Proteomics analysis for identifying proteins responding to low nitrogen application in rice(oryza sativa L.)

Proteomics analysis for identifying proteins responding to low nitrogen application in rice (Oryza sativa L.)

Nitrogen (N) fertilization is essential for alleviating nutrient deficiencies of the world’s population by increasing rice production, one of the most important food crops of our time. Here we established an in vivo hydroponics rice seedling culture system to investigate the physio-biochemical and molecular responses of various rice genotypes to low nitrogen application. Yoshida’s nutrient solution (YS) was used to grow rice seedlings, and at three-week-old the seedlings manifested highly stable and reproducible symptoms, such as reduced shoot growth and length. Out of 12 genetically selected or tested genotypes, almost all (11 genotypes) showed varied degrees of growth reduction response to applied nitrogen (4 and 40 ppm N for treatment and control, respectively), but SR19663-B-B-34-3-3-3-1 showed similar growth as the control though its leaf width was smaller than the control. The leaves of a 11 representative low nitrogen-responsive genotype as BG90-2 were sampled for revealing the protein profiles between low and normal (control) nitrogen application by using two-dimensional gel electrophoresis (2-DGE) followed by staining of separated proteins with silver. Fifty differentially expressed silver stained protein spots were excised from 2-D gels and 41 proteins identified using high-throughput mass spectrometry (MS) using matrix-assisted laser desorption/ionization-time of flight-MS and nano electrospray ionization liquid chromatography tandem MS. These proteins could be assigned as major (energy metabolism, photosynthesis and oxidative stress) and minor functional categories, revealing many novel low N-responsive proteins, including those having energy/photosynthesis, and defense/stress, and iron homeostasis-related functions. Nitrogen (N) fertilization is essential for alleviating nutrient deficiencies of the world’s population by increasing rice production, one of the most important food crops of our time. Here we established an in vivo hydroponics rice seedling culture system to investigate the physio-biochemical and molecular responses of various rice genotypes to low nitrogen application. Yoshida’s nutrient solution (YS) was used to grow rice seedlings, and at three-week-old the seedlings manifested highly stable and reproducible symptoms, such as reduced shoot growth and length. Out of 12 genetically selected or tested genotypes, almost all (11 genotypes) showed varied degrees of growth reduction response to applied nitrogen (4 and 40 ppm N for treatment and control, respectively), but SR19663-B-B-34-3-3-3-1 showed similar growth as the control though its leaf width was smaller than the control. The leaves of a 11 representative low nitrogen-responsive genotype as BG90-2 were sampled for revealing the protein profiles between low and normal (control) nitrogen application by using two-dimensional gel electrophoresis (2-DGE) followed by staining of separated proteins with silver. Fifty differentially expressed silver stained protein spots were excised from 2-D gels and 41 proteins identified using high-throughput mass spectrometry (MS) using matrix-assisted laser desorption/ionization-time of flight-MS and nano electrospray ionization liquid chromatography tandem MS. These proteins could be assigned as major (energy metabolism, photosynthesis and oxidative stress) and minor functional categories, revealing many novel low N-responsive proteins, including those having energy/photosynthesis, and defense/stress, and iron homeostasis-related functions.