Modulating MIOX2 expression in Nicotiana tabacum and impacts on genes involved in cell wall biosynthesis

Cell walls are essential structures for plant development and growth. Apart from its biological functions, the polysaccharides that make cell walls (cellulose, hemicellulose and pectins) are the principal natural fibrous materials, considered the most important renewable resource on earth, used as raw material for many industrial processes among them, for pulp and paper production, charcoal, and biofuels. For all these reasons, the study of molecular composition and biosynthesis of plant cell walls has been a matter of great interest for researchers over the past few years. In this context, a full-length cDNA fragment of Miox2 gene was cloned from Arabidopsis seedlings, using RT-PCR, with an open reading frame of 954 pb and a corresponding protein subunit molecular mass of 37 kDa. The deduced amino acid sequence of the cDNA showed a high degree of homology with myo-Inosytol oxygenases from other organisms. This cDNA was used for genetic transformation of model plants (tobacco), which expressed either antisense or sense RNA. Transgenic homozygous tobacco model plants with either repression or constitutively expressed Miox2 were obtained with the number of copies varying from 1 to 7. Neither, the repression of the endogenous tobacco Miox genes or the constitutive expression of Miox2 gene, caused major impact on plant development, leaf morphology or flowering time. There was however, statistically significant (P<0.05) changes in the arabinan and D-galacturonate contents. These results clearly indicate that the modulation of the myo-Inositol pathway caused no major impact on cell wall polysaccharide biosynthesis.As paredes celulares vegetais são estruturas essenciais para o crescimento e desenvolvimento das plantas. Além de suas diversas funções biológicas, os componentes polissacarídicos constituintes das paredes celulares (celulose, hemiceluloses e pectinas) são de vital importância como fonte natural de fibras, sendo consideradas as fontes principais de recursos renováveis do planeta, utilizados como matéria prima para diversos processos industriais, dentre eles, a produção de papel e celulose, carvão vegetal e biocombustíveis. Todos esses fatores têm despertado grande interesse no estudo da composição e biossíntese das paredes celulares. Neste contexto, um fragmento de cDNA do gene Miox2 foi clonado de plântulas de Arabidopsis, via RT-PCR, com uma região aberta de leitura de 954 pb e sua proteína com massa molecular de 37kDa. A sequência deduzida de aminoácidos do cDNA apresentou alto grau de identidade com mio-Inositol oxigenases de outros organismos. Este cDNA foi usado para transformação genética de plantas modelo (tabaco) que produziram RNA antisense ou sense. Plantas de tabaco homozigotas para o transgene com repressão ou expressão constitutiva do gene Miox2 foram obtidas com um número de cópias do transgene, variando de 1 a 7. A repressão do gene Miox de tabaco endógeno assim como a expressão constitutiva do gene Miox2 de Arabidopsis não causaram alterações no desenvolvimento, morfologia foliar ou tempo de florescimento das plantas. Entretanto, alterações estatisticamente significativas (P<0.05) ocorreram no conteúdo de arabinana e de D-galacturonato. Estes resultados indicam que a modulação do metabolismo do mio-Inositol não causou grandes impactos na biossíntese dos polissacarídeos da parede celular.Fil: Defávari Nascimento, D.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Conti, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Labate, Mônica T. V.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Gutmanis, Gunta. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Bertolo, Ana L. F.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: de Andrade, Alexander. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Bragatto, Juliano. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Pagotto, Luís Otávio. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Damin, Plínio. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Moon, David H.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Labate, Carlos A.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; Brasi

Modulating MIOX2 expression in Nicotiana tabacum and impacts on genes involved in cell wall biosynthesis

Cell walls are essential structures for plant development and growth. Apart from its biological functions, the polysaccharides that make cell walls (cellulose, hemicellulose and pectins) are the principal natural fibrous materials, considered the most important renewable resource on earth, used as raw material for many industrial processes among them, for pulp and paper production, charcoal, and biofuels. For all these reasons, the study of molecular composition and biosynthesis of plant cell walls has been a matter of great interest for researchers over the past few years. In this context, a full-length cDNA fragment of Miox2 gene was cloned from Arabidopsis seedlings, using RT-PCR, with an open reading frame of 954 pb and a corresponding protein subunit molecular mass of 37 kDa. The deduced amino acid sequence of the cDNA showed a high degree of homology with myo-Inosytol oxygenases from other organisms. This cDNA was used for genetic transformation of model plants (tobacco), which expressed either antisense or sense RNA. Transgenic homozygous tobacco model plants with either repression or constitutively expressed Miox2 were obtained with the number of copies varying from 1 to 7. Neither, the repression of the endogenous tobacco Miox genes or the constitutive expression of Miox2 gene, caused major impact on plant development, leaf morphology or flowering time. There was however, statistically significant (P<0.05) changes in the arabinan and D-galacturonate contents. These results clearly indicate that the modulation of the myo-Inositol pathway caused no major impact on cell wall polysaccharide biosynthesis.As paredes celulares vegetais são estruturas essenciais para o crescimento e desenvolvimento das plantas. Além de suas diversas funções biológicas, os componentes polissacarídicos constituintes das paredes celulares (celulose, hemiceluloses e pectinas) são de vital importância como fonte natural de fibras, sendo consideradas as fontes principais de recursos renováveis do planeta, utilizados como matéria prima para diversos processos industriais, dentre eles, a produção de papel e celulose, carvão vegetal e biocombustíveis. Todos esses fatores têm despertado grande interesse no estudo da composição e biossíntese das paredes celulares. Neste contexto, um fragmento de cDNA do gene Miox2 foi clonado de plântulas de Arabidopsis, via RT-PCR, com uma região aberta de leitura de 954 pb e sua proteína com massa molecular de 37kDa. A sequência deduzida de aminoácidos do cDNA apresentou alto grau de identidade com mio-Inositol oxigenases de outros organismos. Este cDNA foi usado para transformação genética de plantas modelo (tabaco) que produziram RNA antisense ou sense. Plantas de tabaco homozigotas para o transgene com repressão ou expressão constitutiva do gene Miox2 foram obtidas com um número de cópias do transgene, variando de 1 a 7. A repressão do gene Miox de tabaco endógeno assim como a expressão constitutiva do gene Miox2 de Arabidopsis não causaram alterações no desenvolvimento, morfologia foliar ou tempo de florescimento das plantas. Entretanto, alterações estatisticamente significativas (P<0.05) ocorreram no conteúdo de arabinana e de D-galacturonato. Estes resultados indicam que a modulação do metabolismo do mio-Inositol não causou grandes impactos na biossíntese dos polissacarídeos da parede celular.Fil: Defávari Nascimento, D.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Conti, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Labate, Mônica T. V.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Gutmanis, Gunta. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Bertolo, Ana L. F.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: de Andrade, Alexander. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Bragatto, Juliano. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Pagotto, Luís Otávio. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Damin, Plínio. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Moon, David H.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Labate, Carlos A.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; Brasi

Malate plays a crucial role in starch metabolism, ripening, and soluble solid content of tomato fruit and affects postharvest softening

Despite the fact that the organic acid content of a fruit is regarded as one of its most commercially important quality traits when assessed by the consumer, relatively little is known concerning the physiological importance of organic acid metabolism for the fruit itself. Here, we evaluate the effect of modifying malate metabolism in a fruit-specific manner, by reduction of the activities of either mitochondrial malate dehydrogenase or fumarase, via targeted antisense approaches in tomato (Solanum lycopersicum). While these genetic perturbations had relatively little effect on the total fruit yield, they had dramatic consequences for fruit metabolism, as well as unanticipated changes in postharvest shelf life and susceptibility to bacterial infection. Detailed characterization suggested that the rate of ripening was essentially unaltered but that lines containing higher malate were characterized by lower levels of transitory starch and a lower soluble sugars content at harvest, whereas those with lower malate contained higher levels of these carbohydrates. Analysis of the activation state of ADP-glucose pyrophosphorylase revealed that it correlated with the accumulation of transitory starch. Taken together with the altered activation state of the plastidial malate dehydrogenase and the modified pigment biosynthesis of the transgenic lines, these results suggest that the phenotypes are due to an altered cellular redox status. The combined data reveal the importance of malate metabolism in tomato fruit metabolism and development and confirm the importance of transitory starch in the determination of agronomic yield in this species.Fil: Centeno, Danilo C.. Max Planck Institute Of Molecular Plant Physiology; AlemaniaFil: Osorio, Sonia. Max Planck Institute Of Molecular Plant Physiology; AlemaniaFil: Nunes Nesi, Adriano. Max Planck Institute Of Molecular Plant Physiology; AlemaniaFil: Bertolo, Ana L. F.. Cornell University; Estados UnidosFil: Carneiro, Raphael T.. Cornell University; Estados UnidosFil: Araújo, Wagner L.. Max Planck Institute Of Molecular Plant Physiology; AlemaniaFil: Steinhauser, Marie Caroline. Max Planck Institute Of Molecular Plant Physiology; AlemaniaFil: Michalska, Justyna. Max Planck Institute Of Molecular Plant Physiology; AlemaniaFil: Rohrmann, Johannes. Max Planck Institute Of Molecular Plant Physiology; AlemaniaFil: Geigenberger, Peter. Technische Universitat München; AlemaniaFil: Oliver, Sandra N.. Max Planck Institute Of Molecular Plant Physiology; AlemaniaFil: Stitt, Mark. Max Planck Institute Of Molecular Plant Physiology; AlemaniaFil: Carrari, Fernando Oscar. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rose, Jocelyn K. C.. Cornell University; Estados UnidosFil: Fernie, Alisdair R.. Max Planck Institute Of Molecular Plant Physiology; Alemani

YES1 drives lung cancer growth and progression and predicts sensitivity to dasatinib

Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non–small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer. Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC. Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples. Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer. Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy

Cloning and endogenous expression of a Eucalyptus grandis UDP-glucose dehydrogenase cDNA

UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis

YES1 drives lung cancer growth and progression and predicts sensitivity to dasatinib

Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non–small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer. Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC. Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples. Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer. Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy