Minisymposium 3: Secondary metabolism

Abs # 13002: Cloning and molecular characterization of the tangerine locus from tomato reveal the pathway of trans-carotenoid biosynthesis in plants

Presenter:	Isaacson, Tal , tisaacson@pob.huji.ac.il
Authors	Isaacson, Tal  (A)   Ronen, Gil  (A)   Amiton, Moshe  (A)   Ohad, Itzhak  (A)   Zamir, Dani  (A)   Beyer, Peter  (B)   Hirschberg, Joseph  (A)
Affiliations:	(A): Departments of Genetics, Biological Chemistry and Field Crops, The Hebrew University of Jerusalem, Israel (B): Center for Applied Biosciences, Universitat Freiburg, Germany

Plant carotenoids are 40-carbon isoprenoids with polyene chains that may contain up to 15 conjugated double bonds. In plants carotenoids are synthesized within the plastids by enzymes that are nuclear encoded. Most enzymes in the centeral pathway of carotenoid biosynthesis in plants has been identified and studied at the molecular level. However, the cis-trans isomerization of carotenoids, which is known to occur in-vivo, has remained unresolved since its discovery over 60 years ago. To elucidate the molecular mechanism of carotenoid isomerization we have taken a map-based cloning approach to clone the tangerine locus from tomato. Fruits of tangerine accumulate prolycopene (7,9,7',9'-tetra-cis-lycopene) instead of all-trans lycopene, which normally accumulates in the wild type. All-trans-lycopene is an essential precursor for cyclic carotenoids (β-carotene) and xanthophylls. The locus tangerine was mapped to long arm of chromosome #10. We have cloned the tangerine gene and designated it CrtISO. The predicted protein encoded by CrtISO contains a di-nucleotide binding motive at its N-terminus, and shows similarity to bacterial and plant carotene desaturases. To study the biochemical properties of CRTISO we have developed an in-vitro assay that is based on purified protein from E. coli cells expressing CrtISO mixed with membranes isolated from E. Coli. We have established that CRTISO is an authentic carotene isomerase. Its catalytic activity of cis to trans isomerization is driven by redox, most probably through a bound flavine suggesting that the mechanism of the reaction involved electron donation to convert the double bond to a single bond which is then desaturated again following rotation of the molecule. Our data suggest that CRTISO functions in the carotenoid biosynthesis pathway in parallel with ζ-carotene desaturation by ZDS, by converting 9,7',9'-cis-neurosporene to 9-cis-neurosporene and 9,7-cis-lycopene to all-trans-lycopene. In mutants that lack functional CRTISO cis to trans isomerization takes place in green tissues only as a result of exposure to light. We conclude that CRTISO is essential fot all-trans-lycopene synthesis in the dark for example during seed germination, and in chromoplasts of flowers and fruits.