Plastid-mediated feedback regulation of Arabidopsis LYCOPENE EPSILON CYCLASE is modulated by the promoter and a 5'UTR structural variant harbouring a conserved IRES.

BACKGROUND: Perturbations in flux through the carotenoid pathway can signal control over nuclear gene expression to maintain metabolic homeostasis in plastids; however, the genetic targets and mechanisms are rarely characterised in plants.

RESULTS: Utilising mutants, over- and exogenous expression lines, and chemical inhibitors of carotenogenesis, we demonstrate that Arabidopsis LYCOPENE EPSILON CYCLASE (εLCY) mRNA levels correlate with changes in the accumulation of cyclic carotenoids generated by the β-branch in the metabolic pathway. In dark- and light-grown seedlings subjected to retrograde feedback signalling triggered by norflurazon and the loss-of-function in CAROTENOID ISOMERASE (CRTISO) activity, changes in εLCY levels were mirrored by FIREFLY LUCIFERASE (FiLUC) levels regulated by the εLCY promoter in isogenic transgenic seedlings. The εLCY promoter enabled three alternative transcription start sites (TSS), and the most prominent 5'UTR fragment generated in dark- and light-grown seedlings harboured a 5' conserved domain (CD) with other Brassicaceae species adjacent to a conserved viral internal ribosome entry site (IRES) sequence proximal to the start codon. In silico modelling predicted the 5'UTR formed two energetically separated RNA structural probabilities distinguished by alternative hairpin structures within the CD that lacked a centroid structure, unlike the adjacent IRES sequence that displayed a prominent centroid peak, higher base-pairing probability, and lower positional entropy indicative of fewer secondary structure outcomes. Unlike the native 5'UTR, site-specific mutations predicted to stabilize the 5'UTR shape triggered post-transcriptional repression of FiLUC activity enabled by the CaMV35S promoter in transient tobacco and stable transgenic Arabidopsis tissues. The mutated 5'UTR RNA shape fragment conferred CaMV35S enabled reporter responsiveness to feedback signalling in crtiso mutant and wild type (WT) etiolated and de-etiolated seedlings treated with norflurazon.

CONCLUSIONS: We propose that the εLCY promoter and 5'UTR harbour an expression platform, whereby alternative TSS and structural definitions enable transcriptional and post-transcriptional regulation in response to feedback signalling during plastid biogenesis and plant development.