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.

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®.

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.

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.

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.