Recent Advancements and Biotechnological Implications of Carotenoid Metabolism of Brassica

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TL;DR

This review summarizes forward-genetic studies of carotenoid metabolism in Brassica crops, identifying key genes (CRTISO, ZEP, CCD4, OR) that control flower and leaf color by altering carotenoid composition. These findings enable biotechnological manipulation, such as CRISPR/Cas9 editing, to enhance nutritional value and ornamental traits. The clinical significance lies in the potential to increase dietary carotenoids (e.g., lutein, β-carotene) linked to reduced risk of age-related macular degeneration, cataracts, and other diseases.

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**Background:** Carotenoids are C40 isoprenoid pigments (e.g., lutein, β-carotene, zeaxanthin) essential for plant photosynthesis and photoprotection, and they provide bright colors to flowers and fruits. In humans, carotenoids serve as vitamin A precursors and are associated with reduced risk of age-related macular degeneration, cataracts, some cancers, and coronary heart disease. The genus Brassica includes many economically important vegetable crops (B. rapa, B. oleracea, B. napus, etc.) that accumulate diverse carotenoids. This review focuses on recent advances in understanding carotenoid metabolism in Brassica through forward genetics, the evolution of key genes, and biotechnological applications.

**Methods:** The authors conducted a narrative review of the literature, synthesizing findings from genetic mapping, QTL analysis, positional cloning, CRISPR/Cas9 mutagenesis, and biochemical characterization of carotenoid metabolism in various Brassica species. They examined studies on flower and leaf color mutants, gene expression analyses, and phylogenetic comparisons.

**Key Results:** The review identifies several major genes controlling carotenoid accumulation and color phenotypes: - **CRTISO**: In B. rapa, the BrCRTISO gene (Bra031539) controls orange pigmentation in inner leaves and flowers. Mutations (e.g., 90-bp promoter deletion, 501-bp 3′ insertion) reduce carotenoid flux, leading to orange leaves. In B. napus, double mutation of BnaA09.CRTISO and BnaC08.CRTISO causes milky white petals and pale yellow leaves, with decreased total carotenoids but increased lycopene, β-carotene, and α-carotene. In Chinese kale (B. oleracea), downregulation of BoaCRTISO reduces all carotenoid biosynthesis genes and turns leaves yellow. - **ZEP**: In B. rapa, a 679-bp insertion in BraA09.ZEP (Bra037130) causes loss of function, increasing total carotenoids and changing petals from yellow to dark yellow. In B. napus, nullification of both BnaA09.ZEP and BnaC09.ZEP (via CRISPR/Cas9) markedly increases lutein and decreases violaxanthin in petals, producing orange flowers. - **CCD4**: In B. napus, a CACTA-like transposable element insertion in BnaC3.CCD4 disrupts expression, leading to yellow flowers; white-flowered lines have higher CCD4 expression. In B. oleracea, BoCCD4 functional failure (e.g., by a 10,608-bp CACTA-like transposon) results in yellow petals. Overexpression of BoCCD4 changes petals from yellow to white or pale yellow. - **OR (Orange protein)**: In cauliflower (B. oleracea), a spontaneous semidominant OR mutant accumulates high β-carotene in normally unpigmented tissues due to a retrotransposon insertion. In B. rapa, the BrGOLDEN allele (BraA09g007080.3C) contains a 4.67-kb LTR insertion, increasing β-carotene by 13.6-fold and lutein in golden leafy heads. - **Other genes**: A mutant in B. napus with yellowish-white flowers (ywf) carries a C-to-T substitution in BnaA08.PDS3 (phytoene desaturase 3), causing premature termination and disrupted carotenoid biosynthesis. In kale, overexpression of BoaNXS (neoxanthin synthase) increases total and individual carotenoids, changing leaf color from yellow-green to green.

Evolutionary analysis shows that Brassica species underwent whole-genome triplication, leading to multiple gene copies. For example, ZEP homologs on chromosomes A07/C07 are leaf-specific, while those on A09/C09 are flower-specific, indicating tissue-specific diversification. The CACTA-like TE insertion in C3.CCD4 occurred before allopolyploidization of B. napus and B. carinata, explaining the prevalence of yellow flowers in these species.

**Clinical Implications:** The review highlights that dietary carotenoids from Brassica crops have documented health benefits, including reduced risk of age-related macular degeneration, cataract, some cancers, and coronary heart disease, as well as potential benefits for cognitive function. By elucidating the genetic basis of carotenoid accumulation, this research enables biotechnological strategies (e.g., CRISPR/Cas9, marker-assisted breeding) to enhance carotenoid content in Brassica vegetables. Such improvements could increase the nutritional value of these widely consumed crops, potentially contributing to public health by boosting dietary intake of lutein, β-carotene, and other carotenoids. The ability to manipulate flower color also has ornamental applications.