Low-intensity electromagnetic fields induce human cryptochrome to modulate intracellular reactive oxygen species.

Exposure to man-made electromagnetic fields (EMFs), which increasingly pollute our environment, have consequences for human health about which there is continuing ignorance and debate. Whereas there is considerable ongoing concern about their harmful effects, magnetic fields are at the same time being applied as therapeutic tools in regenerative medicine, oncology, orthopedics, and neurology. This paradox cannot be resolved until the cellular mechanisms underlying such effects are identified. Here, we show by biochemical and imaging experiments that exposure of mammalian cells to weak pulsed electromagnetic fields (PEMFs) stimulates rapid accumulation of reactive oxygen species (ROS), a potentially toxic metabolite with multiple roles in stress response and cellular ageing. Following exposure to PEMF, cell growth is slowed, and ROS-responsive genes are induced. These effects require the presence of cryptochrome, a putative magnetosensor that synthesizes ROS. We conclude that modulation of intracellular ROS via cryptochromes represents a general response to weak EMFs, which can account for either therapeutic or pathological effects depending on exposure. Clinically, our findings provide a rationale to optimize low field magnetic stimulation for novel therapeutic applications while warning against the possibility of harmful synergistic effects with environmental agents that further increase intracellular ROS.

Genetic Variation and Alleviation of Salinity Stress in Barley (Hordeum vulgare L.).

Barley (Hordeum vulgare L.) represents one of the most important cereals cultivated worldwide. Investigating genetic variability and structure of barley is important for enhancing the crop productivity. This study aimed to investigate the diversity and structure of 40 barley genotypes originated from three European countries (France, the Netherlands, Poland) using amplified fragment length polymorphisms (AFLPs). It also aimed to study 5-aminolevulinic acid (ALA) effect on salinity tolerance of six barley genotypes. The expected heterozygosity (He) diverged from 0.126 to 0.501, with a mean of 0.348. Polymorphic information content (PIC) diverged from 0.103 to 0.482 across barley genotypes, with a mean of 0.316, indicating that barley genotypes are rich in a considerable level of genetic diversity. The 40 barley genotypes were further studied based on their geographical origin (Western Europe and Eastern Europe). The Eastern European region (Poland) has a higher barley variability than the Western European region (France and the Netherlands). Nei's distance-based cluster tree divided the 40 barley accessions into two major clusters; one cluster comprised all the varieties originated from the Eastern European region, while the other major cluster included all accessions originated from the Western European region. Structure analysis results were in a complete concordance with our cluster analysis results. Slaski 2, Damseaux and Urbanowicki genotypes have the highest diversity level, whereas Carmen, Bigo and Cambrinus genotypes have the lowest level. The response of these six varieties to NaCl stress was also investigated. Salt stress (100 mM NaCl) slightly decreased levels of chlorophyll, carotenoid and osmolytes (proteins, soluble sugars, phenolics and flavonoids) in the leaves of Slaski 2, Damseaux and Urbanowicki genotypes at non-significant level, as compared to control samples. However, pigment contents and osmolytes in leaves of Carmen, Bigo and Cambrinus genotypes were significantly decreased by salt stress. Antioxidant enzyme activities were significantly increased in Slaski 2 genotype, but non-significantly increased in Carmen by salt stress. Priming Slaski 2 and Carmen cultivars with ALA under salt stress significantly induced pigment contents, antioxidants enzymes activity and stress-responsive genes expression, relative to NaCl-stressed plants. In conclusion, this study suggested a correlation between variability percentage and degree of salinity resistance. ALA improved salt tolerance in barley.

Analysis of Genetic Variation and Enhancement of Salt Tolerance in French Pea (Pisum Sativum L.).

Pisum sativum L. (field pea) is a crop of a high nutritional value and seed oil content. The characterization of pea germplasm is important to improve yield and quality. This study aimed at using fatty acid profiling and amplified fragment length polymorphism (AFLP) markers to evaluate the variation and relationships of 25 accessions of French pea. It also aimed to conduct a marker-trait associations analysis using the crude oil content as the target trait for this analysis, and to investigate whether 5-aminolevulinic acid (ALA) could enhance salt tolerance in the pea germplasm. The percentage of crude oil of the 25 pea genotypes varied from 2.6 to 3.5%, with a mean of 3.04%. Major fatty acids in all of the accessions were linoleic acid. Moreover, the 12 AFLP markers used were polymorphic. The cluster analysis based on fatty acids data or AFLP data divided the 25 pea germplasm into two main clusters. The gene diversity of the AFLP markers varied from 0.21 to 0.58, with a mean of 0.41. Polymorphic information content (PIC) of pea germplasm varied from 0.184 to 0.416 with a mean of 0.321, and their expected heterozygosity (He) varied from 0.212 to 0.477 with a mean of 0.362. The AFLP results revealed that the Nain Ordinaire cultivar has the highest level of genetic variability, whereas Elatius 3 has the lowest level. Three AFLP markers (E-AAC/M-CAA, E-AAC/M-CAC, and E-ACA/M-CAG) were significantly associated with the crude oil content trait. The response of the Nain Ordinaire and Elatius 3 cultivars to high salinity stress was studied. High salinity (150 mM NaCl) slightly reduced the photosynthetic pigments contents in Nain Ordinaire leaves at a non-significant level, however, the pigments contents in the Elatius 3 leaves were significantly reduced by high salinity. Antioxidant enzymes (APX-ascorbate peroxidase; CAT-catalase; and POD-peroxidase) activities were significantly induced in the Nain Ordinaire cultivar, but non-significantly induced in Elatius 3 by high salinity. Priming the salt-stressed Nain Ordinaire and Elatius 3 plants with ALA significantly enhanced the pigments biosynthesis, antioxidant enzymes activities, and stress-related genes expression, as compared to the plants stressed with salt alone. In conclusion, this study is amongst the first investigations that conducted marker-trait associations in pea, and revealed a sort of correlation between the diversity level and salt tolerance.

Analysis of the Genetic Diversity and Population Structure of Austrian and Belgian Wheat Germplasm within a Regional Context Based on DArT Markers.

Analysis of crop genetic diversity and structure provides valuable information needed to broaden the narrow genetic base as well as to enhance the breeding and conservation strategies of crops. In this study, 95 Austrian and Belgian wheat cultivars maintained at the Centre for Genetic Resources (CGN) in the Netherlands were characterised using 1052 diversity array technology (DArT) markers to evaluate their genetic diversity, relationships and population structure. The rarefacted allelic richness recorded in the Austrian and Belgian breeding pools (A25 = 1.396 and 1.341, respectively) indicated that the Austrian germplasm contained a higher genetic diversity than the Belgian pool. The expected heterozygosity (HE) values of the Austrian and Belgian pools were 0.411 and 0.375, respectively. Moreover, the values of the polymorphic information content (PIC) of the Austrian and Belgian pools were 0.337 and 0.298, respectively. Neighbour-joining tree divided each of the Austrian and Belgian germplasm pools into two genetically distinct groups. The structure analyses of the Austrian and Belgian pools were in a complete concordance with their neighbour-joining trees. Furthermore, the 95 cultivars were compared to 618 wheat genotypes from nine European countries based on a total of 141 common DArT markers in order to place the Austrian and Belgian wheat germplasm in a wider European context. The rarefacted allelic richness (A10) varied from 1.224 (Denmark) to 1.397 (Austria). Cluster and principal coordinates (PCoA) analyses divided the wheat genotypes of the nine European countries into two main clusters. The first cluster comprised the Northern and Western European wheat genotypes, whereas the second included the Central European cultivars. The structure analysis of the 618 European wheat genotypes was in a complete concordance with the results of cluster and PCoA analyses. Interestingly, a highly significant difference was recorded between regions (26.53%). In conclusion, this is the first study to reveal the high diversity levels and structure of the uncharacterised Austrian and Belgian wheat germplasm maintained at the CGN as well as place them in a wider European context. The results should help plant breeders to utilise the most promising wheat genotypes of this study in future breeding programmes for enhancing wheat cultivars.

Genetic Transformation and Hairy Root Induction Enhance the Antioxidant Potential of Lactuca serriola L.

Lactuca serriola L. is a herbaceous species, used for human nutrition and medicinal purposes. The high antioxidant capacity of L. serriola indicates the possibility of enhancing its edible and health potential by increasing the flavonoid and phenolic contents. The present study aimed at enhancing the production of phenolics and flavonoids by hairy root cultures in Lactuca serriola transformed with Agrobacterium rhizogenes strain AR15834 harbouring the rolB gene. The genetic transformation of rolB in transformed roots was validated, and rolB expression level was evaluated using real-time qPCR analysis. Expression levels of flavonoid biosynthesis genes (CHI, PAL, FLS, and CHS) were assessed in the hairy and nontransformed roots. Results showed higher expression levels in the transgenic roots than in the nontransformed ones (p < 0.01). Transgenic hairy roots exhibited a 54.8-96.7% increase in the total phenolic content, 38.1-76.2% increase in the total flavonoid content, and 56.7-96.7% increase in the total reducing power when compared with the nontransgenic roots (p < 0.01). DPPH results also revealed that the transgenic hairy roots exhibited a 31.6-50% increase in antioxidant potential, when compared to normal roots. This study addressed the enhancement of secondary metabolite biosynthesis by hairy root induction in L. serriola.

Blue-light induced accumulation of reactive oxygen species is a consequence of the Drosophila cryptochrome photocycle.

Cryptochromes are evolutionarily conserved blue-light absorbing flavoproteins which participate in many important cellular processes including in entrainment of the circadian clock in plants, Drosophila and humans. Drosophila melanogaster cryptochrome (DmCry) absorbs light through a flavin (FAD) cofactor that undergoes photoreduction to the anionic radical (FAD•-) redox state both in vitro and in vivo. However, recent efforts to link this photoconversion to the initiation of a biological response have remained controversial. Here, we show by kinetic modeling of the DmCry photocycle that the fluence dependence, quantum yield, and half-life of flavin redox state interconversion are consistent with the anionic radical (FAD•-) as the signaling state in vivo. We show by fluorescence detection techniques that illumination of purified DmCry results in enzymatic conversion of molecular oxygen (O2) to reactive oxygen species (ROS). We extend these observations in living cells to demonstrate transient formation of superoxide (O2•-), and accumulation of hydrogen peroxide (H2O2) in the nucleus of insect cell cultures upon DmCry illumination. These results define the kinetic parameters of the Drosophila cryptochrome photocycle and support light-driven electron transfer to the flavin in DmCry signaling. They furthermore raise the intriguing possibility that light-dependent formation of ROS as a byproduct of the cryptochrome photocycle may contribute to its signaling role.

Kinetic Modeling of the Arabidopsis Cryptochrome Photocycle: FADH(o) Accumulation Correlates with Biological Activity.

Cryptochromes are flavoprotein photoreceptors with multiple signaling roles during plant de-etiolation and development. Arabidopsis cryptochromes (cry1 and cry2) absorb light through an oxidized flavin (FADox) cofactor which undergoes reduction to both FADH° and FADH(-) redox states. Since the FADH° redox state has been linked to biological activity, it is important to estimate its concentration formed upon illumination in vivo. Here we model the photocycle of isolated cry1 and cry2 proteins with a three-state kinetic model. Our model fits the experimental data for flavin photoconversion in vitro for both cry1 and cry2, providing calculated quantum yields which are significantly lower in cry1 than for cry2. The model was applied to the cryptochrome photocycle in vivo using biological activity in plants as a readout for FADH° concentration. The fit to the in vivo data provided quantum yields for cry1 and cry2 flavin reduction similar to those obtained in vitro, with decreased cry1 quantum yield as compared to cry2. These results validate our assumption that FADH° concentration correlates with biological activity. This is the first reported attempt at kinetic modeling of the cryptochrome photocycle in relation to macroscopic signaling events in vivo, and thereby provides a theoretical framework to the components of the photocycle that are necessary for cryptochrome response to environmental signals.

Blue-light dependent ROS formation by Arabidopsis cryptochrome-2 may contribute toward its signaling role.

Cryptochromes are blue-light absorbing flavoproteins with many important signaling roles in plants, including in de-etiolation, development, and stress response. They interact with downstream signaling partners such as transcription factors and components of the proteasome, and thereby alter regulation of nuclear gene expression in a light dependent manner. In a prior study, it has also been shown that Arabidopsis cry1 activation by blue light results in direct enzymatic conversion of molecular oxygen (O2) to ROS (reactive oxygen species) in vivo leading to cell death in overexpressing lines. Here we extend these observations to show that Atcry2 is translocated from the cytosol to the nucleus in response to blue light illumination, resulting in nuclear accumulation of ROS in expressing insect cell cultures. These observations suggest that ROS formation may represent a novel means of signaling by Atcry2 distinct from, and perhaps complementary to, the currently known mechanism of light-mediated conformational change.

Blue-light dependent reactive oxygen species formation by Arabidopsis cryptochrome may define a novel evolutionarily conserved signaling mechanism.

Cryptochromes are widespread blue-light absorbing flavoproteins with important signaling roles. In plants they mediate de-etiolation, developmental and stress responses resulting from interaction with downstream signaling partners such as transcription factors and components of the proteasome. Recently, it has been shown that Arabidopsis cry1 activation by blue light also results in direct enzymatic conversion of molecular oxygen (O2 ) to reactive oxygen species (ROS) and hydrogen peroxide (H2 O2 ) in vitro. Here we explored whether direct enzymatic synthesis of ROS by Arabidopsis cry1 can play a physiological role in vivo. ROS formation resulting from cry1 expression was measured by fluorescence assay in insect cell cultures and in Arabidopsis protoplasts from cryptochrome mutant seedlings. Cell death was determined by colorimetric assay. We found that ROS formation results from cry1 activation and induces cell death in insect cell cultures. In plant protoplasts, cryptochrome activation results in rapid increase in ROS formation and cell death. We conclude that ROS formation by cryptochromes may indeed be of physiological relevance and could represent a novel paradigm for cryptochrome signaling.