An upstream open reading frame is essential for feedback regulation of ascorbate biosynthesis in Arabidopsis.
Ascorbate (vitamin C) is an essential antioxidant and enzyme cofactor in both plants and animals. Ascorbate concentration is tightly regulated in plants, partly to respond to stress. Here, we demonstrate that ascorbate concentrations are determined via the posttranscriptional repression of GDP-l-galactose phosphorylase (GGP), a major control enzyme in the ascorbate biosynthesis pathway. This regulation requires a cis-acting upstream open reading frame (uORF) that represses the translation of the downstream GGP open reading frame under high ascorbate concentration. Disruption of this uORF stops the ascorbate feedback regulation of translation and results in increased ascorbate concentrations in leaves. The uORF is predicted to initiate at a noncanonical codon (ACG rather than AUG) and encode a 60- to 65-residue peptide. Analysis of ribosome protection data from Arabidopsis thaliana showed colocation of high levels of ribosomes with both the uORF and the main coding sequence of GGP. Together, our data indicate that the noncanonical uORF is translated and encodes a peptide that functions in the ascorbate inhibition of translation. This posttranslational regulation of ascorbate is likely an ancient mechanism of control as the uORF is conserved in GGP genes from mosses to angiosperms.
The New Zealand Institute for Plant and Food Research, Auckland 1142, New Zealand william.laing@plantandfood.co.nz.
William A
http://orcid.org/0000-0003-0458-6590
WA
Laing
The New Zealand Institute for Plant and Food Research, Auckland 1142, New Zealand.
Marcela
M
Martínez-Sánchez
The New Zealand Institute for Plant and Food Research, Auckland 1142, New Zealand.
Michele A
MA
Wright
The New Zealand Institute for Plant and Food Research, Auckland 1142, New Zealand.
Sean M
SM
Bulley
The New Zealand Institute for Plant and Food Research, Auckland 1142, New Zealand.
Di
D
Brewster
The New Zealand Institute for Plant and Food Research, Auckland 1142, New Zealand.
Andrew P
AP
Dare
The New Zealand Institute for Plant and Food Research, Auckland 1142, New Zealand.
Maysoon
M
Rassam
The New Zealand Institute for Plant and Food Research, Auckland 1142, New Zealand.
Daisy
D
Wang
The New Zealand Institute for Plant and Food Research, Auckland 1142, New Zealand.
Roy
R
Storey
The New Zealand Institute for Plant and Food Research, Auckland 1142, New Zealand Biochemistry Department, University of Otago, Dunedin 9054, New Zealand.
Richard C
RC
Macknight
The New Zealand Institute for Plant and Food Research, Auckland 1142, New Zealand Biochemistry Department, University of Otago, Dunedin 9054, New Zealand.
Roger P
RP
Hellens
D020121:5' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics*; D001205:Ascorbic Acid / Q000096:biosynthesis* / Q000494:pharmacology; D053898:Biosynthetic Pathways / Q000187:drug effects; D003062:Codon / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D025461:Feedback, Physiological / Q000187:drug effects; D005690:Galactose / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D010455:Peptides / Q000737:chemistry; D010770:Phosphotransferases / Q000378:metabolism; D018515:Plant Leaves / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism
D020121:5' Untranslated Regions; D003062:Codon; D010455:Peptides; D008156:Luciferases; D010770:Phosphotransferases; D001205:Ascorbic Acid; D005690:Galactose