Valutazione dello stato nutrizionale e del ruolo della paraoxanosi nella predisposizione dell'anziano al rischio cardiovascolare

Dottorato di ricerca in biochimica e fisiologia della nutrizione. 12. ciclo. A. a. 1998-99. Docente guida L. Sacchetti. Coordinatore G. PizzutiConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Candidate Genes Associated with Abiotic Stress Response in Plants as Tools to Engineer Tolerance to Drought, Salinity and Extreme Temperatures in Wheat: An Overview

Wheat represents one of the most important staple food crops worldwide and its genetic improvement is fundamental to meeting the global demand of the growing population. However, the environmental stresses, worsened by climate change, and the increasing deterioration of arable land make it very difficult to fulfil this demand. In light of this, the tolerance of wheat to abiotic stresses has become a key objective of genetic improvement, as an effective strategy to ensure high yields without increasing the cultivated land. Genetic erosion related to modern agriculture, whereby elite, high-yielding wheat varieties are the product of high selection pressure, has reduced the overall genetic diversity, including the allelic diversity of genes that could be advantageous for adaptation to adverse environmental conditions. This makes traditional breeding a less effective or slower approach to generating new stress-tolerant wheat varieties. Either mining for the diversity of not-adapted large germplasm pools, or generating new diversity, are the mainstream approaches to be pursued. The advent of genetic engineering has opened the possibility to create new plant variability and its application has provided a strong complement to traditional breeding. Genetic engineering strategies such as transgenesis and genome editing have then provided the opportunity to improve environmental tolerance traits of agronomic importance in cultivated species. As for wheat, several laboratories worldwide have successfully produced transgenic wheat lines with enhanced tolerance to abiotic stresses, and, more recently, significant improvements in the CRISPR/Cas9 tools available for targeted variations within the wheat genome have been achieved. In light of this, the present review aims to provide successful examples of genetic engineering applications for the improvement of wheat adaptation to drought, salinity and extreme temperatures, which represent the most frequent and most severe events causing the greatest losses in wheat production worldwide

Transport Pathways—Proton Motive Force Interrelationship in Durum Wheat Mitochondria

In durum wheat mitochondria (DWM) the ATP-inhibited plant mitochondrial potassium channel (PmitoKATP) and the plant uncoupling protein (PUCP) are able to strongly reduce the proton motive force (pmf) to control mitochondrial production of reactive oxygen species; under these conditions, mitochondrial carriers lack the driving force for transport and should be inactive. However, unexpectedly, DWM uncoupling by PmitoKATP neither impairs the exchange of ADP for ATP nor blocks the inward transport of Pi and succinate. This uptake may occur via the plant inner membrane anion channel (PIMAC), which is physiologically inhibited by membrane potential, but unlocks its activity in de-energized mitochondria. Probably, cooperation between PIMAC and carriers may accomplish metabolite movement across the inner membrane under both energized and de-energized conditions. PIMAC may also cooperate with PmitoKATP to transport ammonium salts in DWM. Interestingly, this finding may trouble classical interpretation of in vitro mitochondrial swelling; instead of free passage of ammonia through the inner membrane and proton symport with Pi, that trigger metabolite movements via carriers, transport of ammonium via PmitoKATP and that of the counteranion via PIMAC may occur. Here, we review properties, modulation and function of the above reported DWM channels and carriers to shed new light on the control that they exert on pmf and vice-versa

Carotenoids in Cereal Food Crops: Composition and Retention throughout Grain Storage and Food Processing

Carotenoids are C40 isoprenoids synthesized by plants, as well as some bacteria, fungi and algae, that have been reported to be responsible for a number of benefits conferred on human health. The inability of animals and humans to synthesize de novo these compounds is the reason why they must be introduced from dietary sources. In cereal grains, carotenoids are important phytochemicals responsible for the characteristic yellow colour of the endosperm, which confers nutritional and aesthetic quality to cereal-based products. Cereals are staple foods for a large portion of the world population, and the biofortification of cereal grains with carotenoids may represent a simple way to prevent many human diseases and disorders. Unfortunately, evidence exists that the storage and processing of cereal grains into food products may negatively impact their carotenoid content; so, this loss should be taken into consideration when analysing the potential health benefits of the cereal-based products. Focusing on the recent updates, this review summarizes the chemical composition of the carotenoids in the grains of staple cereals, including wheat, maize, rice and sorghum, the main factors that affect their carotenoid content during storage and processing and the most fruitful strategies used improve the grain carotenoid content and limit the carotenoid post-harvest losses

Candidate Genes Associated with Abiotic Stress Response in Plants as Tools to Engineer Tolerance to Drought, Salinity and Extreme Temperatures in Wheat: An Overview

Wheat represents one of the most important staple food crops worldwide and its genetic improvement is fundamental to meeting the global demand of the growing population. However, the environmental stresses, worsened by climate change, and the increasing deterioration of arable land make it very difficult to fulfil this demand. In light of this, the tolerance of wheat to abiotic stresses has become a key objective of genetic improvement, as an effective strategy to ensure high yields without increasing the cultivated land. Genetic erosion related to modern agriculture, whereby elite, high-yielding wheat varieties are the product of high selection pressure, has reduced the overall genetic diversity, including the allelic diversity of genes that could be advantageous for adaptation to adverse environmental conditions. This makes traditional breeding a less effective or slower approach to generating new stress-tolerant wheat varieties. Either mining for the diversity of not-adapted large germplasm pools, or generating new diversity, are the mainstream approaches to be pursued. The advent of genetic engineering has opened the possibility to create new plant variability and its application has provided a strong complement to traditional breeding. Genetic engineering strategies such as transgenesis and genome editing have then provided the opportunity to improve environmental tolerance traits of agronomic importance in cultivated species. As for wheat, several laboratories worldwide have successfully produced transgenic wheat lines with enhanced tolerance to abiotic stresses, and, more recently, significant improvements in the CRISPR/Cas9 tools available for targeted variations within the wheat genome have been achieved. In light of this, the present review aims to provide successful examples of genetic engineering applications for the improvement of wheat adaptation to drought, salinity and extreme temperatures, which represent the most frequent and most severe events causing the greatest losses in wheat production worldwide

The Molecular and Functional Characterization of the Durum Wheat Lipoxygenase TdLOX2 Suggests Its Role in Hyperosmotic Stress Response

In plants, lipoxygenases (LOXs) are involved in various processes, such as growth, development, and response to stress cues. In the present study, the expression pattern of six durum wheat LOX-encoding genes (TdLpx-B1.1, TdLpx-B1.2, TdLpx-A2, TdLpx-B2, TdLpx-A3 and TdLpx-B3) under hyperosmotic stress was investigated. With osmotic (0.42 M mannitol) and salt (0.21 M NaCl) stress imposed at the early stages of seedling growth, a strong induction of the TdLpx-A2 gene expression in the shoots paralleled an equally strong increase in the LOX activity. Enhanced levels of malondialdehyde (MDA) and increased rates of superoxide anion generation were also observed as a result of the stress imposition. Sequence analysis of the TdLOX2 encoded by the TdLpx-A2 gene revealed that it belonged to the type-1 9-LOX group. When overexpressed in E. coli, TdLOX2 exhibited normal enzyme activity, high sensitivity to specific LOX inhibitors, with 76% and 99% inhibition by salicylhydroxamic and propyl gallate, respectively, and a preference for linoleic acid as substrate, which was converted exclusively to its corresponding 13-hydroperoxide. This unexpected positional specificity could be related to the unusual TV/K motif that in TdLOX2 replaces the canonical TV/R motif of 9-LOXs. Treatment of seedlings with propyl gallate strongly suppressed the increase in LOX activity induced by the hyperosmotic stress; the MDA accumulation was also reduced but less markedly, whereas the rate of superoxide anion generation was even more increased. Overall, our findings suggest that the up-regulation of the TdLpx-A2 gene is a component of the durum wheat response to hyperosmotic stress and that TdLOX2 may act by counteracting the excessive generation of harmful reactive oxygen species responsible for the oxidative damages that occur in plants under stress

Molecular Approaches to Genetically Improve the Accumulation of Health-Promoting Secondary Metabolites in Staple Crops—A Case Study: The Lipoxygenase-B1 Genes and Regulation of the Carotenoid Content in Pasta Products

Secondary metabolites, also known as phytochemicals, represent a large subset of plant molecules that include compounds with health-promoting effects. Indeed, a number of epidemiological studies have shown that, when taken regularly and in adequate amounts, these molecules can have long-term beneficial effects on human health, through reduction of the incidence of degenerative diseases, such as cardiovascular diseases, obesity, diabetes, and cancer. As the dietary intake of these phytochemicals is often inadequate, various strategies are in use to improve their content in staple crops, and the end-products thereof. One of the most effective strategies is crop improvement through genetic approaches, as this is the only way to generate new cultivars in which the high accumulation of a given phytochemical is stably fixed. Efforts to genetically improve quality traits are rapidly evolving, from classical breeding to molecular-assisted approaches; these require sound understanding of the molecular bases underlying the traits, to identify the genes/alleles that control them. This can be achieved through global analysis of the metabolic pathway responsible for phytochemical accumulation, to identify the link between phytochemical content and the activities of key enzymes that regulate the metabolic pathway, and between the key enzymes and their encoding genes/alleles. Once these have been identified, they can be used as markers for selection of new improved genotypes through biotechnological approaches. This review provides an overview of the major health-promoting properties shown to be associated with the dietary intake of phytochemicals, and describes how molecular approaches provide means for improving the health quality of edible crops. Finally, a case study is illustrated, of the identification in durum wheat of the Lipoxygenase-B1 genes that control the final carotenoid content in semolina-based foods, such as pasta products

Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications

Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes

Secretory Phospholipases A2 in Durum Wheat (Triticum durum Desf.): Gene Expression, Enzymatic Activity, and Relation to Drought Stress Adaptation

Phospholipases A2 (PLA2s) are known to mediate signaling cascades during plant growth and development, as well as biotic and abiotic stress responses. In this context, the present study provides extensive characterization of specific PLA2s in durum wheat, and assesses their involvement in durum wheat response to drought stress. In durum wheat leaves, four full-length expressed sequences encoding putative PLA2s were isolated and characterized as belonging to the class of secretory PLA2s (sPLA2s): TdsPLA2I, TdsPLA2II, TdsPLA2III and TdsPLA2IV. PLA2 activity was also detected, the characteristics of which resemble those of previously characterized plant sPLA2s: strong preference for phospholipids; requirement for millimolar Ca2+ concentrations; optimal activity at basic pH; heat stability; and inhibition by the reducing agent dithiothreitol. With drought stress imposed at both the vegetative and reproductive stages, accumulation of TdsPLA2I and TdsPLA2III transcripts, and to a lesser extent of TdsPLA2IV transcript, paralleled increased PLA2 activity; both transcript levels and enzymatic activity decreased as a consequence of stress recovery. Consistently, free fatty acid analysis of drought-stressed leaves revealed increased linoleate, linolenate and palmitate contents, which were reversed by plant re-watering. Overall, these findings strongly suggest that there are inducible sPLA2 isoforms in durum wheat that have roles in orchestrating the plant response to drought stress