Functional Analysis of the b and e Lycopene Cyclase Enzymes of Arabidopsis Reveals a Mechanism for Control of Cyclic Carotenoid Formation

Abstract

Carotenoids with cyclic end groups are essential components of the photosynthetic membranes in all plants, algae, and cyanobacteria. These lipid-soluble compounds protect against photooxidation, harvest light for photosynthesis, and dissipate excess light energy absorbed by the antenna pigments. The cyclization of lycopene (psi, psi-carotene) is a key branch point in the pathway of carotenoid biosynthesis. Two types of cyclic end groups are found in higher plant carotenoids: the beta and epsilon rings. Carotenoids with two beta rings are ubiquitous, and those with one beta and one epsilon ring are common; however, carotenoids with two epsilon rings are rare. We have identified and sequenced cDNAs that encode the enzymes catalyzing the formation of these two rings in Arabidopsis. These beta and epsilon cyclases are encoded by related, single-copy genes, and both enzymes use the linear, symmetrical lycopene as a substrate. However, the epsilon cyclase adds only one ring, forming the monocyclic delta-carotene (epsilon, psi-carotene), whereas the beta cyclase introduces a ring at both ends of lycopene to form the bicyclic beta-carotene (beta, beta-carotene). When combined, the beta and epsilon cyclases convert lycopene to alpha-carotene (beta, epsilon-carotene), a carotenoid with one beta and one epsilon ring. The inability of the epsilon cyclase to catalyze the introduction of a second epsilon ring reveals the mechanism by which production and proportions of beta,beta- and beta, epsilon-carotenoids may be controlled and adjusted in plants and algae, while avoiding the formation of the inappropriate epsilon,epsilon-carotenoids.

Cloning and characterisation of a maize carotenoid cleavage dioxygenase (ZmCCD1) and its involvement in the biosynthesis of apocarotenoids with various roles in mutualistic and parasitic interactions

“…4 GC-MS analysis of ZmCCD1 cleavage products. ZmCCD1 was expressed in E. coli strains accumulating diVerent carotenoid substrates: a lycopene (pACLYC), b -carotene (pACBETA), c zeaxanthin (pACZEAX) (Cunningham et al 1996). Each panel shows a chromatogram for ZmCCD1 (upper chromatogram) and the corresponding empty vector control (lower chromatogram).…”

“…The resulting PCR products were cloned into the BamHI and EcoRI sites in pRSETA (Invitrogen Breda, The Netherlands). Plasmid pRSETA-ZmCCD1A, plasmid pRSETAZmCCD1B, plasmid pRSETA-AtCCD1 as positive control and pRSETA (empty vector) as a negative control were transformed to E. coli BL21 (DE3) pLysS carrying expression plasmids for lycopene, -carotene, and zeaxanthin biosynthesis (Cunningham et al 1996). The transformed E. coli were grown overnight at 37°C on LB solid medium containing 50 g mL ¡1 of ampicillin, 35 g mL ¡1 chloramphenicol and 1% glucose (ampicillin and chloramphenicol from Duchefa, Haarlem, The Netherlands).…”

“…MBS 847 was cloned into the E. coli expression vector pRSETA, which was transformed to E. coli strains engineered to accumulate lycopene, -carotene and zeaxanthin (Cunningham et al 1996). Colonies of these three carotenoid accumulating E. coli strains develop a reddish, orange or yellowish colour, respectively.…”

“…In yellow segments of colonised roots ZmCCD1 transcript levels were 4.1-fold higher than in the white segments of the same mycorrhizal root system. The phosphate transporter ZmPht1.6 was strongly upregulated in colonised total roots (128-fold) compared with (Cunningham et al 1996). Arabidopsis thaliana CCD1 (AtCCD1) was used as positive, an empty vector as negative control; ZmCCD1-A is a variant of ZmCCD1-B having a 27-bp 5Ј extension starting with an ATG in the same reading frame as ZmCCD1-B.…”

The Arabidopsis Spontaneous Cell Death1 gene, encoding a ζ-carotene desaturase essential for carotenoid biosynthesis, is involved in chloroplast development, photoprotection and retrograde signalling

“…In one branch, lycopene undergoes two sequential cyclization reactions to form δ-and α-carotene, followed by the conversion into lutein as the end product. In a second branch, a series of reactions, including cyclization and hydroxylation, result in the production of γ-carotene, β-carotene and zeaxanthin, respectively [19,20]. Epoxidation of zeaxanthin leads to the formation of violaxanthin that can be converted into neoxanthin, the end product of the branch in the plastid.…”