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.

FLOURY ENDOSPERM16 encoding a NAD-dependent cytosolic malate dehydrogenase plays an important role in starch synthesis and seed development in rice.

Starch is the most important form of energy storage in cereal crops. Many key enzymes involved in starch biosynthesis have been identified. However, the molecular mechanisms underlying the regulation of starch biosynthesis are largely unknown. In this study, we isolated a novel floury endosperm rice (Oryza sativa) mutant flo16 with defective starch grain (SG) formation. The amylose content and amylopectin structure were both altered in the flo16 mutant. Map-based cloning and complementation tests demonstrated that FLO16 encodes a NAD-dependent cytosolic malate dehydrogenase (CMDH). The ATP contents were decreased in the mutant, resulting in significant reductions in the activity of starch synthesis-related enzymes. Our results indicated that FLO16 plays a critical role in redox homeostasis that is important for compound SG formation and subsequent starch biosynthesis in rice endosperm. Overexpression of FLO16 significantly improved grain weight, suggesting a possible application of FLO16 in rice breeding. These findings provide a novel insight into the regulation of starch synthesis and seed development in rice.

Rice FLOURY ENDOSPERM10 encodes a pentatricopeptide repeat protein that is essential for the trans-splicing of mitochondrial nad1 intron 1 and endosperm development.

Endosperm, the major storage organ in cereal grains, determines grain yield and quality. Despite the fact that a role for P-type pentatricopeptide repeat (PPR) proteins in the regulation of endosperm development has emerged, molecular functions of many P-type PPR proteins remain obscure. Here, we report a rice endosperm defective mutant, floury endosperm10 (flo10), which developed smaller starch grains in starchy endosperm and abnormal cells in the aleurone layer. Map-based cloning and rescued experiments showed that FLO10 encodes a P-type PPR protein with 26 PPR motifs, which is localized to mitochondria. Loss of function of FLO10 affected the trans-splicing of the mitochondrial nad1 intron 1, which was accompanied by the increased accumulation of the nad1 exon 1 and exons 2-5 precursors. The failed formation of mature nad1 led to a dramatically decreased assembly and activity of complex I, reduced ATP production, and changed mitochondrial morphology. In addition, loss of function of FLO10 significantly induced an alternative respiratory pathway involving alternative oxidase. These results reveal that FLO10 plays an important role in the maintenance of mitochondrial function and endosperm development through its effect on the trans-splicing of the mitochondrial nad1 intron 1 in rice.

FLOURY SHRUNKEN ENDOSPERM1 Connects Phospholipid Metabolism and Amyloplast Development in Rice.

Starch synthesized and stored in amyloplasts serves as the major energy storage molecule in cereal endosperm. To elucidate the molecular mechanisms underlying amyloplast development and starch synthesis, we isolated a series of floury endosperm mutants in rice (Oryza sativa). We identified the rice mutant floury shrunken endosperm1 (fse1), which exhibited obvious defects in the development of compound starch grains, decreased starch content, and altered starch physicochemical features. Map-based cloning showed that FSE1 encodes a phospholipase-like protein homologous to phosphatidic acid-preferring phospholipase A1FSE1 was expressed ubiquitously with abundant levels observed in developing seeds and roots. FSE1 was localized to both the cytosol and intracellular membranes. Lipid profiling indicated that total extra-plastidic lipids and phosphatidic acid were increased in fse1 plants, suggesting that FSE1 may exhibit in vivo phospholipase A1 activity on phosphatidylcholine, phosphatidylinositol, phosphatidyl-Ser, phosphatidylethanolamine, and, in particular, phosphatidic acid. Additionally, the total galactolipid content in developing fse1 endosperm was significantly reduced, which may cause abnormal amyloplast development. Our results identify FSE1 as a phospholipase-like protein that controls the synthesis of galactolipids in rice endosperm and provide a novel connection between lipid metabolism and starch synthesis in rice grains during endosperm development.

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) regulates carbon metabolism during grain filling in rice.

KEY MESSAGE: Decreased PFPase activity in rice perturbs the equilibration of carbon metabolism during grain filling but has no visible phenotypic effects during the vegetative and reproductive growth stages. Starch is a primary energy reserve for various metabolic processes in plant. Despite much advance has been achieved in pathways involved in starch biosynthesis, information was still lacked for precise regulation related to carbon metabolism during seed filling in rice (Oryza sativa). The objective of this study was to identify and characterize new gene associated with carbon metabolism during grain filling. By screening our chemical mutant pool, two allelic mutants exhibiting floury endosperm were isolated. No visible phenotypic defects were observed during both the vegetative and reproductive growth stages, except for the floury-like endosperm of grains with significantly reduced kernel thickness, 1000-grain weight and total starch content. Map-based cloning revealed that the mutant phenotypes were controlled by a gene encoding pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) ß subunit (PFPß), which catalyzes reversible interconversion between fructose-6-phosphate and fructose-1, 6-bisphosphate. The identity of PFP ß was further confirmed by a genetic complementation test. Subcellular analysis demonstrated that PFPß was localized in cytoplasm. Quantitative PCR and histochemical staining indicated PFP ß was ubiquitously expressed in various tissues. Furthermore, we found PFP ß could express in both the early and late phases of starch accumulation during grain filling and decreased activity of PFP ß in pfp mutants resulted in compromised carbon metabolism with increased soluble sugar contents and unfavorable starch biosynthesis. Our results highlight PFPß functions in modulating carbon metabolism during grain filling stage.

FLOURY ENDOSPERM11 encoding a plastid heat shock protein 70 is essential for amyloplast development in rice.

Mutations of stromal Hsp70 cause chloroplast developmental abnormalities and knockout mutants of stromal Hsp70 usually exhibit protein import deficiencies. However, their effects have not been studied in amyloplast development. Here, we identified an amyloplast abnormal development mutant, floury endosperm11 (flo11) that exhibited an opaque phenotype in the inner core and the periphery of grains. Semi-thin section revealed defective amyloplast development in the flo11 endosperm. Map-based cloning and subsequent complementation test demonstrated that FLO11 encoded a plastid-localized heat shock protein 70 (OsHsp70cp-2). OsHsp70cp-2 was abundantly expressed in developing endosperm, whereas its paralogous gene OsHsp70cp-1 was mainly expressed in photosynthetic tissues. Ectopic expression of OsHsp70cp-1 under the control of OsHsp70cp-2 promoter rescued the mutant phenotype of flo11. Moreover, simultaneous knockdown of both OsHsp70cp genes resulted in white stripe leaves and opaque endosperm. BiFC and Co-IP assays revealed that OsHsp70cp-2 was associated with Tic complex. Taken together, OsHsp70cp-2 may regulate protein import into amyloplasts, which is essential for amyloplast development in rice.

OsVPS9A functions cooperatively with OsRAB5A to regulate post-Golgi dense vesicle-mediated storage protein trafficking to the protein storage vacuole in rice endosperm cells.

In the rice endosperm cells, glutelins are synthesized on rough endoplasmic reticulum as proglutelins and are sorted to the protein storage vacuoles (PSVs) called protein body IIs (PBIIs), where they are converted to the mature forms. Dense vesicle (DV)-mediated trafficking of proglutelins in rice seeds has been proposed, but the post-Golgi control of this process is largely unknown. Whether DV can fuse directly with PSV is another matter of debate. In this study, we propose a regulatory mechanism underlying DV-mediated, post-Golgi proglutelin trafficking to PBII (PSV). gpa2, a loss-of-function mutant of OsVPS9A, which encodes a GEF of OsRAB5A, accumulated uncleaved proglutelins. Proglutelins were mis-targeted to the paramural bodies and to the apoplast along the cell wall in the form of DVs, which led to a concomitant reduction in PBII size. Previously reported gpa1, mutated in OsRab5a, has a similar phenotype, while gpa1gpa2 double mutant exacerbated the conditions. In addition, OsVPS9A interacted with OsRAB5A in vitro and in vivo. We concluded that OsVPS9A and OsRAB5A may work together and play a regulatory role in DV-mediated post-Golgi proglutelin trafficking to PBII (PSV). The evidence that DVs might fuse directly to PBII (PSV) to deliver cargos is also presented.