Dynamics of Short-Term Metabolic Profiling in Radish Sprouts (Raphanus sativus L.) in Response to Nitrogen Deficiency

Abstract

Nitrogen (N) is a macronutrient important for the survival of plants. To investigate the effects of N deficiency, a time-course metabolic profiling of radish sprouts was performed. A total of 81 metabolites-including organic acids, inorganic acid, amino acids, sugars, sugar alcohols, amines, amide, sugar phosphates, policosanols, tocopherols, phytosterols, carotenoids, chlorophylls, and glucosinolates-were characterized. Principal component analysis and heat map showed distinction between samples grown under different N conditions, as well as with time. Using PathVisio, metabolic shift in biosynthetic pathways was visualized using the metabolite data obtained for 7 days. The amino acids associated with glucosinolates accumulated as an immediate response against -N condition. The synthesis of pigments and glucosinolates was decreased, but monosaccharides and γ-tocopherol were increased as antioxidants in radish sprouts grown in -N condition. These results indicate that in radish sprouts, response to N deficiency occurred quickly and dynamically. Thus, this metabolic phenotype reveals that radish responds quickly to N deficiency by increasing the content of soluble sugars and γ-tocopherol, which acts as a defense mechanism after the germination of radish seeds.

Keywords: metabolic profiling; nitrogen; nitrogen deficiency; nitrogen metabolism; radish sprouts.

Figure 1

Phenotypic changes in radish sprouts during nitrogen deficient condition. (A) Growth of radish sprouts under nitrogen sufficient (MS) and deficient (–N) conditions. (B) Fresh weight and (C) dry weight of radish sprouts grown under MS and –N conditions. Changes in the length of hypocotyl (D) and root (E) of radish sprout grown under the two nitrogen conditions. Data for different points are the means ± SD (n = 6). * p ≤ 0.05. DAI, day after light incubation began.

Figure 2

(A) Principal component analysis (PCA) score plots and (B) loading plots obtained from metabolite data of radish sprouts. PC1 and PC2 accounted for >59.1% of the total variance. Amino acids, pigments, and glucosinolates are represented as red, green, and orange, respectively; sugars and sugar alcohols are represented as blue; organic acids and inorganic acids are represented as black; policosanols are represented as gray; tocopherols and phytosterols are represented as purple; and amines, amides, and sugar phosphates are represented as yellow. d, day after light incubation began; MS, radish sprouts grown under nitrogen sufficient conditions; –N, radish sprouts grown under nitrogen-deficient conditions; C20, Eicosanol; C21, Heneicosanol; C22, Docosanol; C23, Tricosanol; C24, Tetracosanol; C26, Hexacosanol; C27, Heptacosanol; C28, Octacosanol; C30, Triacontanol.

Figure 3

Heat map of differences in the contents of metabolites between radish sprouts grown under nitrogen sufficient (MS) and nitrogen deficient (–N) conditions. The average of standardized data is indicated in shades of red and blue for increase and decrease, respectively, in the metabolite content. DAI, day after light incubation began; C20, Eicosanol; C21, Heneicosanol; C22, Docosanol; C23, Tricosanol; C24, Tetracosanol; C26, Hexacosanol; C27, Heptacosanol; C28, Octacosanol; C30, Triacontanol.

Figure 4

Metabolite change data mapped onto the schematic pathway of radish sprouts growth. The log 2-fold change (log₂ FC) values of metabolite levels in radish sprouts grown under nitrogen deficient condition relative to those under nitrogen sufficient condition on each day. The log₂ FC values are represented as a gradient and can be visualized per box on the top right (increased abundance is shown in red and decrease in abundance is shown in green). The gray boxes represent the metabolites that could not be detected. The solid-lines represent a direct link and dotted-lines represent an indirect link between the metabolites. The solid-line arrows show the transfer of metabolites to cell organelles. G6P, Glucose 6-phosphate; F6P, Fructose 6-phosphate; F1,6BP, Fructose 1,6-bisphosphate; 3PG, 3-Phosphoglyceric acid; PEP, 2-Phosphoenolpyruvate; MEP, Mevalonate; MVA, Mevalonic acid; DAI, day after light incubation began.