The Roles Of Arabidopsis KEA1 And KEA2 In Regulating PIN5 Function

Na~+,K~+/H~+antiporters are H~+-coupled cotransporters whose biochemical activity is to transfer the Na~+or K~+across a membrane in exchange for protons(H~+).Na~+,K~+/H~+antiporters are critical for diverse cellular processes,including Na~+and K~+movement,salt tolerance,regulation of cell cycle and cell proliferation,vesicle trafficking and membrane fusion,and biogenesis.Arabidopsis KEAs,which encode K~+/H~+antiporters,are homologs of EcKefB and EcKefC in E.Coli.The KEA family contains six genes forming two subgroups:KEA1-3 and KEA4-6.KEA1and KEA2 are located in the chloroplast envelope membranes and KEA3 is located in the chloroplast thylakoid membranes.They function in maintaining chloroplast integrity,regulating chloroplast pH and osmotic hoemostasis.KEA1 and KEA2 play an important role in regulating plant growth and development.However,the mechanism underlying KEA1 and KEA2 function in regulating development remains to be studied.The uniqueness of auxin function in regulating development is its directional transport:auxin needs to be transported from its synthesis site to the action site.PIN5,an ER-localized auxin transporter,plays an important role in regulating auxin homeostasis.Using molecular biology and genetics techniques,we analyzed the growth phenotypes of kea1 kea2 double mutants as well as the roles of KEA1 and KEA2 in reglulating PIN5 function.We aim to understand the mechanism underlying the function of KEA1 and KEA2 in regulating growth and development.The major results are as follows:(1)kea1 or kea2 single mutants displayed no significant growth differences with the wild type plants.kea1 kea2 double mutants showed obvious defects in growth and development:kea1 kea2 was short in roots and hypotocyls,having pale green young leaves,and delayed in flowering.(2)kea1 kea2 was less sensitivity to NAA treatments:under 100 nM NAA,the root length was reduced 30%in wild type,while kea1 kea2 was reduced 17%;kea1 kea2 had fewer lateral roots than Col-0.Overexpression of KEA1 and KEA2 restored the phenotype of kea1 kea2. As shown by DR5::GFP assay,in kea1 kea2,auxin content was decreased in root stele,and increased in the tips of the embryonic cotyledons.DR5::GUS staining found that,in kea1 kea2,auxin contents were decreased both in root tip and leaf tip.These results suggest that KEA1 and KEA2 are important for auxin homeostasis.(3)Overexpression of PIN5 in Col-0,kea1 and kea2 inhibited development:seedlings had shorter roots and hypotocyls,less rosettes and smaller flowers.However,PIN5 lost its suppressing effects in kea1 kea2.These results suggest that PIN5 function depends on KEA1 and KEA2.(4)Using laser confocal microscope,we found that overexpression of PIN5-GFP in kea1,kea2 and kea1 kea2 displayed similar fluorescence intensity to overexpression of PIN5-GFP in Col-0,indicating that KEA1 and KEA2 do not affect the expression and distribution of PIN5-GFP.Also,the fluorescence signals of PIN5-GFP in kea1,kea2,kea1 kea2 were overlapped with the ER marker.These results suggest that KEA1 and KEA2 do not affect PIN5-GFP localization.In conclusion,our study shows that KEA1 and KEA2 regulate PIN5-mediated growth and development in plants.