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First published online April 8, 2005; 10.1104/pp.104.057927 Plant Physiology 138:441-450 (2005) © 2005 American Society of Plant Biologists Why Is Golden Rice Golden (Yellow) Instead of Red?1,[w]Center for Applied Biosciences, University of Freiburg, D–79104 Freiburg, Germany (P.S., S.A.-B., P.B.);and Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom (R.D.)
The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of  -carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase,  -carotene desaturase, carotene cis-trans-isomerase, -lycopene cyclase, and -carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that -carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase,  /-lycopene cyclase, -carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or -carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the -carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed.
Golden Rice (Oryza sativa) denotes a genetically modified rice capable of biosynthesizing and accumulating -carotene (provitamin A) in the endosperm, yielding a characteristic yellow color in the polished grains. Golden rice was developed to help cope with vitamin A deficiency, a problem that prevails in developing countries, affecting millions (Underwood, 2000 ; UNICEF, 2000). Vitamin A deficiency causes mortality and growth retardation in children and frequently impairs vision, leading to blindness. It also contributes indirectly to anemia by interfering with iron bioavailability (for review, see Haschke and Javaid, 1991).
The first Golden Rice (Ye et al., 2000
There are two possible explanations to account for this observation. The first is that enzymes downstream in the pathway, such as lycopene cyclases (LCYs) and - and -carotene hydroxylases (HYDs), are expressed in an enzymatically active form in wild-type rice endosperm, while PSY and one or both of the plant carotene desaturases, phytoene desaturase (PDS) and -carotene desaturase (ZDS), are not. Synthesis of lycopene by PSY and CRTI in the transgenic plant provides the substrate for these downstream enzymes and consequently enables the formation of downstream products. The observation that the expression of PSY alone led to phytoene accumulation but not to desaturated products (Burkhardt et al., 1997) is evidence for the absence of at least one active desaturase, namely PDS. Similarly, the expression of CRTI alone did not produce any color in rice endosperm (A. Klöti and P. Beyer, unpublished data) because of the lack of PSY activity.
The second explanation involves the feedback induction of endogenous carotenoid biosynthetic genes as a result of the presence of transgenes. This was shown to be the case in tomato (Lycopersicon esculentum) fruit upon expression solely of CRTI. Again, To distinguish between these two conceivable explanations, we examined Golden Rice integration events to detect potential alterations in the expression of the endogenous carotenoid biosynthetic genes in response to the transgenes, using TaqMan real-time PCR. We utilized transgenic lines that contained CrtI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, which allowed us to investigate both leaves and the endosperm of developing seeds. We further substantiated our results by using plant model systems expressing CRTI. The results obtained enabled us to explain the Golden Rice phenotype, as described below.
Previously described Golden Rice transformation events expressing CRTI constitutively and PSY in the endosperm (Ye et al., 2000 ; Hoa et al., 2003) were used to carry out expression analyses. The high specificity of TaqMan real-time PCR allowed us to measure the intrinsic rice Psy mRNA levels without interference by the expression of the Psy transgene. Homozygosity of the materials was confirmed by inverse PCR and by the absence of color segregation in T3 seeds. Transgenic and wild-type plants were sown in a randomized plot in the greenhouse, and RNA was extracted from immature milky-stage endosperm. Leaf material was harvested from 16-d-old seedlings. TaqMan real-time PCR was used to characterize mRNA levels in the wild type or to monitor potential changes in rice endogenous mRNA levels in the transgenic samples. The pathway from phytoene to -carotene and -carotene and further on to zeaxanthin was surveyed by monitoring mRNA levels of rice PSY, PDS, ZDS, CRTISO, -LCY,  -LCY, and -HYD. Figure 2 shows the results obtained with wild-type materials.
All carotenoid biosynthetic genes investigated were found to be expressed in rice endosperm, albeit at very low levels. Psy mRNA was the least abundant, 130-fold lower than in leaves, followed by mRNAs for LCY (25-fold), HYD (22-fold), PDS (10-fold), ZDS (8-fold), and CRTISO (4-fold). The barely detectable amount of Psy mRNA found in endosperm explains the need for the Psy transgene to produce carotenoids in rice grains. However, the remaining transcripts, all required to produce -carotene and hydroxylated xanthophylls, were present. This raises questions about the necessity of the CrtI transgene in Golden Rice and may suggest either one or both of the two desaturases, PDS and ZDS, are inactive or that their respective protein levels are too low. To investigate whether carotenoid accumulation in Golden Rice endosperm is due to a modification in this expression pattern of the rice carotenoid biosynthetic genes, a similar set of experiments was carried out using the transgenic plants (Fig. 3). However, no significant changes in expression of the six rice genes investigated was observed, neither in the endosperm nor in leaves from three independent transformation events. These data indicate that the transgenes (especially CrtI) did not cause feedback regulation to alter the mRNA levels of these endogenous genes, suggesting that, in fact, rice LCY and rice HYD are functional in the endosperm and reveal their activity with the availability of lycopene produced by the transgenic PSY and CRTI. Further experiments were necessary to verify the functional presence of rice PDS, ZDS, and CRTISO (see below).
Carotenoid Analysis
Wild-type leaves, in contrast to endosperm, possess an active carotenoid biosynthetic pathway. Pigment changes could be provoked by the constitutive expression of CRTI, but presumably not by PSY, the expression of which was driven by the endosperm-specific gt1 promoter (pCaCar and pB19hpc; see Ye et al., 2000
The Level of CRTI Expression Correlates with the Decrease in Lutein Content in Leaves Western blots were carried out with leaf extracts to investigate CRTI protein levels. Figure 5 shows a band of the expected molecular mass of 55 kD, with varying intensities for the different transgenic events investigated. A second cross-reactive band was always observed, which was absent from the wild-type controls and may represent a degradation product. A clear correlation was observed between the level of RNA and protein of CrtI and the decrease in lutein abundance. For example, the event in lane 13 expresses CRTI strongly and has a lower lutein content, and the event expressing CRTI weakly, shown in lane 12, has approximately wild-type lutein levels (lane 3). The signals of all other lines more or less mirror this inverse relationship.
An evident explanation for this loss of lutein could be a down-regulation of -LCY. When examined by TaqMan real-time PCR, the mRNA changes observed were found to be only minor and did not correlate with the observed lutein-phenotype (Fig. 5E).
To investigate the effects of CRTI expression in a green tissue from a different species, we transformed Arabidopsis (Arabidopsis thaliana) with the vector pSToff. This experiment is analogous to rice transformation with pCaCar or pB19hpc (Ye et al., 2000
Transformation experiments with carotenoid biosynthetic genes targeting photosynthetically inactive tissues often produce more pronounced effects than green tissues, probably because the latter have a tighter regulation at the protein level. We therefore used the transgenic Arabidopsis lines to generate photosynthetically inactive root calli (Banno et al., 2001 ) and investigated their carotenoid patterns. We observed an analogous effect, i.e. a decrease of lutein from 36% in wild-type samples to 24% in line 2, 27% in line 3, 16% in line 4, and 18% in line 5 (line numbering as in Fig. 6).
The experiments described above show that CRTI expression in rice, Arabidopsis, and tobacco BY2 cells (see supplemental data) does not alter the expression of the investigated carotenoid biosynthetic genes. This implies that the carotenoid pattern in Golden Rice is the result of functional endogenous By analysis of the carotenoids in Golden Rice, it is not possible to judge the activity of the rice CRTISO, because the CRTI pathway produces all-trans carotene precursors that do not require cis-to-trans isomerization. The investigation of CRTISO in rice endosperm is only possible in a transgenic background expressing PDS and ZDS, which delivers the plant-specific tetra-cis-lycopene, termed prolycopene.
Transgenic rice producing carotenoid was made using the cultivar Asanohikari expressing PSY, PDS, and ZDS, all from daffodil (Narcissus pseudonarcissus) and each individually under the control of the endosperm-specific Glu-2-promoter with a Ubi1-hpt expression cassette as the selectable marker. A total of 23 events were regenerated and analyzed. Western blots revealed signals for all three proteins that were not detectable in wild-type endosperm (data not shown). The carotenoid content in the resulting yellow grains ranged from 0.2 to 0.8 µg g–1. This is the same range as in Golden Rice, which relies on the same daffodil PSY but on CRTI as the desaturase (Hoa et al., 2003
Analysis of the isomer pattern of the
We have here investigated the effects of introduced bacterial CRTI on the expression of critical endogenous carotenoid biosynthesis genes in rice leaves, rice endosperm, Arabidopsis leaves, and tobacco BY2 cells (see supplemental data). Based on published observations (Al-Babili et al., 1999 ; Römer et al., 2000), it was conceivable that expression of the CrtI transgene provokes transcriptional activation of the carotenoid biosynthetic genes in the target tissue by a feedback regulatory loop. This could be initiated by either the absence of the tetra-cis-lycopene, normally produced by the two plant desaturases (PDS, ZDS), or the constitutive presence of all-trans lycopene, mediated by CRTI. Along these lines of thinking, lycopene isomers would not only represent orthodox intermediates but would also exert a regulatory role. Consequently, the newly identified carotenoid pathway enzyme of plants and cyanobacteria, CRTISO (Isaacson et al., 2002; Park et al., 2002), might play an additional role as regulator. In an extreme scenario, Golden Rice could be yellow due to the transcriptional activation of the entire carotenoid biosynthetic pathway. Alternatively, downstream enzymes, such as cyclases and hydroxylases, might be expressed in wild-type rice in an active form. Their activity would be enabled upon delivery of lycopene mediated by the introduced upstream transgenes.
Wild-type rice endosperm displays low levels of all mRNAs required for xanthophyll formation, i.e. PSY, PDS, ZDS, CRTISO,
The endosperm-specific expression of the PDS/ZDS system instead of CRTI in rice endosperm resulted in the formation of comparable levels of colored carotenoids. Thus, the rice endosperm provides the complex requirements for the activity of the plant desaturases. PDS requires a redox chain, employing quinones, a quinone-reductase, and molecular oxygen as a terminal electron acceptor (Beyer et al., 1989
It has been shown that, in contrast to PSY, CRTI is not rate limiting and is capable of desaturating large amounts of phytoene, thereby increasing
By homology, CRTISO is related to CRTI, and it appears that in evolution CrtIso originated from CrtI (Isaacson et al., 2002
Clearly, LCY activities and the activities of the divergent class of
TaqMan real-time PCR analyses with RNA isolated from transgenic rice endosperm did not give any indication of a feedback regulatory loop that may affect the expression of rice carotenoid biosynthetic genes because all were unchanged in level when compared to the wild type. The same was true in leaves of rice and Arabidopsis, both expressing CRTI constitutively, despite a change in the xanthophyll composition. In both cases, lutein decreased, partially compensated by an increase in
The change in the xanthophyll ratio observed in rice and Arabidopsis leaves varied in an event-dependent manner and was inversely correlated to the CRTI expression level. The flux of substrate into either branch of xanthophyll formation is controlled by the two LCYs, converting lycopene into
The effect described is evident only in tissues exhibiting carotenoid biosynthesis in the wild type, such as in leaves. It cannot occur in transformed endosperm lacking carotenoids in the wild type. However, the decrease in lutein in leaves, as described, may have a negative impact on photosynthetic performance. Lutein, the most abundant xanthophyll, is primarily attached to the light-harvesting complex II. Using mutants of Arabidopsis, it has been shown that decreased lutein content leads to a reduction of PSII antenna size (Lokstein et al., 2002 Taken together, we show that Golden Rice grains show no changes in the level of the carotenogenic mRNAs investigated. To search for any potentially occurring transcriptional alteration on a broader scale, a gene expression analysis using a rice gene chip experiment is being completed.
Vector Construction
Transformation of tobacco (Nicotiana tabacum) BY2 cells and Arabidopsis (Arabidopsis thaliana) plants was performed using the binary vector pSToff containing CrtI under the control of the constitutive CaMV 35S promoter. The vector pSToff was constructed by transferring the CrtI expression cassette (CaMV 35S promoter CrtI-nos terminator) from pBaal2 (Ye et al., 2000 To establish the plant carotene desaturation pathway, PCR was employed to make expression cassettes using the daffodil (Narcissus pseudonarcissus) Psy, Pds, and Zds cDNAs with the rice (Oryza sativa) Gt1 promoter. The native 5' untranslated region was included in all 3 cases (as given in the EMBL database) and 141, 151, and 350 nucleotides, respectively, of the 3' untranslated region. The terminators used were from CaMV 35S (for Psy), potP1-11 (for Pds), and nos (for Zds). The resulting 3 expression cassettes were ligated into pJH0104h, a Bin19-based binary vector containing the hpt marker gene under the control of the Ubi-1 promoter and nos terminator.
Tobacco BY2 cells were cultivated in 100 mL Murashige and Skoog medium (Murashige and Skoog, 1962
Transformation of Arabidopsis plants was done according to the protocol of Bechtold and Pelletier (1998 Seeds of Arabidopsis wild type and 4 CRTI-expressing lines (L11-5, L12-5, L14-2, and L27-3) were germinated on Murashisge and Skoog agar plates containing 30 µg mL–1 hygromycin for 10 d. Twenty seedlings from each line were transferred to soil and grown at 65 µmol m–2 s–1 irradiance and an 8-h/16-h day/night cycle at 21°C for 5 weeks. For each line, 5 leaves of each of the 20 seedlings were harvested and combined to produce RNA pools (see below).
For callus generation, 10-d-old seedlings were transferred to callus induction medium agar plates (Banno et al., 2001
The method used for rice transformation was based on previous protocols (Tanaka et al., 1990 Wild-type seedlings from 2 rice cultivars, TP309 and IR64, as well as several homozygous Golden Rice transformation events, were grown in an incubator (Rumed, Laatzen, Germany) at 50 to 70 µmol m–1 s–2 irradiance and a 16-h/8-h day/night cycle at 28°C for 16 d. Six siblings of each event were harvested and combined for analysis. To minimize variations, seedlings of comparable size (25–30 cm) were used.
Homozygosity of rice transformants was determined either by the segregation of color of propagated grains or by PCR. Using inverse PCR, the integration loci of the T-DNA within the rice genome were identified. Subsequently, homozygosity was investigated by PCR using primers deduced from the flanking genomic DNA and from the T-DNA sequence.
For inverse PCR, circular genomic DNA molecules were produced according to the following protocol: 10 µg genomic DNA were digested with HindIII overnight, purified using the GFX-DNA purification kit (Amersham Biosciences, Freiburg, Germany), and eluted with 100 µL 60°C prewarmed water. Then 40 µL of the digested DNA were ligated using T4-ligase (New England Biolabs, Frankfurt) in a total volume of 300 µL. Before adding the enzyme, the reaction mixture was heated for 5 min at 50°C and chilled on ice. The ligation reaction was then incubated for 2 h at room temperature followed by an overnight incubation at 16°C. The enzyme was then heat inactivated for 10 min at 70°C and DNA was precipitated by adding 30 µL NaAc (3 M, pH 4.7), 800 µL ethanol, and by incubating for 2 h at –20°C. After centrifugation, the pellet of circular genomic DNA was washed with 70% ethanol, dried, and dissolved in 100 µL of distilled water. The inverse PCR reactions were performed in a Mastercycler gradient (Eppendorf, Hamburg, Germany) using the inverse primers SalII, SalII-rev for pCaCar transformants (Hoa et al., 2003
Total RNA was isolated using Trizol (Invitrogen) according to the manufacturer's protocol. Equal amounts of total RNA from at least three independent samples per event were combined and subjected to RNA purification and on-column DNaseI digestion using the Qiagen RNeasy mini kit (Qiagen, Hilden, Germany). First-strand cDNA synthesis was performed using the TaqMan reverse transcription reagents (Applied Biosystems, Darmstadt, Germany), according to the manufacturer's protocol. MGB-TaqMan probes and primers (Supplemental Table 2) were designed based on cDNA sequences from tobacco, Arabidopsis, and rice, using Primer Express 2.0 software (Applied Biosystems). Specific mRNA levels were quantified by TaqMan real-time PCR (ABIPrism 7000) using 18S rRNA for normalization (Human 18S rRNA PDAR; Applied Biosystems). 6FAM and VIC reporter (5' end) dyes were used for the MGB-TaqMan probes and 18S rRNA, respectively. The relative quantities of the transcripts were calculated by using the standard curve method (Livak, 1997
The following transgenic events were used in this study: IR64 37B-2b, TP 309 48–67 (transformed with pCaCar; Hoa et al., 2003
Samples were ground to a fine powder under liquid nitrogen and lyophilized. Lyophilized samples (5 mg for Arabidopsis and rice leaf samples, 150 mg for BY2 and Arabidopsis callus samples) were incubated with 4 mL ethanol/1% 2,6-di-tert-butyl-4-methylphenol (Sigma, Taufkirchen, Germany) at 85°C for 10 min, vigorously mixed once at 5 min. After chilling on ice, 6 mL 1% (w/v) NaCl solution and 3 mL petroleum ether:diethyl ether (2:1, v/v) were added to the sample, followed by brief vigorous mixing. The organic phase was recovered after centrifugation for 10 min at 1,400g. The aqueous phase was extracted a second time as described above. The combined organic phases were dried and dissolved in 50 µL chloroform. Ten microliters were subjected to quantitative analysis using HPLC with a C30 reversed-phase column (YMC Europe GmbH, Schermbeck, Germany) and a gradient system, as described (Hoa et al., 2003
cis-Isomers of
Ground leaf material was incubated at 98°C in sample buffer (65 mM Tris-HCl, pH 6.75, 4% (w/v) SDS, 10% (v/v)
Materials described in this publication are available to members of the Golden Rice licensee network within the research and development strategies of the Golden Rice Humanitarian Board. Proposals may be addressed to P.B., in the first instance, for consideration by the Golden Rice Humanitarian Board.
We thank Jorge Mayer for valuable discussions and Randy Cassada for critically reading the manuscript. Received December 9, 2004; returned for revision February 15, 2005; accepted February 15, 2005.
1 This work was supported by the Rockefeller Foundation, New York, and by HarvestPlus (www.harvestplus.org). 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.057927. * Corresponding author; e-mail peter.beyer{at}biologie.uni-freiburg.de; fax 49–761–203–2675.
Al-Babili S, Hartung W, Kleinig H, Beyer P (1999) CPTA modulates levels of carotenogenic proteins and their mRNAs and affects carotenoid and ABA content as well as chromoplast structure in Narcissus pseudonarcissus flowers. Plant Biol 1: 607–612
Banno H, Ikeda Y, Niu QW, Chua NH (2001) Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration. Plant Cell 13: 2609–2618 Bartley GE, Scolnik PA, Beyer P (1999) Two Arabidopsis thaliana carotene desaturases, phytoene desaturase and zeta-carotene desaturase, expressed in Escherichia coli, catalyze a poly-cis pathway to yield pro-lycopene. Eur J Biochem 259: 396–403[ISI][Medline] Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82: 259–266[Medline] Beyer P, Mayer M, Kleinig H (1989) Molecular oxygen and the state of geometric isomerism are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts. Eur J Biochem 184: 141–150[ISI][Medline] Burkhardt PK, Beyer P, Wünn J, Kloeti A, Armstrong GA, Schledz M, v. Lintig J, Potrykus I (1997) Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis. Plant J 11: 1071–1078[CrossRef][ISI][Medline]
Datta K, Baisakh N, Oliva N, Torrizo L, Abrigo E, Tan J, Rai M, Rehana S, Al-Babili S, Beyer P, et al (2003) Bioengineered "golden" indica rice cultivars with Haschke F, Javaid N (1991) Nutritional anemias. Acta Paediatr Scand 374: 38–44 Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6: 271–282[CrossRef][ISI][Medline] Hoa TTC, Al-Babili S, Schaub P, Potrykus I, Beyer P (2003) Golden indica and japonica rice lines amenable to deregulation. Plant Physiol 113: 161–169
Isaacson T, Ohad I, Beyer P, Hirschberg J (2004) Analysis in vitro of the enzyme CRTISO establishes a poly-cis-carotenoid biosynthesis pathway in plants. Plant Physiol 136: 4246–4255
Isaacson T, Ronen G, Zamir D, Hirschberg J (2002) Cloning of tangerine from tomato reveals a carotenoid isomerase essential for the production of beta-carotene and xanthophylls in plants. Plant Cell 14: 333–342 Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the issue specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204: 383–396[CrossRef][ISI] Kuntz M (2004) Plastid terminal oxidase and its biological significance. Planta 218: 896–899[CrossRef][ISI][Medline] Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Medline] Livak KJ (1997) User Bulletin No. 2: ABI PRISM 7700 Sequence Detection System. PE Applied Biosystems, Foster City, CA, pp 11–15 Lokstein H, Tian L, Polle JE, DellaPenna D (2002) Xanthophyll biosynthetic mutants of Arabidopsis thaliana: altered nonphotochemical quenching of chlorophyll fluorescence is due to changes in photosystem II antenna size and stability. Biochim Biophys Acta 1553: 309–319[Medline] Mayer MP, Beyer P, Kleinig H (1990) Quinone compounds are able to replace molecular oxygen as terminal electron acceptor in phytoene desaturation in chromoplasts of Narcissus pseudonarcissus L. Eur J Biochem 191: 359–363[ISI][Medline] Misawa N, Yamano S, Linden H, de Felipe MR, Lucas M, Ikenaga H, Sandmann G (1993) Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtl in transgenic plants showing an increase of beta-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon. Plant J 4: 833–840[CrossRef][ISI][Medline] Murashige T, Skoog K (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497[CrossRef] Nievelstein V, Vandekerckhove J, Tadros MH, v. Lintig J, Nitschke W, Beyer P (1995) Carotene desaturation is linked to a respiratory redox pathway in N. pseudonarcissus chromoplasts membranes. Involvement of a 23 kDa oxygen-evolving-complex-linked protein. Eur J Biochem 233: 864–872[Medline] Norris SR, Barrette TR, DellaPenna D (1995) Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation. Plant Cell 12: 2139–2149 Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright SY, Hinchliffe E, Drake R (2005) Improving the nutritional value of golden rice through increased pro-vitamin A content. Nat Biotechnol (in press)
Park H, Kreunen SS, Cuttriss AJ, DellaPenna D, Pogson BJ (2002) Identification of the carotenoid isomerase provides insight into carotenoid biosynthesis, prolamellar body formation, and photomorphogenesis. Plant Cell 14: 321–332
Redmond TM, Gentleman S, Duncan T, Yu S, Wiggert B, Gantt E, Cunningham FX Jr (2001) Identification, expression, and substrate specificity of a mammalian beta-carotene 15,15'-dioxygenase. J Biol Chem 276: 6560–6565 Römer S, Fraser PD, Kiano JW, Shipton CA, Misawa N, Schuch W, Bramley PM (2000) Elevation of the provitamin A content of transgenic tomato plants. Nat Biotechnol 18: 666–669[CrossRef][ISI][Medline] Sander LC, Sharpless KE, Craft NE, Wise SA (1994) Development of engineered stationary phases for the separation of carotenoid isomers. Anal Chem 66: 1667–1674
Smith DE, Fisher PA (1984) Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol 99: 20–28 Tanaka A, Mita S, Ohta S, Kyozuka J, Shimamoto K, Nakamura K (1990) Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron. Nucleic Acids Res 11: 6767–6770 Tian L, DellaPenna D (2004) Progress in understanding the origin and functions of carotenoid hydroxylases in plants. Arch Biochem Biophys 430: 22–29[CrossRef][ISI][Medline]
Tian L, Magallanes-Lundback M, Musetti V, DellaPenna D (2003) Functional analysis of beta- and epsilon-ring carotenoid hydroxylases in Arabidopsis. Plant Cell 15: 1320–1332 Underwood BA (2000) Overcoming micronutrient deficiencies in developing countries: Is there a role for agriculture? In NS Scrimshaw, ed, Food and Nutrition Bulletin, Vol 21, No 4. United Nations University Press, New York, pp 356–360 UNICEF (2000) The State of World's Children: Statistical Tables. UNICEF, New York
von Lintig J, Vogt K (2000) Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving beta-carotene to retinal. J Biol Chem 275: 11915–11920 Wyss A, Wirtz G, Woggon W, Brugger R, Wyss M, Friedlein A, Bachmann H, Hunziker W (2000) Cloning and expression of beta,beta-carotene 15,15'-dioxygenase. Biochem Biophys Res Commun 271: 334–336[CrossRef][ISI][Medline] Yan W, Jang GF, Haeseleer F, Esumi N, Chang J, Kerrigan M, Campochiaro M, Campochiaro P, Palczewski K, Zack DJ (2001) Cloning and characterization of a human beta,beta-carotene-15,15'-dioxygenase that is highly expressed in the retinal pigment epithelium. Genomics 72: 193–202[CrossRef][ISI][Medline]
Ye X, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin A ( Zhang J, Xu RJ, Elliott MC, Chen DF (1997) Agrobacterium-mediated transformation of elite indica and japonica rice cultivars. Mol Biotechnol 8: 223–231[Medline] This article has been cited by other articles:
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ABSTRACT
RESULTS
max and underwent a second normalization to correct for their individual molar extinction coefficients relative to echinenone (=1), using violaxanthin/neoxanthin (0.896), lutein (0.969), zeaxanthin (0.891), other xanthophylls (0.911), chlorophyll a (1.311), chlorophyll b (0.885), 
