Accumulation of Flavonols over Hydroxycinnamic Acids Favors Oxidative Damage Protection under Abiotic Stress

Abstract

Efficient detoxification of reactive oxygen species (ROS) is thought to play a key role in enhancing the tolerance of plants to abiotic stresses. Although multiple pathways, enzymes, and antioxidants are present in plants, their exact roles during different stress responses remain unclear. Here, we report on the characterization of the different antioxidant mechanisms of tomato plants subjected to heat stress, salinity stress, or a combination of both stresses. All the treatments applied induced an increase of oxidative stress, with the salinity treatment being the most aggressive, resulting in plants with the lowest biomass, and the highest levels of H2O2 accumulation, lipid peroxidation, and protein oxidation. However, the results obtained from the transcript expression study and enzymatic activities related to the ascorbate-glutathione pathway did not fully explain the differences in the oxidative damage observed between salinity and the combination of salinity and heat. An exhaustive metabolomics study revealed the differential accumulation of phenolic compounds depending on the type of abiotic stress applied. An analysis at gene and enzyme levels of the phenylpropanoid metabolism concluded that under conditions where flavonols accumulated to a greater degree as compared to hydroxycinnamic acids, the oxidative damage was lower, highlighting the importance of flavonols as powerful antioxidants, and their role in abiotic stress tolerance.

Keywords: abiotic stress combination; heat; metabolomics; oxidative metabolism; phenylpropanoid metabolism; salinity; tomato.

Figure 1

Total dry weight of tomato plants grown under control conditions, salinity (80 mM NaCl), heat (35°C), or the combination of salinity and heat. The figure is representative of two independent experiments. Values represent means ± SE (n = 12). Duncan test shows statistical differences among treatments (P < 0.05).

Figure 2

Oxidative metabolism in tomato leaves grown under salinity, heat or the combination of salinity and heat. (A). Expression of oxidative stress metabolism-related transcripts. Scale is log₂ of the mean values after normalization against control plants (n = 6). (B). Activities of the ascorbate-glutathione cycle-related enzymes. Values are represented as the log₂ of the absolute mean values after normalization against control plants (n = 6). (C). Absolute measurements of the oxidative damage-related parameters (H₂O₂ concentration, MDA content, and Protein oxidation). Values represent means ± SE (n = 9). Duncan test shows statistical differences among treatments (P < 0.05). Asterisks mean statistically significant values respect to control at P < 0.05: ^***P < 0.001, ^*0.01 < P > 0.05, and no Asterisk means not significant differences.

Figure 3

Metabolomics study in tomato plants grown under salinity, heat or the combination of salinity and heat. (A). Changes in the accumulation of the different compounds that passed statistical significant tests (see Materials and Methods Section). Green means “down-regulation,” red “up-regulation,” and black “not differentially regulated.” (B). Venn diagram of the significantly altered compounds under the different treatments applied. (C): Classification and changes in accumulation of the molecular features (208) common to all the treatments applied.

Figure 4

HPLC analysis and quantification of some phenylpropanoid compounds in tomato plants grown under salinity, heat or the combination of salinity and heat. (A). Caffeoylquinic acids concentration. (B). Flavonols concentration. Values represent means ± SE (n = 9). Duncan test shows statistical differences among treatments (P < 0.05).

Figure 5

Scheme of the proposed changes in phenolic metabolism in tomato plants subjected to heat, salinity and a combination of heat and salinity. Differences in the activities (circles) of the enzymes involved in the phenylpropanoid pathway, and the expression (squares) of the transcripts that code for these enzymes under salinity, heat, and the combination of salinity and heat compared to control conditions are shown. A red color represents up-regulation, whereas green color shows down-regulation respect to the control treatment. Scale is the log₂ of the mean (expression or activity) values after normalization against control plants (n = 6). Absolute activity values as well as log₂ values of the enzymatic activities and transcript expression can be found in Tables S7–S9, respectively.

Figure 6

Activity of the main enzymes involved in phenolic acids degradation and expression of the transcripts that code for these enzymes in tomato leaves. Scale is the log₂ of the mean (expression or activity) values after normalization against control plants (n = 6). Absolute activity values as well as log₂ values of the enzymatic activities and transcript expression can be found in Supporting Information Tables S8, S9. Duncan test shows statistical differences among treatments (P < 0.05).