Molecular Mochanisms That Arabidopsis Aquaporins AtPIP1;4 And AtPIP2;4 Deploy To Faciliate H₂O₂ Transport And Regulate Disease Resistance

Aquaporins（AQPs）are material transport channels on biological cell membranes or organelle membranes and mediates the transport of small molecule compounds such as H₂O and H₂O₂ in and out of cells or between organelles.Plant AQPs are divided into different protein families,including the plasma membrane intrinsic protein（plasma membrane intrinsic protein,PIP）family,its different members are responsible for different substrates such as H₂O₂ entering and leaving the cells.The burst of reactive oxygen species（ROS）is a hallmark for plants to successful recognize of pathogen-associated molecular patterns（PAMPs）or pathogens.H₂O₂ is an important signaling molecule in the life body,is the main component of ROS,involved in many physiological and pathological reactions.The resulting H₂O₂ induced by PAMPs usually accumulates in the apoplast,PIPs transport of H₂O₂ from apoplast into cytoplasm,interacts with systemic acquired resistance（SAR）and pathogen related molecular patterns triggering immunity（PTI）to regulate plants disease resistance.Previously,the laboratory screened that expressed AtPIP1;4 and AtPIP2;4 of yeast cells were more sensitive to H₂O₂.It has been proved in Arabidopsis that the expressed of AtPIP1;4 and AtPIP2;4 alone can mediate the transport of apoplastic H₂O₂ into cytoplasm,and this plays a key role in signal pathways such as PTI and SAR.Previous laboratories have transferred 13 water channel proteins from Arabidopsis into yeast,we found that yeast cells expressing AtPIP1;4and AtPIP2;4 showed significantly increased sensitivity to H₂O₂,and demonstrated that AtPIP1;4 and AtPIP2;4 expression alone mediated H₂O₂ transport to the cytoplasm is indispensable for signaling pathways such as PTI and SAR.We study mainly investigated the effects of AtPIP1;4 and AtPIP2;4 coexpression on H₂O₂ transport and how to mediate the disease resistance and immune response in plants.In this study,to verify the effect of the coexpression of AtPIP1;4 and AtPIP2;4 on disease resistance,we first hybridize using laboratory existing AtPIP1;4,AtPIP2;4 single mutants and single overexpression transgenic Arabidopsis,harvested and screened three transgenic double mutant lines and three transgenic double overexpression lines.We analyzed the effect of disease resistance to Pst DC3000 in atpip1;4 atpip2;4 double mutant and AtPIP1;4OE AtPIP2;4OE double overexpression plants.Pathogen induced H₂O₂ usually accumulates in the plant plasm.To explore whether coexpression of AtPIP1;4,AtPIP2;4affects H₂O₂ content in Arabidopsis,DAB staining,SOD enzyme activity and reactive oxygen related genes（PPO1,PPO2 and SOD）after Pst DC3000 infection to explore whether AtPIP1;4,AtPIP2;4 coexpression on the production of reactive oxygen species.Infection by Pst DC3000,the transport H₂O₂ capacity of AtPIP1;4 and AtPIP2;4 coexpression was determined by enzyme labeled instrument and visualized using H₂DCF-DA,AR and AUR for cell staining observation.To further explore the immune response of AtPIP1;4 and AtPIP2;4coexpression to PTI signaling pathway,immune response to the PTI signaling pathway were detected by induction of callose deposition in Arabidopsis and gene expression related to MAPKs signaling pathway.The results showed that the disease resistance was significantly improved in the double overexpression compared with the WT,the double mutant were more susceptible.The results were confirmed that the enhanced immune response of AtPIP1;4OE AtPIP2;4OE double overexpressing was not due to large amounts of H₂O₂ accumulation,but due to the coexpression AtPIP1;4 and AtPIP2;4 can promote the transport of apoplast H₂O₂ into cytoplasm.In addition,PAMPs induced AtPIP1;4,AtPIP2;4 double overexpression Arabidopsis significantly higher expression amount of MPK3 and callose deposition also confirmed that AtPIP1;4 and AtPIP2;4 coexpression can positively affect PTI response.