Abstract
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Transcriptome analysis revealed the potential mechanism of nitrogen regulating steviol glycosides synthesis via shifting of leaf carbon metabolic flux or inducing certain transcription factors.
Abstract
Nitrogen (N) plays key regulatory roles in both stevia (Stevia rebaudiana) growth and the synthesis of its functional metabolite steviol glycosides (SGs), but the mechanism by which this nutrient regulates SGs synthesis remains to be elucidated. To address this question, a pot experiment was performed in a greenhouse where stevia plants fertilized with N (the control as CK plants) and compared with plants without the supply of N. Physiological and biochemical analyses were conducted to test the growth and metabolic responses of plants to N regimes. Our results showed that N deficiency significantly inhibited plant growth and leaf photosynthesis, while increased leaf SGs contents in stevia (49.97, 46.64 and 84.80% respectively for rebaudioside A, stevioside, and rebaudioside C), which may be partly due to “concentration effect”. Then, transcriptome analysis was conducted to understand the underlying mechanisms. A total of 535 differentially expressed genes were identified, and carbon metabolism-related events were highlighted by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Many of these genes were significantly upregulated by N-deficiency, including those involved in “phenylpropanoid biosynthesis”, “flavonoid biosynthesis” and “starch and sucrose metabolism”. Our study also analyzed the expression patterns of SGs synthesis-related genes under two N regimes and the potential transcription factors linking N nutrition and SG metabolism. N-deficiency may promote SGs synthesis by changing the carbon metabolism flux or inducing certain transcription factors. Our results provide deeper insight into the relationship between N nutrition and SGs synthesis in stevia plants.
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References
Arsenault PR, Vail DR, Wobbe KK, Weathers PJ (2010) Effect of sugars on artemisinin production in Artemisia annua L.: transcription and metabolite measurements. Molecules 15:2302–2318
Barbet-Massin C, Giuliano S, Alletto L, Dayde J, Berger M (2015) Nitrogen limitation alters biomass production but enhances steviol glycoside concentration in Stevia rebaudiana Bertoni. PLoS One 10:e0133067
Beatty PH, Klein MS, Fischer JJ, Lewis IA, Muench DG, Good AG (2016) Understanding plant nitrogen metabolism through metabolomics and computational approaches. Plants 5:39
Cao W, Wang Y, Shi M, Hao X, Zhao W, Wang Y, Ren J, Kai G (2018) Transcription factor SmWRKY1 positively promotes the biosynthesis of tanshinones in Salvia miltiorrhiza. Front Plant Sci 9:554
Chen J, Hou K, Qin P, Liu H, Yi B, Yang W, Wu W (2014) RNA-Seq for gene identification and transcript profiling of three Stevia rebaudiana genotypes. BMC Genom 15:571
Chen Q, Liu Z, Wang B, Wang X, Lai J, Tian F (2015) Transcriptome sequencing reveals the roles of transcription factors in modulating genotype by nitrogen interaction in maize. Plant Cell Rep 34:1761–1771
Debnath M, Ashwath N, Midmore DJ (2019) Physiological and morphological responses to abiotic stresses in two cultivars of Stevia rebaudiana (Bert.) Bertoni. S Afr J Bot 123:124–132
Ding K, Pei T, Bai Z, Jia Y, Ma P, Liang Z (2017) SmMYB36, a novel R2R3-MYB transcription factor, enhances tanshinone accumulation and decreases phenolic acid content in Salvia miltiorrhiza hairy roots. Sci Rep 7:5104
Foito A, Byrne SL, Hackett CA, Hancock RD, Stewart D, Barth S (2012) Short-term response in leaf metabolism of perennial ryegrass (Lolium perenne) to alterations in nitrogen supply. Metabolomics 9:145–156
Guo J, Wu Y, Wang G, Wang T, Cao F (2020) Integrated analysis of the transcriptome and metabolome in young and mature leaves of Ginkgo biloba L. Ind Crop Prod 143:111906
Hu W, Coomer TD, Loka DA, Oosterhuis DM, Zhou Z (2017) Potassium deficiency affects the carbon–nitrogen balance in cotton leaves. Plant Physiol Biochem 115:408–417
Ibrahim MH, Jaafar HZ, Rahmat A, Rahman ZA (2011) Effects of nitrogen fertilization on synthesis of primary and secondary metabolites in three varieties of kacip Fatimah (Labisia pumila Blume). Int J Mol Sci 12:5238–5254
Jiao F, Luo R, Dai X, Liu H, Yu G, Han S, Lu X, Su C, Chen Q, Song Q, Meng C, Li F, Sun H, Zhang R, Hui T, Qian Y, Zhao A, Jiang Y (2020) Chromosome-level reference genome and population genomic analysis provide insights into the evolution and improvement of domesticated mulberry (Morus alba). Mol Plant 13:1001–1012
Kafle GG, Midmore DJ, Gautam R (2017) Effect of nutrient omission and pH on the biomass and concentration and content of steviol glycosides in stevia (Stevia rebaudiana (Bertoni) Bertoni) under hydroponic conditions. J Appl Res Med Aroma 7:136–142
Karimi M, Moradi K (2018) The response of Stevia (Stevia rebaudiana Bertoni) to nitrogen supply under greenhouse condition. J Plant Nutr 41:1695–1704
Kim D, Langmead B, Salzberg SL (2015a) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357–360
Kim MJ, ** J, Zheng J, Wong L, Chua NH, Jang IC (2015b) Comparative transcriptomics unravel biochemical specialization of leaf tissues of stevia for diterpenoid production. Plant Physiol 169:2462–2480
Koeslin-Findeklee F, Rizi VS, Becker MA, Parra-Londono S, Arif M, Balazadeh S, Mueller-Roeber B, Kunze R, Horst WJ (2015) Transcriptomic analysis of nitrogen starvation- and cultivar-specific leaf senescence in winter oilseed rape (Brassica napus L.). Plant Sci 233:174–185
Koricheva J, Larsson S, Haukioja E, Keinänen M (1998) Regulation of woody plant secondary metabolism by resource availability: hypothesis testing by means of meta-analysis. Oikos 83:212–226
Lea US, Slimestad R, Smedvig P, Lillo C (2007) Nitrogen deficiency enhances expression of specific MYB and bHLH transcription factors and accumulation of end products in the flavonoid pathway. Planta 225:1245–1253
Lemus-Mondaca R, Vega-Galvez A, Zura-Bravo L, Ah-Hen K (2012) Stevia rebaudiana Bertoni, source of a high-potency natural sweetener: a comprehensive review on the biochemical, nutritional and functional aspects. Food Chem 132:1121–1132
Liang X, Zhang T, Lu X, Ellsworth DS, BassiriRad H, You C, Wang D, He P, Deng Q, Liu H, Mo J, Ye Q (2020) Global response patterns of plant photosynthesis to nitrogen addition: a meta-analysis. Glob Change Biol 26:3585–3600
Liu W, Zhu DW, Liu DH, Geng MJ, Zhou WB, Mi WJ, Yang TW, Hamilton D (2010) Influence of nitrogen on the primary and secondary metabolism and synthesis of flavonoids in Chrysanthemum morifolium Ramat. J Plant Nutr 33:240–254
Liu J, Osbourn A, Ma P (2015) MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Mol Plant 8:689–708
Liu T, Luo T, Guo X, Zou X, Zhou D, Afrin S, Li G, Zhang Y, Zhang R, Luo Z (2019) PgMYB2, a MeJA-responsive transcription factor, positively regulates the dammarenediol synthase gene expression in Panax ginseng. Int J Mol Sci 20:2219
Liu Z, Li H, Liu J, Wang Y, Zhuang J (2020) Integrative transcriptome, proteome, and microRNA analysis reveals the effects of nitrogen sufficiency and deficiency conditions on theanine metabolism in the tea plant (Camellia sinensis). Hortic Res 7:65
Lucho SR, do Amaral MN, Benitez LC, Milech C, Kleinowski AM, Bianchi VJ, Braga EJB (2018) Validation of reference genes for RT-qPCR studies in Stevia rebaudiana in response to elicitor agents. Physiol Mol Biol Plants 24:767–779
Ma D, Pu G, Lei C, Ma L, Wang H, Guo Y, Chen J, Du Z, Wang H, Li G, Ye H, Liu B (2009) Isolation and characterization of AaWRKY1, an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene, a key gene of artemisinin biosynthesis. Plant Cell Physiol 50:2146–2161
Makino A (2011) Photosynthesis, grain yield, and nitrogen utilization in rice and wheat. Plant Physiol 155:125–129
Maroufi A, Van Bockstaele E, De Loose M (2010) Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11:15
Massad TJ, Dyer LA, Vega CG (2012) Costs of defense and a test of the carbon-nutrient balance and growth-differentiation balance hypotheses for two co-occurring classes of plant defense. PLoS One 7:e47554
Muller V, Lankes C, Zimmermann BF, Noga G, Hunsche M (2013) Centelloside accumulation in leaves of Centella asiatica is determined by resource partitioning between primary and secondary metabolism while influenced by supply levels of either nitrogen, phosphorus or potassium. J Plant Physiol 170:1165–1175
Nemie-Feyissa D, Olafsdottir SM, Heidari B, Lillo C (2014) Nitrogen depletion and small R3-MYB transcription factors affecting anthocyanin accumulation in Arabidopsis leaves. Phytochemistry 98:34–40
Nunes-Nesi A, Fernie AR, Stitt M (2010) Metabolic and signaling aspects underpinning the regulation of plant carbon nitrogen interactions. Mol Plant 3:973–996
Pal PK, Kumar R, Guleria V, Mahajan M, Prasad R, Pathania V, Gill BS, Singh D, Chand G, Singh B, Singh RD, Ahuja PS (2015) Crop-ecology and nutritional variability influence growth and secondary metabolites of Stevia rebaudiana Bertoni. BMC Plant Biol 15:67
Pariasca-Tanaka J, Satoh K, Rose T, Mauleon R, Wissuwa M (2009) Stress response versus stress tolerance: a transcriptome analysis of two rice lines contrasting in tolerance to phosphorus deficiency. Rice 2:167–185
Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33:290–295
Pott DM, Osorio S, Vallarino JG (2019) From central to specialized metabolism: an overview of some secondary compounds derived from the primary metabolism for their role in conferring nutritional and organoleptic characteristics to fruit. Front Plant Sci 10:835
Prakash I, Markosyan A, Bunders C (2014) Development of next generation stevia sweetener: rebaudioside M. Foods 3:162–175
Qu R, Cao Y, Zhang H, Miao Y, Wei L, Tang X, Sun L (2020) Transcriptome analysis of nitrogen metabolism, transcription factors, and indigoid biosynthesis in Isatis indigotica Fort. response to nitrogen availability. J. Plant Growth Regul 40:1181–1197
Quan X, Zeng J, Ye L, Chen G, Han Z, Shah JM, Zhang G (2016) Transcriptome profiling analysis for two Tibetan wild barley genotypes in responses to low nitrogen. BMC Plant Biol 16:30
Rouached H, Rhee SY (2017) System-level understanding of plant mineral nutrition in the big data era. Curr Opin Syst Biol 4:71–77
Scheible WR, Morcuende R, Czechowski T, Fritz C, Osuna D, Palacios-Rojas N, Schindelasch D, Thimm O, Udvardi MK, Stitt M (2004) Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen. Plant Physiol 136:2483–2499
Schluter U, Mascher M, Colmsee C, Scholz U, Brautigam A, Fahnenstich H, Sonnewald U (2012) Maize source leaf adaptation to nitrogen deficiency affects not only nitrogen and carbon metabolism but also control of phosphate homeostasis. Plant Physiol 160:1384–1406
Scogings PF (2018) Foliar flavonol concentration in Sclerocarya birrea saplings responds to nutrient fertilisation according to growth-differentiation balance hypothesis. Phytochem Lett 23:180–184
Singh G, Singh G, Singh P, Parmar R, Paul N, Vashist R, Swarnkar MK, Kumar A, Singh S, Singh AK, Kumar S, Sharma RK (2017) Molecular dissection of transcriptional reprogramming of steviol glycosides synthesis in leaf tissue during developmental phase transitions in Stevia rebaudiana Bert. Sci Rep 7:11835
Su H, Zhang X, He Y, Li L, Wang Y, Hong G, Xu P (2020) Transcriptomic analysis reveals the molecular adaptation of three major secondary metabolic pathways to multiple macronutrient starvation in Tea (Camellia sinensis). Genes 11:241
Sun W, Zhan J, Zheng T, Sun R, Wang T, Tang Z, Bu T, Li C, Wu Q, Chen H (2018) The jasmonate-responsive transcription factor CbWRKY24 regulates terpenoid biosynthetic genes to promote saponin biosynthesis in Conyza blinii H. Lév. J Genet 97:1379–1388
Sun Y, Hou M, Mur LAJ, Yang Y, Zhang T, Xu X, Huang S, Tong H (2019) Nitrogen drives plant growth to the detriment of leaf sugar and steviol glycosides metabolisms in Stevia (Stevia rebaudiana Bertoni). Plant Physiol Biochem 141:240–249
Sun Y, Guo J, Li Y, Luo G, Li L, Yuan H, Mur LAJ, Guo S (2020a) Negative effects of the simulated nitrogen deposition on plant phenolic metabolism: a meta-analysis. Sci Total Environ 719:137442
Sun Y, Wang M, Mur LAJ, Shen Q, Guo S (2020b) Unravelling the roles of nitrogen nutrition in plant disease defences. Int J Mol Sci 21:572
Sun Y, Yang Y, Hou M, Huang X, Zhang T, Huang S, Xu X, Yuan H (2020c) Optimized nitrogen topdressing strategies enhance steviol glycoside productivity in stevia (Stevia rebaudiana Bertoni) plants. J Soil Sci Plant Nut 20:1133–1143
Tavarini S, Sgherri C, Ranieri AM, Angelini LG (2015) Effect of nitrogen fertilization and harvest time on steviol glycosides, flavonoid composition, and antioxidant properties in Stevia rebaudiana Bertoni. J Agric Food Chem 63:7041–7050
Tiwari JK, Buckseth T, Zinta R, Saraswati A, Singh RK, Rawat S, Dua VK, Chakrabarti SK (2020) Transcriptome analysis of potato shoots, roots and stolons under nitrogen stress. Sci Rep 10:1152
Totté N, Charon L, Rohmer M, Compernolle F, Baboeuf I, Geuns JM (2000) Biosynthesis of the diterpenoid steviol, an ent-kaurene derivative from Stevia rebaudiana Bertoni, via the methylerythritol phosphate pathway. Tetrahedron Lett 41:6407–6410
Wang XK (2006) Plant physiology and biochemistry experiment principle and technology. Higher Education Press, Bei**g, pp 167–173
Wang Y, Weathers P (2007) Sugars proportionately affect artemisinin production. Plant Cell Rep 26:1073–1081
Wang J, Song K, Sun L, Qin Q, Sun Y, Pan J, Xue Y (2019a) Morphological and transcriptome analysis of wheat seedlings response to low nitrogen stress. Plants 8:98
Wang L, Lu W, Ran L, Dou L, Yao S, Hu J, Fan D, Li C, Luo K (2019b) R2R3-MYB transcription factor MYB6 promotes anthocyanin and proanthocyanidin biosynthesis but inhibits secondary cell wall formation in Populus tomentosa. Plant J 99:733–751
Wang M, He X, Jiang B, Liu W, Lin Y, **e D, Liang Z, Chen L, Peng Q (2019c) Transcriptome analysis in different chieh-qua cultivars provides new insights into drought-stress response. Plant Biotechnol Rep 13:663–675
Wang J, Zhao H, Wang Y, Lau H, Zhou W, Chen C, Tan S (2020) A review of stevia as a potential healthcare product: up-to-date functional characteristics, administrative standards and engineering techniques. Trends Food Sci Technol 103:264–281
Ward JL, Baker JM, Llewellyn AM, Hawkins ND, Beale MH (2011) Metabolomic analysis of Arabidopsis reveals hemiterpenoid glycosides as products of a nitrate ion-regulated, carbon flux overflow. Proc Natl Acad Sci 108:10762–10767
Warren CR, Dreyer E, Adams MA (2003) Photosynthesis-Rubisco relationships in foliage of Pinus sylvestris in response to nitrogen supply and the proposed role of Rubisco and amino acids as nitrogen stores. Trees 17:359–366
Wei Z, Zeng X, Qin C, Wang Y, Bai L, Xu Q, Yuan H, Tang Y, Nyima T (2016) Comparative transcriptome analysis revealed genes commonly responsive to varied nitrate stress in leaves of Tibetan hulless barley. Front Plant Sci 7:1067
Yadav SK, Guleria P (2012) Steviol glycosides from Stevia: biosynthesis pathway review and their application in foods and medicine. Crit Rev Food Sci 52:988–998
Yang C, Fang X, Wu X, Mao Y, Wang L, Chen X (2012) Transcriptional regulation of plant secondary metabolism. J Integr Plant Biol 54:703–712
Yang W, Yoon J, Choi H, Fan Y, Chen R, An G (2015a) Transcriptome analysis of nitrogen-starvation-responsive genes in rice. BMC Plant Biol 15:31
Yang Y, Huang S, Han Y, Yuan H, Gu C, Wang Z (2015b) Environmental cues induce changes of steviol glycosides contents and transcription of corresponding biosynthetic genes in Stevia rebaudiana. Plant Physiol Biochem 86:174–180
Yu R, Li D, Du X, **a S, Liu C, Shi G (2017) Comparative transcriptome analysis reveals key cadmium transport-related genes in roots of two pak choi (Brassica rapa L. ssp. chinensis) cultivars. BMC Genomics 18:587
Zhang J, Cun Z, Wu H, Chen J (2020a) Integrated analysis on biochemical profiling and transcriptome revealed nitrogen-driven difference in accumulation of saponins in a medicinal plant Panax notoginseng. Plant Physiol Biochem 154:564–580
Zhang S, Liu Q, Lyu C, Chen J, **ao R, Chen J, Yang Y, Zhang H, Hou K, Wu W (2020b) Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana. BMC Genom 21:794
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This work was supported by the Natural Science Foundation of Jiangsu Province (BK20180312), the National Natural Science Foundation of China (31901597) and Jiangsu Key Laboratory for the Research and Utilization of Plant Resources (JSPKLB201810).
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Sun, Y., Xu, X., Zhang, T. et al. Comparative transcriptome analysis provides insights into steviol glycoside synthesis in stevia (Stevia rebaudiana Bertoni) leaves under nitrogen deficiency. Plant Cell Rep 40, 1709–1722 (2021). https://doi.org/10.1007/s00299-021-02733-1
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DOI: https://doi.org/10.1007/s00299-021-02733-1