Carotenoid cleavage dioxygenase4 is a negative regulator of β-carotene content in Arabidopsis seeds

Abstract

Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed β-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for β-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID cleavage dioxygenase4 (CCD4) as a major negative regulator of seed carotenoid content, especially β-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing β-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with β-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in β-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops.

Figure 1.

Frequency Distribution of Seed Carotenoid Content in the Col-0/Ler and Cvi/Ler RILs. Lutein and total carotenoid levels, indicated by white and black arrows, respectively, are shown for Cvi, Ler, and Col-0 parents grown in parallel with the corresponding Col-0/Ler (A) and Cvi/Ler (B) RIL populations.

Figure 2.

QTL and Fine Mapping of the Chromosome 4 qCRT27 Candidate Gene, At4g19170, CCD4. (A) QTL analysis of 50 CSS4 × Col-0 homozygous F2 recombinant lines. The dashed line indicates the significance cutoff for QTL (LOD > 2.5); percentage values indicate the percentage phenotypic variance explained by each QTL. PDS, 8.19 Mb; NCED2, 10.14 Mb; CCD4, 10.48 Mb; BCH1 (β-CAROTENE HYDROXYLASE1), 13.09 Mb; CCD8, 15.82 Mb. (B) Fine mapping of qCRT27 to a 40.3-kb interval. Lines 6H-4-6 and 10G-1 are CSS4/Col-0 recombinants. Black bars denote the Col-0 genotype, and white bars denote Ler. ^aSeed carotenoid levels as a percentage of Col-0. (C) Location of the T-DNA insertion in the knockout mutant ccd4-1.

Figure 3.

Identification of CCD4 through Genome-Wide Association. (A) Manhattan plot of genome-wide association for seed β-carotene levels using the 6PC+K model. The x axis shows the physical positions of SNPs across the five Arabidopsis chromosomes, which are shown in alternating colors. The y axis shows the negative log of P values, with each dot representing an individual SNP. The candidate region containing CCD4 is indicated by a dashed area, with the arrow indicating the location of the most significant genome-wide SNP, SNP147077, at 10,482,452 bp on chromosome 4. (B) LD analysis relative to SNP147077 (black arrow) showed LD decay to be 8.35 kb encompassing three genes: At4g19160, At4g19170 (CCD4), and At4g19180. Black dashed boxes indicate seven haploblocks (HB) in the interval, with triple asterisks identifying HB2 and HB4, which have statistically (P = 2.33E-07 and P = 1.38E-07) contrasting haplotypes in the association panel. Inverted triangles denote InDels in the 5′ and 3′ regions of CCD4. Of these, only the 3′ InDel is significant (P = 5.49E-05, denoted by triple asterisks). The thick black line at the bottom indicates the region used in allelic complementation of ccd4-1. (C) Thirteen nonsynonymous SNPs (NS SNPs; in green) were located across the 1.7-kb CCD4 coding region. Of these nonsynonymous SNPs, two were within HB4: SNP 10, a Lys-to-Thr conversion, and SNP 11, an Arg-to-Thr conversion. SNP 10 is a rare Ler polymorphism and was present in only 5 of the 307 accessions. Additional details are provided in Supplemental Data Set 3 online. (D) β-Carotene seed levels (nmol/g) in lines with contrasting haplotypes for HB2 and HB4. For HB2, the 59 accessions with the TT haplotype showed β-carotene levels 40% higher than the 92 accessions with the AC haplotype. For HB4, the CT and TC haplotypes were present in 85 and 116 accessions, respectively, and had β-carotene levels 32 and 23% lower than the 105 accessions containing the CC haplotype. Triple asterisks indicate statistically significant differences (P < 0.0001).

Figure 4.

Expression Analysis of Selected Genes for Carotenoid Cleavage Enzymes during Seed Development. (A) Gene expression values for developing Col-0 seeds were obtained from the public AtGenExpress database (http://www.weigelworld.org/resources/microarray/AtGenExpress). Seed stages are as follows: 6, mid to late torpedo embryos; 7, late torpedo to early walking-stick embryos; 8, walking-stick to early curled cotyledon embryos; 9, curled cotyledons to early green cotyledon embryos; 10, green cotyledon embryos. PP2AA3, PROTEIN PHOSPHATASE2A SUBUNIT A3. (B) CCD4 mRNA levels in developing seeds of Col-0, Ler, and the Col-0 knockout line, ccd4-1, at 12, 15, 18, and 21 DAP. Seed stages are as follows: 12 to 15 DAP, green and fully matured seeds; 18 to 21 DAP, seed desiccation coincident with chlorophyll and water loss. Average values are shown with se for n = 3. Asterisks indicate statistically significant differences (**P < 0.001 and ***P < 0.0001) relative to Col-0.

Figure 5.

Fold Increase of Seed Carotenoid Content in Homozygous nced2-3, ccd1-1, ccd4-1, and ccd1-1 ccd4-1 Seeds Relative to the Col-0 Wild Type. The fold change for each carotenoid is expressed relative to the average level in Col-0 seeds (nmol/g): neoxanthin = 1.15, violaxanthin = 3.14, antheraxanthin = 3.11, lutein = 42.74, zeaxanthin = 4.97, β-carotene = 0.26, and total carotenoids = 55.37. Asterisks indicate statistically significant differences (*P < 0.01, **P < 0.001, and ***P < 0.0001).

Figure 6.

Carotenoid Levels during Seed Development and Desiccation in Col-0, ccd1-1, ccd4-1, and ccd1-1 ccd4-1 Lines. Flowers were tagged at anthesis, seeds were harvested at specific DAP, and carotenoids were analyzed by HPLC. Average values (nmol/seed) are shown with se for n = 3 of 150 seeds per biological replicate. Asterisks indicate statistically significant differences (*P < 0.01, **P < 0.001, and ***P < 0.0001) relative to Col-0. Error bars smaller than the symbols are not shown.

Figure 7.

Carotenoid Levels in Leaves of Col-0, ccd1-1, and ccd4-1 Lines Treated for the Indicated Days in Darkness to Induce Leaf Senescence. Individual leaves were covered with foil for 3, 6, and 10 d. Average values (nmol/g) are shown with se for n = 4. Asterisks indicate statistically significant differences (*P < 0.01 and **P < 0.001) relative to Col-0. Error bars smaller than the symbols are not shown.

Figure 8.

Seed Carotenoid Levels When ccd4-1 Is Functionally Complemented with Col-0, Ler, or Cvi CCD4 Alleles. Average values (nmol/g) are shown with se for 11, 14, and 6 ccd4-1 lines homozygous for the introduced Col-0, Ler, and Cvi CCD4 alleles, respectively, relative to wild-type Col-0 seeds. The dashed line indicates the wild-type Col-0 level for each seed carotenoid, with asterisks indicating statistically significant differences (*P < 0.01 and **P < 0.001) compared with wild-type Col-0. Average carotenoid levels in wild-type Col-0 seeds (nmol/g) were as follows: neoxanthin = 1.21, violaxanthin = 3.06, antheraxanthin = 2.75, lutein = 38.81, zeaxanthin = 3.41, β-carotene = 0.19, and total carotenoids = 49.43.