The Roles Of NHX5 And NHX6 In Regulating PIN6-mediated Development In Arabidopsis

Plant Na~+,K~+/H~+antiporters(NHXs)play important roles in maintaining homeostasis of ion and p H,regulating the trafficking of vescular and proteins,and controlling growth and development.NHX5 and NHX6 are located in the endosomal cmpartments including Golgi,TGN(Trans-Golgi Network),PVC(Prevacuolar Compartment)/MVB(Multivescular Bodies)and ER(Endoplasmic Reticulum).Studies have shown that nhx5 nhx6 double mutants exhibited auxin deficient phenotypes,such as dwarfism,long hypocotyls,and epinastic cotyledons,indicating that NHX5 and NHX6 regulate growth and development.Auxin is a key hormone that has polar transport properties;auxin is transported to specific cells or tissues by auxin transporter PIN proteins.Auxin is essential for controlling growth and development.It has been reported that the auxin transporter PIN6 is located at the ER;PIN6 transports auxin from ER to the cytoplasm.Overexpression of PIN6 inhibites growth and thus produces smaller plants with curled and dark-coloured leaves.PIN6 regulates plant development by regulating the cellular auxin homeostasis.In this study,the molecular mechanism of NHX5 and NHX6 in regulating plant development was studied using molecular genetics and cell biology methods.By observing the phenotypes of pin6 nhx5,pin6 nhx6,pin6 nhx5 nhx6 mutants and PIN6-overexpressing plants,we revealed that NHX5 and NHX6 might regulate plant development through controling PIN6-mediated auxin homeostasis.The main findings are as follows:1.pin6 single mutants were screened,and pin6 nhx5,pin6 nhx6 double mutants and pin6 nhx5 nhx6 triple mutants were generated by genetic cross.We also prepared the PIN6 overexpression lines in the Col-0,nhx5,nhx6,and nhx5 nhx6 backgrounds.2.For plants grown in soil,pin6,pin6 nhx5,pin6 nhx6,and pin6 nhx5 nhx6 mutants were increased in rosette leaves compared to Col-0,nhx5,nhx6 and nhx5 nhx6,respectively.In addition,pin6,pin6 nhx5,and pin6 nhx6 mutants were increased in siliques compared to Col-0,nhx5,and nhx6,respectively.However,pin6 nhx5 nhx6 mutants were not different from nhx5 nhx6 in silique numbers.This suggests that NHX5 and NHX6 may regulate plant development by controlling PIN6 function.3.For seedlings grown on 1/2MS medium,overexpression of PIN6 in Col-0,nhx5,and nhx6 reduced the angles between the two cotyledons;there was no changes in angles when expressing PIN6 in nhx5 nhx6.For seedlings grown in soil,overexpression of PIN6 in Col-0,nhx5,and nhx6 produced seedlings with curling leaves and short petioles,and inhibited pollen tube elongation;overexpression of PIN6 in nhx5 nhx6 also produced seedlings with short petioles and inhibited pollen tube elongation,but leaves were uncurled.These suggest that PIN6 is dependent on NHX5 and NHX6.4.Studies form confocal laser scanning microscopy revealed that there was no difference in PIN6-GFP signal between Col-0,nhx5,and nhx6;but PIN6-GFP signal was significantly reduced in nhx5 nhx6.In addition,there was no significant difference in PIN6-GFP signal between Col-0,nhx5,and nhx6 either with or without 2,4-D teatmnets;but PIN6-GFP signal was significantly enhanced in nhx5 nhx6 under 2,4-D teatmnets.These results suggest that mutation of NHX5 and NHX6 genes affects PIN6 function,leading to disruption of cellular auxin homeostasis.In conclusion,this study shows that NHX5 and NHX6 may regulate plant development through controlling PIN6 function and hence cellular auxin homeostasis.