. 1999, Garcion et al. 2006). Besides, AGL23, a type I MADS-box gene, and DG1 (DELAYED GREENING1), a pentatricopeptide repeat (PPR) protein, were also reported to be involved in chloroplast biogenesis during embryogenesis (Chi et al. 2008, Colombo et al. 2008). EMB1303, a chloroplastlocalized protein, is essential for chloroplast development. In the emb1303 mutant embryo development was delayed, and it displayed severe dwarf and albino seedlings with plastids arrested in early developmental stages (Huang et al. 2009). These observations indicate that the normal chloroplast development is required for embryogenesis. To provide more insights into the molecular mechanisms underlying chloroplast biogenesis during embryo development, a screening for Arabidopsis T-DNA insertion mutants bearing white ovules was conducted in this study. One of such mutants developed albino seedling, and a T-DNA was found to be located in a gene encoding a thylakoid membrane-localized protein with three multiple membrane occupation and recognition nexus (MORN) motifs at the N terminus (AT5G22640). When the wild-type ovules began to turn green at the late globular stage the homozygous mutant ovules remained white until before seed maturation. Subsequently, only when plated on culture medium supplemented with 3% sucrose could the mutant seeds develop stunted albino seedlings. Moreover, we also observed some defects in embryogenesis in this mutant, including a severe retardation in embryo development and the abnormal embryo morphology. Transmission electron microscope (TEM) analysis revealed that the chloroplast development in the mutant was severely impaired. Therefore, this MORN motif-containing protein is essential for chloroplast development and thus for embryogenesis, confirming that the functional Physiol. Plant. 2010

chloroplast development is of importance for embryo development.

Materials and methods Plant material and growth conditions The Arabidopsis thaliana ecotype Columbia-0 was used in this study for wild-type analysis. The emb1211-3 mutant allele was isolated from a population of transgenic plants generated in our lab as it displayed white ovules. The emb1211-2 seeds (stock number CS16013) were obtained from the Arabidopsis Biological Resource Center (ABRC, The Ohio State University). Mutant seeds were sterilized in 70% ethanol (with 0.05% Tween 20) for 10 min, and then washed twice in 95% ethanol and 100% ethanol. After ethanol evaporated, seeds were placed on Murashige and Skoog media (MS) agar plates supplemented with 50 μg ml−1 kanamycin, cold treated at 4◦ C for 48 h and then allowed to germinate. Plants were grown at 22◦ C in an air-conditioned greenhouse with light intensity of 300 μmol m−2 s−1 under a 16-h-light/8-h-dark cycle. Lamps were from Philips (TLD Lifemax, 36 w/840). Isolation of T-DNA flanking sequence and segregation analysis The T-DNA flanking sequence was amplified as described by Lu et al. (2008). Heterozygous emb1211-3 mutants were either selfed or reciprocally crossed with wild-type. In both cases, the seeds produced were collected, and the progeny plants were genotyped by polymerase chain reaction (PCR) and phenotyped by analysis of seed development. For genotyping, the genespecific primers LP1 5’-TCAACAACACTCTTCACC-3’ and RP1 5’-GATTGGGATGTTGTTAACGATGAGA-3’ were used to identify wild-type allele, and the T-DNA left border-specific primer LB2 5’-GATCGACCGGCATGCA AG-3’ in combination with the primer RP1 were used to identify the mutant allele emb1211-3. Because the emb1211-2 mutant allele contained a T-DNA insertion in another position of the gene At5g22640 and the T-DNA vector is different from the one that was used to generate the emb1211-3 mutant allele, another set of primers for genotyping were designed as follows. The gene-specific primers LP2 5’-GGATGTCGAGGATAG TGTTG-3’ and RP2 5’-GAGCCTTAGCTGCAGCCACC TC-3’ were used to amplify the wild-type allele, and the T-DNA left border-specific primer LB1 5’-GC CTTTTCAGAAATGGATAAATAGCCTTGCTTCC-3’ and the primer RP2 were used to identify the mutant allele emb1211-2. For segregation analysis of the mutant allele emb1211-3, PCR using primer pairs LP1 + RP1 and Physiol. Plant. 2010

LB2 + RP1 were performed separately, and the PCR products were mixed and loaded in a single lane on an agarose gel. For phenotyping, siliques were opened to score the white seeds: plants producing 25% white seeds were scored as EMB1211/emb1211 and plants producing 100% green seeds were scored as EMB1211/EMB1211. Whole-mount preparation To analyze the defects in embryo development, the siliques of different stages ranging from 1 DAP (day after pollination) to 12 DAP from the heterozygous emb1211-2 and emb1211-3 mutants were dissected with hypodermic needles, and the ovules of 1–4 DAP were immersed in HCG solution (80 g chloral hydrate, 10 ml glycerol, 30 ml H2 O, stirred for 1 h) on microscopy slides for 5–10 min and then observed under LAICA 5500 microscope equipped with differential interference contrast (DIC) optics. The ovules from 5 to 12 DAP were mounted in Hoyer’s solution (7.5 g gum arabic, 100 g chloral hydrate, 5 ml glycerol, 60 ml H2 O, stirred for 1 h) for 2–6 h (the larger the ovules, the longer the time) before observation. Chemicals used were all purchased from Sigma-Aldrich (St. Louis, MO). Transmission electron microscopic analysis of plastid development Embryos and seedlings were fixed and sectioned as described by Takechi et al. (2000). Ultrasections were observed under TEM (model RILI H-7500). Complementation analysis For the molecular complementation experiment, a 6.5-kb genomic region containing the EMB1211 gene was amplified by PCR using the forward primer DF1: 5 -TTCGTCGACGGTGTCCTGGTGTCCTAATATCGT-3 at 2.2 kb upstream of the EMB1211 ATG start codon in combination with the reverse primer DR1: 5 -CCATGGAGACACAGCAGGAGTCTCAGCAAAC-3 . The PCR product was cloned into the binary vector pCAMBIA1302 at KpnI and Pml I sites in correct direction. Constructs were verified by sequencing and were used to transform Arabidopsis plants heterozygous for the mutant allele, using the floral dipping as described previously (Clough and Bent 1998). The collected seeds were plated on 1/2 MS culture medium supplied with 25 mg l−1 Hygromycin B, and the green seedlings were transplanted into soil. The presence of the complementation constructs in these seedlings was confirmed through PCR with the gene-specific primer

CF1: 5 -GAGAAGCTATGGAAGCTCAAG-3 and the vector-specific primer CR1: 5 -TCCCGATCTAGTAACA TAG-3 .

Results

RT-PCR analyses

A T-DNA insertion mutant collection generated in our laboratory was screened for the mutant plants that produced a proportion of white ovules at heart stage of embryogenesis as the greening of a wild-type embryo is indicative of chloroplast development from proplastid progenitors (Mansfield and Briarty 1991, Schulz and Jensen 1968). Consequently, we identified a mutant plant that produced normal ovules in appearance during 1–5 DAP (Fig. 1A), but approximately 25% of which (138 out of 568, 10 siliques) were white while the others in the same siliques turned green at about 6–7 DAP (when the embryos entered the early heart stage) (Fig. 1B). These ovules remained white until the onset of desiccation and dormancy at about 12 DAP (Fig. 1C) and, ultimately, developed into shrunken and dark seeds compared with the others (Fig. 1F). Subsequently, 1000 T2 seeds harvested from the mutant plants were plated on 1/2 MS culture medium supplied with 1% sucrose and 50 mg l−1 kanamycin, and the young seedlings were scored for their sensitivity to kanamycin. These seeds developed 246 yellow seedlings (kanamycin sensitive), 507 green seedlings (kanamycin resistant) and 247 albino seedlings (homozygous for T-DNA insertion, data not shown), with a segregation ratio, as expected, being close to 1:2:1 (P > 0.05). As many as more than 100 of the green seedlings developed normally in soil and the plants produced 25% of white ovules. The results indicate that the T-DNA insertion mutation is monogenic and recessive. To identify the gene that is disrupted in the transgenic, we sought to locate the position of the T-DNA insertion through PCR-based walking (see section Materials and methods). One PCR product was obtained using HpaI-digested genomic DNA as templates. Sequencing of the PCR product confirmed the T-DNA insertion and precisely mapped the T-DNA to a position 179 bp downstream of the ATG start codon of the gene locus At5g22640. Results from an NCBI BLAST search indicate that this locus encodes EMB1211 (EMBRYO DEFECTIVE 1211, www.seedgenes.org; Tzafrir et al. 2004), and is composed of 11 exons and 10 introns, and the T-DNA has inserted into exon 1 (Fig. 2A). The T-DNA insertion was confirmed by amplification of the flanking region using the primer specific to the left border of the T-DNA and the primer specific to the gene (Fig. 2A). To determine whether the T-DNA insertion mutation affects male and/or female gametophyte, the heterozygous mutant was crossed to wild-type as either male or female parent, respectively, and the kanamycin

Total RNA was extracted from the 10-day-old seedlings of wild-type and emb1211 homozygous mutant grown on MS medium supplied with 3% sucrose according to the manufacturer (U-gene Total RNA Kit II). To prepare total RNA from siliques and ovules, EASYspin Plant RNA Kit (Galen Biopharm, Beijing, China) was used. One microgram of the total RNA extracted was treated with RNase-free DNaseI (TaKaRa) and subjected to synthesis of single-stranded cDNA using the First Strand DNA Synthesis Kit (Toyobo, Osaka, Japan). For detection of the EMB1211 transcripts, the genespecific primers LP1 (5’-TCAACAACACTCTTCACC-3’) and RP1 (5’-GATTGGGATGTTGTTAACGATGAGA-3’) were used. Primers were designed to amplify the housekeeping gene ACTIN 2 to equalize RNA loading into the reverse transcription polymerase chain reaction (RT-PCR) reaction. These were ACTIN2F : 5’-CCAACAG AGAGAAGATGACT-3’ and ACTIN2R: 5’-ATGTCTCTTA CAATTTCCCG-3’. After 30 cycles of amplification, PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. Construction of PEMB1211 -GUS fusion and GUS activity assay A 2264-bp (base pair) DNA fragment upstream of the ATG start codon of the EMB1211 gene (At5g22640) was amplified by PCR with primer pairs: PF, 5’-GGTGTCCTG GTGTCCTAATATCGT-3’ and PR, 5’-TAGAGAGAGGG GATAACGAAGAG-3’. The resulting PCR products were cloned into pENTR1A vector at KpnI and Not I sites in correct direction, and then recombined into the destination vector pKGWFS7 using Gateway LR Clonase II Enzyme Mix (Invitrogen, Carlsbad, CA) to generate PEMB1211 – GUS fusion construct. This construct was introduced into Ara bidopsis via Agrobacteriummediated floral dipping. Transgenics were stained with GUS staining solution [0.1 M phosphate buffer, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 1.0 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc), 50 mg ml−1 dimethyl sulfoxide (DMSO)] for 24 h in a 37◦ C shaker at 250 rpm and decolored in 70% ethanol, and then observed under Nikon Digital Camera DXM1200F. For transgenic ovules, siliques were washed with sterilized water after 2-h staining, dissected, and immersed into Hoyer’s solution and observed under LAICA 4500 microscope equipped with DIC optics.

Isolation and characterization of the emb1211 mutants


Table 1. Segregation of the crossing lines between the heterozygous emb1211-3 mutants and wild-type on kanamycin-resistance screening. ∗ χ 2 values are not significantly different at a threshold of P = 0.01 from those expected under the hypothesis of wild-type female gametophyte transmission (i.e. 1:1 segregation). Kanr , kanamycin resistant. Kans , kanamycin sensitive. Parental genotypes Female emb1211-3+/− Wild-type

Fig. 1. Phenotypic characterization of the T-DNA insertion emb1211 mutants. (A) Five-DAP ovules with globular-stage embryos from plants heterozygous for the emb1211 mutation display no difference in appearance between emb1211-2+/− (middle) or emb1211-3+/− (lower) with wild-type (upper). (B) White ovules (arrowheads) are present in 7-DAP immature siliques of the heterozygous emb1211-2+/− (middle) and emb1211-3+/− (lower) when the wild-type embryos (upper) begin to turn green at early heart stage. (C) In 9-DAP siliques of emb12112+/− (middle) and emb1211-3+/− (lower), white ovules (arrowheads) are obviously found intermixed with normal green ovules, indicating that chloroplast development is disrupted during embryogenesis in these mutant alleles. White ovules account for about 25% of the total analyzed (emb1211-2+/−, 123 white ovules out of 497 ovules; emb1211-3+/−, 321 white ovules out of 1295 ovules). (D) Seeds from a mature silique of wild-type (1% shrunken seeds found in 650 seeds scored). (E) and (F) Seeds from a mature silique of the heterozygous emb1211-2 (E) and emb1211-3 (F) mutant plants containing approximately 25% shrunken and dark seeds (arrows). Fifty siliques scored each line. (G) A 12-DAP homozygous emb1211-3 mutant embryo (lower) is white and arrested at torpedo stage compared to the wild-type (upper) develping into green curled-cotyledon embryo stage from the same silique. (H) Five-day-old seedlings of emb1211-2−/− (left) and emb1211-3−/− (middle) show a significant growth arrest with purple cotyledons compared with the wild-type (right). (I) Twelve-day-old seedlings of emb1211-2−/− (left) and emb1211-3−/− (middle) just develop the first pair of true leaves while the wild-type seedlings (right) presented two pairs of true leaves. Bars in (A–F) = 200 μm, in (G) = 50 μm and in (H,I) = 2 mm.


Progeny genotypes

Male

Kanr

Kans

Wild-type

50.7% (n = 538)∗ 50.5% (n = 547)∗

49.7% (n = 523)∗ 49.5% (n = 536)∗

emb1211-3+/−

sensitivity of the progeny seedlings was scored. Analysis of the progeny of the crosses revealed a segregation ratio of kanamycin-resistant vs kanamycin-sensitive being close to 1:1. Moreover, the kanamycin-resistant plants produced approximately 25% white ovules as observed in the mutant parents (data not shown). These observations indicate that this T-DNA insertion mutation neither affects the male nor the female gametophytes (Table 1). To further confirm the identity of the mutation which is responsible for the phenotypes observed, another independent T-DNA insertion mutant line was isolated from the seeds obtained from the ABRC (CS16103), which was previously designated emb1211-2 (www.seedgenes.org; Tzafrir et al. 2004). The T-DNA was found to be located at the border of exon 5 and intron 5 of the EMB1211 gene by PCR amplification and sequencing analysis of the flanking region using the primer specific to the left border of the T-DNA and the primer specific to the gene (Fig. 2A). This mutant showed a phenotype similar to that of the emb1211-3 mutant with white ovules and shrunken seeds observed in the heterozygous mutant plants (Fig. 1B, C, E). As another emb1211 mutant allele obtained from ABRC (CS16102) was previously designated emb1211-1, the mutant allele isolated in our laboratory was then named emb1211-3. Unfortunately, the emb1211-1 seeds germinated neither on medium nor in soil and therefore not included in this study. Homozygous emb1211 mutation causes seedling lethality Fifty-six selfed seeds from a single silique of the mutant emb1211-3 were grown in soil, out of which 13 seeds did not germinate eventually and 43 seeds germinated and developed seedlings with normal morphology. All these seedlings could grow into maturity and set seeds. Among the 43 plants, 28 plants produced white ovules at approximately 25%, and the PCR analyses with the primers specific to the gene and the primer

Fig. 2. Identification and characterization of the gene At5G22640 as EMB1211. (A) Diagram of the T-DNA insertion position in EMB1211 gene. Solid black boxes represent exons. 5 UTR and 3 UTR are shown in open boxes. The two triangles indicate the sites of T-DNA insertion in the two mutant alleles. emb1211-2 contains a T-DNA insertion at the border of exon 5 and intron 5, and emb1211-3 contains a T-DNA insertion located in the first exon at the position 179 bp downstream of the start codon. Arrows show the positions of the gene-specific primers and T-DNA left border-specific primers which were used for genotyping of the mutant alleles. (B) Genotype of the offspring of the emb1211-3+/−. The 548-bp PCR band amplified with primers LP1 and RP1 represents the wild-type allele, and the 378-bp PCR band amplified with primers LB2 and RP1 represents the mutant allele. Samples 1, 2, 5, 7, 8, 10 and 11 are identified as heterozygotes (emb1211-3+/−), 3, 4, 6, 9 as wild-type and 12, 13, 14 as homozygotes (emb1211-3−/−). One hundred and twenty seedlings were genotyped and 14 representatives are shown. M = molecular mass marker. (C) No EMB1211 transcript detected in homozygous emb1211-3 albinos. Gene-specific primers LP1 and RP1 were used to perform RT-PCR analysis. After 30 cycles of PCR reactions, the expected 548-bp band was not detected in the albinos. The ACTIN2 gene was used as control. (D) Conserved domains in EMB1211 protein. Three MORN motifs at the N terminus and a coiled-coil structure at the C terminus are indicated. The amino acid positions of the four domains are numbered. (E) Alignment of the three MORN motifs in EMB1211 protein with other MORN motifs in MORN1 (MORN1-2 and MORN1-4), Hsjunctophilin (Hsjunctophilin-1 and Hsjunctophilin-2), HsALS2 (HsALS2-1 and HsALS2-2), AtARC and AtPIPK1 (seven MORN motifs). Amino acid residues that are conserved across all 17 sequences are shaded yellow; residues that are less conserved are shaded blue or green, respectively. Consensus sequences are shown underneath. The sequences aligned are found in the GenBank under accession numbers DQ181547 for MORN1, NM 020647.2 for Hsjunctophilin, and NM 020919.2 for HsALS2. The sequences of EMB1211 (At5g22640), AtARC (At1g75010) and AtPIPK1 (At1g21980) were from www.arabidopsis.org.

specific to the T-DNA left border revealed that they were all heterozygous, and the other 15 plants, which produced normal green ovules, were all wild-type (data not shown). As expected, none of the 43 plants were genotyped homozygous. Therefore, it is most likely that the seeds that did not germinate in soil were homozygous embryo-lethal mutant seeds. To determine if the emb1211 mutant phenotype can be rescued by tissue culture, 120 selfed seeds (with 29 shrunken seeds included) harvested from the heterozygous emb1211-3 plants were grown on MS

medium supplemented with 3% sucrose under normal growth conditions. Twenty-eight of the 29 shrunken seeds were able to germinate with a 3-day delay and developed small albino seedlings while the other 92 seeds all germinated normally and developed green seedlings (Fig. 1H, I). For example, 5-day-old wild-type had green cotyledons, whereas the emb1211-3 mutants developed slowly into small plantlets with purple cotyledons (Fig. 1H). On the day 12 after germination, wild-type developed first leaf pairs with the second leaf pairs emerging already, whereas the emb1211-3 Physiol. Plant. 2010

Fig. 3. Homozygous emb1211 mutant embryos are retarded and abnormal in morphology. (A–F) Wild-type embryos from heterozygous emb1211-3 plants undergoing normal development. Wild-type embryos from heterozygous emb1211-2 plants are not shown. (A) Globular stage. (B) Early heart stage. (C) Heart stage. (D) Torpedo stage. (E) Linear-cotyledon stage. (F) Curled-cotyledon stage. Mutant embryos from heterozygous emb1211-3 plants (G–L) and from heterozygous emb1211-2 plants (M–R) are retarded in development and morphologically abnormal as compared to wildtype. (G) and (M) Normal-looking globular-stage embryos. (H) and (N) Retarded globular-stage embryos. (I) and (O) Late globular-stage embryos with irregular cell alignment. (J) and (P) Heart-stage embryos with abnormality in the regions that will develop embryo axis and radicle. (K) and (Q) Heart-stage embryos. (L) and (R) Torpedo-stage embryos. Bars = 20 μm.

mutant just shot the first pair of true leaves (Fig. 1I). Later on, the mutant did not grow in size and it began to vitrify and died shortly after they developed the second pair of true leaves. Similar results were obtained with the emb1211-2 mutation allele (Fig. 1H, I). Genotyping of the albino seedlings by PCR amplification from genomic DNA confirmed the absence of the wild-type allele (Fig. 2B), indicating that they were homozygous for the emb1211-3 mutation. Of the 92 normal plants, 60 plants bearing white ovules produced two PCR bands, corresponding to the wild-type allele and the emb1211-3 mutant allele, respectively, and the remaining 32 plants with normal ovules were wild-type (Fig. 2B). Subsequently, the total RNA was extracted from the 10-day-old albinos and the wild-type, and cDNAs were synthesized. RT-PCR analysis using the genespecific primers did not detect any EMB1211 transcripts in the albinos (Fig. 2C). Embryo development is abnormal in the emb1211 mutants Given the fact that the gene of interest was identified as EMB1211, we were interested to determine the developmental basis of the lethal phenotype displayed by the emb1211 mutants in more detail with Physiol. Plant. 2010

respect to the embryogenesis of the white ovules at different developmental stages. Immature seeds from the selfed heterozygous emb1211-2 and emb1211-3 mutants were examined by whole-mount clearing and DIC microscopy. A detailed comparison of embryo development in normal green seeds (Fig. 3A–F) and white seeds (Fig. 3G–L) from the same siliques of emb1211-3+/− plants revealed both retardation in embryo development and abnormal embryo morphology. Up to the globular stage, the emb1211-3 embryos could not be discriminated from the wildtype (Fig. 3A, G). Seventy-five percent of the wild-type ovules that started to turn green signifying the chloroplast formation underwent typical embryo development such as heart, torpedo, linear-cotyledon and curledcotyledon stages (Fig. 3B–F), whereas the white ovules developed slower with abnormal morphology and ended at the torpedo stage (Fig. 3H–L). When the embryos from the green ovules developed to early heart stage with the enation of cotyledonal primordia, the ones from the white ovules were still at the early globular stage (Fig. 3B, H). When the wild-type embryos developed to the heart stage with the growing cotyledons, the mutant embryos were retarded at the late globular stage with irregular cell assignment and with no signs of cotyledon primordial development (Fig. 3C, I). Unlike

the torpedo-stage embryos in the wild-type, the early heart-stage embryos were present in the mutant with abnormal morphology in the regions that will develop into embryo axes and radicles (Fig. 3D, J). When the wild-type embryos were at the linear-cotyledon stage with large cotyledons signifying the storage of rich nutrients, the mutant embryos remained at the heart stage (Fig. 3E, K). At the end, when the wild-type embryos developed to the curled or mature cotyledon stage, the mutant ones were at the torpedo stage with a coneshaped radicle and small slender cotyledons (Fig. 3F, L). We also checked the mutant allele emb1211-2 for its defects in embryogenesis, and the observations revealed a high similarity between these two alleles (Fig. 3M–R). Although the homozygous mutant ovules were white in color, retarded in development and defective in morphology, they were more or less the same in size as the wild-type (Fig. 1A–C). In addition, no development defects were observed in the endosperm of the emb1211 mutants. Furthermore, the heterozygous emb1211-2 and emb1211-3 mutants were crossed reciprocally, and the immature seeds were phenotypically scored for embryo development. Among 676 ovules from 13 siliques, 173 were white and 503 green, with a segregation ratio being close to 1:3 (P > 0.05). A microscopic study was conducted to compare the embryo development between the green ovules (Fig. 4A–D) and the white ovules (Fig. 4E–H). We found that the white ovules displayed the same embryo defects as that of their parents. Taken together, the data indicate that the mutations emb1211-2 and emb1211-3 are allelic to each other and the embryo defects observed are caused by the loss-of-function of the EMB1211 gene.

Fig. 4. Mutant embryos from the crosses between emb1211-2+/− and emb1211-3+/−. (A) and (E), (B) and (F), (C) and (G), (D) and (H) are the ovules from the same siliques, respectively. (A–D) The wild-type embryos from heterozygous emb1211-3 plants. The wild-type embryos from heterozygous emb1211-2 plants are not shown. (A) Heart stage. (B) Late heart or early torpedo stage. (C) Torpedo stage. (D) Curledcotyledon stage. (E–H) Embryos from the white ovules after crossing. (E) Globular stage. (F) Early heart stage. (G) Heart stage. (H) Torpedo stage. Bars = 20 μm.

motifs in EMB1211 and that previously identified in other proteins such as Hsjunctophilin, MORN1, HsALS2, AtARC3 and AtPIPK1 (Fig. 2E). There are four amino acid residues including three glycine residues and one tyrosine residue highly conserved across all the MORN motifs aligned, and another glycine residue is also found present in most of the MORN motifs except for MONR1-4, HsALS2-2, EMB1211-M2 and EMB1211-M3. Besides, some other conserved residues such as tryptophan are also present in the MORN motifs of EMB1211.

EMB1211 comprises conserved MORN motifs

Chloroplast development is impaired in homozygous emb1211 embryos

As predicted by TAIR, the gene EMB1211 (At5g22640) encodes a putative protein that consists of 871 amino acid residues with an estimated molecular mass of 99.9 kDa. A motif scan (http://smart.embl-heidelberg.de/ smart/show motifs.pl) indicated the presence of three conserved MORN motifs toward the N terminus at the residue positions 217–239, 241–259 and 335–356 (23, 18 and 22 amino acids in size, respectively) and a coiled-coil domain at the C terminus (Fig. 2D). The three MORN motifs have a slight difference in amino acid sequences, but all contained a consensus sequence Y(E/A)GX(W/V)(X)6 GXGV(Y/I)(X)7 which encompasses hydrophobic and glycine residues interspersed with several basic amino acids. A multiple sequence alignment (http://www.ebi.ac.uk/Tools/ clustalw2/) was conducted between the three MORN

Because EMB1211 was previously identified as a chloroplast thylakoid membrane-localized protein in a TPP (three-phase partitioning) fractionation experiment (Peltier et al. 2004) and the homozygous emb1211 mutants produced white embryos (Fig. 1B, C, G) and developed small albino seedlings (Fig. 1H, I) on sucrosecontaining MS medium, we were attracted to determine if the chloroplast development was affected in the emb1211 mutants. The torpedo-stage wild-type ovules (green) and mutant ovules (white) from heterozygous emb1211-3 plants were fixed, embedded, sectioned and examined for the presence and morphology of embryo plastids using TEM (see section Materials and methods). Thylakoid membrane in the wild-type embryos developed and began to stack into grana, a significant indicator that plastids started to differentiate into Physiol. Plant. 2010

Rescue of the emb1211 mutant phenotype by complementation

Fig. 5. TEM analysis of the impairment of chloroplast biogenesis in the homozygous emb1211 embryos and seedlings. (A) A wild-type embryo with well-developed chloroplast with thylakoid membranes beginning to stack into grana. (B) A protoplast-like early plastid in mutant embryo with vesicles. (C) The chloroplasts in the cotyledons of 10-day-old wildtype seedlings. These chloroplasts are well developed, with redundant grana interconnected by stroma thylakoids. Enlargement of such a chloroplast is shown in (E). (D) Development-disrupted plastids in the albino cotyledons with no thylakoid membranes. Enlargement of two plastids are shown in (F). gr, grana. ve, vesicle. Bars = 1 μm.

chloroplasts (Fig. 5A). In contrast, only protoplast-like early plastids characterized by the presence of vesicles were detected in the mutant embryos (Fig. 5B). The vesicles derived from invagination of inner envelope membrane will interfuse into thylakoid membrane under sufficient light. In addition, the cotyledons of 12-day-old wild-type and albino were also subjected to TEM analysis. The chloroplasts in green wild-type cotyledons were crescent-shaped and had well-structured thylakoid membranes composed of grana connected by stroma thylakoid (Fig. 5C, E). In contrast, no normally differentiated chloroplasts were detected in the cotyledons of the albino; instead, small plastids showing similarity in structure to the plastids in the white embryos were visualized and localized closely to the plasmalemma (Fig. 5D, F). These results indicated that the differentiation of plastids into chloroplasts was largely impaired in the emb1211 mutant embryos. Physiol. Plant. 2010

To provide additional evidence that the embryodefective phenotype is because of the disruption of the EMB1211 gene a complementation experiment was performed. Plants heterozygous for the emb1211-3 allele were transformed using a binary vector (pCAMBIA1302) carrying the genomic region of EMB1211, which included the sequences 2.2 kb upstream of the translation start site and the complete coding region (Fig.6A). A number of independent T1 transgenic plants were obtained and genotyped for the presence of the emb1211-3 mutation and the complementation construct by PCR (data not shown). Seven of the independent transformants containing the genomic fragment of EMB1211 were heterozygous for the emb1211-3 allele, and their phenotype was scored by dissecting the immature siliques and counting the aborted seeds (Fig.6D). The T1 complemented lines were expected to show a reduction in the number of white seeds (6.25% in theoretical frequency) because of the introduction of the wild-type EMB1211 allele. We found that the seven T1 transgenic plants produced 6.55% of white seeds (Table 2). The T2 plants homozygous for the emb1211-3 allele grew well when the wild-type allele was present in the genome (data not shown). This suggests that the introduced EMB1211 genomic region rescued the embryo-defective phenotype in the emb1211 mutant. Expression profiles of the EMB1211 gene As the embryo development and seedling growth are affected in the emb1211 mutant, we were interested to investigate the temporal and spatial expression pattern of the EMB1211 gene in Arabidopsis. Initially, the expression of the EMB1211 transcripts was examined in different organs including roots, stems, leaves, inflorescences, siliques and ovules at two different development stages (i.e. 4–6 DAP and 7–9 DAP) using RT-PCR with gene-specific primers. As shown in Fig. 7A, the highest transcript expression was detected in the ovules, and the lowest transcript expression was detected in the roots, stems and inflorescences. The siliques and leaves displayed a moderate degree of expression. To gain a further understanding, especially during embryogenesis, of the expression pattern of the EMB1211 gene in more detail, a 2264-bp promoter region upstream of the start codon of the EMB1211 gene was cloned and fused with the chimeric green fluorescence protein (GFP)-β-glucuronidase (GUS) reporter (Fig. 7B). The construct was stably transformed into Arabidopsis and 20 independent transgenic lines were analyzed. One representative line was shown

Fig. 6. Complementation of emb1211+/− mutant with genomic fragment of EMB1211 gene. (A) Schematic representation of the complementation construct. The 6.5-kb genomic DNA of EMB1211 including the sequences 2.2 kb upstream the translation start site and the complete coding region was introduced into pCAMBIA1302 by KpnI and PmlI. (B) The wild-type silique with normal green embryos. (C) The silique of heterozygous emb1211 mutant with approximately 25% white embryos. (D) The silique of T1 complementation line heterozygous for emb1211 allele. It has a reduction of white aborted embryos as indicated by arrowheads. Table 2. Embryo defect in T1 complementation lines heterozygous for emb1211 allele. ∗ Calculated from five to eight siliques of each transformant. T1 plant number 1 2 3 4 5 6 7

T1 plant genotype

Number of T2 white seeds∗

Total number of T2 seeds∗

Frequency of white seeds

Theoretical frequency

χ2

emb1211+/− emb1211+/− emb1211+/− emb1211+/− emb1211+/− emb1211+/− emb1211+/−

28 25 27 18 23 17 22

428 385 395 265 345 276 345

0.0654 0.0649 0.0684 0.0679 0.0667 0.0615 0.0637

0.0625 0.0625 0.0625 0.0625 0.0625 0.0625 0.0625

0.02 0.008 0.14 0.05 0.04 0.001 0.0002

(Fig. 7C–N). A high level of GUS activity was detected in cotyledons and juvenile true leaves (Fig. 7C, D). A strong GUS activity was also detected in rosette leaves (Fig. 7E), stems (Fig. 7F) and cauline leaves (Fig. 7G), with a rather weak expression in petals and anthers (Fig. 7H). In general, GUS gene was preferentially expressed in all the green tissues. We next paid a special attention to GUS expression in the developing embryos of the transgenics. Before globular stage, no GUS staining was visualized in any part of the ovules (Fig. 7I). Since the late globular stage up to the cotyledon stage, the strong GUS expression was specifically detected in the embryos as expected, with GUS staining hardly detected in the endosperm or integument (Fig. 7J–N), suggesting that EMB1211 expression during embryogenesis is embryo-specific. The expression pattern of EMB1211 is in agreement with its essential role for the chloroplast development during embryogenesis and subsequent seedling growth.

Discussion There have been some studies to investigate the developmental inter-relation between chloroplast

biogenesis and plant embryogenesis, and the role of chloroplast in plant embryo development is uncovered. In this study, we describe that an Arabidopsis emb1211 mutant is defective in embryo development. The mutant embryos up to globular stage appear to be morphologically normal, although the embryo development is temporally retarded compared to that of wild-type. The morphological defects in the emb1211/emb1211 embryos are manifested after the heart stage of embryogenesis (Fig. 3). Unlike wildtype, the mature emb1211/emb1211 embryos are white and develop albino seedlings on culture medium (Fig. 1H, I). EMB1211 encodes a putative MORN-repeat containing protein, which, to our knowledge, is the first MORN-motif protein identified to be involved in chloroplast development during embryogenesis in plants. EMB1211 encodes a MORN-repeat containing protein EMB1211 is a MORN motif containing protein in Arabidopsis. The three N-terminal MORN motifs show significant homology with those present in other Physiol. Plant. 2010

Fig. 7. EMB1211 expression using PEMB1211 :GUS reporter lines. (A) RT-PCR analysis of EMB1211 transcripts in different tissues of Arabidopsis. Total RNA was extracted from different tissues of 40-day-old wild-type plants grown in soil. Gene-specific primers LP1 and RP1 were used. The ACTIN2 gene was used as a control. Rt, roots. St, stems. Lf, leaves. Fr, inflorescences. Si, siliques. OV-1, 4–6 DAP ovules. OV-2, 7–9 DAP ovules. (B) Schematic representation of the EMB1211 promoter GFP–GUS fusion construct. The scheme illustrates the construct structure only, and is not to scale. A 2200-bp promoter region was cloned into the binary vector pKGWFS7. (C–N) Histochemical assays for the expression pattern of PEMB1211 :GUS transgenic lines visualized by GUS staining. (C) A 5-day-old seedling. (D) A 12-day-old seedling. (E) A rosette leaf. (F) A stem. (G) A cauline leaf. (H) A flower. (I) No GUS staining detected in eight-cell stage embryo, endosperm or integument. (J) Globular-stage embryo. (K) Heart-stage embryo. (L) Torpedo-stage embryo. (M) Cotyledon-stage embryo. (N) Mature cotyledon. Bars in (C–G) = 1 mm, in (H) = 100 μm, in (I–N) = 20 μm.

proteins, which are essentially characterized by the amino acid residues conserved across all the MORN motifs analyzed (Fig. 2E). Peltier et al. (2004) used TPP fractionation to analyze the thylakoid membrane proteome and found that the EMB1211 protein is localized on the chloroplast thylakoid membranes. Furthermore, disruption of EMB1211 leads to the arrest of chloroplast development in this study (Fig. 5). MORN motifs are first described in Junctophilin, a component of junctional complex between the plasma membrane and endoplasmic reticulum (ER) of excitable cells as an integral ER protein (Takeshima et al. 2000). The eight N-terminal MORN motifs in junctophilin were found to be essential for its binding to plasma membrane phospholipids (Takeshima et al. 2000). Subsequently, MORN motifs have gradually been reported to be involved in membrane–membrane and membrane–cytoskeleton interactions in membrane adhesion or organelle fission in several proteins such as AtARC and MORN1 (Gubbels et al. 2006, Maple et al. 2007, Shimada et al. 2004). MORN1 contains 14 tandem MORN motifs of 23 amino acids each, and is a dynamic Physiol. Plant. 2010

component of the Toxoplasma gondii cell division apparatus and highly conserved among apicomplexans (Gubbels et al. 2006). It is found that MORN1 is specifically localized both to the ring structures at the apical and posterior end of the inner membrane complex and to the centrocone, and is involved in nuclear division and daughter cell budding as a part of protein complex. Furthermore, MORN 1 functions as a linker protein between certain membrane regions and the parasite’s cytoskeleton (Gubbels et al. 2006). Human protein ALS2 (amyotrophic lateral sclerosis 2) and the plant phosphatidylinositol phosphate kinases (PIPKs) are the only MORN motif containing proteins reported to have enzymatic activities (Im et al. 2007). Mutations in the ALS2 gene cause a number of recessive motor neuron diseases, indicating that the ALS2 protein is vital for motor neurons. ALS2 acts as a guanine nucleotide exchange factor (GEF) for Rab5 (Rab5GEF) and is involved in endosomal membrane trafficking (Hadano et al. 2007, Kunita et al. 2007, Otomo et al. 2003). At least, four of the eight MORN motifs at the C terminus of the Rab5GEF are found to be essential for

its function (Hadano et al. 2007). Phosphatidylinositol4-phosphate (PtdInsP)2 5-kinases (PtdInsP 5-kinases) catalyze the synthesis of phosphatidylinositol-(4,5)bisphosphate (PtdIns(4,5)P2), a key component in phosphoinositide (PI) signaling that regulates many cellular processes, for example the opening of stomatal guard cells and salt stress signal transduction (Im et al. 2007, Shimada et al. 2004). At PIPK1–9, the subfamily B PtdInsP 5-kinases, contains repeated 23-amino-acid MORN motifs at N terminus, with seven MORN motifs in At PIPK1–3 and eight motifs in At PIPK4–9, respectively. The MORN motifs have been shown to be essential for PtdOH activation and regulate the accessibility of lipids to the active site in a PtdOH-sensitive manner (Im et al. 2007). EMB1211 is required for chloroplast biogenesis in Arabidopsis Plastid biogenesis is tightly coupled with temporal and spatial stages of plant development, during which plastid volume and composition change as a consequence of acquisition of photosynthetic competence and activation of other biosynthetic processes (Mullet 1988). The acquisition of this competence and the activation of the biosynthetic processes require the activation of both nuclear and chloroplast genes (Bauer et al. 2001, Goldschmidt-Clermont 1998, Harrak et al. 1995). Thus, a key component of plastid development is the coordination of gene expression between the nuclear and plastidic genomes (Leon et al. 1998, Uwer et al. 1998). Several lines of evidence indicate that there exists a complex crosstalk between plastidic genome and nuclear genome. Genetic information from nucleus can regulate the expression of the plastid genome as well as the expression of nuclear genes encoding plastid protein (Uwer et al. 1998, Lopez-Juez ´ 2007, Sakamoto et al. 2008). Likewise, the developmental stage of the chloroplast itself appears to regulate the expression of nuclear genes coding for chloroplast-targeted proteins (Gutiérrez-Nava Mde et al. 2004, Harpster et al. 1984, Mayfield and Taylor 1984, Taylor 1989). To date, there have been a number of genes identified important for chloroplast development (Apuya et al. 2001, Chi et al. 2008, Colombo et al. 2008, Garcion et al. 2006, Huang et al. 2009, Mandel et al. 1996, Motohashi et al. 2001, Reiter et al. 1994, Ruppel and Hangarter 2007, Shimada et al. 2004, Takechi et al. 2000, Tzafrir et al. 2004). Mutations in many of the nuclear genes that regulate chloroplast development have been subjected to molecular analyses. These mutants display defects in a variety of chloroplast functions, including protein targeting to the thylakoid

(Kobayashi et al. 2007, Pesaresi et al. 2006), maintenance of ribosomes within the plastid (Chi et al. 2008), chloroplast divisions (Shimada et al. 2004), translation apparatus (Berg et al. 2005) and communication between chloroplast and mitochondrion (Pesaresi et al. 2006, Yamaoka and leaver 2008). In this study, we show that a T-DNA insertion in the EMB1211 gene causes a developmental arrest of the chloroplasts in mutant embryos, and there are no stacked or well-organized thylakoid membranes (Fig. 5). This indicates that the functional EMB1211 protein is necessary for chloroplast development during embryogenesis. Previously characterized MORN motifs in other proteins function to attach to membrane phospholipids (Gubbels et al. 2006, Ma et al. 2006, Shimada et al. 2004, Takeshima et al. 2000). Therefore, it is possible that EMB1211 protein might function by attaching to thylakoid membranes of chloroplasts. The role of EMB1211 in regulation of chloroplast development awaits further investigation. Embryogenesis is dependent on functional chloroplasts It is well established that the processes of plastid biogenesis and embryo development are tightly linked, presumably because the organelle synthesizes many important products (carbohydrates, fatty acids, amino acids, etc.) that are utilized by the rest of the cell. In addition to providing nourishment for embryo development, it is hypothesized that developing chloroplast also creates a signal required for the regulation of nuclear gene expression, thus affecting embryo development (Oelmuller ¨ 1989, Taylor 1989, Uwer et al. 1998). The emb1211 mutants described in this study and some other mutants previously reported (Apuya et al. 2001, Uwer et al. 1998) that have defects in chloroplast development and are embryo-lethal could not be rescued by tissue culture, indicating an existence of a developmental communication between plastid and nucleus. The Arabidopsis emb1211 mutants are defective in embryo development. The abnormality of embryo development in the homozygous emb1211 mutants becomes evident during the transition from the late globular stage to the heart stage when chloroplast biogenesis takes place with the greening of the ovules being an indicator (Mansfield and Briarty 1991, Ruppel and Hangarter 2007). These embryos are temporally retarded in embryo development and enter into maturity at the torpedo stage with whitening of the embryos (Figs 1 and 3). TEM analysis revealed the presence of undifferentiated plastids containing no well-structured thylakoid membranes both in the retarded embryos and in the albino seedlings which are probably because of the Physiol. Plant. 2010

lack of photosynthetic capacity (Fig. 5). Therefore, it is conceivable that EMB1211 functions in embryogenesis through influencing the construction or maintenance of thylakoid membrane in Arabidopsis. Although many nuclear genes have been identified to play important roles in chloroplast biogenesis, it appears that only those involved in the basal cellular functions such as chloroplast translation machinery and membrane biogenesis are required for the normal embryo development (Apuya et al. 2001, Berg et al. 2005, Chi et al. 2008, Colombo et al. 2008, Garcion et al. 2006, Kobayashi et al. 2007, Ruppel and Hangarter 2007). The biogenesis of thylakoid membranes, the site of photosynthetic reactions, is an indispensable event for the development and functioning of chloroplasts, which requires the co-ordinated synthesis of proteins in the plastidial and cytosolic compartment (GoldschmidtClermont 1998). AKRP and EMB506, two plastidlocalized ANK-repeats containing proteins, act together to play an essential role in the first differentiation of the proplastid during embryo formation, and homozygous emb506 and akr embryos are arrested at the globular stage (Garcion et al. 2006). AminoacyltRNA synthetases (AARSs) are essential components of protein synthesis that catalyze the attachment of amino acids to their cognate tRNAs (O’Donoghue and LutheySchulten 2003). Seven of these genes encode AARSs which are predicted or confirmed to be localized in chloroplasts (Berg et al. 2005). The typical knockout phenotype is an embryo arrest at the globular-to-heart transition stage. Aborted seeds and arrested embryos are white or pale yellow because of the disruption of the chloroplasts. In addition, AGL23, a type I MADS-box gene that controls the chloroplast biogenesis during embryogenesis – the homozygous agl23-1 are albinos with the absence of developed chloroplasts, contributes to the abnormal female gametophyte and embryo development (Colombo et al. 2008). Because AGL23 encodes a transcription factor, there are many possibilities with respect to its molecular basis by which the chloroplast development is controlled. Another example is that the disruption of the biosynthesis of monogalactosyldiacylglycerol (MGDG), the major constituent of the plastid membranes, leads to the complete impairment of photosynthetic ability and arrest of embryo development (Kobayashi et al. 2007). Plastid-localized AtNAP7 represents a conserved SufC protein and is involved in the biogenesis and/or repair of oxidatively damaged plastidic Fe–S clusters during Arabidopsis embryogenesis. Arabidopsis plants deficient in the SufC homolog AtNAP7 show lethality at the globular stage of embryogenesis, and the mutant embryos contain abnormally developing plastids with Physiol. Plant. 2010

disorganized thylakoid structures (Xu et al. 2004). AtcpRRF encodes a chloroplast ribosome recycling factor. The chloroplasts of the hfp108-1 plants deficient in AtcpRRF contain few internal thylakoid membranes and are severely defective in the accumulation of chloroplast-encoded proteins, and the homozygous hfp108-2 embryos are blocked at the heart stage and do not develop further. These results suggest that AtcpRRF is essential for embryogenesis and chloroplast biogenesis (Wang et al. 2010). In conclusion, the results of our analyses indicate that EMB1211, the MORN-repeat containing protein, is required for growth and development in Arabidopsis, and disruption of this protein results in defects in chloroplast development during embryogenesis and seedling lethality. Acknowledgements – This work was supported by the National Basic Research Program of China (grant number 2007CB108801). The plant expression vectors were kindly provided by Flanders Interuniversity Institute for Biotechnology (V. I. B), Belgium. The seeds of CS16103 were obtained from the Arabidopsis Biological Resource Center (ABRC, Ohio State University).

References Albert S, Després B, Guilleminot J, Bechtold N, Pelletier G, Delseny M, Devic M (1999) The EMB 506 gene encodes a novel ankyrin repeat containing protein that is essential for the normal development of Arabidopsis embryos. Plant J 17: 169–179 Alonso AP, Goffman FD, Ohlrogge JB, Shachar-Hill Y (2007) Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos. Plant J 52: 296–308 Apuya NR, Yadegari R, Fischer RL, Harada JJ, Zimmerman JL, Goldberg RB (2001) The Arabidopsis embryo mutant schlepperless has a defect in the chaperonin-60alpha gene. Plant Physiol 126: 717–730 Bauer J, Hiltbrunner A, Kessler F (2001) Molecular biology of chloroplast biogenesis: gene expression, proein import and inraorganellar sorting. Cell Mol Life Sci 58: 420–433 Berg M, Rogers R, Muralla R, Meinke D (2005) Requirement of aminoacyl-tRNA synthetases for gametogenesis and embryo development in Arabidopsis. Plant J 44: 866–878 Chi W, Ma JF, Zhang DY, Guo JK, Chen F, Lu CM, Zhang LX (2008) The pentatricopeptide repeat protein DELAYED GREENING1 is involved in the early chloroplast development and chloroplast gene expression in Arabidopsis. Plant Physiol 147: 573–584

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743 Colombo M, Masiero S, Vanzulli S, Lardelli P, Kater MM, Colombo L (2008) AGL23, a type I MADS-box gene that controls female gametophyte and embryo development in Arabidopsis. Plant J 54: 1037–1048 Garcion C, Guilleminot J, Kroj T, Parcy F, Giraudat J, Devic M (2006) AKRP and EMB506 are two ankyrin repeat proteins essential for plastid differentiation and plant development in Arabidopsis. Plant J 48: 895–906 Goffman FD, Alonso AP, Schwender J, Shachar-Hill Y, Ohlrogge JB (2005) Light enables a very high efficiency of carbon storage in developing embryos of rapeseed. Plant Physiol 138: 2269–2279 Goldberg RB, de Paiva G, Yadegari R (1989) Regulation of gene expression during plant embryogenesis. Cell 56: 149–160 Goldberg RB, de Paiva G, Yadegari R (1994) Plant embryogenesis: zygote to seed. Science 266: 605–614 Goldschmidt-Clermont M (1998) Coordination of nuclear and chloroplast gene expression in plant cells. Int Rev Cytol 177: 115–180 Gutiérrez-Nava Mde L, Gillmor CS, Jiménez LF, Guevara-Garc´ıa A, Leon ´ P (2004) Chloroplast biogenesis genes act cell and noncell autonomously in early chloroplast development. Plant Physiol 135: 471–482 Gubbels MJ, Vaishnava S, Boot N, Dubremetz JF, Striepen B (2006) A MORN-repeat protein is a dynamic component of the Toxoplasma gondii cell division apparatus. J Cell Sci 119: 2236–2245 Hadano S, Kunita R, Otomo A, Suzuki-Utsunomiya K, Ikeda JE (2007) Molecular and cellular function of ALS2/alsin: implication of membrane dynamics in neuronal development and degeneration. Neurochem Int 51: 74–84 Harpster MH, Mayfield SP, Taylor WC (1984) Effects of pigment deficient mutants on accumulation of photosynthetic proteins in maize. Plant Mol Biol 3: 59–71 Harrak H, Lagrange T, Bisanz-Seyer C, Lerbs-Mache S, Mache R (1995) The expression of nuclear genes encoding plastid ribosomal proteins precedes the expression of chloroplast genes during early phases of chloroplast development. Plant Physiol 108: 685–692 Huang X, Zhang X, Yang S (2009) A novel chloroplast-localized protein EMB1303 is required for chloroplast development in Arabidopsis. Cell Res 19: 1205–1216 Im YJ, Davis AJ, Perera IY, Johannes E, Allen NS, Boss WF (2007) The N-terminal membrane occupation and recognition nexus domain of Arabidopsis phosphatidylinositol phosphate kinase 1 regulates enzyme activity. J Biol Chem 282: 5443–5452

Jurgens ¨ G, Mayer U (1994) Arabidopsis. In: Bard JBL (ed) Embryos - Color Atlas of Development. Genetics Unit of Western General Hospital, Edingurgh, Scotland, pp 700–747 Kobayashi K, Kondo M, Fukuda H, Nishimura M, Ohta H (2007) Galactolipid synthesis in chloroplast inner envelope is essential for proper thylakoid biogenesis, photosynthesis, and embryogenesis. Proc Natl Acad Sci USA 104: 17216–17221 Kunita R, Otomo A, Mizumura H, Suzuki-Utsunomiya K, Hadano S, Ikeda JE (2007) The Rab5 activator ALS2/alsin acts as a novel Rac1 effector through Rac1-activated endocytosis. J Biol Chem 282: 16599–16611 Laux T, Jugens ¨ G (1997) Embryogenesis: a new start in life. Plant Cell 9: 989–1000 Leon P, Arroyo A, Mackenzie S (1998) Nuclear control of plastid and mitochondrial development in higher plants. Annu Rev Plant Physiol Plant Mol Biol 49: 453–480 Lopez-Juez ´ E (2007) Plastid biogenesis, between light and shadows. J Exp Bot 58: 11–26 Lu X, Liu X, An L, Zhang W, Sun J, Pei H, Meng H, Fan Y, Zhang C (2008) The Arabidopsis MutS homolog AtMSH5 is required for normal meiosis. Cell Res 18: 589–599 Ma H, Lou Y, Lin WH, Xue HW (2006) MORN motifs in plant PIPKs are involved in the regulation of subcellular localization and phospholipid binding. Cell Res 16: 466–478 Mandel MA, Feldmann KA, Herrera-Estrella L, Rocha-Sosa M, Leon ´ P (1996) CLA1, a novel gene required for chloroplast development, is highly conserved in evolution. Plant J 9: 649–658 Mansfield SG, Briarty LG (1991) Early embryogenesis in Arabidopsis thaliana. II. The developing embryo. Can J Bot 69: 461–476 Maple J, Møller SG (2007) Plastid division: evolution, mechanism and complexity. Ann Bot (Lond) 99: 565–579 Maple J, Vojta L, Soll J, Møller SG (2007) ARC3 is a stromal Z-ring accessory protein essential for plastid division. EMBO Rep 8: 293–299 Mayfield SP, Taylor WC (1984) Carotenoid-deficient maize seedlings fail to accumulate light-harvesting chlorophyll a/b binding protein (LHCP) mRNA. Eur J Biochem 144: 79–84 Meinke DW (1995) Molecular genetics of plant embryogenesis. Annu Rev Plant Physiol Plant Mol Biol 46: 369–394 Motohashi R, Nagata N, Ito T, Takahashi S, Hobo T, Yoshida S, Shinozaki K (2001) An essential role of a TatC homologue of a Delta pH- dependent protein transporter in thylakoid membrane formation during chloroplast development in Arabidopsis thaliana. Proc Natl Acad Sci USA 98: 10499–10504


Mullet J (1988) Chloroplast development and gene expression. Annu Rev Plant Physiol Plant Mol Biol 46: 369–394 O’Donoghue P, Luthey-Schulten Z (2003) On the evolution of structure in aminoacyl-tRNA synthetases. Microbiol Mol Biol Rev 67: 550–573 Oelmuller ¨ R, Kendrick RE, Briggs WR (1989) Blue-light mediated accumulation of nuclear-encoded transcripts coding for proteins of the thylakoid membrane is absent in the phytochrome-deficient aurea mutant of tomato. Plant Mol Biol 13: 223–232 Otomo A, Hadano S, Okada T, Mizumura H, Kunita R, Nishijima H, Showguchi-Miyata J, Yanagisawa Y, Kohiki E, Suga E, Yasuda M, Osuga H, Nishimoto T, Narumiya S, Ikeda JE (2003) ALS2, a novel guanine nucleotide exchange factor for the small GTPase Rab5, is implicated in endosomal dynamics. Hum Mol Genet 12: 1671–1687 Peltier JB, Ytterberg AJ, Sun Q, van Wijk KJ (2004) New functions of the thylakoid membrane proteome of Arabidopsis thaliana revealed by a simple, fast, and versatile fractionation strategy. J Biol Chem 279: 49367–49383 Pesaresi P, Masiero S, Eubel H, Braun HP, Bhushan S, Glaser E, Salamini F, Leister D (2006) Nuclear photosynthetic gene expression is synergistically modulated by rates of protein synthesis in chloroplasts and mitochondria. Plant Cell 18: 970–991 Reiter RS, Coomber SA, Bourett TM, Bartley GE, Scolnik PA (1994) Control of leaf and chloroplast development by the Arabidopsis gene pale cress. Plant Cell 6: 1253–1264 Ruppel NJ, Hangarter RP (2007) Mutations in a plastid-localized elongation factor G alter early stages of plastid development in Arabidopsis thaliana. BMC Plant Biol 7: 37–46 Ruuska SA, Schwender J, Ohlrogge JB (2004) The capacity of green oilseeds to utilize photosynthesis to drive biosynthetic processes. Plant Physiol 136: 2700–2709 Sakamoto W Miyagishima SY, Jarvis P (2008) Chloroplast biogenesis: control of plastid development, protein import, division and inheritance. The Arabidopsis Book. BioOne, Washington DC, USA Schulz P, Jensen WA (1968) Capsella embryogenesis: the early embryo. J Ultrastruct Res 22: 376–392

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Shimada H, Koizumi M, Kuroki K, Mochizuki M, Fujimoto H, Ohta H, Masuda T, Takamiya K (2004) ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase. Plant Cell Physiol 45: 960–967 Sparkes IA, Brandizzi F, Slocombe SP, El-Shami M, Hawes C, Baker A (2003) An Arabidopsis pex10 null mutant is embryo lethal, implicating peroxisomes in an essential role during plant embryogenesis. Plant Physiol 133: 1809–1819 Takechi K, Sodmergen, Murata M, Motoyoshi F, Sakamoto W (2000) The YELLOW VARIEGATED (VAR2) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis. Plant Cell Physiol 41: 1334–1346 Takeshima H, Komazaki S, Nishi M, Iino M, Kangawa K (2000) Junctophilins: a novel family of junctional membrane complex proteins. Mol Cell 6: 11–22 Taylor WC (1989) Regulatory interactions between nuclear and plastid genomes. Annu Rev Plant Physiol Plant Mol Biol 40: 211–233 Tzafrir I, Pena-Muralla R, Dickerman A, Berg M, Rogers R, Hutchens S, Sweeney TC, McElver J, Aux G, Patton D, Meinke D (2004) Identification of genes required for embryo development in Arabidopsis. Plant Physiol 135: 1206–1220 Uwer U, Willmitzer L, Altmann T (1998) Inactivation of a glycyl-tRNA synthetase leads to an arrest in plant embryo development. Plant Cell 10: 1277–1294 Wang L, Ouyang M, Li Q, Zou M, Guo J, Ma J, Lu C, Zhang L (2010) The Arabidopsis chloroplast ribosome recycling factor is essential for embryogenesis and chloroplast biogenesis. Plant Mol Biol 74: 47–59 West MAL, Harada JJ (1993) Embryogenesis in higher plants. An overview. Plant Cell 5: 1361–1369 Xu XM, Møller SG (2004) AtNAP7 is a plastidic SufC-like ATP-binding cassette/ATPase essential for Arabidopsis embryogenesis. Proc Natl Acad Sci USA 101: 9143–9148 Yamaoka S, Leaver CJ (2008) EMB2473/MIRO1, an Arabidopsis Miro GTPase, is required for embryogenesis and influences mitochondrial morphology in pollen. Plant Cell 20: 589–601