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1.
Curr Opin Plant Biol ; 3(6): 488-92, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11074380

ABSTRACT

The size of plant cells is determined by genetic, structural and physical factors as well as by internal and external signals. Our knowledge of the molecular mechanisms of these controls is still rudimentary. Recent studies indicate that ploidy level exerts an important control on cell size. By increasing ploidy, endoreduplication may allow cells to reach extraordinary sizes. This process is widespread in plants and may provide a means to manipulate the cell volume.


Subject(s)
Cell Size , Plant Cells , Ploidies , Cell Cycle , Cell Division , DNA, Plant , Mutation , Plants/genetics
2.
Plant Mol Biol ; 43(5-6): 773-86, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11089876

ABSTRACT

The molecular mechanisms of de novo meristem formation, cell differentiation and the integration of the cell cycle machinery into appropriate stages of the developmental programmes are still largely unknown in plants. Legume root nodules, which house nitrogen-fixing rhizobia, are unique plant organs and their development may serve as a model for organogenetic processes in plants. Nodules form and are essential for the plant only under limitation of combined nitrogen in the soil. Moreover, their development is triggered by external mitogenic signals produced by their symbiotic partners, the rhizobia. These signals, the lipochitooligosaccharide Nod factors, act as host-specific morphogens and induce the re-entry of root cortical cells into mitotic cycles. Maintenance of cell division activity leads to the formation of a persistent nodule meristem from which cells exit continuously and enter the nodule differentiation programme, involving multiple cycles of endoreduplication and enlargement of nuclear and cell volumes. While the small diploid 2C cells remain uninfected, the large polyploid cells can be invaded and, after completing the differentiation programme, host the nitrogen-fixing bacteroids. This review summarizes the present knowledge on cell cycle reactivation and meristem formation in response to Nod factors and reports on a novel plant cell cycle regulator that can switch mitotic cycles to differentiation programmes.


Subject(s)
Medicago sativa/cytology , Sinorhizobium meliloti/physiology , Symbiosis , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Differentiation , Cyclins/metabolism , Growth Substances/metabolism , Lipopolysaccharides/metabolism , Medicago sativa/microbiology , Nitrogen Fixation , Plant Proteins/metabolism , Plant Roots/cytology , Plant Roots/microbiology , Sinorhizobium meliloti/pathogenicity
3.
Plant Physiol ; 124(3): 1373-80, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11080312

ABSTRACT

Using ion-selective microelectrodes, the problem of how signals coming from symbiotic partners or from potential microbial intruders are distinguished was investigated on root hairs of alfalfa (Medicago sativa). The Nod factor, NodRm-IV(C16:2,S), was used to trigger the symbiotic signal and (GlcNAc)(8) was selected from (GlcNAc)(4-8), to elicit defense-related reactions. To both compounds, root hairs responded with initial transient depolarizations and alkalinizations, which were followed by a hyperpolarization and external acidification in the presence of (GlcNAc)(8). We propose that alfalfa recognizes tetrameric Nod factors and N-acetylchitooligosaccharides (n = 4-8) with separate perception sites: (a) (GlcNAc)(4) and (GlcNAc)(6) reduced the depolarization response to (GlcNAc)(8), but not to NodRm-IV(C16:2, S); and (b) depolarization and external alkalization were enhanced when NodRm-IV(C16:2,S) and (GlcNAc)(8) were added jointly without preincubation. We suggest further that changes in cytosolic pH and Ca(2+) are key events in the transduction, as well as in the discrimination of signals leading to symbiotic responses or defense-related reactions. To (GlcNAc)(8), cells responded with a cytosolic acidification, and they responded to NodRm-IV(C16:2,S) with a sustained alkalinization. When both agents were added jointly, the cytosol first alkalized and then acidified. (GlcNAc)(8) and NodRm-IV(C16:2,S) transiently increased cytosolic Ca(2+) activity, whereby the response to (GlcNAc)(8) exceeded the one to NodRm-IV(C16:2,S) by at least a factor of two.


Subject(s)
Lipopolysaccharides/metabolism , Medicago sativa/metabolism , Oligosaccharides/metabolism , Calcium/metabolism , Cell Membrane , Cytosol/metabolism , Hydrogen-Ion Concentration , Medicago sativa/cytology , Models, Biological , Plant Roots/cytology , Plant Roots/metabolism
4.
Plant J ; 23(1): 73-83, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10929103

ABSTRACT

In plants multiple A-type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1-A3), while in animal somatic cells a single type of cyclin A is required for cell-cycle regulation from the S to M phases. We studied the function of an A2-type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A-type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti-Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2-M phase, the protein level after a rapid increase in S-phase reached a plateau during the G2 phase. In the yeast two-hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE-motif-containing cyclin-dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2-associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.


Subject(s)
Cell Cycle , Cyclin A/metabolism , Cyclin-Dependent Kinases/metabolism , Medicago sativa/metabolism , Retinoblastoma Protein/metabolism , Amino Acid Sequence , Base Sequence , Cell Nucleus/metabolism , Cells, Cultured , Cyclin A/chemistry , DNA Primers , Medicago sativa/cytology , Molecular Sequence Data , Protein Binding , Sequence Homology, Amino Acid , Zea mays/metabolism
5.
Mol Plant Microbe Interact ; 13(8): 799-807, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10939251

ABSTRACT

Nod factors (NFs) are rhizobial lipo-chitooligosaccharide signals that trigger root nodule development in legumes. Modifications of NF structures influence their biological activity and affect their degradation by plant chitinases. Nodulation of certain pea cultivars by Rhizobium leguminosarum bv. viciae requires modification of NFs at the reducing end by either an O-acetyl or a fucosyl group. Fucosylated NFs were produced by an in vitro reaction with NodZ fucosyltransferase and purified. Their biological activity on pea was tested by measuring their capacity to stimulate the activity of a hydrolase that cleaves NFs. Nonmodified and fucosylated NFs displayed this activity at nano- to picomolar concentrations, while a sulfated NF from Sinorhizobium meliloti was inactive. In an additional series of experiments, the stability of non-modified and fucosylated NFs in the presence of purified tobacco chitinases was compared. The presence of the fucosyl group affected the degradation rates and the accessibility of specific cleavage sites on the chitooligosaccharide backbone. These results suggest that the fucosyl group in NFs also weakens the interaction of NFs with certain chitinases or chitinase-related proteins in pea roots.


Subject(s)
Chitinases/metabolism , Fucose/metabolism , Lipopolysaccharides/metabolism , Pisum sativum/metabolism , Rhizobium leguminosarum/metabolism , Chromatography, High Pressure Liquid , Kinetics , Lipopolysaccharides/isolation & purification , Plant Roots/metabolism , Plants, Toxic , Nicotiana/enzymology
6.
Mol Plant Microbe Interact ; 13(1): 62-71, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10656586

ABSTRACT

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.


Subject(s)
Expressed Sequence Tags , Medicago sativa/metabolism , Plant Roots/metabolism , Blotting, Northern , Gene Library , Leghemoglobin/metabolism , Medicago sativa/genetics , Medicago sativa/microbiology , Molecular Sequence Data , Plant Roots/microbiology , RNA, Plant/metabolism , Sinorhizobium meliloti/physiology , Symbiosis
7.
Mol Plant Microbe Interact ; 13(1): 72-9, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10656587

ABSTRACT

Nod factors excreted by rhizobia are signal molecules that consist of a chitin oligomer backbone linked with a fatty acid at the nonreducing end. Modifications of the Nod factor structures influence their stability in the rhizosphere and their biological activity. To test the function of N-acetyl groups in Nod factors, NodSm-IV(C16:2,S) from Sinorhizobium meliloti was enzymatically N-deacetylated in vitro with purified chitin deacetylase from Colletotrichum lindemuthianum. A family of partially and completely deacetylated derivatives was produced and purified. The most abundant chemical structures identified by mass spectrometry were GlcN(C16:2)-GlcNAc-GlcNH2-GlcNAc(OH)(S), GlcN(C16,2)-GlcNAc-GlcNH2-GlcNH2(OH)(S), and GlcN(C16:2)-GlcNH2-GlcNH2-GlcNH2(OH)(S). In contrast to NodSm-IV(C16:2,S), the purified N-deacetylated derivatives were stable in the rhizosphere of Medicago sativa, indicating that the N-acetyl groups make the carbohydrate moiety of Nod factors accessible for glycosyl hydrolases of the host plant. The N-deacetylated derivatives displayed only a low level of activity in inducing root hair deformation. Furthermore, the N-deacetylated molecules were not able to stimulate Nod factor degradation by M. sativa roots, a response elicited by active Nod factors. These data show that N-acetyl groups of Nod factors are required for biological activity.


Subject(s)
Medicago sativa/metabolism , Oligosaccharides/metabolism , Sinorhizobium meliloti/metabolism , Amidohydrolases/chemistry , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Mass Spectrometry , Medicago sativa/microbiology , Molecular Sequence Data , Oligosaccharides/chemistry , Oligosaccharides/isolation & purification , Symbiosis
8.
Electrophoresis ; 21(17): 3823-32, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11271500

ABSTRACT

Extractable proteins from Sinorhizobium meliloti strains AK631 and EK698 (a Tn5-induced noIR-deficient mutant of AK631), grown in tryptone agar (TA) medium with or without the addition of the plant signal luteolin, were separated by two-dimensional gel electrophoresis and compared. Analysis of silver-stained gels showed that the noIR mutant had 189 proteins that were significantly altered in their levels (101 protein spots up- and 88 downregulated). Coomassie-stained preparative two-dimensional (2-D) gels or polyvinylidene difluoride (PVDF) membranes blotted from preparative gels showed that at least 52 of the altered proteins could be reproducibly detected and isolated from the noIR mutant. These 52 altered protein spots were classified into five groups based on an assessment of protein abundance by computer analysis and the effect of the presence or absence of luteolin addition to the growth medium. N-terminal microsequencing of 38 proteins revealed that the most striking feature of the consequence of the noIR mutation is the number and broad spectrum of cellular functions that are affected by the loss of the NoIR function. These include proteins involved in the tricarboxylic acid (TCA) cycle, heat shock and cold shock proteins, protein synthesis, a translation elongation factor, oxidative stress and cell growth and maintenance functions. We propose that the NoIR repressor is a global regulatory protein which responds to environmental factors to fine-tune intracellular metabolism.


Subject(s)
Bacterial Proteins/analysis , Sinorhizobium meliloti/chemistry , Bacterial Proteins/classification , Bacterial Proteins/genetics , Electrophoresis, Gel, Two-Dimensional/methods , Genes, Bacterial , Mutagenesis , Peptide Mapping/methods , Proteome
9.
Plant Cell Rep ; 19(7): 647-653, 2000 Jun.
Article in English | MEDLINE | ID: mdl-30754800

ABSTRACT

A high frequency of embryogenesis and transformation from all parts of flowers of two lines of Medicago truncatula R-108-1 and Jemalong J5 were obtained. Using this flower system, we obtained transgenic plants expressing promoter-uidA gene fusions as well as the gfp living cell color reporter gene. Moreover, this method allows us to save time and to use a smaller greenhouse surface for the culture of donor plants. Southern hybridization showed that the internal gfp fragment had the expected size and the number of T-DNA copies integrated in the plant genome varied between one and three. These data suggest that the presence of the GFP protein has no toxic effects, since no rearrangement of the gfp reporter gene was detected in the regenerated plants.

10.
Plant Physiol ; 121(1): 273-80, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10482683

ABSTRACT

In root hairs of alfalfa (Medicago sativa), the requirement of Ca(2+) for Nod factor signaling has been investigated by means of ion-selective microelectrodes. Measured 50 to 100 microm behind the growing tip, 0.1 microM NodRm-IV(C16:2,S) increased the cytosolic free [Ca2+] by about 0.2 pCa, while the same concentration of chitotetraose, the nonactive glucosamine backbone, had no effect. We demonstrate that NodRm-IV(C16:2,S) still depolarized the plasma membrane at external Ca(2+) concentrations below cytosolic values if the free EGTA concentration remained low (

Subject(s)
Calcium Signaling/drug effects , Calcium/metabolism , Lipopolysaccharides/pharmacology , Medicago sativa/metabolism , Calcium/antagonists & inhibitors , Calcium/pharmacology , Calcium-Transporting ATPases/antagonists & inhibitors , Calcium-Transporting ATPases/metabolism , Chelating Agents/pharmacology , Cytosol/metabolism , Dose-Response Relationship, Drug , Egtazic Acid/pharmacology , Hydrogen-Ion Concentration , Hydroquinones/pharmacology , Lipopolysaccharides/antagonists & inhibitors , Medicago sativa/cytology , Medicago sativa/drug effects , Membrane Potentials/drug effects , Microelectrodes , Plant Roots/cytology , Plant Roots/drug effects , Plant Roots/metabolism , Strontium/pharmacology
11.
Planta ; 209(2): 207-212, 1999 Aug 12.
Article in English | MEDLINE | ID: mdl-10436223

ABSTRACT

Using Ca(2+)-selective microelectrodes, the concentration of free calcium ([Ca(2+)]) in the cytosol has been measured in root hair cells of Medicago sativa L. in the presence of nodulation (Nod) factors. Growing root hairs of M. sativa displayed a steep apical [Ca(2+)] gradient, i.e. 604-967 nM in the tip compared with 95-235 nM in the basal region. When tested within the first 5 to 10 µm of the tip, addition of NodRm-IV(C16:2,S) decreased the cytosolic [Ca(2+)], whereas an increase was observed when tested behind the tip. Overall, this led to a partial dissipation of the [Ca(2+)] gradient. The Ca(2+) response was specific: it was equally well observed in the presence of NodRm-IV(Ac,C16:2,S), reduced with NodRm-IV(C16:0,S), but not with chitotetraose, the nonactive glucosamine backbone. In contrast to growing root hairs, non-growing root hairs without a tip-to-base cytosolic [Ca(2+)] gradient responded to NodRm-IV(C16:2,S) with an increase in cytosolic [Ca(2+)] at the tip as well as at the root hair base. We suggest that the response to Nod factors depends on the stage of development of the root hairs, and that changes in cytosolic [Ca(2+)] may play different roles in Nod-factor signaling: changes of cytosolic [Ca(2+)] in the apical part of the root hair may be related to root hair deformation, while the increase in [Ca(2+)] behind the tip may be essential for the amplification of the Nod signal, for its propagation and transduction to trigger downstream events.

12.
EMBO J ; 18(16): 4476-84, 1999 Aug 16.
Article in English | MEDLINE | ID: mdl-10449413

ABSTRACT

Plant organs develop mostly post-embryonically from persistent or newly formed meristems. After cell division arrest, differentiation frequently involves endoreduplication and cell enlargement. Factors controlling transition from mitotic cycles to differentiation programmes have not been identified yet in plants. Here we describe ccs52, a plant homologue of APC activators involved in mitotic cyclin degradation. The ccs52 cDNA clones were isolated from Medicago sativa root nodules, which exhibit the highest degree of endopolyploidy in this plant. ccs52 represents a small multigenic family and appears to be conserved in plants. Overexpression of ccs52 in yeast triggered mitotic cyclin degradation, cell division arrest, endoreduplication and cell enlargement. In Medicago, enhanced expression of ccs52 was found in differentiating cells undergoing endoreduplication. In transgenic M.truncatula plants, overexpression of the ccs52 gene in the antisense orientation resulted in partial suppression of ccs52 expression and decreased the number of endocycles and the volume of the largest cells. Thus, the ccs52 product may switch proliferating cells to differentiation programmes which, in the case of endocycles, result in cell size increments.


Subject(s)
Cell Cycle Proteins/physiology , Genes, Plant , Mitosis , Multigene Family , Plant Proteins/physiology , Schizosaccharomyces pombe Proteins , Amino Acid Sequence , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Differentiation , Cell Division , Cell Size , Cyclin B/metabolism , Fungal Proteins/physiology , Gene Expression Regulation, Plant , Humans , Medicago sativa/genetics , Molecular Sequence Data , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified , Ploidies , Repetitive Sequences, Nucleic Acid , Schizosaccharomyces , Sequence Homology, Amino Acid
13.
Plant J ; 18(4): 431-41, 1999 May.
Article in English | MEDLINE | ID: mdl-10406126

ABSTRACT

We have characterized from the legume plant Medicago a new family of miniature inverted-repeat transposable elements (MITE), called the Bigfoot transposable elements. Two of these insertion elements are present only in a single allele of two different M. sativa genes. Using a PCR strategy we have isolated 19 other Bigfoot elements from the M. sativa and M. truncatula genomes. They differ from the previously characterized MITEs by their sequence, a target site of 9 bp and a partially clustered genomic distribution. In addition, we show that they exhibit a significantly stable secondary structure. These elements may represent up to 0.1% of the genome of the outcrossing Medicago sativa but are present at a reduced copy number in the genome of the autogamous M. truncatula plant, revealing major differences in the genome organization of these two plants.


Subject(s)
DNA Transposable Elements , Medicago sativa/genetics , Plant Proteins/genetics , Amino Acid Sequence , Base Sequence , Genome, Plant , Molecular Sequence Data , Plant Proteins/chemistry , Polymerase Chain Reaction , Promoter Regions, Genetic , Protein Structure, Secondary , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
14.
Plant Cell Rep ; 17(5): 345-355, 1998 Mar.
Article in English | MEDLINE | ID: mdl-30736570

ABSTRACT

We describe a simple and efficient protocol for regeneration-transformation of two diploid Medicago lines: the annual M. truncatula R108-1(c3) and the perennial M. sativa ssp. falcata (L.) Arcangeli PI.564263 selected previously as highly embryogenic genotypes. Here, embryo regeneration of R108-1 to complete plants was further improved by three successive in vitro regeneration cycles resulting in the line R108-1(c3). Agrobacterium tumefaciens-mediated transformation of leaf explants was carried out with promoter-gus constructs of two early nodulins (MsEnod12A and MsEnod12B) and one late nodulin (Srglb3). The transgenic plants thus produced on all explants within 3-4 months remained diploid and were fertile. This protocol appears to be the most efficient and fastest reported so far for leguminous plants.

15.
Proc Natl Acad Sci U S A ; 94(16): 8901-6, 1997 Aug 05.
Article in English | MEDLINE | ID: mdl-11038563

ABSTRACT

Under nitrogen-limiting conditions Rhizobium meliloti can establish symbiosis with Medicago plants to form nitrogen-fixing root nodules. Nodule organogenesis starts with the dedifferentiation and division of root cortical cells. In these cells the early nodulin gene enod40, which encodes an unusually small peptide (12 or 13 amino acids), is induced from the beginning of this process. Herein we show that enod40 expression evokes root nodule initiation. (i) Nitrogen-deprived transgenic Medicago truncatula plants overexpressing enod40 exhibit extensive cortical cell division in their roots in the absence of Rhizobium. (ii) Bombardment of Medicago roots with an enod40-expressing DNA cassette induces dedifferentiation and division of cortical cells and the expression of another early nodulin gene, Msenod12A. Moreover, transient expression of either the enod40 region spanning the oligopeptide sequence or only the downstream region without this sequence induces these responses. Our results suggest that the cell-specific growth response elicited by enod40 is involved in the initiation of root nodule organogenesis.

16.
Plant Mol Biol ; 34(5): 771-80, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9278167

ABSTRACT

Rhizobium meliloti can interact symbiotically with Medicago plants thereby inducing the formation of root nodules. Screening of a young nodule cDNA library led to the isolation of a cDNA from Medicago sativa, Msgbl, that comprises a new member of the RACK1 (Receptor of Activated C Kinase) subfamily of WD-repeat proteins. This subfamily shows homology to the beta-subunit of heterotrimeric G proteins. Besides RACK1, this subfamily contains several plant genes including the well characterized auxin-inducible ArcA of tobacco. The Msgbl gene is strongly expressed in young embryos and in leaves, and is induced upon cytokinin treatment of roots. Whereas northern analysis failed to reveal differences in expression between total RNA from roots and nodules, in situ hybridization demonstrated that the transcript was most abundant in dividing cells of nodule primordia and in the nodule meristem. Msgbl may be related to the signal transduction acting in response to hormone-mediated cell division.


Subject(s)
Cytokinins/physiology , Genes, Plant/physiology , Indoleacetic Acids/physiology , Medicago sativa/genetics , Repetitive Sequences, Nucleic Acid/physiology , Amino Acid Sequence , Base Sequence , Blotting, Southern , Cell Division/drug effects , Cloning, Molecular , Cytokinins/pharmacology , GTP-Binding Proteins/biosynthesis , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Gene Expression Regulation, Plant , Genes, Plant/drug effects , Genome, Plant , Indoleacetic Acids/pharmacology , Medicago sativa/cytology , Medicago sativa/drug effects , Molecular Sequence Data , Plant Proteins/biosynthesis , Plant Proteins/chemistry , Plant Proteins/genetics , Repetitive Sequences, Nucleic Acid/drug effects
17.
Plant Cell ; 9(2): 223-35, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9061953

ABSTRACT

The eukaryotic cell division cycle is coordinated by cyclin-dependent kinases (CDKs), represented by a single major serine/threonine kinase in yeasts (Cdc2/CDC28) and a family of kinases (CDK1 to CDK8) in human cells. Previously, two cdc2 homologs, cdc2MsA and cdc2MsB, have been identified in alfalfa (Medicago sativa). By isolating cDNAs using a cdc2MsA probe, we demonstrate here that at least four additional cdc2 homologous genes are expressed in the tetraploid alfalfa. Proteins encoded by the new cdc2MsC to cdc2MsF cDNAs share the characteristic functional domains of CDKs with the conserved and plant-specific sequence elements. Transcripts from cdc2MsA, cdc2MsB, cdc2MsC, and cdc2MsE genes are synthesized throughout the cell cycle, whereas the amounts of cdc2MsD and cdc2MsF mRNAs peak during G2-to-M phases. The translation of Cdc2MsA/B, Cdc2MsD, and Cdc2MsF proteins follows the pattern of transcript accumulation. The multiplicity of kinase complexes with cell cycle phase-dependent activities was revealed by in vitro phosphorylation experiments. Proteins bound to p13suc1-Sepharose or immunoprecipitated with Cdc2MsA/B antibodies from cells at G1-to-S and G2-to-M phase boundaries showed elevated kinase activities. the Cdc2MsF antibodies separated a G2-to-M phase-related kinase complex. Detection of histone H1 phosphorylation activities in fractions immunoprecipitated with antimitotic cyclin (CyclinMs2) antibodies from G2-to-M phase cells indicates the complex formation between this cyclin and a kinase partner in alfalfa. The observed fluctuation of transcript levels, amounts, and activities of kinases in different cell cycle phases reflects a multilevel regulatory system during cell cycle progression in plants.


Subject(s)
Cyclin-Dependent Kinases/metabolism , Medicago sativa/enzymology , Amino Acid Sequence , Aphidicolin/pharmacology , Cell Cycle , Gene Expression Regulation, Enzymologic , Humans , Medicago sativa/cytology , Molecular Sequence Data , Protein Kinases/metabolism , RNA, Messenger/metabolism , Sequence Alignment
18.
Plant Physiol ; 108(4): 1607-1614, 1995 Aug.
Article in English | MEDLINE | ID: mdl-12228566

ABSTRACT

Extracellular enzymes from alfalfa (Medicago sativa L.) involved in the degradation of nodulation (Nod) factors could be distinguished by their different cleavage specificities and were separated by lectin affinity chromatography. A particular glycoprotein was able to release an acylated lipo-disaccharide from all tested Nod factors having an oligosaccharide chain length of four or five residues. Structural modifications of the basic lipo-chitooligosaccharide did not affect the cleavage site and had only weak influence on the cleavage efficiency of Nod factors tested. The acylated lipo-trisaccharide was resistant to degradation. When alfalfa roots were preincubated with Nod factors at nanomolar concentrations, the activity of the dimer-forming enzyme was stimulated up to 6-fold within a few hours. The inducing activity of Nod factors decreased in the order NodRm-IV(C16:2,Ac,S) > NodRm-IV(C16:2,S) and NodRm-V(C16:2,Ac,S) > NodRm-V(C16:2,S) > NodRm-IV(C16:0,S) > NodRm-IV(C16:2). Pretreatment with NodRm-III(C16:2) as well as unmodified chitooligosaccharides did not stimulate the dimer-forming enzyme. Roots preincubated with Rhizobium meliloti showed similar stimulation of the dimer-forming activity. Mutant strains unable to produce Nod factors did not enhance the hydrolytic activity. These results indicate a rapid feedback inactivation of Nod signals after their perception by the host plant alfalfa.

19.
Plant Mol Biol ; 27(6): 1059-70, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7766889

ABSTRACT

Cyclins in association with the protein kinase p34cdc2 and related cyclin-dependent protein kinases (cdks) are key regulatory elements in controlling the cell division cycle. Here, we describe the identification and characterization of a full-length cDNA clone of alfalfa mitotic cyclin, termed CycIIIMs. Computer analysis of known plant cyclin gene sequences revealed that this cyclin belongs to the same structural group as the other known partial alfalfa cyclin sequences. Genetic segregation analysis based on DNA-DNA hybridization data showed that the CycIIIMs gene(s) locates in a single chromosomal region on linkage group 5 of the alfalfa genetic map between RFLP markers UO89A and CG13. The assignment of this cyclin to the mitotic cyclin class was based on its cDNA-derived sequence and its differential expression during G2/M cell cycle phase transition of a partially synchronized alfalfa cell culture. Sequence analysis indicated common motifs with both the A- and B-types of mitotic cyclins similarly to the newly described B3-type of animal cyclins.


Subject(s)
Chromosome Mapping , Cyclins/genetics , Medicago sativa/genetics , Mitosis/genetics , Plant Proteins/genetics , Amino Acid Sequence , Base Sequence , Cells, Cultured , Cloning, Molecular , DNA, Complementary , Diploidy , Gene Expression , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid
20.
Proc Natl Acad Sci U S A ; 92(7): 2706-9, 1995 Mar 28.
Article in English | MEDLINE | ID: mdl-7708710

ABSTRACT

The Rhizobium common nod gene products NodABC are involved in the synthesis of the core lipochitooligosaccharide (Nod factor) structure, whereas the products of the host-specific nod genes are necessary for diverse structural modifications, which vary in different Rhizobium species. The sulfate group attached to the Rhizobium meliloti Nod signal is necessary for activity on the host plant alfalfa, while its absence renders the Nod factor active on the non-host plant vetch. This substituent is therefore a major determinant of host specificity. The exact biosynthetic pathway of Nod factors has not been fully elucidated. In particular, it is not known why some chemical modifications are introduced with high fidelity whereas others are inaccurate, giving rise to a family of different Nod factor structures produced by a single Rhizobium strain. Using protein extracts and partially purified recombinant NodH protein obtained from Escherichia coli expressing the R. meliloti nodH gene, we demonstrate here NodH-dependent in vitro sulfotransferase activity. Kinetic analyses with Nod factors, chitooligosaccharides, and their deacetylated derivatives revealed that Nod factors are the preferred substrate for the sulfate transfer. Moreover, the tetrameric Nod factor, NodRm-IV, was a better substrate than the trimer, NodRm-III, or the pentamer, NodRm-V. These data suggest that the core lipochitooligosaccharide structure must be synthesized prior to its host-specific modification with a sulfate group. Since in R. meliloti tetrameric Nod factors are the most abundant and the most active ones, high affinity of NodH for the appropriate tetrameric substrate guarantees its modification and thus contributes to the fidelity of host-specific behavior.


Subject(s)
Bacterial Proteins , Lipopolysaccharides/metabolism , Oligosaccharides/chemistry , Sinorhizobium meliloti/enzymology , Sulfotransferases/metabolism , Carbohydrate Sequence , Carbon Radioisotopes , Chromatography, High Pressure Liquid , Kinetics , Lipopolysaccharides/chemistry , Lipopolysaccharides/isolation & purification , Molecular Sequence Data , Oligosaccharides/metabolism , Recombinant Proteins/metabolism , Sinorhizobium meliloti/genetics , Sulfates/metabolism , Sulfotransferases/isolation & purification , Time Factors
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