NADH-glutamate synthase in alfalfa root nodules. Genetic regulation and cellular expression

Abstract

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric beta-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat.

Figure 1

Diagrammatic representation of the portion of the alfalfa NADH-GOGAT gene used for PCR. Exons are indicated by boxed regions, and introns are represented by lines. PCR was performed over three exons (exon 14 [E14], exon 15 [E15], exon 16 [E16]) and two introns (intron 14 [I14] and intron 15 [I15]) of the reported alfalfa gene using primer 1 (AIKQVA) and primer 2 (CEYMTGG). The putative binding for the (Fe-S) cluster is indicated by arrows, and the putative flavin mononucleotide binding site is underlined.

Figure 2

Localization of NADH-GOGAT mRNA in alfalfa root nodules by in situ hybridization. Bright-field (A, C, E, G, I, and K) and dark-field (B, D, F, H, J, and L) photographs of a longitudinal section through 19-d-old effective alfalfa nodules. Nodule ultrastructure was classified based on the nomenclature of Vasse et al. (1990): meristem (zone I), invasion zone (zone II), interzone (*), N₂-fixing zone (zone III), and senescent zone (zone IV). The nodule in A to H was hybridized with NADH-GOGAT ³⁵S-labeled antisense RNA probe. Enlargement of the lower boxed region in A shown in bright-field (C) and dark-field (D) photographs includes a portion of the meristem (zone I), invasion zone (zone II), and interzone (*). Enlargement of the upper box in A shown in bright-field (E) and dark-field (F) photographs includes the central zone (III), including parenchymal tissue (PA). In bright-field (G) and dark-field (H) photographs of infected and uninfected cells, arrows in C through H point to uninfected cells. Bright-field (I and K) and dark-field (J and L) photographs of a longitudinal section through a 19-d-old effective alfalfa nodule hybridized with NADH-GOGAT ³⁵S-labeled sense RNA probe. Bars in B and J = 800 μm; bars in D, F, H, and L = 80 μm.

Figure 3

Localization of NADH-GOGAT transcript in effective and plant-gene-controlled ineffective alfalfa nodules during development. Longitudinal sections through effective (A–H) and plant-controlled ineffective (I–T) alfalfa root nodules. The bright-field and dark-field image pairs are of 7- (A, B, I, and J), 9- (C, D, K, L, M, and N), 19- (E, F, O, P, Q, and R), and 33- (G, H, S, and T) d-old root nodules. Bars = 400 μm.

Figure 4

In situ localization of the NADH-GOGAT, nifH, and leghemoglobin transcripts in 33-d-old alfalfa root nodules using serial sections. The bright-field and dark-field image pairs are of longitudinal sections through 33-d-old root nodules that were hybridized with ³⁵S-labeled NADH-GOGAT (A and B), leghemoglobin (C and D), or nifH (E and F) antisense RNA probe. Bars = 400 μm.

Figure 5

In situ localization of NADH-GOGAT transcripts in nodules induced by S. meliloti F642 and 7154. The bright-field (A and D) and dark-field (B, C, E, and F) images are of longitudinal sections through 19-d-old root nodules induced by S. meliloti F642 (A–C) and S. meliloti 7154 (D–F). The sections in A, B, D, and E were hybridized with ³⁵S-labeled NADH-GOGAT antisense RNA probe, whereas those in C and F were hybridized with sense riboprobes and photographed under dark-field conditions. Bars = 400 μm.

Figure 6

Diagram of the NADH-GOGAT promoter-GUS gene constructs. A, Eight sequences containing the putative NADH-GOGAT promoter were translational fusions to the GUS gene; the length of these truncations is measured with respect to the transcriptional start. The boxed region in construct G1 marks the location of the 88-bp direct repeat. The number of individual transformants tested for each construct is indicated (#). Mean GUS activity (pmol min⁻¹ mg⁻¹ protein) is indicated with the heading “Mean.” lsd values between adjacent deletions were calculated, and the level of significance was set at P < 0.01. N.S., Not significant; *, significant at P < 0.01. B, Distribution of GUS activity per construct measured from individual independently transformed plants.

Figure 7

GUS staining of the NADH-GOGAT promoter deletions. For deletions GLate (A) and G1 (B), individual transformants with GUS activity close to the mean had a GUS staining pattern similar to the results obtained for in situ hybridizations. A loss in tissue specificity was observed in deletion G2 (C), whereas no GUS staining was observed in deletion G7 (D). E, The 409-bp region between deletion G1 and G2. The 88-bp direct repeat is overlined, and the location of the conserved octanucleotide motif is underlined.