PROTEIN OXIDATION IN RESPONSE TO NITROGEN STRESS IN WHEAT SEEDLINGS
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Date
2011
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IARI, DIVISION OF PLANT PHYSIOLOGY
Abstract
Nitrogen is one of the most important nutrients required by plants and its deficiency is also widespread. Sixty percent of the soils in India are deficient in nitrogen (N). This deficiency of N seriously affects plant growth, yield and quality. Lot of work in diverse areas has been done but many questions regarding N nutrition remain unanswered. The nitrogen in wheat is taken up mainly in the form of nitrate, and occasionally in the form of ammonium. But, wheat plants although capable of taking up ammonium are sensitive to ammonium nutrition. In terms of quality the protein content goes down. When plant faces nitrogen stress, it is possible that due to stress in spite of low protein content of tissues, they might be oxidised and carbonylated leading to increased degradation in such plants facing nutrient stress. In the present study the, the effect of different levels and forms of N has been investigated in wheat seedlings grown under controlled conditions. The interaction between cytokinins gene expression and N was also investigated. The extent of protein carbonylation was analyzed and an attempt was made to elucidate the pathway of protein degradation. Wheat seedlings (var. PBW 343) were grown under four different N treatments and various parameters of growth, N-assimilation and stress was studied. Result indicated that growth and nitrogen metabolism was enhanced in seedlings under high NO3- -N when compared to those grown with low and without N. The growth of the wheat seedlings was severely inhibited in terms of biomass accumulation, leaf area and root: shoot ratio was enhanced in zero N and ammonium grown plants as compared to the nitrate N fed wheat seedlings. There was almost 5 fold increase in shoot dry weight of NO3- -N fed seedlings when compared to those grown without N. In seedling grown with 5mM NH4+-N shoot dry weight declined by 63% as compared to the wheat seedlings grown in 5 mM NO3- -N. There was a relative increase in shoot length by 20% to 40% in the seedlings grown in solution having N salts as compared to those grown without N. In zero-N and ammonium fed plants the nitrate levels, total reduced N and total soluble protein content was significantly lower as compared to both low and high NO3- -N grown wheat seedlings. These parameters directly correlated with low NR activity in zero-N and NH4+-N treatments. There was increased production of ROS in terms of increase in superoxide radicals and H2O2 in NH4+-N fed wheat seedlings followed by those grown without N. Increase in protease activity, total free amino acids and TBARS was also observed in these treatments. Under normal conditions, plants contain numerous carbonylated proteins, which are thought to be indicative of oxidative stress damage. Conditions that promote formation of reactive oxygen species (ROS) enhance protein
carbonylation, and protein degradation is required to reverse the damage. However, it is not clear how the degradation of carbonylated proteins is controlled in planta. In this report, we show that detached wheat leaves rapidly and selectively degrade carbonylated proteins when kept in the dark as compared to the detached leaves incubated in light or in the intact seedlings. The loss of carbonylated proteins corresponded to a loss of soluble protein and accumulation of free amino acids. Degradation of carbonylated proteins was blocked by carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132) in dark detached leaves, but not by 3-methyladenine, suggesting that the 26S proteasome pathway rather than the autophagic pathway was involved in degrading protein carbonyls in dark detached leaves. In light incubation pathway the use of inhibitors revealed that both autophagic and proteasomal pathways were involved in degrading protein carbonyls. The study also indicated the role of cytokinins in preventing the the protein carbonyl degradation. The expression of IPT3 gene of cytokinin biosynthetic pathway was almost absent in the seedlings grown at zero N and NH4+-N, and in these treatments the degradation of protein carbonyls was high as supported by low level of total soluble proteins and high levels of free amino acids. Exogenous
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T-8481