Two-Dimensional Gel Electrophoresis in Studies of Flower and Leaf Proteome of Common Buckwheat.

Two-dimensional gel electrophoresis (2-DE) is a proteomic tool used for the separation of protein mixtures according to protein isoelectric point and molecular mass. Although gel-free quantitative and qualitative proteomic study techniques are now available, 2-DE remains a useful analytical tool. The presented protocol was performed to analyze the flower and leaf proteome of common buckwheat using 24 cm immobilized pH gradient strips (pH 4-7) and visualization of proteins on gels via colloidal Coomassie G-250 staining.

Phenol-Based Protein Extraction Method for Plant Proteomic Studies.

The protein extraction method based on the phenol solution and combined with protein precipitation with ammonium acetate in methanol and purification in the same solution, and additionally in acetone and ethanol, is recommended for proteomic studies of plant tissues. The obtained protein samples do not require additional nucleic acid digestion and removal of interfering contaminations. The presented protocol was used to analyze the proteome of common buckwheat flowers and leaves.

New Prospects for Improving Microspore Embryogenesis Induction in Highly Recalcitrant Winter Wheat Lines.

Among various methods stimulating biological progress, double haploid (DH) technology, which utilizes the process of microspore embryogenesis (ME), is potentially the most effective. However, the process depends on complex interactions between many genetic, physiological and environmental variables, and in many cases, e.g.

Comparative proteomic analysis provides new insights into regulation of microspore embryogenesis induction in winter triticale (× Triticosecale Wittm.) after 5-azacytidine treatment.

Effective microspore embryogenesis (ME) requires substantial modifications in gene expression pattern, followed by changes in the cell proteome and its metabolism. Recent studies have awakened also interest in the role of epigenetic factors in microspore de-differentiation and reprogramming. Therefore, demethylating agent (2.

ROS-Scavengers, Osmoprotectants and Violaxanthin De-Epoxidation in Salt-Stressed with Different Tocopherol Composition.

To determine the role of α- and γ-tocopherol (TC), this study compared the response to salt stress (200 mM NaCl) in wild type (WT) (L.) Heynh. And its two mutants: (1) totally TC-deficient ; (2) accumulating γ-TC instead of α-TC; and (3) transgenic line overaccumulating α-TC.

Impact of Ascorbate-Glutathione Cycle Components on the Effectiveness of Embryogenesis Induction in Isolated Microspore Cultures of Barley and Triticale.

Enhanced antioxidant defence plays an essential role in plant survival under stress conditions. However, excessive antioxidant activity sometimes suppresses the signal necessary for the initiation of the desired biological reactions. One such example is microspore embryogenesis (ME)-a process of embryo-like structure formation triggered by stress in immature male gametophytes.

Albino Plant Formation in Androgenic Cultures: An Old Problem and New Facts.

High frequency of albino plant formation in isolated microspore or anther cultures is a great problem limiting the possibility of their exploitation on a wider scale. It is highly inconvenient as androgenesis-based doubled haploid (DH) technology provides the simplest and shortest way to total homozygosity, highly valued by plant geneticists, biotechnologists and especially, plant breeders, and this phenomenon constitutes a serious limitation of these otherwise powerful tools. The genotype-dependent tendency toward albino plant formation is typical for many monocotyledonous plants, including cereals like wheat, barley, rice, triticale, oat and rye - the most important from the economical point of view.

β-1,3-Glucanases and chitinases participate in the stress-related defence mechanisms that are possibly connected with modulation of arabinogalactan proteins (AGP) required for the androgenesis initiation in rye (Secale cereale L.).

This work presents the biochemical, cytochemical and molecular studies on two groups of PR proteins, β-1,3-glucanases and chitinases, and the arabinogalactan proteins (AGP) during the early stages of androgenesis induction in two breeding lines of rye (Secale cereale L.) with different androgenic potential. The process of androgenesis was initiated by tillers pre-treatments with low temperature, mannitol and/or reduced glutathione and resulted in microspores reprogramming and formation of androgenic structures what was associated with high activity of β-1,3-glucanases and chitinases.

Candidate Genes for Freezing and Drought Tolerance Selected on the Basis of Proteome Analysis in Doubled Haploid Lines of Barley.

Plant tolerance to environmental stress is determined by a very complicated network composed of many intra- and extracellular factors. The aim of this study was to select candidate genes involved in responses to freezing and drought in barley on the basis of previous proteomic studies and to analyze changes in their expression caused by application of both stress factors. Six candidate genes for freezing tolerance (namely the genes encoding elongation factor 1 alpha (), ferredoxin-NADP reductase, a 14-3-3a protein, β-fructofuranosidase, CBF2A and CBF4B) and six for drought tolerance (encoding transketolase, periplasmic serine protease, triosephosphate isomerase, a protein with a co-chaperon region (), pfam14200 and actin) were chosen arbitrarily on the basis of in silico bioinformatic analyses.

Chemically-induced DNA de-methylation alters the effectiveness of microspore embryogenesis in triticale.

Microspores exposed to some stress factors may display cell totipotency and could be reprogrammed towards embryogenic development. Plant breeding and genetic engineering widely use haploids/doubled haploids (DHs) derived from in vitro-cultured microspores, but the mechanism of this process remains poorly understood. Recently published data suggest that microspore embryogenesis (ME) is accompanied by changes in DNA methylation and chromatin reorganization.

Glutathione provides antioxidative defence and promotes microspore-derived embryo development in isolated microspore cultures of triticale (× Triticosecale Wittm.).

Depending on the capability for stress adaptation, the role played by glutathione in microspore embryogenesis consists of both antioxidative activity and stimulation of embryo-like structure development. The efficiency of microspore embryogenesis (ME) is determined by the complex network of internal and environmental factors. Among them, the efficient defence against oxidative stress seems to be one of the most important.

Changes in protein abundance and activity involved in freezing tolerance acquisition in winter barley (Hordeum vulgare L.).

Unlabelled: The changes in protein abundance induced by cold hardening were analysed by 2 DE in ten doubled haploid (DH) lines of winter barley, highly differentiated with respect to freezing tolerance level. Among 45 differential proteins identified by MALDI-TOF/TOF, the majority was classified as related to photosynthesis, carbohydrate metabolism, oxidation-reduction reactions and stress response. Among the detected proteins, higher abundance of RuBisCO large and small subunits, RuBisCO activase, two Oxygen-evolving enhancer proteins, Ferredoxin-NADP reductase, Cytochrome P450-dependent fatty acid hydroxylase and 14-3-3 protein was associated with higher freezing tolerance level.

Changes in protein abundance and activity induced by drought during generative development of winter barley (Hordeum vulgare L.).

Unlabelled: The present study investigated drought-induced changes in proteome profiles of ten DH lines of winter barley, relatively varied in water deficit tolerance level. Additionally, the parameters describing the functioning of the photosynthetic apparatus and the activity of the antioxidative system were analysed. Water deficit (3-week growth in soil with water content reduced to ca.

Current insights into hormonal regulation of microspore embryogenesis.

Plant growth regulator (PGR) crosstalk and interaction with the plant's genotype and environmental factors play a crucial role in microspore embryogenesis (ME), controlling microspore-derived embryo differentiation and development as well as haploid/doubled haploid plant regeneration. The complexity of the PGR network which could exist at the level of biosynthesis, distribution, gene expression or signaling pathways, renders the creation of an integrated model of ME-control crosstalk impossible at present. However, the analysis of the published data together with the results received recently with the use of modern analytical techniques brings new insights into hormonal regulation of this process.

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

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 influence of heat stress on auxin distribution in transgenic B. napus microspores and microspore-derived embryos.

Plant embryogenesis is regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients during microspore embryogenesis remain to be identified. For the first time, we describe, using the DR5 or DR5rev reporter gene systems, the GFP- and GUS-based auxin biosensors to monitor auxin during Brassica napus androgenesis at cellular resolution in the initial stages.

Endogenous ABA concentration and cytoplasmic membrane fluidity in microspores of oilseed rape (Brassica napus L.) genotypes differing in responsiveness to androgenesis induction.

A better understanding of androgenesis with a focus on the changes in plasma membrane fluidity and endogenous ABA content affecting embryogenesis induction in microspore suspension of B. napus. Changes in plasma membrane fluidity (MF) and ABA content associated with androgenesis induction were under the study.