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

Background 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. Results 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. Conclusions Our results demonstrated an important role of OsNHX5 in regulating endomembrane luminal pH, which is essential for seed storage protein trafficking in rice. Electronic supplementary material The online version of this article (10.1186/s12870-019-1911-y) contains supplementary material, which is available to authorized users.


Background
Rice seeds accumulate large amount of storage proteins, including glutelin, prolamin, and α-globulin, which supply nutrients for seed germination and seedling growth. Up to 80% of the total seed storage proteins are made up of glutelins which are important protein sources for human consumption because of their easy digestibility. Glutelins are synthesized as 57 kD precursors on the rough endoplasmic reticulum (RER) and transported to PBIIs by DV-mediated post-Golgi transport pathway or ER-derived precursoraccumulating compartments [1][2][3][4][5][6].
In this study, 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-, TGN-and PVClocalized Na + /H + antiporter OsNHX5 which is essential for endomembrane luminal pH homeostasis and proglutelin vacuolar trafficking. Fig. 1 Characterization of the gpa6 Mutant. a Transverse sections of representative wild-type (Indica cultivar N22) and gpa6 mutant dry seeds. Bars = 1 mm. b and c Scanning electron microscopy images of transverse sections of wild-type (b) and gpa6 mutant (c) seeds. Bars = 10 μm. d SDS-PAGE profiles of total seed storage proteins of the wild type and gpa6 mutant. pGlu, 57-kD proglutelins; αGlu, 40-kD glutelin acidic subunits; αGlb, 26-kD α-globulin; βGlu, 20-kD glutelin basic subunits; Pro, prolamins. e Immunoblot analysis of glutelins and the molecular chaperones BiP1 and PDI1-1. Arrowheads represent glutelin basic subunits. Arrows indicate the 57-kD proglutelins (red) and the glutelin acidic subunits (black). EF-1α was used as a loading control Results gpa6 seeds accumulate proglutelins and develop abnormal endosperm A 57H mutant named gpa6 was isolated during our continuous effort to dissect the glutelin trafficking pathway in rice. Unlike the transparent endosperm of wild type, gpa6 mutant endosperm appeared floury (Fig. 1a). Scanning electron microscopy (SEM) analysis revealed that gpa6 endosperm comprised round and loosely packaged compound starch granules instead of the tightly packaged, crystal-like structures observed in the wild type ( Fig. 1b and c). Meanwhile, the 1000-grain weight was significantly decreased, and the amylose content was reduced approximately 20% and the lipid content was increased 81% in the gpa6 mutant. However, the total protein content in the endosperm was not changed (Additional file 8: Table S1). Compared with the wild type, gpa6 accumulated much higher level of unprocessed 57 kD proglutelins, accompanied by concomitant reduction of both 40 kD acidic and 20 kD basic subunits of the mature glutelins (Fig. 1d). The appearance of higher amount of proglutelins was further confirmed by immunoblotting using antibodies against 40-kD glutelin acidic subunits and 20-kD glutelin basic subunits (Fig. 1e). Time course studies revealed that the abnormal accumulation of proglutelins in gpa6 seeds occurred from~12-DAF onwards (Additional file 1: Figure S1). In addition, the expression of representative genes coding for storage proteins, and the protein level of ER lumen BINDING PROTEIN1 (BiP1) and PDI1-1 showed almost no differences between gpa6 and wild-type seeds (Fig. 1e, Additional file 2: Figure S2), indicating that the accumulation of 57 KD proglutelins was not due to an increasement of storage protein coding gene expression and probably protein synthesis was normal as no ER stress was detected. Collectively these data suggested that in the gpa6 mutant might be defective in the trafficking of proglutelins.
The gpa6 mutant is defective in post-Golgi trafficking of storage proteins in developing endosperm To gain an overview of glutelin deposition, semi-thin sections (1 μm) of 12-DAF wild-type and gpa6 mutant endosperm were prepared and subjected to the immunofluoresence staining with specific antibodies against prolamins and glutelin acidic subunits (Fig. 2). In gpa6 mutant, the PBIIs (containing glutelins) were reduced to 53% of the wild type ( Fig. 2a-f, g), while the PBI sizes (containing prolamins) were comparable ( Fig. 2a-f, h). Large glutelin-containing paramural bodies (PMBs) were readily observable (Fig. 2f ). In addition, α-globulins were also transported incorrectly to the PMBs rather than PBIIs in gpa6 (Additional file 3: Figure S3). Consistent with the proglutelin trafficking defects, Pectins labeled with JIM7 were seen to accumulate inside the PMBs in gpa6 rather than display an even distribution along the wild type cell wall (Additional file 4: Figure S4) [36].
Next, subcellular observation by transmission electron microscopy (TEM) was performed using ultra-thin sections of 12-DAF developing endosperm. In wild-type endosperm cells, there are irregularly shaped, fully filled PBIIs and round spherical PBIs (Fig. 3a), however, in the gpa6 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). To determine structural alterations in the endomembrane system that could account for the missorting of storage protein precursors to the apoplast, we analyzed 12-DAF developing endosperm by immunogold labeling. Indeed, in the gpa6 mutant, only part of glutelins was correctly transported to the PBIIs ( Fig. 4a and b), meanwhile, mis-sorted DVs and PMBs were found to contain glutelins ( Fig. 4c and d). These results demonstrated that proglutelin were mis-sorted, and delivered to the apoplast.
Proglutelins are delivered to the PBII via DV-mediated trafficking pathway [5,6], therefore, DV morphology are carefully examined. Statistical analysis showed that the average diameter of DVs near the Golgi apparatus, and presumably just budding from the Golgi apparatus in gpa6 is 193 nm (n = 75, Fig. 3d), which is larger than that of wild type (average 154 nm, n = 56, Fig. 3c), and these DVs tends to cluster in the cytosol (Fig. 3e). In addition, numerous DVs at an average diameter of 198 nm were found just fused with the plasma membrane, and kept spherical shapes (Fig. 3f), gradually, the secreted storage proteins became oval-shaped (Fig. 3g). Occasionally, fusion between two DVs was detected (Fig. 3h), this might account for the slight size increasement of DVs along the plasma membrane (198 nm) versus newly budded ones (193 nm). In short, DVs in gpa6 mutant are enlarged.
Altogether, these results demonstrated that in gpa6 mutant enlarged glutelin-containing DVs were mis-sorted to the apoplast, thus led to the reduction of PBII size.

Map-based cloning of GPA6
The gpa6 mutant was isolated from a 60 Co-irradiated population of indica variety N22. Genetic analysis revealed that the mutant phenotype was inherited as a recessive mutation (Additional file 9: Table S2). For map-based cloning, we crossed gpa6 with the japonica variety Nipponbare to generate 208 F 2 recessive individuals. The GPA6 locus was mapped to chromosome 9 and further fine-mapped to a 98-kb region (Fig. 5a). DNA sequencing revealed a 7 bp deletion in the sixth exon of Os09g0286400, generating a premature stop codon that led to a truncated product with 199 amino acids (Fig. 5b). Three independent transgenic lines bearing Ubiquitin promoter driven Os09g0286400 open reading frame (ORF) rescued the gpa6 mutant Fig. 4 Immunoelectron microscopy localization of glutelins in rice endosperm cells. a Glutelins were accumulated in PBIIs in the wild-type endosperm cells. CW, cell walls. Bars = 500 nm. b Size-reduced PBII containing glutelins. Bars = 500 nm. c Glutelins in DVs (red arrows) and ovalshaped structures (black arrows). Bars = 500 nm. d Glutelins in the PMBs. Bars = 500 nm. 10-nm gold particle conjugated secondary antibodies were used in (a-d) phenotypes, including the floury appearance of endosperm (Fig. 5c), the accumulation of proglutelins (Fig. 5d) and the abnormal glutelin deposit pattern ( Fig. 5e and f). Therefore, Os09g0286400 is the gene responsible for gpa6 mutant phenotypes.
GPA6 encodes OsNHX5 that localizes to the Golgi, TGN and PVC GPA6 encodes a NHX antiporter homologous to the endosomal localized AtNHX5, and was named OsNHX5.
OsNHX5 is predicted to have 9 putative transmembrane domains and the mutation in gpa6 leads to the deletion of the last four. Real-time PCR analysis revealed that OsNHX5 is expressed in all tissues examined (Fig. 6b). During endosperm development, the expression of OsNHX5 was low at the early stages, peaked at~15-DAF, and decreased at~18-DAF (Fig. 6b), which is correlated with the accumulation of glutelins. The rice genome has another putative endosomal antiporter OsNHX6 (Fig. 6a, Additional file 5: Figure S5). Although 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).
To determine the subcellular localization of OsNHX5, OsNHX5 coding sequence was fused to the N-terminus of GFP to obtain p35S:OsNHX5-GFP construct. After transformed into gpa6, it completely rescued the mutant phenotype, indicating that OsNHX5-GFP is functional in vivo (Additional file 6: Figure S6). Unfortunately, GFP fluorescence in the transgenic lines was too weak to be detected. Transiently expressed OsNHX5-GFP in N. benthamiana leaf epidermal cells was examined instead, and OsNHX5-GFP was shown to partially  Fig. 7). Similarly, OsNHX6 was localized to the Golgi, TGN, and PVC as well (Additional file 7: Figure S7).
Given the fact that OsNHX5 mutation alone displayed phenotypes and OsNHX6 is much lower expressed, OsNHX5 might be a predominant endosomal localized NHX antiporter in rice.

The luminal pH of Golgi, TGN and PVC is more acidic in the gpa6 protoplasts
In order to determine whether the cellular pH was affected in gpa6, we use noninvasive live-cell imaging to measure pH by the pHluorin-based pH sensor [37,38]. pH sensors ManI-PRpHluorin, PRpHluorin-BP80 (Y612A) and PRpHluorin-AtVSR2 were used to measure pH of Golgi, TGN and PVC, respectively in the rice protoplasts [38]. The calibration curve was acquired by calculating pH-dependent fluorescence ratios (Fig. 8a). Our results showed that the pH of the Golgi, TGN, and PVC was more acidic in gpa6 (Golgi: 6.45 ± 0.17; TGN: 6.01 ± 0.16; PVC: 5.87 ± 0.19) than in the wild type (Golgi: 6.95 ± 0.21; TGN: 6.36 ± 0.18; PVC: 6.31 ± 0.14) (Fig. 8d) where the △pH = 0.35-0.50. Representative pseudocoloured images of PRpHluorin-BP80 (Y612A) were shown (Fig. 8b and c). These results clearly indicated that OsNHX5 regulates the pH homeostasis of the Golgi, TGN, and PVC in rice protoplasts. Characterization of 57H mutants facilitates us to understand the molecular mechanism of proglutelin trafficking and processing in rice endosperm cells. In this study, we isolated a 57H mutant gpa6 that accumulated large amount of proglutelins in rice endosperm. The comparable protein levels of BiP1 and PDI1-1 between wild type and the gpa6 mutant suggested a normal ER function in gpa6. Consistent with this notion, gpa6 developed normal ER-derived PBIs. In gpa6 mutant, large amount of DVs accumulated in the cytosol, suggesting that the trafficking rate is largely reduced. Furthermore, DVs were mis-sorted to the apoplast, leading to smaller PBIIs. These abnormalities are very similar to gpa1/osrab5a, gpa2/ osvps9a, and gpa3/kelch mutants [5,6,16,18,19]. Taken together, gpa6 is defective in proglutelin post-Golgi vacuolar trafficking pathway in rice endosperm cells.
OsNHX5 is a predominant intracellular NHX antiporter that regulates luminal pH of several endomembrane compartments Similar to animals, plant cells also have specific pH in different endomembrane compartments along the secretory pathway [37][38][39][40]. In this study, using noninvasive live-cell imaging and pH sensors, we found that the pH values of Golgi, TGN and PVC in wildtype rice protoplasts were not significantly different from their counterparts in Arabidopsis protoplasts (Golgi: 6.8; TGN: 6.3; PVC: 6.2) [38]. However, all three compartments had a more acidic pH (△pH = 0.35-0.50) in osnhx5, indicating that OsNHX5 plays an important role in the alkalization of these compartments. Given the fact that atnhx5 atnhx6 had a more acidic pH in those compartments as well (△pH = 0.25-0.40), endosomal antiporters seem to have conserved functions in rice and Arabidopsis cells.
In atnhx5 atnhx6 double mutant, lowered pH led to a compromised receptor-cargo association [33]. The phenotype of storage protein trafficking defects were similar between osnhx5 and atnhx5 atnhx6. Therefore, the acidification of Golgi, TGN, particularly PVC may result in reduced VSR (Vacuolar sorting receptor)-proglutelin association, although the receptors for proglutelins remains to be characterized. Previous studies showed that OsNHX5 has a K + and Na + transport activity [41], thus it is possible that ionic changes might also affect VSR-cargo interactions.
Previous studies implicate that maintaining pH homeostasis of TGN is required for protein trafficking in Arabidopsis [42]. DVs are unique carriers for proglutelin transport in rice presumably budded from the TGN [5,6]. It is worth noting that the average size of DVs newly budded from TGN is enlarged to about 193 nm in gpa6, which is much bigger than 154 nm in the wild type (Fig. 3), 160 nm in gpa2 [5], and 153 nm in gpa3 mutants [6]. Thus, pH of TGN seems to have an impact on DV size control, although the detail mechanism remains to be explored.

Conclusions
In summary, our studies demonstrated that OsNHX5 is localized to the Golgi, TGN and PVC to maintain pH homeostasis, which is important for DV-mediated glutelin trafficking in rice endosperm.

Plant materials and growth conditions
The gpa6 mutant was identified from a 60 Co-irradiated mutant pool of the indica cultivar N22 (Nagina22, an Indian traditional variety). An F 2 population was produced from gpa6 and a japonica variety Nipponbare for mapping. The seeds of all accessions were collected, stored and supplied by the State Key Laboratory of Crop Genetics and Germplasm Enhancement of Nanjing Agricultural University, Jiangsu, China. All plants were grown in the paddy field during the normal growing seasons or in a greenhouse at Nanjing, China. Developing seeds of wild type (N22) and gpa6 at 6-21 days after fertilization (DAF) were used in the experiments.

Protein Extraction from Rice seeds and immunoblot analysis
Total protein extraction and immunoblot assay were performed as described previously [16].

Map-based cloning
To map the GPA6 locus, an F 2 population was generated from a cross between the gpa6 mutant and a japonica variety Nipponbare. Total proteins were extracted from half of an individual rice seed and resolved by SDS-PAGE gel to monitor the accumulation of the proglutelins. Meanwhile, the other half of the identified mutant seeds with embryos was grown for DNA extraction. In total, 208 recessive individuals were used for fine mapping of GPA6. The primers used in fine mapping are listed in Additional file 10: Table S3.

Real-time RT-PCR analysis
Total RNA was extracted from different tissues using an RNA Prep Pure Plant Kit (TIANGEN). First strand cDNA was synthesized using oligo (dT)18 as the primer (TaKaRa). Three biological replicates of real-time RT-PCR were performed with SYBR Premix Ex Taq II (TaKaRa) on an Applied Biosystems 7500 Real-Time PCR System. The primer sequences used for PCR are listed in Additional file 12: Table S5.

Subcellular localization
For transient expression analysis in N. benthamiana leaf epidermal cells, the coding region of OsNHX5 or OsNHX6 was amplified and inserted into the binary vector pCAM-BIA1305GFP to produce the OsNHX5-GFP or OsNHX6-GFP fusion construct (Additional file 11: Table S4). Construct were introduced into the Agrobacterium strain EHA105 and then used to infiltrate N. benthamiana leaves, as described previously [43]. N. benthamiana protoplasts were isolated using the same method used with Arabidopsis [44]. Fluorescence was observed using a confocal laser scanning microscope (Leica TCS-SP8).

pH measurements
The rice protoplasts were isolated from 10-days-old N22 and gpa6 seedlings. pH sensors ManI-PRpHluorin, PRpHluorin-BP80 (Y612A) and PRpHluorin-AtVSR2 were transformed into rice protoplasts as previously described [45,46]. The PRpHluorin signals at emission wavelength of 500 to 550 nm were recorded with dualexcitation wavelength at 405 and 488 nm, respectively, and used to calculate the pH using the calibration curve. In vivo calibration was achieved from the same protoplasts expressing the PRpHluorin for pH measurement. Protoplasts were incubated in WI protoplast buffer (0.5 M mannitol and 20 mM KCl) with 25 μM nigericin, 60 mM KCl, and 10 mM MES/HEPES Bis-Tris-propane adjusted to different pH values ranging from 5.0 to 8.0 for each calibration point [33,37,38,[47][48][49]. Fluorescence was observed using a confocal laser scanning microscope (Leica TCS-SP8). Availability of data and materials All data generated or analyzed during this study are included in this published article and its additional files.
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Consent for publication
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Competing interests
The authors declare that they have no competing interests.