Comparison of the antioxidant capacity in cold-treated recombinant wheat lines

In earlier experiments using 5A chromosome substitution lines of wheat with different freezing tolerance it was shown that this chromosome plays an important role in the regulation of cold-induced changes in the level of antioxidant enzymes and thiols. The purpose of our experiments was to determine the region of chromosome 5A where the genes responsible for the control of stress-induced changes in the level of the antioxidants are localised. Activities of antioxidant enzymes and the glutathione synthesis were compared in lines recombinant for the long arm of chromosome 5A. The chromosome region between the markers Xpsr805 and Xpsr164 was found to influence the cold-induced changes in hydroxymethylglutathione and glutathione levels. The glutathione reductase activity may be affected by the chromosome region near to the marker B-amy-A1

Key Molecular and Metabolic Processes Used for Genetic Engineering to Improve Freezing Tolerance in Cereals

It has been estimated recently that cereals are harvested on 700 million hectares (Mha) worldwide (Dunwell, 2014), and also that, due to low temperature damage, worldwide losses in crop production amount to about US$2 billion each year (Sanghera et al., 2011). In spite of the urgent need for more cold- or frost-tolerant cereal varieties, classical breeding programmes have shown limited progress in improving freezing tolerance (Thomashow, 1999). This lack of success is due mainly to the fact that the physiological process, i.e. the cold acclimation that leads to the development of freezing tolerance, is quite a complex quantitative trait. However, the deeper insight provided by different ‘omics’ technologies has made possible knowledge-based engineering of more stress-resistant plants; while the recent developments in cereal transformation methodology offer the technology to realize these aims. Since many recently published book chapters and reviews summarize our current knowledge on plant abiotic stress tolerance, this chapter focuses particularly on freezing tolerance, especially in cereals

Screening Auxin Response, In Vitro Culture Aptitude and Susceptibility to Agrobacterium-Mediated Transformation of Italian Commercial Durum Wheat Varieties

The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like durum wheat requires the identification and optimization of factors affecting T-DNA delivery and plant regeneration. The purpose of this research was to compare the behavior of diverse durum wheat genotypes during in vitro culture and Agrobacterium tumefaciens-mediated transformation, using immature embryos as explants. Apart from plant genotype, two of the main influencing factors for a successful genetic transformation have been examined here, i.e., auxin source (Dicamba and 2,4-D) and duration of the pre-culture period (one, seven and 21 days). The addition of Dicamba to the media in combination with seven days pre-cultivation resulted in a general enhancement of T-DNA delivery for most of the analyzed cultivars, as revealed by �-glucuronidase (GUS) histochemical assay. Although all genotypes were able to produce calli, significant differences were detected in regeneration and transformation efficiencies, since only two (Karalis and Neolatino) out of 14 cultivars produced fertile transgenic plants. The estimated transformation efficiencies were 6.25% and 1.66% for Karalis and Neolatino, respectively, and �2 analysis revealed the stable integration and segregation of the gus transgene in T1 and T2 progenies. This research has demonstrated that, among the influencing factors, genotype and auxin type play the most important role in the success of durum wheat transformation

A búza fagyállóságban szerepet játszó Cbf gének szerepének bizonyítása transzformációval = Proving the role of Cbf genes in wheat frost tolerance by transformation

A gabonafélék genomja sok CBF gént kódol. Nemrég mutattuk ki, hogy ezek közül melyek játszanak fontos szerepet a búza fagyállóságának kialakításában. A pályázatban direkt módon, transzformációval kívántuk bizonyítani e kulcsfontosságú transzkripciós faktorok szerepét. Búzából izolált génekkel Arabidopsis-t, búzát és árpát transzformáltunk; az ehhez készített konstrukciókban a beépített gént, annak folyamatos túltermelését biztosító szabályozó elem (u.n. konstitutív promóter) vezérelte. Meghatároztuk az előállított transzgénikus vonalakban a beépült gének számát, és igazoltuk azok működését. A gének hatását, a megnövekedett stressz-toleranciát kétféle fagyteszttel vizsgáltuk. Az első során - a természetes viszonyokat szimulálandó - hideg-edzést alkalmaztunk a fagyteszt előtt. A második típusú tesztben ilyet nem alkalmaztunk, mert a transzgén állandóan működött, így feltételezésünk szerint nem volt szükség hideg-indukcióra ahhoz, hogy a hatását kifejtse, azaz a fagyállóságot fokozza. Mindkét típusú fagyteszttel sikerült mindhárom növényfaj esetében olyan transzgénikus vonalakat azonosítanunk, amelyekben a fagyállóság szintje megnőtt a bevitt gén hatására. Mivel a bevitt gének más gének működést befolyásoló ún. transzkripciós faktort kódolnak, ezért vizsgáltuk az általuk szabályozott gének működését is. E kísérletek eredményei is a tanulmányozott CBF gének fagytűrés kialakításában betöltött szerepét igazolták. | Cereal genome encodes numerous CBF genes. Recently we have identified the ones that are involved in the regulation of frost tolerance. Based on these results, this project was aimed at proving directly their function by transformation methods. Arabidopsis, barley and wheat plants were transformed, with constructions that ensured the constitutive expression of the transformed CBF genes. The insertions of the transgenes were proved, the copy number estimated, and their functionality were verified in each transgenic lines developed. The effectiveness of the genes was tested in freezing tests. One type of test was applied with cold hardening period while the other was applied without it. The efficiency of the transgenes was proved in both test types for each plant species, i.e. transgenic Arabidopsis, barley and wheat lines with increased frost tolerance level were indeed identified. Since CBF genes code for transcription factors, the expression patterns of several downstream genes were also recorded. These experiments also proved the effectiveness of the candidate CBF genes in the regulation of cereal frost tolerance

Circadian and Light Regulated Expression of CBFs and their Upstream Signalling Genes in Barley

Abstract: CBF (C-repeat binding factor) transcription factors show high expression levels in response to cold; moreover, they play a key regulatory role in cold acclimation processes. Recently, however, more and more information has led to the conclusion that, apart from cold, light—including its spectra—also has a crucial role in regulating CBF expression. Earlier, studies established that the expression patterns of some of these regulatory genes follow circadian rhythms. To understand more of this complex acclimation process, we studied the expression patterns of the signal transducing pathways, including signal perception, the circadian clock and phospholipid signalling pathways, upstream of the CBF gene regulatory hub. To exclude the confounding effect of cold, experiments were carried out at 22 �C. Our results show that the expression of genes implicated in the phospholipid signalling pathway follow a circadian rhythm. We demonstrated that, from among the tested CBF genes expressed in Hordeum vulgare (Hv) under our conditions, only the members of the HvCBF4-phylogenetic subgroup showed a circadian pattern. We found that the HvCBF4-subgroup genes were expressed late in the afternoon or early in the night. We also determined the expression changes under supplemental far-red illumination and established that the transcript accumulation had appeared four hours earlier and more intensely in several cases. Based on our results, we propose a model to illustrate the effect of the circadian clock and the quality of the light on the elements of signalling pathways upstream of the HvCBFs, thus integrating the complex regulation of the early cellular responses, which finally lead to an elevated abiotic stress tolerance

Circadian and Light Regulated Expression of CBFs and their Upstream Signalling Genes in Barley

CBF (C-repeat binding factor) transcription factors show high expression levels in response to cold; moreover, they play a key regulatory role in cold acclimation processes. Recently, however, more and more information has led to the conclusion that, apart from cold, light-including its spectra-also has a crucial role in regulating CBF expression. Earlier, studies established that the expression patterns of some of these regulatory genes follow circadian rhythms. To understand more of this complex acclimation process, we studied the expression patterns of the signal transducing pathways, including signal perception, the circadian clock and phospholipid signalling pathways, upstream of the CBF gene regulatory hub. To exclude the confounding effect of cold, experiments were carried out at 22 degrees C. Our results show that the expression of genes implicated in the phospholipid signalling pathway follow a circadian rhythm. We demonstrated that, from among the tested CBF genes expressed in Hordeumvulgare (Hv) under our conditions, only the members of the HvCBF4-phylogenetic subgroup showed a circadian pattern. We found that the HvCBF4-subgroup genes were expressed late in the afternoon or early in the night. We also determined the expression changes under supplemental far-red illumination and established that the transcript accumulation had appeared four hours earlier and more intensely in several cases. Based on our results, we propose a model to illustrate the effect of the circadian clock and the quality of the light on the elements of signalling pathways upstream of the HvCBFs, thus integrating the complex regulation of the early cellular responses, which finally lead to an elevated abiotic stress tolerance

Overexpression of Two Upstream Phospholipid Signaling Genes Improves Cold Stress Response and Hypoxia Tolerance, but Leads to Developmental Abnormalities in Barley

Phosphatidylinositol transfer protein (PITP) and phosphatidylinositol 4 kinase (PI4K) are upstream regulatory elements of the phospholipid signaling pathway. PITP and PI4K over expressing transgenic barley lines were developed and studied. We revealed that the over expression of the PITP and PI4K genes increased stress tolerance during hypoxic cold stress, but not during salinity stress, and differences were also found in the level of frost tolerance. On the other hand, we ralised that the over expression of these upstream signaling elements led to more phenotypic abnormalities than in other transgenic studies working with effector genes or even transcription factors. We hypothesize that this high level of abnormalities are the consequence of the modulation of a very upstream signal transduction pathway elements

Identification, Structural and Functional Characterization of Dormancy Regulator Genes in Apricot (Prunus armeniaca L.)

In the present study, we identified and characterized the apricot (Prunus armeniaca L.) homologs of three dormancy-related genes, namely the ParCBF1 (C-repeat binding factor), ParDAM5 (dormancy-associated MADS-BOX) and ParDAM6 genes. All highly conserved structural motifs and the 3D model of the DNA-binding domain indicate an unimpaired DNA-binding ability of ParCBF1. A phylogenetic analysis showed that ParCBF1 was most likely homologous to Prunus mume and Prunus dulcis CBF1. ParDAM5 also contained all characteristic domains of the type II (MIKCC) subfamily of MADS-box transcription factors. The homology modeling of protein domains and a phylogenetic analysis of ParDAM5 suggest its functional integrity. The amino acid positions or small motifs that are diagnostic characteristics of DAM5 and DAM6 were determined. For ParDAM6, only a small part of the cDNA was sequenced, which was sufficient for the quantification of gene expression. The expression of ParCBF1 showed close association with decreasing ambient temperatures in autumn and winter. The expression levels of ParDAM5 and ParDAM6 changed according to CBF1 expression rates and the fulfillment of cultivar chilling requirements (CR). The concomitant decrease of gene expression with endodormancy release is consistent with a role of ParDAM5 and ParDAM6 genes in dormancy induction and maintenance. Cultivars with higher CR and delayed flowering time showed higher expression levels of ParDAM5 and ParDAM6 toward the end of endodormancy. Differences in the timing of anther developmental stages between early- and late-flowering cultivars and two dormant seasons confirmed the genetically and environmentally controlled mechanisms of dormancy release in apricot generative buds. These results support that the newly identified apricot gene homologs have a crucial role in dormancy-associated physiological mechanisms

Effects of ambient temperature in association with photoperiod on phenology and on the expressions of major plant developmental genes in wheat (Triticum aestivum L.)

Abstract In addition to its role in vernalization, temperature is an important environmental stimulus in determining plant growth and development. We used factorial combinations of two photoperiods (16H, 12H) and three temperature levels (11oC, 18oC, and 25oC) to study the temperature responses of 19 wheat cultivars with established genetic relationships. Temperature produced more significant effects on plant development than photoperiod, with This article is protected by copyright. All rights reserved. strong genotypic components. Wheat genotypes with PPD-D1 photoperiod sensitive allele were sensitive to temperature; their development was delayed by higher temperature, which intensified under non-inductive conditions. The effect of temperature on plant development was not proportional; it influenced the stem elongation to the largest extent, warmer temperature lengthened the lag phase between the detection of first node and the beginning of intensive stem elongation. The gene expression patterns of VRN1, VRN2 and PPD1 were also significantly modified by temperature, while VRN3 was more chronologically regulated. The associations between VRN1 and VRN3 gene expression with early apex development were significant in all treatments, but was only significant for later plant developmental phases under optimal conditions (16H and 18oC). Under 16H, the magnitude of the transient peak expression of VRN2 observed at 18oC and 25oC associated with the later developmental phases. Key words: wheat, temperature, photoperiod, apex and plant development, VRN1, VRN2, VRN3 and PPD1 gene expression