A One-Week Laboratory Practice: Introducing the Students to the Study of Plant Biochemistry and Signal Transduction

This report describes a one-week laboratory practice for students. An approach to study a Calcium dependent protein kinase (CDPK) involved in signal transduction processes in potato plants, is undertaken. A number of basic biochemical techniques including the partial purification of a protein kinase, protein kinase activity assays, protein determination, SDS-PAGE analysis of phosphorylated proteins and Western blot assays are described.Fil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Raices, Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Tellez, Maria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

A fluorometric method for the assay of protein kinase activity

Protein kinases constitute one of the largest protein families in nature. Current methods to assay their activity involve the use of radioactive ATP or very expensive reagents. In this work, we developed a highly sensitive, cost-effective and straightforward protocol to measure protein kinase activity using a microplate layout. Released ADP is converted into NAD+, which is quantified by its fluorescent properties after alkaline treatment (linear range 0–10 nmol ADP). To validate our protocol, we characterized a recombinant calcium-dependent protein kinase from potato. Overall, this tool represents a critical step forward in the functional characterization of protein kinases.Fil: Rojas, Bruno Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Santin, Franco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Methylobacterium sp. 2A is a plant growth-promoting rhizobacteria that has the potential to improve potato crop yield under adverse conditions

A Gram-negative pink-pigmented bacillus (named 2A) was isolated from Solanum tuberosum L. cv. Desirée plants that were strikingly more developed, presented increased root hair density, and higher biomass than other potato lines of the same age. The 16S ribosomal DNA sequence, used for comparative gene sequence analysis, indicated that strain 2A belongs to the genus Methylobacterium. Nucleotide identity between Methylobacterium sp. 2A sequenced genome and the rest of the species that belong to the genus suggested that this species has not been described so far. In vitro, potato plants inoculated with Methylobacterium sp. 2A had a better performance when grown under 50 mM NaCl or when infected with Phytophthora infestans. We inoculated Methylobacterium sp. 2A in Arabidopsis thaliana roots and exposed these plants to salt stress (75 mM NaCl). Methylobacterium sp. 2A-inoculated plants, grown in control or salt stress conditions, displayed a higher density of lateral roots (p < 0.05) compared to noninoculated plants. Moreover, under salt stress, they presented a higher number of leaves and larger rosette diameter. In dual confrontation assays, Methylobacterium sp. 2A displayed biocontrol activity against P. infestans, Botrytis cinerea, and Fusarium graminearum, but not against Rhizoctonia solani, and Pythium dissotocum. In addition, we observed that Methylobacterium sp. 2A diminished the size of necrotic lesions and reduced chlorosis when greenhouse potato plants were infected with P. infestans. Methylobacterium sp. 2A produces indole acetic acid, solubilizes mineral phosphate and is able to grow in a N2 free medium. Whole-genome sequencing revealed metabolic pathways associated with its plant growth promoter capacity. Our results suggest that Methylobacterium sp. 2A is a plant growth-promoting rhizobacteria (PGPR) that can alleviate salt stress, and restricts P. infestans infection in potato plants, emerging as a potential strategy to improve crop management.Fil: Grossi, Cecilia Eugenia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Fantino, Elisa Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Serral, Federico. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; ArgentinaFil: Zawoznik, Myriam Sara. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica. Cátedra de Química Biológica Vegetal; ArgentinaFil: Fernández Do Porto, Darío Augusto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; ArgentinaFil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

The protein phosphatase 2A catalytic subunit StPP2Ac2b enhances susceptibility to Phytophthora infestans and senescence in potato

The serine/threonine protein phosphatases type 2A (PP2A) are involved in several physiological responses in plants, playing important roles in developmental programs, stress responses and hormone signaling. Six PP2A catalytic subunits (StPP2Ac) were identified in cultivated potato. Transgenic potato plants constitutively overexpressing the catalytic subunit StPP2Ac2b (StPP2Ac2b-OE) were developed to determine its physiological roles. The response of StPP2Ac2b-OE plants to the oomycete Phytophthora infestans, the causal agent of late blight, was evaluated. We found that overexpression of StPP2Ac2b enhancessusceptibility to the pathogen. Further bioinformatics, biochemical, and molecular analyses revealed that StPP2Ac2b positively regulates developmental and pathogen-induced senescence, and that P. infestans infection promotes senescence, most likely through induction of StPP2Ac2b expression.Fil: Muñiz García, María Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Grossi, Cecilia Eugenia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Capiati, Daniela Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Perspectives of bioremediation of heavy metals with native plants of the Fabaceae family present in Paraguay

Industrial pollution is a worldwide problem because many effluents do not receive proper treatment before being released into watercourses. In Paraguay, leather tanning is a common industrial activity and its main contaminant is Chromium. This heavy metal accumulates in both soil and water and can be harmful to human health in large quantities. This study presents the native Fabaceae found in Paraguay and highlights those plants found in the Sub-humid Flooded Forest of the Paraguay River which could be explored in future bioremediation assays. On the other hand, the phylogenetic study of the rbcL gene sequences present in the databases showed that Glycine max, a model species of the Fabaceae family, has the closest phylogenetic relationship with Erythrina crista galli, for which gene studies could be carried out to propose new strategies for pollution reduction.Fil: Quintana Arrúa, Silverio Andrés. Universidad Nacional de Asunción; Paraguay. Universidad Nacional de General Sarmiento. Instituto de Ciencias; ArgentinaFil: Reyes, Magaliz. Instituto Paraguayo de Tecnología Agraria; ParaguayFil: Mereles, Patricia. Universidad Nacional de Asunción; ParaguayFil: Grossi, Cecilia Eugenia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Plantico, an R statistical analysis package for plant biometric parameters

Los estreses abióticos y bióticos impactan negativamente en el desarrollo de las plantas afectando diferentes parámetros biométricos (por ejemplo, la longitud del vástago o de la raíz) que pueden medirse a distintos tiempos de aplicado el estrés. Sin embargo, es fundamental realizar un adecuado análisis estadístico de los datos numéricos obtenidos para evaluar el efecto del estrés sobre la planta. Por este motivo, a través del uso del lenguaje R, se creó el paquete denominado “Plantico” que realiza el análisis de la varianza (ANOVA) y somete los datos a la prueba de Tukey. Además, permite obtener las gráficas de barra, de puntos, y diagrama de caja para los “t” días del ensayo. Para validar la utilidad del paquete se determinó la longitud de la raíz de plántulas de Arabidopsis crecidas en condiciones control (MS 0,5x) o expuestas a estrés salino (MS 0,5x +75 mM NaCl) u osmótico (MS 0,5x +100 mM manitol) durante 7 días. El análisis estadístico de los datos obtenidos y los gráficos correspondientes se procesaron con Plantico. Esta herramienta puede ser descargada y utilizada de manera gratuita y puede configurarse para incluir otras variables biométricas o diferentes tiempos de estudio. Plantico facilita el análisis estadístico y la visualización de los datos experimentales.Abiotic and biotic stresses negatively impact on plant development affecting biometric parameters, such as root or shoot length, that can be measured at different time points. An adequate statistical analysis of the data is required to evaluate the effect of each stress condition. Thus, Plantico, an open-source R statistical analysis package that performs the analysis of variance (ANOVA) followed by Tukey´s test, as well as the bar, line, and box plots at a given "t” time of the assay was created. An assay was conducted with Arabidopsis seedlings grown in vitro under control (MS 0.5x), saline (MS 0.5X + 75 mM NaCl) or osmotic (MS 0.5 x + 100 mM mannitol) conditions and root length was determined to validate this package. Statistical analysis of the data and the corresponding graphs were performed with Plantico. This tool can be downloaded and used for free and can be modified to include other biometric parameters and time points. Plantico is a user-friendly package that enables statistical analysis and the visualization of experimental data.Fil: Quintana, Silverio Andrés. Universidad Nacional de Asunción; ParaguayFil: Grossi, Cecilia Eugenia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Espinoza, Rodrigo. Universidad Nacional de Asunción; ParaguayFil: Ulloa, Rita Maria. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Signal transduction mechanisms involved in potato developmental processes

Solanum tuberosum L. potato plants undergo several development stages during their life cycle involving stolon formation, tuberization, tuber filling, dormancy and tuber sprouting. Potato tubers are underground sinks originated from stolons in a process that requires the cessation of apex growth, the swelling of the stolon by subapical radial growth, and enlargement of the body. Potato plants produce tubers as a result of the changing balance of endogenous growth regulators, which is brought about by the plant´s ability to perceive changes in the environment. An important aspect of tuber induction is that the stimulus is received on the leaves and is graft-transmissible. Environmental and hormonal signals, such as those mediated by light and gibberellins, are integrated in the leaves and a mobile signal is exported to the underground stolons to initiate tuber formation. This process is accompanied by the accumulation of starch and storage proteins. Tuberization allows the plant to reproduce in a vegetative way and determines that it can be considered a potential perennial plant. With the onset of sprouting, the tuber turns into a source organ; the reducing sugars increase as starch is hydrolyzed, providing carbon and energy for growth of the developing sprout. Tuber development and sprouting require coordinated transcriptional and metabolic changes as well as major changes in gene expression patterns. Signalling cascades are involved in sensing and transducing the environmental and hormonal stimuli that modulate both developmental processes. In this chapter we will review the different external and endogenous factors that regulate both processes and the signal transduction cascades associated to them.Fil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Capiati, Daniela Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Giammaria, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Perspectives of bioremediation of heavy metals with native plants of the Fabaceae family present in Paraguay

Industrial pollution is a worldwide problem because many effluents do not receive proper treatment before being released into watercourses. In Paraguay, leather tanning is a common industrial activity and its main contaminant is Chromium. This heavy metal accumulates in both soil and water and can be harmful to human health in large quantities. This study presents the native Fabaceae found in Paraguay and highlights those plants found in the Sub-humid Flooded Forest of the Paraguay River which could be explored in future bioremediation assays. On the other hand, the phylogenetic study of the rbcL gene sequences present in the databases showed that Glycine max, a model species of the Fabaceae family, has the closest phylogenetic relationship with Erythrina crista galli, for which gene studies could be carried out to propose new strategies for pollution reduction.Fil: Quintana Arrúa, Silverio Andrés. Universidad Nacional de Asunción; Paraguay. Universidad Nacional de General Sarmiento. Instituto de Ciencias; ArgentinaFil: Reyes, Magaliz. Instituto Paraguayo de Tecnología Agraria; ParaguayFil: Mereles, Patricia. Universidad Nacional de Asunción; ParaguayFil: Grossi, Cecilia Eugenia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Water-deficit stress signal transduction pathways in plants: From sensing to response

As sessile organisms plants have to cope with changing environmental conditions. Drought and salinity, which causes water-deficit in plant cells, are common adverse factors that limit plant growth and productivity. Understanding the mechanisms by which plants perceive environmental signals and transmit them to cellular machinery to activate adaptive responses is of great importance to biology and to rational engineering of crop plants. This chapter reviews the signal transduction mechanisms that activate water-deficit stress responses and the regulation of transcription factors that control the expression of stress-responsive genes. The general components of stress signal transduction pathway for drought and salt stress are considered. Signal perception, receptor-coupled phosphorelay, phosphoinositol-induced Ca2+ changes, Ca2+-coupled phosphoprotein cascades,  mitogen-activated protein kinase cascade and transcriptional activation of stress responsive genes are the main signal transduction steps addressed. Abscisic acid (ABA) plays a pivotal role in stress responses in plants. Therefore, the hormone implications are also reviewed. The transcription factors responsible for reprogramming gene expression in response to stress are described. Finally, the physiological and biochemical responses that lead to plant tolerance to water-deficit stress are addressed.Fil: Capiati, Daniela Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Muñiz García, María Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

StCDPK1 is expressed in potato stolon tips and is induced by high sucrose concentration

StCDPK1 encodes a calcium-dependent protein kinase (CDPK) from Solanum tuberosum, which is transiently induced upon tuberization in swelling stolons. In situ hybridization determined that StCDPK1 mRNA is localized in the apical dome of tuberizing stolon tips, close to the region where sucrose was reported to accumulate. The expression of StCDPK1, and other tuber-specific genes was enhanced when in vitro-cultured potato plants were transferred to high sucrose or high sorbitol containing media. Glucose, fructose or a mixture of both showed no effect on CDPK expression. Okadaic acid blocked sucrose-inducible gene expression, suggesting that phosphatases from the PP1/PP2A family could also participate in the regulation of StCDPK1 and other tuberization-related genes.Fil: Raíces, Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: MacIntosh, Gustavo C.. Delaware Biotechnology Institute; Estados UnidosFil: Crespi, Martín. Institut des Sciences Vegetales; FranciaFil: Tellez, Maria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin