A forward genetic screen to explore chloroplast protein import in vivo identifies Moco sulfurase, pivotal for ABA and IAA biosynthesis and purine turnover.
A genetic screen in Arabidopsis was developed to explore the regulation of chloroplast protein import in vivo using two independent reporters representing housekeeping and photosynthetic pre-proteins. We first used 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase*), a key enzyme in the shikimic acid pathway, with a mutation that confers tolerance to the herbicide glyphosate. Because the EPSP synthase* pre-protein must be imported for its function, the loss of glyphosate tolerance provided an initial indication of an import deficiency. Second, the fate of GFP fused to a ferredoxin transit peptide (FD5-GFP) was determined. A class of altered chloroplast import (aci) mutants showed both glyphosate sensitivity and FD5-GFP mislocalized to nuclei. aci2-1 was selected for further study. Yellow fluorescent protein (YFP) fused to the transit peptide of EPSP synthase* or the small subunit of Rubisco was not imported into chloroplasts, but also localized to nuclei during protoplast transient expression. Isolated aci2-1 chloroplasts showed a 50% reduction in pre-protein import efficiency in an in vitro assay. Mutants did not grow photoautotrophically on media without sucrose and were small and dark green in soil. aci2-1 and two alleles code for Moco-sulfurase, which activates the aldehyde oxidases required for the biosynthesis of the plant hormones abscisic acid (ABA) and indole-acetic acid (IAA) and controls purine nucleotide (ATP and GTP) turnover and nitrogen recycling via xanthine dehydrogenase. These enzyme activities were not detected in aci2-1. ABA, IAA and/or purine turnover may play previously unrecognized roles in the regulation of chloroplast protein import in response to developmental, metabolic and environmental cues.
Zhong
Rong
R
Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
Thompson
Jennifer
J
Ottesen
Eric
E
Lamppa
Gayle K
GK
D000040:Abscisic Acid / Q000096:biosynthesis*; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D007210:Indoleacetic Acids / Q000378:metabolism*; D009154:Mutation; D010937:Plant Growth Regulators / Q000096:biosynthesis; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D021381:Protein Transport; D011687:Purines / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D013466:Sulfurtransferases / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes
D029681:Arabidopsis Proteins; D007210:Indoleacetic Acids; D010937:Plant Growth Regulators; D011687:Purines; D011993:Recombinant Fusion Proteins; C030737:indoleacetic acid; D000040:Abscisic Acid; D013466:Sulfurtransferases; D012273:Ribulose-Bisphosphate Carboxylase