Carotenoid biosynthesis in plants has been described at the molecular level for most of the biochemical steps in the pathway. However, the cis-trans isomerization of carotenoids, which is known to occur in vivo, has remained a mystery since its discovery five decades ago. To elucidate the molecular mechanism of carotenoid isomerization, we have taken a genetic map-based approach to clone the tangerine locus from tomato. Fruit of tangerine are orange and accumulate prolycopene (7Z,9Z,7'Z,9'Z-tetra-cis-lycopene) instead of the all-trans-lycopene, which normally is synthesized in the wild type. Our data indicate that the tangerine gene, designated CRTISO, encodes an authentic carotenoid isomerase that is required during carotenoid desaturation. CRTISO is a redox-type enzyme structurally related to the bacterial-type phytoene desaturase CRTI. Two alleles of tangerine have been investigated. In tangerine(mic), loss of function is attributable to a deletion mutation in CRTISO, and in tangerine(3183), expression of this gene is impaired. CRTISO from tomato is expressed in all green tissues but is upregulated during fruit ripening and in flowers. The function of carotene isomerase in plants presumably is to enable carotenoid biosynthesis to occur in the dark and in nonphotosynthetic tissues.

Carotenoids are terpenoid pigments containing systems of conjugated double bonds, each of which can exist in a cis or trans configuration. The existence of a carotenoid isomerase (CrtISO) mediating cis-to-trans isomerization has been suspected ever since the description of the tomato tangerine mutant in the early 1940s, whose fruits accumulate a poly-cis-isomer of lycopene. Recently, CrtISO has been cloned in Synechocystis and in plants, and has been shown to be related to bacterial carotene desaturases.

Most enzymes in the central pathway of carotenoid biosynthesis in plants have been identified and studied at the molecular level. However, the specificity and role of cis-trans-isomerization of carotenoids, which occurs in vivo during carotene biosynthesis, remained unresolved. We have previously cloned from tomato (Solanum lycopersicum) the CrtISO gene, which encodes a carotene cis-trans-isomerase. To study the biochemical properties of the enzyme, we developed an enzymatic in vitro assay in which a purified tomato CRTISO polypeptide overexpressed in Escherichia coli cells is active in the presence of an E. coli lysate that includes membranes. We show that CRTISO is an authentic carotene isomerase. Its catalytic activity of cis-to-trans isomerization requires redox-active components, suggesting that isomerization is achieved by a reversible redox reaction acting at specific double bonds. Our data demonstrate that CRTISO isomerizes adjacent cis-double bonds at C7 and C9 pairwise into the trans-configuration, but is incapable of isomerizing single cis-double bonds at C9 and C9'. We conclude that CRTISO functions in the carotenoid biosynthesis pathway in parallel with zeta-carotene desaturation, by converting 7,9,9'-tri-cis-neurosporene to 9'-cis-neurosporene and 7'9'-di-cis-lycopene into all-trans-lycopene. These results establish that in plants carotene desaturation to lycopene proceeds via cis-carotene intermediates.