A novel carotenoid cleavage activity involved in the
biosynthesis of Citrus fruit-specific apocarotenoid pigments
Item Type Article
Authors Rodrigo, María J.;Alquézar, Berta;Alós, Enriqueta;Medina,
Víctor;Carmona, Lourdes;Bruno, Mark;Al-Babili, Salim;Zacarías, Lorenzo
Citation Rodrigo MJ, Alquezar B, Alos E, Medina V, Carmona L, et al. (2013) A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments. Journal of Experimental Botany 64: 4461-4478. doi:10.1093/jxb/ert260.
Eprint version Publisher's Version/PDF
DOI 10.1093/jxb/ert260
Publisher Oxford University Press (OUP) Journal Journal of Experimental Botany
Rights This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/3.0/), which permits
unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Download date 2024-01-16 17:43:45
Item License http://creativecommons.org/licenses/by/3.0 Link to Item http://hdl.handle.net/10754/325447
Supplemental table S1. Matrix of similarity between the different Citrus clementina CCD4-like family members based on deduced amino acid and sequences alignment (%).
CCD4a CCD4b1 CCD4b2 CCD4c CCD4a 100
CCD4b1 48 100
CCD4b2 48 88 100
CCD4c 60 45 45 100
CCD4d 39 32 30 49
Supplemental Figure S1. Best 3D models of CitrusCCD4b1 by using I-TASSER online platform by using as templates the 3D structure of CCO (PBD: 2biwA) from Synechocystis (A) and VP14 (PBD: 3npeA) from maize (B) which are showed overlapped in purple ribbons. The C-score obtained for each model is indicated below the images. C-score is a confidence score for estimating the quality of predicted models by I-TASSER. It is calculated based on the significance of threading template alignments and the convergence parameters of the structure assembly simulations. C-score is typically in the range of [-5,2], where a C-score of higher value signifies a model with a high confidence and vice-versa.
Zhang Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics. 9: 40;. Roy A., Kucukural A. and Zhang Y.(2010). I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols 5:
725-738.
A B
C-score 0.77 C-score 1.96
Suplemental Figure S2. GC-MS analysis of in vitro enzymatic activity of citrus CCD4b1. Gas phase of in vitro assays performed with -carotene or -cryptoxanthin and crude lysate of thioredoxin-CCD4b1 expressing E. coli cells (CCD4b1) and control lysate obtained from thioredoxin expressing cells (Con) was extracted using an open loop and applied to GC-MS analysis. The total ion current revealed a new compound formed by thioredoxin-CCD4b1, which eluted at min 6.30. Mass spectrum of this product identified it as -cyclocitral.
Relative Abundance
Time (min)
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 5
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
6.77
6.30
7.68
6.07
2.15 6.45
7.00 4.67 7.22
2.953.13 5.205.44
2.322.58 3.16 3.914.194.614.89 5.92 7.37 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100 6.76
7.69
6.076.45
7.01
7.137.62 4.67
2.943.133.37 3.914.61
2.322.58 3.58 4.194.414.905.195.895.966.36 7.40
Relative Abundance
4050 6070 8090100 110 120 130 140 150 160 m/z
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Relative Abundance
66.82 80.82
136.93
40.87 151.95
122.92
90.85 54.83
O O
CsCCD4b1 Con
-cyclocitral
400 500 600 nm mAU
469 I
C22‐dialdehyde
400 500 600
nm 449465
II
C19‐dialdehyde
Apo‐10‘‐lycopenal
Apo‐8‘‐lycopenal
control CcCCD4b1
II
0 5 10 15 20 25 min
mAUat 350-650nm
control CcCCD4b1
I
A
B
A
300 400 500 600 nm
473
B
400 500 nm
459
A
B
Supplemental Figure S3. In vitroCCD4b1 enzymatic assays using apo-lycopenals as subtrates (A, Apo-8´-lycopenal (C30);
B apo-10´-lycopenal (C27).
Sweet orange Navel IG M1 M2 B1 B2 C FC Relative expression CCD4a
IG M B1 B2 C FC
Clementine Hybrid Fortune
IG M B1 B2 C FC 0.5
1.0 1.5 2.5 2.0 3.0
Figure S4. Relative transcript levels of CCD4ain the peel of Navel sweet orange, Clementine mandarin and the hybrid Fortune mandarin during fruit development and ripening.