Glycolytic Flux Is Adjusted to Nitrogenase Activity in Nodules of Detopped and Argon-Treated Alfalfa Plants1

Abstract

To investigate the short-term (30-240 min) interactions among nitrogenase activity, NH 4 ؉ assimilation, and plant glycolysis, we measured the concentrations of selected C and N metabolites in alfalfa (Medicago sativa L.) root nodules after detopping and during continuous exposure of the nodulated roots to Ar:O 2 (80:20, v/v). Both treatments caused an increase in the ratios of glucose-6-phosphate to fructose-1,6-bisphosphate, fructose-6-phosphate to fructose-1,6-bisphosphate, phosphoenolpyruvate (PEP) to pyruvate, and PEP to malate. This suggested that glycolytic flux was inhibited at the steps catalyzed by phosphofructokinase, pyruvate kinase, and PEP carboxylase. In the Ar:O 2 -treated plants the apparent inhibition of glycolytic flux was reversible, whereas in the detopped plants it was not. In both groups of plants the apparent inhibition of glycolytic flux was delayed relative to the decline in nitrogenase activity. The decline in nitrogenase activity was followed by a dramatic increase in the nodular glutamate to glutamine ratio. In the detopped plants this was coincident with the apparent inhibition of glycolytic flux, whereas in the Ar:O 2 -treated plants it preceded the apparent inhibition of glycolytic flux. We propose that the increase in the nodular glutamate to glutamine ratio, which occurs as a result of the decline in nitrogenase activity, may act as a signal to decrease plant glycolytic flux in legume root nodules.N fixation, the reduction of N 2 to NH 4 ϩ by (Brady)rhizobium bacteria, in legume root nodules is catalyzed by the bacterial enzyme nitrogenase. Nitrogenase is O 2 labile, but the root nodule provides an environment that protects it from excess O 2 . Nitrogenase activity in legume root nodules is rapidly inhibited by treatments that affect plant C and N metabolism, such as exposure of the roots to C 2 H 2 , replacement of N 2 in the rhizosphere with Ar, NO 3 Ϫ fertilization, defoliation, detopping, phloem girdling, and drought (Minchin et al

Increase in the concentrations of amino acids in the vascular tissue of white clover and white lupin after defoliation: an indication of a N feedback regulation of symbiotic N2 fixation

“…We found an increase in the concentration of free amino acids in the vascular tissue of the plant and suggest that it triggers a reduced oxygen permeability in the nodules, which leads to the down-regulation of nitrogenase activity [6,11,27]. This may lead to N limitation of the GS/GOGAT cycle and, thus, to an increase in the Glu/Gln ratio and to an appropriate adaptation of the glycolytic pathway [4]. The decrease in the concentration of sucrose in the phloem is considered not to be associated with the decreased nitrogenase activity [4,12,27,28].…”

“…This may lead to N limitation of the GS/GOGAT cycle and, thus, to an increase in the Glu/Gln ratio and to an appropriate adaptation of the glycolytic pathway [4]. The decrease in the concentration of sucrose in the phloem is considered not to be associated with the decreased nitrogenase activity [4,12,27,28].…”

“…This has been shown with white clover [11][12][13][14][15] lucerne [4,6], annual medic (Medicago truncatula L.) [5] and other legume species.…”

Adenylate Gradients and Ar:O2 Effects on Legume Nodules. II. Changes in the Subcellular Adenylate Pools

“…Adenylates and Regulation of Glycolytic Flux in Ar-Exposed Nodules Curioni et al (1999) found direct evidence linking N deprivation to glycolytic flux in alfalfa (Medicago sativa) nodules exposed to Ar:O 2 . The Glu-Gln ratio increased 5-fold during the first 30 min, while malate concentration declined and Suc accumulated.…”

“…The study also showed ratios of Fru-6-phosphate to Fru-1,6-bisphosphate and phosphoenolpyruvate to pyruvate to increase after 30 min Ar-exposure, consistent with a negative effect of N deprivation on the activity of phosphofructokinase (PFK) and pyruvate kinase. Although it was proposed that the increased Glu-Gln ratio, caused by NH 4 1 unavailability, may act as a signal to decrease plant glycolytic flux (Curioni et al, 1999), a role for adenylates, and specifically elevated ATP, can be envisaged. Since the turnover of ATP and ADP is coupled to several steps in both NH 4 1 assimilation and glycolysis, we propose that the observed increase in cytosolic ATP-ADP ratio in Ar (this study) will inhibit the activity of some key glycolytic enzymes, resulting in a reduced C-flux through the pathway.…”