Molecular breeding of water lily: engineering cold stress tolerance into tropical water lily

Abstract

Water lilies (order Nymphaeales) are rich in both economic and cultural values. They grow into aquatic herbs, and are divided into two ecological types: tropical and hardy. Although tropical water lilies have more ornamental and medicinal values compared to the hardy water lily, the study and utilization of tropical water lilies in both landscaping and pharmaceutical use is greatly hindered due to their limited planting area. Tropical water lilies cannot survive the winter in areas beyond 24.3°N latitude. Here, the transgenic pipeline through the pollen-tube pathway was generated for water lily for the first time. To improve cold stress tolerance of tropical water lilies, a gene encoding choline oxidase (CodA) driven by a cold stress-inducible promoter was transformed into a tropical water lily through the pollen-tube transformation. Six independent transgenic lines were tested for survival rate during two winter seasons from 2015 to 2017 in Hangzhou (30.3°N latitude). PCR and southern blot detection revealed that the CodA gene had been integrated into the genome. Reverse transcription PCR showed that CodA gene was induced after cold stress treatment, and further quantitative real-time PCR revealed different expressions among six lines and line 3 had the highest expression. Multiple physiological experiments showed that after cold stress treatment, both the conductivity and malondialdehyde (MDA) levels from transgenic plants were significantly lower than those of non-transgenic plants, whereas the content of betaine and the activity of superoxide dismutase, catalase, and peroxidase were higher than those from non-transgenic plants. These results suggest that expression of exogenous CodA gene significantly improved the cold stress tolerance of tropical water lilies through a wide range of physiological alterations. Our results currently expanded a six-latitude cultivating area of the tropical water lilies. These results not only illuminate the bright future for water lily breeding but will also facilitate the functional genomic studies.

References

[1] Borsch, T. et al. Phylogeny of Nymphaea (Nymphaeaceae): evidence from substitutions and microstructural changes in the chloroplast trnT-trnF region. Int. J. Plant Sci. 168, 639-671 (2007).

[2] Chen, F., Liu, X., Yu, C., Chen, Y. & Tang, H. Water lilies as emerging models for Darwin’s abominable mystery. Hort. Res. 4, 17051 (2017).

[3] Zalzaleh, M., Mathew, M. & Suresh, N. Performance evaluation of water lily varieties (Nymphaea sp.) for landscape beautification in Kuwait. Am. J. Sci. Ind. Res. 2, 122-128 (2011).

[4] Lu, X. M., Lu, P. Z. & Chen, J. J. Nitrogen and phosphorus removal and morphological and physiological response in Nymphaea tetragona under various planting densities. Toxicol. Environ. Chem. 94, 1319-1330 (2012).

[5] Lavid, N., Barkay, Z. & Tel-Or, E. Accumulation of heavy metals in epidermal glands of the water lily (Nymphaeaceae). Planta 212, 313-322 (2001).

[6] Lavid, N., Schwartz, A., Yarden, O. & Tel-Or, E. The involvement of polyphenols and peroxidase activities in heavy-metal accumulation by epidermal glands of the water lily (Nymphaeaceae). Planta 212, 323-331 (2001).

[7] McDonald, J. A. & Stross, B. Water lily and cosmic serpent: equivalent conduits of the Maya spirit realm. J. Ethnobiol. 32, 74-107 (2012).

[8] Yin, D. D. et al. Assessment of flavonoids and volatile compounds in tea infusions of water lily flowers and their antioxidant activities. Food Chem. 187, 20-28 (2015).

[9] Bhandarkar, M. R. & Khan, A. Antihepatotoxic effect of Nymphaea stellata willd., against carbon tetrachloride-induced hepatic damage in albino rats. J. Ethnopharmacol. 91, 61-64 (2004).

[10] Bello, F. H., Maiha, B. B. & Anuka, J. A. The effect of methanol rhizome extract of Nymphaea lotus Linn. (Nymphaeaceae) in animal models of diarrhoea. J. Ethnopharmacol. 190, 13-21 (2016).

[11] Madhusudhanan, N. et al. In vitro antioxidant and free radical scavenging activity of aqueous and ethanolic flower extract of Nymphaea alba. Int. J. Drug Dev. Res. 3, 252-258 (2011).

[12] Zhu, M. et al. Relationship between the composition of flavonoids and flower colors variation in tropical water lily (Nymphaea) cultivars. PLoS ONE 7, e34335 (2012).

[13] Perry, D. S. & Peter Robinson, F. P. Water Gardening-Water Lilies and Lotuses (Timber Press, Portland, OR, 1996).

[14] Kilbane, T. 2016 Plant registrations new water lilies. IWGS Water Gard. J. 31, 14-17 (2016).

[15] Rudall, P. J. et al. Comparative ovule and megagametophyte development in hydatellaceae and water lilies reveal a mosaic of features among the earliest angiosperms. Ann. Bot. 101, 941-956 (2008).

[16] Gombos, Z., Wada, H. & Murata, N. Unsaturation of fatty acids in membrane lipids enhances tolerance of the cyanobacterium Synechocystis PCC6803 to low-temperature photoinhibition. Proc. Natl. Acad. Sci. USA 89, 9959-9963 (1992).

[17] Worrall, D. A carrot leucine-rich-repeat protein that inhibits ice recrystallization. Science 282, 115-117 (1998).

[18] Vigh, L., Los, Da, Horváth, I. & Murata, N. The primary signal in the biological perception of temperature: Pd-catalyzed hydrogenation of membrane lipids stimulated the expression of the desA gene in Synechocystis PCC6803. Proc. Natl. Acad. Sci. USA 90, 9090-9094 (1993).

[19] Wang, M., Zhang, B. & Wang, Q. Cotton transformation via pollen tube pathway. Methods Mol. Biol. 958, 71-77 (2013).

[20] Xiaoming, S. & Yundong Gu, W. Q. Application of a transformation method via the pollen-tube pathway in agriculture molecular breeding. Life Sci. 4, 77-79 (2007).

[21] Niazian, M., Sadatnoori, S. A., Galuszka, P. & Mortazavian, S. M. M. Tissue culture-based Agrobacterium-mediated and in Planta transformation methods. Czech J. Genet. Plant Breed. 53, 133-143 (2017).

[22] Ikuta, S., Imamura, S., Misakl, H. & Horiuti, Y. Purification and characterization of choline oxidase from Arthrobacter globiformis. Biochemistry 82, 1741-1749 (1977).

[23] Huang, J. et al. Genetic engineering of glycinebetaine production toward enhancing stress tolerance in plants: metabolic limitations. Plant Physiol. 122, 747-756 (2000).

[24] Hayashi, H. et al. Transformation of Arabidopsis thaliana with the codA gene for choline oxidase accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J. 12, 133-142 (1997).

[25] Mohanty, A. et al. Transgenics of an elite indica rice variety Pusa Basmati 1 harbouring the codA gene are highly tolerant to salt stress. Theor. Appl. Genet. 106, 51-57 (2002).

[26] Park, E. J. et al. Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage. Plant J. 40, 474-487 (2004).

[27] Goel, D. et al. Transformation of tomato with a bacterial codA gene enhances tolerance to salt and water stresses. J. Plant Physiol. 168, 1286-1294 (2011).

[28] Prasad, K. V. S. K., Sharmila, P., Kumar, P. A. & Saradhi, P. P. Transformation of Brassica juncea (L.) Czern with bacterial codA gene enhances its tolerance to salt stress. Mol. Breed. 6, 489-499 (2000).

[29] Alia et al. Enhanced tolerance to light stress of transgenic Arabidopsis plants that express the codA gene for a bacterial choline oxidase. Plant Mol. Biol. 40, 279-288 (1999).

[30] Li, H. et al. Overexpression of codA gene confers enhanced tolerance to abiotic stresses in alfalfa. Plant Physiol. Biochem. 85, 31-40 (2014).

[31] Qiao, G. et al. Enhanced cold stress tolerance of transgenic Dendrocalamus latiflorus Munro (Ma bamboo) plants expressing a bacterial CodA gene. In Vitro Cell. Dev. Biol. Plant 50, 385-391 (2014).

[32] Erwin H, B., Sebastian, F., Claudia, K. & Katja Hartig, T. B. Specific and unspecific responses of plants to cold and drought stress. J. Biosci. 32, 501-510 (2007).

[33] Doyle, J. J. & Doyle, J. L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull. 19, 11-15 (1987).

[34] Abercrombie, J. M., O’Meara, B. C., Moffatt, A. R. & Williams, J. H. Developmental evolution of flowering plant pollen tube cell walls: callose synthase (CalS) gene expression patterns. Evodevo 2, 14 (2011).

[35] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method . Methods 25, 402-408 (2001).

[36] Fan, L. & Zheng S, W. X. Antisense suppression of phospholipase D alpha retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves. Plant Cell 9, 2183-2196 (1997).

[37] Liu, J. H. et al. Polyamine biosynthesis of apple callus under salt stress: Importance of the arginine decarboxylase pathway in stress response. J. Exp. Bot. 57, 2589-2599 (2006).

[38] Giannopolitis, C. N. & Ries, S. K. Superoxide dismutases: I. Occurrence in higher plants. Plant Physiol. 59, 309-314 (1977).

[39] Castrejón, S. E. & Yatsimirsky, A. K. Cyclodextrin enhanced fluorimetric determination of malonaldehydce by the thiobarbituric acid method. Talanta 44, 951-957 (1997).

[40] Sima, Y. H., Yao, J. M., Hou, Y. S., Wang, L. & Zhao, L. C. Variations of hydrogen peroxide and catalase expression in Bombyx eggs during diapause initiation and termination. Arch. Insect Biochem. Physiol. 77, 72-80 (2011).

[41] Rao, M., Paliyath, G. & Ormrod, D. P. Ultraviolet-B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiol. 110, 125-136 (1996).

[42] Grieve, C. M. & Grattan, S. R. Rapid assay for determination of water soluble quaternary ammonium compounds. Plant Soil 70, 303-307 (1983).

[43] Bonilla-Barbosa, J., Novelo, A., Hornelas Orozco, Y. & Márquez-Guzmán, J. Comparative seed morphology of Mexican Nymphaea species. Aquat. Bot. 68, 189-204 (2000).

[44] Songpanich, P. & Hongtrakul, V. Intersubgeneric cross in Nymphaea spp. L. to develop a blue hardy water lily. Sci. Hortic. (Amst.). 124, 475-481 (2010).

[45] Chen, F., Zhang, X., Liu, X. & Zhang, L. Evolutionary analysis of MIKC^c-type MADS-box genes in gymnosperms and angiosperms . Front. Plant Sci. 8, 1-11 (2017).