Feedback regulation of carotenoid biosynthesis by an apocarotenoid sensing RNA structural switch

  • Yagiz Alagoz

Western Sydney University thesis: Doctoral thesis

Abstract

Carotenoid biosynthesis and accumulation in plants are tightly coordinated to maintain cellular homeostasis by regulating several processes, including plastid development, production of phytohormones and signalling molecules associated with metabolic regulation and environmental stress response. The EPSILON LYCOPENE CYCLASE (IµLCY) and BETA LYCOPENE CYCLASE (I²LCY) enzymes control metabolic flux through the I±- and I²-branches of the carotenoid pathway and regulate the production and accumulation of downstream carotenoids. Previous research showed that the changes in IµLCY gene expression might be associated with an altered abundance of downstream carotenoids such as I²-carotene or controlled with an apocarotenoid retrograde signal originated from cis-carotenes. Here we characterise the substrate of the apocarotenoid signal (ACS) and describe a molecular mechanism regulating IµLCY expression. Analysis using cis-carotene mutants, ccr2, ziso and ccr2 ziso showed that the IµLCY regulatory ACS could not be derived from a cis-carotene intermediate downstream of ZISO metabolic step. The exogenic expression of a bacterial carotenoid isomerase (CrtI) bypassed the metabolic steps between PDS and CRTISO and decreased the cis-carotene content and enhanced the accumulation of I²-branch carotenoids as well as IµLCY expression in dark-grown ccr2 seedlings. The de-etiolation of seedlings under constant light caused higher accumulation of I²-branch carotenoids and enhanced the IµLCY expression. Metabolic and gene expression analysis with green leaves at different stages of development demonstrated that carotenoid accumulation, as well as IµLCY expression, was higher in leaves at an early stage of development. These findings suggest that feedback control of IµLCY expression was required throughout the plant development. Subsequent analysis with norflurazon (NFZ) treated dark-grown wild-type, ccr2 and CrtI expressing transgenic seedlings revealed that cis-carotenes, including phytoene and phytofluene, could not be involved in the regulation of IµLCY expression. The IµLCY expression was upregulated in I²-carotene-rich cotyledon tissues of dark-grown Arabidopsis ccd4 mutants with wild-type and ccr2 background. These pieces of evidence showed that an unknown I²-apocarotenoid retrograde signal might control the IµLCY expression in order to maintain the balance between I±- and I²-carotene accumulation. Treatment with D15, an inhibitor of CCD catalytic activity, revealed that CCDs were not involved in generating an IµLCY regulatory ACS; however, they might have enhanced the I²-carotene substrate accumulation in embryonic tissues to generate I²-apocarotenoids. Therefore, we conclude that the I²-apocarotenoid signal which regulates IµLCY was generated by the oxidative cleavage of I²-carotene rather than by enzymatic catalysis. Previous research shows that the disruption of IµLCY 5'end by mutations or transposon insertion enhances the I²-carotene abundance in crop species. We hypothesized that the IµLCY 5'end may harbour a regulatory unit that might interact with the I²- apocarotenoid signal to control IµLCY expression. In order to test this, we generated Arabidopsis transgenic lines harbouring an IµLCY promoter-reporter gene fusion and confirmed that luciferase transcript levels were reduced in etiolated tissues of ccr2 and NFZ treated seedlings, and up-regulated by 30-fold following light exposure. The regulation of reporter gene expression was parallel to the changes in I²-carotene abundance in plant tissues. In-silico and in-vitro analysis of the IµLCY promoter region revealed three alternative transcription start sites (TSS) separated by a highly conserved 60-70bp AT-rich segments and a putative RNA regulatory switch in the 5' untranslated leader region (UTR) having two alternative secondary structures resembling an RNA riboswitch. A mutated version of the IµLCY 5'UTR having an altered secondary structure reduced CaMV35S promoter-driven luciferase activity in light-grown tissues. In-silico analysis of RNA secondary structures in IµLCY 5'UTR showed that a conserved hairpin motif, which is eliminated in the mutated version of the 5'UTR, might be involved in shifting the RNA structure between transcriptionally active and inhibitor form of RNA structures. We propose that a I²- apocarotenoid signal (ligand) can move out of the chloroplast and regulate IµLCY gene expression through an RNA structural switch (ligand-binding domain) in the 5'UTR in order to fine-tune carotenoid flux through the pathway branch during development and in response to environmental changes.
Date of Award2019
Original languageEnglish

Keywords

  • carotenoids
  • biosynthesis
  • plant hormones
  • growth (plants)
  • plant growth promoting substances

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