Journal: bioRxiv

Article Title: The LYCOPENE EPSILON CYCLASE untranslated mRNA leader modulates carotenoid feedback and post-transcriptional regulation

doi: 10.1101/2024.07.19.604344

Figure Lengend Snippet: A-C) εLCY expression in linear cis -carotene accumulating etiolated tissues from ( A ) MicroTom tangerine ( tang Mic ), ( B ) ccr2 , ziso , and ccr2 ziso , and ( C ) det1-154 , ccr2 , and ccr2 det1-154 . D ) εLCY expression in WT, ccr2 , ccr2 T35ehn :: PaCrtI#11 ( ccr2 transformed with pT35enh::SSU- PaCrtI ), and ccr2 ccd4 etiolated seedlings that accumulate linear cis -carotenes plus cyclic carotenoids. E ) Relative expression of photosynthesis-associated nuclear genes ( RBCS1 , LHCB2.1 ) and εLCY in WT etiolated tissues treated with NFZ. F ) εLCY expression in ccr2 etiolated tissues treated with NFZ. G ) εLCY expression in WT and ccr2 etiolated and de-etiolated seedlings exposed to continuous light for up to 72 hrs. H ) εLCY expression in young emerging leaves (YL) and older mature leaves (OL) from whole rosettes. I ) Model summarising feedback regulations of εLCY expression triggered by chemical (NFZ), genetic ( ccr2 ), developmental (leaf age) and environmental (light) perturbations. Data is representative of two to three independent experiments, and standard error bars of the mean are displayed (n=3-9). Lettering denotes significance by one- or two-way ANOVA statistical analysis with post-hoc Tukey test. Abbreviations: εLCY ; LYCOPENE EPSILON CYCLASE , RBCS1; RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL CHAIN 1A, LHCB2.1; LIGHT-HARVESTING CHLOROPHYLL B-BINDING 2

Article Snippet: Four homozygous ccr2 35S::PaCrtI lines (#5, 6, 9, and 11) that segregated in a typical 3:1 manner (Chi^2 p -value > 0.64) displayed a carotenoid composition similar to that of ccr2 when grown under a long 16 h photoperiod (Table S2).

Techniques: Expressing, Transformation Assay, Binding Assay

Journal: bioRxiv

Article Title: The LYCOPENE EPSILON CYCLASE untranslated mRNA leader modulates carotenoid feedback and post-transcriptional regulation

doi: 10.1101/2024.07.19.604344

Figure Lengend Snippet: A-B ) In planta luminescence emitted by independent WT transgenic plants harbouring the εLCY Prom- εLCY 5′UTR::FiLUC ( εLP::FiLUC #2e; εLP #2e, εLP::FiLUC #4c; εLP #4c) or CaMV35s::FiLUC (35S#C1) transgenes. The colour bar displays the intensity scale where black denotes no light emission and white indicates maximum luminescence. C ) εLCY and FiLUC gene expression levels in young leaves (YL) and old leaves (OL) from WT transgenic lines ( εLP #2e and εLP #4c). D) FiLUC activity (RLU/μg protein) in young and old leaves from WT transgenic lines ( εLP #2e and εLP #4c). E-G ) εLCY (E) and FiLUC (F) mRNA levels, and FiLUC activity (G) in transgenic etiolated and de-etiolated transgenic WT ( εLP #2e, εLP #4c) and ccr2 ( εLP #4e, εLP #1b) seedlings. H-I ) FiLUC activity in 5 μM NFZ-treated etiolated (H) and de-etiolated (I) transgenic WT ( εLP #2e, εLP #4c) and ccr2 ( εLP #4e, εLP #1b) seedlings. ( J ) FiLUC activity in 5 μM NFZ-treated de-etiolated transgenic WT seedlings harboring 35S#C1. Lettering denotes significance by a one- or two-way ANOVA statistical analysis with post-hoc Tukey test. Data is representative of two to three independent experiments, and standard error bars are shown (n=3-9). Abbreviations: RLU; Relative light units, FiLUC; Firefly intron-containing LUCIFEREASE gene.

Article Snippet: Four homozygous ccr2 35S::PaCrtI lines (#5, 6, 9, and 11) that segregated in a typical 3:1 manner (Chi^2 p -value > 0.64) displayed a carotenoid composition similar to that of ccr2 when grown under a long 16 h photoperiod (Table S2).

Techniques: Transgenic Assay, Gene Expression, Activity Assay

Journal: bioRxiv

Article Title: The LYCOPENE EPSILON CYCLASE untranslated mRNA leader modulates carotenoid feedback and post-transcriptional regulation

doi: 10.1101/2024.07.19.604344

Figure Lengend Snippet: A-B ) Percentage (%) FiLUC activity (A) and FiLUC mRNA expression levels (B) in de-etiolated transgenic Arabidopsis seedlings grown on artificial media in the absence (Control; Ctrl) or presence of norflurazon (NFZ; 5μM). Percentages reflect the ratio of NFZ-treated relative to untreated control (Ctrl) seedlings. Transgenic lines harbour the 35S, UTR, Sh-1, εLCY ( εLCYP ) promoter-reporter gene fusions. C-D ) Quantification of FiLUC activity (RLU/mg protein) in mature leaves (C) and de-etiolated seedlings (D) from WT and ccr2 transgenic lines harbouring the Sh-1 fragment. Mean and standard error (n=3) are representative of two independent experiments. Lettering denote significance (one-way ANOVA, p<0.05).

Article Snippet: Four homozygous ccr2 35S::PaCrtI lines (#5, 6, 9, and 11) that segregated in a typical 3:1 manner (Chi^2 p -value > 0.64) displayed a carotenoid composition similar to that of ccr2 when grown under a long 16 h photoperiod (Table S2).

Techniques: Activity Assay, Expressing, Transgenic Assay, Control

Journal: bioRxiv

Article Title: The LYCOPENE EPSILON CYCLASE untranslated mRNA leader modulates carotenoid feedback and post-transcriptional regulation

doi: 10.1101/2024.07.19.604344

Figure Lengend Snippet: β-branch carotenoid levels correlate with εLCY expression levels. Metabolic feedback triggered during etioplast, and chloroplast development regulates εLCY expression in order to balance α/β-carotene homeostasis during seedling morphogenesis and leaf development. β-branch carotenoids are reduced by the loss-of-function CRTISO mutant ( ccr2 ) and by norflurazon (NFZ; inhibits PHYTOENE DESATURASE, PDS) treatment of seedlings. Metabolic feedback via a retrograde signal (e.g., heme or an apocarotenoid signal; ACS) generated in the plastid signals the nucleus to regulate εLCY levels. Mutations that stabilise an alternative distinct RNA structural probability (shape-1) of the εLCY 5′UTR can establish a post-transcriptional expression platform that reduces protein activity without affecting transcript levels. The shape-1 5′UTR fragment harbours an aptamer domain capable of sensing a feedback signal generated in ccr2 or in NFZ-treated seedling tissues that can repress reporter gene expression. The εLCY 5′UTR represents a new conformational RNA regulatory switch that could modulate carotenoid biosynthesis through the branch in the pathway to maintain metabolic homeostasis during plastid biogenesis.

Article Snippet: Four homozygous ccr2 35S::PaCrtI lines (#5, 6, 9, and 11) that segregated in a typical 3:1 manner (Chi^2 p -value > 0.64) displayed a carotenoid composition similar to that of ccr2 when grown under a long 16 h photoperiod (Table S2).

Techniques: Expressing, Mutagenesis, Generated, Activity Assay, Gene Expression