Genetic engineering of AtAOX1a in Saccharomyces cerevisiae prevents oxidative damage and maintains redox homeostasis

Abstract

This study aimed to validate the physiological importance of Arabidopsis thaliana alternative oxidase 1a (AtAOX1a) in alleviating oxidative stress using Saccharomyces cerevisiae as a model organism. The AOX1a transformant (pYES2AtAOX1a) showed cyanide resistant and salicylhydroxamic acid (SHAM)-sensitive respiration, indicating functional expression of AtAOX1a in S. cerevisiae. After exposure to oxidative stress, pYES2AtAOX1a showed better survival and a decrease in reactive oxygen species (ROS) when compared to S. cerevisiae with empty vector (pYES2). Furthermore, pYES2AtAOX1a sustained growth by regulating GPX2 and/or TSA2, and cellular NAD (+)/NADH ratio. Thus, the expression of AtAOX1a in S. cerevisiae enhances its respiratory tolerance which, in turn, maintains cellular redox homeostasis and protects from oxidative damage.

Keywords: Saccharomyces cerevisiae; alternative oxidase 1a; oxidative stress; reactive oxygen species; redox homeostasis; respiration.

Figure 1

Purification profile and molecular mass analysis of a polyhistidine tagged pET28a‐AtAOX1a recombinant protein: (A) 12.5% SDS/PAGE depicting a ~ 36 kDa AtAOX1a protein in different fractions of the purification protocol. Abbreviations used are as follows: M‐marker, P‐pellet (insoluble protein), S‐supernatant (soluble protein), FT‐flow through (supernatant passed through Ni‐NTA column), W‐washing fractions, E‐elute (purified protein). (B) The sequences corresponding to peptide fragments with molecular masses of 1209.819, 1659.057, 1899.286, and 2384.592 Da, respectively, obtained from MALDI‐TOF‐TOF analysis of trypsin digested purified protein showed 100% matching to internal sequences (indicated in red font) of Arabidopsis thaliana AOX1a (AT3G22370), retrieved from NCBI database.

Figure 2

Functional expression of AtAOX1a in Saccharomyces cerevisiae. (A) Western blot showing the AtAOX1a protein (44 kDa) expression in pYES2AtAOX1a (right side) but not in PYES2 (left side); (B) Rates of oxygen uptake by pYES2 and pYES2AtAOX1a in the absence or presence of KCN (1 mm), SHAM (2 mm), and PG (100 μm); (C) Time‐dependent growth curve of pYES2 and pYES2AtAOX1a in the absence or presence of KCN (1 mm) and (D) Growth recovery in pYES2 and pYES2AtAOX1a after KCN (1 mm) treatment for 4 h. Different lowercase alphabetical letters indicate statistically significant difference (P < 0.05).

Figure 3

Effect of H₂O₂ (2 mm) or t‐BOOH (0.25 mm) on the intracellular ROS generation. ROS were monitored in pYES2 and pYES2AtAOX1a at 488 nm (excitation) and 525 nm (emission) wavelengths under a confocal fluorescence microscope as DCF fluorescence produced by the action of esterases on H₂ DCFDA. Sample treated with KCN (1 mm) was used as a positive control.

Figure 4

Effect of H₂O₂ (2 mm) or t‐BOOH (0.25 mm) on (A) the cell survival rate and (B) return to growth assay in pYES2 and pYES2AtAOX1a. Different lowercase alphabetical letters indicate statistically significant difference (P < 0.05).

Figure 5

Relative mRNA profile of the antioxidant genes (A) SOD1, (B) SOD2, (C) GPX2, and (D) TSA2 in pYES2 and pYES2AtAOX1a after exposure to H₂O₂ (2 mm) or t‐BOOH (0.25 mm). ACT1 was used as housekeeping gene. Different lowercase alphabetical letters indicate statistically significant difference (P < 0.05).

Figure 6

Changes in the total cellular pyridine nucleotides (A) NAD ⁺; (B) NADH; and (C) ratio of NAD ⁺ to NADH in pYES2 and pYES2AtAOX1a upon treatment with H₂O₂ (2 mm) or t‐BOOH (0.25 mm). Different lowercase alphabetical letters indicate statistically significant difference (P < 0.05).