Figure 6. The carotenoid cleavage dioxygenase (CCD) inhibitor, D15, restores PLB formation in etiolated ccr2 seedlings, cotyledon greening following de-etiolation and alters cis-carotene accumulation.

(A) Transmission electron micrographs of a representative etioplast from 5-d-old dark grown cotyledons reveal a well-developed PLB in ccr2 treated with the D15, but not in ccr2 treated with ethanol only (control; ctrl). (B) Pchlide levels in Wild Type (WT) and ccr2 treated + /- D15. Fluorescence was measured at 638 nm and 675 nm with an excitation at 440 nm. Net fluorescence of Pchlide was calculated and normalised to protein content. (C) D15 restores chlorophyll accumulation in ccr2 de-etiolated seedlings exposed to continuous light. Twenty seedlings from each of three biological replicates were harvested for chlorophyll determination in every 24 hr under continuous light. Statistical analysis was by ANOVA with a post-hoc Tukey test (n = 20 seedlings). (D) cis-carotene quantification in etiolated cotyledons of ccr2 treated with D15. phytoene (phyt), phytofluene (pflu), tri-cis-ζ-carotene (3ζ-C), di-cis-ζ-carotene (2ζ-C), pro-neurosporene (p-N), tetra-cis-lycopene (p-lyc) and total cis-carotenes were quantified at absorption wavelengths providing maximum detection. Star denotes significance (ANOVA, p<0.05). Error bars show standard error (n = 4). (E) Quantification of carotenoid levels in etiolated tissues of WT treated with D15. Neoxanthin (N); violaxanthin (V); antheraxanthin (A), lutein (L), β-carotene (β-C) and total carotenoids (T) were quantified at a 440 nm absorption wavelength providing maximum detection. Star denotes significance (ANOVA, p<0.05). Data is representative of two independent experiments.

Figure 6—figure supplement 1. The loss-of-function in individual members of the carotenoid cleavage dioxygenase gene family cannot restore plastid development in ccr2 rosettes.

Two-week-old WT, ccr2, ccr2 ccd1, ccr2 ccd4, ccr2 ccd7, and ccr2 ccd8 (F₃ homozygous double mutant lines) plants were shifted from a 16 hr to 8 hr photoperiod until newly formed leaves in the ccr2 rosette displayed a virescent leaf phenotype. (A) Representative images of plants showing newly developed leaves in the rosette. (B) Quantification of yellow leaf virescence in individual rosettes from ccr2 ccd double mutants. Data is representative of multiple independent experiments. Statistical analysis by ANOVA with post-hoc Tukey test showed no significant difference in the number of ccr2 and ccr2 ccd plants displaying a virescent phenotype.