The Study Of The Mechanism That Exogenous Hydrogen Peroxide Influences The Gravitropism Of The Primary Root During The Early Stage Of Germination

Hydrogen peroxide?H₂O₂?is the most stable molecule of the reactive oxygen species?ROS?,which is the key factor in many physiological metabolic processes in the plant,such as stomata behavior,gravitropic growth and programmed cell death?PCD?.Exogenous hydrogen peroxide inhibites root growth,influences gravitropism and induces the bending growth of Arabidopsis primary roots during the early stage of germination.Sensing of the gravitational vector is the key procedure in the development of the plant roots.Then,there comes the question that exogenous hydrogen peroxide causes abnormal growth of plant roots through antioxidant system,gravisensing,IAA transport and cellular signaling,or biosynthesis of ethylene.Thus,we explored the mechanism using some biological techniques,like histological staining,fluorescence labeling,immunohistochemistry,qRT-PCR.The results are follows:1.Exogenous hydrogen peroxide inhibited root growth,influenced gravitropism and induced the bending growth of Arabidopsis primary roots during the early stage of germination.When Arabidopsis seeds were treated with 4 mM H₂O₂,the inhibition of gravitropic growth and induction of bending growth of the primary roots were remarkable.In addition,the germination rate was decreased with the increase of time when treated with 4 mM H₂O₂,and reached the lowest germination rate?43.5%?in 36 h.2.Real-time quantification PCR confirmed that exogenous hydrogen peroxide influenced the antioxidant system of plant,and the relative expression levels of the genes SOD1,APX1 GPX1 and CAT3 were up-regulated to keep the balance of cellular H₂O₂ concentration.3.The histological stain and real-time quantification PCR indicated that exogenous hydrogen peroxide induced the transformation from starch to non-sugar compounds,leading to the degradation of amyloplasts in the root tip column cells and abnormity of gravitropism.4.The staining of calcium ion marked with fluorescence probe?fluo-8?showed that the treatment of exogenous hydrogen peroxide changed the tissue distribution and subcellular localization of calcium ion in the primary root.At bending part of the root,the concentration of calcium ion at the inner side was higher than that in the outer side,and it was increased in the vacuole.5.PINs-GFP expression location indicated that exogenous hydrogen peroxide influenced the polar distribution of PINs protein?PIN1 and PIN2?on the plasmalemma,which resulted in uniform distribution of PINs in the cell membrane.6.Immunofluorescence of IAA and expression location of DR5-GFP confirmed that exogenous hydrogen peroxide changed the distribution of IAA,which was higher at the inner side than the outer side.The results of real-time quantification PCR showed that the auxin signaling molecules were affected by exogenous hydrogen peroxide and the relative expression levels of ABP1,TIR1 and AUX1 were up-regulated.7.As shown by real-time quantification PCR,the relative expression levels of ACS and ACO were up-regulated,indicating that the synthesis of ethylene was changed by exogenous hydrogen peroxide.In conclusion,the loss of gravitropism when treated with H₂O₂ was because that the amyloplasts were converted into other non-sugar,which resulted in the changes of both calcium ion distribution and concentration in the tissue and cells,then leading to the change of the distribution of the PINs protein in the plasma membrane.Subsequently,the signaling pathway of IAA was influenced and IAA was asymmetrical distributed in the opposite sides of the root.Finally,the abnormal gravitropism and physiological feature in the primary root were happened during the early stage of germination.