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At least 973 records · Page 54Linked to original sources
PubMed2000

Identification of alanine dehydrogenase and its role in mixed secretion of ammonium and alanine by pea bacteroids.

N2-fixation by Rhizobium-legume symbionts is of major ecological and agricultural importance, responsible for producing a substantial fraction of the biosphere's nitrogen. On the basis of 15N-labelling studies, it had been generally accepted that ammonium is the sole secretion product of N2-fixation by the bacteroid and that the plant is responsible for assimilating it into amino acids. However, this paradigm has been challenged in a recent 15N-labelling study showing that soybean bacteroids only secrete alanine. Hitherto, nitrogen secretion has only been assessed from in vitro 15N-labelling studies of isolated bacteroids. We show that both ammonium and alanine are secreted by pea bacteroids. The in vitro partitioning between them will depend on whether the system is open or closed, as well as the ammonium concentration and bacteroid density. To overcome these limitations we identified and mutated the gene for alanine dehydrogenase (aldA) and demonstrate that AldA is the primary route for alanine synthesis in isolated bacteroids. Bacteroids of the aldA mutant fix nitrogen but only secrete ammonium at a significant rate, resulting in lower total nitrogen secretion. Peas inoculated with the aldA mutant are green and healthy, demonstrating that ammonium secretion by bacteroids can provide sufficient nitrogen for plant growth. However, plants inoculated with the mutant are reduced in biomass compared with those inoculated with the wild type. The labelling and plant growth studies suggest that alanine synthesis and secretion contributes to the efficiency of N2-fixation and therefore biomass accumulation.

Alanine↗
PubMed1992

Temporal and spatial expression of a thiolprotease gene during pea ovary senescence, and its regulation by gibberellin.

Clones encoding a thiolprotease (tpp) have been isolated from a cDNA library of unpollinated, senescent pea ovaries and its pattern of expression during both ovary senescence and parthenocarpic development have been studied. The sequence of the tpp cDNA displays a high similarity with other plant and animal thiolproteases of the papain group. The homology is highest around the Cys-His of the active centre; a 109 amino acid sequence at the carboxy terminus was found to be homologous only to thiolproteases of plant origin; this part of the mRNA is also present in another pea mRNA that exhibits similar patterns of induction. tpp mRNA shows a temporal pattern of accumulation that precedes that observed for proteolytic activity. Such accumulation did not occur when ovaries were induced to grow parthenocarpically by gibberellic acid (GA) treatment; furthermore the initial low level of expression present in ovaries decreased after GA treatment, indicating that the gene is down-regulated by gibberellins. Spatially, tpp mRNA is localized mainly within the ovule and ovary vascular elements, and transiently within the endocarp of senescent ovaries. This pattern of expression precedes the development of the cytopathogenic effects observed as unpollinated ovaries undergo senescence.

Amino Acid Sequence↗
PubMed1993

Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei.

Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

Cell Nucleus↗
PubMed1993

Lipo-oligosaccharides of Rhizobium induce infection-related early nodulin gene expression in pea root hairs.

This paper shows that lipo-oligosaccharides (Nod factors) synthesized by Rhizobium bacteria elicit the induction of infection-related early nodulin genes (PsENOD5 and PsENOD12) in pea root hairs. R. leguminosarum bv. viciae secretes a mixture of Nod factors containing a C18 fatty acid chain with 4 (C18:4) or 1 double bond (C18:1). Purified Nod factors harbouring either a C18:4 or a C18:1 acyl moiety induce the expression of the pea early nodulin genes, PsENOD5 and PsENOD12, but the kinetics of induction are different. The expression of both early nodulin genes is induced in a transient manner by the purified Nod factors while a mixture of the Nod factors extends the period during which these genes are expressed. In spite of the host-specific nature of the infection process, heterologous Nod factors of R. meliloti also induce the expression of PsENOD5 and PsENOD12 genes, though with a marked delay compared with the homologous compounds.

Base Sequence↗
PubMed1993

The pea plastocyanin promoter directs cell-specific but not full light-regulated expression in transgenic tobacco plants.

A series of 5' deletions of the pea plastocyanin gene (petE) promoter fused to the beta-glucuronidase (GUS) reporter gene has been examined for expression in transgenic tobacco plants. Strong positive and negative cis-elements which modulate quantitative expression of the transgene in the light and the dark have been detected within the petE promoter. Disruption of a negative regulatory element at -784 bp produced the strongest photosynthesis-gene promoter so far described. Histochemical analysis demonstrated that all petE-GUS constructs directed expression in chloroplast-containing cells, and that a region from -176 bp to +4 bp from the translation start site was sufficient for such cell-specific expression. The petE-promoter fusions were expressed at high levels in etiolated transgenic tobacco seedlings but there was no marked induction of GUS activity in the light. The endogenous tobacco plastocyanin genes and the complete pea plastocyanin gene in transgenic tobacco plants were also expressed in the dark, but showed a three- to sevenfold increase in the light. This indicates a requirement for sequences 3' to the promoter for the full light response of the petE gene.

Base Sequence↗
PubMed1995

Induction kinetics of the nuclear proteins encoded by the early indoleacetic acid-inducible genes, PS-IAA4/5 and PS-IAA6, in pea (Pisum sativum L.).

The plant hormone indoleacetic acid (IAA) rapidly induces transcription of two genes, PS-IAA4/5 and PS-IAA6, in pea that encode nuclear proteins. The proteins were expressed in Escherichia coli and polyclonal antibodies were raised. The proteins can neither be detected on immunoblots of pea extracts from IAA-treated epicotyls nor subcellularly localized by immunofluorescence, suggesting that they are of low abundance. However, they can be immunoprecipitated as 35S-methionine-labeled proteins synthesized in vivo from control and IAA-treated tissue segments. Short-term time-course experiments indicate that the amounts of PS-IAA4/5 and PS-IAA6 proteins decrease dramatically in non-IAA-treated tissue. However, the hormone slightly increases the PS-IAA4/5 and significantly enhances the PS-IAA6 proteins compared with the initial amounts present in the tissue, despite a large induction of both mRNAs. A net increase in the amount of the in vivo synthesized PS-IAA6 is observed after a lag period of 30 min after addition of IAA. Little or no PS-IAA4/5 or PS-IAA6 protein is detected after 6 h of induction, even though PS-IAA4/5 and PS-IAA6 mRNAs remain detectable. Immunoprecipitation of in vitro translated polypeptides with mRNAs from various auxin-treated and untreated mono- and dicotyledonous plants reveals that similar proteins are encoded by constitutive or IAA-induced mRNAs. Phylogenetic analysis of 10 PS-IAA4-like proteins from various plant species reveals that the PS-IAA4 and PS-IAA6 proteins belong to different lineages, suggesting that they may have distinct functions. The data suggest that as a primary response to IAA plant tissues produce short-lived nuclear proteins whose synthesis is regulated at the transcriptional and post-transcriptional levels.

Amino Acid Sequence↗
PubMed1997

High mobility group proteins HMG-1 and HMG-I/Y bind to a positive regulatory region of the pea plastocyanin gene promoter.

A 268 bp region (P268) of the pea plastocyanin gene promoter responsible for high-level expression has been shown to interact with the high mobility group proteins HMG-1 and HMG-I/Y isolated from pea shoot chromatin. cDNAs encoding an HMG-1 protein of 154 amino acid residues containing a single HMG-box and a C-terminal acidic tail and an HMG-I/Y-like protein of 197 amino acid residues containing four AT-hooks have been isolated and expressed in Escherichia coli to provide large amounts of full-length proteins. DNase I footprinting identified eight binding sites for HMG-I/Y and six binding sites for HMG-1 in P268. Inhibition of binding by the antibiotic distamycin, which binds in the minor groove of A/T-rich DNA, revealed that HMG-I/Y binding was 400-fold more sensitive than HMG-1 binding. Binding-site selection from a pool of random oligonucleotides indicated that HMG-I/Y binds to oligonucleotides containing stretches of five or more A/T bp and HMG-1 binds preferentially to oligonucleotides enriched in dinucleotides such as TpT and TpG.

Amino Acid Sequence↗
PubMed1997

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants.

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyltransferase (FTase) gene was examined using transgenic expression of the beta-glucuronidase (GUS) gene fused to a 3.2 kb 5' upstream sequence of the gene encoding the pea FTase beta subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase beta subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.

Alkyl and Aryl Transferases↗
PubMed1997

Light-induced transcriptional repression of the pea AS1 gene: identification of cis-elements and transfactors.

Here, we examine the cis-elements and trans-factors affecting the expression of asparagine synthetase (AS) genes whose transcription is negatively regulated by light. The promoters for the AS1 and AS2 genes of pea were isolated, sequenced, and functionally dissected for their ability to confer regulated expression to the GUS reporter gene in transgenic tobacco. Histochemical analysis of transgenic plants demonstrated that the AS1 and AS2 promoters show identical patterns of cell-specific expression. The more highly active AS1 promoter was further demonstrated to confer negative light-regulation to the GUS gene in transgenic tobacco. Deletion analysis and gain-of-function experiments showed that 124 bp of the AS1 promoter was sufficient to confer light-activated repression to a heterologous promoter. Potential conserved transcription regulatory elements, Box B, Box C, and Box C' within this region were shown to bind to nuclear proteins by gel shift analysis. A light-specific DNA:protein interaction was detected with Box B. The nuclear factors that bind to Box C and C' elements of AS1 are competed by a putative repressor element 'RE1' defined previously in the oat phytochrome gene whose transcription is also repressed by light. The Box B and C/C'-Box/RE1-binding factors were found in nuclear extracts of tobacco, pea, and Arabidopsis and may therefore be universal factors involved in light-activated transcriptional repression.

Aspartate-Ammonia Ligase↗
PubMed1998

The characterization of gio, a new pea mutant, shows the role of indoleacetic acid in the control of fruit development by the apical shoot.

Fruit-set and fruit growth in pea (Pisum sativum L.) depend on gibberellins (GAs). The authors have isolated a new pea mutant, gio, which appeared spontaneously within the population of the cultivar Alaska, characterized by unpollinated ovaries much less sensitive to applied GAs. The mutant also has elongated peduncles, and is taller than the wild-type (WT) because the upper plant internodes are longer. Contrary to WT, the gio ovaries respond very little to benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid, but become fully sensitive to GA(3) when this hormone is applied together with BAP. The gio phenotype is determined by a mutation at a single mendelian locus. The mutation is recesive, shows incomplete penetrance, and its expression depends on environmental culture conditions. The sensitivity of the ovaries to GA(3) can be recovered by removing the apical shoot (plant decapitation) and by blocking the transport of indoleacetic acid (IAA) from the apical shoot with 2,3,5-triiodobenzoic acid. The content of IAA in methanolic extracts and phloematic exudates of the apical shoot of gio is about double that in the WT. The rate of transport of [(3)H]IAA applied to the apex of the mutant is also twice that in the WT. This indicates that the insensitivity of the gio ovaries to GAs is due to the inhibitory effect of the higher basipetal IAA transport from the shoot. The interaction between the fruit and the apical shoot mediated by IAA probably also involves cytokinins transported from the basal part of the plant.

Journal Article↗
PubMed1999

Phosphorylation of pea chloroplast acetyl-CoA carboxylase.

We have examined whether chloroplast acetyl-CoA carboxylase is a phosphoprotein. Pea (Pisum sativum) chloroplasts were incubated in the presence of [gamma-33P]-ATP and radiolabeled proteins were examined after immunoprecipitation with antibodies against all four known subunits of heteromeric chloroplast acetyl-CoA carboxylase. The beta-subunit of the carboxyltransferase was found to be labeled by 33P. Phosphoamino acid analysis of the immunoprecipitated beta-subunit of the carboxyltransferase indicates that it is phosphorylated on serine residues. Incorporation of 33P into carboxyltransferase beta-subunit decreased in chloroplasts transferred to dark conditions after labeling in the light. Dephosphorylation of pea chloroplast extracts by an alkaline phosphatase-agarose conjugate reduced in vitro acetyl-CoA carboxylase activity by 67%. Furthermore, while acetyl-CoA carboxylase activity and its carboxyltransferase half-reaction were reduced in dephosphorylated extracts, the biotin carboxylase half-reaction was not inhibited. The evidence presented here points to the carboxyltransferase beta-subunit of chloroplast acetyl-CoA carboxylase as a candidate for regulation by protein phosphorylation/dephosphorylation.

Journal Article↗
PubMed2000

Evidence that auxin promotes gibberellin A1 biosynthesis in pea.

In shoots of the garden pea, the bioactive gibberellin (GA1) is synthesised from GA20, and the enzyme which catalyses this step (a GA 3-oxidase -- PsGA3ox1) is encoded by Mendel's LE gene. It has been reported previously that decapitation of the shoot (excision of the apical bud) dramatically reduces the conversion of [3H]GA20 to [3H]GA1 in stems, and here we show that endogenous GA1 and PsGA3ox1 transcript levels are similarly reduced. We show also that these effects of decapitation are completely reversed by application of the auxin indole-3-acetic acid (IAA) to the 'stump' of decapitated plants. Gibberellin A20 is also converted to an inactive product, GA29, and this step is catalysed by a GA 2-oxidase, PsGA2ox1. In contrast to PsGA3ox1, PsGA2ox1 transcript levels were increased by decapitation and reduced by IAA application. Decapitation and IAA treatment did not markedly affect the level of GA1 precursors. It is suggested that in intact pea plants, auxin from the apical bud moves into the elongating internodes where it (directly or indirectly) maintains PsGA3ox1 transcript levels and, consequently, GA1 biosynthesis.

Catalysis↗
PubMed2000

Temporal and spatial regulation of pectic (1-->4)-beta-D-galactan in cell walls of developing pea cotyledons: implications for mechanical properties.

Modifications in cell wall pectic polysaccharides are thought to influence cell-cell adhesion and the mechanical properties of plant tissues. Monoclonal antibodies to epitopes occurring in homo- galacturonan and side chains of rhamnogalacturonan I have been used in an immunolocalization study of cell wall architecture of developing pea cotyledons. Pectic (1-->4)-beta-D-galactan appears in cotyledon cell walls at a defined stage late in development (approximately 26-30 days after anthesis), whereas homogalacturonan and pectic (1-->5)-alpha-L-arabinan are present in cotyledon cell walls throughout development. (1-->4)-beta-galactan was restricted to a distinct thin layer at the plasma membrane face of the cell wall. Anion exchange and immunoaffinity chromatography indicated that the (1-->4)-beta-galactan was associated with acidic pectic components. Mechanical compressive testing of pea cotyledons, before and after (1-->4)-beta-galactan appearance, indicated that the cotyledons with the galactan-rich cell wall layer were twice as firm as those with no detectable (1-->4)-beta-galactan.

Antibodies, Monoclonal↗
PubMed2000

Modulation by phytochrome of the blue light-induced extracellular acidification by leaf epidermal cells of pea (Pisum sativum l.): a kinetic analysis.

Blue light induces extracellular acidification, a prerequisite of cell expansion, in epidermis cells of young pea leaves, by stimulation of the proton pumping-ATPase activity in the plasma membrane. A transient acidification, reaching a maximum 2.5-5 min after the start of the pulse, could be induced by pulses as short as 30 msec. A pulse of more than 3000 micromol m-2 saturated this response. Responsiveness to a second light pulse was recovered with a time constant of about 7 min. The fluence rate-dependent lag time and sigmoidal increase of the acidification suggested the involvement of several reactions between light perception and activation of the ATPase. In wild-type pea plants, the fluence response relation for short light pulses was biphasic, with a component that saturates at low fluence and one that saturates at high fluence. The phytochrome-deficient mutant pcd2 showed a selective loss of the high-fluence component, suggesting that the high-fluence component is phytochrome-dependent and the low-fluence component is phytochrome-independent. Treatment with the calmodulin inhibitor W7 also led to the elimination of the phytochrome-dependent high-fluence component. Simple models adapted from the one used to simulate blue light-induced guard cell opening failed to explain one or more elements of the experimental data. The hypothesis that phytochrome and a blue light receptor interact in a short-term photoresponse is endorsed by model calculations based upon a three-step signal transduction cascade, of which one component can be modulated by phytochrome.

Acids↗
PubMed2001

Analysis of PEAM4, the pea AP1 functional homologue, supports a model for AP1-like genes controlling both floral meristem and floral organ identity in different plant species.

APETALA1 (AP1) and its homologue SQUAMOSA (SQUA) are key regulatory genes specifying floral meristem identity in the model plants Arabidopsis and Antirrhinum. Despite many similarities in their sequence, expression and functions, only AP1 appears to have the additional role of specifying sepal and petal identity. No true AP1/SQUA-functional homologues from any other plant species have been functionally studied in detail, therefore the question of how the different functions of AP1-like genes are conserved between species has not been addressed. We have isolated and characterized PEAM4, the AP1/SQUA-functional homologue from pea, a plant with a different floral morphology and inflorescence architecture to that of Arabidopsis or Antirrhinum. PEAM4 encodes for a polypeptide 76% identical to AP1, but lacks the C-terminal prenylation motif, common to AP1 and SQUA, that has been suggested to control the activity of AP1. Nevertheless, constitutive expression of PEAM4 caused early flowering in tobacco and Arabidopsis. In Arabidopsis, PEAM4 also caused inflorescence-to-flower transformations similar to constitutive AP1 expression, and was able to rescue the floral organ defects of the strong ap1-1 mutant. Our results suggest that the control of both floral meristem and floral organ identity by AP1 is not restricted to Arabidopsis, but is extended to species with diverse floral morphologies, such as pea.

Amino Acid Sequence↗
PubMed2002

Differential expression and functional analysis of three calmodulin isoforms in germinating pea (Pisum sativum L.) seeds.

Implication of the ubiquitous, highly conserved, Ca2+ sensor calmodulin (CaM) in pea seed germination has been investigated. Mass spectrometry analysis of purified CaM revealed the coexistence in seeds of three protein isoforms, diverging from each other by single amino acid substitution in the N-terminal alpha-helix. CaM was shown to be encoded by a small multigenic family, and full-length cDNAs of the three isoforms (PsCaM1, 2 and 3) were isolated to allow the design of specific primers in more divergent 5' and 3' untranslated regions. Expression studies, performed by semiquantitative RT-PCR, demonstrated differential expression patterns of the three transcripts during germination. PsCaM1 and 2 were detected at different levels in dry axes and cotyledons, and they accumulated during imbibition and prior to radicle protrusion. In contrast, PsCaM3 appeared only upon radicle protrusion, then gradually increased in both tissues. To characterise the biochemical properties of the CaM isoforms, functional analyses were conducted in vitro using recombinant Strep-tagged proteins (CaM1-ST, CaM2-ST and CaM3-ST) expressed in Escherichia coli. Gel mobility shift assays revealed that CaM1-ST exhibited a stoichiometric binding of a synthetic amphiphilic CaM kinase II peptide while CaM2-ST and CaM3-ST affinities for the same peptide were reduced. Affinity differences were also observed for CaM isoform binding to Trp-3, an idealised helical CaM-binding peptide. However, the three proteins activated in the same way the CaM-dependent pea NAD kinase. Finally, the significance of the single substitutions upon CaM interaction with its targets is discussed in a structural context.

Base Sequence↗
PubMed2002

A Ca2+/CaM-dependent kinase from pea is stress regulated and in vitro phosphorylates a protein that binds to AtCaM5 promoter.

An immuno-homologue of maize Ca2+/calmodulin (CaM)-dependent protein kinase with a molecular mass of 72 kDa was identified in pea. The pea kinase (PsCCaMK) was upregulated in roots in response to low temperature and increased salinity. Exogenous Ca2+ application increased the kinase level and the response was faster than that obtained following stress application. Low temperature-mediated, but not salinity-mediated stress kinase increase was inhibited by the application of EGTA and W7, a CaM inhibitor. The purification of PsCCaMK using immuno-affinity chromatography resulted in coelution of the kinase with another polypeptide of molecular mass 40 kDa (p40). Western blot revealed the presence of PsCCaMK in nuclear protein extracts and was found to phosphorylate p40 in vitro. Gel mobility shift and South-Western analysis showed that p40 is a DNA-binding protein and it interacted specifically with one of the cis acting elements of the Arabidopsis CaM5 gene (AtCaM5) promoter. The binding of p40 to the specific elements in the AtCaM5 promoter was dependent of its dephosphorylated state. Our results suggest that p40 could be an upstream signal component of the stress responses.

Arabidopsis↗
PubMed2000

A pea nuclear protein that is induced by dehydration belongs to the vicilin superfamily.

The purification to homogeneity of p16, a protein with an electrophoretic mobility compatible with an apparent molecular mass of 16 kDa, from nuclei of ungerminated pea embryonic axes is described. A cDNA clone of its gene, which was designated psp54, was also isolated. The psp54 cDNA contains an open reading frame coding for a 54.4-kDa polypeptide (p54). p16 corresponds to the C-terminal third of p54, although the mechanisms by which the primary polypeptide could be processed are not yet known. The sequence of p54 is 60% identical with that of the precursor of a sucrose-binding soybean protein, and, to a lesser extent (31-34%), it shares homology with some storage proteins. p16 is also 30% homologous with Nhp2p, a yeast nuclear protein. The psp54 gene, present in a single copy in pea genome, starts being expressed during seed desiccation. Soon after rehydration in seed germination, p54 mRNA disappears and is no longer detectable in vegetative tissues, except in response to hydric stress (exposure to abscisic acid, osmolites or desiccation). p16 can be recovered from nuclei cross-linked to histone H3, when the disulfide bridges that occur in vivo are preserved. On the other hand, p16 shares some properties with dehydrins, which are thought to protect cellular structures against desiccation. We propose that the possible precursor polypeptide p54 belongs to the vicilin superfamily, members of which play a variety of roles. The function of p16 may be related to the protection of chromatin structure against desiccation during seed development.

Amino Acid Sequence↗