Protein Phosphatase (PP2C9) Induces Protein Expression Differentially to Mediate Nitrogen Utilization Efficiency in Rice under Nitrogen-Deficient Condition

Abstract

Nitrogen (N) is an essential element usually limiting in plant growth and a basic factor for increasing the input cost in agriculture. To ensure the food security and environmental sustainability it is urgently required to manage the N fertilizer. The identification or development of genotypes with high nitrogen utilization efficiency (NUE) which can grow efficiently and sustain yield in low N conditions is a possible solution. In this study, two isogenic rice genotypes i.e., wild-type rice kitaake and its transgenic line PP2C9TL overexpressed protein phosphatase gene (PP2C9) were used for comparative proteomics analysis at control and low level of N to identify specific proteins and encoding genes related to high NUE. 2D gel electrophoresis was used to perform the differential proteome analysis. In the leaf proteome, 30 protein spots were differentially expressed between the two isogenic lines under low N level which were involved in the process of energy, photosynthesis, N metabolism, signaling, and defense mechanisms. In addition, we have found that protein phosphatase enhances nitrate reductase activation by downregulation of SnRK1 and 14-3-3 proteins. Furthermore, we showed that PP2C9TL exhibits higher NUE than WT due to higher activity of nitrate reductase. This study provides new insights on the rice proteome which would be useful in the development of new strategies to increase NUE in cereal crops.

Keywords: 14-3-3; 2D; N utilization efficiency; SnRK1; protein phosphatase; proteomics; rice isogenic line.

Figure 1

(A) The leaf dry matter (g) of PP2C9TL and WT at tillering (T), 5, 10, 15, 20, 25, and 30 DAF. (B) The chlorophyll contents of PP2C9TL and WT at tillering (T), 5, 10, 15, 20, 25, and 30 DAF.

Figure 2

(A) The leaf N contents of PP2C9TL and WT at tillering T, 10 DAF, and 30 DAF; (B) the NUE of WT and PP2C9TL at low, control, and high levels of N.

Figure 3

Representative 2-DE gel electrophoresis images of leaf proteins from the WT and PP2C9TL at 10 DAF under control and low N conditions. The MW (kilodaltons) and pI of the proteins are shown on the left and at the top, respectively.

Figure 4

A representative 2-D gel electrophoresis image showing total differentially expressed protein spots in the leaf under low N condition.

Figure 5

Pie graph percentage distribution display of the identified proteins described in (Table 1) based on biological function along with sidewise bar graph depicting up-and down-regulating proteins.

Figure 6

Subcellular indexing of the identified proteins.

Figure 7

Potential molecular pathway based on differentially expressed proteins in PP2C9TL under N deficient conditions. The red color text shows the upregulation and green color text shows downregulation of proteins. Note RUBP (Ribulose 1, 5-bisphosphate), RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase), 1,3 BPG (1,3-Bisphosphoglycerate), FBA (Fructose-bisphosphate aldolase), F6P (Fructose-bisphosphate aldolase), SBP (sedoheptulose-1,7-bisphosphatase), PSII (Photosystem II), OOEs (oxygen evolving enhancer proteins), PSΙ (Photosystem Ι), GLS (Glutaminase), and GS (Glutamate synthase).

Figure 8

PP2C9 role for nitrate reductase activation by dephosphorylation of SnRK. 14-3-3 proteins SnRK (SNF1-related kinase) and CDPK (calcium-dependent protein kinases) can phosphorylate NR in crop plants. After phosphorylation of NR, it binds with the 14-3-3 protein to form a complex which is inactive or has low activity. PP2C9 interacts with 14-3-3 and SnRK and dephosphorylates them to activate NR.

Figure 9

(A)Western blotting of differentially expressed proteins in PP2C9TL and WT at 10 DAF stage. The top photo shows the SDS-PAGE separation of two protein samples used as loading control. The bottom photo shows gel transferred onto nitrocellulose membrane for western blotting to detect actin, anti-Flag-PP2C9, and 14-3-3 protein for WT and PP2C9TL at the 10 DAF stage. (B) Co-immunoprecipitation (Co-IP) assays of PP2C9 associated protein analyzed by SDS-PAGE. Protein molecular weight marker, negative control without antibodies, WT protein, and the third is the PP2C9TL protein. 2.8. Identification of PP2C9 Associated Protein by Co-Immunoprecipitation (CO-IP) and Mass Spectrometry.