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Genetic engineering of eggplant accumulating β-carotene in fruit

Plant Cell Reports volume 39, pages 1029–1039 (2020)Cite this article

Abstract

Key message

Genetic engineering of eggplant using fruit-specific EEF48 promoter-driven bacterial PSY gene, crtB, confers β-carotene accumulation in fruit.

Abstract

Eggplant (Solanum melongena L.) is globally cultivated especially in Asia and is an important source of nutrients in the diets of low-income consumers in developing countries. Since fruits of eggplant have low provitamin A carotenoid content, it is expected to develop eggplant with high carotenoid content for combatting vitamin A deficiency. To achieve this, the present study implemented a metabolic engineering strategy to modify the carotenoid biosynthetic pathway in eggplant. Expression analysis of carotenogenic genes in eggplant tissues showed that the expression of the endogenous phytoene synthase (PSY) was low in fruit and callus. Orange-colored calluses were generated from ectopic expression of crtB gene, which encodes bacterial PSY, in eggplant cells. The orange calluses accumulated > 20 μg g−1 FW of β-carotene, which was approximately 150-fold higher than that of the untransformed calluses. These observations suggest that the PSY expression is the rate-limiting step for β-carotene production in callus and fruit. Since the orange calluses did not regenerate plants, we chose eggplant EEF48 gene, which is presumably expressed in fruit. We amplified its promoter region by TAIL-PCR and showed that the EEF48 promoter is indeed active in eggplant fruit. Subsequently, transgenic eggplant lines having EEF48 promoter-driven crtB were produced. Among the transgenic lines produced, one line set fruit containing 1.50 μg g−1 FW of β-carotene, which was 30-fold higher than that of the untransformed fruits (0.05 μg g−1 FW). The self-pollinated progenies showed a 3:1 segregation ratio for the presence and absence of the transgene, which was linked to the β-carotene accumulation in fruit. These results provide a strategy for improvement of carotenoid content in eggplant fruit.

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Abbreviations

CrtB:

Bacterial phytoene synthase

GGPP:

Geranylgeranyl diphosphate

GUS:

β-Glucuronidase

HPLC:

High-pressure liquid chromatography

PSY:

Phytoene synthase

TP:

Transit peptide

References

  • Azadi P, Otang NV, Chin DP, Nakamura I, Fujisawa M, Harada H, Misawa N, Mii M (2010) Metabolic engineering of Lilium × formolongi using multiple genes of the carotenoid biosynthesis pathway. Plant Biotechnol Rep 4:269–280

    Google Scholar 

  • Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nat Protoc 1:581–585

    CAS  PubMed  Google Scholar 

  • Corona V, Aracri B, Kosturkova G, Bartley GE, Pitto L, Giorgetti L, Scolnik PA, Giuliano G (1996) Regulation of a carotenoid biosynthesis gene promoter during plant development. Plant J 9:505–512

    CAS  PubMed  Google Scholar 

  • Dansako T, Kato K, Satoh J, Sekine M, Yoshida K, Shinmyo A (2003) 5′ Untranslated region of the HSP18.2 gene contributes to efficient translation in plant cells. J Biosci Bioeng 95:52–58

    CAS  PubMed  Google Scholar 

  • Davuluri GR, van Tuinen A, Fraser PD, Manfredonia A, Newman R, Burgess D, Brummell DA, King SR, Palys J, Uhlig J, Bramley PM, Pennings HMJ, Bowler C (2005) Fruit-Specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nat Biotechnol 23:890–895

    CAS  PubMed  Google Scholar 

  • Demmig-Adams B (1990) Carotenoids and photoprotection in plants: a role for the xanthophyll zeaxanthin. BBA Bioenergetics 1020:1–24

    CAS  Google Scholar 

  • Dowling JE, Wald G (1958) Vitamin A deficiency and night blindness. Proc Natl Acad Sci USA 44:648–661

    CAS  PubMed  Google Scholar 

  • Ducreux LJM, Morris WL, Hedley PE, Shepherd T, Davies HV, Millam S, Taylor MA (2005) Metabolic engineering of high carotenoid potato tubers containing enhanced levels of β-carotene and lutein. J Exp Bot 56:81–89

    CAS  PubMed  Google Scholar 

  • Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fraser PD, Romer S, Shipton CA, Mills PB, Kiano JW, Misawa N, Drake RG, Schuch W, Bramley PM (2002) Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner. Proc Natl Acad Sci USA 99:1092–1097

    CAS  PubMed  Google Scholar 

  • Fujisawa M, Watanabe M, Choi SK, Teramoto M, Ohyama K, Misawa N (2008) Enrichment of carotenoids in flaxseed (Linum usitatissimum) by metabolic engineering with introduction of bacterial phytoene synthase gene crtB. J Biosci Bioeng 105:636–641

    CAS  PubMed  Google Scholar 

  • Gürbüz N, Uluişik S, Frary A, Frary A, Doğanlar S (2018) Health benefits and bioactive compounds of eggplant. Food Chem 268:602–610

    PubMed  Google Scholar 

  • Guri A, Sink KC (1988) Agrobacterium transformation of eggplant. J Plant Physiol 133:52–55

    CAS  Google Scholar 

  • Harrison EH (2005) Mechanisms of digestion and absorption of dietary vitamin A. Annu Rev Nutr 25:87–103

    CAS  PubMed  Google Scholar 

  • Hirakawa H, Shirasawa K, Miyatake K, Nunome T, Negoro S, Ohyama A, Yamaguchi H, Sato S, Isobe S, Tabata S, Fukuoka H (2014) Draft genome sequence of eggplant (Solanum melongena L.): the representative Solanum species indigenous to the old world. DNA Res 21:649–660

    CAS  PubMed  PubMed Central  Google Scholar 

  • Höfgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucl Acids Res 16:9877

    PubMed  Google Scholar 

  • Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301

    CAS  PubMed  PubMed Central  Google Scholar 

  • Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231

    CAS  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    CAS  Google Scholar 

  • Jeknić Z, Jeknić S, Jevremović S, Subotić A, Chen THH (2014) Alteration of flower color in Iris germanica L. ‘Fire Bride’ through ectopic expression of phytoene synthase gene (crtB) from Pantoea agglomerans. Plant Cell Rep 33:1307–1321

    PubMed  Google Scholar 

  • Kikuchi K, Honda I, Matsuo S, Fukuda M, Saito T (2008) Stability of fruit set of newly selected parthenocarpic eggplant lines. Sci Hort 115:111–116

    Google Scholar 

  • Lewinsohn E, Sitrit Y, Bar E, Azulay Y, Ibdah M, Meir A, Yosef E, Zamir D, Tadmor Y (2005) Not just colors–carotenoid degradation as a link between pigmentation and aroma in tomato and watermelon fruit. Trends Food Sci Tech 16:407–415

    CAS  Google Scholar 

  • Li L, Yuan H (2013) Chromoplast biogenesis and carotenoid accumulation. Arch Biochem Biophys 539:102–109

    CAS  PubMed  Google Scholar 

  • Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681

    CAS  PubMed  Google Scholar 

  • Misawa N, Nakagawa M, Kobayashi K, Yamano S, Izawa Y, Nakamura K, Harashima K (1990) Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J Bacteriol 172:6704–6712

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mishiba KI, Chin DP, Mii I (2005) Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination. Plant Cell Rep 24:297–303

    CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:437–497

    Google Scholar 

  • Nagasawa M, Sugiyama A, Mori H, Shiratake K, Yamaki S (2001) Analysis of genes preferentially expressed in early stage of pollinated and parthenocarpic fruit in eggplant. J Plant Physiol 158:235–240

    CAS  Google Scholar 

  • Nambara E, Marion-Poll A (2005) Abscisic acid biosynthesis and catabolism. Ann Rev Plant Biol 56:165–185

    CAS  Google Scholar 

  • Olson JA (1994) Needs and sources of carotenoids and vitamin A. Nutr Rev 52:S67–S73

    CAS  PubMed  Google Scholar 

  • Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright SY, Hinchliffe E, Adams JL, Silverstone AL, Drake R (2005) Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nat Biotechnol 23:482–487

    CAS  PubMed  Google Scholar 

  • Rahman S, Rahman AS, Alam N, Ahmed AMS, Ireen S, Chowdhury IA, Chowdhury FP, Rahman SMM, Ahmed T (2016) Vitamin A deficiency and determinants of vitamin A status in Bangladeshi children and women: findings of a national survey. Public Health Nutr 20:1114–1125

    PubMed  Google Scholar 

  • Ravanello MP, Ke D, Alvarez J, Huang B, Shewmaker CK (2003) Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production. Metab Eng 5:255–263

    CAS  PubMed  Google Scholar 

  • Ringli C, Keller B, Ryser U (2001) Glycine-rich proteins as structural components of plant cell walls. Cell Mol Life Sci 58:1430–1441

    CAS  PubMed  Google Scholar 

  • Rotino GL, Gleddie S (1990) Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector. Plant Cell Rep 9:26–29

    CAS  PubMed  Google Scholar 

  • Saini DK, Kaushik P (2019) Visiting eggplant from a biotechnological perspective: a review. Sci Hort 253:327–340

    Google Scholar 

  • San José R, Sánchez-Mata MC, Cámara M, Prohens J (2014) Eggplant fruit composition as affected by the cultivation environment and genetic constitution. J Sci Food Agric 94:2774–2784

    PubMed  Google Scholar 

  • Santino CG, Stanford GL, Conner TW (1997) Developmental and transgenic analysis of two tomato fruit enhanced genes. Plant Mol Biol 33:405–416

    CAS  PubMed  Google Scholar 

  • Tanaka Y, Sasaki N, Ohmiya A (2008) Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J 54:733–749

    CAS  PubMed  Google Scholar 

  • Vervliet G, Holsters M, Teuchy H, Van Montagu M, Schell J (1975) Characterization of different plaque-forming and defective temperate phages in Agrobacterium strains. J Gen Virol 26:33–48

    CAS  PubMed  Google Scholar 

  • Walter MH, Strack D (2011) Carotenoids and their cleavage products: biosynthesis and functions. Nat Prod Rep 28:663–692

    CAS  PubMed  Google Scholar 

  • Weese TL, Bohs L (2010) Eggplant origins: out of Africa, into the orient. Taxon 59:49–56

    Google Scholar 

  • Yang X, Cheng YF, Deng C, Ma Y, Wang ZW, Chen XH, Xue LB (2014) Comparative transcriptome analysis of eggplant (Solanum melongena L.) and turkey berry (Solanum torvum Sw.): phylogenomics and disease resistance analysis. BMC Genom 15:412

    Google Scholar 

  • Ye X, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287:303–305

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank Dr. Norihiko Misawa (Ishikawa Prefectural University) for the pBIScrtB vector,

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Authors and Affiliations

  1. Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen, Nakaku, Sakai, Osaka, 599-8531, Japan

    Kei-Ichiro Mishiba, Kae Nishida, Naoto Inoue, Tomoya Fujiwara, Shunji Teranishi, Yuji Iwata & Nozomu Koizumi

  2. Graduate School of Sciences, Osaka Prefecture University, 1-1 Gakuen, Nakaku, Sakai, Osaka, 599-8531, Japan

    Satomi Takeda

Authors
  1. Kei-Ichiro Mishiba
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  2. Kae Nishida
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  3. Naoto Inoue
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  4. Tomoya Fujiwara
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  5. Shunji Teranishi
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  6. Yuji Iwata
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  7. Satomi Takeda
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  8. Nozomu Koizumi
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Correspondence to Nozomu Koizumi.

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Communicated by Günther Hahne.

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Mishiba, KI., Nishida, K., Inoue, N. et al. Genetic engineering of eggplant accumulating β-carotene in fruit. Plant Cell Rep 39, 1029–1039 (2020). https://doi.org/10.1007/s00299-020-02546-8

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  • DOI: https://doi.org/10.1007/s00299-020-02546-8

Keywords

  • Eggplant
  • Carotenoid
  • Genetic engineering
  • Agrobacterium