Abstract
A cDNA clone (MEL5), encoding a protein homologous to phytoene synthase (PSY), has been isolated from a climacteric melon fruit cDNA library, using the tomato cDNA clone TOM5 [34] as a heterologous probe. MEL5 hybridised to a transcript of 1.65 kb which suggested that the 1.36 kb clone, isolated originally, was not full-length. The missing 5′ end was isolated by a reverse transcriptase-polymerase chain reaction (RT-PCR)-based method. This enabled the full sequence of the protein to be deduced and the cleavage site of the transit peptide for chromoplast import to be predicted. Northern analysis of RNA extracted from fruit samples of different ripening stages as well as from roots, leaves and flower petals was used to examine the expression pattern of the corresponding mRNA. The transcript corresponding to MEL5 is present at low quantities in unripe (green) fruit, reaches its highest levels when the fruit turns from green to orange and persists at lower levels during later ripening stages. A similar transcript was also detected in flower petals and in trace amounts in leaves and roots. Genomic Southern analysis indicates that the clone is homologous to a low-copy-number gene family. Sequence analysis showed a high degree of conservation among plant PSYs.
Similar content being viewed by others
References
Balague C, Watson CF, Turner AJ, Rouge P, Picton S, Pech J, Grierson D: Isolation of a ripening and wound-induced cDNA from Cucumis melo L. encoding a protein with homology to the ethylene-forming enzyme. Eur J Biochem 212: 27–34 (1993).
Bartley GE, Scolnik PA: cDNA cloning, expression during development, and genome mapping of PSY2, a second tomato gene encoding phytoene synthase. J Biol Chem 268: 25718–25721 (1993).
Bartely GE, Scolink PA, Giuliano G: Molecular biology of carotenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 45: 287–301 (1994).
Bartley GE, Viitanen PV, Bacot KO, Scolnik PA: A tomato gene expressed during fruit ripening encodes an enzyme of the carotenoid biosynthesis pathway. J Biol Chem 267: 5036–5039 (1992).
Berry-Lowe SL, Schmidt GW: Chloroplast protein transport. In: Bogorad L, Vasil IK (eds) Cell Culture and Somatic Cell Genetics of Plants, vol. 7A, p. 257. Academic Press, New York (1991).
Bird CR, Ray JA, Fletcher JD, Boniwell JM, Bird AS, Teulieres C, Blain I, Bramley PM, Schuch W: Using antisense RNA to study gene function: Inhibition of carotenoid biosynthesis in transgenic tomatoes. Bio/Technology 9: 635–639 (1991).
Bramley P, Teulieres C, Blain I, Bird C, Schuch W: Biochemical characterisation of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5. Plant J 2: 343–349 (1992).
Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159 (1987).
Clarke BC, Moran LB, Appels R: DNA analyses in wheat breeding. Genome 32: 334–339 (1989).
Dean C, Vandenelzen P, Tamakis S, Dunsmuir P, Bedbrook J: Differential expression of the eight genes of the petunia ribulose bisphosphate carboxylase small subunit multi-gene family. EMBO J 4: 3055–3061 (1985).
Edwards CL, Colbert JT: Regulation of phytochrome mRNA abundance in green oat leaves. Plant Cell Environ 13: 813–819 (1990).
Fraser PD, Truesdale MR, Bird CR, Schuch W, Bramley PM: Carotenoid biosynthesis during tomato fruit development. Evidence for tissue-specific gene expression. Plant Physiol 105: 405–413 (1994).
Fray RG, Grierson D: Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression. Plant Mol Biol 22: 589–602 (1993).
Frohman MA, Dush MK, Martin GR: Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85: 8998–9002 (1988).
Gavel Y, vonHeijne G: A conserved cleavage site motif in chloroplast transit peptides. FEBS Lett 261: 455–458 (1990).
Grierson D, Schuch W: Control of ripening. Phil Trans R Soc Lond B 342: 241–250 (1993).
Grierson D, Tucker GA: Timing of ethylene and polygalacturonase synthesis in relation to the control of tomato ripening. Planta 157: 174–179 (1983).
Gross J: Pigments in Fruits. Academic Press, New York (1987).
Keegstra K, Olsen L: Chloroplastic precursors and their transport across the envelope membranes. Annu Rev Plant Physiol Plant Mol Biol 40: 471–501 (1989).
Kinzer SM, Schwager SJ, Mutschler MA: Mapping of ripening-related or specific cDNA clones of tomato (Lycopersicon esculentum). Theor Appl Genet 79: 489–496 (1990).
Kreuz K, Beyer P, Kleinig H: The site of carotenogenic enzymes in chromoplasts from Narcissus pseudonarcissus L. Planta 154: 66–69 (1982).
Lawrence SD, Cline K, Moore GA: Chromoplasttargeted proteins in tomato (Lycopersicon esculentum Mill) fruit. Plant Physiol 102: 789–794 (1993).
Lutke-Brinkhaus F, Liedvogel B, Kreuz K, Kleinig H: Phytoene synthase and phytoene dehydrogenase associated with envelope membranes from spinach chloroplasts. Planta 156: 176–180 (1982).
Lutke-Brinkhaus F, Kleinig H: Carotenoid and chlorophyll biosynthesis in isolated plastids from mustard seedling cotyledons (Sinapis alba L.) during etioplast-chloroplast conversion. Planta 170: 121–129 (1987).
Mayfield SP, Nelson T, Taylor WC, Malkin R: Carotenoid synthesis and pleiotropic effects in carotenoid-deficient seedlings of maize. Planta 169: 23–32 (1986).
Olson JA: Provitamin-A function of carotenoids: the conversion of β-carotene into vitamin-A. J Nutr 119: 105–108 (1989).
Ray J, Moureau P, Bird C, Bird A, Grierson D, Maunders M, Truesdale M, Bramley P, Schuch W: Cloning and characterisation of a gene involved in phytoene synthesis from tomato. Plant Mol Biol 19: 401–404 (1992).
Romer S, Hugueney P, Bouvier F, Camara B, Kuntz M: Expression of the genes encoding the early carotenoid biosynthetic enzymes in Capsicum annuum. Biochem Biophys Res Comm 196: 1414–1421 (1993).
Sandmann G: Carotenoid biosynthesis in microorganisms and plants. Eur J Biochem 223: 7–24 (1994).
Sandmann G, Boger P: Inhibition of carotenoid biosynthesis by herbicides. In: Boger P, Sandmann G (eds) Target Sites of Herbicide Action, pp. 25–44. CRC Press, Boca Raton, FL (1989).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Scolnik PA, Bartley GE: Nucleotide Sequence of an Arabidopsis cDNA for phytoene synthase. Plant Physiol 104: 1471–1472 (1994).
Shellie KC, Saltveit MEJr: The lack of a respiratory rise in muskmelon fruit ripening on the plant challenges the definition of climacteric behaviour. J Exp Bot 44: 1403–1406 (1993).
Slater A, Maunders MK, Edwards K, Schuch W, Grierson D: Isolation and characterisation of cDNA clones for tomato polygalacturonase and other ripening-related proteins. Plant Mol Biol 5: 137–147 (1985).
Wardsworth GJ, Redinbaugh MG, Scandalios JG: A procedure for the small-scale isolation of RNA suitable for RNA blot analysis. Anal Biochem 172: 279–283 (1988).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Karvouni, Z., John, I., Taylor, J.E. et al. Isolation and characterisation of a melon cDNA clone encoding phytoene synthase. Plant Mol Biol 27, 1153–1162 (1995). https://doi.org/10.1007/BF00020888
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00020888