Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis.

Abstract

N2-fixing root nodules of soybean (Glycine max L. Merr.) convert atmospheric N2 to ammonia(um) in an energy-intensive enzymatic reaction. These nodules synthesize large quantities of purines because nitrogen fixed by bacteria contained within this tissue is transferred to the shoots in the form of ureides, which are degradation products of purines. In animal systems, it has been proposed that proline biosynthesis by pyrroline-5-carboxylate reductase (P5CR) is used to generate the NADP+ required for the synthesis of the purine precursor ribose 5-phosphate. We have examined the levels, properties, and location of P5CR and proline dehydrogenase (ProDH) in soybean nodules. Nodule P5CR was found in the plant cytosol. Its activity was substantially higher than that reported for other animal and plant tissues and is 4-fold higher than in pea (Pisum sativum) nodules (which export amides).The Km for NADPH was lower by a factor of 25 than the Km for NADH, while the Vm. with NADPH was one-third of that with NADH. P5CR activity was diminished by NADP+ but not by proline. These characteristics are consistent with a role for P5CR in supporting nodule purine biosynthesis rather than in producing proline for incorporation into protein. ProDH activity was divided between the bacteroids and plant cytosol, but <2% was in the mitochondria-rich fractions. The specific activity of ProDH in soybean nodule bacteroids was comparable to that in rat liver mitochondria. In addition, we propose that some of the proline synthesized in the plant cytosol by P5CR is catabolized within the bacteroids by ProDH and that this represents a novel mechanism for transferring energy from the plant to its endosymbiont. N2 fixation in legumes is a symbiotic process in which bacteria of the genus Bradyrhizobium or Rhizobium infect root cells and form specialized organs (nodules) within which N2 is reduced to NH'. Fixation of N2 is an energyintensive process, requiring a total of 25-30 ATP per N atom fixed. Of this total, 12-14 ATP per N are required within the bacteroid to reduce N2. As much as 10-30% of the total photosynthetic capacity of the plant is used to support this process (1). The energy-yielding metabolite(s) supplied by the host to the bacteroid, the symbiotic form of the bacterium, is not known. However, one attractive suggestion is that bacteroids import and oxidize a nitrogenous compound, such as glutamate (2). The nitrogen fixed in the bacteroid is exported as ammonia(um) to the infected host cell, where it is packaged for export to the rest of the plant. Legumes of temperate origin (e.g., peas) export nitrogen as amides (principally asparagine), whereas legumes of tropical origin (e.g., soybeans) export the ureides, allantoin, and allantoic acid. Ureide biogenesis proceeds by way of synthesis of purine ribonucleotides (3). This pathway is the same as that found in microorganisms, fungi, and animals. The purines so formed are then oxidatively degraded to ureides (3). The estimated peak rate of de novo purine biosynthesis necessary t...

Removal of Feedback Inhibition of Δ1-Pyrroline-5-Carboxylate Synthetase Results in Increased Proline Accumulation and Protection of Plants from Osmotic Stress

“…Recent studies on Pro synthesis and catabolism genes have provided results that are consistent with diverse functions of Pro as a source of energy, nitrogen and carbon, and as an osmolyte in response to dehydration (Kohl et al, 1988;Kavi Kishor et al, 1995;Peng et al, 1996;Hua et al, 1997;Zhang et al, 1997). Synthesis, accumulation, and catabolism of Pro in plants are highly regulated processes.…”

Molecular Cloning and Evidence for Osmoregulation of the Δ1-Pyrroline-5-Carboxylate Reductase (proC) Gene in Pea (Pisum sativum L.)

“…The Nterminal sequence for the pea P5CR polypeptide does not exhibit the general characteristics of presequences associated with proteins imported into mitochondria or chloroplasts, such as an abundance of hydrophobic residues and a net positive charge (36). Soybean nodule (18) and yeast (5) P5CRs have also been reported to be cytosolic proteins. However, chloroplast-localized P5CR activities have been reported for both greened and etiolated pea tissues (26).…”

“…There are reports of kinetically distinguishable P5CR isozymes (18) and both cytosolic (18) and chloroplastic (26) forms of P5CR in plants.…”

Factors Affecting the Acetylene-Induced Decline during Nitrogenase Assays in Root Nodules of Myrica gale L.

“…It is likely that the introduction of C2H2 would cause a major decrease in the glutamate and aspartate necessary for this shuttle. There are also other amino acids that may be involved in the transfer of carbon and reductant from the host cytoplasm to the nitrogenfixing microsymbiont (5,6,11,16 ase activity during the decline by making more reductant available.…”

“…10), whereas at 20% 02 there is a lag of only 2 to 3 min (Figs. lA, 5 [25°C], 6 [20% 02], 6, and 12 [initial]). …”