A deleterious Sar1c variant in rice inhibits export of seed storage proteins from the endoplasmic reticulum.

KEY MESSAGE: We identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm. Higher plants accumlate large amounts of seed storage proteins (SSPs). However, mechanisms underlying SSP trafficking are largely unknown, especially the ER-Golgi anterograde process. Here, we showed that a rice glutelin precursor accumulation13 (gpa13) mutant exhibited floury endosperm and overaccumulated glutelin precursors, which phenocopied the reported RNAi-Sar1abc line. Molecular cloning revealed that the gpa13 allele encodes a mutated Sar1c (mSar1c) with a deletion of two conserved amino acids Pro134 and Try135. Knockdown or knockout of Sar1c alone caused no obvious phenotype, while overexpression of mSar1c resulted in seedling lethality similar to the gpa13 mutant. Transient expression experiment in tobacco combined with subcellular fractionation experiment in gpa13 demonstrated that the expression of mSar1c affects the subcellular distribution of all Sar1 isoforms and Sec23c. In addition, mSar1c failed to interact with COPII component Sec23. Conversely, mSar1c competed with Sar1a/b/d to interact with guanine nucleotide exchange factor Sec12. Together, we identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm.

Rice FLOURY ENDOSPERM22, encoding a pentatricopeptide repeat protein, is involved in both mitochondrial RNA splicing and editing and is crucial for endosperm development.

Most of the reported P-type pentatricopeptide repeat (PPR) proteins play roles in organelle RNA stabilization and splicing. However, P-type PPRs involved in both RNA splicing and editing have rarely been reported, and their underlying mechanism remains largely unknown. Here, we report a rice floury endosperm22 (flo22) mutant with delayed amyloplast development in endosperm cells. Map-based cloning and complementation tests demonstrated that FLO22 encodes a mitochondrion-localized P-type PPR protein. Mutation of FLO22 resulting in defective trans-splicing of mitochondrial nad1 intron 1 and perhaps causing instability of mature transcripts affected assembly and activity of complex Ⅰ, and mitochondrial morphology and function. RNA-seq analysis showed that expression levels of many genes involved in starch and sucrose metabolism were significantly down-regulated in the flo22 mutant compared with the wild type, whereas genes related to oxidative phosphorylation and the tricarboxylic acid cycle were significantly up-regulated. In addition to involvement in splicing as a P-type PPR protein, we found that FLO22 interacted with DYW3, a DYW-type PPR protein, and they may function synergistically in mitochondrial RNA editing. The present work indicated that FLO22 plays an important role in endosperm development and plant growth by participating in nad1 maturation and multi-site editing of mitochondrial messager RNA.

Fructose-6-phosphate-2-kinase/fructose-2,6-bisphosphatase regulates energy metabolism and synthesis of storage products in developing rice endosperm.

Starch accounts for about 80-85 % of the dry weight of grains and determines yield by impact on grain weight. And, the content and composition of starch also determine appearance, eating, cooking and nutritional quality of rice. By coordinating crucial reactions of the primary carbohydrate metabolism in all eukaryotes, fructose-2,6-bisphosphate (Fru-2,6-P2) is a traffic signal in metabolism. However, the metabolic regulation of starch in plant sink tissues by Fru-2,6-P2 remains unclear. Here we isolated rice mutant floury endosperm23 (flo23) which has opaque endosperm and anomalous compound starch grains (SGs). flo23 mutant grains had reduced contents of starch, lipids and proteins. Map-based cloning and genetic complementation experiments showed that FLO23 encodes a cytoplasmic Fructose-6-phosphate-2-kinase/Fructose-2,6-bisphosphatase (F2KP). Mutation of OsF2KP2 decreased Fru-2,6-P2 content in endosperm cells, leading to drastically reduced phosphoenolpyruvate (PEP) and pyruvate contents and disordered glycolysis and energy metabolism. The results imply that OsF2KP2 participates in the glycolytic pathway by providing precursors and energy for synthesis of grain storage compounds.

Endomembrane-mediated storage protein trafficking in plants: Golgi-dependent or Golgi-independent?

Seed storage proteins (SSPs) accumulated within plant seeds constitute the major protein nutrition sources for human and livestock. SSPs are synthesized on the endoplasmic reticulum and are then deposited in plant-specific protein bodies, including endoplasmic reticulum-derived protein bodies and protein storage vacuoles. Plant seeds have evolved a distinct endomembrane system to accomplish SSP transport. There are two distinct types of trafficking pathways contributing to SSP delivery to protein storage vacuoles: one is Golgi-dependent and the other is Golgi-independent. In recent years, molecular, genetic, and biochemical studies have shed light on the complex network controlling SSP trafficking, to which both evolutionarily conserved molecular machineries and plant-unique regulators contribute. In this review, we discuss current knowledge of protein body biogenesis and endomembrane-mediated SSP transport, focusing on endoplasmic reticulum export and post-Golgi traffic. This knowledge supports a dominant role for the Golgi-dependent pathways in SSP transport in Arabidopsis and rice. In addition, we describe cutting-edge strategies for dissecting the endomembrane trafficking system in plant seeds to advance the field.

Effects of silencing the DUSP1 gene using lentiviral vector-mediated siRNA on the release of proinflammatory cytokines through regulation of the MAPK signaling pathway in mice with acute pancreatitis.

The present study investigated the effects of dual specificity phosphatase 1 (DUSP1) gene silencing using lentiviral vector-mediated small interfering (si)RNA on the release of proinflammatory cytokines through the regulation of the mitogen­activated protein kinase (MAPK) signaling pathway in mice with acute pancreatitis (AP). Two siRNA­DUSP1 sequences and one scramble siRNA sequence were designed, and the expression of DUSP1 was detected using western blot analysis to screen for the one with a higher interference rate. An AP mouse model was established, and KM mice were assigned to either a control, siRNA, AP, AP+PD98059, AP+scramble, AP+siRNA or AP+PD98059+siRNA group. The expression of proinflammatory cytokines, including tumor necrosis factor (TNF)­α, interleukin (IL)­1ß and IL­6, high mobility group box 1 (HMGB1), and S100A12 in serum samples were detected using an enzyme­linked immunosorbent assay at 12, 24 and 48 h post­modeling. The serum amylase levels were also detected. The expression levels of DUSP1, TNF­α, IL­1ß, IL­6, HMGB1, S100A12, phosphorylated (p­) extracellular signal­regulated kinase (ERK), p­c­Jun N­terminal kinase (JNK), p­p38, ERK, JNK and p38 in pancreatic, liver, kidney and lung tissues were detected using reverse transcription­quantitative polymerase chain reaction and western blot analysis. Compared with the control group, the siRNA group demonstrated marginally upregulated serum amylase, lipase, urinary trypsinogen­2, and proinflammatory cytokines, HMGB1 and S100A12 in serum and tissues, with no statistically significant difference, elevated expression levels of p­ERK, p­JNK and p­p38, and decreased expression of DUSP1. The other five groups demonstrated increased expression levels of TNF­α, IL­1ß, IL­6, HMGB1, S100A12, amylase, lipase and urinary trypsinogen­2 in serum, and increased expression levels of DUSP1, TNF­α, IL­1ß, IL­6, HMGB1, S100A12, p­ERK, p­JNK and p­p38 in tissues. Compared with the AP group, the AP+PD98059+siRNA group had decreased expression of DUSP1 in tissues, whereas the AP+PD98059 group had decreased serum expression levels of TNF­α, IL­1ß, IL­6, HMGB1, S100A12 and amylase, lipase and urinary trypsinogen­2. The expression levels of TNF­α, IL­1ß, IL­6, HMGB1, S100A12, p­ERK, p­JNK, p­p38 in tissues, and edema of pancreatic tissue were alleviated, whereas the opposite results were observed in the AP+siRNA group with the decreased expression of DUSP1. The results suggested that DUSP1 gene silencing promoted the release of proinflammatory cytokines through activation of the MAPK signaling pathway in mice with AP.