Isolation and functional characterization of two dioxygenases putatively involved in bixin biosynthesis in annatto ( Bixa orellana L.)

Abstract

Carotenoid cleavage dioxygenases (CCDs) are enzymes that have been implicated in the biosynthesis of a wide diversity of secondary metabolites with important economic value, including bixin. Bixin is the second most used pigment in the world's food industry worldwide, and its main source is the aril of achiote (Bixa orellana L.) seeds. A recent transcriptome analysis of B. orellana identified a new set of eight CCD members (BoCCD4s and BoCCD1s) potentially involved in bixin synthesis. We used several approaches in order to discriminate the best candidates with CCDs genes. A reverse transcription-PCR (RT-qPCR) expression analysis was carried out in five developmental stages of two accessions of B. orellana seeds with different bixin contents: (P13W, low bixin producer and N4P, high bixin producer). The results showed that three BoCCDs (BoCCD4-1, BoCCD4-3, and BoCCD1-1) had an expression pattern consistent with bixin accumulation during seed development. Additionally, an alignment of the CCD enzyme family and homology models of proteins were generated to verify whether the newly proposed CCD enzymes were bona fide CCDs. The study confirmed that these three enzymes were well-preserved and belonged to the CCD family. In a second selection round, the three CCD genes were analyzed by in situ RT-qPCR in seed tissue. Results indicated that BoCCD4-3 and BoCCD1-1 exhibited tissue-specific expressions in the seed aril. To test whether the two selected CCDs had enzymatic activity, they were expressed in Escherichia coli; activity was determined by identifying their products in the crude extract using UHPLC-ESI-QTOF-MS/MS. The cleavage product (bixin aldehyde) was also analyzed by Fourier transform infrared. The results indicated that both BoCCD4-3 and BoCCD1-1 cleave lycopene in vitro at 5,6-5',6'.

Keywords: Annatto; Apocarotenoids; Bixa orellana; Bixin aldehyde; Bixin synthesis; Carotenoid dioxygenase.

Figure 1. Bixin biosynthesis in B. orellana L.

The possible cleavage sites and BoCCDs involved were determined based on the substrates and cleavage sites of type 4 CCDs. The region selectivity has been well-characterized in this and other plants. BoCCD, Carotenoid cleavage dioxygenases; BoALDH, bixin aldehyde dioxygenase; BoMET, norbixin methyltransferase.

Figure 2. Annatto cultivars with contrasting morphological characteristics.

(A) N4P and P13W cultivar characteristics. L, leaf; FB, floral bud; F, flower; IFR, immature fruit; IS, immature seed; MFR, mature fruit; MS: mature seed. (B) General representation of seeds in B. orellana accessions. Stages of seed development: S1–S5. Bar. 100 μm. Photos by Victor Manuel Carballo-Uicab.

Figure 3. In silico analysis of BoCCDs.

(A) Sequence alignments of CCD enzymes, alignment of B. orellana BoCCD-like proteins and subcellular locations of BoCCDs by iPSORT. The phylogenetic tree was inferred using the maximum-likelihood method based on the Jones–Taylor–Thornton (JTT) substitution model and was gamma distributed with Invariant sites (G + I) in MEGA6. Osmanthus fragrans (ABY60887.1), Oryza sativa Japonica group (NP_001047858.1), Crocus sativus (ACD62476.1), Theobroma cacao (XP_007023695.1), Z. mays (O24592), Citrus unshiu (BAE92957.1), Vitis vinifera (AAX48772.1), Coffea canephora (ABA43900.1), O. fragrans (BAJ05401.1), B. orellana L. (CAD71148.1, ABS19630.2, KT359018, KT359019, KT359020, KT359021, KT359022, KT359023, KT359024), and Arabidopsis thaliana (NP_191911.1). Numbers near the branch points represent the bootstrap value produced by 1,000 replications. Phylogenetic trees were rooted with Synechocystis apocarotenoid cleavage oxygenase (ACO) (P74334). Orange diamonds in the tree indicate the sequences in the study. The representative alignment indicates the signal peptide in the green rectangle according to iPSORT. Red letters indicate residues of histidine, and yellow letters indicate residues of aspartate or glutamate. Gene ontology (GO) annotation was performed with Blast2GO software for InterPro scanning to determine potential function of BoCCDs. Top 10 GO description in the three main categories, biological process, molecular function, and cellular component (See Dataset3, Götz et al., 2008). (B) Structural superposition of BoCCD1-1 (green), BoCCD4-1 (light blue), and BoCCD4-3 (lawn green) homology models showing the catalytic iron (red sphere) and α-helical and β-propeller domains. Right inset, close-up of four Fe²⁺-coordinating histidine residues (black cylinders).

Figure 4. Histological analysis of B. orellana L. seeds.

(A) Immature seed, (B) immature seed longitudinal cut, (C) immature seed longitudinal cut in paraffin, (D) S2 structure, (E) S3 structure, (F) S4 structure. BSC, bixin storage cell. Bar 100 μm. Photos by Victor Manuel Carballo-Uicab.

Figure 5. Transcript levels of BoCCD genes throughout B. orellana L. seed development.

(A–H) Transcript levels of BoCCDs in the N4P (red bar) and the P13W (white bar) accession. The expression values are relative to leaf (external control), which was set to 1 and the constitutive 18S gene. The X-axis represent the seed stages: S1, S2, S3, S4, and S5. Error bars are standard errors of the mean from three technical replicates, P = 0.05 level. Bars with similar letters were not significantly different (P = 0.05) by two-way ANOVA followed by a multiple-comparisons T-test.

Figure 6. In situ RT-PCR of selected BoCCDs.

The blue strain indicates gene expression. (A and G) Immature seed without treatment, (B and H) 18S negative control, (C and I) 18S positive control, (D and J) BoCCD1-1 gene, (E and K) BoCCD4-3 gene, and (F and L) BoCCD4-1 gene. white arrow: BSC (bixin storage cells), yellow arrow: endosperm, Bar 100 μm. Photos by Victor Manuel Carballo-Uicab.

Figure 7. Analysis by HPLC, UHPLC, and MS.

Recombinant BoCCDs in E. coli total extracts: (A) pACCRT-EIB, (B) pDEST-BoCCD1-1, (C) pDEST-BoCCD4-3, (D) HPLC chromatograms: lycopene standard (blue line), pACCRT-EIB extract (red line), pDEST-BoCCD1-1 (pink line), pDEST-BoCCD4-3 (green line), box: lycopene UV spectrum, UHPLC-MS of pDEST-BoCCD4-3, (E) lycopene chromatogram (red star), (F) lycopene MS spectrum with lycopene detection (m/z 536.3), (G) bixin aldehyde chromatogram (yellow star), and (H) bixin aldehyde MS spectrum (m/z 349.2), and L = 505 nm, T = 25 °C, running time = 30 min.

Figure 8. Analysis by UHPLC-ESI-QTOF-MS/MS.

Lycopene (m/z 536.3) (red star) and bixin aldehyde detection (m/z 349.2) (orange star) in (A) pACCRT-EIB, (B) pDESTBoCCD1-1, and (C) pDEST-BoCCD4-3.

Figure 9. FTIR spectrum.

(A) pDESTBoCCD1-1, (B) pDESTBoCCD4-3, and (C) bixin standard as a control of harmonic absorptions of NIR (near-infrared overtone absorption): between CHO and CH (*, **) and between CH₂ and CH₃.

Figure 10. Analysis by UHPLC-ESI-QTOF-MS/MS.

Bixin aldehyde detection (m/z 349.2) (orange star) adduct [M+H]⁺ and lycopene (m/z 536.18) (red star) to 1 min (A) pDESTBoCCD1-1, (B) pDESTBoCCD1-1+LYC, (C) pDESTBoCCD4-3, and (D) pDESTBoCCD4-3+LYC. LYC, lycopene.

Figure 11. Bixin biosynthesis model of gene regulation in bixin biosynthesis.

The black solid line represents the cell wall. The double green line represents the plastid. The grey circles represent plastoglobules (PGs). The structure in the form of “S” represent plastid stromules. Membrane-associated plastid complex enzymes: PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; β-LCY, lycopene β-cyclase; ε-LCY, lycopene ε-cyclase; LYC, lycopene; β-Car, β-carotene; Zea, Zeaxanthin, B. ALDH., Bixin aldehyde; C₃₂ (C₃₂ intermediate). Bixin synthesis: BoCCD, Carotenoid cleavage dioxygenases; BoALDH, bixin aldehyde dioxygenase; BoMET norbixin methyltransferase (A) Bixin synthesis, is carried out in plastids: BoCCD4-3 cleaves lycopene symmetrically at position 5,6 (5′,6′) to produce bixin aldehyde and continues to bixin synthesis. Bixin could be transported by plastid stromules into BSCs. (B) Bixin synthesis is partially carried out in plastids and is finished in the cytosol. First BoCCD4-3 cleaves lycopene asymmetrically at position 5,6 or (5′,6′) and the resulting product (C₃₂) diffuses out of the plastid and is used by BoCCD1-1 to form bixin aldehyde.