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 δ-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.Soybean (Glycine max) and numerous other legumes can establish a symbiosis with rhizobia, resulting in the formation of root nodules comprising specialized plant and bacterial cells (for review, see Mylona et al., 1995). Rhizobia reduce atmospheric nitrogen to ammonia within nodules, which is assimilated by the plant host to fulfill its nutritional nitrogen requirement. The high energy requirement for nitrogen fixation necessitates efficient respiration by the prokaryote within the microaerobic milieu of the nodule. The plant host synthesizes a nodule-specific hemoglobin (leghemoglobin) that serves to facilitate oxygen diffusion to the bacterial endosymbiont and to buffer the free oxygen concentration at a low tension (for review, see Appleby, 1992). Both of these functions require that the hemoglobin concentration be high, and, indeed, it exceeds 1 mm in soybean nodules (Appleby, 1984) and is the predominant plant protein in that organ. Once thought to be confined to legume nodules, hemoglobins are found throughout the plant kingdom, and leghemoglobin likely represents a specialization of a general plant phenomenon (for review, see Hardison, 1996). A gene encoding a nonsymbiotic hemoglobin has been identified in soybean and other legumes (Andersson et al., 1996); therefore, expression in nodules involves the specific activation of a subset of genes within a gene family. Leghemoglobin genes may have arisen from gene duplication, followed by specialization (Andersson et al., 1996).Hemes and chlorophyll are tetrapyrroles synthesized from common precursors; chlorophyll is quantitatively the major tetrapyrrole in plants, with heme and other tetrapyrroles being present in minor amounts. Legume root nodules represent an exception, in which heme is synthesized in high quantity in the absence of chlorophyll, thus requiring the activity of enzymes not normally expressed highly in nonphotosynthetic tissues. Heme is synthesized from the universal tetrapyrrole precursor ALA by seven successive enzymatic steps; chlorophyll formation diverges after the synthesis of protoporphyrin, the immediate heme precursor (for review, see O''Brian, 1996). Biochemical and genetic evidence shows that soybean heme biosynthesis genes are strongly induced in root nodules (Sangwan and O''Brian, 1991, 1992, 1993; Madsen et al., 1993; Kaczor et al., 1994; Frustaci et al., 1995; Santana et al., 1998), and immunohistochemical studies demonstrate that induction is concentrated in infected nodule cells (Santana et al., 1998).ALA is synthesized from Glu in plants by a three-step mechanism called the C₅ pathway (Fig. ​(Fig.1);1); the latter two steps are committed to ALA synthesis and are catalyzed by GTR and GSAT, respectively (for review, see Beale and Weinstein, 1990; Jahn et al., 1991). Plant cDNA or genes encoding GTR (Gtr, also called HemA) and GSAT (Gsa) have been identified in several plant species (Grimm, 1990; Sangwan and O''Brian, 1993; Hofgen et al., 1994; Ilag et al., 1994; Frustaci et al., 1995; Wenzlau and Berry-Lowe, 1995; Bougri and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). Two genes for each enzyme have been described, and some genes are reported to be specific to a tissue, tetrapyrrole, or light regimen (Bougri and Grimm, 1996; Kumar et al., 1996; Tanaka et al., 1996). However, soybean Gsa1 is highly expressed in both leaves and nodules and contains a cis-acting element in its promoter that binds to a nuclear factor found in both tissues. (Frustaci et al., 1995). In this study we isolated soybean Gtr1 and characterized the genetic basis of GTR expression in root nodules. Figure 1C₅ pathway for ALA synthesis. The committed steps for ALA synthesis catalyzed by GTR and GSAT are boxed. Glutamyl-tRNA synthetase (GluRS) and glutamyl-tRNA^Glu also participate in protein synthesis. The gene designations in plants are shown in parentheses ...