Tissue-Preferential Activity and Induction of the Pepper Capsaicin Synthase PUN1 Promoter by Wounding, Heat and Metabolic Pathway Precursor in Tobacco and Tomato Plants

  • Justin Kirke
  • Noah Kaplan
  • Stephanie Velez
  • Xiao-Lu Jin
  • Paveena Vichyavichien
  • Xing-Hai Zhang
Original Paper
  • 126 Downloads

Abstract

A promoter is an essential structural component of a gene that controls its transcription activity in different development stages and in response to various environmental stimuli. Knowledge of promoter functionality in heterologous systems is important in the study of gene regulation and biotechnological application. In order to explore the activity of the pepper capsaicin synthase gene (PUN1) promoter, gene constructs of pPUN1::GUS (for β-glucuronidase) and pPUN1::NtKED (for a tobacco wound-responsive protein) were introduced into tobacco and tomato, respectively, and their activities were examined. Higher levels of GUS staining intensity and transcription were detected in ovary, anther and pollen than other tissues or organs in tobacco plants. Likewise, transgenic tomato fruits had a higher level of pPUN1::NtKED gene expression than the leaf and flower. The PUN1-driven gene expression can be transiently induced by wounding, heat (40 °C) and the capsaicinoid biosynthetic pathway precursor phenylalanine. When compared to the reported pPUN1::GUS-expressing Arabidopsis, the PUN1 promoter exhibited a more similar pattern of activities among pepper, tobacco and tomato, all Solanaceae plants. Our results suggest the potential utility of this tissue-preferential and inducible promoter in other non-pungent Solanaceae plants for research of gene function and regulation as well as in the biotechnological applications.

Keywords

β-glucuronidase (GUS) Capsaicinoid biosynthetic pathway Phenylalanine Promoter of pepper capsaicin synthase (PUN1Tobacco (Nicotiana tabacum) wound-responsive protein NtKED Tomato (Solanum lycopersicum

Notes

Acknowledgements

The work was supported in part by a Florida Atlantic University Undergraduate Research Grant to JK and SV. We thank Dr. Yu-Bin Zhang (Guangdong Ocean University, China) for help with statistical analysis, and Dr. Jack Widholm (University of Illinois, USA) for reviewing this manuscript.

Authors’ Contributions

XHZ conceived and designed the experiments. JK, NK, SV, XLJ, PV and XHZ performed the experiments. XHZ wrote the manuscript with comments from all co-authors.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

12033_2018_60_MOESM1_ESM.docx (126 kb)
Supplementary material 1 (DOCX 126 kb)

References

  1. 1.
    Stewart, C., Jr., Kang, B. C., Liu, K., Mazourek, M., Moore, S. L., Yoo, E. Y., et al. (2005). The Pun1 gene for pungency in pepper encodes a putative acyltransferase. The Plant Journal, 42, 675–688.CrossRefGoogle Scholar
  2. 2.
    Ogawa, K., Murota, K., Shimura, H., Furuya, M., Togawa, Y., Matsumura, T., et al. (2015). Evidence of capsaicin synthase activity of the Pun1-encoded protein and its role as a determinant of capsaicinoid accumulation in pepper. BMC Plant Biology, 15, 93.  https://doi.org/10.1186/s12870-015-0476-7.CrossRefGoogle Scholar
  3. 3.
    Keyhaninejad, N., Curry, J., Romero, J., & O’Connell, M. A. (2014). Fruit specific variability in capsaicinoid accumulation and transcription of structural and regulatory genes in Capsicum fruit. Plant Science, 215–216, 59–68.CrossRefGoogle Scholar
  4. 4.
    Tanaka, Y., Sonoyama, T., Muraga, Y., Koeda, S., Goto, T., Yoshida, Y., et al. (2015). Multiple loss-of-function putative aminotransferase alleles contribute to low pungency and capsinoid biosynthesis in Capsicum chinense. Molecular Breeding, 35, 142.CrossRefGoogle Scholar
  5. 5.
    Stewart, C., Jr., Mazourek, M., Stellari, G. M., O’Connell, M., & Jahn, M. (2007). Genetic control of pungency in C. chinense via the Pun1 locus. Journal of Experimental Botany, 58, 979–991.CrossRefGoogle Scholar
  6. 6.
    Kirii, E., Goto, T., Yoshida, Y., Yasuba, K.-I., & Tanaka, Y. (2017). Non-pungency in a Japanese chili pepper landrace (Capsicum annuum) is caused by a novel loss-of-function Pun1 allele. The Horticulture Journal, 86, 61–69.CrossRefGoogle Scholar
  7. 7.
    Kim, J.-S., Park, M., Lee, D. J., & Kim, B.-D. (2009). Characterization of putative capsaicin synthase promoter activity. Molecular Cells, 28, 331–339.CrossRefGoogle Scholar
  8. 8.
    Hara, K., Yagi, M., Koizumi, N., Kusano, T., & Sano, H. (2000). Screening of wound-responsive genes identifies an immediate-early expressed gene encoding a highly charged protein in mechanically wounded tobacco plants. Plant Cell & Physiology, 41, 684–691.CrossRefGoogle Scholar
  9. 9.
    Diet and Fitness Today. (2017). Lysine in tomatoes calculator. www.dietandfitnesstoday.com/lysine-in-tomatoes.php.
  10. 10.
    Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473–497.CrossRefGoogle Scholar
  11. 11.
    Zhang, X.-H., Takagi, H., & Widholm, J. M. (2004). Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination, callus and shoot formation. Plant Cell Reports, 22, 615–622.CrossRefGoogle Scholar
  12. 12.
    Tsai, F.-Y., Zhang, X.-H., Ulanov, A., & Widholm, J. M. (2010). The application of the yeast N-acetyltransferase MPR1 gene and the proline analogue L-azetidine-2-carboxylic acid as a selectable marker system for plant transformation. Journal of Experimental Botany, 61, 2561–2573.CrossRefGoogle Scholar
  13. 13.
    Hill, W., Jin, X.-L., & Zhang, X.-H. (2016). Expression of an arctic chickweed dehydrin, CarDHN, enhances tolerance to abiotic stress in tobacco plants. Plant Growth Regulation, 80, 323–334.CrossRefGoogle Scholar
  14. 14.
    Sun, H.-J., Uchii, S., Watanabe, S., & Ezura, H. (2006). A highly efficient transformation protocol for micro-tom, a model cultivar for tomato functional genomics. Plant Cell & Physiology, 47, 426–431.CrossRefGoogle Scholar
  15. 15.
    Chetty, V. J., Ceballos, N., Garcia, D., Narváez-Vásquez, J., Lopez, W., & Orozco-Cárdenas, M. L. (2012). Evaluation of four Agrobacterium tumefaciens strains for the genetic transformation of tomato (Solanum lycopersicum, cultivar Micro-Tom). Plant Cell Reports, 32, 239–247.CrossRefGoogle Scholar
  16. 16.
    O’Donnell, P. J., Calvert, C., Atzorn, R., Wasternack, C., Leyser, H. M. O., & Bowles, D. J. (1996). Ethylene as a signal mediating the wound response of tomato plants. Science, 274, 1914–1917.CrossRefGoogle Scholar
  17. 17.
    Jefferson, R. A., Kavanagh, T. A., & Bevan, M. W. (1987). GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal, 6, 3901–3907.Google Scholar
  18. 18.
    Schmidt, G. W., & Delaney, S. K. (2010). Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Molecular Genetics and Genomics, 283, 233–241.CrossRefGoogle Scholar
  19. 19.
    Løvdal, T., & Lillo, C. (2009). Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Analytical Biochemistry, 387, 238–242.CrossRefGoogle Scholar
  20. 20.
    Deikman, J., Kline, R., & Fischer, R. L. (1992). Organization of ripening and ethylene regulatory regions in a fruit-specific promoter from tomato (Lycopersicon esculentum). Plant Physiology, 100, 2013–2017.CrossRefGoogle Scholar
  21. 21.
    Van Haaren, M. J. J., & Houck, C. M. (1993). A functional map of the fruit-specific promoter of the tomato 2A11 gene. Plant Molecular Biology, 21, 625–640.CrossRefGoogle Scholar
  22. 22.
    Krasnyanski, S. F., Sandhu, J., Domier, L. I., Buetow, D. E., & Korban, S. S. (2001). Effect of an enhanced CAMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants. In Vitro Cellular & Developmental Biology-Plant, 37, 427–433.CrossRefGoogle Scholar
  23. 23.
    Butelli, E., Titta, L., Giorgio, M., Mock, H.-P., Matros, A., Peterek, S., et al. (2008). Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nature Biotechnology, 26, 1301–1308.CrossRefGoogle Scholar
  24. 24.
    Levy, S., & Barkai, N. (2009). Coordination of gene expression with growth rate: A feedback or a feed-forward strategy? FEBS Letters, 583, 3974–3978.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biological SciencesFlorida Atlantic UniversityBoca RatonUSA

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