Expression of a soybean gene encoding the tetrapyrrole-synthesis enzyme glutamyl-tRNA reductase in symbiotic root nodules.

Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor delta-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3' portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.

gsa1 is a universal tetrapyrrole synthesis gene in soybean and is regulated by a GAGA element.

Expression of plant tetrapyrroles is high in photosynthetic tissues and in legume root nodules in the form of chlorophyll and heme, respectively. The universal tetrapyrrole precursor delta-aminolevulinic acid (ALA) is synthesized from glutamate 1-semialdehyde (GSA) by GSA aminotransferase in plants, which is encoded by gsa. Immunoblot analysis showed that GSA aminotransferase was expressed in soybean leaves and nodules, but not in roots, and that protein correlated with enzyme activity. These observations indicate that GSA aminotransferase expression is controlled in tetrapyrrole formation and argue against significant activity of an enzyme other than the well described aminotransferase for GSA-dependent ALA formation. gas mRNA and protein were induced in soybean nodules, and their activation was temporally intermediate between those of the respective early and late genes endo2 and lb. A GSA aminotransferase gene, designated gsa1, was isolated and appears to be one of two gsa genes in the soybean genome. gsa1 mRNA accumulated to high levels in leaves and nodules, but not in uninfected roots as discerned with a gsa1-specific probe. Message levels were higher in leaves from etiolated plantlets than in mature plants, and expression in the former was slightly elevated by light. The expression pattern of gsa1 mRNA was qualitatively similar to that of total gsa. The data strongly suggest that gsa1 is a universal tetrapyrrole synthesis gene and that a gsa gene specific for a tissue, tetrapyrrole, or light condition is unlikely. The gsa1 promoter contained a genetic element found in numerous Drosophila melanogaster genes; the so-called GAGA element displayed single-stranded character in vitro and formed a complex with nuclear factors from nodules and leaves but not from roots. From these observations we infer that the GAGA element is involved in the transcriptional control of gsa1.

Plant delta-aminolevulinic acid dehydratase. Expression in soybean root nodules and evidence for a bacterial lineage of the Alad gene.

We isolated a soybean (Glycine max) cDNA encoding the heme and chlorophyll synthesis enzyme delta-aminolevulinic acid (ALA) dehydratase by functional complementation of an Escherichia coli hemB mutant, and we designated the gene Alad. ALA dehydratase was strongly expressed in nodules but not in uninfected roots, although Alad mRNA was only 2- to 3-fold greater in the symbiotic tissue. Light was not essential for expression of Alad in leaves of dark-grown etiolated plantlets as discerned by mRNA, protein, and enzyme activity levels; hence, its expression in subterranean nodules was not unique in that regard. The data show that soybean can metabolize the ALA it synthesizes in nodules, which argues in favor of tetrapyrrole formation by the plant host in that organ. Molecular phylogenetic analysis of ALA dehydratases from 11 organisms indicated that plant and bacterial enzymes have a common lineage not shared by animals and yeast. We suggest that plant ALA dehydratase is descended from the bacterial endosymbiont ancestor of chloroplasts and that the Alad gene was transferred to the nucleus during plant evolution.

Expression of the soybean (Glycine max) glutamate 1-semialdehyde aminotransferase gene in symbiotic root nodules.

Extracts of soybean (Glycine max) root nodules and greening etiolated leaves catalyzed radiolabeled delta-aminolevulinic acid (ALA) formation from 3,4-[3H]glutamate but not from 1-[14C]glutamate. Nevertheless, those tissue extracts expressed the activity of glutamate 1-semialdehyde (GSA) aminotransferase, the C5 pathway enzyme that catalyzes ALA synthesis from GSA for tetrapyrrole formation. A soybean nodule cDNA clone that conferred ALA prototrophy, GSA aminotransferase activity, and glutamate-dependent ALA formation activity on an Escherichia coli GSA aminotransferase mutant was isolated. The deduced product of the nodule cDNA shared 79% identity with the GSA aminotransferase expressed in barley leaves, providing, along with the complementation data, strong evidence that the cDNA encodes GSA aminotransferase. GSA aminotransferase mRNA and enzyme activity were expressed in nodules but not in uninfected roots, indicating that the Gsa gene is induced in the symbiotic tissue. The Gsa gene was strongly expressed in leaves of etiolated plantlets independently of light treatment and, to a much lesser extent, in leaves of mature plants. We conclude that GSA aminotransferase, and possibly the C5 pathway, is expressed in a nonphotosynthetic plant organ for nodule heme synthesis and that Gsa is a regulated gene in soybean.

Characterization of delta-Aminolevulinic Acid Formation in Soybean Root Nodules.

Formation of the heme precursor delta-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 60 degrees C. A low level of the plant activity was detected in uninfected roots and was 50-fold greater in nodules from 17-day-old 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 lack of symbiotic induction of B. japonicum ALA synthase indicates either that ALA formation is not rate-limiting, or that ALA synthase is not the only source of ALA for bacterial heme synthesis in nodules. Plant cytosol from nodules catalyzed the formation of radiolabeled ALA from U-[(14)C]glutamate and 3,4-[(3)H]glutamate but not from 1-[(14)C]glutamate, and thus, operation of the C(5) pathway could not be confirmed.

Evidence for an inter-organismic heme biosynthetic pathway in symbiotic soybean root nodules.

The successful symbiosis of soybean with Bradyrhizobium japonicum depends on their complex interactions, culminating in the development and maintenance of root nodules. A B. japonicum mutant defective in heme synthesis in culture was able to produce heme as a result of its symbiotic association with the soybean host. The bacterial mutant was incapable of synthesizing the committed heme precursor delta-aminolevulinic acid (ALA), but nodule plant cells formed ALA from glutamate. In addition, exogenous ALA was taken up by isolated nodule bacteria of the parent strain and of the mutant. It is proposed that bacterial heme found in nodules can be synthesized from plant ALA, hence segments of a single metabolic pathway are spatially separated into two organisms.

Aerobic growth and respiration of a delta-aminolevulinic acid synthase (hemA) mutant of Bradyrhizobium japonicum.

Oxygen-dependent growth of the Bradyrhizobium japonicum hemA mutant MLG1 (M.L. Guerinot and B.K. Chelm, Proc. Natl. Acad. Sci. USA 83:1837-1841, 1986) was demonstrated in cultured cells in the absence of exogenous delta-aminolevulinic acid (ALA), but growth of analogous mutants of Rhizobium meliloti or of Escherichia coli was not observed unless ALA was added to the yeast extract-containing media. No heme could be detected in extracts of strain MLG1 cells as measured by the absorption or by the peroxidase activity of the heme moiety, but the rates of growth and endogenous respiration of the mutant were essentially identical to those found in the parent strain. A role for ALA in the viability of strain MLG1 could not be ruled out since the ALA analog levulinic acid inhibited growth, but neither ALA synthase nor glutamate-dependent ALA synthesis activity was found in the mutant. The data show that the cytochromes normally discerned in wild-type B. japonicum cultured cells by absorption spectroscopy are not essential for aerobic growth or respiration.

Regulation of human immunodeficiency virus env expression by the rev gene product.

A single simian virus 40 late replacement vector which expresses both the rev and envelope (env) genes of human immunodeficiency virus was used to examine the mechanism underlying the dependence of env gene expression on the rev protein. When rev was deleted from the vector, no envelope protein expression could be detected in transfected cells, and the levels of cytoplasmic env mRNA were dramatically reduced. In contrast to this, the levels of env RNA in total cellular RNA preparations were similar with or without rev coexpression, and analysis of nuclear RNA showed that the levels of nuclear env RNA were increased in the absence of rev. These results suggest that rev functions to regulate nuclear export of env mRNA. It was possible to restore env expression from the vector lacking rev by supplying rev in trans, provided that a cis-acting sequence was also present. This sequence was mapped to a 854-base-pair region within the env open reading frame, and it was shown that the sequence could be moved but that it worked only in its original orientation.