Drought induction of AOX level in maize was accompanied by lowering of AOX respiration activity

--Stupnikova, I, Borovskii, G, Korotaeva, N, Antipina, A, Truhin, A, Popov, V

There are two mitochondrial electron transport pathways from ubiquinone to oxygen in plants. The alternative pathway branches from the cytochrome pathway and donates electrons to oxygen directly by alternative oxidase (AOX) (Moore and Siedow, Biochim. Biophys. Acta. 1059:121–140, 1991). The alternative pathway is not coupled with ATP synthesis, but can be induced in response to stress or inhibition of the main electron transfer pathway (Wagner and Moore, Biosci. Rep. 17:319–333, 1997). When plants are exposed to stresses, drought for instance, they produce reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (Prasad et al., Plant Cell. 6:65–74, 1994). ROS damages membranes and other cell components. It has generally been proposed that increased AOX activity could be used to help maintain normal levels of metabolites and to reduce levels of reactive oxygen species (ROS) during the stress. We propose that drought will increase the AOX respiration level and the quantity of the protein in mitochondria of maize seedlings.

The alternative oxidase protein levels in the mitochondria were studied after drought stress at 22 °C for 1 or 2 days. Four day etiolated maize seedlings grown at 22 °C were used as control. Mitochondria were isolated and purified as described earlier (Borovskii et al., BMC Plant Biol. 2:5, 2002). The expression of AOX proteins was performed by immunoblotting with AOX specific antibodies (kindly provided by Dr. T. E. Elthon, University of Nebraska). It had been shown previously that after 24 hours of drought stress, water content in the seedlings decreased from 90.6% to 87.5%, and after 48 hours, water content decreased to 82.7% (Korotaeva et al., MNL 77:34–35, 2003). Respiration of isolated mitochondria was performed as described by Korotaeva et al. (Russ. J. Plant Physiol. 48:798–803, 2001) using malate as substrate and KCN as the cytochrome pathway of respiration inhibitor. To measure potential activity of AOX respiration, it was activated by pyruvate and dithiotreithol.

Immunoblotting analysis revealed that the quantity of AOX protein increased under dehydration stress (Fig. 1). An accumulation of oxidized dimers of AOX was shown. No new isophorm induction appeared. Nevertheless, in spite of the accumulation of AOX protein, the potential alternative respiration of isolated mitochondria decreased during dehydration: from 62±7% at V₄ respiration in control seedlings, to 36±4% at 48h of dehydration. At the same time, the level of respiration without activation was not seriously changed: from 26±4%(control) to 22±5% (48h of dehydration). The results suggest that an increase in the AOX protein level is not enough to provide high alternative respiration activity during drought stress, and made us doubt the role of AOX during dehydration stress itself. It may be that higher AOX activity is favorable in adaptation when oxidative stress, which accompanies dehydration, is not strong.

This work was supported by the Russian Fund of Basic Research (project 02-04-48599).