Effect of chronic radiation on the flax (Linum usitatissimum L.) genome grown for six consecutive generations in the radioactive Chernobyl area.

The growth of plants under chronic radiation stress in the Chernobyl area may cause changes in the genome of plants. To assess the extent of genetic and epigenetic changes in nuclear DNA, seeds of the annual crop flax (Linum usitatissimum L.) of the Kyivskyi variety, sown 21 years after the accident and grown for six generations in radioactive (RAD) and remediated (REM) fields were analysed. Flaxseed used for sowing first generation, which served as a reference (REF), was also analysed. The AFLP (Amplified Fragment Length Polymorphism) revealed a higher number of specific EcoRI-MseI loci (3.4-fold) in pooled flaxseed samples harvested from the RAD field compared with the REM field, indicating a link between the mutation process in the flax genome and the ongoing adaptation process. MSAP (Methylation-Sensitive Amplified Polymorphism) detecting EcoRI-MspI and EcoRI-HpaII loci in flax nuclear DNA genome showed no significant differences in methylation level, reaching about 33% in each of the groups studied. On the other hand, significant changes in the DNA methylation pattern of flaxseed samples harvested from the RAD field compared with controls were detected. Pairwise FST comparison revealed within both, EcoRI-MspI and transformed methylation-Sensitive data sets more than a 3-fold increase of genetic divergence in the RAD field compared with both controls. These results indicate that the nuclear genome of flax exposed to chronic radiation for six generations has more mutations and uses DNA methylation as one of the adaptation mechanisms for sustainability under adverse conditions.

Identification and gene expression analysis of cytosine-5 DNA methyltransferase and demethylase genes in Amaranthus cruentus L. under heavy metal stress.

Amaranth has become increasingly popular due to its highly nutritious grains and ability to tolerate environmental stress. The mechanism underlying defense and adaptation to environmental stress is a complicated process involving DNA methylation and demethylation. These epigenetic features have been well documented to play an important role in plant stress response, including heavy metal-induced stress. This study was aimed at the identification and analysis of cytosine-5 DNA methyltransferase (C5-MTase) and demethylase (DMTase) genes in Amaranthus cruentus. Eight C5-MTase and two DMTase genes were identified and described in response to individual heavy metals (Cd, Pb, Zn, Mn) and their combination (Cd/Pb, Cd/Zn, Pb/Zn) in root and leaf tissues. Studied heavy metals, individually and in combinations, differentially regulated C5-MTase and DMTase gene expression. Interestingly, most of the genes were transcriptionally altered under Zn exposure. Our results suggest that identified amaranth MTase and DMTase genes are involved in heavy metal stress responses through regulating DNA methylation and demethylation level in amaranth plants.

New Mutant Amaranth Varieties as a Potential Source of Biologically Active Substances.

Amaranth species represent a diverse group of plants. Many of them are a rich source of secondary metabolites with many positive biological effects. Total phenolic, total flavonoid and rutin content, antioxidant activity against superoxide and hydroxyl radicals, FRAP (Ferric-reducing ability of plasma) assay and DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging assay were determined in ethanol extracts of dried leaves of the new Slovak amaranth varieties 'Pribina' and 'Zobor'. The amount of total phenolic substances ('Pribina' GAE 38.3 mg.g-1 DM and 'Zobor' GAE 26.1 mg.g-1 DM), content of total flavonoids ('Pribina' QE 26.5 mg.g-1 DM and 'Zobor' QE 20.3 mg.g-1 DM) and rutin ('Pribina' 50.8 mg.g-1 DM and 'Zobor' 15.2 mg.g-1 DM) were higher in the variety 'Pribina', compared to the variety 'Zobor'. A statistically higher antioxidant activity against superoxide radical (1.63%·mg-1g-1 DM), hydroxyl radical (3.20%.mg-1g-1 DM), FRAP assay (292.80 µmol.L-1·mg-1.g-1 DM) and DPPH (54.2 ± 1.78 µg.mL-1 DM) were detected in the 'Pribina' variety. Antiradical and antioxidant activities of both extracts showed high positive correlations in relation to the content of total phenolic substances, total flavonoids and rutin. Amaranth is an undemanding crop on specific environmental conditions and is resistant to abiotic and biotic stress.

Digital Absolute Gene Expression Analysis of Essential Starch-Related Genes in a Radiation Developed Amaranthus cruentus L. Variety in Comparison with Real-Time PCR.

We investigated the expression pattern of four major starch genes at different seed developmental stages in the radiation-bred amaranth variety "Pribina" (Amaranthus cruentus L.) and corresponding control genotype "Ficha" (Amaranthus cruentus L.). Two platforms were used and compared for the gene expression analysis of GBSSI, SSSI, SBE, and DBE amaranth genes, including a standard quantitative real-time PCR (qPCR) technique and relatively novel droplet digital PCR (ddPCR) assay. In our conditions, both methods showed great accuracy and revealed higher expression of the investigated genes in the mutant variety than in the control genotype. Here we report for the first time, a ddPCR gene expression assay for the cultivated grain amaranth, as the most important group of the species in the genus Amaranthus.

Morphological Responses and Gene Expression of Grain Amaranth (Amaranthus spp.) Growing under Cd.

Phytoremediation efficiency depends on the ability of plants to accumulate, translocate and resist high levels of metals without symptoms of toxicity. This study was conducted to evaluate the potential of grain amaranth for remediation of soils contaminated with Cd. Three grain amaranth varieties, "Pribina" (A. cruentus), "Zobor" (A. hypochondriacus x A. hybridus) and Plainsman (A. hypochondriacus x A. hybridus) were tested under different level of Cd (0, 5, 10 and 15 mg/L) in a hydroponic experimental treatment. All could be classified as Cd excluders or Cd-hypertolerant varieties able to grow and accumulate significant amounts of Cd from the hydroponic solution, preferentially in the roots. Under the highest level of Cd exposure, qRT-PCR expression analysis of five stress-related genes was examined in above- and below-ground biomass. The results show that the Cd concentration significantly increased the mRNA level of chitinase 5 (Chit 5) in amaranth roots as the primary site of metal stress. The involvement of phytochelatin synthase (PCS1) in Cd detoxification is suggested. Based on our findings, we can conclude that variety "Pribina" is the most Cd-tolerant among three tested and can be expected to be used in the phytomanagement of Cd loaded soils as an effective phytostabiliser.

Inter-retrotransposon amplified polymorphism markers revealed long terminal repeat retrotransposon insertion polymorphism in flax cultivated on the experimental fields around Chernobyl.

Ionizing radiation in environment comes from various natural and anthropogenic sources. The effect of radioactivity released after the CNPP (Chernobyl Nuclear Power Plant) on plant systems remains of great interest. Even now, more than three decades after the nuclear accident, the long-lived radionuclides represent a strong stress factor. Herein, the emphasis has been placed on analysis of genetic variability represented by activation of LTR (Long Terminal Repeat)-retrotransposons. Polymorphism in LTR-retrotransposon insertions has been investigated throughout the genome of two flax varieties, Kyivskyi and Bethune. For this purpose, two retrotransposon-based marker techniques, IRAP (Inter-Retrotransposon Amplified Polymorphism) and iPBS (inter-Primer Binding Site), have been employed. The hypothesis that chronic radioactive stress may induce mechanism of retransposition has been supported by the activation of FL9, FL11 and FL12 LTR-retrotransposons in flax seeds harvested from radioactive environment. Out of two retrotransposon-based approaches, IRAP appears to be more suitable for identification of LTR-retrotransposon polymorphism. Even though the LTR-retrotransposon polymorphism was identified, the results suggest the high level of plant adaptation in the radioactive Chernobyl area. However, it is not really surprising that plants developed an effective strategy to survive in radio-contaminated environment over the past 30 years.

A Family of Negative Regulators Targets the Committed Step of de Novo Fatty Acid Biosynthesis.

Acetyl-CoA carboxylase (ACCase) catalyzes the committed step of de novo fatty acid biosynthesis. In prokaryotes, green algae, and most plants, this enzyme is a heteromeric complex requiring four different subunits for activity. The plant complex is recalcitrant to conventional purification schemes and hence the structure and composition of the full assembly have been unclear. In vivo coimmunoprecipitation using subunit-specific antibodies identified a novel family of proteins in Arabidopsis thaliana annotated as biotin/lipoyl attachment domain containing (BADC) proteins. Results from yeast two-hybrid and coexpression in Escherichia coli confirmed that all three BADC isoforms interact with the two biotin carboxyl carrier protein (BCCP) isoforms of Arabidopsis ACCase. These proteins resemble BCCP subunits but are not biotinylated due to a mutated biotinylation motif. We demonstrate that BADC proteins significantly inhibit ACCase activity in both E. coli and Arabidopsis. Targeted gene silencing of BADC isoform 1 in Arabidopsis significantly increased seed oil content when normalized to either mass or individual seed. We conclude the BADC proteins are ancestral BCCPs that gained a new function as negative regulators of ACCase after initial loss of the biotinylation motif. A functional model is proposed.