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12 Pág. Centro de Biotecnología y Genómica de Plantas, During barley germination, cysteine proteases are essential in the mobilization of storage compounds providing peptides and amino acids to sustain embryo growth until photosynthesis is completely established. Knockdown barley plants, generated by artificial miRNA, for the cathepsins B- and F-like HvPap-19 and HvPap-1 genes, respectively, showed less cysteine protease activities and consequently lower protein degradation. The functional redundancy between proteases triggered an enzymatic compensation associated with an increase in serine protease activities in both knockdown lines, which was not sufficient to maintain germination rates and behaviour. Concomitantly, these transgenic lines showed alterations in the accumulation of protein and carbohydrates in the grain. While the total amount of protein increased in both transgenic lines, the starch content decreased in HvPap-1 knockdown lines and the sucrose concentration was reduced in silenced HvPap-19 grains. Consequently, phenotypes of HvPap-1 and HvPap-19 artificial miRNA lines showed a delay in the grain germination process. These data demonstrate the potential of exploring the properties of barley proteases for selective modification and use in brewing or in the livestock feeding industry., This research was supported by the Ministerio de Economia, Industria y Competitividad of Spain (projects BIO2014-53508-R, BIO17-83472-R, and RyC17MESFB). RyC grant (RyC2017-21814) from the MEIC and Plan Propio from the UPM-financed MES. We gratefully appreciate comments from Dr Ljudmilla Borisjuk and Dr Volodymyr Radchuk from the IPK-Gatersleben (Germany).

Since surface plays a key role in bioactivity, the response of the host to the biomaterial will determine the success or failure of the prosthesis. The purpose of this study is to make an exhaustive analysis of the histological and histochemical characteristics of new bone tissue around Zr implants anodized at 60 V (Zr60) supported by histomorphometric methods in a rat model. Fibrous tissue was observed around the control implants (Zr0) and osteoblasts were identifed on the trabeculae close to the implantation site that showed typical cytological characteristics of active secretory cells, regardless of the surface condition. The histomorphometrical analysis revealed a signifcant increase in cancellous bone volume, trabecular thickness and in trabecular number together with a decrease in trabecular separation facing Zr60. TRAP staining showed that there was a relative increase in the number of osteoclasts for Zr60. In addition, a larger number of osteoclast with a greater number of nuclei were detected in the tibiae for Zr60. This research demonstrated that the new bone microarchitecture in contact with Zr60 is able to improve the early stages of the osseointegration process and consequently the primary stability of implants which is a crucial factor to reduce recovery time for patients. Fil: Tano de la Hoz, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina Fil: Katunar, Maria Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina Fil: Gonzalez, Ariel. Universidad Nacional de Mar del Plata; Argentina Fil: Gomez Sanchez, Andrea Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentina. Universidad Tecnologica Nacional. Facultad Regional Villa Maria; Argentina Fil: Díaz, Alcira Ofelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Marinas y Costeras. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Marinas y Costeras; Argentina Fil: Ceré, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; Argentina

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy-2 and Icy-4 cystatin genes. Artificial microRNA lines independently silencing the two drought-induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay-green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy-2 and Icy-4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap-1, HvPap-12, and HvPap-16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.

To survive under water deficiency, plants alter gene expression patterns, make structural and physiological adjustments, and optimize the use of water. Rapid degradation and turnover of proteins is required for effective nutrient recycling. Here, we examined the transcriptional responses of the C1A cysteine protease family to drought in barley and found that four genes were up-regulated in stressed plants. Knock-down lines for the protease-encoding genes HvPap-1 and HvPap-19 showed unexpected changes in leaf cuticle thickness and stomatal pore area. The efficiency of photosystem II and the total amount of proteins were almost unaltered in stressed transgenic plants while both parameters decreased in stressed wild-type plants. Although the patterns of proteolytic activities in the knock-down lines did not change, the amino acid accumulation increased in response to drought, concomitant with a higher ABA content. Whilst jasmonic acid (JA) and JA-Ile concentrations increased in stressed leaves of the wild-type and the HvPap-1 knock-down lines, their levels were lower in the HvPap-19 knock-down lines, suggesting the involvement of a specific hormone interaction in the process. Our data indicate that the changes in leaf cuticle thickness and stomatal pore area had advantageous effects on leaf defense against fungal infection and mite feeding mediated by Magnaporthe oryzae and Tetranychus urticae, respectively.

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy-2 and Icy-4 cystatin genes. Artificial microRNA lines independently silencing the two drought-induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay-green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy-2 and Icy-4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap-1, HvPap-12, and HvPap-16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.

Plant senescence is a complex physiological process consequence either of the natural lifespan or externally induced by abiotic and biotic factors. It comprises a coordinated sequence of molecular and biochemical events, phenotypically illustrated by changes in plant colour. Senescence is associated with alterations in chlorophyll and pigment content, reduction of photosynthesis, hydrolysis of macromolecules to produce more simple compounds and dismantling of cell organelles, to finally produce cell death. At the end, relocation of nutrients from the senescent tissues towards sink organs or growing tissues takes place to complete a recycling process. Consequently, the major part of the nitrogen is released as ammonium after being re-assimilated into amino acids to be exported via the phloem to the developing grains, fruits and tubers. During senescence, the reprograming of thousands of genes is triggered in response to specific senescence-promoting factors under a restricted regulatory control. The actual high-throughput omics technologies have led to the generation of integrative information, which has been used to understand the physiological changes during the onset and progression of senescence. This chapter covers an overview on plant senescence, particularly focussed on the senescence of the leaf, including the most recent findings about features, signalling, regulation and pathways involved in this natural or induced process.

Orthopaedic devices for permanent implants require short term fixation and fast bone attachment and healing. Superficial modification of surgical implants is often used as a tool to generate a surface that besides being protective could also allow the integration of the metal to the human body, creating a “bioactive” surface that has the ability of creating a natural bonding between the metal surface and the existing bone. One way of achieving this surface modification is by means anodisation since an increase in the thickness and changes in the topography of the native oxide formed on metals can produce an improvement in bone response. Other possibility in the surface modification of the metallic implants is the coatings with organic-inorganic ceramic or glassy coatings as a way to improve the implant performance.The aim of this charter is review the surface modifications produced on orthopaedic and dentistry metallic materials by anodisation and by hybrid coatings by sol gel technique with the aim of promoting both corrosion resistance in physiological fluids and bioactivity.

Introduction Anodization constitutes a surface modification route that allows the design of the structure, chemistry and electronic properties of the films formed by carefully selecting process variables. This versatility is used to modify the surface of permanent titanium implants to improve osseointegration rate. Post anodization heat treatments are often performed to improve the crystallinity of the surface films and this leads to increase of bioactivity. However, thermal treatments may alter not only the crystal structure of anodized oxide film, but also induce growth of thermal oxide or alter the structure of the anodic films. Since anodic films on titanium may be inhomogeneous and porous, the effect of air heat treatments on such complex structures was evaluated in this work. EXPERIMENTAL Flat specimens of commercially pure titanium Grade 2 were used. Anodising was performed in a two-electrode cell with Pt wire as counter electrode. Two acidic solutions were employed, i) TP: 1 mol L -1 H3PO4, ii) TPF: 1 mol.L -1 H3PO4 + 0.3 % HF; for potentiostatic anodization. Post anodization heat treatments at 553, 623, 673 and 723 K during 3 hours and cooling in furnace were conducted. The surface morphology of the specimens was observed by scanning electron microcopy. The crystalline phases were determined by Raman spectroscopy and graze angle X-ray diffraction. Anodic polarization curves and electrochemical impedance spectroscopy (EIS) tests were performed in borax solution. RESULTS AND DISCUSSION DRX and Raman spectroscopy were complementary used to analyse the crystallographic phases detected and its spatial distribution. Not crystalline phases were detected on mechanical polished titanium (T), anatase was detected in most sites on TP and TPF presents a partial crystalline presence, with sites with anatase, rutile and amorphous film. Changes in colour of heat treated samples are observed in all cases, indicating that a certain degree of change in the surface films has occurred. With Raman spectroscopy an increase in crystalline phase amount was detected in T, TP and TPF samples. The oxide film formed in phosphoric (TP) behaves as a barrier, increasing the corrosion potential, diminishing the current density and increasing the total impedance. On the contrary, anodic films growth in phosphoric and fluoride solution, presents in anodic polarization experiments a similar response than non-anodized samples, but EIS evidences a complex structure of TPF anodic films characterized with two or even three time constants. Thermal treatments in air have different effects on the modified surfaces. On polished titanium, the growth of a thermal oxide is evidenced both on anodic polarization curves and EIS. The thermal oxide layer behaves as a barrier to electronic transfer, acting in a similar way than anodic films. In TP films, the thermal treatments do not produce differences in current density during anodic polarization, while with EIS changes in the inner (compact) and outer (more porous) part of the film are evidenced. Finally, in TPF samples, thermal treatment results in deleterious effects on the barrier efficiency of the anodic layer. Porous TPF films behaves in similar way than polished titanium in anodic polarization tests, since the thermal treatment induce diminish in current densities. However, EIS results indicates a deleterious effect of the thermal treatment on the anodic layer, evidenced by a diminish in the total impedance in Bode plots, and the appearance of a capacitive film governing the EIS response. The electrochemical evidence suggests the rupture of the anodic film of TPF during thermal treatments, corroborating Raman and GDRX results and also SEM evidence. It may be concluded that careful analysis of changes in structure both on the surface and in the substrate inner layer may be due in order to evaluate the influence of thermal treatments on the in vivo or in vitro response of surface modified titanium as a permanent implant, since is clear in this report that not only crystallization of amorphous portion of the surface oxide, but also growth, re-ordering and even destruction of anodic films may be occurring.

Potentiostatic anodizing of commercially pure titanium, using ammonium phosphate and ammonium fluoride solution as electrolyte is studied. The objective is to generate titanium oxides on the surface and phosphor compounds presenting good protective and mechanical properties, and proper adhesion to the metal substrate to remain during surgical orthopedic procedures (implants). Two different applied potentials were used to obtain different surface oxides morphologies (20 and 30V). The characterization and quantification of the generated deposits is presented as a starting point for the future application of these composite types of materials. X-ray Photoelectron Spectroscopy (XPS) and Raman spectroscopy techniques showed the presence of phosphor compounds and anatase (TiO2) as the main constitutive phases. Anodized samples presented less passivity current density with respect to the bare substrate, showing that the film formed by anodizing acts as an effective barrier to the electrolyte entrance to reach the metallic surface. Besides, mechanical properties of bare substrate were improved with the formation of anodic films: hardness value was increased in the oxide zones and the Young’s elastic modulus was decreased in the phosphor-compounds zones in the sample anodized with 30V, generating an interface with different elastic properties that could be able to enhance the prosthesis performance. Nano-scratch tests showed a very ductile behavior and good adhesion of anodic films, without any apparent signs of cracks or delamination. Fil: Games, Lucas Aloia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación en Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina Fil: Gomez Sanchez, Andrea Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación en Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina Fil: Jimenez Piqué, Emilio. Universitat Politècnica de Catalunya; España Fil: Schreiner, Wido H.. Universidade Federal do Paraná; Brasil Fil: Cere, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación en Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina Fil: Ballarre, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigación en Ciencia y Tecnología de Materiales (i); Argentina. Universidad Nacional de Mar del Plata. Facultad de Ingeniería; Argentina