Effect of low oxygen stress on the metabolic responses of tomato fruit cells

Abstract

Postharvest losses of fruits and vegetables can occur due to cell breakdown and browning during controlled atmosphere storage as a result of low oxygen (O₂) stress. Therefore, the study was designed to better understand the underlying mechanisms of the response of isolated tomato fruit cells incubated at low O₂ (hypoxic and anoxic) conditions as a model system. The O₂ stress conditions used for the experiment were based on the results of the Michaelis-Menten constant (K_m) of respiration. A total of 56 polar metabolites belonging mainly to different functional groups, including amino acids, organic acids, sugars and sugar alcohols, were identified using GC-MS. O₂ stress stimulated the biosynthesis of most of the free amino acids while decreasing the synthesis of most of the organic acids (especially those linked to the tricarboxylic acid cycle), sugars (except for ribose) and other nitrogen-containing compounds. The down-regulation of these TCA cycle metabolites served to provide energy to ensure the survival of the cell. Increases in the sugar alcohol levels and induction of fermentative metabolism were observed under low O₂ stress. By employing multivariate statistics, metabolites were identified that were essential to the oxygen stress response and establishing the correlation between metabolite abundance, oxygen levels, and incubation period were achievable. A higher correlation was observed between the O₂ levels and most of the metabolites.

Keywords: Anoxic stress; Cell culture; Hypoxic stress; Metabolomics; Tomato fruit.

Fig. 1

Picture of a viable (A) and a dead (B) cell isolated from intact tomato tissues. Viable tomato cells were capable of excluding Evan's blue dye.

Fig. 2

Oxygen depletion curve for tomato cells at 20 °C.

Fig. 3

Oxygen uptake rates of tomato fruit cells (a) and magnification of the curve to read the K_m value (b).

Fig. 4a

Fig. 4 is divided into two parts. This part shows the changes in the abundance of 30 metabolites in isolated tomato cells following the introduction of low O₂ stress. All values were the average of three replicates, with the error bars indicating the standard error of the mean. Metabolite levels were expressed relative to the starting values.

Fig. 4b

This part of Fig. 4, shows changes in the abundance of another 26 metabolites in isolated tomato cells following the introduction of low O₂ stress. All values were the average of three replicates, with the error bars indicating the standard error of the mean. Metabolite levels were expressed relative to the starting values.

Fig. 5

PLS-DA plots of tomato fruit cells representing different O₂ conditions with time (LV1 versus LV2). Red circle: 0 kPa; green circle: 5 kPa; and blue circle: 21 kPa. The percentage explained variances were indicated in the axes.

Fig. 6

The schematic representation of metabolic pathways (glycolysis, TCA cycle and oxidative pentose phosphate pathway) at time 6 h during O₂ stress. Solid line in the network indicates a single step connecting two metabolites, and the dotted line represent multiple steps in between. Inside of the blocks showing metabolites at three different O₂conditions (left side red color: 21 kPa O₂; middle blue color: 5 kPa O₂ and right-side green color: 0 kPa O₂)