Proteomics Analysis Reveals Non-Controlled Activation of Photosynthesis and Protein Synthesis in a Rice npp1 Mutant under High Temperature and Elevated CO2 Conditions.

Rice nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides including ADP-glucose and ATP. Under high temperature and elevated CO2 conditions (HT + ECO2), the npp1 knockout rice mutant displayed rapid growth and high starch content phenotypes, indicating that NPP1 exerts a negative effect on starch accumulation and growth. To gain further insight into the mechanisms involved in the NPP1 downregulation induced starch overaccumulation, in this study we conducted photosynthesis, leaf proteomic, and chloroplast phosphoproteomic analyses of wild-type (WT) and npp1 plants cultured under HT + ECO2. Photosynthesis in npp1 leaves was significantly higher than in WT. Additionally, npp1 leaves accumulated higher levels of sucrose than WT. The proteomic analyses revealed upregulation of proteins related to carbohydrate metabolism and the protein synthesis system in npp1 plants. Further, our data indicate the induction of 14-3-3 proteins in npp1 plants. Our finding demonstrates a higher level of protein phosphorylation in npp1 chloroplasts, which may play an important role in carbohydrate accumulation. Together, these results offer novel targets and provide additional insights into carbohydrate metabolism regulation under ambient and adverse conditions.

N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate that trans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice.

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1-NPP6 Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.

Nucleotide pyrophosphatase/phosphodiesterase 1 exerts a negative effect on starch accumulation and growth in rice seedlings under high temperature and CO2 concentration conditions.

Nucleotide pyrophosphatase/phosphodiesterase (NPP) is a widely distributed enzymatic activity occurring in both plants and mammals that catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides. Unlike mammalian NPPs, the physiological function of plant NPPs remains largely unknown. Using a complete rice NPP1-encoding cDNA as a probe, in this work we have screened a rice shoot cDNA library and obtained complete cDNAs corresponding to six NPP genes (NPP1-NPP6). As a first step to clarify the role of NPPs, recombinant NPP1, NPP2 and NPP6 were purified from transgenic rice cells constitutively expressing NPP1, NPP2 and NPP6, respectively, and their enzymatic properties were characterized. NPP1 and NPP6 exhibited hydrolytic activities toward ATP, UDP-glucose and the starch precursor molecule, ADP-glucose, whereas NPP2 did not recognize nucleotide sugars as substrates, but hydrolyzed UDP, ADP and adenosine 5'-phosphosulfate. To gain insight into the physiological function of rice NPP1, an npp1 knockout mutant was characterized. The ADP-glucose hydrolytic activities in shoots of npp1 rice seedlings were 8% of those of the wild type (WT), thus indicating that NPP1 is a major determinant of ADP-glucose hydrolytic activity in rice shoots. Importantly, when seedlings were cultured at 160 Pa CO2 under a 28°C/23°C (12 h light/12 h dark) regime, npp1 shoots and roots were larger than those of wild-type (WT) seedlings. Furthermore, the starch content in the npp1 shoots was higher than that of WT shoots. Growth and starch accumulation were also enhanced under an atmospheric CO2 concentration (40 Pa) when plants were cultured under a 33°C/28°C regime. The overall data strongly indicate that NPP1 exerts a negative effect on plant growth and starch accumulation in shoots, especially under high CO2 concentration and high temperature conditions.

Proteomic Analysis of Rice Golgi Membranes Isolated by Floating Through Discontinuous Sucrose Density Gradient.

The Golgi apparatus is an endomembrane system organelle and has roles in glycosylation, sorting, and secretion of proteins in the secretory pathway. It has a central function in living organism and is also essential for plant growth. Proteomic approaches to identify the Golgi membrane proteins have been performed in cell suspension cultures and many Golgi membrane-associated proteins were found, whereas it has well established in rice seedling yet. In this chapter, our recent improving published methods for isolated rice Golgi membranes by floating through a discontinuous sucrose density gradient are provided in detail with proteomic analyses.

Optimized Method of Extracting Rice Chloroplast DNA for High-Quality Plastome Resequencing and de Novo Assembly.

Chloroplasts, which perform photosynthesis, are one of the most important organelles in green plants and algae. Chloroplasts maintain an independent genome that includes important genes encoding their photosynthetic machinery and various housekeeping functions. Owing to its non-recombinant nature, low mutation rates, and uniparental inheritance, the chloroplast genome (plastome) can give insights into plant evolution and ecology and in the development of biotechnological and breeding applications. However, efficient methods to obtain high-quality chloroplast DNA (cpDNA) are currently not available, impeding powerful sequencing and further functional genomics research. To investigate effects on rice chloroplast genome quality, we compared cpDNA extraction by three extraction protocols: liquid nitrogen coupled with sucrose density gradient centrifugation, high-salt buffer, and Percoll gradient centrifugation. The liquid nitrogen-sucrose gradient method gave a high yield of high-quality cpDNA with reliable purity. The cpDNA isolated by this technique was evaluated, resequenced, and assembled de novo to build a robust framework for genomic and genetic studies. Comparison of this high-purity cpDNA with total DNAs revealed the read coverage of the sequenced regions; next-generation sequencing data showed that the high-quality cpDNA eliminated noise derived from contamination by nuclear and mitochondrial DNA, which frequently occurs in total DNA. The assembly process produced highly accurate, long contigs. We summarize the extent to which this improved method of isolating cpDNA from rice can provide practical progress in overcoming challenges related to chloroplast genomes and in further exploring the development of new sequencing technologies.

Proteomic and Glycomic Characterization of Rice Chalky Grains Produced Under Moderate and High-temperature Conditions in Field System.

BACKGROUND: Global climate models predict an increase in global mean temperature and a higher frequency of intense heat spikes during this century. Cereals such as rice (Oryza sativa L.) are more susceptible to heat stress, mainly during the gametogenesis and flowering stages. During periods of high temperatures, grain filling often causes serious damage to the grain quality of rice and, therefore, yield losses. While the genes encoding enzymes involved in carbohydrate metabolism of chalky grains have been established, a significant knowledge gap exists in the proteomic and glycomic responses to warm temperatures in situ. Here, we studied the translucent and opaque characters of high temperature stressed chalky grains of 2009 and 2010 (ripening temperatures: 24.4 and 28.0 °C, respectively). RESULTS: Appearance of chalky grains of both years showed some resemblance, and the high-temperature stress of 2010 remarkably extended the chalking of grain. Scanning electron microscopic observation showed that round-shaped starch granules with numerous small pits were loosely packed in the opaque part of the chalky grains. Proteomic analyzes of rice chalky grains revealed deregulations in the expression of multiple proteins implicated in diverse metabolic and physiological functions, such as protein synthesis, redox homeostasis, lipid metabolism, and starch biosynthesis and degradation. The glycomic profiling has shown slight differences in chain-length distributions of starches in the grains of 2009-to-2010. However, no significant changes were observed in the chain-length distributions between the translucent and opaque parts of perfect and chalky grains in both years. The glucose and soluble starch contents in opaque parts were increased by the high-temperature stress of 2010, though those in perfect grains were not different regardless of the environmental changes of 2009-to-2010. CONCLUSION: Together with previous findings on the increased expression of α-amylases in the endosperm, these results suggested that unusual starch degradation rather than starch synthesis is involved in occurring of chalky grains of rice under the high-temperature stress during grain filling period.