Defoliation-Induced Stress in Nodules of White Clover

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Abstract
Nodule function and protein synthesis were studied in defoliation stressed white clover plants. Uncut control plants (C) were compared with plants from two defoliation treatments: (1) continuous defoliation (CD) where all leaves and petioles were removed each day; and (2) defoliated/recovered (DR) where, after removal of all leaves and petioles, new leaves were then allowed to regrow. After a single defoliation N2 fixation (acetylene reduction activity) and nitrogenase-linked respiration declined by more than 80% within 3 h and by nearly 100% by 24 h. DR plants began to fix nitrogen again at a very low level 3 d later and thereafter rose to control levels by 15 d. Continuously defoliated plants never recovered N2 fixation capacity. Nodule protein complement was assessed by polyacrylamide gel electrophoresis. Major changes occurred in buffer soluble protein band patterns by 6 d in CD plants, but few changes were evident in SDS soluble proteins. By 9 and 14 d significant disruption of all proteins was evident. The prominent host plant protein, leghaemoglobin (Lb) had disappeared by 14 d. In DR plants the intensity of staining was reduced but no major changes in band patterns were evident and by 21 d nodules were rejuvenated. [35S]-labelled methionine was incorporated into nodule proteins from all treatments throughout the experiment. However, continuous defoliation caused increasing variability between replicates in the labelled band patterns. By 21 d CD, much of the labelled protein was present as amorphous low Mr material which suggests either disruption of the protein synthesizing machinery or rapid hydrolysis by proteolytic enzymes. Surprisingly [35S]-methionine was never found in Lb from nodules of any treatment. It is possible that white clover Lb does not contain any methionine residues or that no synthesis of Lb occurred.