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.