Characterization of δ-Aminolevulinic Acid Formation in Soybean Root Nodules

Abstract

Formation of the heme precursor &-aminolevulinic acid (ALA) was studied in soybean root nodules elicited by Bradyrhizobium japonicum. Glutamate-dependent ALA formation activity by soybean (Glycine max) in nodules was maximal at pH 6.5 to 7.0 and at 55 to 600C. A low level of the plant activity was detected in uninfected roots and was 50-fold greater in nodules from 17-dayold plants; this apparent stimulation correlated with increases in both plant and bacterial hemes in nodules compared with the respective asymbiotic cells. The glutamate-dependent ALA formation activity was greatest in nodules from 17-day-old plants and decreased by about one-half in those from 38-day-old plants. Unlike the eukaryotic ALA formation activity, B. japonicum ALA synthase activity was not significantly different in nodules than in cultured cells, and the symbiotic activity was independent of nodule age. The nitrogen-fixing bacterium Bradyrhizobium japonicum elicits root nodule formation on soybean, and the nodule is composed of differentiated plant and bacterial cells. Bacterial and plant hemes increase symbiotically for the synthesis of cytochromes and legume hemoglobin (leghemoglobin), respectively, as these heme proteins are necessary to meet the high energy demand of nitrogen fixation. Some evidence suggests that the heme prosthetic group of plant nodule hemoglobin is a bacterial product, but not all data support that conclusion (reviewed in refs.

Evidence that the Plant Host Synthesizes the Heme Moiety of Leghemoglobin in Root Nodules1

“…4). The time courses of induction were virtually identical for each enzyme and paralleled the increase of both heme and holo-leghemoglobin in the nodule (Nadler and Avissar, 1977;Sangwan and O'Brian, 1992). A similar observation was made for GSA aminotransferase, one of the enzymes of the C5 pathway (Sangwan and O'Brian, 1993), which was undetectable in uninfected roots, and increased to levels greater than in leaves 20 d after infection.…”

“…In particular, it is now known that plant cells synthesize ALA by a completely different route, namely from glutamate via the so-called C5 pathway involving three enzymes (for review, see Kannangara et al, 1988). Glutamate-dependent ALA synthesis has been shown to be present at much higher levels in soybean nodules than in uninfected roots (Sangwan and O'Brian, 1992), whereas bacterial ALA synthase activity was essentially the same in nodules and in free-living bacteria. Furthermore, the plant gene for one of the C5 pathway enzymes, GSA aminotransferase, is induced during nodulation, concomitantly with the increase in enzyme activity (Sangwan and O'Brian, 1993;Frustaci et al, 1995).…”

“…This is also the time at which the rate of heme synthesis is maximal (Nadler and Avissar, 1977;Sangwan and O'Brian, 1992). However, the amount of transcript is not necessarily a direct indication of the amount of enzyme; therefore, we investigated this with western-blot analysis and a determination of enzyme activity over the same time course of nodulation.…”

Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene

“…Cellular hemes of both symbionts are greater in nodules than they are in the respective asymbiotic cells (1,28), and these hemes are necessary to support the large energy requirement of nitrogen fixation. Bacterial cytochromes participate in ATP synthesis which drives nitrogen fixation, and soybean hemoglobin (leghemoglobin) facilitates the diffusion of oxygen to the respiring bacteroids.…”

Transcriptional regulation of delta-aminolevulinic acid dehydratase synthesis by oxygen in Bradyrhizobium japonicum and evidence for developmental control of the hemB gene

“…Accordingly, soybean genes encoding heme synthesis enzymes are strongly induced and maintained at elevated levels within nodules (2,16,21,23,33), and these observations run counter to the long-held view that the host lacks the synthetic capacity for heme formation in the symbiotic organ (reviewed in reference 26). The universal first committed heme precursor ␦-aminolevulinic acid (ALA) is synthesized from glutamate by soybean in nodules, and biochemical and genetic evidence indicate that the so-called C 5 pathway (7) catalyzes that synthesis (16,32,33). Induction of soybean ALA synthesis in nodules is due, at least in part, to activation of gsa1, a gene normally expressed strongly only in photosynthetic tissue in nonsymbiotic plants (16).…”