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First published online July 9, 2004; 10.1104/pp.104.039818 Plant Physiology 135:1776-1783 (2004) © 2004 American Society of Plant Biologists Gene Duplication in the Carotenoid Biosynthetic Pathway Preceded Evolution of the Grasses1Department of Biological Sciences, Lehman College, The City University of New York (CUNY), Bronx, New York 10468 (C.E.G., P.D.M., F.L., E.T.W.); and The Graduate School and University Center-CUNY, New York, New York 10016 (P.D.M., F.L., E.T.W.)
Despite ongoing research on carotenoid biosynthesis in model organisms, there is a paucity of information on pathway regulation operating in the grasses (Poaceae), which include plants of world-wide agronomic importance. As a result, efforts to either breed for or metabolically engineer improvements in carotenoid content or composition in cereal crops have led to unexpected results. In comparison to maize (Zea mays), rice (Oryza sativa) accumulates no endosperm carotenoids, despite having a functional pathway in chloroplasts. To better understand why these two related grasses differ in endosperm carotenoid content, we began to characterize genes encoding phytoene synthase (PSY), since this nuclear-encoded enzyme appeared to catalyze a rate-controlling step in the plastid-localized biosynthetic pathway. The enzyme had been previously associated with the maize Y1 locus thought to be the only functional gene controlling PSY accumulation, though function of the Y1 gene product had never been demonstrated. We show that both maize and rice possess and express products from duplicate PSY genes, PSY1 (Y1) and PSY2; PSY1 transcript accumulation correlates with carotenoid-containing endosperm. Using a heterologous bacterial system, we demonstrate enzyme function of PSY1 and PSY2 that are largely conserved in sequence except for N- and C-terminal domains. By database mining and use of ortholog-specific universal PCR primers, we found that the PSY duplication is prevalent in at least eight subfamilies of the Poaceae, suggesting that this duplication event preceded evolution of the Poaceae. These findings will impact study of grass phylogeny and breeding of enhanced carotenoid content in an entire taxonomic group of plant crops critical for global food security.
Carotenoids, a class of over 600 structures derived from isoprenoids, are synthesized by all photosynthetic organisms, some bacteria, and fungi. In plants, carotenoids are essential for plant growth and development; mutations blocking carotenoid accumulation have pleiotropic effects on chloroplast biogenesis and seed development (Robertson et al., 1978  ; Wurtzel, 1992). Carotenoids function as accessory pigments in photosynthesis, as photoprotectors preventing photooxidative damage, and as precursors to the plant hormone, abscisic acid (Hirschberg, 2001). The presence of carotenoids in plant endosperm tissue adds nutritional value; in humans and animals, dietary carotenoids are essential precursors to vitamin A and to retinoid compounds needed in development (Lee et al., 1981; Bendich and Olson, 1989). Nonprovitamin A carotenoids, such as lycopene, lutein, zeaxanthin, and others, also play beneficial roles in human health (Giovannucci et al., 1995; Kohlmeier et al., 1997; Sommerburg et al., 1998; Krinsky et al., 2003). The various roles of carotenoids affecting plant yield and nutritional potential has made them targets for breeding and metabolic engineering (Shewmaker et al., 1999; Matthews and Wurtzel, 2000; Ye et al., 2000; Davison, 2002; Blott et al., 2003; Gallagher et al., 2003).
In plants, the biosynthesis of carotenoids occurs on membranes of chloroplasts, chromoplasts, and amyloplasts, genetically identical plastids of very different internal membrane architecture. The plant enzymes, which are for the most part well established, are encoded in the nucleus and targeted to the plastids. Despite ongoing research on carotenoid biosynthesis in model organisms or carotenoid accumulating flowers, there is a paucity of information on pathway regulation operating in plants of world-wide agronomic importance, most specifically in the grasses (Poaceae). As a result, efforts to either breed for or metabolically engineer improvements in carotenoid content or composition in cereal crops have led to unexpected results because of the insufficient understanding of how metabolon assembly and function are controlled in plastids of different membrane architectures (Ye et al., 2000
The biosynthesis of all carotenoids begins with the formation of the 40-carbon backbone, phytoene, a step mediated by phytoene synthase (PSY; Cunningham and Gantt, 1998
Isolation and Characterization of the Duplicate PSY Genes in Maize and Rice
In an effort to isolate the rice ortholog of maize Y1, a homologous rice EST (AY024350) was identified and used as a hybridization probe to isolate several rice genomic DNA bacterial artificial chromosome (BAC) clones. In Southern blots against rice genomic DNA, all of these clones shared the same pattern of hybridizing fragments, indicating they were the same gene. One genomic clone was sequenced and deposited as GenBank AY024351. Phylogenetic comparison of the deduced rice PSY against deduced peptides from all available PSY expressed sequence tags (ESTs), indicated that the rice PSY did not cluster with the deduced maize Y1 product, while another rice EST, AU082986, was the closest relative of maize Y1 (Matthews, 2001
Was the PSY Gene Duplication Unique to Rice or Was It Also Present in Maize? Prior maize mapping results identified both the Y1 locus on chromosome 6 L (6.01) and a second locus, termed psy2, on chromosome 8 L (8.07; http://www.maizegdb.org/), suggesting that maize also contained duplicate PSY genes. Though there was no evidence that the second locus encoded a functional PSY enzyme, evidence for transcripts originating from two loci was obtained when we found maize ESTs in GenBank that showed homology to either rice PSY1 or PSY2. We then screened a maize B73 genomic DNA BAC library and identified and sequenced both a Y1 ortholog (denoted PSY1) and a maize ortholog for rice PSY2, for which gene structures are shown in Figure 1. Gene structures showed conservation across species; maize PSY1 was more similar to rice PSY1 than maize PSY1 was to maize PSY2 and the same relationship was seen for the rice genes. All four genes contain six exons and five introns. While the size of each of the six exons is conserved across all four genes, intron size is conserved only between orthologous pairs; PSY1 genes have small first introns (approximately 100 bp), longer second and third introns (greater than 600 bp), while PSY2 genes have long first introns (greater than 700 bp) and short second and third introns (approximately 100 bp). The possibility of the second gene, PSY2, being a pseudogene seemed less likely as suggested by the extensive gene structure conservation spanning two different Poaceae subfamilies. Moreover, there was evidence that all four genes were transcribed as indicated by the presence of ESTs in GenBank. At this point, we could not rule out the possibility that the transcripts might not all encode functional products.
The deduced protein sequences for PSY1 and PSY2 of maize and rice were determined and aligned (data not shown). Rice and maize PSY1 proteins shared 84.3% similar residues compared with 71.4% similar residues shared between maize PSY1 and maize PSY2. What most distinguished PSY1 from PSY2 were the distinct domains found at the carboxy termini; PSY1 proteins had a 15-residue domain distinct from the 7-residue C-terminal domain in PSY2. Using the ChoroP Transit Peptide Predictor, all but rice PSY1 were predicted to have transit peptides (Emanuelsson et al., 1999
To test whether the duplicated genes encoded potentially functional enzymes, we used a common tool for testing functionality of carotenoid biosynthetic enzymes (Matthews et al., 2003
Reverse Transcription-PCR to Test Expression of the PSY1 and PSY2 Transcripts in Maize and Rice Tissues
If either PSY1 or PSY2 transcript accumulation correlates with carotenoid accumulation, we would expect that maize and rice would vary specifically in endosperm transcript levels for one or both genes, given that maize endosperm accumulates carotenoids and rice endosperm does not. To test this possibility, RNA was extracted from young seedlings or endosperm from carotenoid-containing yellow maize, carotenoid-deficient white maize, and rice. Gene-specific primers, pretested on cDNAs to confirm specificity (Fig. 3A), were used to amplify the transcripts corresponding to the two PSY genes. As seen in Figure 3B, transcripts for both PSY genes were present in RNA extracted from either tissue only in the yellow maize endosperm line; for the white endosperm line, PSY1 transcripts were absent in endosperm, but both PSY1 and PSY2 transcripts were found in leaves. Similar to the white maize, PSY1 transcripts were only present in rice leaves, but not in endosperm, while PSY2 transcripts were present in both tissues. In comparison, PDS transcripts were amplified and detected in both tissues for both plants regardless of endosperm phenotype (Li et al., 1996
Duplication of the PSY Genes Preceded Evolution of the Poaceae
Since maize and rice belong to different subfamilies of the Poaceae, Panicoideae and Ehrhartoideae, respectively, it was likely that the gene duplication was more widespread in the Poaceae, a phenomenon proven correct by further GenBank database searching. PSY ESTs were identified for Triticum and Hordeum, species in another Poaceae subfamily, Pooideae. Together with the deduced protein sequences for the maize and rice PSY proteins, these additional sequences were compared with deduced PSY proteins of representative dicots and another monocot, Narcissus. The resulting phylogenetic tree seen in Figure 4 shows that for each of the grasses, sequences either cluster into PSY1-like or PSY2-like groups; the grass duplication appeared to have evolved from a common ancestor prior to the evolution of the grasses. In contrast, the dicot duplication seen for the tomato (Lycopersicon) PSY gene is not found in Arabidopsis, which has been fully sequenced, and generally not the rule for dicot taxa, the only other known exception being tobacco (Nicotiana tabacum; Bartley and Scolnik, 1993
How Widespread within the Poaceae Is the Gene Duplication of PSY? To test further for the distribution of the duplicated PSY genes among other Poaceae subfamilies, ortholog-specific universal primers were designed and tested for specificity. As seen in Figure 5A, we observed the expected PSY1 products of 1,123 bp (maize), and not 387 bp that would be obtained if the corresponding region of PSY2 was nonspecifically amplified; and 838 bp (rice), and not a nonspecific PSY2 product of 370 bp. Similarly for PSY2 universal primers, the expected products for maize and rice, 434 bp and 394 bp, respectively, were observed, and not the nonspecific amplification of PSY1, predicted to be 208 bp and 127 bp, respectively. These universal primers were then used to amplify DNA from representative taxa of the Poaceae. DNA sequences of the PCR amplification products, which have been deposited into GenBank, revealed that some amplified genes contained introns, while others did not. Therefore, we used only the exonic regions in a cluster analysis along with corresponding regions of either PSY1 or PSY2 of maize and rice to confirm that the amplified sequence clustered with either PSY1 or PSY2 (data not shown). Using this simple PCR assay, we detected duplicate genes in 12 taxa representing 8 subfamilies in the Poaceae, as shown in Figure 5B. The use of universal PSY PCR primers will be valuable in assessing the distribution of the PSY duplication among the monocots. These tools will also be useful in phylogenetic analyses within the Poaceae subfamilies to offer improved resolution of evolutionary relationships.
We have found that throughout the Poaceae, the gene for PSY is duplicated, suggesting that this duplication occurred prior to evolution of the grasses. Without evidence of gene product function, Palaisa et al. (2003) previously used associative genetics to correlate endosperm carotenoids with allelic states of maize PSY1 (Y1) but not PSY2 loci. Our data support that study and show that PSY1 but not PSY2 transcripts in endosperm correlate with endosperm carotenoid accumulation. Whereas prior studies did not address whether the genes encoded functional enzymes, we demonstrated that for maize, both PSY1 and PSY2 encode functional enzymes as tested in a bacterial system; rice PSY2 is also functional using this heterologous platform. However, in planta, function requires not only the potential for enzyme activity as demonstrated in the bacterial milieu, but also that the enzyme must localize to a plastid membrane where it gains access to substrate produced by an upstream enzyme. Similarly in Narcissus, where only one PSY gene has been described, PSY was found as an inactive soluble plastid stromal form and as an active plastid membrane-bound enzyme (Schledz et al., 1996). We provide data that suggest that only PSY1 seems to have both demonstrated activity in E. coli and to function in endosperm. While rice PSY2 is functional in E. coli, it is apparently not functional in rice endosperm. The transcript is translated in rice endosperm (data not shown) but the enzyme is not functional given the absence of carotenoids or carotenoid intermediates in rice endosperm (Burkhardt et al., 1997). Therefore, our data suggest that PSY1 and PSY2 are not functionally equivalent in planta and that endosperm carotenoid accumulation requires expression of PSY1. The duplicate grass genes are predicted to encode enzymes with variant N and C termini, suggesting that the grass PSYs may target to different plastid membranes. The difference in membrane architecture between endosperm amyloplasts and leaf chloroplasts may offer a possible explanation of why different PSY isoforms may be associated with presence or absence of endosperm carotenoid accumulation. Further characterization of the two grass enzymes will be needed to define their roles in carotenogenesis in the variety of plastid architectures found in cereals and provide insight into the potential for endosperm carotenoid accumulation throughout the grasses.
The PSY duplication in the grasses predisposed evolution of tissue-specific pathway control, providing a mechanism to modify gene expression in the seed without deleterious effects on photosynthetic organs. Had there been only a single PSY gene, its overexpression would have interfered with the photosynthetic complex, most likely causing photosensitivity in the plant (Busch et al., 2002
Plant Materials Maize (Zea mays; Maize Genetic Stock Center, University of Illinois) was field-grown in Bronx, NY; rice (Oryza sativa indica variety) IR36 was greenhouse-grown with supplemental lighting. Maize endosperm dissected at 20 d after pollination, dissected mature rice endosperm and leaf samples from maize and rice were frozen in liquid nitrogen and stored at –80°C prior to use. For Poaceae subfamily PSY gene amplifications, DNA (Dr. Lynn Clark, Iowa State University) was obtained for Bambusa vulgaris, Pharus lappulaceus, and Zeugites pittieri, or prepared from dried leaves (Dr. Paul Peterson, Smithsonian Institution) for Hordeum muticum J. Presl. collected in Ayacucho, Peru, sample identification (ID): Peterson, P. M., Refulio-Rodriguez, N. 16440, Secale cereale collected in Maryland, ID: Pennington, S. J. 1200, Aristida adscensionis collected in Cajamarca, Peru, ID: Peterson, P.M., Refulio-Rodriguez, N. 15059, Phragmites australis (Cav.) Trin. ex Steud., ID: Peterson, P.M. 17519, Pennisetum tristachyum collected in Cajamarca, Peru, ID: Peterson, P.M. Refulio-Rodriguez, N. 15031, Sorghastrum nutans (L.) Nash collected in Mexico, ID: Peterson, P.M., Gonzalez-Elizondo, S., Brothers, L. E. 16684 and Tripsacum zopilotense Hernández & Randolph collected in Tamaulipas, Mexico, ID: Peterson, P. M., Valdes-Reyna, J. 15903.
A maize B73 genomic BAC library containing 92,160 clones in pECBAC1 and representing 5.2x genome equivalents (Dr. H. Zhang, Texas A & M University) was probed with PSY cDNAs (maize PSY1; GenBank ZMU32636); rice PSY2, GenBank AY024350; Zhang et al., 1996
Plasmid pACCAR25 (Misawa et al., 1990
Carotenoids were separated on a Waters (Millipore, Franklin, MA) HPLC system with 2690 separation module, Millenium version 2.0 software (Waters, Franklin, MA), 996 photodiode array detector (Waters), 717 autosampler, using a Nucleosil 5 C18 (5µ, 250 x 4.6 mm) column (Phenomenex, Torrance, CA) with a Nucleosil C18 (5µ, 4 x 3.0 mm) guard column (Phenomenex). Solvent mixtures used for mobile phases were A, acetonitrile:methanol (9:1, v/v) and B, hexanes:methylene chloride:methanol (4.5:4.5:1, v/v/v; Khachik et al., 1999
Total RNA was isolated from maize leaves and 20 d after pollination endosperm using B73 (a yellow endosperm line) or y1 (a white endosperm line), and from rice IR36 leaves and mature endosperm (RNeasy Plant Mini kit for total RNA isolation, Qiagen, Valencia, CA), concentrations measured spectrophotometrically, and 100 ng of RNA used as template for first strand cDNA synthesis (SuperScript First-Strand Synthesis system for RT-PCR, Invitrogen, Carlsbad, CA). A 2-µL aliquot of the first-strand reaction, 1 to 5 ng of cDNA, was used for PCR amplification under conditions pretested for linearity. Gene-specific primers were designed to flank introns and were tested for specificity using maize PSY1 (GenBank ZMU32636), maize PSY2 (GenBank AY450646), rice PSY1 (pTRPSY1-1, GenBank AY445521), and rice PSY2 (GenBank AY024350). PCR reactions contained 20 mM Tris-HCl pH 8.4, 50 mM KCl, 0.2 mM each dNTP, 1.5 mM MgCl2, 0.4 µM each primer, 0.025 units/µL Taq DNA Polymerase (Invitrogen) and were carried out for one cycle of 3 min at 94°C; followed by 35 cycles of (30 s at 94°C; 30 s at annealing temperature, 45 s at 72°C); and one cycle of 10 min at 72°C. Gene-specific primers, annealing temperatures, and expected products were: maize PSY1, forward (no. 509) 5'-GCATTGCTCAAACGCCAG-3'; reverse (no. 519) 5'-CAGAGAGAGCGGCATCAAG-3', 54°C, 300 bp; maize PSY2, forward (no. 532) 5'-GCGGCAAGTTCCACCACCTGT-3'; reverse (no. 529) 5'-CGAGGTCTGCGCCGAGTA-3', 58°C, 150 bp; rice PSY2, forward (no. 145) 5'-CCTGAAAGGCGCAAAGCTG-3'; reverse (no. 146) 5'-CGATAGCATCAAGGATCTGCC-3', 65°C, 682 bp; rice PDS, forward (no. 151) 5'-GACCATGTTCGCTCTTTGGGTGG-3'; reverse (no. 152) 5'-CGATGATTTCAGTGTCACTCCGTCC-3', 61°C, 430 bp; and maize PDS, as described previously (Matthews et al., 2003
For PCR, genomic DNA from dried grass samples was extracted (REDExtract-N-Amp Plant PCR kit, Sigma, St. Louis) and 4 µL added to 10 µL of REDExtract-N-Amp PCR Ready Mix and 0.5-µM final concentration of each primer in a 20-µL reaction. Universal gene-specific were designed based on conserved sequences between maize and rice: PSY1, forward (no. 530) 5'-TTTGGACCGGTGGGAGAA-3' and reverse (no. 520) 5'-GCCCATCACAGGTACGCTCATT-3' (annealing temperature, 54°C); PSY2, forward (no. 672) 5'-GACGAATATTCTCAGAGACG-3' and reverse (no. 673) 5'-ACTTTCCCTCTGAATATGTC-3' (annealing temperature, 50°C). All reactions were as follows: one cycle of 3 min at 94°C; followed by 40 cycles of (30 s at 94°C; 30 s at annealing temperature, 1 min at 72°C); and one cycle of 10 min at 72°C and products purified using the Qiagen MinElute PCR Purification kit (Qiagen) prior to sequencing. The PSY1 GenBank accession numbers were CG892534 (Phragmites australis), CG892535 (Aristida adscensionis), CG892537 (Hordeum muticum), CG892538 (Pennisetum tristachyum), CG892539 (Secale cereale), CG892540 (Sorghastrum nutans), CG892541 (Tripsacum zopilotense), CG892543 (Pharus lappulaceus), CG892544 (Zeugites pittieri) and CG892545 (Bambusa vulgaris). The PSY2 GenBank accession numbers were CG892547 (Phragmites australis), CG892548 (Aristida adscensionis), CG892549 (Hordeum muticum), CG892550 (Secale cereale), CG892551 (Sorghastrum nutans), CG892552 (Tripsacum zopilotense), CG892553 (Bambusa vulgaris), CG892554 (Pharus lappulaceus), CG892555 (Zeugites pittieri), and CG892559 (Pennisetum tristachyum). Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY024351, ZMU32636, AY024350, AY324431, AY325302, AP005750, AL831803, AY450646, AY452768, ZMU32636, AY450646, AY445521, AY024350, CG892534, CG892535, CG892537, CG892538, CG892539, CG892540, CG892541, CG892543, CG892544, CG892545, CG892547, CG892548, CG892549, CG892550, CG892551, CG892552, CG892553, CG892554, CG892555, CG892559, L23424.1, M84744, Z37543, BT000450.1, AB0379751, BI955682, BM137086, AY325302, AL831803, BE421261, CD862515, AU082986, and X78814.
We thank Drs. A. Vigneswaran and V. Upasani for assistance in some of the earlier work, Dr. F. Khachik (University of Maryland) for valuable advice on carotenoid analysis, Drs. B. Buckner (Truman State University) for the PSY1 cDNA, N. Misawa for Erwinia constructs, S. McCouch (Cornell University) for rice, P. Peterson (Smithsonian Institution), and L. Clark (Iowa State University) for Poaceae subfamily plant and DNA samples, respectively. Received January 27, 2004; returned for revision April 19, 2004; accepted April 28, 2004.
1 This work was supported by the NIH (grant no. S06–GM08225), by PSCUNY, and by the Rockefeller Foundation International Rice Biotechnology Program. 
2 Present address: S.S. Steiner, Inc., 655 Madison Avenue, New York, NY 10021–8078. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.039818. * Corresponding author; e-mail etwlc{at}cunyvm.cuny.edu or wurtzel{at}lehman.cuny.edu; fax 718–960–7348, 718–960–8236.
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RESULTS AND DISCUSSION
crtB + pET23a (empty vector); (C) pACCAR25
-D diglucoside (peak 1), zeaxanthin mono glucoside (peak 2), and zeaxanthin (peak 3) are shown in sections F, G, and H with retention times of 7.2 min, 8.1 min, and 13.1 min, respectively.
