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

Jianping Zhu,Mingming Wu,Yulong Ren,Feng Liu,Yuanyuan Hao,Yunlong Wang,Tian Pan,Xuan Teng,Yihua Wang,Yiqun Bao,Xiaopin Zhu,Yuanyan Zhang,Yanfang Yu,Jie Lei,Jianmin Wan,Yongfei Wang,Mingsheng Zhong,Erchao Duan

doi:10.1186/s12870-019-1911-y

Abstract

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

Highlights

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
We report the functional characterization of a rice gpa6 mutant that accumulated a large amount of proglutelins in the mutant endosperm cells, and demonstrate that GPA6 encodes a Golgi, trans-Golgi network (TGN)- and PVClocalized Na+/H+ antiporter OsNHX5 which is essential for endomembrane luminal pH homeostasis and proglutelin vacuolar trafficking
In wild-type endosperm cells, there are irregularly shaped, fully filled PBIIs and round spherical PBIs (Fig. 3a), in the gpa6 mutant, PBIIs were only partially filled with the storage proteins (Fig. 3b), which was accompanied by the presence of Paramural body (PMB) and secreted oval-shaped structures along the cell wall (Fig. 3g and i)

Summary

Results

Gpa seeds accumulate proglutelins and develop abnormal endosperm A 57H mutant named gpa was isolated during our continuous effort to dissect the glutelin trafficking pathway in rice. Consistent with the proglutelin trafficking defects, Pectins labeled with JIM7 were seen to accumulate inside the PMBs in gpa rather than display an even distribution along the wild type cell wall (Additional file 4: Figure S4) [36]. In wild-type endosperm cells, there are irregularly shaped, fully filled PBIIs and round spherical PBIs (Fig. 3a), in the gpa mutant, PBIIs were only partially filled with the storage proteins (Fig. 3b), which was accompanied by the presence of PMBs (formed by the clustered DVs) and secreted oval-shaped structures along the cell wall (Fig. 3g and i). The rice genome has another putative endosomal antiporter OsNHX6 (Fig. 6a, Additional file 5: Figure S5) It is ubiquitiously expressed, its expression is much lower than OsNHX5 (ratio of expression level of OsNHX5/OsNHX6, root: 91; stem: 25; leaf: 232; leaf sheath: 15; panicle: 18; 12-DAF endosperm: 26) (Fig. 6b). These results clearly indicated that OsNHX5 regulates the pH homeostasis of the Golgi, TGN, and PVC in rice protoplasts

Background

Discussion

Methods