Activation of the native PHYTOENE SYNTHASE 1 promoter by modifying near-miss cis‑acting elements induces carotenoid biosynthesis in embryogenic rice callus

Metabolic engineering in plants typically involves transgene expression or the mutation of endogenous genes. An alternative is promoter modification, where small changes in the promoter sequence allow genes to be switched on or off in particular tissues. To activate silent genes in rice endosperm, we screened native promoters for near-miss cis-acting elements that can be converted to endosperm-active regulatory motifs. We chose rice PHYTOENE SYNTHASE 1 (PSY1), encoding the enzyme responsible for the first committed step in the carotenoid biosynthesis pathway, because it is not expressed in rice endosperm. We identified six motifs within a 120-bp region, upstream of the transcriptional start site, which differed from endosperm-active elements by up to four nucleotides. We mutated four motifs to match functional elements in the endosperm-active BCH2 promoter, and this promoter was able to drive GFP expression in callus and in seeds of regenerated plants. The 4 M promoter was not sufficient to drive PSY1 expression, so we mutated the remaining two elements and used the resulting 6 M promoter to drive PSY1 expression in combination with a PDS transgene. This resulted in deep orange callus tissue indicating the accumulation of carotenoids, which was subsequently confirmed by targeted metabolomics analysis. PSY1 expression driven by the uncorrected or 4 M variants of the promoter plus a PDS transgene produced callus that lacked carotenoids. These results confirm that the adjustment of promoter elements can facilitate the ectopic activation of endogenous plant promoters in rice callus and endosperm and most likely in other tissues and plant species.

We acknowledge the Spanish Ministry of Science, Innovation and Universities and the EU (projects RTI2018-097613- B-I00 and PGC2018-097655-B-I00), the Generalitat de Catalunya, AGAUR (Grups Consolidats 2017-SGR828), and Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR). We acknowledge UKRI for funding via the BBSRC-GCRF NUTRIFOOD project. GSM has an FPI fellowship PRE2019-088852.