Journal: bioRxiv

Article Title: A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development

doi: 10.1101/528331

Figure Lengend Snippet: A model describing how a cis -carotene derived cleavage product controls POR and PLB formation during plastid development. (A) ccr2 can accumulate poly- cis -carotenes that undergo enzymatic cleavage via CCDs to generate an apocarotenoid signal (ACS). Norflurazon (NF) treatment of ccr2 etiolated seedlings or the loss-of-function in ziso-155 block the accumulation of downstream cis-carotenes required for the biosynthesis of ACS. Chemical treatment of etiolated seedlings with D15 inhibits CCD cleavage of pro-neurosporene and/or tetra- cis -lycopene isomers into ACS. (B) During skotomorphogenesis, ACS promotes “Factor X”. Factor X negatively affects PLB formation. Factor X could act to stabilise proteins by disrupting ubiquitination, de-ubiquitination, protease mediated protein degradation, heterodimerization of transcription factors, coactivator concentrations, and/or interact with ligand binding sites of receptors. DET1 is a repressor of photomorphogenesis that post-transcriptionally regulate PIF3 and HY5 protein levels, which control PhANG expression. det1 mutants lack POR and cannot make a PLB. ACS post-transcriptionally enhances POR protein levels, while det1 blocks Factor X, thereby allowing PLB formation in ccr2 det1-154 . det1 reduces cis -carotene accumulation, and downregulates pro-neurosporene and tetra- cis -lycopene to maintain a threshold level of ACS. (C) During photomorphogenesis, extended dark and/or shorter photoperiods, ACS manifests in newly emerged leaves from the ccr2 shoot meristem and perturbed chloroplast development and chlorophyll accumulation causing a leaf variegation phenotype. Green arrows and red lines represent positive and negative regulation, respectively. Abbreviations: PSY, phytoene synthase; PDS, phytoene desaturase, ZDS, ζ-carotene desaturase; Z1SO, ζ-carotene isomerase; CRTISO, carotenoid isomerase; det1-154 , DEETIOLATED1-154; D15, inhibitor of CCD activity; CCD, carotenoid cleavage dioxygenase; ccr2 , CRTISO mutant.

Article Snippet: Inhibition of carotenoid cleavage dioxygenase (CCD) enzyme activity was achieved by adding D15 (aryl-C3N hydroxamic acid) dissolved in ethanol to a final optimal concentration of 100 μM as previously described ( ).

Techniques: Derivative Assay, Blocking Assay, Ligand Binding Assay, Expressing, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development

doi: 10.1101/528331

Figure Lengend Snippet: Altered plastid development in ccr2 is linked with cis -carotene accumulation and not to a perturbation in ABA or SL. (A) Mutants that perturb the levels of lutein, ABA, SL and accumulate cis -carotenes ( ccr2 , ccr1 and ziso ) were grown for two weeks under a 16-h photoperiod and then shifted to a shorter 8-h photoperiod for one week. Representative images showing newly emerged and expanding leaves from multiple experimental and biological repetitions (n > 20 plants per line) are displayed. Genetic alleles tested include Col-0 (WT), ccr2.1 (carotenoid isomerase), lut2.1 (epsilon lycopene cyclase), aba1-3 (Ler background) (zeaxanthin epoxidase), max4/ccd8 (carotenoid cleavage dioxygenase 8), ccr1.1/sdg8 (set domain group 8) and ziso1-3 (ζ-carotene isomerase). (B) Carotenoid profiles in rosette leaves from three-week-old plants grown under a 16-h photoperiod and subjected to 6-d of extended darkness. (C) Carotenoid profiles in three-week-old rosette leaves from plants grown under a constant 8-h light photoperiod. Pigments were profiled in a yellow leaf (YL) and green leaf (GL) from WT and ccr2 . (D) Carotenoid profiles in newly emerged floral bud and rosette leaf tissues harvested from four-week-old plants growing under a 16-h photoperiod. Carotenoid profile traces of various tissue extracts from wild type (WT) and ccr2 show pigments at wavelengths close to the absorption maxima of A 440nm (Neoxanthin; N, violaxanthin; V, antheraxanthin; A, lutein; L, zeaxanthin; Z, β-carotene isomers; β-C, chlorophyll a; Chl a, chlorophyll b; chl b, tetra- cis- lycopene; plyc, neurosporene isomers; neuro, and ζ-carotene; ζ-C), A 348nm (phytofluene; pflu) and A 286nm (phytoene; phyt). HPLC profile y-axis units are in milli-absorbance units (mAU). HPLC traces are representative of multiple leaves from multiple experimental repetitions and retention times vary due to using different columns.

Article Snippet: Inhibition of carotenoid cleavage dioxygenase (CCD) enzyme activity was achieved by adding D15 (aryl-C3N hydroxamic acid) dissolved in ethanol to a final optimal concentration of 100 μM as previously described ( ).

Techniques: High Performance Liquid Chromatography

Journal: bioRxiv

Article Title: A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development

doi: 10.1101/528331

Figure Lengend Snippet: 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 h 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

Article Snippet: Inhibition of carotenoid cleavage dioxygenase (CCD) enzyme activity was achieved by adding D15 (aryl-C3N hydroxamic acid) dissolved in ethanol to a final optimal concentration of 100 μM as previously described ( ).

Techniques: Transmission Assay, Fluorescence

Journal: bioRxiv

Article Title: A cis-carotene derived apocarotenoid regulates etioplast and chloroplast development

doi: 10.1101/528331

Figure Lengend Snippet: The loss-of-function in individual members of the carotenoid cleavage dioxygenase gene family cannot restore plastid development in ccr2 rosettes. Three-week-old WT, ccr2 , ccr2 ccd1, ccr2 ccd4, ccr2 ccd7 , and ccr2 ccd8 (F 3 homozygous double mutant lines) plants were shifted from a 16-h to 8-h 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 leaf variegation 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.

Article Snippet: Inhibition of carotenoid cleavage dioxygenase (CCD) enzyme activity was achieved by adding D15 (aryl-C3N hydroxamic acid) dissolved in ethanol to a final optimal concentration of 100 μM as previously described ( ).

Techniques: Mutagenesis

Journal: Biomolecules

Article Title: Expression of a Chromoplast-Specific Lycopene β-Cyclase Gene (CYC-B) Is Implicated in Carotenoid Accumulation and Coloration in the Loquat

doi: 10.3390/biom9120874

Figure Lengend Snippet: qRT-PCR analysis of genes related to carotenoid metabolic pathways after silencing. Plastids are the sites for the biosynthesis and accumulation of plant carotenoids. PAP, plastid lipid-associated protein; PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; ZISO, 15-cis-ζ-carotene isomerase; CRTISO, carotene isomerase; LCYE, lycopene ε-cyclase; LCYB, lycopene β-cyclase; CYC-B, chromoplast-specific lycopene β-cyclase; BCH, β-carotene hydroxylase; ECH, ε-carotene hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; NXY, neoxanthin synthase; CCD, carotenoid cleavage dioxygenases. Different letters indicate significant differences between negative control and silenced fruits (One-way ANOVA, p

Article Snippet: Carotenoid cleavage dioxygenases (CCD) and nine-cis-epoxycarotenoid dioxygenase (NCED) are involved in the carotenoid cleavage pathway in plants.

Techniques: Quantitative RT-PCR, Negative Control

Journal: Archives of biochemistry and biophysics

Article Title: The carotenoid dioxygenase gene family in maize, sorghum, and rice

doi: 10.1016/j.abb.2010.07.019

Figure Lengend Snippet: Carotenoid dioxygenase gene family in the Grasses

Article Snippet: Ohmiya A, Kishimoto S, Aida R, Yoshioka S, Sumitomo K. Carotenoid cleavage dioxygenase (CmCCD4a) contribute s to white color formation in Chrysanthemum petals.

Techniques:

Journal: Archives of biochemistry and biophysics

Article Title: The carotenoid dioxygenase gene family in maize, sorghum, and rice

doi: 10.1016/j.abb.2010.07.019

Figure Lengend Snippet: Carotenoid cleavage dioxygenase activities in various species

Article Snippet: Ohmiya A, Kishimoto S, Aida R, Yoshioka S, Sumitomo K. Carotenoid cleavage dioxygenase (CmCCD4a) contribute s to white color formation in Chrysanthemum petals.

Techniques: