RESUMO
Auxin response factors (ARFs) are plant transcription factors that activate or repress the expression of auxin-responsive genes and accordingly, play key roles in auxin-mediated developmental processes. Here we identified and characterized the Solanum lycopersicum (tomato) ARF10 homolog (SlARF10), demonstrated that it is posttranscriptionally regulated by Sl-miR160, and investigated the significance of this regulation for tomato development. In wild-type tomato, SlARF10 is primarily expressed in the pericarp of mature and ripened fruit, showing an expression profile complementary to that of Sl-miR160. Constitutive expression of wild-type SlARF10 did not alter tomato development. However, transgenic tomato plants that constitutively expressed the Sl-miR160a-resistant version (mSlARF10) developed narrow leaflet blades, sepals and petals, and abnormally shaped fruit. During compound leaf development, mSlARF10 accumulation specifically inhibited leaflet blade outgrowth without affecting other auxin-driven processes such as leaflet initiation and lobe formation. Moreover, blade size was inversely correlated with mSlARF10 transcript levels, strongly implying that the SlARF10 protein, which was localized to the nucleus, can function as a transcriptional repressor of leaflet lamina outgrowth. Accordingly, known auxin-responsive genes, which promote cell growth, were downregulated in shoot apices that accumulated increased mSlARF10 levels. Taken together, we propose that repression of SlARF10 by Sl-miR160 is essential for auxin-mediated blade outgrowth and early fruit development.
Assuntos
Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/genética , Sequência de Aminoácidos , Sequência de Bases , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Ácidos Indolacéticos/metabolismo , Solanum lycopersicum/metabolismo , MicroRNAs/genética , Dados de Sequência Molecular , Filogenia , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Processamento de Proteína Pós-Traducional , RNA de Plantas/genética , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Expression of bar, a phosphinothricin acetyltransferase, in plant tissues, leads to resistance of these plants to glufosinate ammonium based herbicides. We have created a bar expressing, attenuated zucchini yellow mosaic potyvirus-vector, AGII-Bar, to enable herbicide use in cucurbit fields. The parental vector, ZYMV-AGII, has been rendered environmentally safe by both disease-symptom attenuation and aphid-assisted virus transmission abolishment. The recombinant AGII-Bar virus-encoding cDNA, when inoculated on diverse cucurbits was highly infectious, accumulated to similar levels as AGII, and elicited attenuated AGII-like symptoms. Potted cucurbits inoculated with AGII-Bar became herbicide resistant about a week post-inoculation. Herbicide resistance was sustained in squash over a period of at least 26 days and for at least 60 days in cucumber grown in a net-house under commercial conditions. To test the applicability of AGII-Bar use in a weed-infested field, a controlled experiment including more than 450 plants inoculated with this construct, was performed. Different dosages of glufosinate ammonium were sprayed, 2 weeks after planting, on the foliage of melons, cucumbers, squash, and watermelons. AGII-Bar provided protection to all inoculated plants, of every variety tested, at each dosage applied, including the highest doses that totally eradicated weeds. This study demonstrates that AGII-Bar can be utilized to facilitate weed control in cucurbits and exemplifies the practical potential of attenuated virus-vector use in agriculture.
Assuntos
Agricultura/métodos , Cucurbita/fisiologia , Cucurbita/virologia , Vetores Genéticos , Herbicidas/farmacologia , Potyvirus/genética , Aminobutiratos/farmacologia , Cucurbita/efeitos dos fármacos , Relação Dose-Resposta a DrogaRESUMO
Systematic deletion and peptide tagging of the amino-terminal domain (NT, ~43 amino acids) of an attenuated zucchini yellow mosaic potyvirus (ZYMV-AGII) coat protein (CP) were used to elucidate its role in viral systemic infection. Deletion mutants truncated by 8, 13, and 33 amino acid residues from the CP-NT 5' end were systemically infectious and produced symptoms similar to those of the AGII virus. Tagging these deletion mutants with either human c-Myc (Myc) or hexahistidine peptides maintained viral infectivity. Similarly, addition of these peptides to the intact AGII CP-NT did not affect viral life cycle. To determine which parts, if any, of the CP-NT are essential for viral systemic infection, a series of Myc-tagged mutants with 8 to 43 amino acids removed from the CP-NT were constructed. All Myc-tagged CP-NT deletion mutants, including those from which virtually all the viral CP-NT had been eliminated, were able to encapsidate and cause systemic infection. Furthermore, chimeric viruses with deletions of up to 33 amino acids from CP-NT produced symptoms indistinguishable from those caused by the parental AGII virus. In contrast to CP-NT Myc fusion, addition of the foot-and-mouth disease virus (FMDV) immunogenic epitope to AGII CP-NT did not permit systemic infection. However, fusion of the Myc peptide to the N terminus of the FMDV peptide restored the capability of the virus to spread systemically. We have demonstrated that all CP-NT fused peptides were exposed on the virion surface, masking natural CP immunogenic determinants. Our findings demonstrate that CP-NT is not essential for ZYMV spread and that it can be replaced by an appropriate foreign peptide while maintaining systemic infectivity.
Assuntos
Proteínas do Capsídeo , Capsídeo/fisiologia , Doenças das Plantas/virologia , Potyvirus/fisiologia , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Capsídeo/química , Capsídeo/genética , Deleção de Genes , Genes myc , Teste de Complementação Genética , Histidina/genética , Histidina/metabolismo , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos/química , Potyvirus/química , Replicação ViralRESUMO
PURPOSE: To analyze the postoperative outcome and complication rate after cataract extraction or lensectomy with primary intraocular lens (IOL) implantation for penetrating traumatic cataract. DESIGN: Retrospective, nonconsecutive, noncomparative case series. METHODS: We retrospectively reviewed the files of 21 patients who were admitted to our departments because of traumatic cataract with corneal or scleral laceration caused by penetrating trauma with or without intraocular foreign body (IOFB) from 1992 through 1997. Lens aspiration or manual extracapsular cataract extraction with primary IOL implantation was performed in all patients. Removal of an IOFB was performed in eight patients. MAIN OUTCOME MEASURES: Final visual acuity and deviation of actual refraction from emmetropia and from expected postoperative refraction. RESULTS: The mean follow-up was 20.4 months. Fourteen eyes (67%) achieved final visual acuity of 20/40 or better, 95% obtained 20/60 or better final visual acuity, and all eyes achieved 20/100 or better final visual acuity. Major causes of limited visual acuity were central corneal scar and central retinal injury. Eleven eyes (57%) experienced secondary cataract and underwent neodymium:yytrium-aluminum-garnet capsulotomy. CONCLUSIONS: Primary implantation of posterior chamber lenses after penetrating ocular trauma is associated with favorable visual outcome and a low rate of postoperative complications.
Assuntos
Extração de Catarata , Lesões da Córnea , Corpos Estranhos no Olho/cirurgia , Ferimentos Oculares Penetrantes/cirurgia , Implante de Lente Intraocular , Cristalino/lesões , Esclera/lesões , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Estudos Retrospectivos , Resultado do Tratamento , Acuidade VisualRESUMO
Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and can be transmitted in the field. A novel attenuated zucchini yellow mosaic potyvirus (AG) was used for the development of an environmentally safe non-pathogenic virus vector. The suitability of AG as an expression vector in plants was tested by analysis of two infectious viral constructs, each containing a distinct gene insertion site. Introduction of a foreign viral coat protein gene into AG genome between the P1 and HC-Pro genes, resulted in no expression in planta. In contrast, the same gene was stably expressed when inserted between NIb and CP genes, suggesting that this site is more suitable for a gene vector. Virus-mediated expression of reporter genes was observed in squash and cucumber leaves, stems, roots and edible fruit. Furthermore, AG stably expressed human interferon-alpha 2, an important human anti-viral drug, without affecting plant development and yield. Interferon biological activity was measured in cucumber and squash fruit. Together, these data corroborate a biotechnological utility of AG as a non-pathogenic vector for the expression of a foreign gene, as a benefit trait, in cucurbits and their edible fruit.
Assuntos
Cucurbitaceae/genética , Engenharia Genética/métodos , Potyvirus/genética , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Animais , Afídeos/virologia , Sequência de Bases , Cucumovirus/genética , Cucurbitaceae/virologia , Cisteína Endopeptidases/genética , RNA Polimerases Dirigidas por DNA , Genes Reporter , Proteínas de Fluorescência Verde , Humanos , Interferon-alfa/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Potyvirus/patogenicidade , Proteínas Recombinantes/metabolismo , Proteínas Virais/genéticaRESUMO
Recently we reported on a plasma membrane tobacco protein (designated NtCBP4) that binds calmodulin. When overexpressed in transgenic plants, NtCBP4 confers Pb2+ hypersensitivity associated with enhanced accumulation of this toxic metal. To further investigate possible modulation of Pb2+ tolerance in plants, we prepared transgenic plants that express a truncated version of this protein (designated NtCBP4DeltaC) from which its C-terminal, with the calmodulin-binding domain and part of the putative cyclic nucleotide-binding domain, was removed. In contrast to the phenotype of transgenic plants expressing the full-length gene, transgenic plants expressing the truncated gene showed improved tolerance to Pb2+, in addition to attenuated accumulation of this metal. Furthermore, disruption by T-DNA insertion mutagenesis of the Arabidopsis CNGC1 gene, which encodes a homologous protein, also conferred Pb2+ tolerance. We suggest that NtCBP4 and AtCNGC1 are components of a transport pathway responsible for Pb2+ entry into plant cells.
Assuntos
Arabidopsis/genética , Proteínas de Ligação a Calmodulina/genética , Canais Iônicos/genética , Chumbo/toxicidade , Nicotiana/genética , Proteínas de Plantas , Plantas Tóxicas , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Sequência de Bases , Proteínas de Ligação a Calmodulina/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica de Plantas , Immunoblotting , Chumbo/metabolismo , Dados de Sequência Molecular , Mutação , Desenvolvimento Vegetal , Plantas/efeitos dos fármacos , Plantas/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nicotiana/efeitos dos fármacos , Nicotiana/crescimento & desenvolvimentoRESUMO
Recently we discovered a tobacco protein (designated NtCBP4) that modulates heavy-metal tolerance in transgenic plants. Structurally, NtCBP4 is similar to mammalian cyclic-nucleotide-gated non-selective cation channels containing six putative transmembrane domains, a predicted pore region, a conserved cyclic-nucleotide-binding domain, and a high-affinity calmodulin-binding site that coincides with its cyclic-nucleotide-binding domain. Transgenic tobacco expressing the plasma-membrane-localized NtCBP4 exhibit improved tolerance to Ni(2+) and hypersensitivity to Pb(2+), which are associated with a decreased accumulation of Ni(2+) and an enhanced accumulation of Pb(2+) respectively. Transgenic plants expressing a truncated version of NtCBP4, from which regulatory domains had been removed, have a different phenotype. Here we describe our approach to studying the involvement of NtCBP4 in heavy-metal tolerance and to elucidate its physiological role.
Assuntos
Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/fisiologia , Calmodulina/metabolismo , Canais Iônicos/química , Metais Pesados/metabolismo , Nucleotídeos Cíclicos/metabolismo , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Cátions , Membrana Celular/metabolismo , Sequência Conservada , Canais Iônicos/metabolismo , Chumbo/metabolismo , Chumbo/farmacocinética , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Níquel/metabolismo , Níquel/farmacocinética , Fenótipo , Plantas Geneticamente Modificadas , Plantas Tóxicas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Nicotiana/químicaRESUMO
Recently we isolated a cDNA encoding a tobacco plasma membrane calmodulin-binding channel protein (designated NtCBP4) with a putative cyclic nucleotide-binding domain. Here we analyzed in detail the interaction of NtCBP4 with calmodulin. A full-length recombinant NtCBP4 (81 kDa) expressed in Sf9 insect cells, and the corresponding tobacco membrane protein were solubilized from their respective membrane fractions and partially purified by calmodulin affinity chromatography. NtCBP4 was detected in the eluted fractions using specific antibodies raised against the recombinant protein. By binding [35S]-calmodulin to recombinant NtCBP4 truncations fused to glutathione S-transferase, we identified a single region consisting of 66 amino acids capable of binding calmodulin. A 23 amino acid synthetic peptide from within this region formed a complex with calmodulin in the presence of calcium. We measured the fluorescence of dansyl-calmodulin interacting with this peptide, which revealed a dissociation constant of about 8 nM. The NtCBP4 calmodulin-binding domain was found to perfectly coincide with a phylogenetically conserved alphaC-helix motif of its putative cyclic nucleotide-binding domain. Furthermore, a 23 amino acid region in an equivalent site in the cAMP-binding domain of a mammalian protein kinase regulatory subunit was also found to bind calmodulin. Thus, coinciding calmodulin- and cyclic nucleotide-binding domains may serve as a point of communication between calcium and cyclic nucleotide signal transduction pathways in plants and animals.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Nucleotídeos Cíclicos/metabolismo , Proteínas de Plantas , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Cálcio/farmacologia , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/genética , Bovinos , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Plantas Tóxicas , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Nicotiana/genética , Nicotiana/metabolismoRESUMO
All organisms require a minimal amount of metal ions to sustain their metabolism, growth and development. At the same time, their intrinsic metal-uptake systems render them vulnerable to toxic levels of metals in the biosphere. Using radiolabeled recombinant calmodulin as a probe to screen a tobacco cDNA library, we have discovered a protein designated NtCBP4 (Nicotiana tabacum calmodulin-binding protein) that can modulate plant tolerance to heavy metals. Structurally, NtCBP4 is similar to vertebrate and invertebrate K+ and to non-selective cation channels, as well as to recently reported proteins from barley and Arabidopsis. Here we report on the subcellular localization of NtCBP4 and the phenotype of transgenic plants overexpressing this protein. The localization of NtCBP4 in the plasma membrane was manifested by fractionating tobacco membranes on sucrose gradients or by aqueous two-phase partitioning, and subsequently using immunodetection. Several independent transgenic lines expressing NtCBP4 had higher than normal levels of NtCBP4. These transgenic lines were indistinguishable from wild type under normal growth conditions. However, they exhibited improved tolerance to Ni2+ and hypersensitivity to Pb2+, which are associated with reduced Ni2+ accumulation and enhanced Pb2+ accumulation, respectively. To our knowledge this is the first report of a plant protein that modulates plant tolerance or accumulation of Pb2+. We propose that NtCBP4 is involved in metal uptake across the plant plasma membrane. This gene may prove useful for implementing selective ion tolerance in crops and improving phytoremediation strategies.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Membrana Celular/metabolismo , Chumbo/metabolismo , Níquel/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Plantas Tóxicas , Sequência de Aminoácidos , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/isolamento & purificação , Membrana Celular/genética , Chumbo/toxicidade , Dados de Sequência Molecular , Níquel/toxicidade , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Nicotiana/genéticaRESUMO
The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30-32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca2+/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.
Assuntos
Arabidopsis/genética , Cálcio/metabolismo , Calmodulina/metabolismo , Glutamato Descarboxilase/genética , Isoenzimas/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glutamato Descarboxilase/metabolismo , Isoenzimas/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM-binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.
Assuntos
Calmodulina/metabolismo , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Ácido gama-Aminobutírico/metabolismo , Quelantes/farmacologia , DNA Complementar , Ácido Egtázico/farmacologia , Glutamato Descarboxilase/efeitos dos fármacos , Glutamato Descarboxilase/genética , Dados de Sequência Molecular , Extratos Vegetais , Ligação Proteica , Trifluoperazina/farmacologiaRESUMO
We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.
Assuntos
Arabidopsis/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Glutamato Descarboxilase/química , Glutamato Descarboxilase/metabolismo , Plantas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cálcio/farmacologia , Calmodulina/química , Calmodulina/isolamento & purificação , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/isolamento & purificação , Clonagem Molecular , Escherichia coli , Glutamato Descarboxilase/isolamento & purificação , Glutationa Transferase/biossíntese , Glutationa Transferase/isolamento & purificação , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Recently, we provided preliminary evidence for calcium (Ca2+)/calmodulin (CaM) stimulation of plant glutamate decarboxylase (GAD; EC 4.1.1.15). In the present study, a detailed characterization of the phenomenon is described. GAD was partially purified from various soybean (Glycine max L. Merr.) tissues (developing seed coat and cotyledons, leaf, and root) in the presence of EDTA by a combination of ammonium sulfate precipitation and anion-exchange fast protein liquid chromatography. GAD activity showed a sharp optimum at pH 5.8, with about 12% of maximal activity at pH 7. It was stimulated 2- to 8-fold (depending on the tissue source) in the presence of Ca2+/CaM at pH 7 but not at pH 5.8. Furthermore, when the protease inhibitor phenylmethylsulfonyl fluoride was omitted from the purification procedure, GAD activity was insensitive to Ca2+/CaM but was similar in magnitude to CaM-stimulated activity. The stimulation by Ca2+/CaM was fully inhibited by the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfon-amide and trifluoperazine. With saturating CaM or Ca2+, the concentrations of Ca2+ and CaM required for half-maximal stimulation were about 7 to 11 [mu]M and 25 nM, respectively. The effect of Ca2+ and CaM appeared to be through a 2.4-fold stimulation of Vmax and a 55% reduction in Km. The results suggested that GAD is activated via Ca2+ signal transduction.
RESUMO
Molecular procedures have been applied to isolate plant calmodulin-binding proteins. A petunia cDNA expression library was screened with 35S-labeled recombinant calmodulin as a probe, and a cDNA coding for a Ca(2+)-dependent calmodulin-binding protein was isolated. The deduced amino acid sequence of the petunia protein (500 amino acid residues, 58 kDa) has 67% overall amino acid sequence similarity to glutamate decarboxylase (GAD) from Escherichia coli (466 amino acid residues, 53 kDa). The recombinant protein expressed in E. coli cells displays GAD activity, i.e. catalyzes the conversion of glutamic acid to gamma-aminobutyric acid and binds calmodulin, whereas E. coli GAD does not bind calmodulin. The calmodulin binding domain in the petunia GAD was mapped by binding truncated forms of GAD immobilized on nitrocellulose membranes to recombinant petunia 35S-calmodulin as well as to biotinylated bovine calmodulin and by binding truncated forms of GAD to calmodulin-Sepharose columns. The calmodulin binding domain in petunia GAD is part of a carboxyl end extension that is not present in E. coli GAD. Polyclonal antibodies raised against the recombinant petunia GAD detect a single protein band from plant extracts of gel mobility identical to that of the recombinant GAD. Moreover, the plant protein binds calmodulin in vitro. This is the first report of the isolation of a GAD gene from plants and of a calmodulin-binding GAD from any organism. Our results raise the possibility that intracellular Ca2+ signals via calmodulin are involved in the regulation of gamma-aminobutyric acid synthesis in plants.