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

Abstract

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.

Keywords: (phospho)-proteomics; 14-3-3 proteins; Oryza sativa L.; chloroplast; elevated CO2; heat stress; nucleotide pyrophosphatase/phosphodiesterase; photosynthesis; protein phosphorylation; starch; sucrose.

Figure 1

Leaf temperatures of WT and npp1 mutant plants under different temperatures and CO₂ concentrations in Biotron’s environmental conditions. WT (filled bars) and npp1 (open bars) plants grown for 60 days under normal conditions (14 h light/10 h dark: 28/23 °C, 40 Pa CO₂) after germination were subjected to thermographic analysis of their leaf temperatures under different temperature and CO₂ conditions: (A) 28 °C, 40 Pa, (B) 28 °C, 160 Pa, (C) 33 °C, 40 Pa, and (D) 33 °C, 160 Pa. Values in upper panel show the means ± standard deviation (s.d.) (n = 5). Asterisks indicate significant differences by Student’s t-test (*, p > 0.05; **, p > 0.01). Lower panels represent the thermographic images. WT: wild-type.

Figure 2

Changes in temperatures, g_s, C_i/C_a, and A_n of WT and npp1 mutant plants under different temperatures and CO₂ concentrations in a controlled growth chamber. Top leaves of main culm of WT (■) and npp1 (□) plants grown for 60 days under normal conditions (14 h light/10 h dark: 28/23 °C, 40 Pa CO₂) were subjected to measurements of temperature (A), stomatal conductance to water (g_s) (B), ratios of internal [CO₂] to ambient [CO₂] (C_i/C_a) (C), and photosynthetic rates (A_n) (D) under the following conditions: temperature, 28 and 33 °C; CO₂, 40 to 200 Pa; relative humidity, 50%; light, 1000 μmol·photons·m⁻²·s⁻¹. Values show the means ± s.d. (n = 3).

Figure 2

Changes in temperatures, g_s, C_i/C_a, and A_n of WT and npp1 mutant plants under different temperatures and CO₂ concentrations in a controlled growth chamber. Top leaves of main culm of WT (■) and npp1 (□) plants grown for 60 days under normal conditions (14 h light/10 h dark: 28/23 °C, 40 Pa CO₂) were subjected to measurements of temperature (A), stomatal conductance to water (g_s) (B), ratios of internal [CO₂] to ambient [CO₂] (C_i/C_a) (C), and photosynthetic rates (A_n) (D) under the following conditions: temperature, 28 and 33 °C; CO₂, 40 to 200 Pa; relative humidity, 50%; light, 1000 μmol·photons·m⁻²·s⁻¹. Values show the means ± s.d. (n = 3).

Figure 3

Photosynthetic rates at different intracellular [CO₂] (A_n /C_i curve) of leaves from WT (■) and npp1 (□) plants. The results of Figure 2 were used to draw an A_n/C_i curve. Open and filled arrows represent the data obtained at C_a of 40 and 160 Pa, respectively.

Figure 4

Sucrose accumulation in leaves of WT and npp1 mutant plants. WT (filled bars) and npp1 (open bars) plants grown for 60 days under normal conditions (28/23 °C, 40 Pa CO₂) were further incubated under different temperatures and CO₂ concentrations. At the end of a light cycle, the leaves of the WT and the npp1 mutant plants were subjected to sucrose assays. Values show the means ± s.d. (n = 3~5). Asterisks indicate significant differences by Student’s t-test (*, p > 0.05; **, p > 0.01).

Figure 5

Changes in the expression of carbohydrate- and protein synthesis-related proteins in leaves of WT and npp1 mutant plants under high temperature and elevated CO₂ concentrations. The leaves of WT (B,D) and npp1 (A,C) plants were incubated under normal (28/23 °C, 40 Pa CO₂) and HT + ECO₂ (33/28 °C, 160 Pa CO₂) conditions, and then subjected to a proteomic analysis with Isobaric tags for relative and absolute quantitation (iTRAQ) labeling. Values show the means ± s.d. (n = 3). The red line shows the ratio between HT+ECO₂/normal condition mean equal to 1.

Figure 5

Changes in the expression of carbohydrate- and protein synthesis-related proteins in leaves of WT and npp1 mutant plants under high temperature and elevated CO₂ concentrations. The leaves of WT (B,D) and npp1 (A,C) plants were incubated under normal (28/23 °C, 40 Pa CO₂) and HT + ECO₂ (33/28 °C, 160 Pa CO₂) conditions, and then subjected to a proteomic analysis with Isobaric tags for relative and absolute quantitation (iTRAQ) labeling. Values show the means ± s.d. (n = 3). The red line shows the ratio between HT+ECO₂/normal condition mean equal to 1.

Figure 6

Changes in expression of 14-3-3 proteins in leaves of WT and npp1 mutant plants under high temperature and elevated CO₂ concentrations. Details of incubation conditions via a proteomic analysis were described in Figure 5. The leaves of WT (filled bars) and npp1 (open bars) plants were incubated under normal and HT + ECO₂ conditions. Values show the means ± s.d. (n = 3). Asterisks indicate significant differences by Student’s t-test (**, p > 0.01).