C-Carotene Is Transcriptionally Regulated During Tomato
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Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4962-4966, June 1992 Plant Biology A single polypeptide catalyzing the conversion of phytoene to C-carotene is transcriptionally regulated during tomato fruit ripening (carotenoid biosynthesis/phytoene desaturase/pahtids) IRIS PECKER*, DANIEL CHAMOVITZ*, HARTMUT LINDENt, GERHARD SANDMANNt, AND JOSEPH HIRSCHBERG*t *Department of Genetics, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; and tLehrstuhl ffr Physiologie und Biochemie der Pflanzen, Universitlit Konstanz, Postfach 5560, D-7750 Konstanz, Federal Republic of Germany Communicated by Robert Haselkorn, January 29, 1992 (receivedfor review July 30, 1991) ABSTRACT The cDNA of the gene pds from tomato, ogous genes from higher plants by using the bacterial genes encoding the carotenoid biosynthesis enzyme phytoene desat- as heterologous probes have been unsuccessful. urase, was cloned, and its nucleotide sequence was determined. Recently we cloned the gene pds, which encodes PDS, Cells of Eschenchia coli that expressed the tomato pds gene from the cyanobacterium Synechococcus PCC7942 (13, 14). could convert phytoene to C-carotene. This result sug s that We report here the cloning and nucleotide sequence deter- one polypeptide, the product of the pds gene, can carry out mination of the pds homologue from tomato (Lycopersicon phytoene desaturation in the carotenoid biosynthetic pathway. esculentum).§ Expression of this gene in Escherichia coli Transcripts of thepds gene accumulate in orange tomato fruit, indicates that it codes for PDS that functions in the conver- indicting transcriptional control ofpdy expression during fruit sion of phytoene to C-carotene. Comparison of the deduced ripening. The deduced amino acid sequence of phytoene de- amino acid sequences of pds from a plant, an alga, and a saturase indicates that this enzyme in tomato contains 583 cyanobacterium with the published sequences of crtI gene amino acids that are highly conserved with respect to the products from other prokaryotes and the al-i gene product homologous enzymes in cyanobacteria and algae. The deduced from Neurospora indicates that two distinct, evolutionarily amino acid sequences of the phytoene desaturases from other unrelated types of PDS enzymes exist in nature. microorganisms (purple bacteria and fungi) appear to be evolutionarily unrelated to those from green photosynthetic MATERIALS AND METHODS organisms. Cloning and Nuceotide Sequence Determination of pds. A cDNA library from the green alga Dunaliella bardawil in Carotenoid pigments are widely distributed in nature. They Agtll was provided by Ada Zamir and Amnon Lers from the are essential components of the photosynthetic apparatus, Weizmann Institute. This library was probed with a 32p where they serve in protecting against photooxidative dam- labeled 0.7-kilobase (kb) Acc 1-HindIII fragment of the pds age and also contribute to light harvesting for photosynthesis. gene from Synechococcus PCC7942 (14). A positive clone Carotenoids also serve as antioxidants and colorants in plants was isolated, phage DNA was extracted, and the cDNA and animals (1, 2). They are the major source of vitamin A insert was subcloned into the plasmid vector pUC118. Phage (retinol) in animals (3), are precursors for the synthesis of infection and DNA extraction were according to established abscisic acid in plants (4), and may also function as anticancer procedures (15). agents (5) and as immune system enhancers (6). A cDNA library prepared from ripening tomato fruit Carotenoids are synthesized by all photosynthetic orga- (UC82-B) in A ZAPII, given to us by Lee McIntosh from nisms as well as by several nonphotosynthetic bacteria and Michigan State University, was screened with a 0.8-kb Sac I fungi. The first committed step of carotenoid biosynthesis, internal fragment from the D. bardawilpds gene. Infection of common to all organisms, is the condensation of two mole- E. coli XLI-Blue and screening were according to Stratagene cules of geranylgeranyl pyrophosphate, yielding cis- protocols. Hybridization was done at low stringency: 35% phytoene, a C40 colorless hydrocarbon. Stepwise desatura- (vol/vol) formamide/5x standard saline citrate, 5X Den- tion (dehydrogenation) reactions convert phytoene to lyco- hardt's solution/0.1% SDS/50 mM sodium phosphate for 20 pene via phytofluene, {-carotene, and neurosporene. Two hr at 370C. Filters were washed twice with 2x standard saline cyclization reactions convert lycopene to a-carotene. In citrate at 370C. plants and algae the desaturation and cyclization reactions DNA was sequenced by the dideoxynucleotide chain- occur within plastids and are catalyzed by integral membrane termination reaction (16) using T7 DNA polymerase enzymes (7). The number of different enzymes involved in (Promega). Sac I fragments ofthe D. bardawilpds gene were each of these steps is unknown. subcloned into pUC118 and sequenced by using the universal Clusters of genes devoted to carotenoid biosynthesis have forward and reverse primers. Appropriate oligonucleotides been cloned from the nonsulfur purple photosynthetic bac- were synthesized to use as additional internal sequencing terium Rhodobacter capsulatus (8, 9) and from the nonpho- primers. Overlapping deletions of the tomato pds gene were tosynthetic bacteria Erwinia herbicola (10) and Erwinia ure- generated using the Erase-a-Base kit (Promega). Subclones dovora (11). Among the genes identified in these prokaryotes of restriction fragments, obtained by digestion with EcoRI, is crtI, which codes for phytoene desaturase (PDS). A HindIII, Pst I, and Sph I, were sequenced with the universal homologous gene, al-1, has been cloned from the fungus primers. All regions were sequenced in both directions. Neurospora crassa (12). However, attempts to clone homol- Abbreviation: PDS, phytoene desaturase. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. X59948). 4962 Downloaded by guest on September 29, 2021 Plant Biology: Pecker et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4963 Northern (RNA) Analysis. Total RNA was isolated from 50 (13, 14). The cyanobacterial pds gene hybridized very weakly g of pericarp from mature green and orange tomato fruit and to a tomato genomic DNA blot but hybridized strongly with from 5 g of leaves, according to the guanidine thiocyanate/ DNA from the green alga D. bardawil. We therefore decided cesium chloride protocol described in ref. 15 with the fol- to first clone the algal gene and then to use it as a probe to lowing modifications: the tissue was first pulverized in liquid isolate the plant pds gene. A portion of the cyanobacterial N2; the homogenization buffer contained 5 M guanidine gene was therefore used to screen a cDNA library from D. thiocyanate and 8% (vol/vol) 2-mercaptoethanol; sodium bardawil. A positive clone was isolated, and the cDNA insert lauryl sulfate was not added to the homogenate. Poly(A)+ was subcloned into the plasmid vector pUC118. Sequence mRNA was isolated by binding to oligo(dT)-cellulose beads analysis revealed an open reading frame coding for a poly- (17) (Boehringer Mannheim) according to the manufacturer's peptide, which had an amino acid sequence highly similar to protocol. Twenty-five micrograms of poly(A)+-enriched that of the cyanobacterial PDS (data not shown). The cloned RNA of each sample was separated by electrophoresis in a D. bardawil pds cDNA served as a molecular probe for formaldehyde-containing agarose gel and transferred to a screening the tomato cDNA library. Four recombinant Blue- GeneScreen membrane (DuPont). The entire cDNA clone of script SK- plasmids that contained the same DNA sequence pds was labeled with 32P and used as probe. Hybridization were identified. The nucleotide sequence of the longest was done in 35% (vol/vol) formamide/3 x standard saline cDNA clone, in a plasmid designated pTPD, was determined. citrate/1% SDS/2.5 x Denhardt's solution/salmon sperm An open reading frame of583 codons was found, which codes DNA at 100 gg/ml at 370C. After hybridization, the filter was for a polypeptide with a calculated molecular mass of 64.9 washed in lx SSC/0.1% SDS at 370C. kDa (Fig. 1). This coding region is flanked at its 5' end by an Amino Acid Sequence Comparisons. Amino acid sequence untranslated sequence of >440 nucleotides. comparisons were done using the GAP program of the Ge- Expression of the Tomato pds Gene in E. coli. Normally, E. netics Computer Group sequence analysis software package coli cells do not synthesize carotenoids. However, cells ofE. (version 6.2, June 1990) and HIBIO PROSIS (Hitachi Software coli JM109, which carry-the plasmid pACCRT-BE, produce Engineering, Tokyo). high quantities of phytoene (19). This plasmid contains the Analysis of Carotenoid Biosynthetic Intermediates. Cells of genes crtE and crtB from E. uredovora (11), which code for E. coli strain JM109, carrying carotenoid biosynthetic genes geranylgeranyl pyrophosphate synthase and phytoene syn- on the plasmids pACCRT-BE and pTPDEX (see text and Fig. thase, respectively, on the vector pACYC184 (Fig. 2). An 2 for details), were grown to stationary phase. Extraction of expression vector carrying the pds gene from tomato, pTP- carotenoids and HPLC analysis were according to the