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.

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.