TY - JOUR
AU - Pugliesi,, Claudio
AB - Abstract The non dormant-1 (nd-1) mutant of sunflower (Helianthus annuus L.) is characterized by an albino and viviparous phenotype. Pigment analysis by spectrophotometer and HPLC demonstrated in nd-1 cotyledons the absence of β-carotene, lutein and violaxanthin. Additionally, we found a strong accumulation of ζ-carotene and, to a lesser extent, of phytofluene and cis-phytoene in nd-1 seedlings grown in very dim light (1 µmol m–2 s–1). These results suggested that ζ-carotene desaturation was impaired in the mutant plants. To understand the molecular basis of the nd-1 mutation, we cloned and characterized the ζ-carotene desaturase (Zds) gene from sunflower. A reconstructed full-length sequence (1,916 bp) of the Zds cDNA was obtained from homozygous Nd-1/Nd-1 wild-type plants. It contains a 1,761-bp CDS, 62 nucleotides of 5′-untranslated region (UTR), and 77 nucleotides of 3′-UTR. The predicted protein (64.9 kDa) consists of 587 amino acid residues with a putative transit sequence for plastid targeting in the N-terminal region and a typical amino oxidase domain that includes the flavin adenosine dinucleotide (FAD) binding motif. The phylogenetic analysis demonstrated that the sunflower Zds was clustered to marigold (Tagetes) Zds gene, for which it showed an overall aminoacidic identity of 96.6% and resulted strictly correlated with other Zds sequences of higher plants. Interestingly, RT-PCR analyses showed that nd-1 plants were unable to accumulate Zds transcripts. Sequence information from the Zds cDNA was used to design specific primers and to isolate the full-length exons/introns region of the gene. The sunflower Zds gene (HaZds) comprises 14 exons and 13 introns scattered in a ca. 5.0-kb region. Also, HaZds showed a high conservation of the distribution and size of the exons with rice Zds gene. Based on genomic Southern analysis, the nd-1 genome disclosed a large deficiency at the Zds locus. (Received December 5, 2003; Accepted February 2, 2004) Introduction In higher plants, carotenoids are tetraterpenoid pigments of chloroplast and chromoplast membranes characterized by a C40 backbone with polyene chains, that may contain up to 15 conjugated double bonds. Because of their chemical properties, carotenoids are essential components of all photosynthetic organisms. In green tissues, such as leaves, carotenoids are fundamental to the light-harvesting function and protection against the effects of singlet oxygen and free radicals generated in the presence of light and endogenous photosensitizers such as chlorophylls, heme, and protoporphyrin IX. In addition, carotenoids also function as a redox intermediate in the electron-transfer processes of photosystem II (Tracewell et al. 2001). In chromoplasts this class of isoprenoids is responsible, in many cases, for the yellow, orange or red colors of petals and fruits to attract pollinators and agents of seed dispersal (reviewed in Armstrong and Hearst 1996, Sandmann 2002). Carotenoids are also precursors for the synthesis of the plant hormone abscisic acid (ABA). Metabolic engineering of carotenoids is a topic of relevant interest both for human health and for the production of animal feed additives (Römer et al. 2000, Dharmapuri et al. 2002). The consumption of carotenoids in human diet provides a source of vitamin A, while their antioxidant properties are believed to alleviate the incidence of chronic diseases such as cardiovascular disease and certain cancers (Handelmann 2001). Moreover, the macular pigment of the primate retina depends on two dietary oxygenated carotenoids, lutein and zeaxanthin. Nutritional benefits of carotenoids have led to metabolic engineering of crops, deficient in carotenoids, to increase the production of these compounds (Ye et al. 2000, reviewed in Hirschberg 1999, Giuliano et al. 2000, Sandmann 2001). The biochemistry of carotenoid biosynthesis has been well established. Genes encoding some of the carotenogenic enzyme have been isolated in bacteria, algae, fungi and higher plants (reviewed in Armstrong and Hearst 1996, Cunningham and Gantt 1998, Takaichi 1999, Hirschberg 2001, Sandmann 2001, Giuliano et al. 2002). Carotenoid biosynthesis takes place in the plastid, but all known enzymes in the pathway are nuclear-encoded and post-translationally imported into the organelle. In plastids, the biosynthesis of carotenoids depends on the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway (Rodríquez-Concepción and Boronat 2002, Nagata et al. 2002). The fundamental steps of carotenoid biosynthesis are the assembly of C40 backbone, the desaturation and cyclization and finally, the xanthophyll formation. The first committed step of carotenoid synthesis, the head-to-head condensation of geranylgeranyl diphosphate (GGPP) molecules to produce phytoene (colorless), is mediated by the soluble enzyme phytoene synthase (Fig. 1). Membrane-localized enzymes carry out subsequent steps of the pathway leading to the colored carotenoids. The phytoene undergoes four desaturation reactions with the production of lycopene (pink). In this part of the pathway, phytofluene, ζ-carotene and neurosporene are the intermediates (Sandmann 2001). It is generally accepted that most carotenoid biosynthetic enzymes arose independently in oxygenic, photosynthetic organisms (cyanobacteria and plants) and anoxygenic or non-photosynthetic organisms (Hirschberg et al. 1997, Takaichi 1999, Sandmann 2002). In heterotrophic and some purple photosynthetic bacteria and in fungi, a single phytoene desaturase (CRTI) encoded by the gene CrtI is responsible for the entire desaturation sequence, shown in Fig. 1 (Misawa et al. 1995, Takaichi 1999, Harada et al. 2001, Sandmann 2001). By contrast, two structurally unrelated genes for ζ-carotene desaturase were cloned from cyanobacteria. One CrtQa (CrtQ-1) from Anabaena PCC 7120 is related to the bacterial CrtI gene (Linden et al. 1993, Linden et al. 1994), whereas the second CrtQb (CrtQ-2) from Synechocystis PCC 6803 is quite similar to cyanobacterial phytoene desaturase (CrtP) gene and it is very closely related to the plant ζ-carotene desaturase (Zds) gene (Breitenbach et al. 1998). In higher plants, the four desaturations are performed by two FAD-containing enzymes, the phytoene desaturase (PDS) and the ZDS, which are unrelated to the bacterial CRTI-like desaturase (Sandmann 2001). Catalytic differences make it possible to create herbicide-resistant plants by introducing bacterial or fungous desaturase (Misawa et al. 1993). The expression of CrtI in E. coli yields all-trans-lycopene, whereas the expression of Pds plus Zds yields poly-cis-lycopene (pro-lycopene) (Bartley et al. 1999, Sandmann 2002). The two symmetric dehydrogenation reactions catalyzed by plant desaturase required plastoquinone and a plastid terminal oxidase (encoded by the Ptox gene) as electron acceptors (Norris et al. 1995, Carol et al. 1999, Carol and Kuntz 2001). The PDS inserts trans double bonds at the 11,11′ positions, whereas ZDS inserts cis double bonds at the 7,7′ positions (Giuliano et al. 2002). In the following step, the carotenoid isomerase (CRTISO) enzyme converts pro-lycopene to all-trans-lycopene (Isaacson et al. 2002, Park et al. 2002). Similarly, a gene of a pro-lycopene isomerase (CrtH) has been cloned from cyanobacteria (Breitenbach et al. 2001, Masamoto et al. 2001). Lycopene is converted to cyclic carotenoids β-carotene and α-carotene by cyclases such as lycopene β-cyclase (Pecker et al. 1996) and lycopene ε-cyclase (Ronen et al. 1999). Subsequently, substitutions by hydroxyl, oxo, and/or epoxy groups produce xanthophylls with bright orange/yellow colors. Defective mutants in thylakoid biogenesis, photosynthetic activity, and/or pigment biosynthesis have been frequently used in attempts to identify genes with a relevant role in chloroplast function and to define the biochemical nature of photosynthetic apparatus (reviewed in Somerville 1986, Cunningham and Gantt 1998, Sandmann 2002). In sunflower (Helianthus annuus L.), pigment-deficient mutations are common as a major feature of inbreeding depression; however, most of these mutants have been recovered after mutagenic treatments (Wallace and Habermann 1959, Triboush et al. 1999), or isolated from progenies of regenerated plants (Pugliesi et al. 1991, Fambrini et al. 1993, Fambrini et al. 2002, Barotti et al. 1995). However, the biochemical and molecular characterization of these mutants is often incomplete. The nuclear non dormant-1 (nd-1) mutant, characterized by an albino and viviparous phenotype, was isolated by selfing an in vitro-regenerated plants (Pugliesi et al. 1991). A preliminary analysis of the biochemical nature of this mutation suggested the impairment at an early step of the carotenoid biosynthesis (Fambrini et al. 1993). Here, we isolated and characterized the Zds gene of sunflower and we provided biochemical and molecular evidences that desaturation of ζ-carotene is not functional in nd-1 mutant, as a consequence of a deficiency in the genomic region which includes the Zds gene. Results Spectrophotometric and HPLC pigment analyses The lethal nd-1 mutant developed small white cotyledons under high light intensity (100 µmol m–2 s–1) and pale-green cotyledons in very dim light (1 µmol m–2 s–1) and, under such conditions, nd-1 cotyledons accumulated chlorophyll (Fig. 2, Table 1). The absorption maxima in petroleum ether (379, 400 and 426 nm), shown by the extract of nd-1 seedlings grown in very dim light (Fig. 2), suggested the accumulation of ζ-carotene. Pigment analysis by HPLC demonstrated the absence of lutein, β-carotene, violaxanthin and cis-neoxanthin in nd-1 cotyledons grown in both light conditions (Table 1). Moreover, nd-1 seedlings grown in very dim light showed a strong accumulation of linear carotenes, ζ-carotene isomers with trans spectral characteristics, and, to a lesser extent, of phytofluene and cis-phytoene. The results suggested a deficient activity of the enzyme ZDS in the desaturation of the phytoene desaturase (PDS) product, that is ζ-carotene. Therefore, our attention was focused on the molecular analysis of the Zds gene of H. annuus. Isolation of a cDNA corresponding to the ζ-carotene desaturase (Zds) gene of sunflower and phylogenetic analysis Primers recognizing conserved features of the Zds genes from Arabidopsis, pepper, tomato, marigold, maize and daffodil (Narcissus) were used to amplify cDNAs derived from cotyledons of H. annuus (Materials and Methods). Amplification products were electrophoresed on a 2% agarose gel, from which a band corresponding to the expected size of 540 bp was purified, cloned and sequenced. This H. annuus fragment showed a high degree of sequence similarity to the conserved positions of the consensus sequences created from the six Zds genes (data not shown). The reconstructed full-length cDNA sequence (GenBank accession number AJ438587), obtained from 3′ and 5′ RACE (1,916 bp), was confirmed from the sequence of the clone Ha3Zds (1,841 bp) and it contained a 1,761-bp CDS, 62 nucleotides of 5′-untranslated region (UTR), and 77 nucleotides of 3′-UTR. The predicted protein displayed a sequence of 587 amino acid residues (Fig. 3A) with a calculated molecular mass of 64.9 kDa. The deduced amino acid sequence of sunflower ZDS revealed a typical amino oxidase domain represented by GenBank pfam01593, that includes the pyridine dinucleotide binding motif, FAD_binding_3 represented by GenBank pfam 01494 (Fig. 3A). A putative transit sequence for plastid targeting was predicted to be the N-terminal residues 1 to 52 by comparing sunflower ZDS with the N-terminal sequence of the cyanobacterium Synechocystis ZDS proteins (GenBank D90914) and according to the predicted transit peptides of daffodil, bell pepper (Albrecht et al. 1995), Arabidopsis (Scolnik and Bartley 1995) and maize (Matthews et al. 2003) which are 61, 49, 59 and 30 residues, respectively (Fig. 3A). A ChloroP 1.1 neural network-based method (Emanuelsson et al. 1999) predicts a transit peptide cleavage site between residues 52 and 53. In order to analyse the correspondence of the H. annuus gene with Zds genes from other species, a phylogenetic analysis employing protein sequences from several monocots and dicots was constructed (Fig. 3B). In particular, sunflower ZDS was closely related to Tagetes ZDS (Moehs et al. 2001), with which it showed an overall identity of 96.6%. A high degree of overall identity was also observed with other ZDS proteins, including an 84.8% identity to Capsicum ZDS (Albrecht et al. 1995) and an 83.0% identity to Medicagotruncatula ZDS. A lower identity was shown for Arabidopsis (Scolnik and Bartley 1995) and maize (Matthews et al. 2003) ZDS, 77.2% and 76.6% respectively. A significant identity (54.2%) was also noticeable with the ζ-carotene desaturase CRTQb of the cyanobacterium Synechocystis PCC 6803 (Breitenbach et al. 1998). By contrast, a low identity (12.9%) was displayed with the CRTI-type desaturase from the cyanobacterium Anabaena PCC 7120 (Linden et al. 1993). Genomic organization of the sunflower Zds gene The full-length exons/introns region of the Zds gene (GenBank accession number, AJ514406) were reconstructed by PCR amplified products, obtained with the gene specific primer pair P-5DNA and P-3INTR and the primer pair P-1Z and P-PI. The H. annuus gene contained 14 exons and 13 introns, 44 nucleotides of 5′-untranslated region (UTR), and 26 nucleotides of 3′-UTR scattered in a 5,020-bp region. The genomic organization of sunflower (HaZds), rice (OsZds) and Arabidopsis (AtZds) Zds genes is reported in Fig. 4 from the translation start to the stop codon. The AtZds has 13 exons and 12 introns scattered in a ca. 3.3-kb region. By contrast, there are 14 exons and 13 introns scattered in a ca. 4.9 kb region in the OsZds. The length between Arabidopsis and sunflower genes is distinguished particularly by introns 1 (561 bp in the Arabidopsis gene vs. 1,040 bp in the HaZds) and 8 (146 bp in the AtZds vs. 830 bp in the sunflower gene). The size of the first exon from the translation start is 118 bp for the AtZds, 160 bp for the OsZds and 196 for the HaZds. The size from the second to the 8th exon is highly conserved in all the three genes; by contrast, in Arabidopsis the exon 9 (200 bp) is likely to be the result from the fusion of the corresponding exons 9 (43 bp) and 10 (157 bp), which are found in both rice and sunflower Zds genes (Fig. 4). The exons 11 and 12 from Zds of rice and sunflower and the corresponding exons 10 and 11 from the AtZds displayed the same length. The size of exon 13 from OsZds is 165 bp like the corresponding 12th of AtZds; by contrast, the exon 13 from the HaZds is 162 bp. The size of the last exon (until the stop codon) from Zds of Arabidopsis, rice and sunflower is similar: 147 bp, 159 bp and 156 bp, respectively. All introns follow the GT/AG splicing rule. RT-PCR and Southern analyses RT-PCR experiments were performed to evaluate the presence of Zds transcripts in the nd-1 seedlings (Fig. 5A–C). In the first experiments, RT-PCR essays were performed to yield a full-length Zds cDNA of H. annuus (1,841 bp) by using the Zds-specific primers P-5DNA and P-PI, located in proximity to the 5′ and 3′ends, respectively. The amplification of a Psy cDNA fragment by the specific primers, P-A21 and P-1105, was employed as control of PCR reaction. Psy (437 bp) and Zds (1,841) transcripts were normally accumulated in wild-type (WT) plants. By contrast, no Zds products were visually amplified from mutant plants (Fig. 5B). The absence of Zds transcripts from nd-1 plants was confirmed in other RT-PCR experiments conducted with Zds-specific primers (Materials and Methods), and a high cycle number (45), to amplify partial regions (508, 553 and 582 bp) of the Zds cDNA (Fig. 5C). In order to evaluate whether the absence of RT-PCR products from nd-1 plants is related to mutation in the DNA sequence, we have conducted a Southern analysis. Genomic DNA extracted from WT and nd-1 leaves was digested with rarely cutting enzymes (NcoI, BclI) for the sunflower genome and probed at high stringency with a DIG-labeled PCR fragment (1,004 bp) amplified between the specific HaZds primers: P-PA and P-PI (probe PAPI). The HaZds had one recognition site for NcoI and BclI located at 2,287 bp and 4,591 bp from the start translation codon, respectively (Fig. 5D). The Southern analysis showed that, in genomic DNA of WT, there are one band with NcoI digestion and two with the BclI digestion (Fig. 5E). Therefore, these results indicate either that, in the H. annuus genome, there is a single Zds gene or that there is a small gene family with no restriction fragment size variation produced by these two enzymes. In the nd-1, the absence of signals after hybridization with the probe PAPI could be consistent with a large deficiency in the structural locus for ZDS. Discussion In this study, we have presented evidences on the biochemical and molecular basis of the nd-1 mutation induced in sunflower, by in vitro tissue culture (Pugliesi et al. 1991). Preliminary analyses of the nd-1 mutant suggested the impairment at an early step of the carotenoid biosynthesis (Fambrini et al. 1993). Here, we demonstrated by spectrophotometric and HPLC analyses an accumulation of ζ-carotene isomers and, to a lesser extent, of cis-phytoene and phytofluene in nd-1 cotyledons grown at very dim light conditions (1 µmol m–2 s–1). ζ-carotene, the product of PDS activity, is the first carotenoid displaying yellow color and absorption maxima in the long wavelength ultraviolet and the visible region of the spectrum. However, in mutant plants grown in very dim light the yellow color was hidden by the concomitant chlorophyll accumulation. The content of ζ-carotene isomers was reduced at higher light intensity (100 µmol m–2 s–1), probably by a photo-oxidation process, as observed in carotenoid-deficient mutants of maize (Horváth et al. 1972). Light is required to drive the carotene desaturation pathway towards completion (Beyer et al. 1989, Bartley et al. 1999). At the ζ-carotene stage, illumination leads to activation of ZDS through a photoisomerization of ζ-carotene isomers carrying a cis-configured central C15-C15′ double bond into a species that exhibits trans spectral characteristics (Beyer et al. 1989, Bartley et al. 1999). These results suggested a stereoisomeric preference of the enzyme ZDS with respect to its substrate (Bartley et al. 1999). Such a photostimulation was not seen for Capsicum (Breitenbach et al. 1999) and maize (Matthews et al. 2003). Moreover, the application of different geometric isomers as substrates revealed that ZDS of C. annuum had no preference for certain isomers (Breitenbach et al. 1999), and that ZDS stereospecificity remains a contentious issue (Giuliano et al. 2002). Nevertheless, it has been hypothesized that, beside photoisomerization, the activities of a putative carotenoid isomerase could occur between the reaction of the two-plant desaturase PDS and ZDS (Bartley et al. 1999, Giuliano et al. 2002, Matthews et al. 2003). Some algal mutants seemingly underscore this view. The Scenedesmus obliquus mutant C6-D accumulates 15-cis-ζ-carotene which is isomerized and then further metabolized upon illumination (Powls and Britton 1977). Furthermore, in all systems showing inefficient isomerization as in dark grown plants of the ccr2 mutant of Arabidopsis (Park et al. 2002) or dark-grown sll033Synechocystis (Breitenbach et al. 2001), the accumulation of pro-ζ-carotene, in addition to pro-lycopene, suggests that isomerization might be required also to allow ZDS action. The spectrophotometric analysis of the ζ-carotene accumulated in nd-1 plants indicated trans spectral characteristics for the main product and suggested that a normal isomerization of PDS products had occurred. Thus, the nd-1 phenotype was probably the result of a peculiar deficient activity of ZDS. Several genes encoding ZDS were previously cloned from higher plants and cyanobacteria (Linden et al. 1993, Breitenbach et al. 1998, Bartley et al. 1999, Moehs et al. 2001, Sandmann 2001, Zhu et al. 2002, Matthews et al. 2003). The deduced amino acid sequence of sunflower ZDS revealed a putative transit sequence for plastid targeting in the N-terminal region and a typical amino oxidase domain that includes the pyridine dinucleotide binding motif. The FAD appears to feed an electron transport chain, involving quinones and the plastid terminal oxidase (PTOX), which ultimately reduces oxygen (Norris et al. 1995, Carol and Kuntz 2001). Southern blot hybridization analysis of genomic DNA with a DNA fragment of HaZds indicated the presence of a single copy of the HaZds gene or a small gene family in the sunflower genome. The phylogenetic analysis clearly demonstrated that the sunflower Zds is clustered to Tagetes Zds gene (Moehs et al. 2001), with which it showed an overall amino acid identity of 96.6%, and closely correlated with other Zds sequences of higher plants. Despite the evolutionary distances between some genera, dicot and monocot genes occur in this family without very clear demarcations by subfamilies indicating the maintenance of a high degree of sequence conservation throughout the plant evolution. The conservation of the distribution and size of the exons in the sunflower and rice Zds genes reflects this close evolutionary relationship. As expected, HaZds is more closely related to CrtQ-2 (formerly CrtQb) from the cyanobacterium Synechocystis PCC 6803 (Breitenbach et al. 1998) than to the CrtQa of Anabaena PCC 7120 (Linden et al. 1993), which is related to the bacterial CrtI-type phytoene desaturase. Only few carotenoid deficient mutants with defective ZDS activity have been described. In Citrus sinensis, the accumulation of linear carotenes (phytoene, phytofluene and ζ-carotene) in the flavedo of the Pinalate mutant suggested a defect in Zds or in ζ-carotene desaturase-associated factors (Rodrigo et al. 2003). In maize, Viviparous9 (Vp9) was suggested as a candidate locus for structural Zds gene by using a combination of RI-RFLP analysis, RT-PCR semi-quantification of transcripts accumulation, and HPLC analyses (Matthews et al. 2003). The decrease in Zds transcripts, observed in the vp9 mutant with respect to control plants, corroborated the assertion of Vp9 as the structural locus for ZDS, but as pointed out by the authors, it is still likely that Vp9 is a regulator of the Zds gene (Matthews et al. 2003). By using RT-PCR approaches, it was possible to demonstrate that nd-1 plants of sunflower were unable to accumulate functional Zds transcripts. This result agrees with the hybridization Southern analysis that underlined a deficiency in the 3′ region of the Zds gene. We cannot rule out that the nd-1 mutation involved other genes, closely linked to the Zds locus. However, the results of genetic analysis showed a monogenic nuclear control of the nd-1 mutation (Fambrini et al. 1993), which suggests the absence of gonial lethality and probably the smallness of the nd-1 deficiency. Unequivocal evidences that some recessive mutations are the consequence of homozygous small deficiencies were previously obtained in Drosophila and maize (Ephrussi 1934, McClintock 1941). The segregation of hemizygous-derived progenies followed the Mendelian monogenic ratio. In conclusion, the reported results indicate that the pigment-deficient phenotype of the nd-1 mutant is caused by a defect in ZDS activity due to a chromosomal deficiency, that includes at least a region of the structural locus for ZDS. Further investigation is needed to map the nd-1 mutation, while chromosomal walking experiments may provide information on the amplitude of this deficiency in the nd-1 genome. Materials and Methods Plant materials and growth conditions Sunflower (H. annuus L.) seeds of WT (Nd-1/Nd-1) and nd-1 mutant (Fambrini et al. 1993) were used as starting material. Achenes were germinated in Petri dishes on distilled water and after 3 d were transferred to 8-cm diameter pots containing Mannaflor S compost (Manna Laives, Bolzano, Italy). Seedlings were grown in a growth chamber at 23±1°C with a light schedule of 16 h of white light (WL) and a photon fluence rate of 100 or 1 µmol m–2 s–1 as standard conditions. The WL was obtained from Philips TLM 40 W/33 RS fluorescent tubes (Philips, Eindhoven, The Netherlands). Pigment extraction and spectrophotometric analysis Pigment extraction for spectrophotometric analysis was performed, as previously described (Fambrini et al. 1993), from 10-day-old cotyledons of WT and nd-1 seedlings grown under WL at the standard fluence rate of 100 or 1 µmol m–2 s–1. Samples of both genotypes were extracted in the dark with 3 ml g–1 tissue of petroleum ether, and clarified by centrifugation (Fambrini et al. 1993). Then, the upper phase was dried under a stream of nitrogen and resuspended in petroleum ether (b.p. 35–65). Absorption spectra were obtained with an UV-visible light spectrophotometer (UV-2101PC, Shimazu, S.R.L, Milan, Italy). Pigment extraction and HPLC analysis Pigment extraction for HPLC analysis was performed as previously described by Bonora et al. (2000). Briefly, 10-day-old cotyledons of WT and nd-1 seedlings grown under WL at the standard fluence rate of 100 or 1 µmol m–2 s–1 were ground with a tissue mixer (Sorvall Omni-Mixer) in cold absolute ethanol. After centrifugation at 3,000×g for 5 min, the residues were re-extracted to remove the pigment completely. To verify the effectiveness of the ethanolic extraction, further extraction of the residues with cold n-hexane was carried out. After mixing, the crude ethanolic fractions were purified through an octadecyl silica cartridge (Water C-18 Sep-Pak) and eluted with a few milliliters of ethyl acetate. The solvents were removed on a rotary evaporator (Buchi 461 rotavapor) at 30°C and the residues dissolved in ethanol. The solutions were filtered through a 0.45 µm HVLP Millipore filter. HPLC analysis, preparation of carotenoid reference compounds, saponification procedure, epoxide and iodine-catalysed photoisomerization test were performed according to Bonora et al. (2000). The liquid chromatograph apparatus consisted of a Varian 5020 system equipped with a Rheodyne 7126 injector and a Varian UV-100 visible-UV-variable wavelength detector set at 287 nm (phytoene), 348 nm (phytofluene) and 425 nm (according to Minguez-Mosquera et al. 1992). Chromatograms were recorded on a Merk-Hitachi D-2000 chromato-integrator. Portions of the ethanolic extracts were chromatographed by using a Hibar-Lichosorb Rp 18 column (5 µm, 4×250 mm) with a precolumn Lichrocart-Lichrosorb Rp 18 (5 µm, 4×4 mm). A Gilson 201 fraction collector was employed for collecting pigment peaks. Each analytical estimation was replicated three times. The mobile phase composition, at a flow rate of 1 ml min–1, was: A = water; B = acetonitrile, methanol, and 2-propanil (80 : 15 : 5; by vol.). Gradient mode elution was: from 0 min to 17 min = 92% B; from 17 min to 23 min = 92–97% B; from 23 min to 35 min = 97–100% B. The various carotenoids were identified by comparing retention time and spectral characteristics of separated peaks with reference standards prepared as reported by Bonora et al. (2000). Cloning of ζ-carotene desaturase gene from H. annuus Amino acid sequences of cloned Zds genes from Tagetes erecta (GenBank AF251013), Arabidopsis thaliana (AF121947), Narcissus pseudonarcissus (AJ224683), Lycopersicon esculentum (AF195507), Capsicum annuum (X89897) and Zea mays (AF047490) were aligned. Regions with highest conservation were identified and two oligonucleotides primers were designed: P-357, 5′-GAGCTGGGCTTGCAGGCATGTC-3′ (forward) and P-RT, 5′-TCAATGAATCCAAGAGCATA-3′ (reverse). Total RNA was extracted from sunflower leaves of 15-day-old seedlings of both genotypes grown under 1 µmol m–2 s–1 (Chomczynski and Sacchi 1987). First-strand cDNA was made with 5 µg of total RNA using the Superscript preamplification kit (Invitrogen S.R.L, Life Technologies, Milan, Italy) in conditions recommended by manufacturer. The cDNA was amplified with P-357 and P-RT primers. PCRs were performed with Gene Amp® PCR System 2700 (Applied Biosystems, Applera Italia, Monza, Italy) thermocycler in 50 µl of 1× buffer (Applied Biosystems) containing 0.2 mM dNTPs, 0.2 µM of each primer, 2 mM MgCl2, 0.25 U AmpliTaq DNA polymerase (Applied Biosystems) and 0.2 µl of single-strand cDNA. After an initial 4 min/94°C denaturation step, 30 cycles were run, each with 30 s of denaturation at 94°C, followed by 30 s annealing at 37°C and 30 s extension at 72°C. Final extension was at 72°C for 7 min. The PCR reaction yielded a product of about 540 bp, which was cloned into the pCRII vector (Invitrogen) and sequenced by the dideoxy method with an automated sequencer (Ha1Zds). The rapid amplification of cDNA ends (RACE) approach was used to isolate the 5′ and 3′ ends of the Zds cDNA. All reactions were performed with kits according to the manufacturer’s instructions (Invitrogen). First-strand synthesis was performed with Superscript II retrotranscriptase in the presence of the Adapter Primer (5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′). Terminal deoxynucleotidyl transferase (TdT) was used to add, at the 5′ end of cDNA, a poly-C tail that allowed the coupling of the 5′ Race Abridged Anchor Primer (5′-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3′). After C-tailing, PCR amplification was performed with the Zds specific primer P-5Z (5′-CCACCGATAAAGGTCCTTGAC-3′) and anchor primers provided in the kit. The 3′ end of the Zds cDNA was amplified using first-strand cDNAs made with the Adapter Primer. Each PCR was done with the Zds specific primer P-3Z (5′-CGGAACCTCGATAACATTAGC-3′), and the Abridged Universal Amplification Primer provided in the kit. Specific cDNAs from 3′ and 5′ RACE were subcloned into the TOPO pCR 2.1 Vector (Invitrogen) and sequenced; the overlapping region with the first clone (Ha1Zds) was confirmed. A putative full-length Zds cDNA clone was obtained by PCR using specific primers at the extreme 5′ end (P-5DNA, located 44 nucleotides upstream the ATG: 5′-GAGAACACAAAAGCGGTTGC-3′) and 3′end (P-PI, located nine nucleotides downstream from the translational stop codon: 5′-CCCAATCACTTACACTATTAACC-3′). The PCR reactions were carried out in 50 µl of 1× buffer (Promega, Madison, WI, U.S.A.) containing 0.2 mM dNTPs, 0.4 µM of each primer, 0.5 U Pfu-DNA polymerase (Promega) and 0.4 µl of single-strand cDNA. A product of 1,841 bp was cloned into the pCRII vector (Invitrogen). A clone, named Ha3Zds and containing the entire sequence, was selected and sequenced. To isolate the full-length exons/introns region of the Zds gene, sequence information from the cDNA was used to design specific primers. Forward (P-5DNA and P-1Z: 5′-GAGCGATGACACAAATACGG-3′) and reverse (P-PI and P-3INTR: 5′-GAATACTTGTTCGGGTTCC-3′) gene-specific primers were used for PCRs with 7- to 10-day-old sunflower cotyledon genomic DNA (Dellaporta et al. 1983). The PCR reactions were performed in 50 µl of 1× buffer (Promega) containing 0.2 mM dNTPs, 1.5 µM of each primer, 0.5 U Pfu-DNA polymerase (Promega) and 0.1 µg genomic DNA. After denaturation at 94°C for 4 min, amplification of DNA was carried out for 30 cycles (30 s at 94°C, 30 s at 58°C, 5 min at 72°C). Final extension was at 72°C for 10 min. The PCR product obtained with the primers P-5DNA and P-3INTR combination (about 2,500 bp) and the PCR product obtained with primers P1Z and P-PI combination (about 2,750 bp) were cloned into the pCRII vector and sequenced on both strands. The overlapping region between the two clones (167 bp) was confirmed. DNA and amino acid sequence analysis Database searches were carried out with the Blast program at the National Center for Biotechnology Information (NCBI) (Altschul et al. 1997). A ChloroP1.1 neural network-based method for predicting chloroplast transit peptides was used to estimate a putative signal of transit peptide (Emanuelsson et al. 1999). The PROSITE and PFAM databases were used to identify conserved domains (Bateman et al. 2002, Falquet et al. 2002). The deduced amino acid sequence of the H. annuus ZDS was compared to other ZDS sequences of higher plants. The sequences were aligned by ClustalW version 1.7 (Thompson et al. 1994) and manually adjusted. Phylogenetic analysis was performed using programs from the PHYLIP group, PHYLogeny Inference Package, Version 3.6 (Felsenstein 1985). As support for the trees obtained, a bootstrap analysis, with 100 replicates, was performed by SEQBOOT. The search for most-parsimonious trees was done by PROTPARS and strict consensus trees were obtained by CONSENSE. RT-PCR experiments Total RNAs were extracted from leaves of 15-day-old seedlings of both genotypes grown under 1 µmol m–2 s–1. Aliquots of 5 µg of total RNA were reverse-transcripted as described above for Zds cDNA cloning. PCRs were carried out for 30 or 45 cycles by using gene-specific primers for Zds and phytoene synthase (Psy, GenBank accession number AJ304825) of sunflower. The primers P-A21 (forward, 5′-GCATCGCATATAACTCCCAAAGC-3′) and P-1105 (reverse, 5′-ATGTCTTCATCTGACAATCCGGC-3′) were designed to yield a 437 bp Psy cDNA fragment. The primers, P-5DNA (forward) and P-PI (reverse) were designed to yield a full-length Zds cDNA. PCRs were performed in 50 µl of 1× buffer (Applied Biosystems) containing 0.2 mM dNTPs, 0.2 µM of each primer (1 primer pair for Zds or 1 primer pair for Psy), 2 mM MgCl2, 0.25 U AmpliTaq DNA polymerase (Applied Biosystems) and 0.2 µl of single-strand cDNA. After denaturation at 94°C for 4 min, amplification of DNA was carried out for 30 cycles (94°C for 30 s, 55°C for 30 s, 72°C for 2 min). Final extension was at 72°C for 7 min. In a second RT-PCR experiment, specific primers for Zds were used to amplify partial regions of the cDNA in the following combinations: primer P-TO (forward, 5′-CGTTGTGACCGTGCAACTTCG-3′) with the primer P-7Z (reverse, 5′-AGACTCAGCTCATCAACT-3′) to yield a 582 bp Zds cDNA fragment; primer P-PA (forward, 5′-GCTGGGTAACAGAATTGCAGG-3′) with the primer P-7Z (reverse) to yield a 553 bp Zds cDNA fragment; primer P-4Z (forward, 5′-GGCAAGCTGCAGGGTTGG-3′) with the primer P-7Z (reverse) to yield a 508 bp Zds cDNA fragment. PCRs were performed in 50 µl of 1× buffer (Applied Biosystems) containing 0.2 mM dNTPs, 0.2 µM of each primer (1 primer pair for Zds or 1 primer pair for Psy), 2mM MgCl2, 0.25 U AmpliTaq DNA polymerase (Applied Biosystems) and 0.2 µl of single-strand cDNA. After denaturation at 94°C for 4 min, amplification of DNA was carried out for 45 cycles (94°C for 30 s, 55°C for 30 s, 72°C for 50 s). Final extension was at 72°C for 7 min. PCR reaction products were separated by 2.0% (w/v) Tris-acetate-EDTA (TAE)-agarose gel electrophoresis and stained with ethidium bromide. Southern blot analysis Genomic DNA was isolated from nd-1 and WT cotyledons, following standard protocols (Dellaporta et al. 1983). Genomic DNA was digested by restriction enzymes BclI and NcoI and the resulting fragments separated on a 0.8% (w/v) agarose gel and capillary blotted onto a nylon membrane positively charged (Roche Diagnostics GmbH, Mannheim, Germany). Hybridization was performed at 38°C in DIG-Easy Hyb with a DIG-labeled (DIG-DNA Labeling Kit, Roche) PCR fragment amplified between the primers P-PA and P-PI (probe PAPI, 1,004 bp), according to the manufacturer’s recommendations. The filter was washed twice in 2× SSC, 0.1% SDS at room temperature for 5 min, and twice in 0.2× SSC, 0.1% SDS at 37°C for 10 min. Acknowledgments We would like to thank Alberto Cenci for help in sequence analysis and Daniele Bertini for technical assistance. This work was partially supported by a Scuola Normale Superiore grant to M. Salvini. 4 Corresponding author: E-mail, cpuglies@agr.unipi.it; Fax, +39-50-576750. Open in new tabDownload slide Fig. 1 Schematic pathway for lycopene biosynthesis from geranylgeranyl diphosphate. Selected enzymes (PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; CRTI, bacterial-type phytoene desaturase; CRTISO, carotenoid isomerase) are shown. A hypothetical carotenoid isomerase (ISOMERASE) is supposed to occur between the reactions of PDS and ZDS. The genes from bacteria, cyanobacteria and higher plants encoding the relative enzymes are indicated in brackets. Open in new tabDownload slide Fig. 1 Schematic pathway for lycopene biosynthesis from geranylgeranyl diphosphate. Selected enzymes (PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; CRTI, bacterial-type phytoene desaturase; CRTISO, carotenoid isomerase) are shown. A hypothetical carotenoid isomerase (ISOMERASE) is supposed to occur between the reactions of PDS and ZDS. The genes from bacteria, cyanobacteria and higher plants encoding the relative enzymes are indicated in brackets. Open in new tabDownload slide Fig. 2 Absorption spectra of the total pigment extract from cotyledons of 10-day-old nd-1 sunflower seedlings that were grown in high (100 µmol m–2 s–1) or very dim (1 µmol m–2 s–1) light intensities. Open in new tabDownload slide Fig. 2 Absorption spectra of the total pigment extract from cotyledons of 10-day-old nd-1 sunflower seedlings that were grown in high (100 µmol m–2 s–1) or very dim (1 µmol m–2 s–1) light intensities. Open in new tabDownload slide Fig. 3 Sequence analysis of the sunflower ζ-carotene desaturase (ZDS). (A) Deduced amino acid sequence of Zds cDNA. The underlined region shows the putative signal of chloroplastic transit peptide. The putative dinucleotide-binding domain, FAD_binding_3: pfam 01494 (Bateman et al. 2002) is double underlined. The putative aminooxidase domain: pfam 01593 is in bold. Arrows mark the boundaries of low complexity domain. (B) Bootstrap consensus tree based on maximum parsimony, generated using Protpars program (Phylip package 3.572 version). The bootstrap replicates were 100 (values are given at the nodes); the deduced amino acid sequence of the ζ-carotene desaturase (Zds) gene of H. annuus (accession number AJ43885) was compared to other ZDS amino acid sequences selected from higher plants, and with amino acid sequences of CRTQa (CRT1-like desaturase) and CRTQb from cyanobacteria. The sequences were from Citrus unshiu (AB071342), Citrus sinensis (AJ319762), Citrus x paradisi (AF372617), Tagetes erecta (AF251013), Lycopersicon esculentum (AF195507), Capsicum annuum (X89897), Narcissus pseudonarcissus (AJ224683), Zea mays (AF047490), Arabidopsis thaliana (AF121947), Medicago truncatula (TC78514), Sorghum bicolor (TC68058), Oryza sativa (AP004273), Glycine max (TC188592), Anabaena PCC 7120 (CAB56041.1), Anabaena PCC 7120 (BAA05091.1, CRTI-like desaturase), Synechocystis PCC 6803 (P74306, CRTQb). The amino acid sequence of CRTI from Erwinia herbicola (Pantoea agglomerans) (P22871) was used as out-group. Open in new tabDownload slide Fig. 3 Sequence analysis of the sunflower ζ-carotene desaturase (ZDS). (A) Deduced amino acid sequence of Zds cDNA. The underlined region shows the putative signal of chloroplastic transit peptide. The putative dinucleotide-binding domain, FAD_binding_3: pfam 01494 (Bateman et al. 2002) is double underlined. The putative aminooxidase domain: pfam 01593 is in bold. Arrows mark the boundaries of low complexity domain. (B) Bootstrap consensus tree based on maximum parsimony, generated using Protpars program (Phylip package 3.572 version). The bootstrap replicates were 100 (values are given at the nodes); the deduced amino acid sequence of the ζ-carotene desaturase (Zds) gene of H. annuus (accession number AJ43885) was compared to other ZDS amino acid sequences selected from higher plants, and with amino acid sequences of CRTQa (CRT1-like desaturase) and CRTQb from cyanobacteria. The sequences were from Citrus unshiu (AB071342), Citrus sinensis (AJ319762), Citrus x paradisi (AF372617), Tagetes erecta (AF251013), Lycopersicon esculentum (AF195507), Capsicum annuum (X89897), Narcissus pseudonarcissus (AJ224683), Zea mays (AF047490), Arabidopsis thaliana (AF121947), Medicago truncatula (TC78514), Sorghum bicolor (TC68058), Oryza sativa (AP004273), Glycine max (TC188592), Anabaena PCC 7120 (CAB56041.1), Anabaena PCC 7120 (BAA05091.1, CRTI-like desaturase), Synechocystis PCC 6803 (P74306, CRTQb). The amino acid sequence of CRTI from Erwinia herbicola (Pantoea agglomerans) (P22871) was used as out-group. Open in new tabDownload slide Fig. 4 Schematic comparison of ζ-carotene desaturase (Zds) genomic sequence of Arabidopsis thaliana (AtZds, accession number AY096583), Oryza sativa (OsZds, AP004273) and Helianthus annuus (HaZds, AJ514406). The size of exons and introns of genomic DNA from the presumed translation start and the stop codons is shown. Introns (shown as lines) and exons (shown as numbered boxes) are drawn to scale. The exon 9 of the Arabidopsis gene, as well as exons 9 and 10 in rice and sunflower genes, are denoted by asterisks. Open in new tabDownload slide Fig. 4 Schematic comparison of ζ-carotene desaturase (Zds) genomic sequence of Arabidopsis thaliana (AtZds, accession number AY096583), Oryza sativa (OsZds, AP004273) and Helianthus annuus (HaZds, AJ514406). The size of exons and introns of genomic DNA from the presumed translation start and the stop codons is shown. Introns (shown as lines) and exons (shown as numbered boxes) are drawn to scale. The exon 9 of the Arabidopsis gene, as well as exons 9 and 10 in rice and sunflower genes, are denoted by asterisks. Open in new tabDownload slide Fig. 5 RT-PCR and Southern analyses. (A) Schematic representation of primer recognition sites within the Zds cDNA of H. annuus. The position of the first nucleotide (5′) of each primer sequence is indicated in brackets. (B) PCRs performed using gene-specific primers to yield a 437 bp fragment for phytoene synthase (Psy) and a full-length (1,841 bp) ζ-carotene desaturase (Zds) cDNA. Amplifications were carried out with 30 cycles. (C) PCRs performed using gene-specific primers to amplify partial regions of Zds (508 bp, 553 bp and 582 bp) and Psy (437 bp) cDNAs. Amplifications were carried out with 45 cycles. The PCR products were resolved on TAE 2.0% (w/v) agarose gels. Primer combinations are shown below the gels. (D) Schematic representation of NcoI and BclI recognition sites within the ζ-carotene desaturase (Zds) gene of H. annuus. The location of enzyme site from the presumed start translation codon is indicated in brackets. The position of the probe PAPI (shaded box), a DIG-labeled PCR fragment (1,004 bp) amplified between the specific primers for Zds of sunflower, P-PA and P-PI, is shown. (E) Genomic DNA (30 µg) was digested with NcoI or BclI. The fragments were separated by TAE 1.0% (w/v) agarose gel electrophoresis and were hybridized with the PAPI-specific probe. WT = wild type; nd-1 = mutant; M = markers. Open in new tabDownload slide Fig. 5 RT-PCR and Southern analyses. (A) Schematic representation of primer recognition sites within the Zds cDNA of H. annuus. The position of the first nucleotide (5′) of each primer sequence is indicated in brackets. (B) PCRs performed using gene-specific primers to yield a 437 bp fragment for phytoene synthase (Psy) and a full-length (1,841 bp) ζ-carotene desaturase (Zds) cDNA. Amplifications were carried out with 30 cycles. (C) PCRs performed using gene-specific primers to amplify partial regions of Zds (508 bp, 553 bp and 582 bp) and Psy (437 bp) cDNAs. Amplifications were carried out with 45 cycles. The PCR products were resolved on TAE 2.0% (w/v) agarose gels. Primer combinations are shown below the gels. (D) Schematic representation of NcoI and BclI recognition sites within the ζ-carotene desaturase (Zds) gene of H. annuus. The location of enzyme site from the presumed start translation codon is indicated in brackets. The position of the probe PAPI (shaded box), a DIG-labeled PCR fragment (1,004 bp) amplified between the specific primers for Zds of sunflower, P-PA and P-PI, is shown. (E) Genomic DNA (30 µg) was digested with NcoI or BclI. The fragments were separated by TAE 1.0% (w/v) agarose gel electrophoresis and were hybridized with the PAPI-specific probe. WT = wild type; nd-1 = mutant; M = markers. Table 1 Quantitative determination by HPLC analysis of cotyledon pigments (nmol (g FW)–1) in wild type (WT) and nd-1 mutant of sunflower (Helianthus annuus L.) grown at different light conditions Pigment Light 100 (µmol m–2 s–1) Light 1 (µmol m–2 s–1) WT nd-1 WT nd-1 Chlorophyll a 536.5 0.0 *** a 164.2 26.4 *** Chlorophyll b 218.1 0.0 *** 89.3 6.7 *** Chlorophyll (a + b) 754.6 0.0 *** 253.6 33.1 *** cis-Neoxanthin 32.2 0.0 *** 8.6 0.0 * Violaxanthin 45.0 0.0 *** 16.1 0.0 * Lutein 105.9 0.0 *** 36.0 0.0 ** β-Carotene 71.9 0.0 *** 17.4 0.0 ** ζ-Carotene 0.0 2.6 * 0.0 34.3 *** Phytofluene 0.0 3.7 * 0.0 2.6 * cis-Phytoene 0.0 8.1 * 0.0 1.9 * Pigment Light 100 (µmol m–2 s–1) Light 1 (µmol m–2 s–1) WT nd-1 WT nd-1 Chlorophyll a 536.5 0.0 *** a 164.2 26.4 *** Chlorophyll b 218.1 0.0 *** 89.3 6.7 *** Chlorophyll (a + b) 754.6 0.0 *** 253.6 33.1 *** cis-Neoxanthin 32.2 0.0 *** 8.6 0.0 * Violaxanthin 45.0 0.0 *** 16.1 0.0 * Lutein 105.9 0.0 *** 36.0 0.0 ** β-Carotene 71.9 0.0 *** 17.4 0.0 ** ζ-Carotene 0.0 2.6 * 0.0 34.3 *** Phytofluene 0.0 3.7 * 0.0 2.6 * cis-Phytoene 0.0 8.1 * 0.0 1.9 * a Statistical evaluation was done separately for the two light conditions. Differences from the control values were significant at: * P <0.05, ** P <0.01, *** P <0.001 levels according to Student’s t-test. Open in new tab Table 1 Quantitative determination by HPLC analysis of cotyledon pigments (nmol (g FW)–1) in wild type (WT) and nd-1 mutant of sunflower (Helianthus annuus L.) grown at different light conditions Pigment Light 100 (µmol m–2 s–1) Light 1 (µmol m–2 s–1) WT nd-1 WT nd-1 Chlorophyll a 536.5 0.0 *** a 164.2 26.4 *** Chlorophyll b 218.1 0.0 *** 89.3 6.7 *** Chlorophyll (a + b) 754.6 0.0 *** 253.6 33.1 *** cis-Neoxanthin 32.2 0.0 *** 8.6 0.0 * Violaxanthin 45.0 0.0 *** 16.1 0.0 * Lutein 105.9 0.0 *** 36.0 0.0 ** β-Carotene 71.9 0.0 *** 17.4 0.0 ** ζ-Carotene 0.0 2.6 * 0.0 34.3 *** Phytofluene 0.0 3.7 * 0.0 2.6 * cis-Phytoene 0.0 8.1 * 0.0 1.9 * Pigment Light 100 (µmol m–2 s–1) Light 1 (µmol m–2 s–1) WT nd-1 WT nd-1 Chlorophyll a 536.5 0.0 *** a 164.2 26.4 *** Chlorophyll b 218.1 0.0 *** 89.3 6.7 *** Chlorophyll (a + b) 754.6 0.0 *** 253.6 33.1 *** cis-Neoxanthin 32.2 0.0 *** 8.6 0.0 * Violaxanthin 45.0 0.0 *** 16.1 0.0 * Lutein 105.9 0.0 *** 36.0 0.0 ** β-Carotene 71.9 0.0 *** 17.4 0.0 ** ζ-Carotene 0.0 2.6 * 0.0 34.3 *** Phytofluene 0.0 3.7 * 0.0 2.6 * cis-Phytoene 0.0 8.1 * 0.0 1.9 * a Statistical evaluation was done separately for the two light conditions. Differences from the control values were significant at: * P <0.05, ** P <0.01, *** P <0.001 levels according to Student’s t-test. Open in new tab Abbreviations ABA abscisic acid CrtB phytoene synthase gene CRTI bacterial-type phytoene desaturase CrtI phytoene desaturase gene CRTISO carotenoid isomerase CrtH CrtISO carotenoid isomerase genes CrtP phytoene desaturase gene CrtQa CrtQb ζ-carotene desaturase genes FAD flavin adenosine dinucleotide GGPP geranylgeranyl diphosphate MEP 2-C-methyl-d-erythritol 4-phosphate RACE rapid amplification of cDNA ends RT-PCR reverse transcriptase polymerase chain reaction PDS phytoene desaturase Pds phytoene desaturase gene PSY phytoene synthase Psy phytoene synthase gene WL white light ZDS ζ-carotene desaturase Zds ζ-carotene desaturase gene. " The nucleotide sequences reported in this paper have been submitted to the GenBank EMBL under the accession numbers AJ438587 (Zds cDNA) and AJ514406 (HaZds). 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TI - A Deficiency at the Gene Coding for ζ-Carotene Desaturase Characterizes the Sunflower non dormant-1 Mutant
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pch052
DA - 2004-04-15
UR - https://www.deepdyve.com/lp/oxford-university-press/a-deficiency-at-the-gene-coding-for-carotene-desaturase-characterizes-W6mDCACYri
SP - 445
EP - 455
VL - 45
IS - 4
DP - DeepDyve
ER -