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Plant Physiology 135:1314-1323 (2004) © 2004 American Society of Plant Biologists Characterization of Arabidopsis Glutamine Phosphoribosyl Pyrophosphate Amidotransferase-Deficient Mutants1Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan (W.-F.H.); Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (W.-F.H., L.-J.C., C.-W.S., H.-m.L.); and Department of Plant Physiology, University of Rostock, 18051 Rostock, Germany (R.B.)
Using a transgene-based screening, we previously isolated several Arabidopsis mutants defective in protein import into chloroplasts. Positional cloning of one of the loci, CIA1, revealed that CIA1 encodes Gln phosphoribosyl pyrophosphate amidotransferase 2 (ATase2), one of the three ATase isozymes responsible for the first committed step of de novo purine biosynthesis. The cia1 mutant had normal green cotyledons but small and albino/pale-green mosaic leaves. Adding AMP, but not cytokinin or NADH, to plant liquid cultures partially complemented the mutant phenotypes. Both ATase1 and ATase2 were localized to chloroplasts. Overexpression of ATase1 fully complemented the ATase2-deficient phenotypes. A T-DNA insertion knockout mutant of the ATase1 gene was also obtained. The mutant was indistinguishable from the wild type. A double mutant of cia1/ATase1-knockout had the same phenotype as cia1, suggesting at least partial gene redundancy between ATase1 and ATase2. Characterizations of the cia1 mutant revealed that mutant leaves had slightly smaller cell size but only half the cell number of wild-type leaves. This phenotype confirms the role of de novo purine biosynthesis in cell division. Chloroplasts isolated from the cia1 mutant imported proteins at an efficiency less than 50% that of wild-type chloroplasts. Adding ATP and GTP to isolated mutant chloroplasts could not restore the import efficiency. We conclude that de novo purine biosynthesis is not only important for cell division, but also for chloroplast biogenesis.
De novo biosynthesis of the purine ring is essential for plant growth and development. The major products, AMP and GMP, are the building blocks for DNA and RNA. AMP, when converted into ATP, is the major energy source for multiple cellular processes. Several important coenzymes, e.g. NAD and FAD, are also derived from the same pathway. In nodules of N-fixing tropical legumes, such as soybean (Glycine max) and cowpea (Vigna unguiculata), the pathway also plays a dominant role in primary nitrogen metabolism. The activity of enzymes in the purine biosynthesis pathway is enhanced considerably in nodules compared to other tissues. Therefore, most studies of purine biosynthesis in plants have used these legume nodules as materials and focused on the function of purine biosynthesis in nitrogen assimilation (for review, see Smith and Atkins, 2002  ). Studies on the role of purine biosynthesis in normal plant physiology or in non-N-fixing plants have been relatively few.
The location of purine biosynthesis within plant cells is still in dispute. The plant enzymes in the pathway are similar to those in Escherichia coli, except that each plant enzyme has an N-terminal extension that is presumed to function as an organelle-targeting signal (Smith and Atkins, 2002
The enzyme Gln phosporibosyl pyrophosphate (PRPP) amidotransferase (EC 2.4.2.14) catalyzes the first committed step of de novo purine biosynthesis, the conversion of 5-phosphoribosyl-(
Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol as higher Mr precursors with N-terminal extensions called transit peptides. Import of these proteins into chloroplasts requires transit peptides and a translocon complex in the chloroplast envelope (for review, see Schleiff and Soll, 2000
We have designed a transgene-based screening strategy to isolate Arabidopsis mutants defective in chloroplast protein import (Sun et al., 2001
The cia1 Mutant Phenotypes The cia1 mutant has normal green cotyledons but small and albino/pale-green mosaic leaves (Fig. 1, A and B). To determine the nature of the mutation, cia1 was back-crossed to the unmutagenized parental line. F1 progeny were wild type in appearance. F2 progeny segregated at a wild type to mutant ratio of 3:1 (333:114), indicating that cia1 was a single-gene recessive mutation. Another mutant was discovered in an unrelated screen and had an identical phenotype to cia1. Complementation crosses indicated that these two mutants are allelic. The original cia1 was named cia1-1 and the new mutant cia1-2. Because a back-crossed line of cia1-2 was obtained first, all experiments described in this article were performed with the cia1-2 allele. The cia1 mutant was much smaller than the wild type but was still viable and fertile even when directly germinated on soil. The leaves showed an irregular mosaic of green sectors among white portions. The mosaic pattern varied considerably, depending on growth, nutrient, and light conditions (Fig. 1B; data not shown).
Chloroplasts were isolated from cia1 and wild-type plants and used for in vitro protein import experiments. Protein import efficiency of the mutant chloroplasts was about 50% that of wild-type chloroplasts (Fig. 1C). Import of precursors to the stroma-located small subunit of Rubisco (RBCS) and thylakoid-located chlorophyll a/b-binding protein (CAB) of PSII and plastocyanin (PC) were all reduced in the mutant, indicating a general defect in protein import into chloroplasts.
The cia1 mutation was mapped to the lower arm of chromosome VI between the cleaved amplified polymorphic markers (CAPS) AG and DHS1 (Konieczny and Ausubel, 1993
ATase converts PRPP to phosphoribosylamin, which is the first committed step in de novo purine biosynthesis (Fig. 3A). Nucleotide measurements revealed that the cia1 mutant indeed had reduced levels of adenine and guanine nucleotides (Fig. 3B). The residual purine nucleotides in the mutant, and the fact that the mutant was still viable, are most likely due to the presence of the two other isozymes.
In non-N-fixing plants, de novo purine biosynthesis leads to three major groups of products: ATP/GTP, cytokinin, and NAD/NADP and coenzymes (Smith and Atkins, 2002 ). We next investigated whether the mutant phenotypes could be complemented biochemically by exogenous addition of the three groups of products. Addition of AMP to plant liquid cultures restored the chlorophyll content and partially restored the size of mutant plants (Fig. 4, A and B). AMP added to agar plate media had no effect (data not shown), suggesting that roots were unable to absorb AMP directly. The rescuing effect of AMP in liquid cultures may be due to the fact that AMP could be hydrolyzed to adenosine and adenine by phosphatases and nucleosidases, and possibly salvaged to adenine nucleotides by adenosine kinase and adenine phosphoribosyltransferase. IMP, GMP, FAD, and NADH had no effect on rescuing the mutant phenotypes even when added to plant liquid cultures (data not shown). Arabidopsis seedlings may not be able to absorb these compounds exogenously, or a deficiency in these compounds was not the major reason for the phenotypes of cia1. Low concentrations (1 and 10 nM) of synthetic cytokinin benzylaminopurine (BA) could not rescue the mutant phenotypes (Fig. 4C). At a higher concentration (100 nM), BA inhibited plant growth and made the mutant artificially resemble the wild type.
ATase2 Was Localized to the Stroma of Chloroplasts We next investigated the localization of ATase2 in Arabidopsis leaf cells. The cia1 mutant was transformed with the full-length ATase2 cDNA fused in front of five tandem repeats of cMyc tag, and the fusion construct was driven by the CaMV 35S promoter. The transformants were restored to wild-type appearance (Fig. 1D), indicating the fusion protein was functioning properly. Using the ATase2-cMyc-complemented transgenic plants, we first investigated the localization of ATase2 by fractionation. The protein detected in whole-cell extract was about 10 kD smaller than the in vitro-translated ATase2-cMyc fusion protein (Fig. 5, compare lanes 1 and 2), suggesting that cleavage of an N-terminal targeting signal had occurred within cells. When mitochondria and chloroplasts were isolated from the transgenic plants, ATase2-cMyc was detected only in chloroplasts, not in mitochondria (Fig. 5, lanes 3 and 4). Control hybridization with anti-mitochondrial porin antibodies showed that the chloroplast fraction was not contaminated by mitochondria. ATase2-cMyc fusion protein was further localized to the soluble fraction of chloroplasts (Fig. 5, lane 5), indicating the protein was localized in the stroma of chloroplasts. Control hybridization with antibodies against the stroma-located small subunit of ADP-Glc pyrophosphorylase and the inner envelope membrane-located Tic110 showed that the chloroplast membrane and soluble fractions were not contaminated with each other.
We further confirmed the fractionation data using electron microscopy and immunogold labeling (Fig. 6). Leaf tissue sections were hybridized with anti-cMyc antibodies and then with gold particle-conjugated secondary antibodies. Gold particles were only detected in chloroplasts of the ATase2-cMyc transgenic plants, not in other organelles, cytosol, or extracellular space (Fig. 6A). No specific hybridization was detected in nontransformed control plants (Fig. 6C).
Expression of the Three ATase Genes in Cotyledons
It has been shown that ATase2 is expressed in roots, leaves, and flowers (Ito et al., 1994
Functional Relationship of ATase1 and ATase2
In Arabidopsis, all enzymes in the de novo purine biosynthesis pathway are encoded by single genes, except those for ATase (Smith and Atkins, 2002 We then obtained a T-DNA insertion knockout mutant of the ATase1 gene (Salk_008888) from the Arabidopsis Biological Resource Center (ABRC; Fig. 2C). The mutant was indistinguishable from the wild type (Fig. 1A). A double mutant of cia1/ATase1 knockout was indistinguishable from cia1 (Fig. 1A), suggesting that ATase2 was the major isozyme for plant growth.
We further investigated whether the smaller size of mutant leaves was due to reduced cell numbers, cell size, or both. Scanning electron microscopy revealed no major difference in cell size or numbers between the wild type and the mutant in cotyledons (data not shown). By contrast, in mature true leaves, cia1 mutant had slightly smaller cell size but only about half the cell number of wild-type plants (Table I). This result confirmed the role of ATase in cell division.
We further characterized the protein import defect of cia1. We first tested the effect of supplementing ATP and GTP during the in vitro import reaction. Under sufficient light, when the wild-type chloroplasts had sufficient ATP from photosynthesis, addition of extra ATP or GTP had little effect on the import efficiency of wild-type chloroplasts (Fig. 8A). The same treatment slightly increased the amount of proteins imported in the mutant chloroplasts, suggesting that mutant chloroplasts were indeed deficient in ATP and GTP. However, the amount of proteins imported was still much less than that of wild-type chloroplasts. We further checked the amount of translocon proteins present in the mutant plastids by immunoblots. When equal numbers of plastids were analyzed, cia1 plastids had slightly lower amounts of outer membrane translocon proteins like Toc159 and Toc33 (Fig. 8B). However, it is unclear whether this small reduction in some translocon components could result in the import defect observed in cia1 mutant chloroplasts.
Although de novo purine biosynthesis has been studied extensively in animals and legumes of higher plants, studies in other plant species have been relatively few. Arabidopsis offers the advantage of a fully sequenced genome and vast resources of genetic and genomic tools. Our characterization of the Arabidopsis ATase mutants offered insights into the roles of de novo purine biosynthesis in normal plant physiology in non-N-fixing plants. We showed that both ATase1 and ATase2 were located in chloroplasts of leaf cells. We confirmed the role of de novo purine biosynthesis in cell division and further showed that its deficiency has a detrimental effect in chloroplast biogenesis. Because the cia1 mutant plants have very low levels of ATP and GTP (Fig. 3B), a decrease in energy charge may be altering growth and chloroplast protein import significantly. The exact mechanism linking de novo purine biosynthesis to chloroplast biogenesis requires further study. In Arabidopsis, ATase is one of the few enzymes in the de novo purine biosynthesis pathway that is encoded by multiple genes. Therefore, it is interesting to study the role played by each isozyme. From the expression pattern and our mutant analysis, ATase2 is clearly the major isoform. However, although the cia1/ATase1 knockout double mutant has the phenotype of cia1 and the expression level of ATase3 is very low, our preliminary data indicate that a cia1 mutant transformed with an ATase1 antisense construct is lethal (data not shown), suggesting ATase3 or a yet unknown mechanism of purine biosynthesis is responsible for the survival of the cia1/ATase1 knockout double mutant. The mosaic patterns of cia1 mutant leaves are irregular and variable. We have observed that, under conditions that favor rapid growth, e.g. high light or supplementing Suc to growth media, more white sectors are produced. By contrast, under low light or on media without Suc, although cia1 plants grow even smaller, cia1 leaves can be evenly pale green without white sectors (data not shown). It is possible that there is a threshold level of purine nucleotides that needs to be met for normal chloroplast biogenesis. Under rapid growth conditions, purine nucleotides are channeled into cell division, the purine nucleotide level in the mutant cytosol drops below the threshold, and chloroplasts fail to develop normally. White sectors are then observed on mutant leaves. When the division and growth rate of leaf cells are slower, purine nucleotides have time to accumulate in the cytosol of mutant cells, eventually reaching the threshold level.
The isolation of cia1-1 from our screen indicated that a deficiency in ATase2 would decrease chloroplast protein import efficiency since our screen is based on accumulation or transient retention of chloroplast precursor proteins in the cytosol (Sun et al., 2001
Molecular Cloning of CIA1
The cia1-1 mutant was isolated by a transgene-based screening strategy as described by Sun et al. (2001)
The plasmids containing cDNA clones for the precursors to RBCS, PC, and CAB of PSII were first transcribed in vitro and then translated with [35S]Met (Perry et al., 1991
For chloroplast isolation, Arabidopsis plants were grown on 1x Murashige and Skoog agar medium with Gamborg's vitamins and 2% Suc at 22°C under a 16-h-light/8-h-dark cycle for 4 weeks. Chloroplasts were isolated using the protoplast procedure as described by Fitzpatrick and Keegstra (2001)
The number of chloroplasts was counted on a hemocytometer under a phase-contrast microscope. After electrophoresis, proteins were transferred to Immobilon-P PVDF membranes (Millipore, Bredford, MA). The membranes were blocked for 30 min with Tris-buffered saline plus Tween 20 (TBST; 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, and 0.05% Tween 20) containing 5% (w/v) nonfat dry milk. Antibodies against Toc75, Arabidopsis Toc33, Toc159, Tic110, and Tic40 were generated against Escherichia coli overexpressed and purified full-length pea Toc75 precursor, Arabidopsis Toc33 residues 1 to 256, Arabidopsis Toc159 residues 1,224 to 1,503, Arabidopsis Tic110 residues 431 to 1,016, and Arabidopsis Tic40 full-length precursor (Chou et al., 2003
Wild-type and mutant plants were grown in 1x Murashige and Skoog liquid media with Gamborg's vitamins, 2% Suc, and 0.05% MES under continuous light at 22°C with constant orbital shaking at 125 rpm. Various amounts of AMP or BA, as indicated in the figures, or 0.1, 1, or 5 mM of IMP, GMP, or NADP were added to the liquid media. Continual observation of phenotypes was made during 4 weeks. Seedling chlorophyll contents were measured as described by Lichtenthaler (1987)
Wild-type and mutant plant leaf materials (500–600 mg) were frozen in liquid nitrogen and homogenized. Nucleotides were extracted by the trichloroacetic acid (TCA) method described by Hajirezaei et al. (2003)
DNA encoding five tandem copies of cMyc was amplified from plasmid Myc6mNotch1C and cloned into the EcoRV site of pSP72 (Promega), producing the plasmid pSP72-cMyc. The ATase2 full-length cDNA was amplified from first-strand cDNA of Arabidopsis leaf mRNA (Sun et al., 2001 DNA encoding five tandem copies of cMyc was amplified from plasmid Myc6mNotch1C and cloned into the EcoRV site of pBluescript SK (Stratagene), producing plasmid pBluescript-cMyc. ATase1 full-length cDNA was amplified from first-strand cDNA of Arabidopsis leaf mRNA with an N-terminal primer containing an XbaI site at the end and a C-terminal primer containing a BamHI site at the end and mutating the stop codon to Gly. The amplified fragment was subcloned into the BamHI/XbaI site of pBluescript-cMyc. The ATase1-cMyc fragment was then excised with HincII and XbaI, and cloned into the SmaI/XbaI site of pCAMBIA 1390 containing a 35S promoter. The ATase1-cMyc and ATase2-cMyc pCAMBIA 1390-35S constructs were introduced into Arabidopsis by Agrobacterium-mediated transformation. Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes.
Protoplasts were isolated from 4-week-old ATase2-cMyc transgenic plants and lysed by forcing them through two layers of nylon mesh (20 and 10 µm; Fitzpatrick and Keegstra, 2001
Leaves from wild-type and transgenic plants were cut into small sections (about 2 mm2) and then fixed in 0.1 M phosphate buffer (pH 7.0) with 4% (w/v) paraformaldehyde and 0.1% (v/v) glutaraldehyde overnight at 4°C. The leaf sections were washed with 0.1 M phosphate buffer (pH 7.0), dehydrated with a series of ethanol solutions, and infiltrated with London Resin Gold (Electron Microscopy Sciences, Fort Washington, PA). Polymerization was conducted with UV (360 nm) at –20°C for 24 h and then at room temperature for 48 h. Ultrathin sections were put on nickel grids. The grids were first blocked with TBST buffer containing 4% (w/v) bovine serum albumin at room temperature for 1 h, and then incubated with 1:25 diluted anti-cMyc rabbit polyclonal antibodies (Santa Cruz Biotechnology) for 1 h. The grids were then washed in TBS, incubated for 1 h with 20 nm gold-conjugated goat anti-rabbit IgG (dilution 1:25; British Biocell International, Cardiff, UK), and washed in TBS and then distilled water. Selected sections were further stained with uranyl acetate. Cell numbers and size were examined by cutting the first true leaves of 24- or 35-d-old wild-type or mutant seedlings at the base of petioles. The lower side of leaves was examined directly with a Zeiss (Jena, Germany) LSM 510 laser scanning confocal microscope. Three rows of cells along the vein were measured and counted from the base of the petiole to two-thirds of the length of the excised leaves. Cell sizes were determined using the program Metamorph (Adobe, Mountain View, CA). Cell numbers were counted by assembling continuous images into one single picture of the leaves.
Amounts of ATase transcripts were analyzed by RT-PCR. RNA was isolated from cotyledons and the first true leaves of 5- and 10-d-old seedlings using Trizol (Invitrogen, Carlsbad, CA) and reverse transcribed into first-strand cDNA (Sun et al., 2001
We thank the Arabidopsis Biological Resource Center for the ATase1 knockout mutant, Dr. H. W. Chen for the Myc6mNotch1C plasmid containing five tandem copies of cMyc coding sequence, Dr. Jychian Chen for antibodies against APS, and Dr. Dai Hwa for antibodies against maize mitochondria porin. Further, we would like to thank Dr. R. Zrenner (MPI-Golm, Germany) for supporting the nucleotide measurements. Received February 14, 2004; returned for revision May 3, 2004; accepted May 6, 2004.
1 This work was supported by grants from the National Science Council (NSC 90–2321–B001–006) and the Academia Sinica of Taiwan (to H.-m.L.), and from the Deutsche Forschungsgemeinschaft (BO 1236/3–1 to R.B.) as part of the The Arabidopsis Functional Genomics Network (AFGN). 
2 Present address: Department of Life Sciences, National Normal University, Taipei 116, Taiwan. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.040956. * Corresponding author; e-mail mbhmli{at}ccvax.sinica.edu.tw; fax 886–2–2782–6085.
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ABSTRACT
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) 1-pyrophosphate to 5-phosphoribosyl-(
) 1-amine. It is subjected to feed-back regulation and forms the rate-limiting step in de novo purine biosynthesis (Zalkin and Nygaard, 1996
