The Role Of AtNHX5 And AtNHX6 In The Trafficking Of Seed Storage Proteins In Arabidopsis Thaliana

Na+,K+/H+ antiporters(NHX antiporters) are H+-coupled transporters catalyzing the transport of Na+ or K+ across a membrane in exchange for protons(H+). Plant NHX antiporters are critical for ion homeostasis, p H regulation, membrane fusion, osmotic adjustment, stress responses,protein sorting, and plant growth and development. The Arabidopsis NHX gene family contains 8members that can be categorized into three subclasses based on their subcellular localizations:vacuole(At NHX1-At NHX4), endosomal(At NHX5 and At NHX6), and plasma membrane(At NHX7/SOS1 and At NHX8). While the plasma membrane and vacuolar NHXs have been studied extensively, the endosomal NHXs are just beginning to be discovered. At NHX5 and At NHX6 are endosomal Na+,K+/H+ antiporters that share high sequence similarity. At NHX5 and At NHX6 localized in the Golgi, trans-Golgi network(TGN), and prevacuolear compartment(PVC). Studies have shown that At NHX5 and At NHX6 mediate K+ and Na+ transport, regulate cellular p H homeostasis and protein trafficking. However, the mechanism underlying the role of At NHX5 and At NHX6 in protein trafficking remains largely unknown.We examined the growth and development of seedlings and seeds of nhx5 nhx6 double mutants, and analyzed the trafficking of seed storage proteins toward protein storage vacuoles(PSV) in nhx5 nhx6 using the techniques of molecular genetics, cell biology and molecular biology. We further examined the growth of nhx5 nhx6 syp22 triple mutants, analyzed subcellular localizations of the SNARE complex as well as the interreactions between At NHX5 or At NHX6 and the SNARE complex. We aim to understand the mechanism behind the role of At NHX5 and At NHX6 in regulating the trafficking of seed storage proteins in Arabidopsis.The major findings are listed below:(1) We first generated nhx5 nhx6 double mutants and found that nhx5 nhx6 not only showed profound defects in growth and development, but also exhibited smaller siliques and reduced seed production, indicating that At NHX5 and At NHX6 regulate the growth and development of both seedlings and seeds in Arabidopsis.(2) We tracked the trafficking of the artificial seed protein GFP-CT24 to the PSV, and found that GFP-CT24 appeared in the extracellular spaces in nhx5 nhx6 and failed to reach to the PSV.SDS-PAGE and immunoblot analysis showed that nhx5 nhx6 accumulated precursor proteins of both 12 S globulin and 2S albumin. The immune EM showed unprocessed precursors of12 S globulin were detected in the intercellular space of nhx5 nhx6. Meanwhile, A chimeric LV-targetd protein Spo:GFP was defective in trafficking to LV in nhx5 nhx6. These results suggest that At NHX5 and At NHX6 regulate protein trafficking toward the vacuole. Using a confocal microscope, we found that PSV’s size was reduced but its number was increased in nhx5 nhx6, indicating that At NHX5 and At NHX6 are vital for PSV biogenesis in the seeds of Arabidopsis.(3) At NHX5 and At NHX6 contain four conserved acidic amino acid residues in the transmembrane domains(D164, E188, D193, E320 in At NHX5, and D165, E189, D194,E320 in At NHX6).We expressed At NHX5 and At NHX6 genes mutated in these four conserved residues in nhx5 nhx6. SDS-PAGE and immunoblot showed that the trafficking of the seed storage proteins was impaired in three of the point mutations in both At NHX5 and At NHX6 genes(D164N, E188 Q and D193 N of At NHX5, and D165 N, E189 Q and D194 N of At NHX6), suggesting that At NHX5 and At NHX6 regulate the transport of seed storage proteins by their transport activity.(4) We generated nhx5 nhx6 syp22 triple mutants, and found that nhx5 nhx6 syp22 showed much more severe defects in growth and development. nhx5 nhx6 syp22 showed dwarfism, smaller rosettes, late flowering, short siliques, reduced seed sets, aborted seeds and much larger seeds,suggesting that At NHX5, At NHX6 and At SYP22 have an overlapped function in plant growth and development, particularly in silique growth and seed development. Morphology analysis of the PSV by confocal microscope showed that nhx5 nhx6 syp22 had a significantly increased number of PSV and reduced average PSV area, suggesting that At NHX5, At NHX6 and At SYP22 may involve in the PSV biogenesis in the seeds of Arabidopsis. SDS-PAGE and immunoblot analysis showed that nhx5 nhx6 syp22 accumulated the precursor proteins of both 12 S globulin and 2S albumin, indicating that protein transport is impaired in nhx5 nhx6syp22.(5) Transient expression of Arabidopsis protoplasts showed that the PVC localization of SNARE components At SYP22 and At VAMP727 was repressed; At SYP22 and At VAMP727 were trapped in the Golgi or TGN in nhx5 nhx6, suggesting that At NHX5 and At NHX6 are required for the localization of the SNARE complex. In addition, transient expression of Arabidopsis protoplasts showed that At NHX5 and At NHX6 were co-localized with At SYP22 and At VAMP727 on the PVC. However, co-immunoprecipitation exhibited no interaction between At NHX5 or At NHX6 and SNARE components At SYP22, At VTI11 or At SYP51,indicating that there is no physical interactions between the SNARE complex and At NHX5 or At NHX6.In summary, our results demonstrate that At NHX5 and At NHX6 regulate the trafficking of seed storage proteins in Arabidopsis. At NHX5 and At NHX6 are required for the subcellular localizations of the SNARE complex that mediates the trafficking of seed storage proteins into the PSV. At NHX5 and At NHX6 may regulate protein trafficking via their transport activity.