ABSTRACT
Metabolic 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 Arabidopsis LYCOPENE EPSILON CYCLASE (εLCY) mRNA levels correlate with changes in β-carotenoid accumulation. Transgenic seedlings harbouring the εLCY 5’ leader sequence fused to FIREFLY LUCIFERASE (FiLUC) showed reporter responsiveness to metabolic feedback triggered by norflurazon or loss-of-function in the CAROTENOID ISOMERASE (CRTISO). The εLCY 5’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 other Brassicaceae species and a viral internal ribosome entry site (IRES) proximal to the start codon. In silico modelling 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 in crtiso mutant etiolated and de-etiolated seedlings. The εLCY 5’UTR resembles a conformational RNA regulatory switch harbouring a posttranscriptional expression platform and aptamer domain responsive to carotenoid-mediated feedback signalling.
Footnotes
The authors declare no conflict of interest.
One-sentence summary: The untranslated leader region of the LYCOPENE EPSILON CYCLASE gene harbours an RNA structural switch modulating post-transcriptional regulation and responsiveness to carotenoid feedback signalling.
Abbreviations
- ACS
- apocarotenoid signal
- β-ACS
- β-carotene derived apocarotenoid signal
- cis-ACS
- cis-carotene derived apocarotenoid signal
- FiLUC
- intron modified FIREFLY (Photinus pyralis) LUCIFERASE gene
- MutDels
- mutations and/or deletions
- NFZ
- norflurazon
- Nost
- nopaline synthase terminator
- uORF
- upstream open reading frame
- RLU
- relative light units
- TSS
- transcription start site
- 5’UTR
- 5’ Untranslated Region