Expression of symbiotic and nonsymbiotic globin genes responding to microsymbionts on Lotus japonicus

Leguminous plants have both symbiotic and nonsymbiotic hemoglobin (sym- and nonsym-Hb) genes. Three symbiotic (LjLb1, 2, 3) and one nonsymbiotic (LjNSG1) Hb genes were isolated from a genomic library of Lotus japonicus MG20 Miyakojima. Two motif sequences (AAAGAT and CTCTT) critical for nodule specific expression were conserved on the 5'-upstream sequence of LjLb1, 2 and 3. The 5'-upstream region of LjNSG1 contained the sequence consensus for nonsym-Hb. RT-PCR with specific primer sets for each LjLb gene showed that all the sym-Hb genes (LjLb1, 2, 3) were expressed specifically and strongly in root nodules. The expression of LjLb1, 2 and 3 could not be detected in root, leaf or stem of a mature plant, whereas low level expression was detected in seedlings by RT-PCR. This suggests that sym-Hbs may have another unknown function besides being oxygen transporter for the microsymbiont in symbiotic nitrogen fixation. The expression of LjNSG1, examined with RT-PCR, was detected at low level in root, leaf and stem. The expression of LjNSG1 was enhanced in root nodules, whereas it was repressed in roots colonized by mycorrhizal fungi Glomus sp. R10. The repression of the nonsym-Hb gene was also observed in the roots of Medicago sativa colonized by Glomus sp. R10.     

Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene. Revision of the ARE consensus sequence

Constitutive and inducible expression of the gene encoding the modulator subunit of human glutamate-cysteine ligase (GCLM) is regulated by either of two regions of the promoter; an antioxidant response element (ARE) at -302:-291 and a 44-bp fragment (-346:-303) upstream of the ARE. This second region includes a consensus AP-1 site previously considered responsible for the enhancer activity of the upstream fragment. Deletion of a 165-bp fragment (-348:-183) including the ARE and upstream 44-bp fragment totally ablated t-butyl hydroquinone (tBHQ) inducibility of a GCLM promoter/luciferase transgene. Mutation analyses confirmed that both the ARE and the -346:-303 fragment could support induction following tBHQ exposure but demonstrated that induction in the latter case did not involve the AP-1 site at -341:-335. A region sharing significant homology with the consensus ARE sequence except for a single nucleotide mismatch at -330 (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') was identified at the 5'-end of the 44-bp fragment immediately adjacent to the AP-1 site. A G in this position has been considered an invariant requirement of functional ARE sequences. Mutation of T(-330) to A (a substitution known to ablate ARE function) or C eliminated basal and inducible expression. Substitution of a G at -330 enhanced basal expression relative to the wild-type sequence, but induction following tBHQ exposure was comparable, indicating that either sequence (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') may function as an ARE, although the former sequence is less effective at directing basal expression. This possibility was confirmed by similar mutational analyses of the core sequence of hNQO1, a prototypic ARE. Electrophoretic mobility shift competition assays revealed that the 5'-TTACnnnGCA-3' sequence could compete with the hNQO1 ARE for protein binding but was less effective than a similar probe containing the 5'-TGACnnnGCA-3' motif. Probes including the T(-330)A or T(-330)C mutations were ineffective. These results reveal that the GCLM promoter includes two functional AREs, one having a variant sequence. The results indicate that the consensus ARE sequence should be revised to 5'-RTKAYnnnGCR-3'.     

Primary structure of the soybean nodulin-23 gene and potential regulatory elements in the 5''-flanking regions of nodulin and leghemoglobin genes.

The nodulin-23 gene of soybean is one of the most abundantly transcribed genes induced during symbiosis with Rhizobium. Using a plasmid (pNod25) from a nodule cDNA library, we have isolated the nodulin-23 gene from a soybean genomic library. Nucleotide sequence analysis of the cDNA and of the genomic clone indicated that the coding region of this gene is 669 bp long and is interrupted by a single intron of about 530 bp. The deduced protein sequence suggests that nodulin-23 may have a signal sequence. The 5'-flanking sequence of two other nodulin genes, nodulin-24 encoding for a membrane polypeptide and one of the leghemoglobin genes (LbC3), were obtained. Comparison of these sequences revealed three conserved regions, one of which, an octanucleotide (GTTTCCCT), has 100% homology. The conserved sequences are arranged in a unique fashion and have a spatial organization with respect to order and position, which may suggest a potential regulatory role in controlling the expression of nodulin and leghemoglobin genes during symbiosis.     

A two-component nodule-specific enhancer in the soybean N23 gene promoter.

The two positive cis elements in the soybean nodulin N23 gene promoter were investigated in transgenic Lotus corniculatus plants and shown to constitute a two-component nodule-specific enhancer. Equal quantitative contributions from the two components were suggested by the similar expression level of chimeric N23-chloramphenicol acetyltransferase genes after deletion of either the distal positive element (PE-A, -320 to -298) or the proximal positive element (PE-B, -257 to -165). A combined effect of the two elements was indicated by orientation-dependent effects in the N23 promoter, and by the observation that neither PE-A nor PE-B separately was able to confer any activity to the cauliflower mosaic virus 35S minimal promoter. Reactivation of the minimal N23 and the minimal cauliflower mosaic virus 35S promoters by the inverted complete element (PE-AB) further suggested that PE-AB is a nodule-specific enhancer containing two equally strong enhancer components. Two 12-bp sequence motifs, InvA and InvB, constituting an inverted repeat, were identified as the core of the enhancer components PE-A and PE-B, respectively. Point mutations in InvA or InvB resulted in lower expression levels and mutations in both abolished enhancer activity. Point mutations in two nodulin consensus sequences, 5'-CTCTT and 5'-AAAGAT located downstream of PE-AB, resulted in a decreased level of expression, confirming the involvement of these two motifs in nodulin gene expression. The binding site for the nodule-specific trans-acting factor, NAT2, present in the PE-A segment, was removed without affecting expression significantly. This interaction is, therefore, dispensable for enhancer activity.     

Nuclear factors interact with conserved A/T-rich elements upstream of a nodule-enhanced glutamine synthetase gene from French bean.

The gln-gamma gene, encoding the gamma subunit of glutamine synthetase in French bean (Phaseolus vulgaris), is strongly induced during nodule development. We have determined the nucleotide sequence of a 1.3-kilobase region at its 5' end and have identified several sequences common to the promoter regions of late nodulin genes from other legume species. The 5'-flanking region was analyzed for sequence-specific interactions with nuclear factors from French bean. A factor from nodules (PNF-1) was identified that binds to multiple sites between -860 and -154, and a related but distinct factor (PRF-1) was detected in extracts from uninfected roots. PNF-1 and PRF-1 bound strongly to a synthetic oligonucleotide containing the sequence of an A/T-rich 21-base pair imperfect repeat found at positions -516 and -466. The same factors also had a high affinity for a protein binding site from a soybean leghemoglobin gene and appeared to be closely related to the soybean nodule factor NAT2, which binds to A/T-rich sequences in the lbc3 and nodulin 23 genes [Jacobsen et al. (1990). Plant Cell 2, 85-94]. Comparison of NAT2/PNF-1 binding sites from a variety of nodulin genes revealed the conservation of the short consensus core motif TATTTWAT, and evidence was obtained that this sequence is important for protein recognition. Cross-recognition by PNF-1 of a protein binding site in a soybean seed protein gene points to the existence of a ubiquitous family of factors with related binding affinities. Our data suggest that PNF-1 and PRF-1 belong to an evolutionarily conserved group of nuclear factors that interact with specific A/T-rich sequences in a diverse set of plant genes. We consider the possible role of these factors in coregulating the expression of gln-gamma and other late nodulin genes.     

Root nodule specific gene regulation: analysis of the soybean nodulin N23 gene promoter in heterologous symbiotic systems.

The nodulin N23 gene promoter was analysed in transgenic plants using the chloramphenicol acetyltransferase (CAT) coding sequence as a reporter. A 5' flanking region of less than 1 kb was sufficient for the organ-specific expression of a chimeric N23-CAT-3'lbc3 gene in root nodules formed on Lotus corniculatus and Trifolium repens after infection by their respective Rhizobium symbionts. Expression was regulated at the level of RNA in both species of transgenic plants. Promoter deletion analysis defined the 5' region required for high level expression and delimited two putative regulatory sequences involved in positive control of the N23 gene in L. corniculatus.     

Interaction of the H4 autonomously replicating sequence core consensus sequence and its 3''-flanking domain.

Yeast autonomously replicating sequence (ARS) elements are composed of a conserved 11-base-pair (bp) core consensus sequence and a less well defined 3'-flanking region. We have investigated the relationship between the H4 ARS core consensus sequence and its 3'-flanking domain. The minimal sequences necessary and sufficient for function were determined by combining external 3' and 5' deletions to produce a nested set of ARS fragments. Sequences 5' of the core consensus were dispensable for function, but at least 66 bp of 3'-flanking domain DNA was required for full ARS function. The importance of the relative orientation of the core consensus element with respect to the 3'-flanking domain was tested by precisely inverting 14 bp of DNA including the core consensus sequence by oligonucleotide mutagenesis. This core inversion mutant was defective for all ARS function, showing that a fixed relative orientation of the core consensus and 3'-flanking domain is required for function. The 3'-flanking domain of the minimal functional H4 ARS fragment contains three sequences with a 9-of-11-bp match to the core consensus. The role of these near-match sequences was tested by directed mutagenesis. When all near-match sequences with an 8-of-11-bp match or better were simultaneously disrupted by point mutations, the resulting ARS construct retained full replication function. Therefore, multiple copies of a sequence closely related to the core consensus element are not required for H4 ARS function.     

Site-specific mutagenesis of the nodule-infected cell expression (NICE) element and the AT-rich element ATRE-BS2* of the Sesbania rostrata leghemoglobin glb3 promoter.

Sesbania rostrata leghemoglobin glb3 (Srglb3) promoter sequences responsible for expression in infected cells of transgenic Lotus corniculatus nodules were delimited to a 78-bp Dral-Hinfl fragment. This region, which is located between coordinates -194 to -116 relative to the start codon of the Srglb3 gene, was named the nodule-infected cell expression (NICE) element. Insertion of the NICE element into the truncated nopaline synthase promoter was found to confer a nodule-specific expression pattern on this normally root-enhanced promoter. Within the NICE element, three distinct motifs ([A]AAAGAT, TTGTCTCTT, and CACCC[T]) were identified; they are highly conserved in the promoter regions of a variety of plant (leg)hemoglobin genes. The NICE element and the adjacent AT-rich element (ATRE-BS2*) were subjected to site-directed mutagenesis. The expression patterns of nine selected Srglb3 promoter fragments carrying mutations in ATRE-BS2* and 19 with mutations in the NICE element were examined. Mutations in ATRE-BS2* had varying effects on Srglb3 promoter activity, ranging from a two- to threefold reduction to a slight stimulation of activity. Mutations in the highly conserved (A)AAAGAT motif of the NICE element reduced Srglb3 promoter activity two- to fourfold, whereas mutations in the TCTT portion of the TTGTCTCTT motif virtually abolished promoter activity, demonstrating the essential nature of these motifs for Srglb3 gene expression. An A-to-T substitution in the CACCC(T) motif of the NICE element also abolished Srglb3 promoter activity, while a C-to-T mutation at position 4 resulted in a threefold reduction of promoter strength. The latter phenotypes resemble the effect of similar mutations in the conserved CACCC motif located in the promoter region of mammalian beta-globin genes. The possible analogies between these two systems will be discussed.