Physiological, Nutritional and Metabolomic Responses of Tomato Plants After the Foliar Application of Amino Acids Aspartic Acid, Glutamic Acid and Alanine

Abstract

Agriculture is facing a great number of different pressures due to the increase in population and the greater amount of food it demands, the environmental impact due to the excessive use of conventional fertilizers, and climate change, which subjects the crops to extreme environmental conditions. One of the solutions to these problems could be the use of biostimulant products that are rich in amino acids (AAs), which substitute and/or complement conventional fertilizers and help plants adapt to climate change. To formulate these products, it is first necessary to understand the role of the application of AAs (individually or as a mixture) in the physiological and metabolic processes of crops. For this, research was conducted to assess the effects of the application of different amino acids (Aspartic acid (Asp), Glutamic acid (Glu), L-Alanine (Ala) and their mixtures Asp + Glu and Asp + Glu + Ala on tomato seedlings (Solanum lycopersicum L.). To understand the effect of these treatments, morphological, physiological, ionomic and metabolomic studies were performed. The results showed that the application of Asp + Glu increased the growth of the plants, while those plants that received Ala had a decreased dry biomass of the shoots. The greatest increase in the growth of the plants with Asp + Glu was related with the increase in the net CO₂ assimilation, the increase of proline, isoleucine and glucose with respect to the rest of the treatments. These data allow us to conclude that there is a synergistic effect between Aspartic acid and Glutamic acid, and the amino acid Alanine produces phytotoxicity when applied at 15 mM. The application of this amino acid altered the synthesis of proline and the pentose-phosphate route, and increased GABA and trigonelline.

Keywords: 1H-NMR; gas exchange parameters; metabolites; minerals; nutrients; organic acids; sugars.

FIGURE 1

Growth parameters: height (cm) (A), diameter stem (mm) (B) and shoot (g dw) (C), measured in the ‘Optima’ variety of tomato after a week from the exogenous application of the treatment with AAs: Control (without AAs), Aspartic acid (Asp ac.), Glutamic acid (Glu ac.), L-Alanine (L-Ala), combination Asp + Glu and combination Asp + Glu + Ala. In the ANOVA: ** and *** indicate significant differences at p < 0.01 and p < 0.001, respectively. The different lower case letters indicate significant differences (p < 0.05) between the means established by Duncan’s test (n = 4).

FIGURE 2

Gas exchange parameters: net CO₂ assimilation rate (A_CO2) (A), stomatal conductance (g_s) (B), Ci/Ca ratio (Ci corresponds to the substomatal CO₂ and Ca correspond to external CO₂) (C) and water use efficiency (WUE) (D), measured in the ‘Optima’ variety of tomato after a week from the exogenous application of the treatment with AAs: Control (without AAs), Aspartic acid (Asp ac.), Glutamic acid (Glu ac.), L-Alanine (L-Ala), combination Asp + Glu and combination Asp + Glu + Ala. In the ANOVA: *** indicate significant differences for p < 0.001. The different lower case letters indicate significant differences (p < 0.05) between the means established by Duncan’s test (n = 4).

FIGURE 3

Chlorophyll fluorescence: antennae efficiency of PSII (Fv′/Fm′) (A), quantum efficiency of PSII (Φ_PSII) (B), photochemical quenching co-efficient (qP) (C) and chlorophylls (Chl) measured in completely developed leaves (DL) (D) and leaf buds (LB) (E), parameters measured in the ‘Optima’ variety of tomato after a week from the exogenous application of the treatment with AAs: Control (without AAs), Aspartic acid (Asp ac.), Glutamic acid (Glu ac.), L-Alanine (L-Ala), combination Asp + Glu and combination Asp + Glu + Ala. In the ANOVA: ‘ns’ indicates non-significant differences for a confidence interval of 95%; on their part, *, **, and *** indicate significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively. The different lower case letters indicate significant differences (p < 0.05) between the means established by Duncan’s test (n = 4).

FIGURE 4

Concentration of organic acids (mg g^{– 1} dw): malate (A), citrate (B), fumarate (C), and formate (D), quantified by NMR in tomato leaves from the ‘Optima’ variety of tomato after a week from the exogenous application of the treatment with AAs: Control (without AAs), Aspartic acid (Asp ac.), Glutamic acid (Glu ac.), L-Alanine (L-Ala), combination Asp + Glu and combination Asp + Glu + Ala. In the ANOVA: ** and *** indicate significant differences for p < 0.01 and 0.001, respectively. The different lower case letters indicate significant differences (p < 0.05) between the means established by Ducan’s test. The vertical bar indicates the standard error of the mean (n = 4).

FIGURE 5

Concentration of sugars (mg g^{– 1} dw): fructose (A), Sucrose (B), and glucose (C), quantified by NMR in tomato leaves from the ‘Optima’ variety of tomato after a week from the exogenous application of the treatment with AAs: Control (without AAs), Aspartic acid (Asp ac.), Glutamic acid (Glu ac.), L-Alanine (L-Ala), combination Asp + Glu and combination Asp + Glu + Ala. In the ANOVA: * and *** indicate significant differences for p < 0.05 and 0.001, respectively. The different lower case letters indicate significant differences (p < 0.05) between the means established by Ducan’s test. The vertical bar indicates the standard error of the mean (n = 4).

FIGURE 6

Concentration of GABA (A) and Trigonelline (B) (mg g^{– 1} dw) quantified by NMR in tomato leaves from the ‘Optima’ variety of tomato after a week from the exogenous application of the treatment with AAs: Control (without AAs), Aspartic acid (Asp ac.), Glutamic acid (Glu ac.), L-Alanine (L-Ala), combination Asp + Glu and combination Asp + Glu + Ala. In the ANOVA: *** indicates significant differences for p < 0.001. The different lower case letters indicate significant differences (p < 0.05) between the means established by Ducan’s test. The vertical bar indicates the standard error of the mean (n = 4).

FIGURE 7

Principal component analysis (PC1 and PC2) (A) and Cluster analysis (CA) (B) in tomato leaves from the ‘Optima’ variety of tomato after a week from the exogenous application of the treatment with AAs: Control (without AAs), Aspartic acid (Asp ac.), Glutamic acid (Glu ac.), L-Alanine (L-Ala), combination Asp + Glu and combination Asp + Glu + Ala.

FIGURE 8

Summary of the relative results with respect to the control obtained after the foliar application of the AA treatments: Control (without AAs), Aspartic acid (Asp ac.), Glutamic acid (Glu ac.), L-Alanine (L-Ala), combination Asp + Glu and combination Asp + Glu + Ala. The red or green color indicate a significant increase or reduction, respectively, of the concentration of the metabolite in plants from the AA treatments compared with the control treatment. The white color indicates that no significant differences were found between the control and the corresponding AA treatment.