Abstract
The characteristic yellow fruit phenotype of the r,r mutant and Psy-1 (phytoene synthase-1) antisense tomatoes is due to a mutated or down-regulated phytoene synthase protein, respectively, resulting in the virtual absence of carotenoids. Based on detailed carotenoid determinations Psy-1 appeared to barely contribute to the formation of carotenoids in chloroplast-containing tissues. Despite the virtual absence of carotenoids in ripe fruit the formation of phytoene in vitro was detected in fruit of both mutants. When [14C]isopentenyl pyrophosphate (IPP) was used as the substrate for phytoene synthase a reduction (e.g. r,r mutant, 5-fold) in the formation of phytoene was observed with an accumulation (e.g. r,r mutant, 2-fold) of the immediate precursor geranylgeranyl pyrophosphate (GGPP). Contrastingly, reduced phytoene synthase activity was not detected when [3H]GGPP was used as the substrate. The profile of phytoene formation during ripening was also different in the down-regulated mutants compared to the wild-type. Using specific primers, RT-PCR analysis detected the presence of Psy-2 transcripts in the down-regulated mutants and wild-type throughout fruit development and ripening. These data were supported by the detection of phytoene synthase protein on western blots. Both GGPP formation and phytoene desaturation were elevated in these mutants. Therefore, it appears that despite the absence of carotenoids in ripe fruit, both the mutants have the enzymic capability to synthesize carotenoids in this tissue. Implications of the data with respect to the regulation of carotenoid formation and the channelling of prenyl lipid precursors in tomato (and its potential manipulation) are discussed.
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Anderson, I.C. and Robertson, D.S. 1960. Role of carotenoids in photoprotecting chlorophyll from photodestruction. Plant Physiol. 35: 531–534.
Bartley, G.E. and Scolnik, P.A. 1993. cDNA cloning, expression during development and genome mapping of Psy-2, a second tomato gene encoding phytoene synthase. J. Biol. Chem. 268: 25718–25721.
Bartley, G.E., Vitanen, P.V., Bacot, K.O. and Scolnik P.A. 1992. A tomato gene expressed during fruit ripening encodes an enzyme of the carotenoid biosynthesis pathway. J. Biol. Chem. 267: 5036–5039.
Bathgate, B., Purton, M.E., Grierson, D. and Goodenough, P.W. 1985. Plastid changes during the conversion of chloroplasts to chromoplasts in ripening tomatoes. Planta 165: 197–204.
Bird, C.R., Ray, J.A., Fletcher, J.D., Boniwell, J.M., Bird, A.S., Teulieres, C., Blain, I., Bramley, P.M. and Schuch, W. 1991. Using antisense RNA to study gene function: inhibition of carotenoid biosynthesis in transgenic tomatoes. Bio/technology 9: 635–639.
Bonk, M., Hoffmann, B., Von Lintig, J., Schledz, M., Al-Babili, S., Hobeika, E., Kleinig, H. and Beyer, P. 1997. Chloroplast import of four carotenoid biosynthetic enzymes in vitro reveals differential fates prior to membrane binding and oligomeric assembly. Eur. J. Biochem. 247: 942–950.
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.
Bramley, P.M., Davies, B.H. and Rees, A.F. 1974. Colour quenching by carotenoids. In: M.A Crook and P. Johnson (Eds.), Liquid Scintillation Counting, Vol 3. Heyden, London, pp. 76–85.
Bramley, P.M., Teulieres, C., Blain, I., Bird, C. and Schuch, W. 1992. Biochemical characterisation of transgenic tomato plants in which carotenoid synthesis has been inhibited through expression of antisense RNA to pTOM5. Plant J. 2: 343–349.
Britton, G. 1991. Carotenoids. In: B.V. Charlwood and D.V. Banthorpe (Eds.), Methods in Plant Biochemistry, Vol. 7. Academic Press, London, pp. 473–518.
Corona, V., Benedetto, A., Kosturkova, G., Bartley, G., Pitto, L., Giorgetti, L., Scolnik, P. and Giuliano, G. 1996. Regulation of a carotenoid biosynthesis gene promoter during plant development. Plant. J. 9: 241–247.
Davis, K.M. and Grierson, D. 1989. Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene. Planta 179: 73–80.
Dixon, R., Lamb, C.J., Masoud, S., Sewalt, V.J.H. and Paiva, N.L. 1996. Metabolic engineering: prospects for crop improvement through the genetic manipulation of phenyl propanoid biosynthesis and defense responses. Gene 179: 61–71.
Dogbo, O., Lafferiere, A., D'Harlingue, A. and Camara, B. 1988. Carotenoid biosynthesis: Isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene. Proc. Natl. Acad. Sci. USA 85: 7054–7058.
Fraser, P.D., de las Rivas, J., Mackenzie, A. and Bramley, P.M. 1991. Phycomyces blakesleeanus carB mutants: their use in assays of phytoene desaturase. Phytochemistry 12: 3971–3976.
Fraser, P.D., Misawa, N., Linden, H., Yamano, S., Kobayashi, K. and Sandmann, G. 1992. Expression in Escherichia coli, purifi-cation, and reactivation of the recombinant Erwinia uredovora phytoene desaturase. J. Biol. Chem. 267: 19891–19895.
Fraser, P.D., Albrecht, M., Sandmann, G. 1993a. Development of high performance liquid chromatographic systems for the sepa698 ration of radiolabeled carotenes and precursors formed in specific enzymatic reactions. J. Chromatog. 645: 265–272.
Fraser, P.D., Linden, H. and Sandmann, G. 1993b Purification and reactivation of recombinant Synechococcus phytoene desaturase from an overexpressing strain of Escherichia coli. Biochem. J. 291: 687–692.
Fraser, P.D., Truesdale, M., Bird, C.R., Schuch, W. and Bramley, P.M. 1994. Carotenoid biosynthesis during tomato fruit development. Plant Physiol. 105: 405–413.
Fray, R.G. and Grierson, D. 1993. Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression. PlantMol. Biol. 22: 589–602.
Giuliano, G., Bartley, G.E. and Scolnik, P.A. 1993. Regulation of carotenoid biosynthesis during tomato development. Plant Cell 5: 379–387.
Goodwin, T.W. 1980. In: T.W. Goodwin (Ed.), The Biochemistry of Carotenoids, Vol. 1, Plants. Chapman and Hall, New York.
Gottlob-McHugh, S.G., Sangwan, R.S., Blakeley, S.D., Vandeburghe, G.C., Ko, K., Turpin, D.H., Plaxton, W.C., Miki, B.L. and Dennis, D.T. 1992. Normal growth of transgenic tobacco plants in the absence of cytosolic pyruvate kinase. Plant Physiol. 100: 820–825.
Hajirezaei, M., Sonnewald, U., Viola, R., Carlisle, S., Dennis, D. and Stitt, M. 1994. Transgenic potato plants with strongly decreased expression of pyrophosphate:fructose-6-phophate phosphotransferase show no visible phenotype and minor changes in metabolic fluxes in their tubers. Planta 192: 16–30.
Hirschberg, H., Cohen, M., Harker, M., Lotan, T., Mann, V. and Pecker, I. 1997. Molecular genetics of the carotenoid biosynthesis pathway in plants and algae. Pure Appl. Chem. 69: 2145–2150.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Lichtenthaler, H.K. and Wellburn, A.R. 1983. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. 11: 591–592.
Misawa, N., Truesdale, M.R., Sandmann, G., Fraser, P.D., Bird, C., Schuch, W. and Bramley, P.M. 1994. Expression of a tomato cDNA coding for phytoene synthase in Escherichia coli, phytoene formation in vivo and in vitro, and functional analysis of the various truncated gene products. J. Biochem. 116: 980–985.
Murphy, L.D., Herzog, C.E., Rudick, J., Fojo, A.T. and Bates, S.E. 1990. Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry 29: 10351–10356.
Pecker, I., Gabby, R., Cunningham, F.X. and Hirschberg, J. 1996. Cloning and characterization of the cDNA for lycopene _-cyclase from tomato reveals decrease in its expression during fruit ripening. Plant Mol. Biol. 30: 807–819.
Porter, J. and Spurgeon, S. 1979. Enzymatic synthesis of carotenes. Pure Appl. Chem. 51: 609–622.
Ray, J., Bird, C.R., Maunders, M., Grierson, D. and Schuch, W. 1987. Sequence of pTOM5, a ripening related cDNA from tomato. Nucl. Acids Res. 15: 10587.
Römer, S., Hugueney, P., Bouvier, F., Camara, B. and Kuntz, M. 1993. Expression of the genes encoding the early carotenoid biosynthetic enzymes in Capsicum annuum. Biochem. Biophys. Res. Comm. 196: 1414–1421.
Schultz, D.J., Craig, R., Cox-Foster, D.L., Mumma, R.O. and Medford, J.I. 1994. RNA isolation from recalcitrant plant tissue. Plant Mol. Biol. Rep. 12: 310–316.
Scolnik, P.A. and Bartley, G.E. 1996. A table of some cloned plant genes involved in isoprenoid biosynthesis. Plant Mol. Biol. Rep. 14: 305–319.
Sreve, P.A. 1987. Complexes of sequential metabolic enzymes. Annu. Rev. Biochem. 56: 89–124.
Wessel, D. and Flügge, U.I. 1984. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138: 141–143.
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Fraser, P.D., Kiano, J.W., Truesdale, M.R. et al. Phytoene synthase-2 enzyme activity in tomato does not contribute to carotenoid synthesis in ripening fruit. Plant Mol Biol 40, 687–698 (1999). https://doi.org/10.1023/A:1006256302570
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DOI: https://doi.org/10.1023/A:1006256302570