The Role And Up-regulation Mechanism Of Mitochondrial Alternative Oxidase Pathway In Plant Photoprotection

Alternative oxidase(AOX)respiration pathway diverts electrons from ubiquinone pool and reduces oxygen to water without any proton translocation or ATP synthesis in plant mitochondria.AOX pathway contributes to photoprotection under stress.The widely accepted model by which AOX pathway protects photosystems is that AOX pathway dissipates excess reducing equivalents exported from chloroplast through malate/oxaloacetic acid(MAL-OAA)shuttle to avoid over-reduction of the electron transport chain in chloroplast.However,previous studies regarding the photoprotection function of AOX pathway were conducted using C3 plants,and no attention has been paid to the photoprotection function of AOX pathway in C4 plants.Moreover,there are still some unsolved questions about AOX photoprotection mechanisms,such as,the capacity of AOX pathway is far below the photosynthetic O2 evolution rate(only approximately 0.5-2.5% of the photosynthetic O2 evolution),but the photoprotection of AOX pathway in C3 plants is irreplaceable,why can the AOX pathway with such a low capacity significantly influence photosynthesis? The contribution of MAL-OAA shuttle to photoprotection could be replaced or compensated by other ways,why does the irreplaceable photoprotection function of the AOX pathway dependent on the MAL-OAA shuttle? As an extra-chloroplastic defense pathway,the mechanism of AOX pathway in photoprotection is still unclear.In this study,the physiological function of the AOX pathway in photoprotection of plants was confirmed in both C3 and C4 plants and in an aox1 a mutant.And the up-regulation mechanism of AOX pathway by light was discussed.The main results obtained are as follows:(1)Under high light condition,pretreatment with salicylhydroxamic acid(SHAM)decreased the maximum quantum yield of PSII(Fv/Fm),which was significantly aggravated in C3(Cucumis sativus,Rumex K-1,Salix babylonica and Arabidopsis thaliana)leaves,and the extent of the decrease was aggravated by an increase in the SHAM concentration.However,the SHAM pretreatment had no observable influence on the Fv/Fm in leaves of the three types of C4 plants with different decarboxylation enzymes,such as NADP-malic enzyme(NADP-ME;in Zea mays,Sorghum bicolor and Euchlaena mexicana),NADP-malic enzyme(NAD-ME;in Portulaca oleracea)and phosphoenolpyruvate carboxykinase(PPCK;in Salvia farinacea).These results showed that the AOX pathway plays an important and irreplaceable photoprotection role in C3 but not in C4 leaves.(2)After exposed to high light,the total respiration rate obviously increased in both C3 and C4 leaves.The increase in the AOX pathway capacity contributed to most of the increase in the total respiration rate in the C3 leaves,while the SHAM-resistant respiration rate increased slightly.Simlar with the change of AOX pathway activity,the transcript levels and the protein content of AOX gene in C3 leaves significantly increased during the high light exposure.In contrast,the SHAM-resistant respiration contributed to most of the increase in the total respiration rate in the C4 leaves,while the AOX pathway capacity increased slightly.The AOX pathway capacity and the transcript levels and the protein content of AOX gene had no significant changes in the C4 leaves.Under high light condition,the AOX pathway was significantly activated and played an important and irreplaceable role in photoprotection in C3 leaves,but it was insensitive to high light and does not contribute to photoprotection in C4 leaves.It was well known that,in C4 plants,especially in NADP-ME and NAD-ME types C4 plants,the capacity of MAL-OAA shuttle is much greater than that in C3 plants,however,AOX did not contribute to photoinhibition in C4 plants,indicating that the photoprotection of AOX pathway does not only depend on MAL-OAA shuttle,and there maybe another mechanism of AOX pathway to contribute to photoprotection.(3)Under high light condition,when the photorespiration was inhibited by different inhibitors or under low O2(2%)or low CO2(50ppm)condition,the Fv/Fm was no longer decreased in SHAM-treated Cucumis sativus and Rumex K-1 leaves.However,SHAM treatment did not lead to an obvious difference in C4 leaves with the inhibitors treatment or under low O2(2%)or low CO2(50ppm)condition.The results demonstrated that with photorespiration inhibited in different ways,the AOX pathway no longer plays an important role in photoprotection in C3 leaves.(4)In C3 leaves,the pretreatment of SHAM decreased the post-illumination CO2 burst(PIB)levels and the O2 inhibition of Pn but increased the GLY/SER ratio under high light.However,the PIB levels,O2 inhibition of Pn and GLY/SER ratio in C4 leaves were insensitive to SHAM pretreatment.The results indicate that the inhibition of AOX pathway significantly restrains photorespiration in C3 leaves rather than in C4 leaves.The photorespiration supports PSII photoprotection by two ways: Export of excess reducing equivalents from chloroplasts and detoxification of glycolate.So under high light,the up-regulated AOX pathway is important to maintain the photorespiration to contribute to PSII photoprotection.(5)Under high-light conditions,the aox1 a Arobidopsis mutant showed more severe photoinhibition than the wild-type plants,but when the photorespiration was inhibited by low CO2 or low O2,or by an inhibitor,there was no obvious difference between the aox1 a mutant and the wild type plants.Morever,the PIB levels and the O2 inhibition of Pn were lower in aox1 a mutant and the GLY/SER ratio was higher in aox1 a mutant under high light.These results supported our speculation that the AOX pathway contributed to the photoprotecion by maintaining the photorespiration.(6)Light increased the capacity of AOX pathway in C3 leaves.But the capacity of AOX pathway did not increase when the photosynthetic electron transport from QA to QB was inhibited by DCMU in the light,which induced severe accumulation of ROS in chloroplast.This result indicated that ROS accumulated in chloroplast was not involved in the up-regulation in AOX pathway in the light.The IA(Iodoacetic Acid)pretreatment,which induced the accumulation of ROS and NADPH in chloroplast under highlight by inhibiting the activity of Rubisco,did not enhance the capacity of the AOX pathway under light either.This result suggests that ROS and NADPH accumulated in chloroplast were not the direct signals in induction of AOX pathway in the light.(7)The content of photorespiration metabolite glycine increased in C3 leaves under high light.Exogenously-application of glycine increased the AOX pathway in C3 leaves and in tobacco BY-2 cell suspension without functional chloroplast.And after the inhibition of glycine oxidation by Aminoacetonitrile(ANN),exogenous glycine still enhanced the AOX pathway both in C3 leaves and BY-2 cells.These results indicated that glycine increased the AOX pathway capacity,which was not dependent of the photorespiration and photosynthetic metabolism.The AOX pathway was not enhanced in C4 leaves after high light treatment,but treatment with exogenous glycine,a photorespiration metabolite,significantly induced the AOX pathway.It is reasonable to suggest that GLY,a photorespiration intermediate,plays a major role in up-regulation of AOX pathway in C3 leaves under high light,and the insensitivity of AOX in C4 plant to high light is due to that C4 plants have little photorespiration,and glycine does not accumulate in mitochondria under high light.