Erik Østergaard Jensen

Interaction of a nodule specific, trans-acting factor with distinct DNA elements in the soybean leghaemoglobin Ibc(3) 5' upstream region.

Nuclear extracts from soybean nodules, leaves and roots were used to investigate protein–DNA interactions in the 5′ upstream (promoter) region of the soybean leghaemoglobin lbc3 gene. Two distinct regions were identified which strongly bind a nodule specific factor. A Bal31 deletion analysis delimited the DNA elements responsible for the binding of this factor, which map at nucleotides –223 to –246 (element 1) and –161 to –176 (element 2), relative to the start point of transcription. Competition experiments strongly suggest that both elements bind to the same nodule specific factor, but with different affinities. Elements 1 and 2 share a common motif, although their AT‐rich DNA sequences differ. Element 2 is highly conserved at an analogous position in other soybean lb gene 5′ upstream regions.

Primary structure and promoter analysis of leghemoglobin genes of the stem-nodulated tropical legume Sesbania rostrata: conserved coding sequences, cis-elements and trans-acting factors

SummaryThe primary structure of a leghemoglobin (lb) gene from the stem-nodulated, tropical legume Sesbania ostrata and two lb gene promoter regions was analysed. The S. rostrata lb gene structure and Lb amino acid composition were found to be highly conserved with previously described lb genes and Lb proteins. Distinct DNA elements were identified in the S. rostrata lb promoter regions, which share a high degree of homology with cis-active regulatory elements found in the soybean (Glycine max) lbc3 promoter. One conserved DNA element was found to interact specifically with an apparently universal, trans-acting factor present in nuclear extracts of nodules. These results suggest a conserved mechanism for nodule specific induction of lb genes in leguminous plants.

Transcription of the soybean leghemoglobin genes during nodule development.

During the early stages of soybean nodule development the leghemoglobin (Lb) genes are activated sequentially in the opposite order to which they are arranged in the soybean genome. At a specific stage after the initial activation of all the Lb genes, a large increment occurs in the transcription of the Lbc1, Lbc3 and Lba genes while the transcription of the Lbc2 gene is not amplified to a similar extent. All the Lb genes retain significant activity for a long period during the lifetime of a nodule. Consequently the soybean Lb genes are not regulated by a developmental gene switching mechanism as is the case for vertebrate globin genes. Concomitantly with the increase in Lb gene transcription some of the other nodule specific plant genes are activated. These specific changes in the activities of the Lb and nodulin genes precede the activation of the bacterial nitrogenase gene. Thus the alteration in bacterial metabolism due to nitrogen fixation is not responsible for the observed changes in the transcriptional activities of the Lb and nodule‐specific genes.

Expression of NO scavenging hemoglobin is involved in the timing of bolting in Arabidopsis thaliana

Plants contain three classes of hemoglobin genes of which two, class 1 and class 2, have a structure similar to classical vertebrate globins. We investigated the effect of silencing the class 1 non-symbiotic hemoglobin gene, GLB1, and the effect of overexpression of GLB1 or the class 2 non-symbiotic hemoglobin gene, GLB2, in Arabidopsis thaliana. Lines with GLB1 silencing had a significant delay of bolting and after bolting, shoots reverted to the rosette vegetative phase by formation of aerial rosettes at lateral meristems. Lines with overexpression of GLB1 or GLB2 bolted earlier than wild type plants. By germinating the lines in a medium containing the nitric oxide (NO) donor, sodium nitroprusside (SNP), it was demonstrated that both GLB1 and GLB2 promote bolting by antagonizing the effect of NO, suggesting that non-symbiotic plant hemoglobin controls bolting by scavenging the floral transition signal molecule, NO. So far, NO scavenging has only been demonstrated for class 1 non-symbiotic hemoglobins. A direct assay in Arabidopsis leaf cells shows that GLB1 as well as the class 2 non-symbiotic hemoglobin, GLB2, scavenge NO in vivo. NO has also been demonstrated to be a growth stimulating signal with an optimum at low concentrations. It was observed that overexpression of either GLB1 or GLB2 shifts the optimum for NO growth stimulation to a higher concentration. In conclusion, we have found that expression of NO scavenging plant hemoglobin is involved in the control of bolting in Arabidopsis.

Changes of rat kidney AQP2 and Na,K-ATPase mRNA expression in lithium-induced nephrogenic diabetes insipidus

Background/Aim: In a rat model, lithium treatment is associated with polyuria and severe downregulation of aquaporin-2 (AQP2) protein in the inner medulla (IM) or in the whole kidney. However, it is not known (1) to what extent this downregulation occurs at the mRNA level; (2) whether the main sodium transporter of the nephron, Na,K-ATPase, is regulated in parallel at the mRNA level, and (3) whether lithium treatment induces zonal or segmental differences in AQP2 and Na,K-ATPase mRNA levels. Method: We examined the changes in mRNA expression levels for AQP2 and Na,K-ATPase in kidney cortex, inner stripe of the outer medulla (ISOM), and IM of rats treated with lithium orally using semiquantitative Northern blot analyses and in situ hybridization at the light and electron microscopic levels. Results: The AQP2 mRNA levels decreased significantly (p < 0.01) in lithium-treated rats to 37 ± 4% in the cortex, to 17 ± 4% in the ISOM, and to 23 ± 5% in the IM, while the Na,K-ATPase mRNA levels were not altered in the cortex, but were significantly (p < 0.05) altered in the ISOM (144 ± 15% after 10 days, but 68 ± 4% after 4 weeks) and in the IM (63 ± 8% after 10 days, but normalized after 4 weeks). In situ hybridization showed reduced levels of AQP2 mRNA in all zones of the kidney, but the Na,K-ATPase mRNA expressions were slightly decreased only in IM collecting ducts. At the ultrastructural level, principal cells in the IM collecting ducts showed slight hypertrophy, but no cell damage after 4 weeks of lithium treatment. The results demonstrate substantial downregulation of AQP2 at the mRNA level throughout the collecting duct in experimental lithium-induced nephrogenic dabetes insipidus and moderately decreased Na,K-ATPase mRNA levels in the ISOM and in the IM. Conclusion: The results suggest that decreased mRNA expressions of AQP2 and Na,K-ATPase contribute to the development of lithium-induced nephrogenic diabetes insipidus.

A 200 bp region of the pea ENOD12 promoter is sufficient for nodule-specific and nod factor induced expression.

ENOD12 is one of the first nodulin genes expressed upon inoculation with Rhizobium and also purified Nod factors are able to induce ENOD12 expression. The ENOD12 gene family in pea (Pisum sativum) has two members. A cDNA clone representing PsENOD12A [26] and a PsENOD12B genomic clone [7] have been previously described. The isolation and characterization of a PsENOD12A genomic clone is presented in this paper. By using a Vicia hirsuta-Agrobacterium rhizogenes transformation system it is shown that both genes have a similar expression pattern in transgenic V. hirsuta root nodules. Promoter analyses of both PsENOD12 promoters showed that the 200 bp immediately upstream of the transcription start are sufficient to direct nodule-specific and Nod factor-induced gene expression.

A single hemoglobin gene in Myrica gale retains both symbiotic and non-symbiotic specificity.

Here, a hemoglobin gene from the nitrogen-fixing actinorhizal plant Myrica gale was isolated, cloned and sequenced. The gene (MgHb) was a class I hemoglobin with strong sequence homology to non-symbiotic hemoglobin genes. MgHb is highly expressed in symbiotic root nodules, but transcripts and protein were also detected in leaves of M. gale. In Arabidopsis thaliana the MgHb promoter, linked to a β-glucuronidase coding region, directed expression in the vascular tissue, in shoot meristem and at root branch point - a pattern very similar to the combined expression pattern of the two non-symbiotic A. thaliana hemoglobin promoters AHb1 and AHb2. The results points to a symbiotic as well as a non-symbiotic specificity of MgHb similar to a hemoglobin gene identified in Parasponia andersonii, but different from the situation in Casuarina glauca - a close actinorhizal relative of M. gale.

The chromosomal arrangement of six soybean leghemoglobin genes

Clones containing six leghemoglobin (Lb) genes have been isolated from two genomic libraries of soybean. They encompass two independent DNA regions: a 40‐kb region containing four genes in the order 5′ Lba‐Lbc1‐[unk]Lb‐Lbc3 3′ with the same transcriptional polarity, and another 40‐kb region containing two genes in the order 5′ Lbc4‐Lbc2 3′ with the same polarity. The order in which the Lb genes are arranged in the soybean genome imply that they are activated in the opposite order to which they are arranged on the chromosome. There is a close similarity between corresponding DNA regions outside the Lb genes in the two clusters. Thus, a moderately repetitive DNA element is present in corresponding positions in each cluster. In addition, at least two different non‐Lb genes are linked to each Lb gene cluster in corresponding positions. These genes are apparently regulated in a way which differs from that of the Lb genes. The existence of two very similar Lb gene clusters in soybean suggest that soybean may have evolved from an ancestral form by genome duplication.

A novel type of DNA-binding protein interacts with a conserved sequence in an early nodulin ENOD12 promoter.

The pea genes PsENOD12A and PsENOD12B are expressed in the root hairs shortly after infection with the nitrogen-fixing bacterium Rhizobium leguminosarum bv. viciae or after application of purified Nod factors. A 199 bp promoter fragment of the PsENOD12B gene contains sufficient information for Nod factor-induced tissue-specific expression. We have isolated a Vicia sativa cDNA encoding a 1641 amino acid protein, ENBP1, that interacts with the 199 bp ENOD12 promoter. Two different DNA-binding domains were identified in ENBP1. A domain containing six AT-hooks interacts specifically with an AT-rich sequence located between positions −95 and −77 in the PsENOD12B promoter. A second domain in ENBP1 is a cysteine-rich region that binds to the ENOD12 promoter in a sequence non-specific but metal-dependent way. ENBP1 is expressed in the same cell types as ENOD12. However, additional expression is observed in the nodule parenchyma and meristem. The presence of three small overlapping ORFs in the 5′-untranslated region of the ENBP1 cDNA indicates that ENBP1 expression might be regulated at the translational level. The interaction of ENBP1 with a conserved AT-rich element within the ENOD12 promoter and the presence of the ENBP1 transcript in cells expressing ENOD12 strongly suggest that ENBP1 is a transcription factor involved in the regulation of ENOD12. Finally, the C-terminal region of ENBP1 shows strong homology to a protein from rat that is specifically expressed in testis tissue.

The Lotus japonicus ndxgene family is involved in nodule function and maintenance

To elucidate the function of the ndx homeobox genes during the Rhizobium-legume symbiosis, two Lotus japonicus ndxgenes were expressed in the antisense orientation under the control of the nodule-expressed promoter Psenod12 in transgenic Lotus japonicus plants. Many of the transformants obtained segregated into plants that failed to sustain proper development and maintenance of root nodules concomitant with down-regulation of the two ndx genes. The root nodules were actively fixing nitrogen 3 weeks after inoculation, but the plants exhibited a stunted growth phenotype. The nodules on such antisense plants had under-developed vasculature and lenticels when grown on medium lacking nitrogen sources. These nodules furthermore entered senescence earlier than the wild-type nodules. Normal plant growth was resumed upon external addition of nitrogen. This suggests that assimilated nitrogen is not properly supplied to the plants in which the two ndx genes are down-regulated. The results presented here, indicate that the ndx genes play a role in the development of structural nodule features, required for proper gas diffusion into the nodule and/or transport of the assimilated nitrogen to the plant.