Enhancement of carotenoid biosynthesis in transplastomic tomatoes by induced lycopene-to-provitamin A conversion

Abstract

Carotenoids are essential pigments of the photosynthetic apparatus and an indispensable component of the human diet. In addition to being potent antioxidants, they also provide the vitamin A precursor beta-carotene. In tomato (Solanum lycopersicum) fruits, carotenoids accumulate in specialized plastids, the chromoplasts. How the carotenoid biosynthetic pathway is regulated and what limits total carotenoid accumulation in fruit chromoplasts is not well understood. Here, we have introduced the lycopene beta-cyclase genes from the eubacterium Erwinia herbicola and the higher plant daffodil (Narcissus pseudonarcissus) into the tomato plastid genome. While expression of the bacterial enzyme did not strongly alter carotenoid composition, expression of the plant enzyme efficiently converted lycopene, the major storage carotenoid of the tomato fruit, into provitamin A (beta-carotene). In green leaves of the transplastomic tomato plants, more lycopene was channeled into the beta-branch of carotenoid biosynthesis, resulting in increased accumulation of xanthophyll cycle pigments and correspondingly reduced accumulation of the alpha-branch xanthophyll lutein. In fruits, most of the lycopene was converted into beta-carotene with provitamin A levels reaching 1 mg per g dry weight. Unexpectedly, transplastomic tomatoes also showed a >50% increase in total carotenoid accumulation, indicating that lycopene beta-cyclase expression enhanced the flux through the pathway in chromoplasts. Our results provide new insights into the regulation of carotenoid biosynthesis and demonstrate the potential of plastids genome engineering for the nutritional enhancement of food crops.

Figure 1.

Engineering of the carotenoid biosynthetic pathway by plastid transformation. A, Carotenoid biosynthetic pathway in higher plants. The pathway splits into an α-branch and a β-branch immediately downstream of lycopene, the major storage carotenoid of tomato fruits. The enzyme expressed from the tomato plastid genome in this study, lycopene β-cyclase, leads into the β-branch. B, Physical maps of the targeting region in the plastid genome (ptDNA) and the plastid transformation vectors pEcrtY and pNLyc constructed in this study. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. The transgenes are targeted to the intergenic region between the trnfM and trnfG genes (Ruf et al., 2001). The selectable marker gene aadA is driven by a chimeric rRNA operon promoter (Prrn; Svab and Maliga, 1993), fused to the 3′-UTR from the psbA gene (TpsbA), and flanked by two loxP sites to allow marker removal by Cre-mediated site-specific recombination (Zhou et al., 2008). The transgene expression cassette consists of the ribosomal RNA operon promoter fused to the 5′ leader from the gene 10 of phage T7 (Prrn-G10L; Kuroda and Maliga, 2001) and the 3′-UTR of the rps16 gene (Trps16). Restriction sites used for cloning or RFLP analysis are indicated, and the psaB-derived hybridization probe is denoted by a horizontal bar. Sites lost due to ligation to heterologous ends are in parentheses. C, Southern-blot analysis of tomato transplastomic lines carrying the lycopene β-cyclase gene from daffodil (S.l.-pNLyc) or from E. herbicola (S.l.-pEcrtY). Total cellular DNA was digested with BglII and hybridized to a radioactively labeled probe detecting the psaB region of the plastid genome, which flanks the transgene insertion site (section B). Fragment sizes are given in kb. wt, Wild type. D, Alignment (produced with ClustalW2) of the amino acid sequences of the lycopin β-cyclases from daffodil (Np) and E. herbicola (Eh). Asterisk (*) denotes residues identical in both sequences (marked in bold), colon (:) indicates conserved substitutions, and a dot indicates semiconserved substitutions. The N-terminal extension of the Np sequence is likely to harbor the transit peptide for protein import into plastids. The amino acids that changed due to correction of the Lyc sequence from daffodil (published sequence: GenBank accession no. X98796.1; corrected sequence: accession no. GQ327929) are underlined. The corrections improve the sequence similarity in the N-terminal domains of the Np and Eh sequences.

Figure 2.

Homoplasmy and phenotypes of transplastomic tomato lines. A, Examples of seed tests to confirm homoplasmy. Seeds from the wild-type (S.l.-wt) and transplastomic plants generated with constructs pEcrtY and pNLyc were germinated on medium with spectinomycin (100 mg/L). Antibiotic resistance and lack of segregation in the T1 generation confirms the homoplasmic state of the transplastomic lines. B, Wild-type-like phenotype of homoplasmic tomato lines expressing lycopene β-cyclase transgenes from their plastid genomes.

Figure 3.

Analysis of lycopene β-cyclase mRNA accumulation in leaves (A) and ripe fruits (B) of transplastomic tomato plants. Total cellular RNA was hybridized to radiolabeled probes corresponding to the coding regions of crtY or Lyc. With each probe, two major transcript species are detected. While the bottom band represents mature monocistronic lycopene β-cyclase mRNA (1.3 kb for crtY and 1.7 kb for Lyc), the top band most probably represents a stable read-through transcript, as has been observed before with pKP9-derived vectors (Zhou et al., 2008; Oey et al., 2009). Sizes of marker bands are indicated in kb. Note that crtY and Lyc do not cross-hybridize due to insufficient sequence similarity (Fig. 1D). wt, Wild type.

Figure 4.

Herbicide resistance assays to test for lycopene β-cyclase expression. The herbicide CPTA was used as specific inhibitor of the lycopin β-cyclase activity. Aseptically grown tomato plants were watered with 2.5 mL CPTA solution to obtain the final concentrations indicated (50, 100, 150, 250, or 500 μm). Phenotypic comparison with a water-treated control (0 μm) was done after 7 d. wt, Wild type.

Figure 5.

Phenotypes of tomato fruits from transplastomic tomato plants expressing lycopene β-cyclase transgenes. Fruits from a wild-type plant (S.l.-wt), an S.l.-pEcrtY line, and an S.l.-pNLyc line were harvested at different ripening stages and photographed from the side (top row) and from the bottom (bottom row). The orange color of ripe S.l.-pNLyc fruits indicates efficient conversion of red lycopene into orange β-carotene (provitamin A).

Figure 6.

HPLC analysis of pigment accumulation in fruits and leaves from wild-type plants (S.l.-wt) and transplastomic S.l.-pEcrtY and S.l.-pNLyc plants. A, Comparison of lycopene and β-carotene contents (in ng/mg dry weight [DW]) in ripe fruits as determined by HPLC. Values represent means from 12 measurements: three fruits harvested from three to four independent plant lines per construct. B, Comparison of carotenoid contents in leaves. Values represent means from nine measurements, which included leaves harvested from at least four independent transplastomic lines per construct. The sd is shown as error bar. Asterisks indicate significant differences compared to the wild type (P value < 0.05).