Structural Basis Of Arabidopsis Plasma Membrane Intrinsic Proteins To Hydrogen Peroxide Transport And Plant Immunity

Aquaporins are channel proteins located in membranes,which transport H2O and other uncharged small molecules.Aquaporins are found in all species,including archaea,bacterium,fungi,plant and animal.Plasma membrane intrinsic proteins(PIPs)are water channel proteins located in the plasma membrane of plant cells,which are mainly responsible for regulating the flux of compounds such as H2O,H2O2 and CO2.Although the physiological functions of PIPs have been extensively and profoundly studied,their effects on plant immune processes have only gradually attracted attention in recent years.In the plant immune response,PIPs participate in regulating stomatal closure,forming a physical barrier to leaf diseases.Studies in this laboratory have confirmed that PIPs can also act as immune receptors,regulating pathogens’ infection of hosts.The burst of-H2O2,the substrate of PIPs,is a marker for plants to successfully recognize pathogen-associated molecular patterns(PAMPs)or pathogens.PIPs also regulate a series of immune responses by mediating the transport of H2O2 from apoplasts into cytoplasm.Based on the extensive participation of PIPs in plant immunity,we did three aspects of research work on PIPs from Arabidopsis thaliana.1.AtPIP2;4,AtPIP2;5 and AtPIP2;7 cooperate to mediate the apoplastic H2O2 into cytoplasm in the process of plant immunity;2.The three-dimensional structure of AtPIP2;4 and its structural analysis when conducting H2O2;The transmembrane helices 2(TM2)and TM3 in PIPs of Arabidopsis determine the subcellular localization of the AtPIP1 and AtPIP2 subfamilies.Arabidopsis has 13 PIPs,which are divided into two subfamilies,named AtPIP1 and AtPIP2 subfamily.AtPIP1 subfamily members include AtPIP1;1-AtPIP1;5;AtPIPs include AtPIP2;1-AtPIP2;8.It was found that the yeast cells expressing AtPIP2;4,AtPIP2;5 and AtPIP2;7 had significantly improved sensitivity to H2O2 when all the 13 PIP were expressed in yeast.Therefore,it is speculated that AtPIP2;4,AtPIP2;5 and AtPIP2;7 are involved in the basic immune response of Arabidopsis.The bacterial disease Pseudomonas syringae pv.Tomato DC3000(Pst DC3000)was used to treat the leaves of Arabidopsis thaliana,and as a result,the expression of AtPIP2;4,AtPIP2;5 and AtPIP2;7 was up-regulated,suggesting AtPIP2;4,AtPIP2;5 and AtPIP2;7 play a role in the Pst DC3000 infection of Arabidopsis.Through transgenic manipulations of Arabidopsis,we obtained the mutant,complementary and overexpression plants of AtPIP2;4,AtPIP2;5 and AtPIP2;7.Arabidopsis treated with Pst DC3000 showed that the overexpression of the three genes AtPIP2;4,AtPIP2;5 and AtPIP2;7 significantly increased the resistance of Arabidopsis to Pst DC3000,and mutation of these three genes significantly reduced Arabidopsis resistance to Pst DC3000.In addition,compared to wild-type plants,mutations and overexpression resulted in reduced and increased callose deposition in Arabidopsis,respectively.The above results indicate that AtPIP2;4,AtPIP2;5 and AtPIP2;7 participate in the basic immune response of Arabidopsis,respectively.In order to further explore the synergistic effects of AtPIP2;4,AtPIP2;5,and AtPIP2;7 in plant immunity,virus-induced gene silencing(VIGS)technology was used to silence AtPIP2;4,AtPIP2;5,and AtPIP2;7 in combination.Pst DC3000 was used to infect the silenced plants and it was found that any silencing combination of AtPIP2;4,AtPIP2;5,and AtPIP2;7 significantly reduced Arabidopsis resistance to Pst DC3000.Moreover,plants that silenced three genes at the same time were more susceptible to Pst DC3000 than plants that silenced only two of them.So how do AtPIP2;4,AtPIP2;5 and AtPIP2;7 participate in the immune response of Arabidopsis?To answer this question,H2O2 was testified as a substrate of AtPIP2;4,AtPIP2;5,and AtPIP2;7 in yeast cells using H2O2 fluorescent dyes in yeast cells.Further plant experiments confirmed that in the face of bacterial infection,Arabidopsis utilizes AtPIP2;4,AtPIP2;5,and AtPIP2;7 to transfer extracellular H2O2 into the cell,thereby effectively regulating the downstream response.This study has confirmed that AtPIP2;4 mediates the transport of extracellular H2O2 into the cell and thus acts as a molecular switch for plant immune responses.In addition to transporting H2O2,AtPIP2;4 can also efficiently transport H2O.In order to confirm this,a fermentor was used to express AtPIP2;4 in a large scale,and then AtPIP2;4 was isolated and purified.The protein was encapsulated on liposomes,and the permeation efficiency(Pf)of AtPIP2;4 was measured using a stop flow instrument,which confirmed that AtPIP2;4 was a water transport channel.Using proteoliposomes,a method for quantitatively detecting aquaporins transporting H2O2 was also established.This method assembles fluorescent dyes into proteoliposome vesicles.And after applying H2O2,analyzes the efficiency of aquaporins in transporting H2O2 by detecting fluorescence intensity.Using this method,this study also confirmed that AtPIP2;4 and spinach aquaporin SoPIP2;1 can efficiently transport H2O2,with a significantly higher efficiency than the human aquaporin hAQP1.The structural study of the aquaporins makes the molecular basis of its transport substrates clear.Aquaporins are highly homologous even among different species,and their structural models are also very similar.But the structural details of different aquaporins show complex differentiation.In order to further study the functional basis of AtPIP2;4,a structural model of AtPIP2;4 was constructed by protein crystal diffraction in this study.First,the protein crystals of AtPIP2;4 were obtained by the hanging drop method.After X-ray diffraction,analysis,and structural reconstruction,the protein structure of AtPIP2;4 was obtained.AtPIP2;4,like other aquaporins,are stacked together in the form of tetramers,each of monomers contains 6 transmembrane helices and 5 loops.By comparing the amino acid sequences and structures of AtPIP2;4,SoPIP2;1,and hAQP1,it was found that there are differences in the key sites and domains which control the closure and open of the channel.Further analysis found that a joint-like site on the loop was significantly different,which may cause differences in the H2O2 transport efficiency of the three.We already know that aquaporins mediate the transmembrane transport of a large variety of uncharged small molecules,but the premise of PIPs to function as a transporter is that they can be correctly positioned in the plasma membrane of the cell.PIPs of Arabidopsis include two families,PIP1 and PIP2.PIP2s can localize to the plasma membrane of plants by itself,but PIP1s cannot,unless assisted by a specific PIP2.In this study,AtPIP1;1 and AtPIP2;4 were determined to significant differences in subcellular localization by expressing AtPIP1;1 and AtPIP2;4 in yeast cells and Xenopus oocytes and protoplast cells of Arabidopsis thaliana.Whether in yeast cells or Xenopus oocytes,when expressed alone,most of AtPIP2;4 was successfully located in the plasma membrane of the cell,while AtPIPI;1 could not realize that.AtPIP2;4 mutants formed by replacing AtPIP2;4’s transmembrane helix TM2 or TM3 with AtPIP1;1’s TM2 or TM3 correspondingly were failed to localize in the plasma membrane like wild-type AtPIP2;4,indicating that TM2 and TM3 are in a certain position in this process.Surprisingly,the replacement of AtPIP2;4’s TM2 and TM3 to AtPIP1;I at the corresponding regions resulted in an artificial aquaporin,which was constructed based on AtPIP1;1,but with the same localization as AtPIP2;4.In order to fully prove the role of TM2 and TM3 in localization,we replaced TM2 and TM3 of AtPIP2;4 to all proteins in the AtPIP1 family,and found that(TM2+TM3)AtPIP2;4 can change the location of all AtPIP1s.Sequence and protein structure analysis of AtPIP1s and AtPIP2s revealed that a hydrophilic amino acid residue in TM2 and a defective LxxxA motif in TM3 make it difficult for AtPIPls to perform efficient plasma membrane localization.The above results confirm that TM2 and TM3 are essential for the plasma membrane localization of AtPIPs.Taken together,this study proved for the first time that AtPIP2;4,AtPIP2;5,and AtPIP2;7 work together in the immune process of plants to jointly promote the transport of extracellular H2O2 into the cell,therefore,enhanced plant disease resistance.It will significantly facilitate the excavation of plant resistance genes.Further,this study firstly resolved the three-dimensional structure of the Arabidopsis plasma membrane intrinsic protein,and provided the necessary structural basis for subsequent functional studies of PIPs.At the same time,this study also issued a method for quantitative analysis of aquaporins in efficiency of transporting H2O2.In addition,the effect of TM2 and TM3 on the localization of aquaporins was also clarified in this study,which is very important for studying the function of aquaporins.