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Vesicular trafficking plays critical roles in cell expansion in yeast and mammals, but information linking vesicular trafficking and cell expansion in plants is limited. Here, we isolated and characterized a rice (Oryza sativa) mutant, decreased plant height 1-1 (dph1-1), which exhibited a wide spectrum of developmental phenotypes, including reduced plant height and smaller panicles and grains. Cytological analysis revealed that limited cell expansion was responsible for the dph1-1 mutant phenotype compared to the wild-type. Map-based cloning revealed that DPH1 encodes a plant-specific protein, OsSCD2, which is homologous to Arabidopsis (Arabidopsis thaliana) STOMATAL CYTOKINESIS DEFECTIVE2 (SCD2). Subcellular localization revealed that OsSCD2 is associated with clathrin. Confocal microscopy showed that the dph1-1 mutant has defective endocytosis and post-Golgi trafficking. Biochemical and confocal data indicated that OsSCD2 physically interacts with OsSCD1 and that they are associated with intracellular structures that colocalize with microtubules. Furthermore, we found that cellulose synthesis was affected in the dph1-1 mutant, evidenced by reduced cellulose synthase gene accumulation at the transcript and protein levels, most likely resulting from an impaired localization pattern. Our results suggest that OsSCD2 is involved in clathrin-related vesicular trafficking with an important role in maintaining plant growth in rice.

Protein storage vacuoles (PSVs) are unique organelles that accumulate storage proteins in plant seeds. Although morphological evidence points to the existence of multiple PSV-trafficking pathways for storage protein targeting, the molecular mechanisms that regulate these processes remain mostly unknown. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation7 (gpa7) mutant, which over-accumulates 57-kDa glutelin precursors in dry seeds. Cytological and immunocytochemistry studies revealed that the gpa7 mutant exhibits abnormal accumulation of storage prevacuolar compartment-like structures, accompanied by the partial mistargeting of glutelins to the extracellular space. The gpa7 mutant was altered in the CCZ1 locus, which encodes the rice homolog of Arabidopsis (Arabidopsis thaliana) CALCIUM CAFFEINE ZINC SENSITIVITY1a (CCZ1a) and CCZ1b. Biochemical evidence showed that rice CCZ1 interacts with MONENSIN SENSITIVITY1 (MON1) and that these proteins function together as the Rat brain 5 (Rab5) effector and the Rab7 guanine nucleotide exchange factor (GEF). Notably, loss of CCZ1 function promoted the endosomal localization of vacuolar protein sorting-associated protein 9 (VPS9), which is the GEF for Rab5 in plants. Together, our results indicate that the MON1–CCZ1 complex is involved in post-Golgi trafficking of rice storage protein through a Rab5- and Rab7-dependent pathway.

Ubiquitination and deubiquitination are reversible processes that play crucial roles in regulating organ size in plants. However, information linking deubiquitination and seed size in rice (Oryza sativa) is limited. Here, we characterized a dominant large-grain mutant, large grain1-D (lg1-D), with a 30.8% increase in seed width and a 34.5% increase in 1,000-grain weight relative to the wild type. The lg1-D mutant had more cells oriented in the lateral direction of the spikelet hull compared with the wild type. Map-based cloning showed that LG1 encodes a constitutively expressed ubiquitin-specific protease15 (OsUBP15) that possesses deubiquitination activity in vitro. Loss-of-function and down-regulated expression of OsUBP15 produced narrower and smaller grains than the control. A set of in vivo experiments indicated that the mutant Osubp15 had enhanced protein stability relative to wild-type OsUBP15. Further experiments verified that OsDA1 directly interacted with OsUBP15. Genetic data indicated that OsUBP15 and GRAIN WIDTH 2 (GW2) were not independent in regulating grain width and size. In summary, we identified OsUBP15 as a positive regulator of grain width and size in rice and provide a promising strategy for improvement of grain yield by pyramiding OsUBP15 and gw2.

Dense vesicles (DVs) are Golgi-derived plant-specific carriers that mediate post-Golgi transport of seed storage proteins in angiosperms. How this process is regulated remains elusive. Here, we report a rice (Oryza sativa) mutant, named glutelin precursor accumulation8 (gpa8) that abnormally accumulates 57-kDa proglutelins in the mature endosperm. Cytological analyses of the gpa8 mutant revealed that proglutelin-containing DVs were mistargeted to the apoplast forming electron-dense aggregates and paramural bodies in developing endosperm cells. Differing from previously reported gpa mutants with post-Golgi trafficking defects, the gpa8 mutant showed bent Golgi bodies, defective trans-Golgi network (TGN), and enlarged DVs, suggesting a specific role of GPA8 in DV biogenesis. We demonstrated that GPA8 encodes a subunit E isoform 1 of vacuolar H+-ATPase (OsVHA-E1) that mainly localizes to TGN and the tonoplast. Further analysis revealed that the luminal pH of the TGN and vacuole is dramatically increased in the gpa8 mutant. Moreover, the colocalization of GPA1 and GPA3 with TGN marker protein in gpa8 protoplasts was obviously decreased. Our data indicated that OsVHA-E1 is involved in endomembrane luminal pH homeostasis, as well as maintenance of Golgi morphology and TGN required for DV biogenesis and subsequent protein trafficking in rice endosperm cells.