Identification and expression pattern of a new carotenoid cleavage dioxygenase gene member from Bixa orellana

Abstract

Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes involved in the biosynthesis of a broad diversity of secondary metabolites known as apocarotenoids. In plants, CCDs are part of a genetic family with members which cleave specific double bonds of carotenoid molecules. CCDs are involved in the production of diverse and important metabolites such as vitamin A and abscisic acid (ABA). Bixa orellana L. is the main source of the natural pigment annatto or bixin, an apocarotenoid accumulated in large quantities in its seeds. Bixin biosynthesis has been studied and the involvement of a CCD has been confirmed in vitro. However, the CCD genes involved in the biosynthesis of the wide variety of apocarotenoids found in this plant have not been well documented. In this study, a new CCD1 gene member (BoCCD1) was identified and its expression was charaterized in different plant tissues of B. orellana plantlets and adult plants. The BoCCD1 sequence showed high homology with plant CCD1s involved mainly in the cleavage of carotenoids in several sites to generate multiple apocarotenoid products. Here, the expression profiles of the BoCCD1 gene were analysed and discussed in relation to total carotenoids and other important apocarotenoids such as bixin.

Fig. 1.

Histological analysis of different bixin-accumulating Bixa orellana plant organs. (A–D) Plantlet root (A, longitudinal section; B–D, transversal section). (E–H) Stalk (E, longitudinal section; F and G, transversal sections). (I–L) Immature seed (I, longitudinal section; J, aril cells; K and L, transversal sections). (M–P) Leaves (M and N, blade section; O and P, transversal section). A, E, I, J, M, and N are unstained tissue sections. Arrows indicate bixin accumulation. Scale bars in G, M, N, O, P=400 μm; A, B, F, I, K=200 μm; C, D, E, H, L=100 μm.

Fig. 2.

(A) Translated sequence alignment of the CCD probe isolated with other plant dioxygenases. Boxes indicate the location of the RPE65 domain. (B) Phenogram obtained from the Neighbor–Joining phylogenetic analysis of the B. orellana CCD isolated. The percentage of parsimonious trees in which the associated taxa clustered together (bootstrap value >1000 repetitions) is shown next to the branches. The arrow indicates the gene under study. Brackets indicate the main carotenoid cleavage dioxygenase families. Accession numbers are in parentheses.

Fig. 3.

BoCCD1 expression of different organs of B. orellana plants. (A) Images of the different organs used for semi-quantitative RT-PCR analysis. L, leaf; FB, floral bud; F, flower; IF, immature fruit; IS, immature seed; MS, mature seed. (B) BoCCD1 RT-PCR expression profile obtained for the tissues shown in (A). (C) Six different developmental stages of the immature seeds. (D) Expression profile of BoCCD1 obtained during seed development. (E) Photograph of a 30-day-old plantlet. (F) BoCCD1 expression profile obtained for B. orellana plantlets. BoCCD1 and 18S rRNA gene RT-PCR analyses were conducted with 30 and 25 cycles, respectively. R, root; S, stalk; L, leaf.

Fig. 4.

BoCCD1 gene location in seed preparations (A–D) and in the dissected aril layer (E–H) from the immature seeds of B. orellana. Seed and dissected fresh aril preparations are shown in A and E. In situ RT-PCR tissue preparations are shown in B–D and F–H, with the following treatments: (B) and (F) BoCCD1 amplification; (C) and (G) actin amplification; (D) and (H) digestion with RNase prior to BoCCD1 amplification. Arrowheads point to RT-PCR-positive cells. A, B, and E are unstained tissue sections Tissue abbreviations: ar, aril; oc, oily cells; en, endosperm. Scale bars=400 μm.

Fig. 5.

BoCCD1 gene location in stalk preparations. (A and B) BoCCD1; (C and D) actin; (E and F) RNase+BoCCD1; (G and H) PAS staining. All panels show transversal sections. Scale bars=100 μm. pc, pericycle; ph, phloem; xl, xylem; cp, cells of parenchyma; pd, peridermis; fb, fibres; pt, pith; ph, phloem; bx, bixin inclusions. Arrowheads: RT-PCR-positive cells.

Fig. 6.

Hypothetical biosynthesis pathway of linear apocarotenoids reported for Bixa orellana. The possible cleavage sites and CCDs involved were determined based on the substrates and cleavage sites of type 1 CCDs (such as the one isolated in this study) and type 4 (BoLCD; Bouvier et al., 2003), the regioselectivity of which has been well charaterized in this and in other plants. The question marks relate to possible precursors and routes of biosynthesis inferred on the basis of the chemical structure observed. The small dotted lines indicate the possible cleavage sites of the CCDs, whose hypothetical function is indicated.