A model for extracellular freezing based on observations on Equisetum hyemale.

In frost hardy plants, the lethal intracellular formation of ice crystals has to be prevented during frost periods. Besides the ability for supercooling and pre-frost dehydration of tissues, extracellular ice formation is another strategy to control ice development in tissues. During extracellular ice formation, partially large ice bodies accumulate in intercellular spaces, often at preferred sites which can also be expandable. In this contribution, the physico-chemical processes underlying the water movements towards the sites of extracellular ice formation are studied theoretically, based on observations on the frost hardy horsetail species Equisetum hyemale, with the overall aim to obtain a better understanding of the physical processes involved in extracellular ice formation. In E. hyemale, ice accumulates in the extensive internal canal system. The study focuses on the processes which are triggered in the cellular osmotic-mechanic system by falling, and especially subzero temperatures. It can be shown that when the temperature falls, (1) water flow out of cells is actuated and (2) "stiff-walled" cells lose less water than "soft-walled" cells. Furthermore, (3) cell water loss increases with increasing (= less negative) turgor loss point. These processes are not related to any specific activities of the cell but are solely a consequence of the structure of the cellular osmotic system. On this basis, a directed water flow can be initiated triggered by subzero temperatures. The suggested mechanism may be quite common in frost hardy species with extracellular ice formation.

Morphometric and mechanical characteristics of Equisetum hyemale stem enhance its vibration.

MAIN CONCLUSION: The order of the internodes, and their geometry and mechanical characteristics influence the capability of the Equisetum stem to vibrate, potentially stimulating spore liberation at the optimum stress setting along the stem. Equisetum hyemale L. plants represent a special example of cellular solid construction with mechanical stability achieved by a high second moment of area and relatively high resistance against local buckling. We proposed the hypothesis that the order of E. hyemale L. stem internodes, their geometry and mechanical characteristics influence the capability of the stem to vibrate, stimulating spore liberation at the minimum stress setting value along the stem. An analysis of apex vibration was done based on videos presenting the behavior of an Equisetum clump filmed in a wind tunnel and also as a result of excitation by bending the stem by 20°. We compared these data with the vibrations of stems of the same size but deprived of the three topmost internodes. Also, we created a finite element model (FEM), upon which we have based the 'natural' stem vibration as a copy of the real object, 'random' with reshuffled internodes and 'uniform', created as one tube with the characters averaged from all internodes. The natural internode arrangement influences the frequency and amplitude of the apex vibration, maintaining an equal stress distribution in the stem, which may influence the capability for efficient spore spreading.

Potencial de preparados de cavalinha (Equisetum sp. ) na síntese de metabólitos de defesa em cotilédones de soja (Glycine max L. ) e o efeito sobre o crescimento de Rhizoctonia solani Kuhn, in vitro / Potential of horsetail (Equisetum sp. ) derivatives on the synthesis of defense metabolites using soybean ( Glycine max L. ) cotyledons and their effect on the in vitro growth of Rhizoctonia solani Kuhn

Foram desenvolvidos dois experimentos com objetivo de avaliar o potencial de preparados de cavalinha (Equisetum sp.) na síntese de metabólitos de defesa em cotilédones de soja (Glycinemax L.) e o efeito sobre o crescimento de Rhizoctonia solani, in vitro. O delineamento experimental utilizado para os experimentos foi inteiramente casualizado em esquema fatorial 3x5 (formas de extração x concentrações), com quatro repetições. As formas de extração foram extrato alcoólico, infusão e maceração, nas concentrações de zero; 1; 10, 20 e 40%. No primeiro experimento foi avaliada a indução de compostos de defesa vegetal em cotilédones de soja em resposta aos derivados a base de cavalinha, sendo quantificada a atividade da enzima fenilalanina amônia-liase (FAL), via espectofotometria, a fitoalexina gliceolina, e o teor de fenóis totais. No segundo experimento, in vitro, a unidade experimental foi uma placa de Petri, sendo os preparados de cavalinha incorporados ao meio BDA (Batata-dextrose e Agar) e avaliado o crescimento micelial de R. Solani. Os preparados de extrato alcoólico, infusão e maceração de cavalinha apresentaram capacidade de indução das fitoalexinas gliceolinas em cotilédones de soja, bem como, ativaram o metabolismo de compostos fenólicos. Entre os preparados, o extrato alcoólico e a maceração, se sobressaem sobre a infusão. Os preparados de extrato alcoólico, infusão e maceração de cavalinha em todas as suas concentrações inibem o crescimento do fungo R. solani, in vitro. .

Two experiments were carried out in the Federal Technological University of Paraná - Dois Vizinhos Campus - with the aim to evaluate the potential of horsetail (Equisetum sp.) derivatives for the synthesis of defense metabolites in soybean (Glycine max L.) cotyledons and their effect on the in vitro growth of Rhizoctonia solani. The experimental design was completely randomized in a 3 x 5 factorial design (extraction form x concentration), with four replications. The extraction forms were alcoholic extract, infusion and maceration and the concentrations tested were zero, 1, 10, 20 and 40%. In the first experiment, we evaluated the induction of plant defense in soybean cotyledons as a response to horsetail derivatives through spectrophotometry according to phytoalexin glyceollin, phenylalanine ammonia lyase enzyme activity (PAL) and total phenols. In the second experiment, in vitro, the experimental unit was a Petri dish, and the horsetail derivatives were incorporated into medium culture (potato dextrose agar), and we evaluated the mycelial growth of R. solani. The alcoholic extract, infusion and maceration of horsetail derivatives presented phytoalexin glyceolin induction in soybean cotyledons, in addition to activating the metabolism of phenolic compounds. Among the derivatives, the alcoholic extract and the maceration form of extraction were superior in relation to the infusion. The alcoholic extract, infusion and maceration of horsetail derivatives inhibited the in vitro growth of R. solani in all concentrations.

The walk and jump of Equisetum spores.

Equisetum plants (horsetails) reproduce by producing tiny spherical spores that are typically 50 µm in diameter. The spores have four elaters, which are flexible ribbon-like appendages that are initially wrapped around the main spore body and that deploy upon drying or fold back in humid air. If elaters are believed to help dispersal, the exact mechanism for spore motion remains unclear in the literature. In this manuscript, we present observations of the 'walks' and 'jumps' of Equisetum spores, which are novel types of spore locomotion mechanisms compared to the ones of other spores. Walks are driven by humidity cycles, each cycle inducing a small step in a random direction. The dispersal range from the walk is limited, but the walk provides key steps to either exit the sporangium or to reorient and refold. Jumps occur when the spores suddenly thrust themselves after being tightly folded. They result in a very efficient dispersal: even spores jumping from the ground can catch the wind again, whereas non-jumping spores stay on the ground. The understanding of these movements, which are solely driven by humidity variations, conveys biomimetic inspiration for a new class of self-propelled objects.

On the robustness of the geometrical model for cell wall deposition.

All plant cells are provided with the necessary rigidity to withstand the turgor by an exterior cell wall. This wall is composed of long crystalline cellulose microfibrils embedded in a matrix of other polysaccharides. The cellulose microfibrils are deposited by mobile membrane bound protein complexes in remarkably ordered lamellar textures. The mechanism by which these ordered textures arise, however, is still under debate. The geometrical model for cell wall deposition proposed by Emons and Mulder (Proc. Natl. Acad. Sci. 95, 7215-7219, 1998) provides a detailed approach to the case of cell wall deposition in non-growing cells, where there is no evidence for the direct influence of other cellular components such as microtubules. The model successfully reproduces even the so-called helicoidal wall; the most intricate texture observed. However, a number of simplifying assumptions were made in the original calculations. The present work addresses the issue of the robustness of the model to relaxation of these assumptions, by considering whether the helicoidal solutions survive when three aspects of the model are varied. These are: (i) the shape of the insertion domain, (ii) the distribution of lifetimes of individual CSCs, and (iii) fluctuations and overcrowding. Although details of the solutions do change, we find that in all cases the overall character of the helicoidal solutions is preserved.

[Chemical signaling in plant microspore cells].

Chemical signal transduction from the cell surface to organelles was studied in unicellular vegetative (Equisetum arvense) and generative (Hippeastrum hybridum pollen) microspores of plants. Neurotransmitters acetylcholine, dopamine, and serotonin, their agonists and antagonists, Na+, K+, and Ca2+ channel blockers, as well as forskolin and theophylline (agents increasing the intracellular level of cyclic adenosine monophosphate) were used as chemical signals. Both types of microspores exposed to neurotransmitters, their agonists, forskolin, and theophylline demonstrated growth activation, while neurotransmitter antagonists and ion channel blockers inhibited this process. No stimulating effects of neurotransmitters were observed for cells pretreated with the antagonists and ion channel blockers. Pretreatment with ion channel blockers and then by anticontractile agents (cytochalasin B or colchicine) either had no effect or increased the inhibition of microspore growth. Pathways of chemical signal transduction from the cell surface to organelles are discussed.

[Physiological metabolism and protective enzyme activity of Equisetum ramosissimum under Cu stress].

The study with pot culture experiment showed that Equisetum ramosissirmunm did not appear obvious poisoning symptoms when treated with low concentration Cu (500 mg x kg(-1)), while serious injuries were found when treated with high concentration Cu (1000 to approximately 3000 mg x kg(-1)), which reflected in the severe damage of cell membrane and cytoarchitecture as well as the structure and function of main organelles, and the significant decrease of the contents of leaf chlorophyll a and b and stem soluble monosaccharose. The cell membrane osmolarity and the average MDA content of the plant exposed to heavy copper pollution was 1 to approximately 2 and 1 to approximately 3 times greater than the control, respectively. It could be concluded that high concentration Cu disturbed the physiological metabolism, and critically threatened the normal growth of E. ramosissimum. The activities of protective enzyme, especially of SOD and POD, were enhanced with increasing Cu concentration, and had a positive correlation with Cu concentration (rPOD = 0.978, rSOD = 0.926, P < 0.05).

Specificity patterns indicate that auxin exporters and receptors are the same proteins.

A study of transport and action of synthetic auxin analogues can help to identify transporters and receptors of this plant hormone. Both aspects--transportability and action on growth--were tested with 2-naphthoxyacetic acid (2-NOA) and compared across several plant species. 2-NOA stimulates elongation effectively at low concentrations in petioles of the gymnosperm Ginkgo biloba L., in hypocotyls or internodes of the dicot legumes, mung bean (Vigna mungo L.) and pea (Pisum sativum L.), in cotyledons of onion (Allium cepa L.) and in leaf bases of chive (Allium schoenoprasum L.), the latter two of the monocot order Asparagales. In contrast, elongation of coleoptile segments of maize (Zea mays L.) is poorly responsive to 2-NOA. Significant auxin-like transport of 2-NOA was observed in segments of mung bean hypocotyls, pea internodes, and chive leaf bases, but not in segments of the grass coleoptiles. Thus, for the two assays, elongation and polar transportability, the same difference in ligand specificity was observed between the grass and all other species assayed. This finding supports the hypothesis that a common protein mediates auxin efflux as well as auxin action on elongation.

[Reactive oxygen forms and luminescence of intact microspore cells]. / Aktivnye formy kisloroda i liuminestsentsiia intaktnykh kletok mikrospor.

The participation of reactive oxygen species (ROS) in luminescence (chemiluminescence and autofluorescence induced by ultraviolet light of 360-380 nm) was analyzed. Microspores, the pollen (male gametophyte) of Hippeastrum hybridum, Philadelphus grandiflorus, and Betula verrucosa and vegetative microspores of the spore-breeding plant Equisetum arvense served as models. It was found that the addition of the chemiluminescent probe lucigenin, which luminesces in the presence of superoxide anionradicals, leads to intensive chemiluminescence of microspores. No emission was observed in the absence of lucigenin and in the presence of the dye luminol as a chemiluminescent probe. The emission decreased significantly if superoxide dismutase, an enzyme of the superoxide anionradical dismutation during which this radical disappeared, was added before the dye addition. The autofluorescence intensity of microspores decreased in the presence of both superoxide dismutase and peroxidase, an enzyme destroying hydrogen peroxide and organic peroxides. The most significant effect was noted after the addition of peroxidase, which indicates a greater contribution of peroxides to this type of emission. The fumigation with ozone, which increases the amount of ROS on the cell surface, enhanced the intensity of the chemiluminescence of microspores with lucigenin, but decreased the intensity of the autofluorescence of microspores. Exogenous peroxides (hydrogen peroxide and tert-butylhydroperoxide) stimulated the autofluorescence of pollen and vegetative spores in a concentration-dependent manner. It was shown that the formation of ROS contributes to the luminescence of plant microspores, which reflects their functional state.

[Autofluorescence of intact Equisetum arvense L. spores during their development]. / Avtofluorestsentsiia intaktnykh spor khvoshcha Equisetum arvense L. v protsesse razvitiia.

The autofluorescence of horsetail Equisetum arvense spores excited with UV-light of 360-380 nm was studied by microspectrofluorimetry during their development from an individual cell to the formation of a multicellular thallus with the generative organs. The investigation involved the registration of the fluorescence spectra of individual intact developing cells and the measurement of the ratio of cell fluorescence intensities in the blue and red regions of the spectrum. Dry blue-fluorescing microspores showed the maxima at 460 and 530 nm and a small maximum at 680 nm. Thirty minutes after moistening in water, red-fluorescing cells arose among blue-fluorescing microspores, indicating the onset of development. Red fluorescence with a maximum at 680 nm enhanced as cells put off their cover, which brightly fluoresced in the blue region of the spectrum with the main maximum at 460 nm. By estimating the ratio of autofluorescence intensities in the blue region of the spectrum to red lightening of microspores at the first stages of development up to 24 h (in particular, their first division, the formation of nonfluorescencing rhizoid, etc.), nonviable (only blue-lightening) cells were distinguished from viable cells, in which red fluorescence began to prevail. After 25-40 days of development, the gametophyte fluorescing mainly at 680 nm formed male organs, antheridia, with blue-green-fluorescing spermatozoids. Then female generative organs archegonia with the egg cell appeared, which fluoresced blue, whereas the surrounding cells fluoresced red. It was supposed that the lightening in the blue and green regions of the spectrum is due to the presence of phenols, terpenoids, and azulenes, whereas the emission in the red region is associated with the presence of chlorophyll and azulenes. The observation of autofluorescence makes it possible to easily distinguish generative cells without additional staining.