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Engineering the Provitamin A (β-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm

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Science  14 Jan 2000:
Vol. 287, Issue 5451, pp. 303-305
DOI: 10.1126/science.287.5451.303

Abstract

Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.

Vitamin A deficiency causes symptoms ranging from night blindness to those of xerophthalmia and keratomalacia, leading to total blindness. In Southeast Asia, it is estimated that a quarter of a million children go blind each year because of this nutritional deficiency (1). Furthermore, vitamin A deficiency exacerbates afflictions such as diarrhea, respiratory diseases, and childhood diseases such as measles (2,3). It is estimated that 124 million children worldwide are deficient in vitamin A (4) and that improved nutrition could prevent 1 million to 2 million deaths annually among children (3). Oral delivery of vitamin A is problematic (5, 6), mainly due to the lack of infrastructure, so alternatives are urgently required. Success might be found in supplementation of a major staple food, rice, with provitamin A. Because no rice cultivars produce this provitamin in the endosperm, recombinant technologies rather than conventional breeding are required.

Immature rice endosperm is capable of synthesizing the early intermediate geranylgeranyl diphosphate, which can be used to produce the uncolored carotene phytoene by expressing the enzyme phytoene synthase in rice endosperm (7). The synthesis of β-carotene requires the complementation with three additional plant enzymes: phytoene desaturase and ζ-carotene desaturase, each catalyzing the introduction of two double bonds, and lycopene β-cyclase, encoded by the lcy gene. To reduce the transformation effort, a bacterial carotene desaturase, capable of introducing all four double bonds required, can be used.

We used Agrobacterium-mediated transformation to introduce the entire β-carotene biosynthetic pathway into rice endosperm in a single transformation effort with three vectors (Fig. 1) (8). The vector pB19hpc combines the sequences for a plant phytoene synthase (psy) originating from daffodil (9) (Narcissus pseudonarcissus; GenBank accession number X78814) with the sequence coding for a bacterial phytoene desaturase (crtI) originating from Erwinia uredovora(GenBank accession number D90087) placed under control of the endosperm-specific glutelin (Gt1) and the constitutive CaMV (cauliflower mosaic virus) 35S promoter, respectively. The phytoene synthase cDNA contained a 5′-sequence coding for a functional transit peptide (10), and the crtI gene contained the transit peptide (tp) sequence of the pea Rubisco small subunit (11). This plasmid should direct the formation of lycopene in the endosperm plastids, the site of geranylgeranyl-diphosphate formation.

Figure 1

Structures of the T-DNA region of pB19hpc used in single transformations, and of pZPsC and pZLcyH used in co-transformations. Representative Southern blots of independent transgenic T0-plants are given below the respectiveAgrobacterium vectors. LB, left border; RB, right border; “!”, polyadenylation signals; p, promoters; psy, phytoene synthase; crtI, bacterial phytoene desaturase;lcy, lycopene β-cyclase; tp, transit peptide.

To complete the β-carotene biosynthetic pathway, we co-transformed with vectors pZPsC and pZLcyH. Vector pZPsC carries psy and crtI, as in plasmid pB19hpc, but lacks the selectable marker aphIV expression cassette. Vector pZLcyH provides lycopene β-cyclase fromNarcissus pseudonarcissus (12) (GenBank accession number X98796) controlled by rice glutelin promoter and theaphIV gene controlled by the CaMV 35S promoter as a selectable marker. Lycopene β-cyclase carried a functional transit peptide allowing plastid import (10).

Precultured immature rice embryos (n = 800) were inoculated with AgrobacteriumLBA4404/pB19hpc. Hygromycin-resistant plants (n = 50) were analyzed for the presence ofpsy and crtI genes (Fig. 2). Meganuclease I–Sce I digestion released the ∼10-kb insertion containing the aphIV,psy, and crtI expression cassettes. Kpn I was used to estimate the insertion copy number. All samples analyzed carried the transgenes and revealed mostly single insertions.

Figure 2

Phenotypes of transgenic rice seeds. Bar, 1 cm. (A) Panel 1, untransformed control; panels 2 through 4, pB19hpc single transformants lines h11a (panel 2), h15b (panel 3), h6 (panel 4). (B) pZPsC/pZLcyH co-transformants lines z5 (panel 1), z11b (panel 2), z4a (panel 3), z18 (panel 4).

Immature rice embryos (n = 500) were inoculated with a mixture of Agrobacterium LBA4404/pZPsC and LBA4404/pZLcyH. Co-transformed plants were identified by Southern hybridization, and the presence of pZPsC was analyzed by restriction digestion. Presence of the pZLcyH expression cassettes was determined by probing I-Sce I–and Spe I–digested genomic DNA with internallcy fragments. Of 60 randomly selected regenerated lines, all were positive for lcy and 12 contained pZPsC as shown by the presence of the expected fragments: 6.6 kb for the I-Sce I–excised psy and crtI expression cassettes from pZPsC and 9.5 kb for the lcy and aphIV genes from pZCycH (Fig. 1). One to three transgene copies were found in co-transformed plants. Ten plants harboring all four introduced genes were transferred into the greenhouse for setting seeds. All transformed plants described here showed a normal vegetative phenotype and were fertile.

Mature seeds from T0 transgenic lines and from control plants were air dried, dehusked, and, in order to isolate the endosperm, polished with emery paper. In most cases, the transformed endosperms were yellow, indicating carotenoid formation. The pB19hpc single transformants (Fig. 2A) showed a 3:1 (colored/noncolored) segregation pattern, whereas the pZPsC/pZLcyH co-transformants (Fig. 2B) showed variable segregation. The pB19hpc single transformants, engineered to synthesize only lycopene (red), were similar in color to the pZPsC/pZLcyH co-transformants engineered for β-carotene (yellow) synthesis.

Seeds from individual lines (1 g for each line) were analyzed for carotenoids by photometric and by high-performance liquid chromatography (HPLC) analyses (13). The carotenoids found in the pB19hpc single transformants accounted for the color; none of these lines accumulated detectable amounts of lycopene. Instead, β-carotene, and to some extent lutein and zeaxanthin, were formed (Fig. 3). Thus, the lycopene α(ɛ)- and β-cyclases and the hydroxylase are either constitutively expressed in normal rice endosperm or induced upon lycopene formation.

Figure 3

The carotenoid extracts from seeds (1 g for each line) were subjected quantitatively to HPLC analysis. (A) Control seeds, (B) line h2b (single transformant), (C) line z11b (co-transformant), and (D) z4b (co-transformant). The site of lycopene elution in the chromatogram is indicated by an arrow.

The pZPsC/pZLcyH co-transformants had a more variable carotenoid pattern ranging from phenotypes similar to those from single transformations to others that contain β-carotene as almost the only carotenoid. Line z11b is such an example (Fig. 3C and Fig. 2B, panel 2) with 1.6 μg/g carotenoid in the endosperm. However, reliable quantitations must await homozygous lines with uniformly colored grains. Considering that extracts from the sum of (colored/noncolored) segregating grains were analyzed, the goal of providing at least 2 μg/g provitamin A in homozygous lines (corresponding to 100 μg retinol equivalents at a daily intake of 300 g of rice per day), seems to be realistic (7). It is not yet clear whether lines producing provitamin A (β-carotene) or lines possessing additionally zeaxanthin and lutein would be more nutritious, because the latter have been implicated in the maintenance of a healthy macula within the retina (14).

  • * These authors contributed equally to this work.

  • Present address: Agracetus Monsanto, 8520 University Green, Middleton, WI 53562, USA.

  • Present address: Paradigm Genetics, 104 Alexander Drive, Research Triangle Park, NC 27709–4528, USA.

  • § To whom correspondence should be addressed. E-mail: beyer{at}uni-freiburg.de (P.B.) and ingo.potrykus{at}ipw.biol.ethz.ch(I.P.)

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