Ascorbate is an essential biochemical for plants and animals, as it a powerful antioxidant and an enzyme co-factor. In higher plants, ascorbate is synthesised via the Smirnoff-Wheeler pathway, which is primarily regulated by GDP-L-galactose phosphorylase (GGP). An upstream open reading frame (uORF), with a non-canonical ACG start codon, negatively regulates the translation of GGP in response to high ascorbate levels, in a cis-regulatory manner (Laing et al., 2015). Ribosome profiling data suggested that the uORF induced ribosome stalling in the presence of high ascorbate to inhibit GGP translation (Laing et al., 2015). This study aimed to investigate the uORF non-canonical ACG start codon, and the uORF negative regulatory mechanism that controls translation of GGP.
Translation initiation inefficiently occurs from the uORF non-canonical ACG start codon in the presence and absence of high ascorbate. This showed that the non-canonical start codon does not respond to high ascorbate levels to increase translation of the stalling uORF peptide. Of the investigated uORF 5’ leader elements, only the Kozak sequence promoted uORF translation initiation. Removal of all stop codons between the uORF and GGP did not alter levels of GGP translation. These results indicate that ribosomes rarely initiate at the uORF, and primarily scan to GGP to initiate translation.
The uORF peptide was hypothesised to recognise ascorbate and inhibit GGP translation via ribosome stalling. Site-directed mutagenesis of uORF residue Arg33, suggested that it was essential for recognising ascorbate. When some elements of the uORF less highly conserved region were disrupted, or when proline residues of the uORF highly conserved region (HCR) were mutated, inhibition of GGP translation occurred in the absence of high ascorbate. These results enabled a model for the uORF negative regulatory mechanism to be proposed: Positively charged His30 and Arg33 residues of the HCR bind the ascorbate anion, inducing a conformational change that is reinforced by HCR proline residues within the ribosome tunnel; this causes a disruption to nucleotides of the ribosome peptidyl-transferase centre, leading to ribosome stalling, and inhibition of GGP translation.
Developing a detailed understanding into the mechanisms regulating the expression of GGP, and the impact of this regulation on ascorbate levels, will enable the biofortification of crop plants to have increased levels of ascorbate.
