Roles for Plant Mitochondrial Alternative Oxidase Under Normoxia, Hypoxia, and Reoxygenation Conditions

Abstract

Alternative oxidase (AOX) is a non-energy conserving terminal oxidase in the plant mitochondrial electron transport chain (ETC) that has a lower affinity for oxygen than does cytochrome (cyt) oxidase. To investigate the role(s) of AOX under different oxygen conditions, wild-type (WT) Nicotiana tabacum plants were compared with AOX knockdown and overexpression plants under normoxia, hypoxia (near-anoxia), and during a reoxygenation period following hypoxia. Paradoxically, under all the conditions tested, the AOX amount across plant lines correlated positively with leaf energy status (ATP/ADP ratio). Under normoxia, AOX was important to maintain respiratory carbon flow, to prevent the mitochondrial generation of superoxide and nitric oxide (NO), to control lipid peroxidation and protein S-nitrosylation, and possibly to reduce the inhibition of cyt oxidase by NO. Under hypoxia, AOX was again important in preventing superoxide generation and lipid peroxidation, but now contributed positively to NO amount. This may indicate an ability of AOX to generate NO under hypoxia, similar to the nitrite reductase activity of cyt oxidase under hypoxia. Alternatively, it may indicate that AOX activity simply reduces the amount of superoxide scavenging of NO, by reducing the availability of superoxide. The amount of inactivation of mitochondrial aconitase during hypoxia was also dependent upon AOX amount, perhaps through its effects on NO amount, and this influenced carbon flow under hypoxia. Finally, AOX was particularly important in preventing nitro-oxidative stress during the reoxygenation period, thereby contributing positively to the recovery of energy status following hypoxia. Overall, the results suggest that AOX plays a beneficial role in low oxygen metabolism, despite its lower affinity for oxygen than cytochrome oxidase.

Keywords: alternative oxidase; hypoxia; mitochondria; nitric oxide; reactive oxygen species; reoxygenation.

FIGURE 1

Leaf O₂^– amount (A) and H₂O₂ amount (B) in tobacco plants treated under either normoxia conditions, hypoxia conditions (for 4 h), or reoxygenation conditions (for 15 or 120 min) following a 4 h hypoxia treatment. Data are presented for WT (gray bar), alternative oxidase overexpressors (B8, left closed bar; B7, right closed bar) and alternative oxidase knockdowns (RI9, left open bar; RI29, right open bar). Each experiment included single individuals for each plant line and treatment. Data are the mean ± SD of three independent experiments (n = 3). Within a treatment, plant lines not sharing a common letter are significantly different from one another (P < 0.05).

FIGURE 2

Leaf R-SNO amount (A) and MDA amount (B) in tobacco plants treated under either normoxia conditions, hypoxia conditions (for 4 h), or reoxygenation conditions (for 15 or 120 min) following a 4 h hypoxia treatment. Data are presented for WT (gray bar), alternative oxidase overexpressors (B8, left closed bar; B7, right closed bar) and alternative oxidase knockdowns (RI9, left open bar; RI29, right open bar). Each experiment included single individuals for each plant line and treatment. Data are the mean ± SD of three independent experiments (n = 3). Within a treatment, plant lines not sharing a common letter are significantly different from one another (P < 0.05).

FIGURE 3

Leaf ATP/ADP ratio in tobacco plants treated under either normoxia conditions, hypoxia conditions (for 4 h), or reoxygenation conditions (for 15 or 120 min) following a 4 h hypoxia treatment. Data are presented for WT (gray bar), alternative oxidase overexpressors (B8, left closed bar; B7, right closed bar) and alternative oxidase knockdowns (RI9, left open bar; RI29, right open bar). Each experiment included single individuals for each plant line and treatment. Data are the mean ± SD of three independent experiments (n = 3). Within a treatment, plant lines not sharing a common letter are significantly different from one another (P < 0.05).

FIGURE 4

Typical appearance of wild-type (WT), AOX knockdown (RI9, RI29), and AOX overexpression (B7, B8) plants following either the normoxia (A) or 4 h hypoxia (B) treatment.

FIGURE 5

Nitric oxide (NO) emission rates from tobacco leaves in the presence of 50 mM sodium nitrate (control) or 50 mM sodium nitrate plus 5 mM SHAM. NO emission was measured in an atmosphere containing approximately 0.001% oxygen. Data are presented for WT (gray bar), alternative oxidase overexpressors (B8, left closed bar; B7, right closed bar) and alternative oxidase knockdowns (RI9, left open bar; RI29, right open bar). Each experiment included single individuals for each plant line and treatment. Data are the mean ± SD of three independent experiments (n = 3). Within a treatment, plant lines not sharing a common letter are significantly different from one another (P < 0.05).

FIGURE 6

Leaf mitochondrial aconitase activity (A) and leaf cytosolic aconitase activity (B) in tobacco plants following either a normoxia or 3 h hypoxia treatment. Data are presented for WT (gray bar), alternative oxidase overexpressor (B7, closed bar), and alternative oxidase knockdown (RI9, open bar) plants. Each experiment included single individuals for each plant line and treatment. Data are the mean ± SD of three independent experiments (n = 3). Within a treatment, plant lines not sharing a common letter are significantly different from one another (P < 0.05).

FIGURE 7

Leaf metabolite amounts in tobacco plants following either a normoxia or 3 h hypoxia treatment. Data are presented for WT (gray bar), alternative oxidase overexpressor (B7, closed bar) and alternative oxidase knockdown (RI9, open bar) plants. The metabolites include various sugars (A,B), organic acids (C–H), and amino acids (I–P). Each experiment included single individuals for each plant line and treatment. Data are the mean ± SD of three independent experiments (n = 3). Within a treatment, plant lines not sharing a common letter are significantly different from one another (P < 0.05).

FIGURE 8

Total amount of leaf phenolics (A) and total amount of leaf flavonoids (B) in tobacco plants following either a normoxia or 3 h hypoxia treatment. Data are presented for WT (gray bar), alternative oxidase overexpressor (B7, closed bar) and alternative oxidase knockdown (RI9, open bar) plants. Each experiment included single individuals for each plant line and treatment. Data are the mean ± SD of three independent experiments (n = 3). Within a treatment, plant lines not sharing a common letter are significantly different from one another (P < 0.05).