Function of C1A barley peptidases and their inhibitors (cystatins) in barley seed germination : Funcion de las peptidasas C1A de cebada y sus inhibidores (cistatinas) en la germinacion de la semilla

Plant proteolysis is a metabolic process where specific enzymes called peptidases degrade proteins. In plants, this complex process involves broad metabolic networks and different sub-cellular compartments. Several types of peptidases take part in the proteolytic process, mainly cysteine-, serine-, aspartyl- and metallo- peptidases. Among the cysteine-peptidases, the papain-like or C1A peptidases (family C1, clan CA) are extensively present in land plants and are classified into catepsins L-, B-, H- and Flike. The catalytic mechanism of these C1A peptidases is highly conserved and involves the three amino acids Cys, His and Asn in the catalytic triad, and a Gln residue which seems essential for maintaining an active enzyme conformation. These proteins are synthesized as inactive precursors, which comprise an N-terminal signal peptide, a propeptide, and the mature protein. In barley, we have identified 33 cysteine-peptidases from the papain-like family, classifying them into 8 different groups. Five of them corresponded to cathepsins L-like (5 subgroups), 1 cathepsin B-like group, 1 cathepsin F-like group and 1 cathepsin H-like group. Besides, C1A peptidases are the specific targets of the plant proteinaceous inhibitors known as phytocystatins (PhyCys). The cystatin inhibitory mechanism is produced by a tight and reversible interaction with their target enzymes. In barley, the cystatin gene family is comprised by 13 members. In this work we have tried to elucidate the role of the C1A cysteine-peptidases and their specific inhibitors (cystatins) in the germination process of the barley grain. Therefore, we selected a representative member of each group/subgroup of C1A peptidases (1 cathepsin B-like, 1 cathepsin F-like, 1 cathepsin H-like and 5 cathepsins L-like). The molecular characterization of the cysteine-peptidases was done and the peptidase-inhibitor interaction was analyzed in vitro and in vivo. A study in the structural basis for specificity of pro-peptide/enzyme interaction in barley C1A cysteine-peptidases has been also carried out by inhibitory assays and the modeling of the three-dimensional structures. The barley grain maturation produces the accumulation of storage proteins (prolamins) in the endosperm which are mobilized during germination to supply the required nutrients until the photosynthesis is fully established. In this work, we have demonstrated the participation of the cysteine-peptidases and their inhibitors in the degradation of the different storage protein fractions (hordeins, albumins and globulins) present in the barley grain. Besides, transgenic barley plants overexpressing or silencing cysteine-peptidases or cystatins were obtained by Agrobacterium-mediated transformation of barley immature embryos to analyze their physiological function in vivo. Preliminary assays were carried out with the T1 grains of several transgenic lines. Comparing the knock-out and the overexpressing lines with the WT, alterations in the germination process were detected and were correlated with their grain hordein content. These data will be validated with the homozygous grains that are being produced through the double haploid technique by microspore culture. Resumen La proteólisis es un proceso metabólico por el cual se lleva a cabo la degradación de las proteínas de un organismo a través de enzimas específicas llamadas proteasas. En plantas, este complejo proceso comprende un entramado de rutas metabólicas que implican, además, diferentes compartimentos subcelulares. En la proteólisis participan numerosas proteasas, principalmente cisteín-, serín-, aspartil-, y metalo-proteasas. Dentro de las cisteín-proteasas, las proteasas tipo papaína o C1A (familia C1, clan CA) están extensamente representadas en plantas terrestres, y se clasifican en catepsinas tipo L, B, H y F. El mecanismo catalítico de estas proteasas está altamente conservado y la triada catalítica formada por los aminoácidos Cys, His y Asn, y a un aminoácido Gln, que parece esencial para el mantenimiento de la conformación activa de la proteína. Las proteasas C1A se sintetizan como precursores inactivos y comprenden un péptido señal en el extremo N-terminal, un pro-péptido y la proteína madura. En cebada hemos identificado 33 cisteín-proteasas de tipo papaína y las hemos clasificado filogenéticamente en 8 grupos diferentes. Cinco de ellos pertenecen a las catepsinas tipo L (5 subgrupos), un grupo a las catepsinas tipo-B, otro a las catepsinas tipo-F y un último a las catepsinas tipo-H. Las proteasas C1A son además las dianas específicas de los inhibidores protéicos de plantas denominados fitocistatinas. El mecanismo de inhibición de las cistatinas está basado en una fuerte interacción reversible. En cebada, se conoce la familia génica completa de las cistatinas, que está formada por 13 miembros. En el presente trabajo se ha investigado el papel de las cisteín-proteasas de cebada y sus inhibidores específicos en el proceso de la germinación de la semilla. Para ello, se seleccionó una proteasa representante de cada grupo/subgrupo (1 catepsina tipo- B, 1 tipo-F, 1 tipo-H, y 5 tipo-L, una por cada subgrupo). Se ha llevado a cabo su caracterización molecular y se ha analizado la interacción enzima-inhibidor tanto in vivo como in vitro. También se han realizado estudios sobre las bases estructurales que demuestran la especificidad en la interacción enzima/propéptido en las proteasas C1A de cebada, mediante ensayos de inhibición y la predicción de modelos estructurales de la interacción. Finalmente, y dado que durante la maduración de la semilla se almacenan proteínas de reserva (prolaminas) en el endospermo que son movilizadas durante la germinación para suministrar los nutrientes necesarios hasta que la nueva planta pueda realizar la fotosíntesis, en este trabajo se ha demostrado la participación de las cisteínproteasas y sus inhibidores en la degradación de las diferentes tipos de proteínas de reserva (hordeinas, albúmins y globulinas) presentes en el grano de cebada. Además, se han obtenido plantas transgénicas de cebada que sobre-expresan o silencian cistatinas y cisteín-proteasas con el fin de analizar la función fisiológica in vivo. Se han realizado análisis preliminares en las semillas T1 de varias líneas tránsgenicas de cebada y al comparar las líneas knock-out y las líneas de sobre-expresión con las silvestres, se han detectado alteraciones en la germinación que están además correlacionadas con el contenido de hordeinas de las semillas. Estos datos serán validados en las semillas homocigotas que se están generando mediante la técnica de dobles haploides a partir del cultivo de microesporas.

A Fluctuation-Dissipation Model for Electrical Noise

This paper shows that today’s modelling of electrical noise as coming from noisy resistances is a non sense one contradicting their nature as systems bearing an electrical noise. We present a new model for electrical noise that including Johnson and Nyquist work also agrees with the Quantum Mechanical description of noisy systems done by Callen and Welton, where electrical energy fluctuates and is dissipated with time. By the two currents the Admittance function links in frequency domain with their common voltage, this new model shows the connection Cause-Effect that exists between Fluctuation and Dissipation of energy in time domain. In spite of its radical departure from today’s belief on electrical noise in resistors, this Complex model for electrical noise is obtained from Nyquist result by basic concepts of Circuit Theory and Thermo- dynamics that also apply to capacitors and inductors.

Adaptive control system based on lineal control theory for the path-following problem of a car-like mobile robot

The objective of this paper is to design a path following control system for a car-like mobile robot using classical linear control techniques, so that it adapts on-line to varying conditions during the trajectory following task. The main advantages of the proposed control structure is that well known linear control theory can be applied in calculating the PID controllers to full control requirements, while at the same time it is exible to be applied in non-linear changing conditions of the path following task. For this purpose the Frenet frame kinematic model of the robot is linearised at a varying working point that is calculated as a function of the actual velocity, the path curvature and kinematic parameters of the robot, yielding a transfer function that varies during the trajectory. The proposed controller is formed by a combination of an adaptive PID and a feed-forward controller, which varies accordingly with the working conditions and compensates the non-linearity of the system. The good features and exibility of the proposed control structure have been demonstrated through realistic simulations that include both kinematics and dynamics of the car-like robot.

Geometric diagnostics of complex patterns: Spiral defect chaos

Motivated by the observation of spiral patterns in a wide range of physical, chemical, and biological systems, we present an automated approach that aims at characterizing quantitatively spiral-like elements in complex stripelike patterns. The approach provides the location of the spiral tip and the size of the spiral arms in terms of their arc length and their winding number. In addition, it yields the number of pattern components (Betti number of order 1), as well as their size and certain aspects of their shape. We apply the method to spiral defect chaos in thermally driven Rayleigh- Bénard convection and find that the arc length of spirals decreases monotonically with decreasing Prandtl number of the fluid and increasing heating. By contrast, the winding number of the spirals is nonmonotonic in the heating. The distribution function for the number of spirals is significantly narrower than a Poisson distribution. The distribution function for the winding number shows approximately an exponential decay. It depends only weakly on the heating, but strongly on the Prandtl number. Large spirals arise only for larger Prandtl numbers. In this regime the joint distribution for the spiral length and the winding number exhibits a three-peak structure, indicating the dominance of Archimedean spirals of opposite sign and relatively straight sections. For small Prandtl numbers the distribution function reveals a large number of small compact pattern components.

Low work-function coating for an entirely propellantless bare electrodynamic tether

We present the possibility of a low work-function material, calcium aluminate electride, being used for a coating on a bare electrodynamic tether system. Analyses suggest that the coating would eliminate the need for an active cathodic device like a hollow cathode and, consequently, eliminate the need for an expellant to the hollow cathode, thus resulting in an electrodynamic tether system that requires no consumables. Applications include on-orbit power generation and deorbiting debris from low Earth orbit in a simple and trouble-free manner.