The Arabidopsis intracellular Na+/H+ antiporters NHX5 and NHX6 are endosome associated and necessary for plant growth and development.

Intracellular Na(+)/H(+) antiporters (NHXs) play important roles in cellular pH and Na(+) and K(+) homeostasis in all eukaryotes. Based on sequence similarity, the six intracellular Arabidopsis thaliana members are divided into two groups. Unlike the vacuolar NHX1-4, NHX5 and NHX6 are believed to be endosomal; however, little data exist to support either their function or localization. Using reverse genetics, we show that whereas single knockouts nhx5 or nhx6 did not differ from the wild type, the double knockout nhx5 nhx6 showed reduced growth, with smaller and fewer cells and increased sensitivity to salinity. Reduced growth of nhx5 nhx6 was due to slowed cell expansion. Transcriptome analysis indicated that nhx5, nhx6, and the wild type had similar gene expression profiles, whereas transcripts related to vesicular trafficking and abiotic stress were enriched in nhx5 nhx6. We show that unlike other intracellular NHX proteins, NHX5 and NHX6 are associated with punctate, motile cytosolic vesicles, sensitive to Brefeldin A, that colocalize to known Golgi and trans-Golgi network markers. We provide data to show that vacuolar trafficking is affected in nhx5 nhx6. Possible involvements of NHX5 and NHX6 in maintaining organelle pH and ion homeostasis with implications in endosomal sorting and cellular stress responses are discussed.