Grazia M. Borrelli

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.

The colours of durum wheat: a review

Abstract. Pigments are essential to the life of all living organisms. Animals and plants have been the subjects of basic and applied research with the aim of determining the basis of the accumulation and physiological roles of pigments. In crop species, the edible organs show large variations in colour. In durum wheat grain, which is a staple food for humans, the colour is mainly due to two natural classes of pigment: carotenoids and anthocyanins. The carotenoids provide the yellow pigmentation of the durum wheat endosperm, and consequently of the semolina, which has important implications for the marketing of end products based on durum wheat. Anthocyanins accumulate in the aleurone or pericarp of durum wheat and provide the blue, purple and red colours of the grain. Both the carotenoids and the anthocyanins are known to provide benefits for human health, in terms of decreased risks of certain diseases. Therefore, accumulation of these pigments in the grain represents an important trait in breeding programs aimed at improving the nutritional value of durum wheat grain and its end products. This review focuses on the biochemical and genetic bases of pigment accumulation in durum wheat grain, and on the breeding strategies aimed at modifying grain colour.

Novel durum wheat genes up-regulated in response to a combination of heat and drought stress

We report the effect of heat, drought and combined stress on the expression of a group of genes that are up-regulated under these conditions in durum wheat (Triticum turgidum subsp. durum) plants. Modulation of gene expression was studied by cDNA-AFLP performed on RNAs extracted from flag leaves. By this approach, we identified several novel durum wheat genes whose expression is modulated under different stress conditions. We focused on a group of hitherto undescribed up-regulated genes in durum wheat, among these, 7 are up-regulated by heat, 8 by drought stress, 15 by combined heat and drought stress, 4 are up-regulated by both heat and combined stress, and 3 by both drought and combined stress. The functional characterization of these genes will provide new data that could help the developing of strategies aimed at improving durum wheat tolerance to field stress.

Different stress responsive strategies to drought and heat in two durum wheat cultivars with contrasting water use efficiency

BackgroundDurum wheat often faces water scarcity and high temperatures, two events that usually occur simultaneously in the fields. Here we report on the stress responsive strategy of two durum wheat cultivars, characterized by different water use efficiency, subjected to drought, heat and a combination of both stresses.ResultsThe cv Ofanto (lower water use efficiency) activated a large set of well-known drought-related genes after drought treatment, while Cappelli (higher water use efficiency) showed the constitutive expression of several genes induced by drought in Ofanto and a modulation of a limited number of genes in response to stress. At molecular level the two cvs differed for the activation of molecular messengers, genes involved in the regulation of chromatin condensation, nuclear speckles and stomatal closure. Noteworthy, the heat response in Cappelli involved also the up-regulation of genes belonging to fatty acid β-oxidation pathway, glyoxylate cycle and senescence, suggesting an early activation of senescence in this cv. A gene of unknown function having the greatest expression difference between the two cultivars was selected and used for expression QTL analysis, the corresponding QTL was mapped on chromosome 6B.ConclusionOfanto and Cappelli are characterized by two opposite stress-responsive strategies. In Ofanto the combination of drought and heat stress led to an increased number of modulated genes, exceeding the simple cumulative effects of the two single stresses, whereas in Cappelli the same treatment triggered a number of differentially expressed genes lower than those altered in response to heat stress alone. This work provides clear evidences that the genetic system based on Cappelli and Ofanto represents an ideal tool for the genetic dissection of the molecular response to drought and other abiotic stresses.

Stay-green trait-antioxidant status interrelationship in durum wheat (Triticum durum) flag leaf during post-flowering

Three independent durum wheat mutant lines that show delayed leaf senescence or stay-green (SG) phenotype, SG196, SG310 and SG504, were compared to the parental genotype, cv. Trinakria, with respect to the photosynthetic parameters and the cellular redox state of the flag leaf in the period from flowering to senescence. The SG mutants maintained their chlorophyll content and net photosynthetic rate for longer than Trinakria, thus revealing a functional SG phenotype. They also showed a better redox state as demonstrated by: (1) a lower rate of superoxide anion production due to generally higher activity of the antioxidant enzymes superoxide dismutase and catalase in all of the SG mutants and also of the total peroxidase in SG196; (2) a higher thiol content that can be ascribed to a higher activity of the NADPH-providing enzyme glucose-6-phosphate dehydrogenase in all of the SG mutants and also of the NADP+-dependent malic enzyme in SG196; (3) a lower pro-oxidant activity of lipoxygenase that characterises SG196 and SG504 mutants close to leaf senescence. Overall, these results show a general relationship in durum wheat between the SG phenotype and a better redox state. This relationship differs across the different SG mutants, probably as a consequence of the different set of altered genes underlying the SG trait in these independent mutant lines.

Integrated views in plant breeding: from the perspective of biotechnology

Conventional plant breeding has typically used phenotypic observations and statistical analysis to select for improved germplasm in segregating populations and the improvements in yield were likely associated with the unconscious pyramiding of yield-related traits. Modern breeding relies on genetics and genomics to identify superior alleles and to introduce them into new high-yielding varieties and hybrids. The recent advances in genomics and the diffusion of low-cost sequencing platforms have implications for both current and future plant breeding. New high-throughput genotyping facilities are allowing a fast identification of marker-trait associations and make possible a genome-wide selection process (genomic selection). Resequencing projects, leading to the discovery of new alleles and new molecular breeding methods based on site specific mutagenesis in combination with the knowledge on gene functions, open new perspectives for genetically modified (GM) and non-GM plant improvement.

Integrated Views in Plant Breeding: Modern Approaches for an Old Topic

This chapter provides an overview of the breeding progress for some of the most important agronomic traits and summarizes molecular technologies associated with plant breeding, highlighting future perspectives that could result from the integration of recent advances in physiology, genomics, and biotechnology. Plant breeding can be described as the continuous accumulation of superior alleles (i.e., genes encoding useful traits) in the gene pool of the cultivated elite lines. The cultivated germplasm represents only a minimal part of the whole germplasm of a given crop species. Under these conditions, breeding has two main targets: to search for new useful alleles worthy to be introduced into the elite lines (also called “prebreeding”) and to promote the recombination within the elite germplasm to find the best combinations among the best alleles. Prebreeding refers to the transfer or introgression of genes and gene combinations from unadapted sources into breeding materials. There are two distinct approaches for using wild species, exotic, and landrace germplasm in plant breeding: introgression and incorporation. Introgression indicates the transfer of one or a few alleles from exotic genotypes to adapted bred cultivars that lack the allele(s) controlling a specific trait. Incorporation refers to a large-scale effort aiming at developing locally adapted genotypes using exotic germplasm, which is likely to broaden the genetic base of new breeding materials.

Regulation and Evolution of NLR Genes: A Close Interconnection for Plant Immunity

NLR (NOD-like receptor) genes belong to one of the largest gene families in plants. Their role in plants’ resistance to pathogens has been clearly described for many members of this gene family, and dysregulation or overexpression of some of these genes has been shown to induce an autoimmunity state that strongly affects plant growth and yield. For this reason, these genes have to be tightly regulated in their expression and activity, and several regulatory mechanisms are described here that tune their gene expression and protein levels. This gene family is subjected to rapid evolution, and to maintain diversity at NLRs, a plethora of genetic mechanisms have been identified as sources of variation. Interestingly, regulation of gene expression and evolution of this gene family are two strictly interconnected aspects. Indeed, some examples have been reported in which mechanisms of gene expression regulation have roles in promotion of the evolution of this gene family. Moreover, co-evolution of the NLR gene family and other gene families devoted to their control has been recently demonstrated, as in the case of miRNAs.

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.

Integrated Views in Plant Breeding

This chapter provides an overview of the breeding progress for some of the most important agronomic traits and summarizes molecular technologies associated with plant breeding, highlighting future perspectives that could result from the integration of recent advances in physiology, genomics, and biotechnology. Plant breeding can be described as the continuous accumulation of superior alleles (i.e., genes encoding useful traits) in the gene pool of the cultivated elite lines. The cultivated germplasm represents only a minimal part of the whole germplasm of a given crop species. Under these conditions, breeding has two main targets: to search for new useful alleles worthy to be introduced into the elite lines (also called “prebreeding”) and to promote the recombination within the elite germplasm to find the best combinations among the best alleles. Prebreeding refers to the transfer or introgression of genes and gene combinations from unadapted sources into breeding materials. There are two distinct approaches for using wild species, exotic, and landrace germplasm in plant breeding: introgression and incorporation. Introgression indicates the transfer of one or a few alleles from exotic genotypes to adapted bred cultivars that lack the allele(s) controlling a specific trait. Incorporation refers to a large-scale effort aiming at developing locally adapted genotypes using exotic germplasm, which is likely to broaden the genetic base of new breeding materials.