Giovanni Pontecorvo

Salinity Stress and Salt Tolerance

Salinity is one of the most serious factors limiting the productivity of agricultural crops, with adverse effects on germination, plant vigour and crop yield (R Munns & Tester, 2008). Salinization affects many irrigated areas mainly due to the use of brackish water. Worldwide, more than 45 million hectares of irrigated land have been damaged by salt, and 1.5 million hectares are taken out of production each year as a result of high salinity levels in the soil (R Munns & Tester, 2008). High salinity affects plants in several ways: water stress, ion toxicity, nutritional disorders, oxidative stress, alteration of metabolic processes, membrane disorganization, reduction of cell division and expansion, genotoxicity (Hasegawa, Bressan, Zhu, & Bohnert, 2000; R. Munns, 2002; Zhu, 2007). Together, these effects reduce plant growth, development and survival. During the onset and development of salt stress within a plant, all the major processes such as photosynthesis, protein synthesis and energy and lipid metabolism are affected (Parida & Das, 2005). During initial exposure to salinity, plants experience water stress, which in turn reduces leaf expansion. The osmotic effects of salinity stress can be observed immediately after salt application and are believed to continue for the duration of exposure, resulting in inhibited cell expansion and cell division, as well as stomatal closure (T. J. Flowers, 2004; R. Munns, 2002). During long-term exposure to salinity, plants experience ionic stress, which can lead to premature senescence of adult leaves, and thus a reduction in the photosynthetic area available to support continued growth (Cramer & Nowak, 1992). In fact, excess sodium and more importantly chloride has the potential to affect plant enzymes and cause cell swelling, resulting in reduced energy production and other physiological changes (Larcher 1980). Ionic stress results in premature senescence of older leaves and in toxicity symptoms (chlorosis, necrosis) in mature leaves due to high Na+ which affects plants by disrupting protein synthesis and interfering with enzyme activity (Hasegawa, Bressan, Zhu, & Bohnert, 2000; R. Munns, 2002; R Munns & Termaat, 1986). Many plants have evolved several mechanisms either to exclude salt from their cells or to tolerate its presence within the cells. In this chapter, we mainly discuss about soil salinity, its effects on plants and tolerance mechanisms which permit the plants to withstand stress, with particular emphasis on ion homeostasis, Na+ exclusion and tissue tolerance. Moreover we give a synthetic overview of the two major approaches that have been used to improve stress tolerance: exploitation of natural genetic variations and generation of transgenic plants with novel genes or altered expression levels of the existing genes. A fundamental biological understanding and knowledge of the effects of salt stress on plants is necessary to provide additional

Polymorphism of a new Ty1-copia retrotransposon in durum wheat under salt and light stresses

Long terminal repeat retrotransposons are the most abundant mobile elements in the plant genome and play an important role in the genome reorganization induced by environmental challenges. Their success depends on the ability of their promoters to respond to different signaling pathways that regulate plant adaptation to biotic and abiotic stresses. We have isolated a new Ty1-copia-like retrotransposon, named Ttd1a from the Triticum durum L. genome. To get insight into stress activation pathways in Ttd1a, we investigated the effect of salt and light stresses by RT-PCR and S-SAP profiling. We screened for Ttd1a insertion polymorphisms in plants grown to stress and showed that one new insertion was located near the resistance gene. Our analysis showed that the activation and mobilization of Ttd1a was controlled by salt and light stresses, which strengthened the hypothesis that stress mobilization of this element might play a role in the defense response to environmental stresses.

A PCR based SNPs marker for specific characterization of English walnut (Juglans regia L.) cultivars

English walnut (Juglans regia L.) is the most economically important species from all the 21 species belonging to the genus Juglans and is an important and healthy food as well as base material for timber industry. The aim of this study was to develop a simple technique for specific characterization of English walnut using DNA method. The first and second internal transcribed spacers (ITS1 and ITS2) as well as the intervening 5.8S coding region of the rRNA gene for 18 cultivars of J. regia L. isolated from different geographic origins were characterized. The size of the spacers sequences ranged from 257 to 263 bases for ITS1 and from 217 to 219 bases for ITS2. Variation of GC contents has also been observed and scored as 55–56.7 and 57.1–58.9% for ITS1 and ITS2, respectively. This data exhibited the presence of polymorphism among cultivars. Alignment of the ITS1-5.8S-ITS2 sequences from 18 walnut cultivars showed that there were 244 single nucleotide polymorphisms (SNPs) and 1 short insertion–deletion (indel) at 5′ end ITS1. Amplification refractory mutation system strategy was successfully applied to the SNP markers of the ITS1 and ITS2 sequences for the fingerprinting analysis of 17 on 18 walnut cultivars. The prediction of ITS1 and ITS2 RNA secondary structure from each cultivar was improved by detecting key functional elements shared by all sequences in the alignments. Phylogenetic analysis of the ITS1-5.8S-ITS2 region clearly separated the isolated sequences into two clusters. The results showed that ITS1 and ITS2 region could be used to discriminate these walnut cultivars.

Ttd1a promoter is involved in DNA–protein binding by salt and light stresses

Stress modulation of retrotransposons may play a role in generating host genetic plasticity in response to environmental stress. Transposable elements have been suggested to contribute to the evolution of genes, by providing cis-regulatory elements leading to changes in expression patterns. Indeed, their promoter elements are similar to those of plant defence genes and may bind similar defence-induced transcription factors. We previously isolated a new Ty1-copia retrontrasposon named Ttd1a and showed its activation and mobilization in salt and light stresses. Here, using a retard mobility assay in Triticum durum L. crude extracts, we showed that the CAAT motif present in the Ttd1a retrotransposon promoter, is involved in DNA–protein binding under salt and light stresses and therefore in the regulation of Ttd1a activity. Data presented in this paper suggest that nuclear proteins can interact with the CAAT motif either directly or indirectly and enhance Ttd1a by a specific ligand-dependent activation under stress.

Plant Genes for Abiotic Stress

Abiotic stress is the primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50%. Plants as sessile organisms are constantly exposed to changes in environmental conditions. When these changes are rapid and extreme, plants generally perceive them as stresses. However stresses are not necessarily a problem for plants because they have evolved effective mechanisms to avoid or reduce the possible damages. The response to changes in environment can be rapid, depending on the type of stress and can involve either adaptation mechanisms, which allow them to survive the adverse conditions, or specific growth habitus to avoid stress conditions. In fact, plants can perceive abiotic stresses and elicit appropriate responses with altered metabolism, growth and development. The regulatory circuits include stress sensors, signalling pathways comprising a network of protein-protein interactions, transcription factors and promoters, and finally the output proteins or metabolites (table 1). A number of abiotic stresses such as extreme temperatures, high light intensity, osmotic stresses, heavy metals and a number of herbicides and toxins lead to over production of reactive oxygen species (ROS) including H2O2 causing extensive cellular damage and inhibition of photosynthesis. Normally, ROS are rapidly removed by antioxidative mechanisms, but this removal can be impaired by stresses themselves (Allan & Fluhr, 2007), causing a rise in their intracellular concentration and an increase of the damage. To prevent or repair these damages, plant cells use a complex defence system, involving a number of antioxidative stress-related defence genes that, in turn, induce changes in the biochemical plant machinery. Studies have shown that ROS probably require additional molecules to transduce and amplify defence signals. ROS production and anti-oxidant processes, all act in a synergistic, additive or antagonistic way, related to the control of oxidative stress. Responses to stress are not linear pathways, but are complex integrated circuits involving multiple pathways and in specific cellular compartments, tissues, and the interaction of additional cofactors and/or signalling molecules to coordinate a specified response to a given stimulus (Dombrowski, 2009). Onset of a stress triggers some (mostly unknown) initial sensors, which then activate cytoplasmic Ca2+ and protein signalling pathways, leading to stress-responsive gene expression and physiological changes (Bressan et al., 1998;

Transcription Factors and Genes in Abiotic Stress

Plants are constantly exposed to changes in environmental conditions. When these changes are rapid and extreme, plants generally perceive them as stresses. Abiotic stresses are the most serious factors limiting the productivity of agricultural crops, with adverse effects on germination, plant vigour and crop yield. Responses to abiotic stresses are not linear pathways, but are complicated integrated circuits involving the interaction of additional cofactors and/or signalling molecules to coordinate a specified response to a given stimulus. The regulation of these responses requires proteins operating in signal transduction pathways, such as transcriptional factors, which modulate gene expression by binding to specific DNA sequences in the promoters of respective target genes. This type of transcriptional regulatory system is called regulon. At least four different regulons that are active in response to abiotic stresses have been identified. Dehydration-responsive element binding protein 1 (DREB1)/C-repeat binding factor (CBF) and DREB2 regulons function in ABA-independent gene expression, whereas the ABA-responsive element (ABRE) binding protein (AREB)/ABRE binding factor (ABF) regulon functions in ABA-dependent gene expression. In addition to these major pathways, other regulons, including the NAC and MYB/MYC regulons are involved in abiotic stress-responsive gene expression. Transcription factors (TFs) are powerful targets for genetic engineering in abiotic stress resistance in crop plants and many studies have been done in the last two decades on this topic. The aim of this book chapter is to give a comprehensive and up-to-date literature review in this field.

Novel methylation at GpC dinucleotide in the fish Sparus aurata genome.

To date, vertebrate DNA has been found methylated at the 5′ position of cytosine exclusively in dinucleotide CpG or CpNpG stretches. On the the other hand, we determined that cytosine was methylated unusually in dinucleotide GpC at 5′-GGCC-3′ sequences in the teleost Sparus aurataEcoRI satellite DNA family. This finding is the first example of methylated GpC sequences in the eukaryotic genomes. At this regard, we have examined the relative methylation levels at this site of the highly repetitive EcoRI satellite DNA family from Sparus aurata different tissues. The EcoRI repeat was remarkably more methylated in male germ cells but hypomethylated in female germ cells at the Hae III restriction site ( GpC). The novel modification and the differential methylation pattern suggest that EcoRI satellite could have a structural and/or functional role at the centromeres of Sparus aurata.

Use of Nuclear and Mitochondrial Single Nucleotide Polymorphisms to Characterize English Walnut ( Juglans regia L.) Genotypes

English walnut (Juglans regia L.) is the most economically important species, for both food and timber, of the 21 species belonging to the genus Juglans. This study was undertaken to analyze and compare DNA sequences of the mitochondrial cytochrome oxidase subunit II (COX2) and ribosomal DNA (rDNA) genes in the molecular characterization of 30 English walnut genotypes. rDNA sequences revealed the presence of 402 variations, including 101 in 3′ ends of 18S, 21 in internal transcribed spacer 1(ITS1), 170 in ITS2, 30 in 5.8S, and 80 in 5′ ends of 28S regions. Cox2 intron I sequences showed 769 variable positions and GG insertion/deletion at 3′ end regions. Based on single nucleotide polymorphism markers of rDNA and cox2 intron I sequences, an amplification refractory mutation system was used to fingerprint 18 out of 30 walnut genotypes. The findings revealed that the cox2 intron I region, either alone or in conjunction with rDNA, could be used effectively in identifying these walnut genotypes.

Molecular characterization of the first satellite DNA with CENP‐B and CDEIII motifs in the bat Pipistrellus kuhli

The centromere is an essential structure in the chromosomes of all eukariotes and is central to the mechanism that ensures proper segregation during mitosis and meiosis. The comparison of DNA sequence motifs, organization and kinetocore components from yeast to man is beginning to indicate that, although centromeres are highly variable DNA elements, a conserved pattern of sequence arrangement and function is emerging. We have identified and characterized the first satellite DNA (P.k.SAT) from microbat species Pipistrellus kuhli. The presence of mammalian CENP‐B box and yeast CDEIII box could indicate the participation of P.k.SAT in centromere organization.

Variability of DNA methylation pattern in somatic and germ cells in male newt (Amphibia, Urodela) Triturus cristatus carnifex

In a survey of several mammalian genomes, namely humans, rodents and bovines, the differences in the 5‐methylcytosine (m5C) content show that repeated DNA sequences from sperm were undermethylated and from various somatic tissues were heavily methylated. This report shows a pattern of methylation in male newt (Amphibia, Urodela) Triturus cristatus carnifex (T. c. c.) unlike that so far described by other authors in mammals. Using methylation sensitive and insensitive enzymes (HpaII and MspI) and successive 3′ terminal labelling (fill‐in), we found a greater degree of DNA methylation in premeiotic germ and sperm cells compared to somatic tissue such as hepatocytes. Furthermore the degree of total DNA methylation in spermatozoa appears somewhere between premeiotic germ cells and somatic tissue. Blot hybridization shows that two highly conserved repetitive sequences in amphibian T. c. c., pTvm1 and pTvm8, contribute significantly to the degree of DNA methylation, suggesting a function for these sequences, such as a role in transcriptional regulation.