Metabolome and molecular basis for carbohydrate increase and nitrate reduction in burley tobacco seedlings by glycerol through upregulating carbon and nitrogen metabolism

Abstract

Burley tobacco (Nicotiana Tabacum) is a chlorophyll-deficiency mutant. Nitrate is one precursor of tobacco-specific nitrosamines (TSNAs) and is largely accumulated in burley tobacco. To decrease nitrate accumulation in burley tobacco, glycerol, a polyhydric alcohol compound and physiological regulating material, was sprayed and its effects were investigated based on metabolomic technology and molecular biology. The results showed that glucose, glutamine and glutamic acid increased by 2.6, 5.1 and 196, folds, respectively, in tobacco leaves after glycerol application. Nitrate content was significantly decreased by 12-16% and expression of eight genes responsible for carbon and nitrogen metabolism were up-regulated with glycerol applications under both normal and 20% reduced nitrogen levels (P < 0.01). Leaf biomass of plants sprayed with glycerol and 20% nitrogen reduction was equivalent to that of no glycerol control with normal nitrogen application. Carbohydrates biosynthesis, nitrate transport and nitrate assimilation were enhanced in glycerol sprayed burley tobacco seedlings which might contribute to reduced nitrate and increased carbohydrates contents. In conclusion, glyerol spray coupled with 20% nitrogen reduction would be an effective method to reduce nitrate accumulation in burley tobacco.

Figure 1

Transcriptome analysis strategies and GO enrichment of target DEGs and leaf biomass (a) Venn diagram of down-regulated DEGs of deficient nitrogen condition vs sufficient nitrogen condition in TN90 and TN86 (b) GO enrichment in common down-regulated DEGs between TN90 and TN86 (c) Venn diagram of up-regulated DEGs of deficient nitrogen condition vs sufficient nitrogen condition in TN90 and TN86 (d) GO enrichment in common up-regulated DEGs between TN90 and TN86 (e) Leaf biomass (f) RNA-seq results confirmed by quantitative qRT-PCR. Nd-TN90: TN90 cultivated under deficient nitrogen condition; Ns-TN90: TN90 cultivated under sufficient nitrogen condition; Nd-TN86: TN86 cultivated under deficient nitrogen condition; Ns-TN86: TN86 cultivated under sufficient nitrogen condition. Error bars of leaf biomass indicate standard error of the means (n = 3). Error bars of qRT-PCR indicate standard error of the means (n = 6).

Figure 2

PCA of metabolome of spraying glycerol at normal nitrogen level. The blue boxes indicate that control group with no spraying glycerol. The red circles indicate that treatments with spaying glycerol.

Figure 3

Light stimulus, carbon fixation, sucrose biosynthesis and nitrate assimilation induced by spraying glycerol at 2 days after spray. Expression levels of ten genes were obtained by qRT-PCR. (a) Genes in response to light stimulus. (b) Genes in carbon fixation pathways (c) Genes in sucrose biosynthesis pathways. (d) Genes in nitrate assimilation pathways. CK: control; Gl: glycerol. EGY1 (Probable zinc metalloprotease, Gene ID 833476), CP12-2 (Calvin cycle protein, Gene ID 825414), PGK (Phosphoglycerate kinase, Gene ID 107787830), PPC16 (Phosphoenolpyruvate carboxylase, Gene ID 547769), GLPK (glycerol kinase [Nicotiana attenuata], Gene ID 109221820), SPS (sucrose-phosphate synthase, Gene ID 107766133), SUS2_2 (sucrose synthase [Nicotiana attenuata], Gene ID 109214879), NIA1 (nitrate reductase [NADH] 1 [Nicotiana attenuata], Gene ID 107823732), NLP7 (Protein NLP7, Gene ID 828502), NPF7.3 (Protein NRT1/ PTR family 7.3, Gene ID 840139). Error bars of qRT-PCR indicate standard error of the means (n = 3). Symbols **and * indicate that the significant differences between treatment of control and treatment of spraying glycerol are at 0.01 and 0.05, respectively.

Figure 4

Glycerol enhanced carbon and nitrogen metabolism in burley tobacco 7 days after spray under different nitrogen levels. Expression levels of nine genes were quantified by qRT-PCR. Carbon metabolism: GLPK (glycerol kinase [Nicotiana attenuata], Gene ID 109221820), gpmA (2,3-bisphosphoglycerate-dependent phosphoglycerate mutase, accession number BX908798), PGK (Phosphoglycerate kinase, Gene ID 107787830), SPS (sucrose-phosphate synthase, Gene ID 107766133), SUS2_2 (sucrose synthase [Nicotiana attenuata], Gene ID 109214879). Nitrogen metabolism: NLP7 (Protein NLP7, Gene ID 828502), NPF3.1 (Protein NRT1/ PTR family 3.1, Gene ID 843186), NPF7.3 (Protein NRT1/ PTR family 7.3, Gene ID 840139), NIA1 (nitrate reductase [NADH] 1 [Nicotiana attenuata], Gene ID 107823732).

Figure 5

Effect of pigment content and photosynthesis rate by spraying glycerol under different nitrogen conditions (a,d) Pn (b,e) Chlorophyll a (c,f): Pigment. CK: control; Gl: glycerol. Pn: photosynthesis rate. Error bars of photosynthesis rate indicate standard error of the means (N = 6, “N” means the number of individuals), and error bars of chlorophyll a content and pigment content indicate standard error of the means (n = 3, three biological replicates). Symbols ** ¹and * ¹indicate that the significant differences between treatment of control and treatment of spraying glycerol under sufficient nitrogen condition (N100) are at 0.01 and 0.05, respectively. Symbols ** ²and * ²indicate that the significant differences between treatment of control and treatment of spraying glycerol under 20% less nitrogen application condition (N80) are at 0.01and 0.05, respectively (a–c) TN90 (d–f), TN86.

Figure 6

Effect of spraying glycerol on total soluble sugar (a,d), reducing sugar content (b,e) and biomass accumulation (c,f) in burley tobacco seedlings under different nitrogen conditions. CK: control; Gl: glycerol. TS: total soluble sugar content. RS: reducing sugar content. Error bars indicate standard error of the means (n = 3). Symbols ** ¹indicate that the significant differences between treatment of control and treatment of spraying glycerol under sufficient nitrogen application (N100) are at 0.01. Symbols ** ²indicate that the significant differences between treatment of control and treatment of spraying glycerol under 20% less nitrogen application condition (N80) are at 0.01 (a–c) TN90 (d–f) TN86.

Figure 7

Effect of spraying glycerol on NO₃-N (a,d), total nitrogen content (b,e) and soluble protein content (c,f) in burley tobacco seedlings under different nitrogen conditions. CK: control; Gl: glycerol. TN: total nitrogen content. Error bars indicate standard error of the means (n = 3). Symbols ** ¹indicate that the significant differences between treatment of control and treatment of spraying glycerol under sufficient nitrogen condition (N100) are at 0.01. Symbols ** ²and * ²indicate that the significant differences between treatment of control and treatment of spraying glycerol under 20% less nitrogen application condition (N80) are at 0.01 and 0.05, respectively. (a–c), TN90; (d–f), TN86.