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Overexpression of CrtR-b2 (carotene beta hydroxylase 2) from S. lycopersicum L. differentially affects xanthophyll synthesis and accumulation in transgenic tomato plants

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Abstract

Plant chloroplasts are enriched in xanthophylls which participate in photosynthesis as light-absorbing pigments and as dissipaters of excess light. In comparison, chromoplasts have evolved the capacity to synthesize and store brightly coloured carotenoid pigments to give flowers and fruits the power to attract pollinators and fruit dispersers. The best performing accumulator of xanthophylls in tomato is the petal chromoplast in contrast to the fruit chromoplast which only seems able to store carotenes. We have generated genetically engineered tomato lines carrying the tomato CrtR-b2 transgene with the aim of forcing the fruit to accumulate beta-xanthophylls. Both chloroplast- and chromoplast-containing tissues of hemizygous transgenic plants were found to contain elevated xanthophyll contents as a direct consequence of the increased number of CrtR-b2 transcripts. Hemizygous transgenic leaves contained fourfold more violaxanthin than control leaves. Developing fruits were yellow instead of green since they lacked chlorophyll a, and their violaxanthin and neoxanthin contents were seven- and threefold higher, respectively, than those of the control. Ripe fruits of hemizygous transgenic plants contained free violaxanthin and significant amounts of esterified xanthophylls. Esterified xanthophylls were present also in ripe fruits of control and homozygous plants. However, in transgenic homozygous plants, we observed a reduction in transcript content in most tissues, particularly in petals, due to a post-transcriptional gene silencing process. These findings demonstrate that tomato fruit chromoplasts can accumulate xanthophylls with the same sequestration mechanism (esterification) as that exploited by chromoplasts of the tomato petal and pepper fruit. This study on transgenic plants overexpressing an important carotenoid gene (CrtR-b2) provides an interesting model for future investigations on perturbations in beta-carotene-derived xanthophyll synthesis which in turn may provide insights into the molecular mechanisms controlling carotenoid metabolism in tomato.

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Acknowledgements

We gratefully acknowledge many colleagues of Metapontum Agrobios and Luciana Pace for their help. This project was conducted within the ITA.LYCO programme financed by the “Ministero dell’Istruzione, dell’Univerità e della Ricerca” of Italy.

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Authors and Affiliations

  1. Metapontum Agrobios, SS Jonica 106, Km 448.2, 75012, Metaponto, Italy

    Caterina D’Ambrosio, Adriana Lucia Stigliani & Giovanni Giorio

Authors
  1. Caterina D’Ambrosio
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  2. Adriana Lucia Stigliani
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  3. Giovanni Giorio
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Corresponding author

Correspondence to Giovanni Giorio.

Electronic supplementary material

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11248_2010_9387_MOESM1_ESM.doc

Fig. 1S Relative transcript content of psy1, psy2, CrtR-b1 and CrtR-b2 genes in various tissues of the three genotypes. The RT-qPCR analyses were performed with gene-specific TaqMan probes. Relative quantification of target gene transcripts was performed using the Standard curve method. Standard curves were prepared for both the target genes and the endogenous reference 18S rRNA gene Three replicated reactions were performed for each sample, both in the construction of the standard curve and in the quantification of samples. Relative starting quantities (RSQs) of transcripts were then divided by the corresponding 18S rRNA starting quantities to normalise for the amount of cDNA used for the PCR reactions. The derived Normalized Relative Quantities (NRQ) of transgenic UO and UU genotype samples were compared to that of the control OO sample (calibrator) across all gene-by-tissue combinations. The only exception was the leaf CrtR-b2 since this transcript is not detectable in the control OO sample. In this case, the UO sample was chosen as the calibrator. The estimates are expressed as the mean ± standard deviation. Fruits are at the Immature Green (IG) stage of control cv. Red Setter and CrtR-b2 transgenic plants (DOC 40 kb)

11248_2010_9387_MOESM2_ESM.doc

Fig. 2S HPLC profiles of carotenoids in ripe fruits of transgenic hemizygous genotype. (a) Unsaponified extract. (b) Saponified extract. Chromatograms were extracted at 450 nm. Peak identification: 1, cis-violaxanthin; 2, all-trans-violaxanthin; 3, neoxanthin; 4, luteoxanthin; 5, anteraxanthin; 6, lutein; 7, zeaxanthin; I.S., Internal Standard (trans-β-apo-8′-carotenal); 8, phytofluene; 9-10, neoxanthin ester; 11, all-trans- β-carotene; 12, δ-carotene derived; 13-14-15, lycopene isomers; 16, all-trans-lycopene. 17: 15-cis-lycopene (DOC 1678 kb)

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D’Ambrosio, C., Stigliani, A.L. & Giorio, G. Overexpression of CrtR-b2 (carotene beta hydroxylase 2) from S. lycopersicum L. differentially affects xanthophyll synthesis and accumulation in transgenic tomato plants. Transgenic Res 20, 47–60 (2011). https://doi.org/10.1007/s11248-010-9387-4

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