Mitochondrial alternative NAD(P)H dehydrogenases A1 and A2 control levels of reactive oxygen species and improve plant survival during reoxygenation

Flooding is a biphasic stress with an initial reduction of molecular oxygen during submergence followed by reoxygenation post-flooding. Survival of this compound stress and growth vigor of the plant rely on efficient control of reactive oxygen species (ROS) homeostasis. Mitochondria are central to cellular energy supply and redox homeostasis. The mitochondrial electron transport chain (mETC) produces ATP under oxygen-sufficient conditions while under limited oxygen level, the mETC gets reduced as electrons are not transferred to molecular oxygen. Upon reoxygenation of flooded plants, the reductive power can lead to excess ROS formation. Plant mitochondria possess an alternative ETC. The alternative NADH dehydrogenases A1 and A2 (NDA1 and NDA2) are plant-specific mitochondrial non-proton pumping respiratory bypass proteins located at the matrix side of the inner mitochondrial membrane. NDAs act in concert with the mitochondrial alternative oxidase (AOX) to dissipate energy conserved in NADH as heat. Based on the hypothesis that the alternative ETC helps avoid excessive ROS production, we studied the role of NDAs in post-anoxic recovery in Arabidopsis thaliana. The results show that NDAs limit ROS formation, and their knockout in nda1 nda2 plants resulted in elevated ROS levels and a much reduced survival rate compared to wild type while overexpression of NDAs led to reduced ROS and a higher survival rate indicating that the alternative ETC is important for plant survival during post-anoxic recovery. The energy and redox metabolism of the cells, in terms of the ATP production and NADH/NAD+ redox balance showed no obvious differences during anoxia and reoxygenation in wild type and nda1-1 nda2-2, NDA1ox3 and NDA2ox2. A lower Fv/Fm ratio in shoots of nda1-1 nda2-2 seedlings after 8 h of reoxygenation indicated that NDAs are essential for photosynthetic recovery during reoxygenation. On the other hand, reoxygenation in 1% CO2 and in ambient air with 0.039% CO2 resulted in the same survival rate indicating that photorespiration was not a crucial factor. Analysis of of NDA gene regulation revealed that the hypoxia-responsive ethylene response factor 2 (HRE2) acts as a transcriptional repressor of NDA1 and NDA2. The expression of NDA1 and NDA2 increased 5-fold and 4-fold after 2 h of reoxygenation in wild type and 7-fold in erfVII and hre2-2. Conversely, expression of NDAs remained at control levels in HRE2ox1 and HRE2ox5 seedlings. The Arabidopsis transcriptional regulators No apical meristem/Arabidopsis transcription activation factor/ Cup-shaped cotyledon (ANAC013), ANAC016, and ANAC017 were shown to bind to the mitochondrial dysfunctional motif (MDM) in the NDA1 promoter and activated the NDA1 gene. More so, the anac017 mutant showed reduced expression of NDA1 and NDA2 during reoxygenation in comparison to wild type indicating positive regulation of NDAs by ANAC017 and possibly ANAC013 and ANAC016. The results indicate that HRE2 and ANAC017 antagonistically regulate expression of NDA1 and NDA2 during anoxia and reoxygenation. Taken together, this study revealed a central function of mitochondrial NDAs in ROS regulation and plant survival of reoxygenation stress. Based on our findings, we propose that control of mitochondrial ROS levels during recovery from low oxygen stress is central to plant survival of flooding.


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