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First published online May 7, 2004; 10.1104/pp.103.036996 Plant Physiology 135:471-482 (2004) © 2004 American Society of Plant Biologists CHLOROPLAST BIOGENESIS Genes Act Cell and Noncell Autonomously in Early Chloroplast Development1Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de Mexico, Cuernavaca, Morelos 62271, Mexico (M.G.-N., A.G.-G., P.L.); Department of Plant Biology, Carnegie Institution of Washington (C.S.G.) and Department of Biological Sciences (C.S.G.), Stanford University, Stanford, California, 94305; and Laboratorio de Microscopía Electrónica, Facultad de Ciencias, Universidad Nacional Autónoma de Mexico, Mexico D.F. 04510, Mexico (L.F.J.)
In order to identify nuclear genes required for early chloroplast development, a collection of photosynthetic pigment mutants of Arabidopsis was assembled and screened for lines with extremely low levels of chlorophyll. Nine chloroplast biogenesis (clb) mutants that affect proplastid growth and thylakoid membrane formation and result in an albino seedling phenotype were identified. These mutations identify six new genes as well as a novel allele of cla1. clb mutants have less than 2% of wild-type chlorophyll levels, and little or no expression of nuclear and plastid-encoded genes required for chloroplast development and function. In all but one mutant, proplastids do not differentiate enough to form elongated stroma thylakoid membranes. Analysis of mutants during embryogenesis allows differentiation between CLB genes that act noncell autonomously, where partial maternal complementation of chloroplast development is observed in embryos, and those that act cell autonomously, where complementation during embryogenesis is not observed. Molecular characterization of the noncell autonomous clb4 mutant established that the CLB4 gene encodes for hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), the next to the last enzyme of the methylerythritol 4-phosphate (MEP) pathway for the synthesis of plastidic isoprenoids. The noncell autonomous nature of the clb4 mutant suggests that products of the MEP pathway can travel between tissues, and provides in vivo evidence that some movement of MEP intermediates exists from the cytoplasm to the plastid. The isolation and characterization of clb mutants represents the first systematic study of genes required for early chloroplast development in Arabidopsis.
Chloroplasts are responsible for essential plant functions such as the fixation of CO2, manufacture of carbon skeletons, fatty acids and pigments, and the synthesis of amino acids from inorganic nitrogen, among others (Staehelin and Newcomb, 2000  ). In higher plants, chloroplasts develop from proplastids, small organelles (0.2–0.5 µm diameter) that lack thylakoid membranes and that are present primarily in meristematic cells and in young postmitotic cells (Vothknecht and Westhoff, 2001). As meristematic cells begin to differentiate into mesophyll and palisade cells, proplastids coordinately differentiate into chloroplasts. This differentiation process is also modulated by environmental cues such as light, and thus certain Arabidopsis mutants with an altered light signal transduction pathway are able to develop leaves even in the absence of chloroplast development (Li et al., 1994).
During development, the increase in plastid volume can be greater than 100-fold. When the proplastid reaches an average size of 1.0 µm the inner layer of the membrane begins to invaginate into the stroma (Mühlethaler and Frey-Wyssling, 1959
The conversion of proplastids into chloroplasts is accompanied by high transcription levels of plastid- and nuclear-encoded genes involved in the transcription/translation apparatus (Baumgartner et al., 1989
Mutations that interfere with or block different stages of chloroplast development have been isolated from a variety of plant species, including maize, barley, tobacco, tomato, Antirrhinum, and Arabidopsis (Mascia and Robertson, 1978
Plant isoprenoids serve essential roles in photosynthesis, respiration, growth and development. These compounds include an enormous variety of natural products synthesized through the consecutive condensation and modification of two basic 5-carbon units, the isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants the biosynthesis of these two structural blocks takes place by two independent pathways localized in different cellular compartments. The well-characterized mevalonic pathway operates in the cytoplasm, while in plastids IPP and DMAPP are synthesized by the recently discovered methylerythritol 4-phosphate (MEP) pathway (Lichtenthaler, 1999 To identify new genes necessary for early steps of chloroplast biogenesis, we assembled a collection of pigmentation lines, and focused our subsequent analysis on the mutants with extremely low levels of chlorophyll. These albino lines define six novel genes, which we have named CHLOROPLAST BIOGENESIS (CLB) 1–6. Growth of clb seedlings under high and low light conditions demonstrated that the albino phenotype of clb seedlings is not a secondary effect of photooxidative damage. Our analysis demonstrates that CLB genes are required for plastid growth and the formation of thylakoid membranes, as well as for the expression of plastid- and nuclear-encoded genes required for early chloroplast biogenesis. By comparing the phenotype of clb mutants during embryogenesis and seedling growth, we show that CLB genes encode factors required for early chloroplast biogenesis that act both cell and noncell autonomously. Further, we found that CLB4 corresponds to the ISPG gene, which encodes the enzyme that participates in the next to last step in the plastidic isoprenoid biosynthesis pathway. clb4 corresponds to the first loss-of-function mutant for this enzyme in plants, and underscores the importance of isoprenoids in chloroplast development.
Survey of Photosynthetic Pigment Lines from Arabidopsis Stock Center To identify new mutants that affect early stages of chloroplast development, 22 lines segregating seedling pigment mutations (classified as albino) were obtained from the ABRC. Lines were grown in tissue culture media supplemented with Suc and examined visually. Seedlings representative of the observed phenotypic spectrum are shown in Figure 1 . Surprisingly, the majority of the ABRC mutants classified as albino had a considerable amount of chlorophyll (Fig. 1B) or carotenoid (Fig. 1C) pigments, and therefore are referred to here as pale green or yellow phenotypes, respectively. Only 2 of these lines, CS27 and CS213 (Fig. 1D), were visually severely lacking in photosynthetic pigments and thus fit our phenotypic criteria for albino mutants. The fact that only 2 out of 22 ABRC pigment lines displayed a true albino seedling phenotype suggested that the number of genes that when mutated render an albino phenotype might be relatively small. In view of this, a genetic screen to isolate more albino mutants was performed.
Identification of chloroplast biogenesis Mutants
Since wild-type Arabidopsis embryos develop chloroplasts during embryogenesis (Mansfield and Briarty, 1991 Based on subsequent visual inspection of seedlings of the 99 pigment lines in our collection (22 from the ABRC and 77 from our screen), 61 mutant lines were classified as pale green, 29 as yellow, and only 9 as albino (Table I). As our specific interest is in early chloroplast biogenesis, subsequent analysis was focused on the albino mutant lines (Table II). We chose the name chloroplast biogenesis (clb) for these mutants, to reflect the requirement of these genes for early chloroplast development. To corroborate the visual observation that our clb mutants were severely lacking in chlorophylls and carotenoids, both pigment levels were quantified. It was found that all clb mutants contained 2% or less chlorophyll levels per wet weight compared with wild-type seedlings (Table III). The carotenoid content in these mutants is also severely reduced, although not as much as the chlorophylls (Table III).
Complementation analysis of the nine clb mutants demonstrated that these mutations identified seven different genetic loci (see Table II). The mutation in ABRC stock center line CS213, was found to be allelic to the previously described cla1-1 (Mandel et al., 1996 ), and was designated cla1-2. Three additional mutants were found to be allelic and were designated clb5-1, clb5-2, and clb5-3. The remaining five nonallelic mutant lines were designated as clb1-1, clb2-1, clb3-1, clb4-1, and clb6-1. Plants heterozygous for all nine clb mutations segregated albino embryos as monogenic, nuclear recessive alleles with normal transmission (data not shown). clb mutants were mapped using cosegregation analysis of PCR-based molecular markers, and the map positions for CLB genes are shown in Figure 2
.
Seedling Morphology of clb Mutants
It is well known that the absence of carotenoid pigments can result in photo-oxidative stress under high/normal light conditions, resulting in pleiotropic effects on chloroplast development (Oelmüller, 1989
After 10 d of growth on solidified germination media (GM) under high light conditions, all six clb mutants present a similar morphological phenotype. Seedling growth of clb mutants is retarded compared to wild-type. A typical 10-d-old wild-type seedling has four leaves and two cotyledons, while clb mutants have two cotyledons and only two small leaves (Fig. 3, B–G). Cotyledons of clb mutants have many swollen epidermal cells, and tend to curl under at the ends. With the exception of clb5, all clb mutants produce trichomes on the adaxial side of their first two leaves, similar to wild-type seedlings. clb seedlings accumulate considerable amounts of anthocyanin under high light conditions, but not when grown under low light conditions, presumably due to the decrease in photooxidative stress. With the exception of the difference in the accumulation of anthocyanin pigments, the effect of growing clb mutants under low light parallels that of wild-type seedlings grown under low light: seedlings show increased expansion of hypocotyls and petioles, and more oval shaped cotyledons (compare Fig. 3H with 3, I–N). Under low light conditions, clb4 (Fig. 3L) and clb6 (Fig. 3N) develop a subtle yellow color.
To assess the effect of clb mutations on chloroplast development, plastids from the first leaf of 3-week-old seedlings were examined by electron microscopy (EM). Compared with the wild-type chloroplast (Fig. 4A ), the plastids of clb mutants are all arrested at an early stage of differentiation. Plastids of clb2 (Fig. 4C), clb3 (Fig. 4D), and clb5 (Fig. 4F) lack appressed internal membranes and have large vesicle-like structures with unknown contents, similar to those found in proplastids. By contrast, the chloropasts of clb1 (Fig. 4B) and clb6 (Fig. 4G) mutants contain short, linear appressed membranes, while clb4 (Fig. 4E) contains long appressed membranes. Based on plastid morphology, chloroplast development seems to be arrested earliest in clb5, clb2, and clb3, and slightly later in clb1, clb6, and clb4.
CLB Genes Are Required for Expression of Nuclear- and Chloroplast-Encoded Genes Involved in Plastid Transcription, Translation, and Photosynthesis EM analysis of plastids in clb mutants suggested that chloroplast differentiation is arrested at an early stage. To further characterize the plastid differentiation stage in clb mutants, the expression of several nuclear- and chloroplast-encoded genes known to be expressed at different moments of chloroplast development was determined.
The rrn16S (16S rRNA) gene is the most highly expressed plastid-encoded gene in proplastids (Bisanz-Seyer et al., 1989
We also quantified transcript levels for accD, the chloroplast-encoded subunit of acetyl-CoA carboxylase, involved in lipid biosynthesis in the plastid. accD is exclusively transcribed by a nuclear-encoded plastid-localized RNA polymerase, required for proplastid maintenance, and is thus a marker of plastid transcription (Hajdukiewicz et al., 1997 ). accD transcript levels are detected at near wild-type levels in clb6, at about 70% of wild type in clb1 and clb4, at about 50% of wild type in clb2 and clb3, and at about 20% of wild type in clb5. The low accumulation of nuclear- and plastid-encoded ribosomal genes detected in clb5 suggests that plastids in this mutant are severely affected in translational competence.
Nuclear- and chloroplast-encoded genes required for photosynthetic reactions are late molecular markers for chloroplast biosynthesis and function. Thus, some of these genes were also chosen as markers for the molecular analysis of the clb mutants. The transcript level of nuclear-encoded RBCS, the essential enzyme of the Calvin cycle, and chloroplast-encoded psbA, which encodes the D1 protein of the reaction center of photosystem II (Bruick and Mayfield, 1999
During Arabidopsis embryo development, differentiated chloroplasts are observed beginning at the heart stage, and their number continues to increase until the end of embryo growth (Mansfield and Briarty, 1991
CLA1 is an essential gene in Arabidopsis that encodes for the DXS enzyme required in the first biosynthetic step of the MEP pathway (Estévez et al., 2000 ). It has previously been demonstrated that the albino phenotype of cla1-1 mutant seedlings can be complemented noncell autonomously by the addition of a synthetic version of the product of the DXS enzyme, 1-deoxyxylulose 5-P (DXP), to seedling growth medium (Estévez et al., 2000). Thus, diffusion of DXP or a downstream compound from the mother plant to cla1 embryos is probably responsible for the partial complementation that results in the observed chlorophyll accumulation during embryogenesis. Since clb4 and clb6 embryos also accumulate significant levels of chlorophyll, while clb4 and clb6 seedlings have almost no chlorophyll, the CLB4 and CLB6 genes can also be defined as acting noncell autonomously. By contrast, as shown in Figure 6, E–H, chlorophyll fluorescence was never detected in clb1, clb2, clb3, and clb5 embryos. These mutations can be defined as cell autonomous, as they are not complemented by maternal factors during embryogenesis.
The observation that the CLA1, CLB4, and CLB6 genes behave noncell autonomously suggested that (similar to CLA1) CLB4 and CLB6 might also encode enzymes required for the MEP isoprenoid biosynthesis. To test this hypothesis, a candidate gene approach was used to identify the CLB4 gene. The mapping experiments described in Figure 2 placed the CLB4 gene approximately 4 cM north of the SSLP marker ciw10 on chromosome 5. Localization of genes involved in the MEP pathway showed the Arabidopsis homolog of the ISPG/GCPE gene (At5g60600) to be located in this region. In bacteria GcpE encodes for the 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) synthase (HDS) enzyme, involved in the conversion of methylerythritol 2,4- cyclodiphosphate (ME-cPP) into HMBPP, which is the next to the last step of the MEP pathway (Hecht et al., 2001 Sequencing of the ISPG gene from genomic DNA of clb4-1 showed that this gene contained a cytosine to thymine change compared to wild type at bp 1264 of the ISPG cDNA, resulting in TAA stop at codon 371 (Fig. 7A ). We subsequently identified a T-DNA insertion (SALK 017595) into the 16th exon of the ISPG gene (At5g60600). Plants heterozygous for this T-DNA insertion segregated albino seedlings with a phenotype identical to clb4-1, and crosses between clb4-1 and SALK 017595 heterozygous plants showed that this line failed to complement the clb4-1 mutation. Thus, the mutation in SALK 017595 was named clb4-2. To provide further evidence that clb4-1 and clb4-2 alleles affect the expression of the ISPG gene, the transcript levels of this gene were analyzed in both mutants. As shown in Figure 7B, while a transcript of the predicted molecular size for the ISPG gene was observed in wild-type seedlings, no transcript was detected in either clb4-1 or clb4-2 alleles. These results together demonstrate that both mutants behave as null alleles for the Arabidopsis ISPG gene, and strongly suggest that the disruption of this gene is responsible for the albino phenotype in both mutant lines.
To examine whether the effect observed at the transcript level was reflected at the protein level, we probed extracts of wild-type, cla1-1, clb4-1, and clb4-2 seedlings with polyclonal antibodies raised against a synthetic peptide of the HDS protein, the product of the ISPG gene from Arabidopsis. In wild-type and cla1-1 seedlings, this antibody recognized two bands of similar size, in the expected size range for the HDS protein. In protein extracts from clb4-1 and clb4-2 seedlings, the lower of these two bands was undetectable (Fig. 7C). While the identity of the upper band is unclear, these results indicate that the lower band corresponds to the HDS protein and that both mutant alleles have undetectable levels of the HDS protein.
Although there has been important progress in the elucidation of the MEP pathway genes in recent years, little is known about the expression profile and regulation of most of the genes of the pathway in plants. It has been reported that the first enzymes of the pathway (DXS and DXR) are expressed in most plant tissues but with highest levels in young photosynthetic tissues (Estévez et al., 2000
In this work we report the isolation and characterization of mutations in six independent CHLOROPLAST BIOGENESIS genes, identified in a systematic visual screen for pigment mutants of Arabidopsis. Analysis of photosynthetic pigment levels, chloroplast ultrastructure, and expression of genes required for chloroplast transcription, translation, and photosynthesis demonstrated that chloroplasts in clb mutants are arrested at early stages of development with phenotypes similar to proplastids. It was further demonstrated that the albino phenotype of clb mutants is not due to photooxidation. The CLB4 gene was identified based on its map position, and demonstrated to correspond to the ISPG gene, which encodes for the HDS, the second-to-last acting enzyme in the MEP isoprenoid biosynthesis pathway. The noncell autonomous nature of the clb4 mutation provides evidence that products of the MEP pathway can move between tissue types, and suggests that there is at least some movement of MEP products from the cytoplasm to the chloroplast.
Isoprenoids play a fundamental role in plant development as they include molecules such as chlorophyll and carotenoids, as well as the plant hormones GA3 and abscisic acid (Rodríguez-Concepción and Boronat, 2002
The work described here demonstrated that CLB4 corresponds to the ISPG gene, which encodes the HDS enzyme, and is the second-to-last acting enzyme in the MEP plastidic isoprenoid biosynthesis pathway. Although a previous report has shown that the ISPG gene homolog from Arabidopsis complements a bacterial mutant affected in the HDS enzyme (Querol et al., 2002
CLB4 and CLB6 genes were proposed to act noncell autonomously based on the observation that chlorophyll fluorescence accumulates in embryos of clb4 and clb6. A similar pattern of chlorophyll accumulation was also seen in cla1 embryos, and it was previously shown that cla1 seedlings can be rescued by growth on medium containing the MEP intermediate produced by the CLA1 gene product, demonstrating that CLA1 can act noncell autonomously (Estévez et al., 2000
The protein expression found for CMS and HDS is similar to the one observed for DXS and DXR (Estévez et al., 2000 The phenotype of clb4 and clb6 seedlings may be alleviated by the presence of some functional chloroplasts during embryogenesis, allowing mutant embryos to produce a low level of essential compounds required during embryogenesis. These molecules, including MEP-derived isoprenoid such as hormones, could persist during the first stages of seedling growth, lessening the consequences of lack of CLB4 or CLB6 activity. To our knowledge, this is the first time that partial complementation of a mutation during embryogenesis by diffusion of maternal factors has been observed in plants. Conceivably, this could occur with any embryo defective mutant lacking a molecule that can diffuse or is actively transported from the mother plant to the embryo. If this is a general phenomenon, mutations affecting gene products that act noncell autonomously and that are required both during embryogenesis and seedling growth may only express a fully penetrant mutant phenotype beginning at the seedling stage.
It is intriguing that the three noncell autonomous mutants identified in this analysis affect the same biosynthetic pathway. Although the size of our mutant screen could be far from saturation, it is difficult to believe that the isolation of three different noncell autonomous mutants in the MEP pathway might result from a random chance. One explanation is that few noncell autonomous biosynthetic pathways render true albino phenotypes in Arabidopsis. Interestingly, similar results have been also obtained in a large-scale genetic screen to identify seedling lethal mutants (Budziszewski et al., 2001
Although the seedling phenotype of all six clb mutants is similar, the mutants can be grouped by severity when plastid morphology and marker gene expression are taken into consideration. clb5 and clb2 show the most severe phenotypes, as their plastids lack appressed internal membranes and resemble proplastids. Both mutants had undetectable or nearly undetectable levels of the photosynthetic genes RBCS and psbA. While clb5 also had low or undetectable expression of early-expressed plastid genes related to transcription and translation, clb2 was less severe in showing significant levels of rrn16S and accD transcripts. clb3 plastids are similar to those of clb5 and clb2, in that they lack appressed internal membranes, and have large electron translucent cavities. However, clb3 differs in that photosynthetic genes show detectable expression, and early plastid genes are expressed at about 50% of wild-type levels. clb1 mutants have a similar gene expression profile to clb3, but clb1 plastids show a less severe phenotype in that some partially appressed internal membranes are visible. Therefore, with respect to chloroplast development, the phenotype of clb1 and clb3 can be considered intermediate among the clb mutants. Finally, clb4 and clb6 mutants have the highest marker gene expression, with early genes expressed slightly less than in wild type, and photosynthetic genes expressed at about 20% of wild-type levels. Plastids of clb6 show short, appressed internal membranes, while clb4 plastids have long single membranes. Therefore, the mutants clb1, clb2, clb3, and clb5 show more severe albino phenotypes than clb4 and clb6. Based on these phenotypic observations and on the fact that clb4 and clb6 carry null mutations in their respective genes, we can conclude that the cell autonomous genes CLB1, CLB2, CLB3, and CLB5 are required earlier in chloroplast biogenesis than the noncell autonomous genes CLB4 and CLB6. Due to their cell autonomous nature, these genes are more likely to be required for processes such as plastid transcription or translation, or the biogenesis of thylakoid membranes. The systematic isolation and analysis of mutants of Arabidopsis that affect early chloroplast differentiation represents an important step forward in studies of chloroplast biogenesis. The extreme phenotype and early arrest of chloroplast differentiation in cell autonomous clb mutants suggests that one or more of these CLB genes may be involved in the early signaling that initiates the chloroplast differentiation process in plastids. Cloning and characterization of these cell autonomous CLB genes, a current focus of research in our laboratory, will begin to allow the determination of their precise molecular role in chloroplast biogenesis, and should contribute to the overall understanding of this fascinating area of biology.
Plant Material and Growth Conditions Arabidopsis L. Heyhn. ecotypes Landsberg erecta (Ler), Columbia (Col), Wassilewskija (WS), C24, and Dijon were used in this study. For experiments involving plants grown under sterile conditions, seeds were surface-sterilized and plated on GM containing 1x Murashige and Skoog basal salts (Gibco BRL, Grand Island, NY), 2% (w/v) Suc, 1x B5 vitamin solution (Gamborg's, Sigma, St. Louis ), 0.05% (w/v) MES [2-(N-morpholino) ethanesulfonic acid], solidified with 0.8% (w/v) phytoagar. Adult plants were grown in Metro-Mix 200 (Grace Sierra, Milpitas, CA) under 16 h light:8 h dark at 24°C. Seedlings were grown under 16 h light:8 h dark cycle at 120 µE for high light conditions or at 5µE for low light conditions, at 22°C in growth chambers. Seeds were incubated at 4°C for 5 d to break dormancy prior to germination. Pigmentation mutant lines CS27, CS213, CS214, CS215, CS2751, CS2771, CS2790, CS2791, CS2793, CS2794, CS2795, CS2797, CS2798, CS2800, CS2801, CS2802, CS2804, CS2805, CS2806, CS2807, CS2808, and CS2809, T-DNA pools, and SALK insertion line 017595 were obtained from the Arabidopsis Biological Resource Center (ABRC, http://www.biosci.ohio-state.edu/plantbio/Facilities/abrc/abrchome.htm). EMS mutagenized seed, fast neutron mutagenized lines, and some T-DNA pools were generously provided by Chris Somerville (Carnegie Institute of Washington, Stanford, CA).
For the analysis of mutant embryos during development, each clb mutant line was crossed to the homozygous green fluorescent protein marker line 29-1 (Ler ecotype). Plants heterozygous for the corresponding clb mutation and homozygous for the 29-1 transgene were selected in the F2 generation, and used for confocal analysis. Seed expressing plasma membrane-localized GFP (line 29-1) were obtained from Sean Cutler, Joel Griffits, and David Ehrhardt (Carnegie Institution of Washington, Stanford, CA; Cutler et al., 2000
Complementation analysis was done by crossing heterozygous mutant plants in all possible combinations and scoring resulting F1 embryos for the mutant phenotype. Genetic mapping was performed according to Lukowitz et al. (2000)
Total carotenoids and chlorophylls were determined according to Lichtenthaler and Wellburn (1983)
Total RNA was prepared from 18-d-old seedlings grown on GM medium plates by extraction with Trizol Reagent (Sigma). 15 µg of total RNA was fractionated by electrophoresis in 1.2% (w/v) agarose gel and transferred onto Gene Screen nylon membrane (New Life Science Products, Boston). Hybridization was done with PSE buffer (1 M sodium phosphate buffer, pH 7.2, plus 10% sodium dodecyl sulfate), and washes were done under high stringency conditions according to standard procedures (Sambrook et al., 1989 For sequencing of the ISPG gene (At5g60600) from the clb4-1 allele, the entire At5g60600 gene was amplified from genomic DNA isolated from homozygous clb4-1 seedlings, and directly sequenced using the ABI Big Dye Terminator 2.0 cycle sequencing kit. Samples were analyzed on an ABI 377 sequencer. The position of the T-DNA insertion site into the At5g60600 gene in the clb4-2 allele was verified by amplifying and sequencing the genomic T-DNA border.
Total protein samples were obtained from frozen tissue ground in liquid nitrogen and thawed in SDS sample buffer (0.125 M Tris-Cl pH 6.8, 20% v/v glycerol, 4% w/v SDS, 2% v/v 2-mercaptoethanol). The protein concentration was determined with Bradford reagent (Bio-Rad, Hercules, CA) using BSA as a standard, and then separated by SDS-PAGE (SDS-PAGE). To verify equal protein loading, a parallel gel was run and stained with Coomassie Brilliant Blue R-250. The proteins were transferred onto nitrocellulose (Hybond C, Amersham) by electroblotting for 1 h at 200 mA in 25 mM Tris, 0.2 M Gly, and 20% (w/v) methanol. Immunodetection was performed using a 1:1000 dilution of a polyclonal antibody against DXS1 (Estévez et al., 2000
For confocal microscopy, late stage embryos were dissected from heterozygous plants, mounted in a solution of 300 mm mannitol on large coverslips, and imaged on a Nikon inverted fluorescence microscope equipped with a 20x Nikon objective (Tokyo) and a Bio-Rad MRC 1024 confocal head. Confocal reconstructions were made from approximately 1 µm optical sections using the public domain NIH Image program (developed at the United States National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). Digital images of seedlings were obtained with a Sony DSC-F707 digital camera using a Wild M3Z dissecting microscope. Transmission electron micrographs were obtained exactly as described in Mandel et al. (1996)
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AF434673, At5g60600, At1g29930, and NC 002202.
Many thanks to Chris Somerville for providing laboratory and greenhouse space, reagents, and support during the course of this work. Thanks to Drs. Shinjiro Yamaguchi, Hiroyuki Kasahara, and Yuji Kamiya, from the Plant Science Research Center RIKEN, for providing the antibodies against the HDS protein. Thanks to Rogene Gillmor for expert assistance with mapping, Carolina San Roman for her assistance in protein analysis, Araceli Cantero for plant maintenance, and Daniel Grimanelli and Olivier Leblanc for support during the preparation of this manuscript. We gratefully acknowledge the Salk T-DNA insertion project for the production of sequenced T-DNA insertion lines and the Arabidopsis Biological Resource Center for providing seeds. Received November 30, 2003; returned for revision February 3, 2004; accepted February 11, 2004.
1 This work was supported by CONACYT (31791–N), DGAPA IN210200, BASF, and Howard Hughes grants. M.G.-N. was supported by fellowships from CONACYT, SNI, the Wood-Whelan Research Foundation, and by a UNESCO Short-Term Biotechnology Fellowship. C.S.G. was partially supported by a U.S. Department of Energy/National Science Foundation/U.S. Department of Agriculture tri-agency training grant, and by a grant from the U.S. Department of Energy (DE–FG02–97ER20133) to C.R. Somerville (Carnegie Institution of Washington, Department of Plant Biology). 
2 These authors contributed equally to the paper.
3 Present address: Applied Biotechnology Center, CIMMYT (International Maize and Wheat Improvement Center), Apartado 6–641, Mexico D.F. 06600, Mexico. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.036996. * Corresponding author; e-mail patricia{at}ibt.unam.mx; fax 52–73–139988.
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