Defoliation-induced stress in nodules of white clover. II. Immunological and enzymic measurements of key proteins

Changes in key nodule proteins following defoliation of white clover plants were assessed by measurements of enzyme activities and by use of antibodies to specific nodule proteins. Defoliation caused major declines in protein and leghaemoglobin levels and in the activities of invertase, sucrose synthase, UDP glucose pyrophosphorylase, aspartate amino transferase, glutamine synthetase, phosphoenolpyruvate carboxylase, and malate dehydrogenase. In continuously defoliated plants the activities of these nodule enzymes continued to decline throughout the course of the 17 d experiment. In plants defoliated once and then allowed to regrow new leaves, nodule enzyme activities declined for 7 to 10 d before increasing again to control levels by the end of the experiment. In this single defoliation/recovery treatment only the activities of PEP carboxylase and glutamine synthetase declined to a greater extent than the general decline in protein content. Alcohol dehydrogenase, increased in specific activity following a single defoliation and only declined in nodules of continuously defoliated plants. Amino peptidase activity declined in concert with other enzyme activities described above. Endopeptidase activity, in contrast, increased significantly after 4 d following either a single or continuous defoliation. In the plants allowed to regrow new leaves endopeptidase activity declined to control levels again, whereas in plants continuously defoliated the activity rose 5-fold. However, endopeptidase increased only after significant declines in protein and other enzyme activities had already occurred. Western immunoblotting confirmed that the declines in glutamine synthetase activity and leghaemoglobin levels were due to the disappearance of antigen. Declines in nitrogenase components I and II indicated that bacteroid proteins were affected by defoliation over the same time-scale as host plant encoded proteins. As new leaves grew and nodule N2 fixation was reestablished, these specific nodule proteins were again detectable by immunoblotting.