TheLYCOPENE EPSILON CYCLASEuntranslated mRNA leader modulates carotenoid feedback and post-transcriptional regulation

Abstract

ABSTRACTMetabolic feedback is proposed to modulate nuclear gene expression and carotenoid biosynthesis in plastids, however few mechanisms have been identified so far in plants. Utilising mutants, overexpression lines, and chemical inhibitors, we demonstrate that ArabidopsisLYCOPENE EPSILON CYCLASE(εLCY) mRNA levels correlate with changes in β-carotenoid accumulation. Transgenic seedlings harbouring theεLCY5’ leader sequence fused toFIREFLY LUCIFERASE(FiLUC) showed reporter responsiveness to metabolic feedback triggered by norflurazon or loss-of-function in the CAROTENOID ISOMERASE (CRTISO). TheεLCY5’UTR harboured three alternative transcription start sites (TSS). The most abundant -133bp sequence generated in dark and light grown seedlings harboured a 5’ conserved domain (CD) with otherBrassicaceaespecies and a viral internal ribosome entry site (IRES) proximal to the start codon.In silicomodelling predicted the 5’UTR formed two energetically separated RNA structural probabilities having a minimal free energy consistent with metabolite-binding RNA riboswitches that was distinguished by hairpin structures within the CD. Site-specific mutations were used to stabilize the 5’UTR into a single RNA shape definition having negligible separation between the mountain plot structure prediction curves and a distal terminator-like hairpin structure. Stabilizing the 5’UTR shape triggered the posttranscriptional repression of FiLUC activity enabled by the CaMV35S promoter in tobacco transient assays and stable transgenic Arabidopsis lines. The stabilised shape fragment became responsive to metabolic feedback induced by norflurazon and incrtisomutant etiolated and de-etiolated seedlings. TheεLCY5’UTR resembles a conformational RNA regulatory switch harbouring a posttranscriptional expression platform and aptamer domain responsive to carotenoid-mediated feedback signalling.