Effect of Induced Nodule Senescence on Parameters Related to Dinitrogen Fixation, Bacteroid Size and Nucleic Acid Content

Abstract

Darkness and treatment with combined nitrogen (NH,Cl or KN03) were used to induce nodule senescence in alfalfa and soybeans. Nodule senescence was assessed by determinations of the acetylene-reducing activity and leghaemoglobin and sugar contents of the nodules. Bacteroids from nodules of the treated plants were compared using flow microfluorimetry. Upon induced nodule senescence, alfalfa bacteroids decreased both in nucleic acid content and cell size while the soybean bacteroids were essentially unaffected. INTRODUCTIONDinitrogen fixation by Rhizobium-legume associations involves a complex physiological interplay between the symbiotic rhizobia (bacteroids) and the host plants. The reduction of dinitrogen occurs in the bacteroids while the required energy and reductants are supplied by the plant through photosynthesis. Ching et al. (1975) Rigaud, 1976;Rigaud & Puppo, 1977).Previous research from this laboratory (Paau et al., 1978) has shown that the basal portion of the 6 week or older alfalfa nodule has very low acetylene reduction capability in comparison with the middle nodule region and that bacteroids in this basal region have considerably less nucleic acid than bacteroids of similar size in the middle nodule regions. The bacteroids in the basal region apparently have begun senescence. While the effects of limited light and combined nitrogen have been shown to reduce parameters related to dinitrogen fixation in the nodules, their specific effect on bacteroid senescence has not been reported. In this communication, we demonstrate the effect of darkness and treatment with combined nitrogen on the nucleic acid content of bacteroids from alfalfa and soybean nodules. Changes in the acetylene-reducing activity of the nodules and in the leghaemoglobin and sugar contents of the nodule cytosol are also reported. METHODSThe maintenance and growth conditions of free-living Rhizobium meliloti F28 and Rhizobium japonicum 705 were as previously described (Cowles et ul., 1969). Alfalfa (Medicagu sativu Buffalo) and soybean (Glycine max Chippewa) plants were inoculated 1 week after germination with cultures of R. meliloti F28 and R. japonicum 705, respectively. The soybean and alfalfa nodules were harvested 8 and 6 weeks after inoculation, respectively. The times at which treatments were initiated were chosen so that the control and treated plants were harvested on the same day. For dark treatments, the plants were covered with black plastic bags (NAPCO, 1 x 0.9 m) on the appropriate day before harvest. The control plants were covered with transparent plastic bags. The black bags transmitted 4,6 and 7.5 % of incident light at 450,550 and 680 nm, respectively; the transparent bags transmitted 62, 60 and 727; of incident light at these wavelengths. For combined nitrogen treatments, 500 ml of 100 mM-NH,Cl or KN03 was applied to the plants on the appropriate initial day and continued every other day until harvest.The harvested nodules were first assayed for acetylene-reducing activity by gas chromatography (Hardy et al., 1968)...

Reversible-Dark Induced Senescence of Soybean Root Nodules

“…Dark treatment of soybeans for 8 to 11 d resulted in a 50% decline in total Lb3 which indicated substantial changes occurring in host cell function. In contrast, no differences were detected in bacteroid nucleic acid content or size (17), which suggests that during dark-induced senescence of soybean nodules bacteroid integrity was maintained to a greater extent than host cell function.…”

“…Dark treatment of soybean plants caused an immediate decline in the level of nitrogenase activity, although at least 4 to 6 d of continuous darkness were required to completely inhibit N2 fixation (17,28). Declining glucose concentrations were closely correlated to the complete inhibition of nitrogenase activity (17), and although poly-/8-hydroxybutyrate did not appear to be used as an alternate energy source for N2 fixation, it might serve as a source of four carbon intermediates for bacteroid respiration (28). Dark treatment of soybeans for 8 to 11 d resulted in a 50% decline in total Lb3 which indicated substantial changes occurring in host cell function.…”

“…Foliar dark treatment has been used to induce metabolic changes in legume nodules which are characteristic of nodule senescence (3,15,17,28). Dark treatment of soybean plants caused an immediate decline in the level of nitrogenase activity, although at least 4 to 6 d of continuous darkness were required to completely inhibit N2 fixation (17,28).…”

Reversible Dark-Induced Senescence of Soybean Root Nodules

“…Foliar dark treatment has been used to induce metabolic changes in legume nodules which are characteristic of nodule senescence (3,15,17, 28). Dark treatment of soybean plants caused an immediate decline in the level of nitrogenase activity, although at least 4 to 6 d of continuous darkness were required to completely inhibit N2 fixation (17, 28).…”

“…Senescence of alfalfa nodules can be accelerated as a result of defoliation or prolonged darkness (4,26,27), and the capacity of a single nodule to fix nitrogen can be regained by virtue of reinfection of the meristematic tissue (27 Initial experiments in this laboratory suggested that the metabolic changes observed in soybean nodules as a result of foliar dark treatment, including loss of nitrogenase activity, could be reversed. This study was conducted to investigate the changes in nodular metabolism which occurred as a result of dark-induced senescence, and to document the reversal of this process in soybean nodules.Foliar dark treatment has been used to induce metabolic changes in legume nodules which are characteristic of nodule senescence (3,15,17, 28). Dark treatment of soybean plants caused an immediate decline in the level of nitrogenase activity, although at least 4 to 6 d of continuous darkness were required to completely inhibit N2 fixation (17, 28).…”