The effect of excess manganese on nitrate assimilation in lettuce

Growth of lettuce may be limited in acid soils by manganese toxicity which may be alleviated by applications of lime, phosphate fertilisers, iron and molybdenum. However few investigations have been undertaken to determine the biochemical effect of excess manganese in plants. The aim of this project was to demonstrate that manganese toxicity could reduce lettuce yield by inhibiting nitrate reductase activity. Manganese toxicity inhibited nitrate reductase activity in lettuce plants grown in an acid krasnozem soil. The yield of affected plants, which was significantly lower than the yield of control plants, was significantly correlated with the decrease in nitrate reductase activity. Associated with the decrease in nitrate reductase activity was an accumulation of nitrate in leaves and a decrease in the total nitrogen content (mgN/plant). Following applications of molybdenum foliar sprays there were significant increases in yield and nitrate reductase activity. The molybdenum content of plants increased in plants which had high tissue levels of manganese. The increase was probably due to the lower yield in these plants. It is not known how molybdenum reverses the manganese inhibition of nitrate reductase. Similar responses were observed in lettuce grown in nutrient solution. The Critical solution concentration of manganese,at which there was a decrease in yield and in nitrate reductase activity, was found to be 36uM. Nitrate reductase activity of young and mature leaves was significantly correlated with leaf manganese levels. Plants grown in nitrate-nitrogen nutrient solutions were more susceptible to manganese toxicity than plants grown in ammonium nitrogen nutrient solutions. A study of the time-course of manganese inhibition of nitrate reductase in vivo (indicated that after a short lag period, manganese significantly decreased nitrate reductase activity. The half life for this decrease in enzyme activity was found to be 2.5 hours. These results indicated that manganese was not inducing molybdenum deficiency and that manganese may have inhibited nitrate reductase by binding to the enzyme. Enzyme kinetic analysis of the manganese inhibition of nitrate reductase (in vitro) showed that manganese was a competitive inhibitor of nitrate reductase with respect to NADH and a non-competitive inhibitor of nitrate reductase with respect to nitrate. These results indicated that manganese was bound to the enzyme at or near the binding site for NADH. When manganese was added to the enzyme before NADH the degree of inhibition was greater than when the NADH was added to the enzyme before manganese. These results also indicated that manganese was interfering with the binding of NADH to the enzyme. EPR studies supported the results obtained with the kinetic analysis of the inhibition of nitrate reductase. Results of the EPR study showed that manganese was not bound by NADH but could be bound by the partially purified enzyme extract.