Biochemical, Physiological and Transcriptomic Comparison between Burley and Flue-Cured Tobacco Seedlings in Relation to Carbohydrates and Nitrate Content

Abstract

Burley tobacco is a genotype of chloroplast-deficient mutant with accumulates high levels of tobacco-specific nitrosamines (TSNAs) which would induce malignant tumors in animals. Nitrate is a principle precursor of tobacco-specific nitrosamines. Nitrate content in burley tobacco was significantly higher than that in flue-cured tobacco. The present study investigated differences between the two tobacco types to explore the mechanisms of nitrate accumulation in burley tobacco. transcripts (3079) related to the nitrogen and carbon metabolism were observed. Expression of genes involved in carbon fixation, glucose and starch biosynthesis, nitrate translocation and assimilation were significantly low in burley tobacco than flue-cured tobacco. Being relative to flue-cured tobacco, burley tobacco was significantly lower at total nitrogen and carbohydrate content, nitrate reductase and glutamine synthetase activities, chlorophyll content and photosynthetic rate (Pn), but higher nitrate content. Burley tobacco required six-fold more nitrogen fertilizers than flue-cured tobacco, but both tobaccos had a similar leaf biomass. Reduced chlorophyll content and photosynthetic rate (Pn) might result in low carbohydrate formation, and low capacity of nitrogen assimilation and translocation might lead to nitrate accumulation in burley tobacco.

Keywords: burley tobacco; carbohydrate; chlorophyll; nitrate; nitrogen assimilation.

Figure 1

Transcriptome analysis strategies and GO enrichment of target DEGs. (a) Leaf biomass in burley tobacco and flue-cured tobacco; (b) Venn diagram of DEGs of flue-cured tobacco (NL_G1_G2) vs burley tobacco (NL_Y1_Y2) at the same nitrogen application level and flue-cured tobacco (NL_G1_G2) vs. burley tobacco (NH_Y1_Y2) at the same leaf biomass accumulation condition; (c) PCA analysis of treatments; (d) Genes represented in Profile 73; (e) GO enrichment in Profile 73; (f) Genes represented in Profile 38; (g) GO enrichment in Profile 38. NL_G1: low nitrogen level, HD; NL_G2: low nitrogen level, Z100; NL_Y1: low nitrogen level, TN90; NL_Y2: low nitrogen level, TN86; NH_Y1: high nitrogen level, TN90; NH_Y2: high nitrogen level, TN86.

Figure 2

Expressed pattern analysis of genes involved in nitrogen and carbon metabolism in flue-cured tobacco and burley tobacco. (a) Primary transcripts correlated with carbon and nitrogen metabolism; (b) Starch and sucrose metabolism; (c) Nitrate response, transport and assimilation; (d) Expression pattern of genes related to carbon metabolism; (e) Expression pattern of genes related to nitrogen metabolism. NL_G1: low nitrogen level, HD; NL_G2: low nitrogen level, Z100; NL_Y1: low nitrogen level, TN90; NL_Y2: low nitrogen level, TN86; NH_Y1: high nitrogen level, TN90; NH_Y2: high nitrogen level, TN86. Box-whisker plot represents dispersity of minimum, first quartile, median, third quartile in genes expression level of treatments. Y-axis represents normalized expression level (log 10 (FPKM + 1)). Brown represents genes expression pattern of flue-cured tobacco grown at low nitrogen level. Yellow represents genes expression pattern of burley tobacco grown at low nitrogen level. Green represents genes expression pattern of burley tobacco grown at high nitrogen level. Date are means of three biological replications. Symbol * indicated that gene expressed pattern was significant enrichment in profile (0, 0, −1, −1, −1, −1) performed by STEM. The same gene labels represent the different transcripts of the same gene.

Figure 3

RNA-seq results confirmed by quantitative qRT-PCR. (a) qRT-PCR validation for nine selected genes in flue-cures tobacco and burley tobacco at same nitrogen level; (b) qRT-PCR validation for nine selected genes in flue-cures tobacco and burley tobacco at same leaf biomass condition. Error bars represent standard error (n = 6).

Figure 4

Differences in pigment content (a), carotene content (b), chlorophyll a (c), chlorophyll b (d) in flue-cured tobacco and burley tobacco at the same nitrogen level and same leaf biomass accumulation condition. NL_G1: low nitrogen level, HD; NL_G2: low nitrogen level, Z100; NL_Y1: low nitrogen level, TN90; NL_Y2: low nitrogen level, TN86; NH_Y1: high nitrogen level, TN90; NH_Y2: high nitrogen level, TN86. Error bars indicate standard error of the means (n = 3). Symbols ** ¹ indicate that the significant differences between flue-cured tobacco and burley tobacco at the same nitrogen application are at 0.01, respectively. Symbols ** ² indicate that the significant differences between flue-cured tobacco and burley tobacco at the same leaf biomass accumulation condition are at 0.01, respectively.

Figure 5

Differences in Fv/Fm (a), Pn (b), reducing sugar content (c), total soluble sugar content (d) in flue-cured tobacco and burley tobacco at the same nitrogen level and same leaf biomass accumulation condition. Fv/Fm: Maximum quantum yield of PS II photochemistry; Pn: Photosynthetic rate. NL_G1: low nitrogen level, HD; NL_G2: low nitrogen level, Z100; NL_Y1: low nitrogen level, TN90; NL_Y2: low nitrogen level, TN86; NH_Y1: high nitrogen level, TN90; NH_Y2: high nitrogen level, TN86. Error bars of chlorophyll a fluorescence and photosynthesis rate indicate standard error of the means (N = 20, “N” means the number of individuals), and error bars of reducing sugar content and total soluble sugar content indicate standard error of the means (n = 3, three biological replicates). Symbols ** ¹ and * ¹ indicate that the significant differences between flue-cured tobacco and burley tobacco at the same nitrogen application are at 0.01 and 0.05, respectively. Symbols ** ² indicate that the significant differences between flue-cured tobacco and burley tobacco at the same leaf biomass accumulation condition are at 0.01, respectively.

Figure 6

Differences in NRA/NA (a), GSA/NA (b), NH₄⁺ content (c) and soluble protein content (d) between the two types at the same nitrogen level and same leaf biomass accumulation condition. NA: Nitrogen application level; NRA/NA: Ratio of nitrate reductase activity to nitrogen application level; GSA/NA: Ratio of glutamine synthetase activity to nitrogen application level. NL_G1: low nitrogen level, HD; NL_G2: low nitrogen level, Z100; NL_Y1: low nitrogen level, TN90; NL_Y2: low nitrogen level, TN86; NH_Y1: high nitrogen level, TN90; NH_Y2: high nitrogen level, TN86. Error bars indicate standard error of the means (n = 3). Symbols ** ¹ and * ¹ indicate that the significant differences between flue-cured tobacco and burley tobacco at the same nitrogen application are at 0.01 and 0.05, respectively. Symbols ** ² indicate that the significant differences between flue-cured tobacco and burley tobacco at the same leaf biomass accumulation condition are at 0.01, respectively.

Figure 7

Differences in nitrogen compound between the two types at the same nitrogen level and same leaf biomass accumulation condition. (a) Nitrogen accumulation per plant; (b) Total nitrogen content (TN); (c) NO₃-N content; (d) Ratio of NO₃-N to total nitrogen content (TN) between the two types of different treatments. NL_G1: low nitrogen level, HD; NL_G2: low nitrogen level, Z100; NL_Y1: low nitrogen level, TN90; NL_Y2: low nitrogen level, TN86; NH_Y1: high nitrogen level, TN90; NH_Y2: high nitrogen level, TN86. Error bars indicate standard error of the means (n = 3). Symbols * ¹ indicate that the significant differences between flue-cured tobacco and burley tobacco at the same nitrogen application are at 0.05, respectively. Symbols ** ² indicate that the significant differences between flue-cured tobacco and burley tobacco at the same leaf biomass accumulation condition are at 0.01, respectively.