Characterization of Arabidopsis glutamine phosphoribosyl pyrophosphate amidotransferase-deficient mutants

Abstract

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.

Figure 1.

Phenotypes of various mutants. A, Morphology of wild-type (WT), cia1-1 and cia1-2, ATase1 knockout (ATase1 KO), and cia1-2/ATase1 KO double mutant plants at 13 d and 20 d. Bar = 0.5 cm. B, Various mosaic patterns of the cia1-2 mutant leaves. C, Import of precursor proteins into isolated wild-type and cia1 chloroplasts. RBCS, small subunit of Rubisco; CAB, chlorophyll a/b-binding protein of PSII; PC, plastocyanin. White triangle, Precursor form of each protein bound on the chloroplast surface. Black circle, Imported mature form of each protein. D, Complementation of cia1 by cDNA encoding ATase2-cMyc or ATase1-cMyc driven by the CaMV 35S promoter. Plants were grown on Murashige and Skoog agar plates for 18 d. Bar = 0.5 cm.

Figure 2.

Molecular cloning of CIA1 and positions of mutations or T-DNA insertion in cia1-1, cia1-2, and the ATase1 knockout line. A, Summary of positional cloning of CIA1. Vertical lines indicate positions of PCR-based markers. Values beneath the lines indicate number of recombinants. The direction of transcription of ATase2 ORF is indicated (arrow). B, Comparison of the three Arabidopsis ATases (AtATase) with ATases from other species. Arrowheads indicate the positions of mutations in cia1-1 and cia1-2. Predicted transit peptides in the three Arabidopsis ATases are underlined. C, Schematic representation of T-DNA insertion in the ATase1 gene in the Salk_008888 line. The T-DNA inserts in the coding sequence of Phe residue 282. The ATase1 gene has no intron. LB, T-DNA left border.

Figure 3.

ATase and de novo purine biosynthesis. A, The reaction carried out by ATase. B, Purine nucleotide levels in wild type (WT) and cia1. Nucleotides were extracted by TCA and measured by HPLC, as described in “Materials and Methods.” FW, fresh weight. Except for GDP measurement in the wild type, for which six different plants were measured, seven different wild-type plants and nine different cia1 mutant plants were individually measured. Data points represent the average ± se. For all four forms of nucleotides, wild type had significantly higher amounts of nucleotides than cia1 (P < 0.05).

Figure 4.

Complementation analysis of cia1 by AMP and cytokinin. A, Morphology of wild-type (WT) and cia1 mutant seedlings after growth in Murashige and Skoog liquid medium supplemented with various concentrations of AMP for 2 weeks. B, Chlorophyll content of the plants shown in A. Data points represent the average ± sd of three independent experiments except for wild type at 0.1 mm, which are from two independent experiments. At 0, 0.1, and 1 mm AMP conditions, wild type had significantly higher amounts of chlorophyll than cia1 under the same AMP concentration (P < 0.05). At 5 mm ATP, the chlorophyll content of the wild type was not significantly higher than cia1. C, Morphology of wild-type and cia1 mutant seedlings after growth on Murashige and Skoog agar plates supplemented with various concentrations of BA for 17 d.

Figure 5.

Localization of ATase 2-cMyc by fractionation. Chloroplasts and mitochondria (lanes 3 and 4, 6 μg of proteins in each lane) were isolated from the same batch of ATase2-cMyc-complemented cia1 transformants. A portion of the chloroplasts was lysed and further separated into soluble and membrane fractions (lanes 5 and 6, 5 μg of proteins in each lane). Samples were analyzed by SDS-PAGE and immunoblots probed with antibodies against various proteins as indicated on the right. Lane 1, in vitro translated protein derived from the ATase2-cMyc cDNA and analyzed by SDS-PAGE and immunoblots probed with anti-cMyc monoclonal antibodies together with the chloroplast and mitochondrial fractions. Molecular masses for marker proteins ran on the anti-cMyc blot are indicated on the left. APS, Small subunit of ADP-Glc pyrophosphorylase.

Figure 6.

Localization of ATase2-cMyc and ATase1-cMyc by immunogold-labeling electron microscopy. Leaf sections from ATase2-cMyc- (A) and ATase1-cMyc-complemented (B) cia1 transformants and the nontransformant control plant (C; wild type) were hybridized with anti-cMyc antibodies and gold-conjugated secondary antibodies. Bars = 0.5 μm.

Figure 7.

Expression of three ATase genes in cotyledons (Cot) from 5-and 10-d-old seedlings and the first true leaf from 10-d-old seedlings of wild-type plants. Total RNA was isolated and reverse transcribed into first-strand cDNA. Amounts of transcripts were analyzed by gene-specific primers and semiquantitative RT-PCR, and normalized to the amount of UBQ10 transcript. Data points represent the average ± se of three independent experiments. ATase2 had a higher expression level than ATase1 in 5- and 10-d-old cotyledons (P < 0.05).

Figure 8.

Analysis of the protein import defect of cia1 chloroplasts. A, Effect of adding ATP and GTP to import reactions. Chloroplasts were isolated from 28-d-old wild-type (WT) and cia1 mutant plants and used to perform in vitro protein import experiments supplemented with the indicated amounts of either ATP, GTP, or both. Positions of the precursor form of RBCS (prRBCS) bound on the chloroplast surface or mature RBCS imported are indicated. B, Amount of translocon proteins in wild-type and cia1 mutant chloroplasts. Chloroplasts were isolated from 28-d-old wild-type and cia1 mutant plants and analyzed by SDS-PAGE and immunoblots probed with antibodies against various translocon proteins as indicated on the left. Equal numbers of plastids were loaded in each lane.