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First published online April 25, 2008; 10.1104/pp.108.120014 Plant Physiology 147:650-660 (2008) © 2008 American Society of Plant Biologists
pur4 Mutations Are Lethal to the Male, But Not the Female, Gametophyte and Affect Sporophyte Development in Arabidopsis[C],[W]Station de Génétique et d'Amélioration des Plantes, Institut Jean-Pierre Bourgin, INRA UR254, 78026 Versailles cedex, France
Purine metabolism is crucial in living cells and involves three complex pathways in plants: the de novo synthesis, the salvage, and the degradation pathways. The relative importance of each pathway in plant development and reproduction, however, is still unclear. We identified two T-DNA insertions in the Arabidopsis (Arabidopsis thaliana) PUR4 gene (At1g74260) that encodes formylglycinamidine ribonucleotide synthase (EC 6.3.5.3), the fourth enzyme in the de novo purine biosynthesis pathway. The mutated alleles were never transmitted through the pollen of heterozygous plants but could be inherited through the female gametophyte, indicating that de novo purine synthesis is specifically necessary for pollen development. Because the pur4 mutations were lethal to the male gametophyte, homozygous pur4 plants could not be obtained. However, the reproductive phenotype of hetererozygous plants carrying the pur4-2 mutated allele was more severe than that carrying the pur4-1 mutated allele, and pur4-2/+ plants showed slightly delayed early development. We showed that the pur4-2 allele produces an antisense transcript and that the amount of PUR4 mRNA is reduced in these plants. Transient expression of a translational fusion with the green fluorescent protein in Arabidopsis plantlets showed that the formylglycinamidine ribonucleotide synthase protein is dually targeted to chloroplast and mitochondria, suggesting that at least some steps of the de novo purine biosynthesis pathway can take place in both organelles in Arabidopsis, a dual location previously thought to be a peculiarity of ureide-forming tropical legumes.
Purine nucleotides are major metabolites in living cells, at the crossroads of energy metabolism and the synthesis of essential cofactors and nucleic acids. In plants, purine molecules are also precursors for cytokinin metabolism and for the production of secondary metabolites such as ureides and alkaloids. Purine metabolism is complex, comprising a de novo synthesis pathway, which produces IMP, the direct precursor for AMP and GMP, from simple molecules; a salvage pathway allowing the recycling of purine rings at lower energetic cost; and a degradation pathway, which plays a central role in nitrogen metabolism (for review, see Moffatt and Ashihara, 2002  ; Stasolla et al., 2003; Zrenner et al., 2006).
De novo purine synthesis is thought to be crucial in dividing cells, because of the need for nucleotides in genome replication. However, in plants, high activity of salvage enzymes was reported in the cell proliferation phase of somatic embryogenesis (Stasolla et al., 2003
The genes encoding the enzymes that catalyze the first 10 steps of the purine de novo synthesis pathway have been identified in the Arabidopsis (Arabidopsis thaliana) genome (Boldt and Zrenner, 2003
In plants, seven of the steps involve monofunctional enzymes, as in prokaryotes, whereas other eukaryotes have additional bifunctional enzymes (Smith and Atkins, 2002
In Arabidopsis, insertional and ethyl methanesulfonate mutations for ATase2 and an insertional mutation for ATase1 have been described, and in tobacco (Nicotiana tabacum), transgenic plants with reduced ATase were generated by expression of an antisense construct (Hung et al., 2004 Here, we report the characterization of two insertional mutations in the single copy gene (PUR4) encoding formylglycinamidine ribonucleotide synthase (FGAMS; EC 6.3.5.3), the fourth enzyme of the de novo purine biosynthesis pathway in Arabidopsis. This study provides strong evidence that de novo purine synthesis is crucial for male, but not female, gametophyte development. In addition, due to the suppression of PUR4 expression by one of the mutations, the effect of impaired de novo purine synthesis could also be observed in the sporophyte. Furthermore, we show that Arabidopsis FGAMS is dually targeted to mitochondria and plastids, indicating that the de novo purine synthesis pathway, or at least a part of it, can be dually located in a nonlegume plant.
In a study aimed at identifying mutations that affect genes encoding proteins targeted to mitochondria or plastids and impaired in sexual reproduction (Berthomé et al., 2003 ), we identified two mutations in the PUR4 gene (At1g74260). pur4-1, in the Columbia (Col0) genotype, was obtained from the Joe Ecker T-DNA insertional mutant collection (Salk Institute; SALK 050980); pur4-2 was previously identified in the Wassilewskija (Ws) genotype by a screen designed to isolate gametophytic lethal mutations (Bonhomme et al., 1998). We determined the complete sequence of the PUR4 mRNA by sequencing overlapping amplification products from the cDNA and performing 3' and 5' RACE experiments. We detected a previously unreported intron in the 5' untranslated region (UTR) of the gene and determined the 5' and 3' ends of the mRNA. The resulting sequence is aligned with the genomic sequence in Supplemental Figure S1. The PUR4 gene structure is presented in Figure 1 , showing the positions of the T-DNA insertions in the two mutant alleles. We analyzed both mutations by DNA hybridization analyses and sequencing (Supplemental Fig. S2). The pur4-1 T-DNA insertion is complex, but its 5' flanking sequence was confirmed by sequencing. The pur4-2 T-DNA insertion is simple and was confirmed by sequencing the 5' and 3' flanking regions. In both cases, DNA hybridization showed that no other copy of the T-DNA was present in the plants carrying the mutated alleles, since the nptII probe on EcoRI digestions (only one site in each T-DNA) gave only one hybridization signal for both insertions.
Genetic Analyses of the pur4-1 and pur4-2 Mutations The selfed progeny of hemizygous plants from both T-DNA lines was screened for the T-DNA insertion, either on kanamycin-containing medium or by direct PCR genotyping. Plants homozygous for the pur4 mutations were never recovered for either allele. The transmission efficiency of both mutations in selfed plants is shown in Figure 2 (see Supplemental Tables S1 and S2 for detailed results). The transmission rate of both mutant alleles was dramatically lower than expected for a Mendelian trait. Two types of mutations result in lower transmission rates in selfed progeny and the absence of homozygous mutants: (1) embryo-lethal mutations, which are transmitted to two-thirds of the viable progeny because of the loss of homozygous mutants; and (2) gametophyte-lethal mutations, which are transmitted to only half of the progeny because of the loss of either the male or the female gametes carrying the mutation. If the mutation is lethal for both gametes, it is not transmitted to offspring and is completely lost.
We found that the pur4 mutations were transmitted even less efficiently than expected for a gametophyte-lethal mutation and that the pur4-2 allele was transmitted less efficiently than the pur4-1 allele. These results suggest that at least one of the two gametophytes is unable to transmit the mutated pur4 alleles. In order to determine the cause of this transmission deficiency more precisely, we made reciprocal crosses with wild-type plants (Table I ). When the pur4/+ plants were used as pollen donors, none of the resulting progeny carried either of the pur4 mutations, showing that the male gametophyte could not transmit the pur4 mutations. We conclude that the pur4 mutations are male gametophyte lethal. Following crosses using the pur4/+ plants as the female parent, the two mutations were recovered, although often in less than half of the progeny (Table I). Therefore, we suspect that the pur4 mutations also have an effect on the female gametophyte.
Overall, the transmission of the pur4 mutant alleles was analyzed in progeny from 36 pur4-1/+ and 71 pur4-2/+ individual plants.
Although all were heterozygous, the kanamycin-resistant plantlets carrying the pur4-2 mutation showed delayed germination and early development, regardless of whether they resulted from selfing or outcrossing or were produced by pur4-2/+ plants themselves issued from selfing or backcrossing. In addition, the germination efficiency of seeds from pur4-2/+ plants was extremely variable (25%–100%) and often quite poor, whether produced by selfing or outcrossing or from plants themselves originating from selfing or backcrossing (data not shown). Sometimes, a few pur4-2/+ plantlets appeared to be chlorotic (Fig. 3A ). The proportion (0%–2%) of these pale plants in (selfed or backcrossed) progeny was variable between individual progenies. When transferred to the greenhouse, pale plants grew slowly but finally reached the reproductive stage and set seeds at roughly the same time as wild-type siblings (Fig. 3B). All of the plants carrying the pur4-1 mutation were indistinguishable from wild-type plants (Fig. 3A).
We observed that pur4-2/+ plants, issued either from selfing or backcrossing, had smaller anthers and produced less pollen than pur4-1/+ or wild-type plants (Fig. 3C). We Alexander stained and examined pollen in mature anthers of pur4-1/+ and pur4-2/+ plants issued from backcrosses (Fig. 3C) and scored viable (red-stained) or aborted (green-stained) pollen grains (Fig. 3D). In both wild-type controls, no aborted pollen was observed. In pur4-1/+ plants, however, half of the pollen was aborted, and because the mutation is not transmitted through pollen, we inferred that the dead pollen carries the pur4-1 mutation. In pur4-2/+ anthers, more than half of the pollen had aborted. We concluded that some of the pollen grains carrying the wild-type PUR4 allele also died in the pur4-2/+ background. We carried out further cytological observations on anthers from pur4-1/+ and pur4-2/+ plants issued from backcrosses in order to define the precise stage at which pollen development is impaired (Fig. 4 ). In both heterozygous genotypes, pollen development was indistinguishable from that of the wild type until the vacuolated microspore stage (buds with a 1-mm pistil; data not shown). In buds with a 1.25-mm pistil, corresponding to pollen mitosis I (PMI) or soon after, we observed poorly stained cytoplasm and a remaining large vacuole in a large proportion (roughly half) of pollen grains in both heterozygous genotypes (Fig. 4A). In pur4-1/+ and pur4-2/+ buds with a 1.5-mm pistil, corresponding to pollen mitosis II (PMII) or soon after, about half of the pollen grains had almost completely collapsed. In addition, in pur4-2/+ anthers, some of the remaining normally shaped pollen grains also showed poorly stained cytoplasm (arrows in Fig. 4B). Again, the proportion of these poorly stained pollen grains was variable among pur4-2/+ plants. Next, we stained mature pollen with 4',6-diamino-phenylindole (DAPI) and found that some of the nonaborted pollen grains in pur4-2/+ anthers had abnormal nuclei numbers, suggesting impaired pollen mitoses (Fig. 4C).
Finally, we scored the number of fully developed seeds and the number of empty spaces (holes) in mature siliques of plants issued from backcrosses. The results are shown in Fig. 5 . Both heterozygous mutants showed lower seed set than their wild-type siblings. Again, the phenotype was more severe for the pur4-2 allele.
The pur4-2 Mutation Leads to the Suppression of PUR4 mRNA We analyzed the expression of the PUR4 gene in pur4-2/+ plants issued from selfing compared with wild-type plants by performing quantitative reverse transcription (RT)-PCR analyses on total RNA from different organs (data not shown). In all organs, the PUR4 mRNA was accumulated at less than 20% of the wild-type level in pur4-2/+ plants. We repeated quantitative RT-PCR analysis on plantlets issued from the backcross. Results of the expression level of PUR4 in several individual plants are shown in Figure 6A . We observed that pur-4/+ plantlets accumulated between 15% and 30% of the amount of PUR4 mRNA found in their wild-type siblings. Seedlings carrying the pur4-1 mutation accumulated approximately 50% of the wild-type level of PUR4 mRNA (data not shown).
The low level of PUR4 mRNA in pur4-2/+ plants suggests that the accumulation of the PUR4 transcript is affected by a suppression mechanism in these plants. A possible explanation could be that an aberrant RNA is produced from the mutated allele, leading to a decrease of the PUR4 transcript. To test this hypothesis, we performed RT-PCR experiments with primers allowing the detection of sense and antisense transcripts in plants issued from the backcross. The results are shown in Figure 6, B and C. The sense transcript originating from the PUR4 allele was detected in both pur4-2/+ and wild-type plants (Fig. 6B). Oppositely, we detected an antisense transcript only in pur4-2/+ plants (Fig. 6C). A partial sequence of the pur4-2-specific antisense RNA was obtained by sequencing several RT-PCR products. Alignment of the resulting sequences with the PUR4 gene and the pGKB5 T-DNA sequences is shown in Supplemental Figure S1. We conclude that the pur4-2 allele produces an antisense chimeric transcript, overlapping the PUR4 and T-DNA sequences, which may interfere with PUR4 mRNA accumulation.
The enzymes for de novo purine synthesis are predicted to be targeted to plastids, and the Arabidopsis ATase, which catalyzes the first step of the pathway, was shown to be present in chloroplasts (Hung et al., 2004
Arabidopsis FGAMS Is Targeted to Mitochondria and Chloroplasts
Our results strongly suggest that the N-terminal portion of the Arabidopsis PUR4 protein, most probably containing targeting sequences, directed GFP to mitochondria as well as chloroplasts. Until now, the de novo purine synthesis pathway was thought to occur in plastids, with the exception of the nodules of ureide-forming legumes, in which the pathway was detected in both mitochondria and plastids (Atkins et al., 1997
We characterized two insertional mutations in the single copy gene encoding the enzyme that catalyzes the fourth step of de novo purine synthesis in Arabidopsis. From our results, it appears that the pur4-1 allele is most likely a null mutation. First, the insertion is in the second protein-encoding exon; second, it appears to be associated with a partial deletion of this exon, together with a rearrangement of a small part of the gene (Supplemental Fig. S2). We showed that the pur4-2 allele produces an antisense RNA that overlaps the T-DNA and the PUR4 gene and is spliced at abnormal positions (Fig. 6; Supplemental Fig. S1). Abnormal splicing of the pur4-2 transcript is consistent with its antisense direction. Due to the presence of the antisense RNA from the pur4-2 allele, it is likely that the decrease in PUR4 mRNA observed in pur4-2/+ heterozygous plants (Fig. 6) is caused by a suppression mechanism.
We observed no pollen transmission of either pur4 mutation in the progeny of more than 100 heterozygous plants issued either from selfing or backcrossing. These results show unambiguously that pur4 null alleles are male gametophyte lethal. In addition, staining of mature pollen showed that at least half of the pollen from pur4-1/+ and pur4-2/+ plants aborted (Fig. 3, C and D). These results show that FGAMS activity is essential for proper pollen development. FGAMS, therefore, is the second enzyme involved in a metabolic pathway that is necessary for male gametophyte development. Recently, Ser palmitoyltransferase, an enzyme involved in the de novo synthesis of sphingolipids, was shown to be essential for pollen development (Teng et al., 2008
Our results tend to indicate that de novo purine biosynthesis is necessary for the formation of normal, viable pollen grains. The only other step of de novo purine biosynthesis for which mutants have been described is the first step of the pathway, catalyzed by ATase. Arabidopsis plants carrying mutations in the two genes encoding ATase were reported to have delayed growth but no pollen abnormalities (Hung et al., 2004
Interestingly, both pur4 mutations are successfully transmitted through the ovules, ensuring that the mutant alleles were not directly lost after the first generation. However, for both mutants, the efficiency of transmission through the female gametophyte was sometimes lower than for the wild-type allele (Table I), and heterozygous pur4/+ plants produce fewer seeds than their wild-type counterparts, regardless of whether they are issued from selfing or backcrossing (Fig. 5). These results suggest that the pur4 mutations also affect the viability of the female gametophyte, but with lower penetrance than for the male gametophyte. Therefore, we infer that FGAM synthesis is less limiting during female than male gametophyte development. It is unlikely that embryo sac development requires fewer purines than pollen development; for example, it involves three successive divisions, whereas male gametophyte development involves only two divisions. Consequently, we propose that the female gametophyte benefits from purine inputs from surrounding cells, conferring tolerance to purine synthesis deficiency. Purines could be supplied into the developing embryo sac from the three other meiosis products, ensuring recycling of their contents, perhaps through the purine salvage pathway, to the advantage of the cells entering gametophytic development, before their collapse. Alternatively, sporophytic cells, which tightly surround the female gametophyte throughout development, could be a direct source of purines. Another possibility, which cannot be excluded at the moment, is that FGAM accumulates in the megaspore, reducing the need for FGAM synthesis during embryo sac development.
Gametophyte-lethal mutations can be of two types, as pointed out by Feldmann et al. (1997) We observed that plants carrying the pur4-2 mutation accumulated lower levels of PUR4 mRNA than wild-type or pur4-1/+ plants (Fig. 6A; data not shown). The detection of an antisense RNA transcript originating from the pur4-2 allele provides a possible explanation for this observation (Fig. 6B). The sequence analysis of this antisense RNA shows that it results from a transcriptional fusion of the PUR4 gene and the T-DNA and displays aberrant splicing (Supplemental Fig. S1), indicating that its transcription starts in the T-DNA sequence. Its presence, therefore, could lead to the partial degradation of PUR4 mRNA, resulting in lower expression of the gene in pur4-2/+ plants.
The metabolic function of the PUR4 product implies that its depletion in sporophytic tissues would have severe consequences, at least in parts of the plant and/or at time points in development where large amounts of purine nucleotides are needed. However, the purine synthesis salvage pathway can provide cells with purine nucleotides and is activated in important phases of development, such as embryo formation and seed germination (Stasolla et al., 2003
Interestingly, all pur4-2/+ plants, originating from selfing or backcrosses, displayed a more severe pollen phenotype than pur4-1/+ plants (Fig. 3, C and D). Indeed, pur4-2/+ plants have smaller anthers and produce less pollen than wild-type and pur4-1/+ plants. This suggests that although the vegetative phenotype of most of these plants was nearly normal, the amount of remaining PUR4 mRNA in sporophytic tissues of reproductive organs is limiting for pollen development. Analysis of PUR4 mRNA accumulation in anther tissues would be necessary to test this hypothesis. In addition, our cytological analysis suggests that, in pur4-2/+ anthers, some wild-type pollen grains aborted at a later stage than pur4 pollen (Figs. 3, C and D, and 4). DAPI staining of pollen in pur4-2/+ plants suggested that PMII (and maybe PMI) was affected in some pollen of these plants (Fig. 4C). Considering our findings, it is likely that, although pollen development needs active de novo purine synthesis in the gametophyte, it is also dependent on the purine synthesis pathway in the surrounding sporophyte. Therefore, our results, together with those obtained regarding the aprt mutant (Gaillard et al., 1998 Altogether, our results demonstrate that de novo purine synthesis, and particularly the fourth step, catalyzed by FGAMS, is crucial for plant reproduction, particularly in male gametophyte development, but probably also in the sporophytic tissues sustaining pollen and embryo sac developments. Together with other reports, this suggests that both salvage and de novo synthesis pathways play an important role in the anther during pollen development. Further studies are needed to decipher their respective roles and to what extent reciprocal compensation occurs between the two pathways.
Plant Material and Growth Conditions
Seeds of Arabidopsis (Arabidopsis thaliana) ecotype Col0 carrying the pur4-1 mutation (SALK 050980) and seeds of Arabidopsis ecotype Ws carrying the pur4-2 mutation (FST 064G02) were obtained from the Salk Institute collection (Alonso et al., 2003
Seeds grown in vitro for genetic analyses and for kanamycin resistance assays were surface sterilized for 10 min with a solution containing 10% (v/v) commercial bleach (Minichlor; Hygiena) diluted in 95°C ethyl alcohol and 0.05% (v/v) Teepol 610 (Serva). Seeds were rinsed thoroughly with 95°C ethyl alcohol and dried overnight in a sterile hood. Seeds were sown on Arabidopsis medium (Estelle and Somerville, 1987
Primers used in this study are shown in Supplemental Table S3, and the positions of those targeted to the PUR4 gene are shown in Figure 1.
Extraction of Nucleic Acids
PCR Genotyping
Sequencing of the Transcripts To detect the antisense mRNA PCR product, a first PCR was run with 5 µL of first-strand cDNA obtained as above from pur4-2/+ plants using primers 28 and 33. The extension of the antisense mRNA into the T-DNA was demonstrated using primers 27 and 3 (targeting the pGKB5 left border). The sequences of both RT-PCR products were assembled to give the partial sequence given in Supplemental Figure S1. For both RNAs, each PCR product was purified using the NucleoTrap kit (Macherey-Nagel) and cloned using the TOPO TA cloning kit (Invitrogen). For each PCR product, four independent clones were sequenced.
Real-time RT-PCR was carried out using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). A specific primer set (21/22) was designed to amplify a 211-bp fragment on the PUR4 cDNA. A standard curve was generated from duplicate series of five DNA template dilutions to test PCR efficiencies. PCR was conducted in duplicate in the presence of 1 ng of cDNA, 1.2 µL of each primer (2.5 µM), 5 µL of SYBR Green Mastermix, and distilled water to a final volume of 10 µL. PCR conditions were as described above, with 10 min at 95°C and 40 cycles at 95°C for 10 s and 60°C for 10 s. The results were standardized by comparing the data obtained for the ACTIN2 gene (primer set 23/24). The quantification of gene expression was performed using the comparative cycle threshold number method.
The subcellular localization of the PUR4 protein was predicted in silico using four different software programs: TargetP version 1.1 (Emanuelsson et al., 2000
All cytological analyses were performed on pur4/+ plants issued from backcrosses.
Pollen viability was evaluated after Alexander staining (Alexander, 1969
Toluidine blue O staining was carried out as described by Trump et al. (1961)
DAPI staining of mature pollen was done according to Coleman and Goff (1985) Samples were visualized with a Leitz DIAPLAN microscope under UV light for DAPI staining and in bright-field conditions for toluidine blue O and Alexander staining. Photographs were captured with a Leica DFC 480 camera. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number EU091297.
The following materials are available in the online version of this article.
We gratefully thank S. Bonhomme for a thorough critical reading of the manuscript and advice on gametophytic mutants. We thank M. Laloue and C. Mézard for stimulating discussions. We are grateful to S. Bonhomme, C. Horlow, and M. Grelon for generously providing information from their previous screen. The generous gift of the FDH-DsRed2 fusion by C. Colas des Francs-Small is gratefully acknowledged. We thank A. Martin-Canadell, who took care of the plants in the greenhouse. We are grateful to O. Grandjean for his valuable help in confocal microscopy. The constant support of P. Guerche, head of the laboratory, is gratefully appreciated. Received March 28, 2008; accepted April 22, 2008; published April 25, 2008.
1 Present address: INRA-UMRGV, 2 Rue Gaston Crémieux, CP 5708, 91057 Evry, France. 
2 Present address: Unité de Recherche Amélioration, Génétique et Physiologie Forestières, INRA, Avenue de la Pomme de Pin, Ardon-BP 20619, 45166 Olivet cedex, France.
3 Present address: Laboratoire de Reproduction et du Développement des Plantes, Ecole Normale Supérieure de Lyon, UMR 5667, INRA, ENS Lyon UCB Lyon, 46 allée d'Italie, 69364 Lyon cedex 07, France. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Richard Berthomé (berthome{at}evry.inra.fr).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.108.120014 * Corresponding author; e-mail budar{at}versailles.inra.fr.
Alexander MP (1969) Differential staining of aborted and non aborted pollen. Stain Technol 44: 117–122[Web of Science][Medline] Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al (2003) Genome-wide insertional mutagenesis of Arabidopsis. Science 301: 653–657 Ambard-Bretteville F, Small I, Grandjean O, Colas des Francs-Small C (2003) Discrete mutations in the presequence of potato formate dehydrogenase inhibit the in vivo targeting of GFP fusions into mitochondria. Biochem Biophys Res Commun 311: 966–971[CrossRef][Web of Science][Medline] Atkins CA, Smith P, Storer PJ (1997) Reexamination of the intracellular localization of de novo purine synthesis in cowpea nodules. Plant Physiol 113: 127–135[Abstract] Bannai H, Tamada Y, Maruyama O, Nakai K, Miyano S (2002) Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18: 298–305 Bechtold N, Ellis J, Pelletier G (1993) In planta, Agrobacterium mediated gene transfer by integration of adult Arabidopsis plants. C R Acad Sci Ser III Sci Vie 316: 1194–1199 Berthomé R, Froger N, Hiard S, Balasse H, Martin-Canadell A, Budar F (2003) The involvement of organelles in plant sexual reproduction: a post-genomic approach. Acta Biologica Cracoviensia Series Botanica 45: 119–124[Web of Science] Boldt R, Zrenner R (2003) Purine and pyrimidine biosynthesis in higher plants. Physiol Plant 117: 297–304[CrossRef][Medline] Bonhomme S, Horlow C, Vezon D, de Laissardiere S, Guyon A, Ferault M, Marchand M, Bechtold N, Pelletier G (1998) T-DNA mediated disruption of essential gametophytic genes in Arabidopsis is unexpectedly rare and cannot be inferred from segregation distortion alone. Mol Gen Genet 260: 444–452[CrossRef][Web of Science][Medline] Bouchez D, Vittorioso P, Courtila B, Camilleri C (1996) Kanamycin rescue: a simple technique for the recovery of T-DNA flanking sequences. Plant Mol Biol 14: 115–123[CrossRef][Web of Science] Claros MG, Vincens P (1996) Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem 241: 779–786[Web of Science][Medline] Coleman AW, Goff LJ (1985) Applications of fluorochromes to pollen biology. 1. Mithramycin and 4',6-diamidino-2-phenylindole (DAPI) as vital stains and for quantification of nuclear DNA. Stain Technol 60: 145–154[Web of Science][Medline] Duchene AM, Giritch A, Hoffmann B, Cognat V, Lancelin D, Peeters NM, Zaepfel M, Marechal-Drouard L, Small ID (2005) Dual targeting is the rule for organellar aminoacyl-tRNA synthetases in Arabidopsis. Proc Natl Acad Sci USA 102: 16484–16489 Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19: 1349 Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300: 1005–1016[CrossRef][Web of Science][Medline] Estelle MA, Somerville CR (1987) Auxin-resistant mutants of Arabidopsis with an altered morphology. Mol Gen Genet 206: 200–206[CrossRef][Web of Science] Feldmann KA, Coury DA, Christianson ML (1997) Exceptional segregation of a selectable marker (KanR) in Arabidopsis identifies genes important for gametophytic growth and development. Genetics 147: 1411–1422[Abstract] Gaillard C, Moffatt BA, Blacker M, Laloue M (1998) Male sterility associated with APRT deficiency in Arabidopsis results from a mutation in the gene APT1. Mol Gen Genet 257: 348–353[CrossRef][Web of Science][Medline] Golovkin M, Reddy AS (2003) Expression of U1 small nuclear ribonucleoprotein 70K antisense transcript using APETALA3 promoter suppresses the development of sepals and petals. Plant Physiol 132: 1884–1891 Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42: 819–832[CrossRef][Web of Science][Medline] Hung WF, Chen LJ, Boldt R, Sun CW, Li HM (2004) Characterization of Arabidopsis glutamine phosphoribosyl pyrophosphate amidotransferase-deficient mutants. Plant Physiol 135: 1314–1323 Ito J, Heazlewood JL, Millar AH (2006) Analysis of the soluble ATP-binding proteome of plant mitochondria identifies new proteins and nucleotide triphosphate interactions within the matrix. J Proteome Res 5: 3459–3469[CrossRef][Web of Science][Medline] Jiang L, Yang SL, Xie LF, Puah CS, Zhang XQ, Yang WC, Sundaresan V, Ye D (2005) VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract. Plant Cell 17: 584–596 Karniely S, Pines O (2005) Single translation-dual destination: mechanisms of dual protein targeting in eukaryotes. EMBO Rep 6: 420–425[CrossRef][Web of Science][Medline] Kleffmann T, Russenberger D, von Zychlinski A, Christopher W, Sjolander K, Gruissem W, Baginsky S (2004) The Arabidopsis chloroplast proteome reveals pathway abundance and novel protein functions. Curr Biol 14: 354–362[CrossRef][Web of Science][Medline] Koncz C, Kreuzaler F, Kalman Z, Schell J (1984) A simple method to transfer, integrate and study expression of foreign genes, such as chicken ovalbumin and alpha-actin in plant tumors. EMBO J 3: 1029–1037[Web of Science][Medline] Li H, Shen JJ, Zheng ZL, Lin Y, Yang Z (2001) The Rop GTPase switch controls multiple developmental processes in Arabidopsis. Plant Physiol 126: 670–684 Lurin C, Andres C, Aubourg S, Bellaoui M, Bitton F, Bruyere C, Caboche M, Debast C, Gualberto J, Hoffmann B, et al (2004) Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16: 2089–2103 Marion J, Bach L, Bellec Y, Meyer C, Gissot L, Faure J-D (2008) Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J (in press) Millar AH, Whelan J, Small I (2006) Recent surprises in protein targeting to mitochondria and plastids. Curr Opin Plant Biol 9: 610–615[CrossRef][Web of Science][Medline] Moffatt BA, Ashihara H (2002) Purine and pyrimidine nucleotide synthesis and metabolism. In C Somerville, E Meyerowitz, eds, The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD, doi/10.1199/tab.0018, www.aspb.org/publications/arabidopsis/ Niewiadomski P, Knappe S, Geimer S, Fischer K, Schulz B, Unte US, Rosso MG, Ache P, Flugge UI, Schneider A (2005) The Arabidopsis plastidic glucose 6-phosphate/phosphate translocator GPT1 is essential for pollen maturation and embryo sac development. Plant Cell 17: 760–775 Peltier JB, Cai Y, Sun Q, Zabrouskov V, Giacomelli L, Rudella A, Ytterberg AJ, Rutschow H, van Wijk KJ (2006) The oligomeric stromal proteome of Arabidopsis chloroplasts. Mol Cell Proteomics 5: 114–133 Pujol C, Marechal-Drouard L, Duchene AM (2007) How can organellar protein N-terminal sequences be dual targeting signals? In silico analysis and mutagenesis approach. J Mol Biol 369: 356–367[CrossRef][Web of Science][Medline] Robertson WR, Clark K, Young JC, Sussman MR (2004) An Arabidopsis plasma membrane proton pump is essential for pollen development. Genetics 168: 1677–1687 Schnorr KM, Nygaard P, Laloue M (1994) Molecular characterization of Arabidopsis cDNAs encoding three purine biosynthetic enzymes. Plant J 6: 113–121[CrossRef][Web of Science][Medline] Senecoff JF, McKinney EC, Meagher RB (1996) De novo purine synthesis in Arabidopsis. II. The PUR7 gene encoding 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole synthetase is expressed in rapidly dividing tissues. Plant Physiol 112: 905–917[Abstract] Senecoff JF, Meagher RB (1993) Isolating the Arabidopsis genes for de novo purine synthesis by suppression of Escherichia coli mutants. I. 5'-Phosphoribosyl-5-aminoimidazole synthetase. Plant Physiol 102: 387–399[Abstract] Small I, Peeters N, Legeai F, Lurin C (2004) Predotar: a tool for rapidly screening proteomes for N-terminal targeting sequences. Proteomics 4: 1581–1590[CrossRef][Web of Science][Medline] Smith PM, Atkins CA (2002) Purine biosynthesis: big in cell division, even bigger in nitrogen assimilation. Plant Physiol 128: 793–802 Stasolla C, Katahira R, Thorpe TA, Ashihara H (2003) Purine and pyrimidine nucleotide metabolism in higher plants. J Plant Physiol 160: 1271–1295[CrossRef][Web of Science][Medline] Taira M, Valtersson U, Burkhardt B, Ludwig RA (2004) Arabidopsis GLN2-encoded glutamine synthetase is dual targeted to leaf mitochondria and chloroplasts. Plant Cell 16: 2048–2058 Teng C, Dong H, Shi L, Deng Y, Mu J, Zhang J, Yang X, Zuo J (2008) Serine palmitoyltransferase, a key enzyme for de novo synthesis of sphingolipids, is essential for male gametophyte development in Arabidopsis. Plant Physiol 146: 1322–1332 Trump BF, Smuckler EA, Benditt EP (1961) A method for staining epoxy sections for light microscopy. J Ultrastruct Res 5: 343–348[CrossRef][Web of Science][Medline] Twell D, Oh S-A, Honys D (2006) Pollen development, a genetic and transcriptomic view. In R Malho, ed, The Pollen Tube, a Cellular and Molecular Perspective, Vol 3. Springer-Verlag, Berlin, pp 15–45 van der Graaff E, Hooykaas P, Lein W, Lerchl J, Kunze G, Sonnewald U, Boldt R (2004) Molecular analysis of "de novo" purine biosynthesis in solanaceous species and in Arabidopsis. Front Biosci 9: 1803–1816[CrossRef][Web of Science][Medline] von Braun SS, Sabetti A, Hanic-Joyce PJ, Gu J, Schleiff E, Joyce PB (2007) Dual targeting of the tRNA nucleotidyltransferase in plants: not just the signal. J Exp Bot 58: 4083–4093 Zrenner R, Stitt M, Sonnewald U, Boldt R (2006) Pyrimidine and purine biosynthesis and degradation in plants. Annu Rev Plant Biol 57: 805–836[CrossRef][Medline]
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