Characterization of Arabidopsis glutamine phosphoribosyl pyrophosphate amidotransferase-deficient mutants.
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.
Hung
Wei-Fon
WF
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan.
Chen
Lih-Jen
LJ
Boldt
Ralf
R
Sun
Chih-Wen
CW
Li
Hsou-Min
HM
D000582:Amidophosphoribosyltransferase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D000582:Amidophosphoribosyltransferase