Study On The Founction Of AtNHX5 And AtNHX6 In Regulating Auxin Transporter AtPIN8 In Arabidopsis

Plant Na~+,K~+/H~+ exchangers(NHX)play important roles in ion homeostasis,p H regulation,stress response,and growth and development.The Arabidopsis AtNHX5 and AtNHX6 localized in the Golgi,trans Golgi network(TGN),and prevacuolear compartment(PVC).Studies have shown that nhx5 nhx6 double mutants are defective in development,having smaller siliques and reduced seed yields,indicating that AtNHX5 and AtNHX6 regulate plant growth and development.However,the mechanism underlying the function of AtNHX5 and AtNHX6 in regulating growth and development remains largely unknown.Auxin is critical for pollen development.Studies have shown that AtPIN8,an auxin transporter specifically expressed in male gametophytes,regulates pollen development.Using molecular genetic methods,we observed pollen germination and pollen tube growth for nhx5 nhx6,nhx5 nhx6 pin8 and AtPIN8-overexpressing plants.We also analyzed pollen transmission and pollen morphology for nhx5 nhx6 and nhx5 nhx6 pin8 mutant plants.We aim to understand the mechanism underlying the role of AtNHX5 and AtNHX6 in regulating pollen growth and development.The major results are listed below:1.Assays with pollen germination and growth found that nhx5 nhx6 was defective in pollen germination and pollen tube growth.At15 m M KCl,pollen germination and pollen tube growth of Col-0 plants were decreased by 30% and 50%,respectively,while nhx5 nhx6 decreased by 25% and 38%,respectively;At20 m M Na Cl,pollen germination and pollen tube growth of Col-0 plants were decreased by 42% and 45%,respectively,while nhx5 nhx6 decreased by 28% and 38%,respectively;pollen tube growth of Col-0 plants was increased by 24% at acidic p H(4.5)but decreased by 23% at alkaline p H(7.5).The pollen tube growth of nhx5 nhx6 was unchanged at either acidic or alkaline p H.These results suggest that AtNHX5 and AtNHX6 regulate pollen growth and development,and are cortical for pollen response to the changes in cellular ion and p H.2.The nhx5 nhx6 pin8 triple mutants as well as the nhx5 pin8 and nhx6 pin8 double mutants were generated by genetic crossing.The pollen germination rate of Col-0 plants was decreased by 18% at 100 m M NAA;the germination rate of pin8-1,nhx5 nhx6,nhx5 pin8,nhx6 pin8,and nhx5 nhx6 pin8,which were similar with each other,was decreased by 25%,21%,23%,20%,and 23%,respectively;Similarly,pollen tube growth of Col-0 plants was decreased by 20%;pollen tube growth of pin8-1,nhx5 nhx6,nhx5 pin8,nhx6 pin8,and nhx5 nhx6 pin8,which were similar with each other,was decreased by 29%,28%,29%,24%,and 30%,respectively.These results suggest that there is no genetic addition between AtNHX5 and AtNHX6 and AtPIN8,further indicating that AtNHX5 and AtNHX6 may regulate AtPIN8 activity.3.Using pollen specific promoter LAT52,we generated PIN8-overexpressing plants.When LAT52::PIN8 was expressed in Col-0,pollen tube growth was increased by 10% while pollen germination was unchanged when there was no NAA in the medium;At100 m M NAA,pollen germination and pollen tube growth of Col-0 plants were increased by 18% and 19%,respectively.However,when LAT52:: PIN8 was expressed in nhx5 nhx6,no significant changes were observed in pollen germination and pollen tube growth with or without NAA treatment.These results suggest that AtPIN8 function depends on AtNHX5 and AtNHX6.4.We generated PIN8-overexpressing plants driven by 35 S promoter,and observed their growth and development.We found that the 35S::PIN8(Col-0)plants were enhanced in hypocotyl and root growth,and were bolting and flowering early.However,the growth of hypocotyls and roots as well as bolting and flowering of 35S::PIN8/NHX5(Col-0)and 35S::PIN8/NHX6(Col-0)were unchanged,which were remained the same as Col-0 plants,suggesting that AtNHX5 and AtNH6 regulate AtPIN8 function.5.Confocal microscopy assay showed that AtNHX5 and AtNHX6 were co-localized with AtPIN8,suggesting that there may be interaction between the AtNHX5 or AtNHX6 and AtPIN8.6.DR5 staining showed that Col-0,nhx5 and nhx6 had a higher levels of auxin in inflorescence,while auxin was low in anthers of nhx5 nhx6.7.We found that Col-0 and nhx5 nhx6 were similar in pollen transmission,having a transmission rate of 51% and 50%,respectively;however,pin8-1 and nhx5 nhx6 pin8 were low in transmission,having a transmission rate of 38% and 37%,respectively;DAPI staining showed that the number of normal pollen morphology in pin8-1,nhx5 pin8,nhx6 pin8 and nhx5 nhx6 pin8 is similar,lower than Col-0 and nhx5 nhx6.These results suggest that AtNHX5 and AtNHX6 do not affect pollen transmission.In summary,our results demonstrate that AtNHX5 and AtNHX6 regulate pollen growth and development.AtNHX5 and AtNHX6 may regulate pollen growth and development by regulating AtPIN8 activity and hence cellular auxin levels.