Time-course of the phosphinothricin effect on gas exchange and nitrate reduction in Medicago sativa

The long-time effect of phosphinothricin (PPT) on gas exchange and nitrate metabolism in intact plants of lucerne ( Medicago sativa L. cv. Aragón) was investigated. Photosynthetic CO₂ uptake, stomatal conductance, and transpiration were measured with an Infra-Red Gas Analyzer (IRGA). Under photorespiratory conditions, CO₂ uptake continuously decreased after PPT treatment. The decrease of photosynthesis led to an increase in the internal CO₂ concentration, which in turn caused stomatal closure and a reduction of transpiration rate. Nitrate reduction from plants sprayed with PPT was assayed both in vitro and in vivo. In vivo nitrate reductase was measured with and without nitrate in the infiltration medium. Both types of nitrate reductase assays indicated that the enzyme was inhibited in plants treated with PPT; however, the enzyme appeared more affected when the in vivo assay was used than when the one in vitro was applied. The nitrate reduction was pronouncedly affected after 24 h of PPT treatment, when glutamine synthetase (GS, EC 6.3.1.2.) activity and gas exchange were inhibited by more than 60%. The data suggest that the inhibition of GS leads to inhibition of photosynthesis, which, in turn, means lack of NADPH and nitrate, the substrates for nitrate reductase. The inhibition of GS also leads to a high ammonia level, which will produce a secondary inhibition of nitrate reductase activity.