OsNHX5-mediated pH homeostasis is required for post-Golgi trafficking of seed storage proteins in rice endosperm cells

As the major storage protein in rice seeds, glutelins are synthesized at the endoplasmic reticulum (ER) as proglutelins and transported to protein storage vacuoles (PSVs) called PBIIs (Protein body IIs), where they are cleaved into mature forms by the vacuolar processing enzymes. However, the molecular mechanisms underlying glutelin trafficking are largely unknown.

In this study, we report a rice mutant, named glutelin precursor accumulation6 (gpa6), which abnormally accumulates massive proglutelins. Cytological analyses revealed that in gpa6 endosperm cells, proglutelins were mis-sorted, leading to the presence of dense vesicles (DVs) and the formation paramural bodies (PMBs) at the apoplast, consequently, smaller PBII were observed. Mutated gene in gpa6 was found to encode a Na⁺/H⁺ antiporter, OsNHX5. OsNHX5 is expressed in all tissues analyzed, and its expression level is much higher than its closest paralog OsNHX6. The OsNHX5 protein colocalizes to the Golgi, the trans-Golgi network (TGN) and the pre-vacuolar compartment (PVC) in tobacco leaf epidermal cells. In vivo pH measurements indicated that the lumens of Golgi, TGN and PVC became more acidic in gpa6.

Our results demonstrated an important role of OsNHX5 in regulating endomembrane luminal pH, which is essential for seed storage protein trafficking in rice.