Hydrogen Metabolism Of Mitochondrial Complex ? In Higher Plants

In recent years,molecular hydrogen(H₂)has been reported to have potential therapeutic effects on many diseases.In addition to the beneficial roles of hydrogen reported in animals,hydrogen also has an important regulatory effect on the physiological function of plants.Hydrogen could enhance the plant tolerance and resistance to several abiotic stresses such as high salinity,aluminum,cadmium,mercury and oxidative damage caused by paraquat.However,in the face of such a wide range of biological effects,the mechanism of hydrogen molecules remains unclear.We speculate that the broad botanical effects exhibited by hydrogen may be related to hydrogen metabolism in higher plants.However,previous studies on hydrogenases have focused on algae and anaerobic microorganisms,and little research has been done on hydrogen metabolism in higher plants.Therefore,the study of hydrogen metabolism in higher plants is great significance for illuminating the broad biological mechanism of H₂.In this study,Vigna radiata seedlings were used as research objects to study hydrogen production law and hydrogen metabolism mechanism.The main results are as follows:1.Establish hydrogen detection method.The methods for the measurement of hydrogen concentration in aqueous phase by headspace gas chromatography(GC)or hydrogen microsensor were established.The method of headspace was used to transfer hydrogen into gas phase.After SH-Rt?-Msieve 5A column separation,there was a good linear relationship between the hydrogen concentration and the peak area of GC in the range of 0?1.62 mg/L and 0?0.2025 mg/L,the method detection limit(MDL)was 8.7x10-4 mg/L.A hydrogen microsensor method was established for the direct determination of hydrogen concentration in aqueous phase.There was a good linear relationship between the hydrogen concentration and microsensor signal value in the range of 0?1.62 mg/Land 0?0.2025 mg/L,the MDL was 4.3x10-3 mg/L.2.Study on the activity of hydrogen production during the germination/growth of V.radiata seedlings.Gas chromatography was used to detect the endogenous hydrogen content of plants.The results showed that V.radiata seedlings could release hydrogen during the growth process,and the hydrogen content reached to the maximum when it was grown to 96h.Different tissues including roots,hypocotyls,cotyledons and true leaves all had the ability of hydrogen production.Different cell components were separated by differential centrifugation,and the hydrogen production activity of different cell components was determined by gas chromatography.It was found that 12000 g precipitated and 80000 g precipitated components had high hydrogen production activities.3.It was first proved that V.radiata mitochondria seedlings had high hydrogen production activity.Optimum condition for hydrogen production of V.radiata mitochondria was 10 m M MOPS,0.3 M mannitol,0.1%(w/v)BSA,PH6.0,35?.Further study showed that the mitochondria of soybean,Arabidopsis and wheat had hydrogen production activity.4.Study on the mechanism of mitochondrial hydrogen production.In order to determine whether the mitochondria of higher plants have hydrogen production activity and the specific site of hydrogen production,the mechanism of hydrogen production of mitochondria was studied by adding mitochondrial inhibitors in vitro and using LC-MS to determine the change of metabolic substrate in the process of hydrogen production of mitochondria of V.radiata.The results showed that rotenone at low concentration could significantly inhibit the hydrogen production activity of mitochondria,which indicated that the hydrogen production activity of mitochondria in higher plants was closely related to mitochondrial complex I.The results of LC-MS showed that the accumulation of succinic acid and?-ketoglutarate during the process of mitochondrial hydrogen production.The addition of substrate and coenzyme Q in vitro further indicated that higher plant mitochondrial complex I had hydrogen production activity,which was closely related to coenzyme Q binding site on mitochondrial complex I.In conclusion,higher plants can release hydrogen during the growth process,and the mitochondria of higher plants have hydrogen production activity.The activity of hydrogen production in mitochondria of higher plants may be related to the process of electron and proton transfer,especially the electron and proton transfer at the binding site of coenzyme Q of mitochondrial complex I.We speculate that hydrogen appears to participate in regulating redox homeostasis of the organism via hydrogen metabolism pathway,thereby improving the ability of plants to resist stress.This is the first study on the relationship between mitochondria and hydrogen metabolism in higher plants.