Characterising Biological and Physiological Drought Signals in Diverse Parents of a Wheat Mapping Population.

Water deficit affects the growth as well as physiological and biochemical processes in plants. The aim of this study was to determine differences in physiological and biochemical responses to drought stress in two wheat cultivars-Chinese Spring (CS) and SQ1 (which are parents of a mapping population of doubled haploid lines)-and to relate these responses to final yield and agronomic traits. Drought stress was induced by withholding water for 14 days, after which plants were re-watered and maintained until harvest. Instantaneous gas exchange parameters were evaluated on the 3rd, 5th, 10th, and 14th days of seedling growth under drought. After 14 days, water content and levels of chlorophyll a+b, carotenoids, malondialdehyde, soluble carbohydrates, phenolics, salicylic acid, abscisic acid (ABA), and polyamines were measured. At final maturity, yield components (grain number and weight), biomass, straw weight, and harvest index were evaluated. Physiological and biochemical parameters of CS responded more than those of SQ1 to the 14-day drought, reflected in a greater reduction in final biomass and yield in CS. Marked biochemical differences between responses of CS and SQ1 to the drought were found for soluble carbohydrates and polyamines. These would be good candidates for testing in the mapping population for the coincidence of the genetic control of these traits and final biomass and yield.

Comparative Assessment of Lignan Profiling and Biological Activities of Schisandra henryi Leaf and In Vitro PlantForm Bioreactor-Grown Culture Extracts.

This research's scope encompassed biotechnological, phytochemical, and biological studies of Schisandra henryi, including investigations into its in vitro microshoot culture grown in PlantForm bioreactors (temporary immersion systems, TISs), as well as extracts from leaves of the parent plant, focusing on anti-inflammatory, antioxidant, anticancer, and antimicrobial activities. The phytochemical analysis included the isolation and quantification of 17 compounds from dibenzocyclooctadiene, aryltetralin lignans, and neolignans using centrifugal partition chromatography (CPC), HPLC-DAD, and UHPLC-MS/MS tandem mass spectrometry with triple quadrupole mass filter methods. Higher contents of compounds were found in microshoots extracts (max. 543.99 mg/100 g DW). The major compound was schisantherin B both in the extracts from microshoots and the leaves (390.16 and 361.24 mg/100 g DW, respectively). The results of the anti-inflammatory activity in terms of the inhibition of COX-1, COX-2, sPLA2, and LOX-15 enzymes indicated that PlantForm microshoot extracts showed strong activity against COX-1 and COX-2 (for 177 mg/mL the inhibition percentage was 76% and 66%, respectively). The antioxidant potential assessed using FRAP, CUPRAC, and DPPH assays showed that extracts from microshoot cultures had 5.6, 3.8, and 3.3 times higher power compared to extracts from the leaves of the parent plant, respectively. The total polyphenol content (TPC) was 4.1 times higher in extracts from the in vitro culture compared to the leaves. The antiproliferative activity against T-cell lymphoblast line Jurkat, breast adenocarcinoma cultures (MCF-7), colon adenocarcinoma (HT-29), and cervical adenocarcinoma (HeLa), showed that both extracts have considerable effects on the tested cell lines. The antimicrobial activity tested against strains of Gram-positive and Gram-negative bacteria and fungi showed the highest activity towards H. pylori (MIC and MBC 0.625 mg/mL).

Untargeted metabolomic in basil cell cultures - a case study of Ocimum basilicum L. var. minimum Alef.

AIMS: Due to the lack of experimental databases, together with the chemical complexity and the dynamic nature of plants' metabolome, most of the metabolites in complex biological materials (like plant in vitro tissue cultures) are not-annotated, unidentified metabolites. In this study, a method for further metabolite characterization and classification based on the UPLC-HESI-HRMS/MS approach for small-leaved basil (Ocimum basilicum L. var. minimum Alef.) callus and cell suspension culture is presented. KEY RESULTS: A total of 2168 metabolic features were detected, out of which the database for exact mass metabolic profiling for 1949 metabolites is presented here since there is no available database dedicated to O. basilicum. We further focused on secondary metabolites (particularly phenolic compounds). The presence of 60 different phenolic compounds belonging mainly to the groups of flavonoids, glycosides, terpenoids, and phenolic acids is confirmed. By comparing relative abundances of phenolic compounds from callus culture and cell suspension culture, both grown on two types of media, via svd-PCА, univariate analysis, post-hoc tests, and heatmapping of metabolites, we provided a practical example of how resources presented here can be further applied in tissue culture-based basil metabolomics studies. UTILITY: This study represents the first approach toward routine targeted investigation of secondary metabolites in basil in vitro cultures and provides various opportunities for new-generation analyses.

Phenolic Acids and Amaryllidaceae Alkaloids Profiles in Leucojum aestivum L. In Vitro Plants Grown under Different Light Conditions.

Light-emitting diodes (LEDs) have emerged as efficient light sources for promoting in vitro plant growth and primary and secondary metabolite biosynthesis. This study investigated the effects of blue, red, and white-red LED lights on plant biomass growth, photosynthetic pigments, soluble sugars, phenolic compounds, the production of Amaryllidaceae alkaloids, and the activities of antioxidant enzymes in Leucojum aestivum L. cultures. A white fluorescent light was used as a control. The plants that were grown under white-red and red light showed the highest fresh biomass increments. The blue light stimulated chlorophyll a, carotenoid, and flavonoid production. The white-red and blue lights were favourable for phenolic acid biosynthesis. Chlorogenic, p-hydroxybenzoic, caffeic, syringic, p-coumaric, ferulic, sinapic, and benzoic acids were identified in plant materials, with ferulic acid dominating. The blue light had a significant beneficial effect both on galanthamine (4.67 µg/g of dry weight (DW)) and lycorine (115 µg/g DW) biosynthesis. Red light treatment increased catalase and superoxide dismutase activities, and high catalase activity was also observed in plants treated with white-red and blue light. This is the first report to provide evidence of the effects of LED light on the biosynthesis of phenolic acid and Amaryllidaceae alkaloids in L. aestivum cultures, which is of pharmacological importance and can propose new strategies for their production.

Changes in physio-biochemical parameters and expression of metallothioneins in Avena sativa L. in response to drought.

Drought is one of the major threats to food security. Among several mechanisms involved in plant stress tolerance, one protein family-the plant metallothioneins (MTs)-shows great promise for enhancing drought resistance. Plant metallothioneins in oat (Avena sativa L.) have not yet been deeply analysed, and the literature lacks a comprehensive study of the whole family of plant MTs in response to drought. In this study, we showed that the number and nature of cis-elements linked with stress response in promoters of AsMTs1-3 differed depending on the MT type. Drought stress in oat plants caused an increase in the expression of AsMT2 and AsMT3 and a decrease in the expression of AsMT1 compared to well-watered plants. Moreover, the low values of relative water content, water use efficiency, net photosynthesis (PN), transpiration (E), stomatal conductance (gs), chlorophyll a, and carotenoid were accompanied by high levels of electrolyte leakage, internal CO2 concentration (Ci) and abscisic acid content, and high activity of antioxidants enzymes in plants under drought stress. The present study puts forward the idea that AsMTs are crucial for oat response to drought stress not only by regulating antioxidant activity but also by changing the plant water regime and photosynthesis. Our results support the hypothesis that structural differences among types of plant MTs reflect their diversified physiological roles.

1,2-Hydrogenation and Transhydrogenation Catalyzed by 3-Ketosteroid Δ1-Dehydrogenase from Sterolibacterium denitrificans-Kinetics, Isotope Labelling and QM:MM Modelling Studies.

Bacteria and fungi that are able to metabolize steroids express 3-ketosteroid-Δ1-dehydrogenases (KstDs). KstDs such as AcmB form Sterolibacterium denitrificans Chol-1 catalyze the enantioselective 1α,2ß-dehydrogenation of steroids to their desaturated analogues, e.g., the formation of 1,4-androstadiene-3,17-dione (ADD) from 4-androsten-3,17-dione (AD). The reaction catalyzed by KstD can be reversed if the appropriate electron donor, such as benzyl viologen radical cation, is present. Furthermore, KstDs can also catalyze transhydrogenation, which is the transfer of H atoms between 3-ketosteroids and 1-dehydrosteroids. In this paper, we showed that AcmB exhibits lower pH optima for hydrogenation and dehydrogenation by 3.5-4 pH units than those observed for KstD from Nocardia corallina. We confirmed the enantiospecificity of 1α,2ß-hydrogenation and 1α,2ß-transhydrogenation catalyzed by AcmB and showed that, under acidic pH conditions, deuterons are introduced not only at 2ß but also at the 1α position. We observed a higher degree of H/D exchange at Y363, which activates the C2-H bond, compared to that at FAD, which is responsible for redox at the C1 position. Furthermore, for the first time, we observed the introduction of the third deuteron into the steroid core. This effect was explained through a combination of LC-MS experiments and QM:MM modelling, and we attribute it to a decrease in the enantioselectivity of C2-H activation upon the deuteration of the 2ß position. The increase in the activation barrier resulting from isotopic substitution increases the chance of the formation of d3-substituted 3-ketosteroids. Finally, we demonstrate a method for the synthesis of 3-ketosteroids chirally deuterated at 1α,2ß positions, obtaining 1α,2ß-d2-4-androsten-3,17-dione with a 51% yield (8.61 mg).

Regulation of the Bud Dormancy Development and Release in Micropropagated Rhubarb 'Malinowy'.

Culinary rhubarb is a vegetable crop, valued for its stalks, very rich in different natural bioactive ingredients. In commercial rhubarb stalk production, the bud dormancy development and release are crucial processes that determine the yields and quality of stalks. To date, reports on rhubarb bud dormancy regulation, however, are lacking. It is known that dormancy status depends on cultivars. The study aimed to determine the dormancy regulation in a valuable selection of rhubarb 'Malinowy'. Changes in carbohydrate, total phenolic, endogenous hormone levels, and gene expression levels during dormancy development and release were studied in micropropagated rhubarb plantlets. Dormancy developed at high temperature (25.5 °C), and long day. Leaf senescence and dying were consistent with a significant increase in starch, total phenolics, ABA, IAA and SA levels. Five weeks of cooling at 4 °C were sufficient to break dormancy, but rhizomes stored for a longer duration showed faster and more uniformity leaf growing, and higher stalk length. No growth response was observed for non-cooled rhizomes. The low temperature activated carbohydrate and hormone metabolism and signalling in the buds. The increased expression of AMY3, BMY3, SUS3, BGLU17, GAMYB genes were consistent with a decrease in starch and increase in soluble sugars levels during dormancy release. Moreover, some genes (ZEP, ABF2, GASA4, GA2OX8) related to ABA and GA metabolism and signal transduction were activated. The relationship between auxin (IAA, IBA, 5-Cl-IAA), and phenolic, including SA levels and dormancy status was also observed.

Growth Cessation and Dormancy Induction in Micropropagated Plantlets of Rheum rhaponticum 'Raspberry' Influenced by Photoperiod and Temperature.

Dormancy development in micropropagated plantlets at the acclimatization stage and early growth ex vitro is undesirable as it lowers their survival rate and restricts the efficient year-round production of planting material. Thus far, little is known about the factors and mechanisms involved in the dormancy development of micropropagated herbaceous perennials, including rhubarb. This study determined physiological and molecular changes in the Rheum rhaponticum (culinary rhubarb) 'Raspberry' planting material in response to photoperiod and temperature. We found that the rhubarb plantlets that were grown under a 16-h photoperiod (LD) and a temperature within the normal growth range (17-23 °C) showed active growth of leaves and rhizomes and did not develop dormancy. Rapid growth cessation and dormancy development were observed in response to a 10-h photoperiod (SD) or elevated temperature under LD. These morphological changes were accompanied by enhanced abscisic acid (ABA) and starch levels and also the upregulation of various genes involved in carbohydrate synthesis and transport (SUS3, AMY3, BMY3, BGLU17) and ABA synthesis and signaling (ZEP and ABF2). We also found enhanced expression levels of heat shock transcription factors (HSFA2 and HSFA6B), heat shock proteins (HSP22, HSP70.1, HSP90.2 and HSP101) and antioxidant enzymes (PRX12, APX2 and GPX). This may suggest that dormancy induction in micropropagated rhubarb plantlets is a stress response to light deficiency and high temperatures and is endogenously coordinated by the ABA, carbohydrate and ROS pathways.

Herbal Additives Substantially Modify Antioxidant Properties and Tocopherol Content of Cold-Pressed Oils.

The aim of the study was to examine combinations of base oils and herbal additives with a view to obtaining macerates with improved health benefits. Base oils were cold-pressed from the seeds of black cumin, borage, evening primrose, safflower, walnut, common hazel, and oilseed rape, as well as the flesh of sea-buckthorn fruits. They were then supplemented with herbs, including basil, thyme, and sage, in order to create macerates. Total antioxidant activity and tocopherol level were analyzed in oils, macerates, and oil cakes. Additionally, chemical properties of oil cakes-such as the level of fibre, vitamin C, ß-carotene, and lutein-were also examined. Supplementation with herbs caused diversified effects on antioxidant activity and tocopherol level in macerates depending on the base oil, herb, and supplementation method. The obtained results indicate that tocopherol level does not play a decisive role in determining the antioxidant properties of oils, macerates, and oil cakes, suggesting significant involvement of other antioxidants. Among the tested macerates, the most promising one seems to be oilseed rape oil enriched with sage or basil to maximize its health benefits. The study can serve as a starting point for the development and implementation of functional macerates and oil cakes in healthy nutrition.

Exogenous Polyamines Only Indirectly Induce Stress Tolerance in Wheat Growing in Hydroponic Culture under Polyethylene Glycol-Induced Osmotic Stress.

The aim of the present study was to evaluate the effect of osmotic stress caused by polyethylene glycol (PEG) 6000 in hydroponic culture on wheat seedlings of drought-resistant Chinese Spring (CS) and drought-susceptible SQ1 cultivar, and to examine the alleviative role of exogenous polyamines (PAs) applied to the medium. The assessment was based on physiological (chlorophyll a fluorescence kinetics, chlorophyll and water content) as well as biochemical (content of carbohydrates, phenols, proline, salicylic and abscisic acid, activity of low molecular weight antioxidants) parameters, measured after supplementation with PAs (putrescine, spermidine and spermine) on the 3rd, 5th and 7th day of the treatment. The results indicate that PAs ameliorate the effects of stress, indirectly and conditionally inducing stress tolerance of wheat seedlings. In contrast to the susceptible SQ1, the resistant CS cultivar activated its protective mechanisms, adjusting the degree of their activation to the level of the stress, depending on the genetic resources of the plant. Increased accumulation of antioxidants in the resistant CS in response to stress after the application of PAs confirms the hypothesis that PAs are involved in the signaling pathway determining the antioxidative response and the tolerance of wheat plants to drought stress.

Investigation of quaternary structure of aggregating 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans: In the pursuit of consensus of various biophysical techniques.

In this work we analyzed the quaternary structure of FAD-dependent 3-ketosteroid dehydrogenase (AcmB) from Sterolibacterium denitrificans, the protein that in solution forms massive aggregates (>600 kDa). Using size-excursion chromatography (SEC), dynamic light scattering (DLS), native-PAGE and atomic force microscopy (AFM) we studied the nature of enzyme aggregation. Partial protein de-aggregation was facilitated by the presence of non-ionic detergent such as Tween 20 or by a high degree of protein dilution but not by addition of a reducing agent or an increase of ionic strength. De-aggregating influence of Tween 20 had no impact on either enzyme's specific activity or FAD reconstitution to recombinant AcmB. The joint experimental (DLS, isoelectric focusing) and theoretical investigations demonstrated gradual shift of enzyme's isoelectric point upon aggregation from 8.6 for a monomeric form to even 5.0. The AFM imaging on mica or highly oriented pyrolytic graphite (HOPG) surface enabled observation of individual protein monomers deposited from a highly diluted solution (0.2 µg/ml). Such approach revealed that native AcmB can indeed be monomeric. AFM imaging supported by theoretical random sequential adsorption (RSA) kinetics allowed estimation of distribution enzyme forms in the bulk solution: 5%, monomer, 11.4% dimer and 12% trimer. Finally, based on results of AFM as well as analysis of the surface of AcmB homology models we have observed that aggregation is most probably initiated by hydrophobic forces and then assisted by electrostatic attraction between negatively charged aggregates and positively charged monomers.

Long-Term Effects of Cold on Growth, Development and Yield of Narrow-Leaf Lupine May Be Alleviated by Seed Hydropriming or Butenolide.

In this article, the effects of cold on the development of Lupine angustifolius and the possibility of mitigating it, via seed hydropriming or pre-treatment with butenolide (10-6 M⁻10-4 M), are investigated in two cultivars, differing in their ability to germinate at low temperature. Physiological background of plant development after cold stress was investigated in imbibed seeds. For the first four weeks, the seedlings grew at 7 °C or 13 °C. Seeds well germinating at 7 °C demonstrated higher activity of α-amylase and higher levels of gibberellins, IAA and kinetin. Germination ability at low temperature correlated with dehydrogenase activity and membrane permeability. Seed pre-treatment improved germination at low temperature by decreasing abscisic acid content. Seed hydropriming alleviated cold effects on plant development rate and yield, while butenolide accelerated vegetative development but delayed the generative phase. Potential seed yield may be predicted based on the seed germination vigour and the photosynthetic efficiency measured before flowering.

Influence of salicylic acid pretreatment on seeds germination and some defence mechanisms of Zea mays plants under copper stress.

The study was focused on the influence of salicylic acid (SA) on maize seeds germination and on some physiological and biochemical processes in maize plants growing in the hydroponic culture under copper (Cu) stress. A significant influence of SA pretreatment on the advanced induction of the maize seeds metabolic activity and the level of the endogenous SA in germinated seeds and developing roots have been stated. Although, the ability of maize seeds to uptake SA and accumulate it in the germinated roots was confirmed, the growth inhibition of Cu-stressed maize seedlings was not ameliorated by SA seeds pretreatment. Cu-stressed plants exhibited a decrease in the photosynthetic pigment concentration and the increase in non-photochemical quenching (NPQ) - an indicator of an excess energy in PSII antenna assemblies lost as a heat. The amelioration effect of SA application was found only for carotenoids content which increased in stressed plants. It was also shown that maize roots growing in stress conditions significantly differed in the chemical composition in comparison to the roots of control plants, but the SA pretreatment did not affect these differences. On the other hand, it was found that SA seed pretreatment significantly influenced the ability of stressed plants to accumulate copper in the roots. It was stated that a higher level of exogenous SA application led to a lower accumulation of Cu ions in maize roots. Cu-stressed plants exhibited higher oxidative stress in roots than in leaves which was manifested as an increase in the concentration of hydrogen peroxide due to stress factor application. We observed an increase in catalase (CAT) activity in leaves of Cu-stressed plants which corresponded with a lower H2O2 content when compared with roots where the hydrogen peroxide level was higher, and the inhibition of the CAT activity was found. Furthermore, we found that the SA seed pretreatment led to a decrease in the H2O2 content in the roots of the Cu-stressed plants, but it did not influence the H2O2 level in leaves. The increase in hydrogen peroxide content in the roots of Cu-stressed plants correlated with a higher activity of the MnSODI and MnSODII isoforms. It was found that SA pretreatment caused a decrease in MnSODII activity accompanied by the decrease in H2O2 concentration. Achieved results indicated also that the changes in the chemical composition of the root tissue under copper stress constituted protection mechanisms of blocking copper flow into other plant organs. However, it might be assumed that the root tissue remodelling under Cu stress did not only prevent against the Cu ions uptake but also limited the absorption of minerals required for the normal growth leading to the inhibition of the plant development.

Physiological and biochemical characterisation of watered and drought-stressed barley mutants in the HvDWARF gene encoding C6-oxidase involved in brassinosteroid biosynthesis.

Brassinosteroids (BR) are plant steroid hormones that were discovered more than thirty years ago, but their physiological function has yet to be fully explained. The aim of the study was to answer the question of whether/how disturbances in the production of BR in barley affects the plant's metabolism and development under conditions of optimal watering and drought. Mutants with an impaired production of BR are one of the best tools in research aimed at understanding the mechanisms of action of these hormones. The study used barley cultivars with a normal BR synthesis (wild type) and semi-dwarf allelic mutants with an impaired activity of C6-oxidase (mutation in HvDWARF), which resulted in a decreased BR synthesis. Half of the plants were subjected to drought stress in the seedling stage and the other half were watered optimally. Plants with impaired BR production were characterised by a lower height and developmental retardation. Under both optimal watering and drought, BR synthesis disorders caused the reduced production of ABA and cytokinins, but not auxins. The BR mutants also produced less osmoprotectant (proline). The optimally watered and drought-stressed mutants accumulated less sucrose, which was accompanied by changes in the production of other soluble sugars. The increased content of fructooligosaccharide (kestose) in optimally watered mutants would suggest that BR is a negative regulator of kestose production. The decreased level of nystose in the drought-stressed mutants also suggests BR involvement in the regulation of the production of this fructooligosaccharide. The accumulation of the transcripts of genes associated with stress response (hsp90) was lower in the watered and drought-stressed BR-deficient mutants. In turn, the lower efficiency of photosystem II and the net photosynthetic rate in mutants was revealed only under drought conditions. The presented research allows for the physiological and biochemical traits of two BR-barley mutants to be characterised, which helps BR function to be understood. The knowledge can also be a good starting point for some breeding companies that are interested in introducing new semi-dwarf barley cultivars.

Auxin and chloroplast movements.

Auxin is involved in a wide spectrum of physiological processes in plants, including responses controlled by the blue light photoreceptors phototropins: phototropic bending and stomatal movement. However, the role of auxin in phototropin-mediated chloroplast movements has never been studied. To address this question we searched for potential interactions between auxin and the chloroplast movement signaling pathway using different experimental approaches and two model plants, Arabidopsis thaliana and Nicotiana tabacum. We observed that the disturbance of auxin homeostasis by shoot decapitation caused a decrease in chloroplast movement parameters, which could be rescued by exogenous auxin application. In several cases, the impairment of polar auxin transport, by chemical inhibitors or in auxin carrier mutants, had a similar negative effect on chloroplast movements. This inhibition was not correlated with changes in auxin levels. Chloroplast relocations were also affected by the antiauxin p-chlorophenoxyisobutyric acid and mutations in genes encoding some of the elements of the SCF(TIR1)-Aux/IAA auxin receptor complex. The observed changes in chloroplast movement parameters are not prominent, which points to a modulatory role of auxin in this process. Taken together, the obtained results suggest that auxin acts indirectly to regulate chloroplast movements, presumably by regulating gene expression via the SCF(TIR1)-Aux/IAA-ARF pathway. Auxin does not seem to be involved in controlling the expression of phototropins.

Influence of Oil Contamination on Physical and Biological Properties of Forest Soil After Chainsaw Use.

Forestry works using chainsaws result in up to 7 million liters of various mineral oils being soaked annually into forest soils. These substances, containing a complex mixture of polycyclic aromatic hydrocarbons (PAHs), are highly toxic. The aim of the study was to determine the effect of oil contamination with PAHs on the physical and biological properties of forest soils. The study area was located in southern Poland in the Miechów forest district. The experiment was conducted on four treatment blocks with various amounts of oil addition. The study included the determination of PAH content, dehydrogenase and urease activity, and biomass of earthworms. Physical properties were determined using the dryer method and Kopecky rings of 250 cm3 volume. The results obtained confirmed the hypothesis that oil contamination with PAHs modified the physical properties of forest soils and oil had a negative impact on enzyme activity in soil. Enzyme activity in the studied soils was negatively correlated with PAH content. Earthworm population density reflected the contamination level of oil-contaminated soils.

Hormonal requirements for effective induction of microspore embryogenesis in triticale (× Triticosecale Wittm.) anther cultures.

KEY MESSAGE: Effective microspore embryogenesis in triticale is determined by a specific hormonal homeostasis: low value of IAA/cytokinins, IAA/ABA and cytokinins/ABA ratios as well as proper endogenous/exogenous auxin balance, which favours androgenic structure formation and green plant regeneration ability. The concentration of plant growth regulators (PGRs): auxins (Auxs), cytokinins (CKs) and abscisic acid (ABA) was measured in anthers of eight DH lines of triticale (× Triticosecale Wittm.), and associated with microspore embryogenesis (ME) responsiveness. The analysis was conducted on anthers excised from control tillers at the phase optimal for ME induction and then after ME-initiating tillers treatment (21 days at 4 °C). In control, IAA predominated among Auxs (11-39 nmol g(-1) DW), with IBA constituting only 1 % of total Auxs content. The prevailing isoforms of CKs were cis isomers of zeatin (121-424 pmol g(-1) DW) and zeatin ryboside (cZR, 146-432 pmol g(-1) DW). Surprisingly, a relatively high level (10-64 pmol g(-1) DW) of kinetin (KIN) was detected. Cold treatment significantly changed the levels of all analysed PGRs. The anthers of 'responsive' DH lines contained higher concentrations of IBA, cis and trans zeatin, cZR and ABA, and lower amount of IAA and KIN in comparison with 'recalcitrant' genotypes. However, the effects of exogenous ABA, p-chlorophenoxyisobutyric acid (PCIB) and 2,3,5-triiodobenzoic acid treatments suggest that none of the studied PGRs acts alone in the acquisition of embryogenic competency, which seems to be an effect of concerted PGRs crosstalk. The initiation of ME required a certain threshold level of ABA. A crucial prerequisite for high ME effectiveness was a specific PGRs homeostasis: lower Auxs level in comparison with CKs and ABA, and lower CKs/ABA ratio. A proper balance between endogenous Auxs in anthers and exogenous Auxs supplied by culture media was also essential.

UV-induced spectral shift and protonation of DNA fluorescent dye Hoechst 33258.

DNA-bound Hoechst 33258 is readily excited with UV light and emits blue fluorescence, however, upon exposure to UV, the dye undergoes photobleaching as well as photoconversion to a blue-excited green-emitting form. We demonstrate that the UV-generated green-emitting form of Hoechst 33258 exhibits spectral properties very similar to the form of the dye that can be obtained by subjecting it to an acidic environment (pH 0.5-3.0). We also demonstrate that exposure of Hoechst 33258 to UV light (or hydrogen peroxide) leads to generation of the protonated (1+, 2+, 3+ and possibly the 4+) forms of the dye. Photoconversion of Hoechst 33258 has recently been exploited in single molecule localisation microscopy, thus understanding photophysics of this process can facilitate further development of high resolution optical imaging.

Alleviation of osmotic stress effects by exogenous application of salicylic or abscisic acid on wheat seedlings.

The aim of the study was to assess the role of salicylic acid (SA) and abscisic acid (ABA) in osmotic stress tolerance of wheat seedlings. This was accomplished by determining the impact of the acids applied exogenously on seedlings grown under osmotic stress in hydroponics. The investigation was unique in its comprehensiveness, examining changes under osmotic stress and other conditions, and testing a number of parameters simultaneously. In both drought susceptible (SQ1) and drought resistant (CS) wheat cultivars, significant physiological and biochemical changes were observed upon the addition of SA (0.05 mM) or ABA (0.1 µM) to solutions containing half-strength Hoagland medium and PEG 6000 (-0.75 MPa). The most noticeable result of supplementing SA or ABA to the medium (PEG + SA and PEG + ABA) was a decrease in the length of leaves and roots in both cultivars. While PEG treatment reduced gas exchange parameters, chlorophyll content in CS, and osmotic potential, and conversely, increased lipid peroxidation, soluble carbohydrates in SQ1, proline content in both cultivars and total antioxidants activity in SQ1, PEG + SA or PEG + ABA did not change the values of these parameters. Furthermore, PEG caused a two-fold increase of endogenous ABA content in SQ1 and a four-fold increase in CS. PEG + ABA increased endogenous ABA only in SQ1, whereas PEG + SA caused a greater increase of ABA content in both cultivars compared to PEG. In PEG-treated plants growing until the harvest, a greater decrease of yield components was observed in SQ1 than in CS. PEG + SA, and particularly PEG + ABA, caused a greater increase of these yield parameters in CS compared to SQ1. In conclusion, SA and ABA ameliorate, particularly in the tolerant wheat cultivar, the harmful effects and after effects of osmotic stress induced by PEG in hydroponics through better osmotic adjustment achieved by an increase in proline and carbohydrate content as well as by an increase in antioxidant activity.