Fig 2: A forward genetics screen identified revertant lines of ccr2 having reduced lutein and normal chlorophyll accumulation when grown under a shorter photoperiod.(A) Representative images of rccr2-155 and rccr2-154 rosettes one week after shifting two-week old plants from a 16 hr to 8 hr photoperiod. (B) Percentage lutein relative to total carotenoids in immature leaves from WT, ccr2 and rccr2 lines. (C) The degree of leaf virescence detected in rosettes following a reduction in photoperiod. Leaf virescence (% of yellow relative to RGB; Red-Green-Blue) in WT, ccr2, rccr2-154 and rccr2-155 rosettes was quantified using the Lemnatec Scanalyser system and software. (D) Total chlorophyll content in rosette leaves from WT, ccr2, rccr2-154 and rccr2-155 plants exposed to a shorter photoperiod. (E) Segregation ratios of rccr2-154 and rccr2-155 after backcrossing to the ccr2 parent in both Columbia (Col-0) and Landsberg erecta (Ler) ecotypes. (NGS; next generation sequencing) (F) and (G). Leaves were pooled from a segregating F2 progeny of rccr2-155 (F) and rccr2-154 (G) plants and genomic DNA purified for NGS. Bars reflect independent polymorphisms for Ler and/or Columbia SNPs across the Chromosome. The SNP desert indicates there is only Columbia SNP, indicating linkage disequilibrium and less recombination around the location of the causative mutation for ziso-155 (3606630 bp; G to A) and det1-154 (6347991 bp; G to A). Error bars denote standard error of means (SEM) and stars denote statistical significance (ANOVA; p<0.05).

Fig 3: Chemical inhibition of CCD activity revealed how a ccr2 generated apocarotenoid signal transcriptionally up-regulates POR and PIF3 in parallel to det1-154 during skotomorphogenesis.(A) Transcript levels of PORA, PIF3 and HY5 in WT, ccr2, ccr2 det1-154 and det1-154 etiolated seedlings growing on MS media (+ /- D15). Statistical analysis denoted as a star was performed by a pair-wise t-test (p<0.05). Error bars represent standard error of means. (B), (C) and (D) Representative western blot images showing POR, DET1, PIF3 and HY5 protein levels, respectively. Proteins were extracted from WT, ccr2 and ccr2 det1-154 etiolated seedlings grown on MS media without (control; Ctrl) or with the chemical inhibitor of CCD activity (D15). The membrane was re-probed using anti-Actin antibody as an internal loading control. Lattice-like symbol below POR western (B), represents formation of a PLB in etiolated cotyledons from that genotype and treatment. (E) Model describing how a cis-carotene derived cleavage product, ACS, regulates POR, HY5, PIF3 and PLB formation during skotomorphogenesis. DET1 maintains skotomorphogenesis by post-transcriptionally maintaining a higher and lower PIF3 and HY5 protein levels, respectively. HY5 promotes and PIF3 represses PhANG expression. det1 mutants trigger photomorphogenesis in that they lack POR mRNA transcripts, protein and a PLB. ccr2 generates ACS that enhances POR mRNA transcript and protein levels that enable PLB formation in det1-154. det1-154 restores PLB formation in ccr2 by blocking a signalling pathway acting independent of POR.

Fig 4: det1 restores PLB formation, plastid development and cotyledon greening in ccr2.(A) Schematic structure of the wild type DET1 gDNA, DET1 protein and alternative spliced DET1-154 protein. A G->A mutation at the end of exon 4 (1449 bp) of AT4G10180 (6347991 bp) was confirmed by Sanger sequencing that leads to the skipping of exon 4 (69 bp). The DET1-154 splice variant produces a shorter protein (521 aa). Exon 4 comprises 23 amino acids in-frame, having homology to the six-hairpin glycosidase-like (IPR008928) domain. (B) Rosette images of WT, ccr2, ccr2 det1-154, and ccr2 det1-154::DET1-OE showing leaf pigmentations in newly emerged leaves from plants shifted from a 16 hr photoperiod (2 weeks old) to an 8 hr photoperiod for 1 week. Images are representative of 122/149 T1 generation ccr2 det1-154 plants from 12 independent lines surviving Basta herbicide selection after being transformed with pEARLEY::DET1-OE. (C) Carotenoid profiles of 7-d-old dark grown cotyledons from WT, ccr2, ccr2 det1-154 and det1-1 etiolated seedlings. Wavelengths close to the absorption maxima of A440 (major carotenoids and ?-carotene isomers) show neoxanthin (N); violaxanthin (V); lutein (L), ß-carotene (ß-C) in WT and neurosporene isomers (1 and 2) tetra-cis-lycopene (3); pro-neurosporene (4), and pro-?-carotene (5) in ccr2 and to a less extent in ccr2 det1-154. (D) Etiolated seedling morphology of WT, ccr2, ccr2 det1-154 and det1-154. Seedlings were grown in the dark for 7 d on MS media without sucrose. Representative images (>100 seedlings from independent experiments) depict a typical apical hook for WT and ccr2, and shorter hypocotyl with open cotyledons for ccr2 det1-154 and det1-154. (E) Chlorophyll levels in cotyledons following de-etiolation. ccr2, ccr2 det1-154 and WT were etiolated for 4 d in darkness and thereafter exposed to continuous white light. Chlorophyll measurements were taken at 0, 24, 48 and 72 hr after de-etiolation. Letters within a time point denote statistical analysis by one-way ANOVA with a post-hoc Tukey test (n > 20 seedlings). Error bars denote standard error of means.