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Daniel M. Roberts - One of the best experts on this subject based on the ideXlab platform.
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arabidopsis nip2 1 a major intrinsic protein transporter of lactic acid induced by anoxic stress
Journal of Biological Chemistry, 2007Co-Authors: Won Gyu Choi, Daniel M. RobertsAbstract:Nodulin 26 intrinsic proteins (NIPs) are plant-specific, highly conserved water and solute transport proteins with structural and functional homology to soybean Nodulin 26. Arabidopsis thaliana contains nine NIP genes. In this study, it is shown that one of these, AtNIP2;1, is exquisitely sensitive to water logging and anoxia stress. Based on quantitative PCR and promoter::GUS experiments, AtNIP2;1 is expressed at a low basal level in the root tips and the vascular bundle of differentiated roots. Transcript levels are elevated acutely and rapidly upon water logging of root or leaf tissues, increasing 70-fold in roots within the 1st h of submersion. After this large initial increase, mRNA levels decline to steady state levels that remain over 10-fold higher by 6 h post-submersion. An even greater induction of AtNIP2;1 expression was observed upon anoxia challenge of Arabidopsis seedlings, with a 300-fold increase in AtNIP2;1 transcript observed by 2 h after the initiation of oxygen deprivation. Functional analysis of AtNIP2;1 expressed in Xenopus oocytes shows that the protein differs from soybean Nodulin 26, showing minimal water and glycerol transport. Instead, AtNIP2;1 displays transport of lactic acid, with a preference for the protonated acidic form of this weak acid. Overall, the data suggest that AtNIP2;1 is an anaerobic-induced gene that encodes a lactic acid transporter and may play a role in adaptation to lactic fermentation under anaerobic stress.
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Phosphorylation of Soybean Nodulin 26 on Serine 262 Enhances Water Permeability and Is Regulated Developmentally and by Osmotic Signals
The Plant cell, 2003Co-Authors: James F. Guenther, Nouth Chanmanivone, Manker P. Galetovic, Ian S. Wallace, Jennifer A. Cobb, Daniel M. RobertsAbstract:Soybean Nodulin 26 is expressed and targeted to the symbiosome membrane of nitrogen-fixing nodules, where it forms an aquaporin channel with a modest water transport rate. In this study, we show that the phosphorylation of Nodulin 26 on Ser-262, which is catalyzed by a symbiosome membrane–associated calcium-dependent protein kinase, stimulates its intrinsic water transport rate. Furthermore, using a phosphospecific antibody, we have elucidated the developmental appearance and regulation of Nodulin 26 phosphorylation in vivo. Although Nodulin 26 protein is detected first in differentiating infected cells (16 days), phosphorylated Nodulin 26 does not become pronounced until infected cell maturation (25 days). Phosphorylation is sustained at steady state levels until entry into senescence. Nodulin 26 phosphorylation is enhanced further by osmotic stresses (water deprivation and salinity). Thus, the phosphorylation of Nodulin 26 coincides with the establishment of mature nitrogen-fixing symbiosomes, is regulated by osmotic stresses that induce calcium-signaling pathways, and appears to be part of the adaptive responses of infected cells to osmotic challenge.
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Functional selectivity for glycerol of the Nodulin 26 subfamily of plant membrane intrinsic proteins.
FEBS letters, 2002Co-Authors: Ian S. Wallace, James F. Guenther, David M Wills, Daniel M. RobertsAbstract:The Nodulin-like intrinsic protein (NIP) subfamily of water and solute channels in plants is named for Nodulin 26 of legume nodules. Two NIPs, soybean Nodulin 26 and Lotus japonicus LIMP2, show a distinct functional profile with a low intrinsic osmotic water permeability (P(f)) and the ability to flux uncharged polyols such as glycerol. NIPs have a conserved signature sequence within the 'aromatic/arginine' region that forms the selectivity filter for major intrinsic proteins. This sequence is a hybrid of glyceroporin and aquaporin residues as well as exhibiting substitutions unique to the NIP subfamily. Site-directed mutagenesis of a conserved tryptophan in helix 2 of LIMP2 shows that this is a major determinant of glycerol selectivity.
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Functional selectivity for glycerol of the Nodulin 26 subfamily of plant membrane intrinsic proteins
FEBS Letters, 2002Co-Authors: Ian S. Wallace, James F. Guenther, David M Wills, Daniel M. RobertsAbstract:The Nodulin-like intrinsic protein (NIP) subfamily of water and solute channels in plants is named for Nodulin 26 of legume nodules.Two NIPs, soybean Nodulin 26 and Lotus ja- ponicus LIMP2, show a distinct functional pro¢le with a low intrinsic osmotic water permeability (Pf) and the ability to £ux uncharged polyols such as glycerol.NIPs have a conserved sig- nature sequence within the 'aromatic/arginine' region that forms the selectivity ¢lter for major intrinsic proteins.This sequence is a hybrid of glyceroporin and aquaporin residues as well as ex- hibiting substitutions unique to the NIP subfamily.Site-directed mutagenesis of a conserved tryptophan in helix 2 of LIMP2 shows that this is a major determinant of glycerol selec- tivity.. 2002 Published by Elsevier Science B. on behalf of the Federation of European Biochemical Societies.
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Purification and functional reconstitution of soybean Nodulin 26. An aquaporin with water and glycerol transport properties
Biochemistry, 1999Co-Authors: Robert M. Dean, Ricky L. Rivers, Mark L. Zeidel, Daniel M. RobertsAbstract:Infection of soybean roots by nitrogen-fixing Bradyrhizobium japonicum leads to expression of plant nodule-specific genes known as Nodulins. Nodulin 26, a member of the major intrinsic protein/aquaporin (AQP) channel family, is a major component of the soybean symbiosome membrane (SM) that encloses the rhizobium bacteroid. To investigate the water and solute transport characteristics of Nodulin 26, we purified the protein from SMs and reconstituted it into carboxyfluorescein-loaded liposomes for transport studies using stopped-flow spectrofluorimetry. Liposomes containing Nodulin 26 exhibited a high osmotic permeability (Pf = 0. 012 +/- 0.0013 cm/s), a value fivefold higher than that obtained with control liposomes. Water flux through Nodulin 26 showed a low activation energy (Ea) (4.07 kcal/mol) and was reduced 70% upon addition of 1 mM HgCl2. Reconstituted Nodulin 26 exhibited a single-channel conductance of 3.8 +/- 2.5 x 10(-)15 cm3/s (n = 3), a value that is lower than other characterized AQPs. Nodulin 26 proteoliposomes also facilitate glycerol transport, showing a 43-fold higher rate of glycerol flux than control liposomes. This observation was supported by expression experiments in Xenopus oocytes that showed that Nodulin 26 facilitated glycerol flux in a manner indistinguishable from the Escherichia coli GlpF glycerol facilitator. Consistent with the results of water transport, glycerol transport was inhibited by HgCl2 and showed a low Ea (4.43 kcal/mol). These results indicate that Nodulin 26 is a multifunctional AQP that confers water and glycerol transport to the SM, and likely plays a role in osmoregulation during legume/rhizobia symbioses.
Andreas M. Perlick - One of the best experts on this subject based on the ideXlab platform.
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A small gene family of broad bean codes for late Nodulins containing conserved cysteine clusters
Plant Science, 2000Co-Authors: Martin Frühling, U. Albus, Natalija Hohnjec, Gerhard Geise, Alfred Pühler, Andreas M. PerlickAbstract:Five transcripts encoding different members of a Nodulin family with conserved cysteine clusters (Cys-X-4-Asp-Cys and Cys-X-4-Cys) were identified in broad bean root nodules. They displayed homologies to the early Nodulins PsENOD3 and PsENOD14 and the late Nodulin PsNOD6 from pea. In addition to the occurence of putative secretory signal peptides, the spatial distribution of the cysteine residues was comparable in both the broad bean and the pea Nodulins. Based on tissue print hybridizations, we found that the corresponding broad bean genes VfNOD-CCP1, VfNOD-CCP3 and VfNOD-CCP5 were expressed in the interzone II-III and the nitrogen fixing zone III of mature nodules whereas the gene VfNOD-CCP4 was first induced in the prefixing zone II. A strong expression of the VfNOD-CCP2 gene only could be detected the interzone II-III region. Sequence analysis of a genomic VfNOD-CCP1 clone isolated revealed the presence of one intron seperating a first exon encoding the signal peptide from a second exon encoding the cysteine cluster domain of this Nodulin. Apart from the multiple presence of the common Nodulin motifs AAAGAT and CTCTT on both DNA strands of the putative VfNOD-CCP1 promoter region a sequence element resembling the organ specific element of the soybean lbc3 gene promoter was identified. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
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Analysis of genes encoding modular Nodulins from Vicia hirsuta and Vicia faba
Plant Science, 1999Co-Authors: Helge Küster, Alfred Pühler, Andreas M. PerlickAbstract:Abstract The expression of genes encoding modular Nodulins was analysed in the Vicia hirsuta and the Vicia faba system. From V. hirsuta root nodules, we isolated two families of transcript sequences designated VhNOD28/32-A and VhNOD28/32-B encoding Nodulins homologous to the V. faba Nodulins Nvf-28/32 and the Medicago sativa Nodulin-25. The modular proteins encoded by the V. hirsuta transcript sequences consisted of N- and C-terminal unique modules flanking two types of repetitive modules. Specific repetitive modules were deleted from individual VhNOD28/32 transcripts. Southern hybridizations indicated the presence of either a small VhNOD28/32 gene family or a single copy gene comprising at least two alleles. VhNOD28/32 transcripts were expressed specifically in root nodules, where they were localized in the nitrogen-fixing zone III. In the Vicia faba system, we isolated the promoter of two alleles of the VfNOD28/32 gene by PCR-based DNA walking. The promoter sequences were similar to the promoter of the M. sativa Nodulin-25 gene and contained an organ-specific element at position −68/−55 from the transcriptional start. An analysis of transgenic V. hirsut a root nodules expressing the gus Aint reporter gene under the control of the two VfNOD28/32 promoters demonstrated that both promoters were active in the central region of transgenic V . hirsuta nodules.
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Isolation of Two Transcript Sequences of Different Sizes Encoding Modular Nodulins of Vicia hirsuta : Expression Properties and Analysis of Transgenic Nodules Containing Antisense Constructs
Biological Nitrogen Fixation for the 21st Century, 1998Co-Authors: Helge Küster, Alfred Pühler, M. G. Finke, Jörg D. Becker, Andreas M. PerlickAbstract:Nodule-specific transcript sequences of the Vicia faba VfNOD28/32 gene encode six different late Nodulins composed of two types of repetitive sequence modules flanked by unique N- and C-terminal modules (Kuster et al. 1994, 1996). In Medicago sativa, Kiss et al. (1990) and Vegh et al. (1990) identified the MsNOD25 gene encoding the late Nodulin Nms-25. Some repetitive and the unique modules were homologous between the Nms-25 and the broad bean Nodulins, but the number and order of repetitive modules was different. This was also the case for the deduced protein sequence of the nodule-specific cDNA Ngo40 from Galega orientalis (S. Karjalainen, personal communication).
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the nodule specific vfenod grp3 gene encoding a glycine rich early Nodulin is located on chromosome i of vicia faba l and is predominantly expressed in the interzone ii iii of root nodules
Plant Molecular Biology, 1995Co-Authors: Helge Küster, Martin Frühling, Andreas M. Perlick, Gerald Schroder, Uta Pich, Mechthild Rieping, Ingo Schubert, Alfred PühlerAbstract:A nodule-specific cDNA was isolated from a Vicia faba L. nodule cDNA library. Since time course experiments revealed an early expression of this transcript in the nodule, this cDNA coded for an early Nodulin and was designated VfENOD-GRP3. Based on tissue print hybridizations, we found a predominant expression of VfENOD-GRP3 transcripts in the interzone II-III region of broad bean root nodules. The encoded early Nodulin ENOD-GRP3 was characterized by an N-terminal signal peptide and a C-terminal domain displaying a glycine content of 31%. Sequence analysis of a genomic VfENOD-GRP3 clone revealed that the signal peptide and the glycine-rich domain were specified by two separate exons. Primer extension experiments identified two adjacent transcription start sites for VfENOD-GRP3 transcripts. The common Nodulin sequences ‘AAAGAT’ and ‘CTCTT’ were present five and three times on both DNA strands of the putative VfENOD-GRP3 promoter, respectively. Additionally, three sequence motifs resembling organ-specific elements of the soybean lbc3 gene promoter and a sequence similar to the binding site 1 for the nodule trans-acting factor Nat2 were identified. From Southern blot data and from sequence analysis of genomic PCR fragments, the presence of a VfENOD-GRP3 gene family was inferred. By PCR experiments using sequence-specific primers and DNA of microisolated chromosomes as a template, this family was located on the long arm of chromosome I.
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Members of a broadbean Nodulin family with partial homologies to the alfalfa Nodulin 25 are composed of two types of amino acid repeats flanked by unique amino acid sequence termini.
Plant molecular biology, 1994Co-Authors: Helge Küster, Andreas M. Perlick, Alfred PühlerAbstract:Five cross-hybridizing cDNAs from clone group ‘VfNDS-L’ of a broadbean nodule-specific cDNA library differed by five specific in-frame deletions within the coding region. Northern blot analysis revealed that the transcripts represented by these clones were expressed in a nodule-specific way and therefore encoded a new family of broadbean Nodulins. These Nodulins have been designated Nvf-28/32. The Nvf-28/32 proteins were between 269 and 299 amino acids long with a high proportion of charged amino acids and contained a putative signal peptide of 20 amino acids. Sequence analysis indicated that the central part of the Nvf-28/32 proteins was composed of two different types of repeating amino acid stretches designated ‘repeat 1’ and ‘repeat 2’, whereas the N- and C-termini were unique. In contrast to ‘repeat 1’ stretches, which contained both positively and negatively charged amino acid residues, ‘repeat 2’ sequences did not contain any positively, but a total of 13 negatively charged amino acid residues. We could demonstrate that the five deletions identified exactly corresponded to complete repeats. The unique sequence termini of the Nvf-28/32 proteins displayed strong homologies to the late Nodulin 25 from alfalfa. In addition, the repeating units identified were significantly homologous to several, but not all exon parts of this protein. We speculate that the ‘VfNDS-L’ transcripts were derived from a differential splicing mechanism and that the Nvf-28/32 proteins fulfil a structural rather than an enzymatic function within the broadbean root nodule.
Helge Küster - One of the best experts on this subject based on the ideXlab platform.
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the signal peptide of the medicago truncatula modular Nodulin mtnod25 operates as an address label for the specific targeting of proteins to nitrogen fixing symbiosomes
Molecular Plant-microbe Interactions, 2009Co-Authors: Natalija Hohnjec, Frauke Lenz, Vera Fehlberg, Martin F Vieweg, Markus C Baier, Bettina Hause, Helge KüsterAbstract:The nodule-specific MtNOD25 gene of the model legume Medicago truncatula encodes a modular Nodulin composed of different repetitive modules flanked by distinct N- and C-termini. Although similarities are low with respect to all repetitive modules, both the N-terminal signal peptide (SP) and the C-terminus are highly conserved in modular Nodulins from different legumes. On the cellular level, MtNOD25 is only transcribed in the infected cells of root nodules, and this activation is mediated by a 299-bp minimal promoter containing an organ-specific element. By expressing mGFP6 translational fusions in transgenic nodules, we show that MtNOD25 proteins are exclusively translocated to the symbiosomes of infected cells. This specific targeting only requires an N-terminal MtNOD25 SP that is highly conserved across a family of legume-specific symbiosome proteins. Our finding sheds light on one possible mechanism for the delivery of host proteins to the symbiosomes of infected root nodule cells and, in addition, defines a short molecular address label of only 24 amino acids whose N-terminal presence is sufficient to translocate proteins across the peribacteroid membrane.
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Analysis of genes encoding modular Nodulins from Vicia hirsuta and Vicia faba
Plant Science, 1999Co-Authors: Helge Küster, Alfred Pühler, Andreas M. PerlickAbstract:Abstract The expression of genes encoding modular Nodulins was analysed in the Vicia hirsuta and the Vicia faba system. From V. hirsuta root nodules, we isolated two families of transcript sequences designated VhNOD28/32-A and VhNOD28/32-B encoding Nodulins homologous to the V. faba Nodulins Nvf-28/32 and the Medicago sativa Nodulin-25. The modular proteins encoded by the V. hirsuta transcript sequences consisted of N- and C-terminal unique modules flanking two types of repetitive modules. Specific repetitive modules were deleted from individual VhNOD28/32 transcripts. Southern hybridizations indicated the presence of either a small VhNOD28/32 gene family or a single copy gene comprising at least two alleles. VhNOD28/32 transcripts were expressed specifically in root nodules, where they were localized in the nitrogen-fixing zone III. In the Vicia faba system, we isolated the promoter of two alleles of the VfNOD28/32 gene by PCR-based DNA walking. The promoter sequences were similar to the promoter of the M. sativa Nodulin-25 gene and contained an organ-specific element at position −68/−55 from the transcriptional start. An analysis of transgenic V. hirsut a root nodules expressing the gus Aint reporter gene under the control of the two VfNOD28/32 promoters demonstrated that both promoters were active in the central region of transgenic V . hirsuta nodules.
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Isolation of Two Transcript Sequences of Different Sizes Encoding Modular Nodulins of Vicia hirsuta : Expression Properties and Analysis of Transgenic Nodules Containing Antisense Constructs
Biological Nitrogen Fixation for the 21st Century, 1998Co-Authors: Helge Küster, Alfred Pühler, M. G. Finke, Jörg D. Becker, Andreas M. PerlickAbstract:Nodule-specific transcript sequences of the Vicia faba VfNOD28/32 gene encode six different late Nodulins composed of two types of repetitive sequence modules flanked by unique N- and C-terminal modules (Kuster et al. 1994, 1996). In Medicago sativa, Kiss et al. (1990) and Vegh et al. (1990) identified the MsNOD25 gene encoding the late Nodulin Nms-25. Some repetitive and the unique modules were homologous between the Nms-25 and the broad bean Nodulins, but the number and order of repetitive modules was different. This was also the case for the deduced protein sequence of the nodule-specific cDNA Ngo40 from Galega orientalis (S. Karjalainen, personal communication).
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the nodule specific vfenod grp3 gene encoding a glycine rich early Nodulin is located on chromosome i of vicia faba l and is predominantly expressed in the interzone ii iii of root nodules
Plant Molecular Biology, 1995Co-Authors: Helge Küster, Martin Frühling, Andreas M. Perlick, Gerald Schroder, Uta Pich, Mechthild Rieping, Ingo Schubert, Alfred PühlerAbstract:A nodule-specific cDNA was isolated from a Vicia faba L. nodule cDNA library. Since time course experiments revealed an early expression of this transcript in the nodule, this cDNA coded for an early Nodulin and was designated VfENOD-GRP3. Based on tissue print hybridizations, we found a predominant expression of VfENOD-GRP3 transcripts in the interzone II-III region of broad bean root nodules. The encoded early Nodulin ENOD-GRP3 was characterized by an N-terminal signal peptide and a C-terminal domain displaying a glycine content of 31%. Sequence analysis of a genomic VfENOD-GRP3 clone revealed that the signal peptide and the glycine-rich domain were specified by two separate exons. Primer extension experiments identified two adjacent transcription start sites for VfENOD-GRP3 transcripts. The common Nodulin sequences ‘AAAGAT’ and ‘CTCTT’ were present five and three times on both DNA strands of the putative VfENOD-GRP3 promoter, respectively. Additionally, three sequence motifs resembling organ-specific elements of the soybean lbc3 gene promoter and a sequence similar to the binding site 1 for the nodule trans-acting factor Nat2 were identified. From Southern blot data and from sequence analysis of genomic PCR fragments, the presence of a VfENOD-GRP3 gene family was inferred. By PCR experiments using sequence-specific primers and DNA of microisolated chromosomes as a template, this family was located on the long arm of chromosome I.
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Members of a broadbean Nodulin family with partial homologies to the alfalfa Nodulin 25 are composed of two types of amino acid repeats flanked by unique amino acid sequence termini.
Plant molecular biology, 1994Co-Authors: Helge Küster, Andreas M. Perlick, Alfred PühlerAbstract:Five cross-hybridizing cDNAs from clone group ‘VfNDS-L’ of a broadbean nodule-specific cDNA library differed by five specific in-frame deletions within the coding region. Northern blot analysis revealed that the transcripts represented by these clones were expressed in a nodule-specific way and therefore encoded a new family of broadbean Nodulins. These Nodulins have been designated Nvf-28/32. The Nvf-28/32 proteins were between 269 and 299 amino acids long with a high proportion of charged amino acids and contained a putative signal peptide of 20 amino acids. Sequence analysis indicated that the central part of the Nvf-28/32 proteins was composed of two different types of repeating amino acid stretches designated ‘repeat 1’ and ‘repeat 2’, whereas the N- and C-termini were unique. In contrast to ‘repeat 1’ stretches, which contained both positively and negatively charged amino acid residues, ‘repeat 2’ sequences did not contain any positively, but a total of 13 negatively charged amino acid residues. We could demonstrate that the five deletions identified exactly corresponded to complete repeats. The unique sequence termini of the Nvf-28/32 proteins displayed strong homologies to the late Nodulin 25 from alfalfa. In addition, the repeating units identified were significantly homologous to several, but not all exon parts of this protein. We speculate that the ‘VfNDS-L’ transcripts were derived from a differential splicing mechanism and that the Nvf-28/32 proteins fulfil a structural rather than an enzymatic function within the broadbean root nodule.
Alfred Pühler - One of the best experts on this subject based on the ideXlab platform.
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A small gene family of broad bean codes for late Nodulins containing conserved cysteine clusters
Plant Science, 2000Co-Authors: Martin Frühling, U. Albus, Natalija Hohnjec, Gerhard Geise, Alfred Pühler, Andreas M. PerlickAbstract:Five transcripts encoding different members of a Nodulin family with conserved cysteine clusters (Cys-X-4-Asp-Cys and Cys-X-4-Cys) were identified in broad bean root nodules. They displayed homologies to the early Nodulins PsENOD3 and PsENOD14 and the late Nodulin PsNOD6 from pea. In addition to the occurence of putative secretory signal peptides, the spatial distribution of the cysteine residues was comparable in both the broad bean and the pea Nodulins. Based on tissue print hybridizations, we found that the corresponding broad bean genes VfNOD-CCP1, VfNOD-CCP3 and VfNOD-CCP5 were expressed in the interzone II-III and the nitrogen fixing zone III of mature nodules whereas the gene VfNOD-CCP4 was first induced in the prefixing zone II. A strong expression of the VfNOD-CCP2 gene only could be detected the interzone II-III region. Sequence analysis of a genomic VfNOD-CCP1 clone isolated revealed the presence of one intron seperating a first exon encoding the signal peptide from a second exon encoding the cysteine cluster domain of this Nodulin. Apart from the multiple presence of the common Nodulin motifs AAAGAT and CTCTT on both DNA strands of the putative VfNOD-CCP1 promoter region a sequence element resembling the organ specific element of the soybean lbc3 gene promoter was identified. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
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Analysis of genes encoding modular Nodulins from Vicia hirsuta and Vicia faba
Plant Science, 1999Co-Authors: Helge Küster, Alfred Pühler, Andreas M. PerlickAbstract:Abstract The expression of genes encoding modular Nodulins was analysed in the Vicia hirsuta and the Vicia faba system. From V. hirsuta root nodules, we isolated two families of transcript sequences designated VhNOD28/32-A and VhNOD28/32-B encoding Nodulins homologous to the V. faba Nodulins Nvf-28/32 and the Medicago sativa Nodulin-25. The modular proteins encoded by the V. hirsuta transcript sequences consisted of N- and C-terminal unique modules flanking two types of repetitive modules. Specific repetitive modules were deleted from individual VhNOD28/32 transcripts. Southern hybridizations indicated the presence of either a small VhNOD28/32 gene family or a single copy gene comprising at least two alleles. VhNOD28/32 transcripts were expressed specifically in root nodules, where they were localized in the nitrogen-fixing zone III. In the Vicia faba system, we isolated the promoter of two alleles of the VfNOD28/32 gene by PCR-based DNA walking. The promoter sequences were similar to the promoter of the M. sativa Nodulin-25 gene and contained an organ-specific element at position −68/−55 from the transcriptional start. An analysis of transgenic V. hirsut a root nodules expressing the gus Aint reporter gene under the control of the two VfNOD28/32 promoters demonstrated that both promoters were active in the central region of transgenic V . hirsuta nodules.
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Isolation of Two Transcript Sequences of Different Sizes Encoding Modular Nodulins of Vicia hirsuta : Expression Properties and Analysis of Transgenic Nodules Containing Antisense Constructs
Biological Nitrogen Fixation for the 21st Century, 1998Co-Authors: Helge Küster, Alfred Pühler, M. G. Finke, Jörg D. Becker, Andreas M. PerlickAbstract:Nodule-specific transcript sequences of the Vicia faba VfNOD28/32 gene encode six different late Nodulins composed of two types of repetitive sequence modules flanked by unique N- and C-terminal modules (Kuster et al. 1994, 1996). In Medicago sativa, Kiss et al. (1990) and Vegh et al. (1990) identified the MsNOD25 gene encoding the late Nodulin Nms-25. Some repetitive and the unique modules were homologous between the Nms-25 and the broad bean Nodulins, but the number and order of repetitive modules was different. This was also the case for the deduced protein sequence of the nodule-specific cDNA Ngo40 from Galega orientalis (S. Karjalainen, personal communication).
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the nodule specific vfenod grp3 gene encoding a glycine rich early Nodulin is located on chromosome i of vicia faba l and is predominantly expressed in the interzone ii iii of root nodules
Plant Molecular Biology, 1995Co-Authors: Helge Küster, Martin Frühling, Andreas M. Perlick, Gerald Schroder, Uta Pich, Mechthild Rieping, Ingo Schubert, Alfred PühlerAbstract:A nodule-specific cDNA was isolated from a Vicia faba L. nodule cDNA library. Since time course experiments revealed an early expression of this transcript in the nodule, this cDNA coded for an early Nodulin and was designated VfENOD-GRP3. Based on tissue print hybridizations, we found a predominant expression of VfENOD-GRP3 transcripts in the interzone II-III region of broad bean root nodules. The encoded early Nodulin ENOD-GRP3 was characterized by an N-terminal signal peptide and a C-terminal domain displaying a glycine content of 31%. Sequence analysis of a genomic VfENOD-GRP3 clone revealed that the signal peptide and the glycine-rich domain were specified by two separate exons. Primer extension experiments identified two adjacent transcription start sites for VfENOD-GRP3 transcripts. The common Nodulin sequences ‘AAAGAT’ and ‘CTCTT’ were present five and three times on both DNA strands of the putative VfENOD-GRP3 promoter, respectively. Additionally, three sequence motifs resembling organ-specific elements of the soybean lbc3 gene promoter and a sequence similar to the binding site 1 for the nodule trans-acting factor Nat2 were identified. From Southern blot data and from sequence analysis of genomic PCR fragments, the presence of a VfENOD-GRP3 gene family was inferred. By PCR experiments using sequence-specific primers and DNA of microisolated chromosomes as a template, this family was located on the long arm of chromosome I.
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Members of a broadbean Nodulin family with partial homologies to the alfalfa Nodulin 25 are composed of two types of amino acid repeats flanked by unique amino acid sequence termini.
Plant molecular biology, 1994Co-Authors: Helge Küster, Andreas M. Perlick, Alfred PühlerAbstract:Five cross-hybridizing cDNAs from clone group ‘VfNDS-L’ of a broadbean nodule-specific cDNA library differed by five specific in-frame deletions within the coding region. Northern blot analysis revealed that the transcripts represented by these clones were expressed in a nodule-specific way and therefore encoded a new family of broadbean Nodulins. These Nodulins have been designated Nvf-28/32. The Nvf-28/32 proteins were between 269 and 299 amino acids long with a high proportion of charged amino acids and contained a putative signal peptide of 20 amino acids. Sequence analysis indicated that the central part of the Nvf-28/32 proteins was composed of two different types of repeating amino acid stretches designated ‘repeat 1’ and ‘repeat 2’, whereas the N- and C-termini were unique. In contrast to ‘repeat 1’ stretches, which contained both positively and negatively charged amino acid residues, ‘repeat 2’ sequences did not contain any positively, but a total of 13 negatively charged amino acid residues. We could demonstrate that the five deletions identified exactly corresponded to complete repeats. The unique sequence termini of the Nvf-28/32 proteins displayed strong homologies to the late Nodulin 25 from alfalfa. In addition, the repeating units identified were significantly homologous to several, but not all exon parts of this protein. We speculate that the ‘VfNDS-L’ transcripts were derived from a differential splicing mechanism and that the Nvf-28/32 proteins fulfil a structural rather than an enzymatic function within the broadbean root nodule.
Hiroshi Kouchi - One of the best experts on this subject based on the ideXlab platform.
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Expression of genes encoding late Nodulins characterized by a putative signal peptide and conserved cysteine residues is reduced in ineffective pea nodules.
Molecular plant-microbe interactions : MPMI, 2002Co-Authors: Takashi Kato, Masanori Tamaoki, Hiroshi Kouchi, Kazuya Kawashima, Masami Miwa, Yoshifumi Mimura, Norio SuganumaAbstract:Five Nodulin genes, PsN1, PsN6, PsN314, PsN335, and PsN466, with reduced expression in ineffective nodules on the pea (Pisum sativum) mutant E135 (sym13) were characterized. They encode small polypeptides containing a putative signal peptide and conserved cysteine residues and show homology to the Nodulins PsENOD3/14 and PsNOD6. For each gene, multiple bands were detected by genomic Southern analysis. Northern analysis showed that all five genes were expressed exclusively in nodules and that their temporal expression patterns were similar to that of the leghemoglobin (Lb) gene during nodule development. Their transcripts were localized predominantly from the interzone II-III to the distal part of nitrogen-fixing zone III in effective nodules, resembling the Lb gene. However, transcripts in ineffective E135 nodules were localized in narrower regions than those in the effective nodules. These results indicate that these Nodulins are abundant in pea nodules and that their successive expression during nodule development is associated with nitrogen-fixing activity.
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Widespread Occurrence of the Homologues of the Early Nodulin (ENOD) Genes in Oryza Species and Related Grasses
Biochemical and biophysical research communications, 1999Co-Authors: Pallavolu M. Reddy, Ramesh K. Aggarwal, M. C. Ramos, Jagdish K. Ladha, Darshan S. Brar, Hiroshi KouchiAbstract:Abstract Eighty accessions representing 23 species from the genus Oryza were examined for the presence of homologues of early Nodulin ( ENOD ) genes. Southern analyses indicated a widespread distribution of homologues of ENOD genes across all the genomes of rice as well as other monocots. The degree of cross-hybridization of the legume ENOD genes with sequences in the genomes of various species, as revealed by hybridization differentials measured in terms of signal intensities, however, suggests that the homologues of ENOD genes are conserved to varied extents in different Oryza species. The presence of homologues of ENOD genes in a wide variety of plant species denotes that the biological functions of early Nodulins may be diverse, and not restricted to nodule organogenesis alone. The fact that ENOD gene homologues exist widely both in dicots and monocots provides evidence that these homologues have arisen from a common ancestral plant.
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Uninfected cell specific enhancement of a Nodulin-35 genomic gene expression in soybean nodules. Evidences by RT-PCR and chimeric promoter region: GUS reporter gene analysis using transgenic hairy root system of soybean
Plant Nutrition for Sustainable Food Production and Environment, 1997Co-Authors: Shigeyuki Tajima, Kenichi Takane, Hiroshi KouchiAbstract:Uricase II (Nodulin-35) is believed to regulate allantoin biosynthesis in nodules of various legumes. The mechanisms of nodule-specific enhancement of Nodulin-35 gene expression were studied in soybeans (Glycine max. L. cv. akisengoku). Northern blot hybridization assay detected only very low levels of Nodulin-35 gene expression in various non-symbiotic tissues of soybean plants; only in nodule tissues was the expression enhanced significantly. RT-PCR assay showing enhanced Nodulin-35 expression was observed in only one (UR9) of two Nodulin-35 genes (UR2 and UR9) in the soybean genome.
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Two Distinct Uricase II (Nodulin 35) Genes Are Differentially Expressed in Soybean Plants
Molecular plant-microbe interactions : MPMI, 1997Co-Authors: Kenichi Takane, Shigeyuki Tajima, Hiroshi KouchiAbstract:Nodule-specific uricase (uricase II) is a homotetramer of a 33-kDa polypeptide, Nodulin 35, and plays a key role in the assimilation of nitrogen fixed by microsymbionts in most legumes that have determinate nodules. We have isolated two distinct genes, UR2 and UR9, that encode for Nodulin 35 from a soybean genomic library. Their corresponding cDNAs were also isolated from a nodule cDNA library. UR2 and UR9 both encode for 309 amino acid proteins with 12 amino acid differences. The expression of these two genes in various organs of soybean was examined by reverse transcription-polymerase chain reaction with primers specific to each cDNA sequences. Expression of UR9 was almost specific in root nodules, although it was expressed in roots, primary leaves, and developing seed at very low levels. In contrast, the UR2 transcripts were present in almost all plant organs at low levels, but no enhancement of the expression was observed in nodules. Thus, UR9 behaves as a Nodulin gene, whereas UR2 is a nonsymbiotic u...
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Expression of Nodulin genes in plant-determined ineffective nodules of pea
Plant molecular biology, 1995Co-Authors: Norio Suganuma, Masanori Tamaoki, Hiroshi KouchiAbstract:The mutant E135 (sym 13) of pea (Pisum sativum L.) forms a normal number of small white nodules that contain bacteroids, but these bacteroids lack nitrogenase activity. To evaluate the effects of the sym 13 gene on the expression of Nodulin genes, cDNA clones for Nodulins were isolated from pea nodules and the expression of Nodulin genes in ineffective E135 nodules was compared with that in nitrogen-fixing nodules on the wild-type parent, cv. Sparkle. Nineteen cDNA clones for Nodulins, including ENOD2 and cDNAs for two distinct leghemoglobins (Lbs), were isolated from Sparkle nodules by a subtractive hybridization procedure. All the Nodulin genes examined were expressed in nodules on both E135 and Sparkle plants. However, the level of expression of seven genes, one of which was an Lb gene that corresponded to PsN5, was significantly lower in E135 nodules. The levels of Lb apo-proteins, with the exception of Lb-III and Lb-IV, in E135 nodules resembled those in Sparkle nodules, but the level of heme in E135 nodules was lower than that in Sparkle nodules. Although the expression of the two Lb genes that corresponded to PsN5 and PsN120 in E135 nodules was slightly derepressed by exogenous ammonia, the level of the PsN5 transcript was still lower than the control level in Sparkle nodules. Our results indicate that the plant gene sym 13 does not influence the induction of Nodulin genes but does influence the level of the expression of some genes, one of which is a gene for Lb, as well as the level of heme.