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.

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.

Climate Change-The Rise of Climate-Resilient Crops.

Climate change disrupts food production in many regions of the world. The accompanying extreme weather events, such as droughts, floods, heat waves, and cold snaps, pose threats to crops. The concentration of carbon dioxide also increases in the atmosphere. The United Nations is implementing the climate-smart agriculture initiative to ensure food security. An element of this project involves the breeding of climate-resilient crops or plant cultivars with enhanced resistance to unfavorable environmental conditions. Modern agriculture, which is currently homogeneous, needs to diversify the species and cultivars of cultivated plants. Plant breeding programs should extensively incorporate new molecular technologies, supported by the development of field phenotyping techniques. Breeders should closely cooperate with scientists from various fields of science.

Redox regulation of the Calvin-Benson-Bassham cycle during cold acclimation.

NADPH-dependent thioredoxin reductase C (NTRC) redox interaction with protein CP12 plays a role in cold acclimation. A recent study by Teh et al. describes the underlying molecular mechanisms that leads to dissociation of the autoinhibitory PRK/CP12/GAPDH (phosphoribulokinase/CP12/glyceraldehyde-3-phosphate dehydrogenase) supracomplex. We propose that chloroplast-to-nucleus retrograde signaling precedes the described mechanism.

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., winter wheat, does not operate with the efficiency required for commercial use. Stress associated with low-temperature treatment, isolation and transfer to in vitro culture has been shown to disturb redox homeostasis and generate relatively high levels of reactive oxygen species (ROS), affecting microspore vitality. The aim of this study was to investigate whether controlled plant growth, specific tiller pre-treatment and culture conditions could improve the potential of microspores to cope with stress and effectively induce ME. To understand the mechanism of the stress response, hydrogen peroxide levels, total activity and the content of the most important low-molecular-weight antioxidants (glutathione and ascorbate), as well as the content of selected macro- (Mg, Ca, NA, K) and micronutrients (Mn, Zn, Fe, Cu, Mo) were determined. These analyses, combined with the cytological characteristics of the microspore suspensions, allowed us to demonstrate that an increased microspore vitality and stronger response to ME induction were associated with higher stress resistance based on more efficient ROS scavenging and nutrient management. It was shown that a modified procedure, combining a low temperature with mannitol and sodium selenate tiller pre-treatment, reduced oxidative stress and improved the effectiveness of ME in winter wheat lines.

The yield of common buckwheat (Fagopyrum esculentum Moench) depends on the genotype but not on the Pin-to-Thrum ratio.

Common buckwheat has a complicated flowering biology. It is characterized by a strong self-incompatibility resulting from heterostyly, i.e. the occurrence of two types of flowers: Pin and Thrum, differing in the length of pistils and stamens. Fertilization occurs only as a result of cross-pollination between these morphs. Suspicions exist that the disturbed ratio between plants producing Pin and Thrum flowers (with the latter type generating more seeds) causes low seed yield. The aim of the study was to analyze: (1) the ratio between plants with Pin and Thrum morphs, (2) flower and seed production, as well as abortion of flowers, (3) the composition of nectar collected at an early flowering stage and during full flowering. The study was performed under semi-controlled and field conditions on six Polish accessions. The results indicated that under semi-controlled conditions the Pin-to-Thrum ratio was indeed disproportionate; such a phenomenon is called anisoplethy. In the field, however, the Pin-to-Thrum ratio was well-balanced (isoplethy). The plants with both morphs aborted a similar percentage of flowers and produced a comparable number of empty seeds. The number of flowers, their abortion, and ripe seed production were independent of flower type, however, they were genotypically controlled. A strong correlation between the number of flowers produced by a plant, flower abortion and the number of empty seeds was found. The percentage of aborted flowers correlated positively with the weight of ripe seeds. Nectar composition was similar for all buckwheat genotypes, but we found some differences in the amount of individual sugars depending on the blooming stage. In the majority of accessions, the nectar produced at the early blooming stage was characterized by a greater mass and volume, and contained more individual sugars than at the full-flowering stage.

The Effect of Stimulants on Nectar Composition, Flowering, and Seed Yield of Common Buckwheat (Fagopyrum esculentum Moench).

Common buckwheat is a valuable plant producing seeds containing a number of health-promoting compounds and elements. Buckwheat does not contain gluten and is characterized by an excellent composition of amino acids. This species is also a melliferous plant. Despite many advantages, the area of buckwheat cultivation is decreasing due to unstable yields. One of the reasons for low seed yield is its sensitivity to drought, high temperatures, and assimilate deficiencies. These factors have a significant impact on the nectar composition, which is important for visiting pollinators and thus for pollination. High temperature during flowering increases the degeneration of embryo sacs and embryos, which is high anyway (genetic determination) in common buckwheat. This phenomenon seems to be unbreakable by breeding methods. The authors aimed to determine whether stimulants commonly used in agriculture could increase the seed yield of this plant species. The aim of the work was to choose from eight different stimulants the most effective one that would improve the seed yield of two accessions of common buckwheat by increasing the efficiency of nectar production and reducing the number of empty seeds. The plants were sprayed at either the beginning of flowering or at full bloom. The content of sugars and amino acids was higher in the nectar produced at the beginning of flowering. The nectar of both lines included also polyamines. The level of sugars in the nectar increased mainly after spraying with the stimulants in the second phase of flowering. A positive correlation between the total amount of sugars and amino acids in the nectar and seed yield was found. All the stimulants used reduced the number of empty seeds in both accessions. Seed production in the PA15 line increased significantly under the influence of all stimulants used at the beginning of flowering, and the most effective were ASAHI SL and TYTANIT®.

Physiological and Biochemical Parameters of Salinity Resistance of Three Durum Wheat Genotypes.

The area of farming lands affected by increasing soil salinity is growing significantly worldwide. For this reason, breeding works are conducted to improve the salinity tolerance of important crop species. The goal of the present study was to indicate physiological or biochemical parameters characterizing three durum wheat accessions with various tolerance to salinity. The study was carried out on germinating seeds and mature plants of a Polish SMH87 line, an Australian cultivar 'Tamaroi' (salt-sensitive), and the BC5Nax2 line (salt-tolerant) exposed to 0-150 mM NaCl. Germination parameters, electrolyte leakage (EL), and salt susceptibility index were determined in the germinating caryopses, whereas photosynthetic parameters, carbohydrate and phenolic content, antioxidant activity as well as yield were measured in fully developed plants. The parameters that most differentiated the examined accessions in the germination phase were the percentage of germinating seeds (PGS) and germination vigor (Vi). In the fully developed plants, parameters included whether the plants had the maximum efficiency of the water-splitting reaction on the donor side of photosystem II (PSII)-Fv/F0, energy dissipation from PSII-DIo/CSm, and the content of photosynthetic pigments and hydrogen peroxide, which differentiated studied genotypes in terms of salinity tolerance degree. Salinity has a negative impact on grain yield by reducing the number of seeds per spike and the mass of one thousand seeds (MTS), which can be used as the most suitable parameter for determining tolerance to salinity stress. The most salt-tolerant BC5Nax2 line was characterized by the highest PGS, and Vi for NaCl concentration of 100-150 mM, content of chlorophyll a, b, carotenoids, and also MTS at all applied salt concentrations as compared with the other accessions. The most salt-sensitive cv. 'Tamaroi' demonstrated higher H2O2 concentration which proves considerable oxidative damage caused by salinity stress. Mentioned parameters can be helpful for breeders in the selection of genotypes the most resistant to this stress.

Post-translational activation of CBF for inducing freezing tolerance.

Plants can acquire increased freezing tolerance through cold-acclimation involving the ICE1-CBF-COR pathway. Recently, Lee et al. investigated a potential link between the functional activation of CBF and cellular redox state. We propose that redox-mediated CBF activation could be a hub of low temperature as well as light signaling in the cold-acclimation process.

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 (2.5-10 µM 5-azacytidine, AC) together with low temperature (3 weeks at 4 °C) were used as ME-inducing tiller treatment in two doubled haploid (DH) lines of triticale and its effect was analyzed in respect of anther protein profiles, expression of selected genes (TAPETUM DETERMINANT1 (TaTPD1-like), SOMATIC EMBRYOGENESIS RECEPTOR KINASE 2 (SERK2) and GLUTATHIONE S-TRANSFERASE (GSTF2)) and ME efficiency. Tiller treatment with 5.0 µM AC was the most effective in ME induction; it was associated with (1) suppression of intensive anabolic processes-mainly photosynthesis and light-dependent reactions, (2) transition to effective catabolism and mobilization of carbohydrate reserve to meet the high energy demand of cells during microspore reprograming and (3) effective defense against stress-inducing treatment, i.e. protection of proper folding during protein biosynthesis and effective degradation of dysfunctional or damaged proteins. Additionally, 5.0 µM AC enhanced the expression of all genes previously identified as being associated with embryogenic potential of microspores (TaTPD1-like, SERK and GSTF2).

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. The study focused on the role of reactive oxygen species and antioxidant defence in triticale (×Triticosecale Wittm.) and barley (Hordeum vulgare L.) microspore reprogramming. ME was induced through various stress treatments of tillers and its effectiveness was analysed in terms of ascorbate and glutathione contents, total activity of low molecular weight antioxidants and activities of glutathione-ascorbate cycle enzymes. The most effective treatment for both species was a combination of low temperature and exogenous application of 0.3 M mannitol, with or without 0.3 mM reduced glutathione. The applied treatments induced genotype-specific defence responses. In triticale, both ascorbate and glutathione were associated with ME induction, though the role of glutathione did not seem to be related to its function as a reducing agent. In barley, effective ME was accompanied by an accumulation of ascorbate and high activity of enzymes regulating its redox status, without direct relation to glutathione content.

Physiological and Biochemical Response to Fusarium culmorum Infection in Three Durum Wheat Genotypes at Seedling and Full Anthesis Stage.

Fusarium culmorum is a worldwide, soil-borne plant pathogen. It causes diseases of cereals, reduces their yield, and fills the grain with toxins. The main direction of modern breeding is to select wheat genotypes the most resistant to Fusarium diseases. This study uses seedlings and plants at the anthesis stage to analyze total soluble carbohydrates, total and cell-wall bound phenolics, chlorophyll content, antioxidant activity, hydrogen peroxide content, mycotoxin accumulation, visual symptoms of the disease, and Fusarium head blight index (FHBi). These results determine the resistance of three durum wheat accessions. We identify physiological or biochemical markers of durum wheat resistance to F. culmorum. Our results confirm correlations between FHBi and mycotoxin accumulation in the grain, which results in grain yield decrease. The degree of spike infection (FHBi) may indicate accumulation mainly of deoxynivalenol and nivalenol in the grain. High catalase activity in the infected leaves could be considered a biochemical marker of durum sensitivity to this fungus. These findings allowed us to formulate a strategy for rapid evaluation of the disease severity and the selection of plants with higher level, or resistance to F. culmorum infection.

Changes in the Flower and Leaf Proteome of Common Buckwheat (Fagopyrum esculentum Moench) under High Temperature.

Common buckwheat (Fagopyrum esculentum Moench), a pseudocereal crop, produces a large number of flowers, but this does not guarantee high seed yields. This species demonstrates strong abortion of flowers and embryos. High temperatures during the generative growth phase result in an increase in the degeneration of embryo sacs. The aim of this study was to investigate proteomic changes in flowers and leaves of two common buckwheat accessions with different degrees of heat tolerance, Panda and PA15. Two-dimensional gel electrophoresis and mass spectrometry techniques were used to analyze the proteome profiles. Analyses were conducted for flower buds, open flowers capable of fertilization, and wilted flowers, as well as donor leaves, i.e., those growing closest to the inflorescences. High temperature up-regulated the expression of 182 proteins. The proteomic response to heat stress differed between the accessions and among their organs. In the Panda accession, we observed a change in abundance of 17, 13, 28, and 11 proteins, in buds, open and wilted flowers, and leaves, respectively. However, in the PA15 accession there were 34, 21, 63, and 21 such proteins, respectively. Fifteen heat-affected proteins were common to both accessions. The indole-3-glycerol phosphate synthase chloroplastic-like isoform X2 accumulated in the open flowers of the heat-sensitive cultivar Panda in response to high temperature, and may be a candidate protein as a marker of heat sensitivity in buckwheat plants.

Identification of the Genetic Basis of Response to De-Acclimation in Winter Barley.

Mechanisms involved in the de-acclimation of herbaceous plants caused by warm periods during winter are poorly understood. This study identifies the genes associated with this mechanism in winter barley. Seedlings of eight accessions (four tolerant and four susceptible to de-acclimation cultivars and advanced breeding lines) were cold acclimated for three weeks and de-acclimated at 12 °C/5 °C (day/night) for one week. We performed differential expression analysis using RNA sequencing. In addition, reverse-transcription quantitative real-time PCR and enzyme activity analyses were used to investigate changes in the expression of selected genes. The number of transcripts with accumulation level changed in opposite directions during acclimation and de-acclimation was much lower than the number of transcripts with level changed exclusively during one of these processes. The de-acclimation-susceptible accessions showed changes in the expression of a higher number of functionally diverse genes during de-acclimation. Transcripts associated with stress response, especially oxidoreductases, were the most abundant in this group. The results provide novel evidence for the distinct molecular regulation of cold acclimation and de-acclimation. Upregulation of genes controlling developmental changes, typical for spring de-acclimation, was not observed during mid-winter de-acclimation. Mid-winter de-acclimation seems to be perceived as an opportunity to regenerate after stress. Unfortunately, it is competitive to remain in the cold-acclimated state. This study shows that the response to mid-winter de-acclimation is far more expansive in de-acclimation-susceptible cultivars, suggesting that a reduced response to the rising temperature is crucial for de-acclimation tolerance.

Reducing Flower Competition for Assimilates by Half Results in Higher Yield of Fagopyrum esculentum.

Despite abundant flowering throughout the season, common buckwheat develops a very low number of kernels probably due to competition for assimilates. We hypothesized that plants with a shorter flowering period may give a higher seed yield. To verify the hypothesis, we studied nutrient stress in vitro and in planta and analyzed different embryological and yield parameters, including hormone profile in the flowers. In vitro cultivated flowers on media with strongly reduced nutrient content demonstrated a drastic increase in degenerated embryo sacs. In in planta experiments, where 50% or 75% of flowers or all lateral ramifications were removed, the reduction of the flower competition by half turned out to be the most promising treatment for improving yield. This treatment increased the frequency of properly developed embryo sacs, the average number of mature seeds per plant, and their mass. Strong seed compensation under 50% inflorescence removal could result from increased production of salicylic and jasmonic acid that both favor more effective pollinator attraction. Plants in single-shoot cultivation finished their vegetation earlier, and they demonstrated greater single seed mass per plant than in control. This result suggests that plants of common buckwheat with shorter blooming period could deliver higher seed yield.

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 (EF1A), ferredoxin-NADP reductase, a 14-3-3a protein, ß-fructofuranosidase, CBF2A and CBF4B) and six for drought tolerance (encoding transketolase, periplasmic serine protease, triosephosphate isomerase, a protein with a co-chaperon region (GroEs), pfam14200 and actin) were chosen arbitrarily on the basis of in silico bioinformatic analyses. The expression levels of these genes were measured under control and stress conditions in six DH (doubled haploid) lines with differing freezing and drought tolerance. The results of gene expression analysis confirmed the roles of the candidate genes preselected in this study on the basis of previous proteome analysis in contributing to the differences in freezing and drought tolerance observed in the studied population of DH lines of winter barley.

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. Here, we used two triticale DH lines (DH19 and DH28), significantly different with respect to embryogenic potential. To change DNA methylation levels, we applied two cytosine-analogs: 5-azacytidine (AC) and 2'-deoxy-5-azacytidine (DAC) treatments. We found that chemically-induced DNA demethylation caused chromatin relaxation and dysregulation of marker genes (TaTPD1-like, GSTF2, GSTA2, CHI3, Tad1, TaNF-YA7, SERK2, TaME1) related to ME. Both drugs showed significant cytotoxicity in a dose-dependent manner. We noticed that lines varied in terms of overall DNA methylation levels and responded in a different way to hypomethylation caused by the drugs. DH19 (low embryogenic) after inhibitors treatment, showed higher microspore viability, but its recalcitrancy was not overcome. For highly embryogenic DH28, we noted significantly higher effectiveness of embryo-like structure production and plant regeneration. In summary, our study provides new insight into the role of DNA methylation in ME initiation. They suggest potential benefits resulting from the utilization of epigenetic inhibitors to improve the process of DHs production.

Effects of High Temperature on Embryological Development and Hormone Profile in Flowers and Leaves of Common Buckwheat (Fagopyrum esculentum Moench).

Common buckwheat is a valuable crop, mainly due to the beneficial chemical composition of its seeds. However, buckwheat cultivation is limited because of unstable seed yield. The most important reasons for the low yield include embryo and flower abortion. The aim of this work is to verify whether high temperature affects embryological development in this plant species. The experiment was conducted on plants of a Polish cultivar 'Panda' and strain PA15, in which the percentage of degenerating embryo sacs was previously determined and amounted to 32% and 10%, respectively. The plants were cultivated in phytotronic conditions at 20 °C (control), and 30 °C (thermal stress). The embryological processes and hormonal profiles in flowers at various developmental stages (buds, open flowers, and wilted flowers) and in donor leaves were analyzed in two-month-old plants. Significant effects of thermal stress on the defective development of female gametophytes and hormone content in flowers and leaves were observed. Ovules were much more sensitive to high temperature than pollen grains in both genotypes. Pollen viability remained unaffected at 30 °C in both genotypes. The effect of temperature on female gametophyte development was visible in cv. Panda but not in PA15 buds. A drastic reduction in the number of properly developed embryo sacs was clear in open flowers at 30 °C in both genotypes. A considerable increase in abscisic acid in open flowers ready for fertilization may serve as a signal inducing flower senescence observed in the next few days. Based on embryological analyses and hormone profiles in flowers, we conclude that cv. 'Panda' is more sensitive to thermal stress than strain PA15, mainly due to a much earlier response to thermal stress involving impairment of embryological processes already in the flower buds.

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.

Seed Hydropriming and Smoke Water Significantly Improve Low-Temperature Germination of Lupinus angustifolius L.

Seed imbibition under cold temperature is dangerous when dry seeds have relatively low water content. The aim of this study was to investigate germination of 20 lines/cultivars of narrow-leaf lupine at 7 °C (cold) and 13 °C (control) under the influence of smoke water and following seed hydropriming for 3 h at 20 °C. The efficacy of individual treatments was examined with regard to seed protection during low-temperature germination. Based on seed germination, vigour at cold was evaluated four days after sowing by means of hypocotyl length, the studied lines/cultivars were divided into three groups with low, high and very high germination rates. Germination vigour correlated with cell membrane permeability, dehydrogenase activity and abscisic acid (ABA) content and was analysed in the seeds one day after sowing. Gibberellin content did not correlate with germination vigour. The seeds of weakly germinating lines/cultivars had the highest cell permeability and ABA content as well as the lowest amylolytic activity at both studied temperatures. Additionally, the vigour of weakly germinating seeds at 7 °C correlated with dehydrogenase activity. Three-hour hydropriming was the most effective for seed germination under cold due to reduced cell membrane permeability and ABA level. Stimulating effects of smoke water on germination under cold could be explained by enhanced dehydrogenase activity.