The Photoprotection And Mechanism Of Alternative Oxidase Pathway In Mitochondria At Low Temperature

Light is necessary to drive photosynthesis,however,when the abosorbed light excesses the need of plant,it will reduce the light use efficiency of leaves,this phenomenon was called “photoinhibition”.When high light combined with other stresses,the photosynthetic apparatus will be more sensitive to high light.In order to adapte the high light environmental and other various stresses,the plant evolved a series of photoinhibition defense systems,such as non-photochemical quenching,cyclic electron transport and water-water cycle.These photoinhibition defense systems are all located in the chloroplast to be close to the site where the excess reducing power and reactive oxygen species produced.However,there is also photoprotection mechanism outside the chloroplast.The AOX pathway is one of the photoprotection mechanisms outside the chloroplast that attract many people's attention recently.AOX pathway is an extra electron transport pathway in addition to the cytochrome oxidase pathway(COX)in plant mitochondrion.This pathway diverts electrons from the ubiquinol to molecular oxygen.Electron transport through the AOX pathway occurs without proton translocation,consequently,it is not limited by the ATP synthesis or energy conservation,so the AOX pathway can consume the reduction powers quickly.Previous research noticed that AOX has the important photoprotection function under high-light,the photoprotection function of AOX is more obvious under drought stress.However,most of the current researches on AOX photoprotection are carried on under room temperature.The photoprotection function of AOX and and it's mechanism under low temperature are still unknown.It is reported that the growth of aox1 a mutant is slower than the wild type plant at low temperature,while the growth of aox1 a mutant and wild type plants are similar at normal temperature.It is well known that the photosynthesis is critical for growth of plant but low temperature will cause the photoinhibition in leaves.So we speculated that the photoinhibition may be aggravated and thus the growth is inhibited in aox1 a mutant at low temperature.The aim of the research was to clarify the photoprotection contribution of AOX to PSII at low and normal temperature and the photoprotection mechanism by which the AOX pathway works.Here,the aox1 a mutant,nadp-mdh mutant,aox1 a nadp-mdh double mutant Arabidopsis thaliana plants,as well as the typical chilling sensitive C3 and C4 plants: cucumber and maize were used as experimental material.The plant physiology,biochemistry,and molecular biological research techniques were used to comprehensively analyze the respiration,photosynthesis,reactive oxygen metabolism under low and normal temperature.The main results and conclusions are as follow:(1)The AOX protein expression was strongly induced by highlight under normal temperature,however,the induction of AOX protein expression by highlight was attenuated by low temperature in wild type Arabidopsis thaliana leaves.The increase of AOX pathway respiration rate caused by high light was also attenuated by low temperature.There results indicate that the sensitivity of AOX pathway to high light decreased under low temperature.In addition,it was observed that the PSII photoinhibition caused by high light was severer in aox1 a matant than in wild type Arabidopsis thaliana leaves at normal temperature,while the PSII photoinhibition in aox1 a mutant was similar to that in wild type plant under low temperature and high light.The Arabidopsis thaliana is chilling-resistant species.It was also observed in leaves of chilling-sensitive species,cucumber,the pretreatment with AOX pathway inhibitor(SHAM)deteriorated the PSII photoinhibition under high light and normal temperature rather than under high light and low temperature.There results indicate that the contribution of AOX to PSII photoprotection was obvious under normal temperature rather than under low temperature in both chilling-resistant and chilling-sensitive species.The results were opposite to our hypothesis.The slower growth and high mortality under low temperature in aox1 a mutant than WT plants was not due to the higher PSII photoprotection.(2)The most popular model of AOX pathway explaining PSII photoprotection is malate-acetoacetic acid(Mal-OAA)shuttle model.The excess reducing power in the chloroplast can be transported into mitochondria through Mal-OAA shuttle,which is consumed through the AOX pathway in the mitochondria,therefor alleviating the overreduction in the chloroplasts and thus alleviating the PSII photoinhibition.According to the Mal-OAA shuttle model,the Calvin cycle is suppressed at low temperature and the accumulation of reducing power in the chloroplast is higher than that at normal temperature.This is beneficial to the function of the Mal-OAA shuttle,and AOX should play a more significant photoprotection function under low temperature than under normal temperature,which is in contradiction with our results.In order to clarify the relationship between Mal-OAA shuttle and photoprotection function of AOX pathway,we examined the activity of NADP-MDH enzyme under different temperatures.The results showed that the activity of NADP-MDH enzyme increased with the decrease of temperature,while the photoprotection function of AOX decreased with the decrease of temperature,which implies that photoprotection function of AOX pathway does not depend on the Mal-OAA shuttle.In order to further explore the relationship between the AOX photoprotection and Mal-OAA shuttle,we constructed the aox1 a nadp-mdh double mutant.It was observed that high light significantly induced the expression of AOX protein and increased the respiratory rate of AOX pathway in nadp-mdh mutant,and the upregulation was similar to that in wild type.This factor indicates that the lack of nadp-mdh gene does not affect the sensitivity of AOX pathway to high light.In addition,the PSII photoinhibition in nadp-mdh aox1 a double mutant under strong light is obviously severer than that in the nadp-mdh mutant,and the increase of PSII photoinhibition caused by the lack of AOX pathway in nadp-mdh mutant and wild type is similar.This factor shows that when Mal-OAA shuttle is lacked,AOX still has an important role in photoprotection.Based on the above results,we believe that the photoprotection role of AOX pathway does not depend on the Mal-OAA shuttle.(3)To explore the mechanism of AOX pathway in photoprotection,the leaves were exposed to high light under different gas conditions.We found that under low oxygen,high carbon dioxide,zero carbon dioxide atmosphere and other non photorespiration condition,the photoinhibition in leaves of aox1 a mutant was greatly alleviated,the photoinhibition in aox1 a leaves was closed to it in WT leaves.Moreover,the photoinhibition in leaves of aox1 a nadp-mdh double mutant was also alleviated to the same level as the nadp-mdh mutant.The PSII photoinhibition of the cucumber leaves treated by SHAM decreased significantly,reaching the same level as that of the control leaves.These results indicate that the photoprotective effect of AOX is closely related to photorespiration.The photorespiration rate of leaves in the presence or absence of AOX pathway was also analyzed.The results showed that the lack of AOX pathway in the wild or nadp-mdh Arabidopsis thaliana mutants as well as in cucumber leaves,could decrease the rate of photorespiration.In addition,it was also found that the AOX pathway only contributes to photoprotection in C3 plants rather than in C4 plants without photorespiration.Based on these results and other literatures,we established a new model of AOX pathway contributes to photoprotection,namely AOX-photorespiration model.In photorespiration,glycine converts to serine in mitochondrial and produces massive reducing power(NADH).The partial NADH is converged to Mal by mitochondrial NAD-MDH,the Mal was transferred to peroxisome and is reconverged to NADH by peroxisomal NAD-MDH and used to convert hydroxypyruvic acid to glyceric acid.But the remaining reducing power in mitochondrial needed to be consumed by the AOX pathway.When the AOX is inhibited,the reducing power is accumulated in the mitochondrial and blocked the convert from glycine to serine,which would induce the accumulation of glycine and feedback inhibits the photorespiration and finally induces the accumulation of phosphoglycolate in chloroplast.Meanwhile,when conversion from glycine to serine is blocked,the downstream metabolite such as hydroxypyruvate will decrease.The conversion from hydroxypyruvate to glyceric consume massive NADH,which is the main sink of NADH from mitochondria,so the decreased content of hydroxypyruvate would decrease the requirement of NADH in peroxisome and induces the accumulation of reducing power in mitochondrial.It would further aggravate the accumulation of phosphoglycolate in chloroplast.Phosphoglycolate is toxic,even if a little accumulation would aggravate the PSII photoinhibition.(4)The new AOX-photorespiration model could explain why the AOX pathway contributes to photoprotection only under normal temperature but not in low temperature: the photorespiration is suppressed and the NADH produced during the conversion from glycine to serine decreases under low temperature,therefor,the AOX pathway is no longer necessary to consume NADH.In addition,the new AOX-photorespiration model could clarify another puzzle that why the capacity of AOX pathway consumes NADH is only about 2-5% of NADH production in photosynthesis,but the AOX pathway has significant photoprotection function.It is due to that the AOX pathway is necessary to maintain the conversion from glycine to serine,and the the conversion from glycine to serine is sole pathway to detoxify the phosphoglycolate,phosphoglycolate is toxic,even if a little accumulation would aggravate the PSII photoinhibition.