The Physiological Mechanism Of Brassinosteroids Enhanceing Resistance To Manganese Toxicity In Malus Robusta Rehd.

With the development of industrial society and the acceleration of urbanization,as well as the long-term manganese（Mn）mining,the content of Mn in soil in many areas increases sharply,and Mn poisoning poses a serious threat to the normal growth and development of plants.Brassinosteroids（BRs）have been confirmed to actively participate in drought,high temperature,salt and other abiotic stresses,but the physiological mechanism of BR alleviating plant Mn stress is still unclear.In this experiment,Malus robusta Rehd.was used as the research object to simulate Mn stress in hydroponic experiments,and 2,4-epibrassinolide（EBL）was applied exogenous under Mn stress.The effects of EBL on growth,endogenous polyamine content,chemical morphology,subcellular distribution,absorption and transport,and avoidance mechanism of Mn in plants of Malus robusta Rehd.were revealed,so as to reveal the physiological regulatory mechanism of EBL to improve Mn tolerance of Malus robusta Rehd.seedlings.The main results obtained are as follows:1.EBL promoted the growth rate（root length and plant height）of Malus robusta Rehd.under Mn stress,increased the photosynthetic efficiency(Fv/Fm andΦ_PSII)of leaves,and reduced the growth inhibition under Mn stress.EBL significantly reduced the content of superoxide anion(O₂^·-)and hydrogen peroxide（H₂O₂）by 71.87%and 71.39%,respectively,compared with Mn treatment alone.The activities of superoxide dismutase（SOD）,peroxidase（POD）and glutathione reductase（GR）and the contents of ascorbic acid（AsA）and glutathione（GsH）were significantly increased to ensure the redox homeostasis in plants.EBL also reduced the content of mineral elements（K,Ca,Mg）and maintained the ion balance under Mn stress.2.The external application of EBL significantly reduced the contents of polyamine precursors（Orn and Arg）,improved the activities of ornithine decarboxylase（ODC）and arginine decarboxylase（ADC）,up-regulated the transcription levels of MdSPMS and MdSPDS genes,promoted the conversion of putsumine（Put）to spermidine（Spd）and spermidine（Spm）,and promoted the accumulation of spermidine content.Finally,the ratio of（Spd+Spm）/Put was increased,and the ratio of external application of EBL was increased by 54.80%under Mn treatment.3.Exogenous EBL reduced the accumulation of Mn in organelles by regulating the distribution of Mn in subcellular level（cell wall and vacuole）,and reduced the contents of free Mn（dd-H₂O extraction state and 80%ethanol extraction state by 17.34%and9.91%,respectively）by increasing the content and proportion of pectin and protein-bound Mn（NaCl extraction state）.The damage to cells caused by Mn stress was reduced.In addition,the relative expression levels of MdMTP,MdCAX and MdYSL genes were significantly increased by applying EBL,and Mn transported to cell wall and vacuole was further increased.The transcription level of MdNRAMP gene was decreased,and the transport of Mn between organelles was reduced.4.Mn stress induced the increase of the contents of many organic acids（oxalic acid,tartaric acid,malic acid and citric acid）and free amino acids（glutamic acid,alanine,aspartic acid,valine,methionine,tyrosine and proline）,which acted as the chelating and binding ligand of Mn²⁺to reduce the damage of free Mn²⁺.At the same time,EBL increased the expression levels of MdPCS and MdMT genes,the expression levels of MdPCS1,MdPCS2 and MdPCS3 increased by 21.05%,52.11%and 17.98%,respectively,and the expression levels of MdMT increased by 30.51%,promoting the formation of less toxic chelating compounds with Mn²⁺.In conclusion,EBL obviously alleviates the growth damage caused by Mn toxicity by affecting the cell wall regionalization of endogenous polyamines,endogenous Mn,metal transporters and avoidance strategies of Malus robusta Rehd.The results not only analyzed the physiological mechanism of EBL alleviating Mn stress,but also provided theoretical basis for the application of EBL in the prevention and control of heavy metals stress.