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This paper reports on the role of proteases secreted by roots in nitrogen capture by plants. The study was conducted on aseptically cultivated wheat seedlings ( Triticum aestivum cv. Tacher) obtained from embryos isolated from grains. Seedlings were cultivated for 21 days on deionised water, Murashige Skoog medium (MS), MS without inorganic nitrogen (IN), and MS without IN, in which IN was replaced by casein (0.01%, 0.1% or 1%). Comparison of seedlings grown on these media showed that casein entirely compensated for the lack of inorganic nitrogen in the medium. Shoots and roots of seedlings cultivated on MS medium with this protein had higher fresh weight than those cultivated on MS medium without casein. The increase in fresh weight of seedlings was correlated with casein concentration and proteolytic activity in the medium. In conclusion, wheat that uses proteases secreted by the roots can directly utilise proteins in the medium as a source of nitrogen without prior digestion by microbial proteases and without protein mineralisation. These results suggest the important role of organic nitrogen fertilisers in increasing wheat yield. [ABSTRACT FROM AUTHOR]

An in vitro system for the initiation of somatic embryogenesis and plant regeneration of spring- and winter-type rye has been developed. The optimal development stage of immature embryos to be used as explants is the late stage of spherical coleoptile when the tissue turns milky in colour and the coleoptile is enlarged but still spherical. The type and concentration of auxin in the induction medium is of importance for obtaining high efficiency of somatic embryogenesis. CC-medium with 30 μM Dicamba resulted in well-developed somatic embryos in large numbers. Positive effects on the efficiency, intensity and speed of development of somatic embryos have been observed by the addition of coconut water to CC-medium, replacing agar by agarose and culturing the explants in low light intensity. Following a "step by step" optimization, culture conditions have been defined giving rise to a 90-100 % efficiency of somatic embryogenesis and a high number of regenerated plants. [ABSTRACT FROM AUTHOR]

Fertile transgenic Triticale ( × Triticosecale Wittmack) plants expressing the β-glucuronidase ( uidA) and phosphinothricin acetyltransferase ( bar) genes were obtained after microprojectile bombardment of scutellar tissue with the plasmid pDB1 containing the uidA gene under the control of the actin-1 promoter (Act1) from rice and the selectable marker gene bar under the control of the CaMV 35S promoter. From 465 bombarded scutella about 4000 plantlets were regenerated; 300 plants survived the selection. These regenerants were screened for enzyme activity by the histological GUS assay and by spraying the plants with a herbicide (Basta). Twenty-five regenerants showed GUS activity and survived repeated Basta spraying. Southern blot analysis showed the presence of both marker genes introduced into the genome of analysed plants. All transgenic plants were fertile. They were grown to maturity and set seed. Pollen and progeny analyses provided evidence for inheritance of the introduced genes to the next generation. [ABSTRACT FROM AUTHOR]

Background: Microspore embryogenesis is a process that produces doubled haploids in tissue culture environments and is widely used in cereal plants. The efficient production of green regenerants requires stresses that could be sensed at the level of glycolysis, followed by the Krebs cycle and electron transfer chain. The latter can be affected by Cu(II) ion concentration in the induction media acting as cofactors of biochemical reactions, indirectly influencing the production of glutathione (GSH) and S-adenosyl-L-methionine (SAM) and thereby affecting epigenetic mechanisms involving DNA methylation (demethylation-DM, de novo methylation-DNM). The conclusions mentioned were acquired from research on triticale regenerants, but there is no similar research on barley. In this way, the study looks at how DNM, DM, Cu(II), SAM, GSH, and β-glucan affect the ability of green plant regeneration efficiency (GPRE)., Results: The experiment involved spring barley regenerants obtained through anther culture. Nine variants (trials) of induction media were created by adding copper (CuSO 4 : 0.1; 5; 10 µM) and silver salts (AgNO 3 : 0; 10; 60 µM), with varying incubation times for the anthers (21, 28, and 35 days). Changes in DNA methylation were estimated using the DArTseqMet molecular marker method, which also detects cytosine methylation. Phenotype variability in β-glucans, SAM and GSH induced by the nutrient treatments was assessed using tentative assignments based on the Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The effectiveness of green plant regeneration ranged from 0.1 to 2.91 plants per 100 plated anthers. The level of demethylation ranged from 7.61 to 32.29, while de novo methylation reached values ranging from 6.83 to 32.27. The paper demonstrates that the samples from specific in vitro conditions (trials) formed tight groups linked to the factors contributing to the two main components responsible for 55.05% of the variance (to the first component DNM, DM, to the second component GSH, β-glucans, Cu(II), GPRE)., Conclusions: We can conclude that in vitro tissue culture conditions affect biochemical levels, DNA methylation changes, and GPRE. Increasing Cu(II) concentration in the IM impacts the metabolism and DNA methylation, elevating GPRE. Thus, changing Cu(II) concentration in the IM is fair to expect to boost GPRE., (© 2024. The Author(s).)

Background: The development of the plant in vitro techniques has brought about the variation identified in regenerants known as somaclonal or tissue culture-induced variation (TCIV). S-adenosyl-L-methionine (SAM), glutathione (GSH), low methylated pectins (LMP), and Cu(II) ions may be implicated in green plant regeneration efficiency (GPRE) and TCIV, according to studies in barley (Hordeum vulgare L.) and partially in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927). Using structural equation models (SEM), these metabolites have been connected to the metabolic pathways (Krebs and Yang cycles, glycolysis, transsulfuration), but not for triticale. Using metabolomic and (epi)genetic data, the study sought to develop a triticale regeneration efficiency statistical model. The culture's induction medium was supplemented with various quantities of Cu(II) and Ag(I) ions for regeneration. The period of plant regeneration has also changed. The donor plant, anther-derived regenerants, and metAFLP were utilized to analyze TCIV concerning DNA in symmetric (CG, CHG) and asymmetric (CHH) sequence contexts. Attenuated Total Reflectance-Fourier Transfer Infrared (ATR-FTIR) spectroscopy was used to gather the metabolomic information on LMP, SAM, and GSH. To frame the data, a structural equation model was employed., Results: According to metAFLP analysis, the average sequence change in the CHH context was 8.65%, and 0.58% was de novo methylation. Absorbances of FTIR spectra in regions specific for LMP, SAM, and GSH were used as variables values introduced to the SEM model. The average number of green regenerants per 100 plated anthers was 2.55., Conclusions: The amounts of pectin demethylation, SAM, de novo methylation, and GSH are connected in the model to explain GPRE. By altering the concentration of Cu(II) ions in the medium, which influences the amount of pectin, triticale's GPRE can be increased., (© 2024. The Author(s).)

Recent advances in knowledge suggest that micro- and nanoplastics pose a threat to plant health, however, the responses of plants to this stressor are not well-known. Here we examined the response of plant cell defence mechanisms to nanoparticles of commonly used plastic, polystyrene. We used plant cell cultures of widely cultivated plants, the monocots wheat and barley (Triticum aestivum L., Hordeum vulgare L.) and the dicots carrot and tomato (Daucus carota L., Solanum lycopersicum L.). We measured the activities of enzymes involved in the scavenging of reactive oxygen species and nonenzymatic antioxidants and we estimated potential damages in plant cell structures and functioning via lipid peroxidation and DNA methylation levels. Our results demonstrate that the mode of action of polystyrene nanoparticles on plant cells involves oxidative stress. However, the changes in plant defence mechanisms are dependent on plant species, exposure time and nanoplastic concentrations. In general, both monocots showed similar responses to nanoplastics, but the carrot followed more the response of monocots than a second dicot, a tomato. Higher H 2 O 2 , lipid peroxidation and lower enzyme activities scavenging H 2 O 2 suggest that tomato cells may be more susceptible to polystyrene-induced stress. In conclusion, polystyrene nanoplastics induce oxidative stress and the response of the plant defense mechanisms involving several chain reactions leading to oxidoreductive homeostasis., (© 2023. The Author(s).)

Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium -mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8'-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective means of Sanger sequencing and bioinformatic analysis. We discuss advantages and potential future developments of this method in contrast to other in vitro approaches., Competing Interests: The authors declare no conflict of interest.

The biological improvement of triticale, a cereal of increasing importance in agriculture, may be accelerated via the production of doubled haploid lines using in vitro culture. Among the relevant factors affecting the culture efficiency are Cu(II) or Ag(I) acting, e.g., as cofactors of enzymes. The copper ions are known to positively affect green plant regeneration efficiency. However, the biochemical basis, mainly its role in the generation of in vitro-induced genetic and epigenetic variation and green plant regeneration efficiency, is not well understood. Here, we employed structural equation modeling to evaluate the relationship between de novo DNA methylation affecting the asymmetric context of CHH sequences, the methylation-sensitive Amplified Fragment Length Polymorphism related sequence variation, and the concentration of Cu(II) and Ag(I) ions in induction media, as well as their effect on S-adenosyl-L-methionine perturbations, observed using FTIR spectroscopy, and the green plant regeneration efficiency. Our results allowed the construction of a theory-based model reflecting the biological phenomena associated with green plant regeneration efficiency. Furthermore, it is shown that Cu(II) ions in induction media affect plant regeneration, and by manipulating their concentration, the regeneration efficiency can be altered. Additionally, S-adenosyl-L-methionine is involved in the efficiency of green plant regeneration through methylation of the asymmetric CHH sequence related to de novo methylation. This shows that the Yang cycle may impact the production of green regenerants.

Plant tissue culture techniques are handy tools for obtaining unique plant materials that are difficult to propagate or important for agriculture. Homozygous materials derived through in vitro cultures are invaluable and significantly accelerate the evaluation of new varieties, e.g., cereals. The induction of somatic embryogenesis/androgenesis and the regeneration and its efficiency can be influenced by the external conditions of tissue culture, such as the ingredients present in the induction or regeneration media. We have developed an approach based on biological system, molecular markers, Fourier Transform Infrared spectroscopy, and structural equation modeling technique to establish links between changes in sequence and DNA methylation at specific symmetric (CG, CHG) and asymmetric (CHH) sequences, glutathione, and green plant regeneration efficiency in the presence of variable supplementation of induction medium with copper ions. The methylation-sensitive Amplified Fragment Length Polymorphism was used to assess tissue culture-induced variation, Fourier Transform Infrared spectroscopy to describe the glutathione spectrum, and a structural equation model to develop the relationship between sequence variation , de novo DNA methylation within asymmetric sequence contexts, and copper ions in the induction medium, as well as, glutathione, and green plant efficiency. An essential aspect of the study is demonstrating the contribution of glutathione to green plant regeneration efficiency and indicating the critical role of copper ions in influencing tissue culture-induced variation, glutathione, and obtaining green regenerants. The model presented here also has practical implications, showing that manipulating the concentration of copper ions in the induction medium may influence cell function and increases green plant regeneration efficiency., Competing Interests: The authors declare that the research was conducted without any commercial or financial relationships construed as a potential conflict of interest., (Copyright © 2022 Bednarek, Orłowska, Mańkowski, Zimny, Kowalczyk, Nowak and Zebrowski.)

The present study focuses on exploring the differences and relationship between well-being and experience of pastoral and psychological service of religious denomination based on religious affiliation during the first wave of the pandemic in Czechia. Our research has been focused on the investigation, comparison, and correlation between the level of well-being and pastoral and psychological service. The research sample ( n = 1126) consisted of the Czech health population with age over 16 years, of which 42.4% were men ( n = 478) and 57.5% were women ( n = 648). From the perspective of religiosity, the study sample was divided in terms of religion into two groups-51.9% participants with religious affiliation ( n = 584) and 48.1% participants without religious affiliation ( n = 542). The level of well-being was identified by means of The Satisfaction with Life Scale (Diener, Emmons, Larsen, & Griffin, 1985). The level of experience with pastoral and psychological service was measured using our non-standardised questionnaire. The results confirmed the differences between the variables of well-being and positive experience with pastoral and psychological service based on religious affiliation. Moreover, we confirmed the hypothesis of a positive correlation between well-being and positive experience with pastoral and psychological service in Czechia.

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9 /guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9 /gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8'-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticalecan beincreased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes., Competing Interests: The authors declare no competing interest.

In vitro tissue culture could be exploited to study cellular mechanisms that induce sequence variation. Altering the metal ion composition of tissue culture medium affects biochemical pathways involved in tissue culture-induced variation. Copper ions are involved in the mitochondrial respiratory chain and Yang cycle. Copper ions may participate in oxidative mutations, which may contribute to DNA sequence variation. Silver ions compete with copper ions to bind to the complex IV subunit of the respiratory chain, thus affecting the Yang cycle and DNA methylation. The mechanisms underlying somaclonal variation are unknown. In this study, we evaluated embryo-derived barley regenerants obtained from a single double-haploid plant via embryo culture under varying copper and silver ion concentrations and different durations of in vitro culture. Morphological variation among regenerants and the donor plant was not evaluated. Methylation-sensitive Amplified Fragment Length Polymorphism analysis of DNA samples showed DNA methylation pattern variation in CG and CHG (H = A, C, or T) sequence contexts. Furthermore, modification of in vitro culture conditions explained DNA sequence variation, demethylation, and de novo methylation in the CHG context, as indicated by analysis of variance. Linear regression indicated that DNA sequence variation was related to de novo DNA methylation in the CHG context. Mediation analysis showed the role of copper ions as a mediator of sequence variation in the CHG context. No other contexts showed a significant sequence variation in mediation analysis. Silver ions did not act as a mediator between any methylation contexts and sequence variation. Thus, incorporating copper ions in the induction medium should be treated with caution., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Orłowska, Zimny and Bednarek.)

Key Message: A single division meiosis mechanism of meiotic restitution is incompletely penetrant but significantly associated with restored fertility in triticale haploids (n = 21, genome formula ABR). Meiotic restitution, or failure of meiosis to produce gametes with a reduced chromosome number, can lead to the restoration of fertility in allohaploids. Meiotic restitution is of major interest for producing doubled haploids, as haploid plants undergoing meiotic restitution can often form seeds without the need to apply mitosis inhibitors to double chromosome number. We aimed to characterize meiotic restitution in a population of 183 haploids (n = 21, genome formula ABR) derived from an F 1 wheat-rye hybrid where one parent was known to carry factors responsible for restoration of fertility in wide-cross haploids. Based on cytological analysis, approximately half of the plants analyzed were characterized by normal meiosis, while half showed at least some cytological evidence of meiotic restitution. However, this mechanism was incompletely penetrant in the population, with no individual plant showing 100% unreduced gamete formation: restitution occurred sectorially within each anther and was not observed in all the anthers of a given plant. Hence, the absence of meiotic restitution could not be confirmed conclusively for any individual plant, confounding this analysis. However, cytological observation of meiotic restitution was significantly associated with seed set, further confirming the role of meiotic restitution in fertility restoration. Our results provide insight into this mechanism of unreduced gamete formation, and provide a basis for future work identifying the genetic factors responsible for this trait.

Plants are often challenged by abiotic and biotic stresses acting in combination and the response to combinatorial stress differs from that triggered by each factor individually. Although salinity and pathogens are major stressors limiting plant growth and productivity worldwide, their interaction is poorly understood. The reactions to pathogens overlap with those to abiotic stresses, and reactive oxygen species (ROS) and stress hormones represent central nodes in the interacting signaling pathways. Usually, abiotic stress negatively affects plant susceptibility to disease. Specific focus of this review is on cucumber plants exposed to salt stress and thereafter infected with Pseudomonas syringae pv lachrymans ( Psl ). We addressed this problem by discussing the changes in photochemistry, the antioxidant system, primary carbon metabolism, salicylic acid (SA) and abscisic acid (ABA) contents. Salt-treated plants were more prone to infection and this effect was determined by changes in the hormonal and redox balance as well as the carboxylate metabolism and activities of some NADPH-generating enzymes. Our detailed understanding of the interactive effects of biotic and abiotic stresses is fundamental to achieve enhanced tolerance to combination stress in agronomically important crops.

Background: In vitro plant regeneration via androgenesis or somatic embryogenesis is capable of inducing (epi)mutations that may affect sexual progenies. While epimutations are associated with DNA methylation, mutations could be due to the movement of transposons. The common notion is that both processes are linked. It is being assumed that demethylation activates transposable elements (TEs). Analysis of methylation changes and their relation with TEs activation in tissue cultures requires uniquely derived donor plants (Ds), their regenerants (Rs) and respective progeny (Ps) that would allow discrimination of processes not related to changes introduced via in vitro cultures. Moreover, a set of methods (RP-HPLC, SSAP, and MSTD) is needed to study whether different TEs families are being activated during in vitro tissue culture plant regeneration and whether their activity could be linked to DNA methylation changes or alternative explanations should be considered., Results: The in vitro tissue culture plant regeneration in barley was responsible for the induction of DNA methylation in regenerants and conservation of the methylation level in the progeny as shown by the RP-HPLC approach. No difference between andro- and embryo-derived Rs and Ps was observed. The SSAP and MSTD approach revealed that Ds and Rs were more polymorphic than Ps. Moreover, Rs individuals exhibited more polymorphisms with the MSTD than SSAP approach. The differences between Ds, Rs and Ps were also evaluated via ANOVA and AMOVA., Conclusions: Stressful conditions during plant regeneration via in vitro tissue cultures affect regenerants and their sexual progeny leading to an increase in global DNA methylation of Rs and Ps compared to Ds in barley. The increased methylation level noted among regenerants remains unchanged in the Ps as indicated via RP-HPLC data. Marker-based experiments suggest that TEs are activated via in vitro tissue cultures and that, independently of the increased methylation, their activity in Rs is greater than in Ps. Thus, the increased methylation level may not correspond to the stabilization of TEs movement at least at the level of regenerants. The presence of TEs variation among Ds that were genetically and epigenetically uniform may suggest that at least some mobile elements may be active, and they may mask variation related to tissue cultures. Thus, tissue cultures may activate some TEs whereas the others remain intact, or their level of movement is changed. Finally, we suggest that sexual reproduction may be responsible for the stabilization of TEs.

Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

Fragile histidine triad (HIT) proteins (Fhits) occur in all eukaryotes but their function is largely unknown. Human Fhit is presumed to function as a tumour suppressor. Previously, we demonstrated that Fhits catalyse hydrolysis of not only dinucleoside triphosphates but also natural adenosine 5'-phosphoramidate (NH2-pA) and adenosine 5'-phosphosulfate (SO4-pA) as well as synthetic adenosine 5'-phosphorofluoridate (F-pA). In the present study, we describe an Fhit-catalysed displacement of the amino group of nucleoside 5'-phosphoramidates (NH2-pNs) or the sulfate moiety of nucleoside 5'-phosphosulfates (SO4-pNs) by fluoride anion. This results in transient accumulation of the corresponding nucleoside 5'-phosphorofluoridates (F-pNs). Substrate specificity and kinetic characterization of the fluorolytic reactions catalysed by the human Fhit and other examples of involvement of fluoride in the biochemistry of nucleotides are described. Among other HIT proteins, human histidine triad nucleotide-binding protein (Hint1) catalysed fluorolysis of NH2-pA 20 times and human Hint2 40 times more slowly than human Fhit.

We present the development of the theoretical background of the metAFLP approach which allows for partition of complex variation into sequence changes, de novo methylation and demethylation of the regenerants derived via in vitro tissue culture methods in the case of triticale. It was demonstrated that, independent of whether andro- or embryogenesis was used for plant regeneration, the level of sequence changes identified between regenerants is about 10 %. Moreover, DNA demethylation prevails over de novo methylation of the regenerants compared to the donor plant. The metAFLP approach allows for the evaluation of numerous quantitative characteristics. For instance, one may quantify the number of sites unaffected by tissue culture approaches, global site DNA methylation etc. It is suggested that the approach could be useful for breeders in order to control plant material uniformity or for the evaluation of modified in vitro tissue culture approaches allowing for control of the (epi)mutation level. The extended metAFLP approach presented here delivers sufficient background for the evaluation of software that could facilitate analyses of the tissue culture induced variation.

The formation of homocysteine thiolactone (HcyTl) from homocysteine occurs in all examined so far organisms including bacteria, yeast, and humans. Protein N-homocysteinylation at the ε-amino group of lysine is an adverse result of HcyTl accumulation. Since tagging of proteins by ubiquitination before their proteasomal degradation takes place at the same residue, we wondered how N-homocysteinylation may affect the ubiquitination of proteins. We used different yeast strains carrying mutations in genes involved in the homocysteine metabolism. We found positive correlation between the concentration of endogenous HcyTl and the concentration of ubiquitinated proteins. This suggests that N-homocysteinylation of proteins apparently does not preclude but rather promotes their decomposition.

Background: While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species., Results: Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy., Conclusions: The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants.

Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human pathologies. However, the role of homocysteinylation of lysyl residues is still poorly known. In order to study the features of homocysteinylation of intrinsically unstructured proteins (IUP) bovine caseins were used as a model. α(S1)-, β- and κ-caseins, showing different aggregations and micelle formation, were modified with homocysteine-thiolactone and their physico-chemical properties were studied. Efficiency of homocysteine incorporation was estimated to be about 1.5, 2.1 and 1.3 homocysteyl residues per one β-, α(S1)-, and κ-casein molecule, respectively. Use of intrinsic and extrinsic fluorescent markers such as Trp, thioflavin T and ANS, reveal structural changes of casein structures after homocysteinylation reflected by an increase in beta-sheet content, which in some cases may be characteristic of amyloid-like transformations. CD spectra also show an increase in beta-sheet content of homocysteinylated caseins. Casein homocysteinylation leads in all cases to aggregation. The sizes of aggregates and aggregation rates were dependent on homocysteine thiolactone concentration and temperature. DLS and microscopic studies have revealed the formation of large aggregates of about 1-3μm. Homocysteinylation of α(S1)- and β-caseins results in formation of regular spheres. Homocysteinylated κ-casein forms thin unbranched fibrils about 400-800nm long. In case of κ-casein amyloidogenic effect of homocysteinylation was confirmed by Congo red spectra. Taken together, data indicate that N-homocysteinylation provokes significant changes in properties of native caseins. A comparison of amyloidogenic transformation of 3 different casein types, belonging to the IUP protein family, shows that the efficiency of amyloidogenic transformation upon homocysteinylation depends on micellization capacity, additional disulphide bonds and other structural features., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Doubled haploids are an established tool in plant breeding and research. Of several methods for their production, androgenesis is technically simple and can efficiently produce substantial numbers of lines. It is well suited to such crops as hexaploid triticale. Owing to meiotic irregularities of triticale hybrids, aneuploidy may affect the efficiency of androgenesis more severely than in meiotically stable crops. This study addresses the issue of aneuploidy among androgenic regenerants of triticale. Plant morphology, seed set and seed quality were better predictors of aneuploidy, as determined cytologically, than flow cytometry. Most aneuploids were hypoploids and these included nullisomics, telosomics, and translocation lines; among 42 chromosome plants were nulli-tetrasomics. Rye chromosomes involved in aneuploidy greatly outnumbered wheat chromosomes; in C(0) rye chromosomes 2R and 5R were most frequently involved. While the frequency of nullisomy 2R was fairly constant in most cross combinations, nullisomy 5R was more frequent in the most recalcitrant combination, and its frequency increased with time spent in culture with up to 70% of green plants recovered late being nullisomic 5R. Given that 5R was not involved in meiotic aberrations with an above-average frequency, it is possible that its absence promotes androgenesis or green plant regeneration. Overall, aneuploidy among tested combinations reduced the average efficiency of double haploid production by 35% and by 69% in one recalcitrant combination, seriously reducing the yield of useful lines.

Histidine triad (HIT)-family proteins interact with different mono- and dinucleotides and catalyze their hydrolysis. During a study of the substrate specificity of seven HIT-family proteins, we have shown that each can act as a sulfohydrolase, catalyzing the liberation of AMP from adenosine 5'-phosphosulfate (APS or SO(4)-pA). However, in the presence of orthophosphate, Arabidopsis thaliana Hint4 and Caenorhabditis elegans DcpS also behaved as APS phosphorylases, forming ADP. Low pH promoted the phosphorolytic and high pH the hydrolytic activities. These proteins, and in particular Hint4, also catalyzed hydrolysis or phosphorolysis of some other adenylyl-derivatives but at lower rates than those for APS cleavage. A mechanism for these activities is proposed and the possible role of some HIT-proteins in APS metabolism is discussed.

The safety assessment of genetically modified (GM) food and feed is performed to identify the possible effects upon animal and human health, also the long-term, multigenerational influence upon functioning of different organs and systems, such as the immune system. In this study C57BL/6J mice were fed for five consecutive generations with pellets containing 20% of conventional triticale grain (control) vs. pellets containing 20% of the transgenic triticale grain resistant to BASTA herbicide (experimental). The F5 experimental animals showed enlarged inguinal and axillary lymph nodes, but not spleens, and increased WBC counts in blood (but within the norm for Mus musculus). Immunophenotyped cell suspensions derived from spleens, inguinal and axillaris lymph nodes and PBMCs from blood showed the significant decrease in the percentage of T cells in spleen and lymph nodes and the B cells in lymph nodes and blood of the F5 experimental mice in comparison to the control F5 mice. Immunoblotting analysis of IL-2, IL-4, IL-10, IL-12, IL- 6, IFN-gamma levels in serum showed significantly increased IL-2 levels and decreased IL-6 levels in the F5-experimental mice sera. No significant changes in the levels of IgE in sera in both mice groups were observed. The obtained results indicate that multigenerational use of feeds for rodents containing the GM-triticale leads to expansion of the B cell compartment in the secondary lymphoid organs, but it is not caused by malignant processes or the allergic response.

As a member of intrinsically unstructured protein family, beta-casein (beta-CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native beta-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant beta-CNs wild type (WT) beta-CN, C4 beta-CN (with cysteinyl residue in position 4) and C208 beta-CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native beta-CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (beta-CND) of C4-beta-CN and C208 beta-CN were also studied and their chaperone-like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT beta-CN, C208 beta-CN, C4 beta-CN and C4 beta-CND exhibited significantly lower chaperone-like activities than native beta-CN. Dimerization of C208 beta-CN with two distal hydrophilic domains considerably improved its chaperone-like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N-terminal hydrophilic domain as important functional elements in enhancing the chaperone-like activity of native beta-CN. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 623-632, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.

Background: When plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic) change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level., Results: A variant of methylation sensitive AFLP, based on the isoschizomeric combinations Acc65I/MseI and KpnI/MseI was applied to analyze, at both the sequence and methylation levels, the outcomes of regeneration from tissue culture in barley. Both sequence mutation and alteration in methylation pattern were detected. Two sets of regenerants from each of five DH donor lines were compared. One set was derived via androgenesis, and the other via somatic embryogenesis, developed from immature embryos. These comparisons delivered a quantitative assessment of the various types of somaclonal variation induced. The average level of variation was 6%, of which almost 1.7% could be accounted for by nucleotide mutation, and the remainder by changes in methylation state. The nucleotide mutation rates and the rate of epimutations were substantially similar between the andro- and embryo-derived sets of regenerants across all the donors., Conclusion: We have developed an AFLP based approach that is capable of describing the qualitative and quantitative characteristics of the tissue culture-induced variation. We believe that this approach will find particular value in the study of patterns of inheritance of somaclonal variation, since non-heritable variation is of little interest for the improvement of plant species which are sexually propagated. Of significant biological interest is the conclusion that the mode of regeneration has no significant effect on the balance between sequence and methylation state change induced by the tissue culture process.

Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcy-thiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone.

Amplified fragment length polymorphism of DNA has been used to analyse the equality of plants obtained from isolated microspores. Although the control parental material was regarded as being highly homozygous, the analysis of the banding patterns of single plants showed a certain level of polymorphism. The analysis of regenerants with a doubled chromosome number did not show any diversity within the progeny of a single line. The differences in banding patterns coming from single plants were only observed in microspore donor lines. These results have proven the high purity of homozygous lines obtained via androgenesis from isolated microspores.

Root-tip derived suspended callus of Oryza sativa cv. Thaipei showed the capacity for plant regeneration via organogenesis. Cell cultures were induced in liquid Murashige-Skoog medium containing 2 mg/l 2.4-dichlorophenoxyacetic acid. Dicamba or Picloram were effective for induction of organogenesis. Shoots and roots differentiated following subculture on medium lacking auxins but containing kinetin. At 1 and 4 mg/l Dicamba and 1 mg/l Picloram normal green plants were regenerated whereas with 7 mg/l Dicamba in the medium only albino plantlets were obtained. Regenerated plantlets were grown to maturity and set seed. Cell suspension cultures, initiated from the root-tip derived calli, provided suitable material for protoplast isolation.

Transient expression of chimaeric genes (neomycin phosphotransferase fused to four different promoters) was detected in suspension culture derived protoplasts of maize, barley and rice, in mesophyll protoplasts of maize, rice, rye, and root protoplasts of maize. The introduction and expression of foreign genes could be performed with both dividing and non-dividing protoplasts by applying the PEG transformation method. The significance of this method for the functional analysis of genes was demonstrated by the differential expression of a regulated gene in protoplasts of different tissues in agreement with its expression in the donor tissue.