Comprehensive Embryological Analyses of Common Buckwheat (Fagopyrym esculentum Moench).

Knowledge of detailed reproductive biology of cultivated species is important as requirements for fruit and seed production allow the development of effective management strategies and a sustainable use. Embryological processes of common buckwheat (Fagopyrum esculentum Moench) are difficult to interpret due to the influence of genetic determinants, i.e., dimorphic heterostyly resulting in the production of long- and short-styled flowers, and environmental predisposition, i.e., sensitivity of ovules to thermal stress. Furthermore, the situation is complicated by overproduction of flowers and depletion of resources as the plant ages. Herein we provide protocols that allow to visualize both basic and more specific embryological features and also disturbances in sexual reproduction of common buckwheat resulting from external and internal factors. All stages of plant material fixation, preparation, staining, and observation are described and explained in detail. Technical tips and pictures of properly prepared microscopic sections are also provided.

Innate, High Tolerance to Zinc and Lead in Violets Confirmed at the Suspended Cell Level.

Many species of the Viola L. genus (violets) colonize areas with high concentrations of trace elements in the soil, e.g., nickel, cadmium, zinc, and lead. Although tolerance to heavy metals is a common phenomenon in violets, it is not clear whether this is the result of gradual microevolutionary processes as a part of the adaptation to the specific conditions, or whether the tolerance was inherited from the ancestor(s). We developed cell suspension cultures of five plant species: two non-metallophytes-Arabidopsis thaliana (Col-0) and Viola · wittrockiana, and three metallophytes-V. philippica, V. tricolor, and Silene vulgaris subsp. humilis for tolerance tests. The aim of the study was to measure the level of tolerance of violets in comparison with species from the other genera to verify the hypothesis of the high, innate tolerance of the former. We measured cell viability, non-enzymatic antioxidant content, and the accumulation of heavy metals after cell treatment with Zn or Pb. The results indicate they are innate and independent on the ecological status (metallophyte vs. non-metallophyte) and high in comparison with other species tolerance to Zn and Pb in violets. Viability of the cells after Zn and Pb (1000 µM) exposure for 72 h was the highest in violets. Antioxidant content, after heavy metal treatment, increased significantly, particularly in metallophyte violets, indicating their high responsivity to metals. In all species, lead was detected in the protoplasm of the cells, not in the vacuole or cell wall. All violets were characterized by the accumulation capacity of lead. Here, we clearly show that the physiological and biochemical studies conducted with the use of heavy metals on plant cells translate into the heavy metal tolerance of the species.

Insights into Plant Programmed Cell Death Induced by Heavy Metals-Discovering a Terra Incognita.

Programmed cell death (PCD) is a process that plays a fundamental role in plant development and responses to biotic and abiotic stresses. Knowledge of plant PCD mechanisms is still very scarce and is incomparable to the large number of studies on PCD mechanisms in animals. Quick and accurate assays, e.g., the TUNEL assay, comet assay, and analysis of caspase-like enzyme activity, enable the differentiation of PCD from necrosis. Two main types of plant PCD, developmental (dPCD) regulated by internal factors, and environmental (ePCD) induced by external stimuli, are distinguished based on the differences in the expression of the conserved PCD-inducing genes. Abiotic stress factors, including heavy metals, induce necrosis or ePCD. Heavy metals induce PCD by triggering oxidative stress via reactive oxygen species (ROS) overproduction. ROS that are mainly produced by mitochondria modulate phytotoxicity mechanisms induced by heavy metals. Complex crosstalk between ROS, hormones (ethylene), nitric oxide (NO), and calcium ions evokes PCD, with proteases with caspase-like activity executing PCD in plant cells exposed to heavy metals. This pathway leads to very similar cytological hallmarks of heavy metal induced PCD to PCD induced by other abiotic factors. The forms, hallmarks, mechanisms, and genetic regulation of plant ePCD induced by abiotic stress are reviewed here in detail, with an emphasis on plant cell culture as a suitable model for PCD studies. The similarities and differences between plant and animal PCD are also discussed.

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.

Differences in the functioning of photosynthetic electron transport between metallicolous and non-metallicolous populations of the pseudometallophyte Viola tricolor.

The objective of this study was to assess the effect of metalliferous conditions on the functioning of photosynthetic electron transport in waste heap populations of a pseudometallophyte, Viola tricolor L. Measurements of chlorophyll a fluorescence and the absorbance changes at 830 nm enabled a non-invasive assessment of photosynthetic apparatus performance. This was complemented by the evaluation of the chlorophyll content. Low temperature chlorophyll fluorescence emission spectra were also recorded. Based on the OJIP test performed in situ, we demonstrated a disturbed condition of photosystem II (PSII) in three metalliferous populations in comparison with a non-metallicolous one. The combined effects of elevated concentrations of zinc, cadmium and lead in soil resulted in the decline of some parameters describing the efficiency and electron flow through PSII. The differences between waste heap populations seemed to be partly correlated with the concentration of heavy metals in the soil. The characteristic of electron transport at photosystem I (PSI) in the light-adapted state revealed increased values of PSI donor-side limitation (YND) and a declined PSI quantum efficiency (YI). It was also demonstrated that the waste heap conditions negatively affect the total chlorophyll content in leaves and led to an increased ratio of fluorescence emission at 77 K (F730/F685). The obtained data indicate that, regardless of the high adaptation of metallicolous populations, photosynthetic electron transport is hampered in V. tricolor plants at metal polluted sites.

From cells highly tolerant to Zn and Pb to fully fertile plants - Selection of tolerant lines with in vitro culture.

Viola arvensis cells were selected after treatment with Zn or Pb and regenerated into plants likely to have higher tolerance levels than the initial plant. The surviving cells in the suspension treated with 2000 µM of Zn, 2000 µM of Pb or 0 µM for 72 h were maintained on a solidified half-strength MS medium supplemented with 0.5 mg L-1 TDZ to induce divisions and organogenesis. The adventitious shoots obtained were rooted on a half-strength MS medium with 1 mg L-1 IBA. Regenerants derived from the Zn- and Pb-treated cells were vigorous and fully fertile. The in vitro conditions and metal impact generated a low genome alteration and overall low genetic diversity of regenerants compared to the initial plant and plants from the natural population. The cells of regenerants obtained after Pb treatment represented an approximately 12% higher tolerance level to Pb than the cells of the initial plant. This is the first report of plant regeneration from highly tolerant cells selected by heavy metal treatment. Regenerants successfully obtained in vitro could be considered as a source material for the recultivation of areas polluted with heavy metals.

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.

Suspended cells of metallicolous and nonmetallicolous Viola species tolerate, accumulate and detoxify zinc and lead.

We studied the zinc and lead accumulation and tolerance level of suspended cells of four Viola species with different metallophyte statuses: Viola lutea ssp. westfalica (obligate metallophyte), V. tricolor (facultative metallophyte), V. arvensis (accidental metallophyte) and V. uliginosa (nonmetallophyte), in order to determine the correlation between cell and plant tolerance. Cells of all studied species/genotypes were tolerant to metal concentrations applied to the medium for 24, 48 and 72 h, more for zinc than for lead, as estimated by cell viability using the alamarBlue assay. Viable cells of each analyzed species/genotype accumulated zinc and particularly lead in very high amounts after treatment with 2000 µM for 72 h (1500-4500 mg kg-1, 24 000-32 000 mg kg-1, respectively), determined by atomic absorption spectrometry. The bioaccumulation factor values confirmed the cells' hyperaccumulation strategy. The cell-activated detoxification mechanism, consisting in deposition of metals in the cell wall and vacuoles, as shown by transmission electron microscopy with X-ray microanalysis, allows the cells to survive despite the high level of metal accumulation. These results indicate innate high tolerance to zinc and lead in violets with different metallophyte statuses and also in the nonmetallophyte, suggesting that evolutionarily developed hypertolerance may occurs in this group as a whole.