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1.
Plant Mol Biol ; 81(3): 273-86, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23242917

RESUMO

Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of L-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H(2)O(2) production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species.


Assuntos
Agrobacterium tumefaciens/genética , Glutamina/farmacologia , Inositol/farmacologia , Lolium/genética , Oryza/genética , Transformação Genética , Agrobacterium tumefaciens/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/fisiologia , Parede Celular/metabolismo , Temperatura Baixa , Meios de Cultura , Técnicas de Transferência de Genes , Vetores Genéticos , Proteínas de Fluorescência Verde , Peróxido de Hidrogênio/metabolismo , Lignina/metabolismo , Lolium/efeitos dos fármacos , Lolium/imunologia , Lolium/fisiologia , Oryza/efeitos dos fármacos , Oryza/imunologia , Oryza/fisiologia , Ácido Fítico/metabolismo , Imunidade Vegetal , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/imunologia , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Regeneração , Sementes/efeitos dos fármacos , Sementes/genética , Sementes/imunologia , Sementes/fisiologia , Amido/metabolismo , Técnicas de Cultura de Tecidos
2.
Plant Physiol ; 151(2): 893-904, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19684226

RESUMO

Plants produce reactive oxygen species (ROS) in response to environmental stresses sending signaling cues, which, if uncontrolled, result in cell death. Like other aerobic organisms, plants have ROS-scavenging enzymes, such as superoxide dismutase (SOD), which removes superoxide anion radical (O(2)(-)) and prevents the production and buildup of toxic free radicals. However, increasing the expression of cytosolic SODs is complex, and increasing their production in vivo has proven to be challenging. To avoid problems with endogenous regulation of gene expression, we expressed a gene from the archaeal hyperthermophile Pyrococcus furiosus that reduces O(2)(-). P. furiosus uses superoxide reductase (SOR) rather than SOD to remove superoxide. SOR is a thermostable enzyme that reduces O(2)(-) in a one-electron reduction without producing oxygen. We show that P. furiosus SOR can be produced as a functional enzyme in planta and that plants producing SOR have enhanced tolerance to heat, light, and chemically induced ROS. Stress tolerance in the SOR-producing plants correlates positively with a delayed increase in ROS-sensitive transcripts and a decrease in ascorbate peroxidase activity. The SOR plants provide a good model system to study the impact of cytosolic ROS on downstream signaling in plant growth and development. Furthermore, this work demonstrates that this synthetic approach for reducing cytosolic ROS holds promise as a means for improving stress tolerance in crop plants.


Assuntos
Adaptação Fisiológica , Arabidopsis/metabolismo , Temperatura Alta , Oxirredutases/metabolismo , Pyrococcus furiosus/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ascorbato Peroxidases , Clorofila/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação , Resposta ao Choque Térmico , Peróxido de Hidrogênio/metabolismo , Imunoprecipitação , Oxirredutases/genética , Peroxidases/metabolismo , Extratos Vegetais/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/enzimologia , Sementes/metabolismo
3.
Plant Physiol ; 133(3): 1385-96, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14563927

RESUMO

Calreticulin (CRT) is a multifunctional protein mainly localized to the endoplasmic reticulum in eukaryotic cells. Here, we present the first analysis, to our knowledge, of evolutionary diversity and expression profiling among different plant CRT isoforms. Phylogenetic studies and expression analysis show that higher plants contain two distinct groups of CRTs: a CRT1/CRT2 group and a CRT3 group. To corroborate the existence of these isoform groups, we cloned a putative CRT3 ortholog from Brassica rapa. The CRT3 gene appears to be most closely related to the ancestral CRT gene in higher plants. Distinct tissue-dependent expression patterns and stress-related regulation were observed for the isoform groups. Furthermore, analysis of posttranslational modifications revealed differences in the glycosylation status among members within the CRT1/CRT2 isoform group. Based on evolutionary relationship, a new nomenclature for plant CRTs is suggested. The presence of two distinct CRT isoform groups, with distinct expression patterns and posttranslational modifications, supports functional specificity among plant CRTs and could account for the multiple functional roles assigned to CRTs.


Assuntos
Calreticulina/genética , Perfilação da Expressão Gênica/métodos , Filogenia , Proteínas de Plantas/genética , Plantas/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Calreticulina/metabolismo , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Evolução Molecular , Glicosilação , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Zea mays/genética , Zea mays/metabolismo
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