Cell shape can be uncoupled from formononetin induction in a novel cell line from Callerya speciosa.

KEY MESSAGE: It is the first time that formononetin produced by cell culture and its accumulation was shown to be triggered by specific stress signalling linked jasmonate pathway. Callerya speciosa, an endangered traditional Chinese medicine plant, is intensively used in traditional folk medicine. To develop sustainable alternatives for the overexploitation of natural resources, a suspension cell line was created from C. speciosa. Ingredients of C. speciosa, for instance the isoflavone formononetin, are formed during a peculiar swelling response of the root, which is considered as a quality trait for commercial application. A cell strain with elongated cells was obtained by using synthetic cytokinin 6-benzylaminopurine (6-BA) and synthetic auxin picloram. Both, picloram and 6-BA, promote cell division, whereas picloram was shown to be crucial for the maintenance of axial cell expansion. We addressed the question, whether the loss of axiality observed in the maturating root is necessary and sufficient for the accumulation of formononetin. While we were able to mimic a loss of axiality for cell expansion, either by specific combinations of 6-BA and picloram, or by treatment with the anti-microtubular compound oryzalin, formononetin was not detectable. However, formononetin could be induced by the stress hormone methyl jasmonate (MeJA), as well as by the bacterial elicitor flagellin peptide (flg22), but not by a necrosis inducing protein. Combined the fact that none of these treatments induced the loss of axiality, we conclude that formononetin accumulates in response to basal defence and unrelated with cell swelling.

Determination of Botanical Origin of Propolis from Monte Region of Argentina by Histological and Chemical Methods.

Propolis production by honey bees is the result of a selective harvest of exudates from plants in the neighborhood of the hive. This product is used in Argentina as a food supplement and alternative medicine. The aim of this study was to determine the botanical origin of propolis from the arid regions of Monte of Argentina using rapid histochemical techniques and by comparison of TLC and HPLC-DAD chromatographic profiles with extract profiles obtained from Zuccagnia punctata, Larrea divaricata and Larrea cuneifolia, plant species that grow in the study area as a natural community named "jarillal". Microscopical analysis revealed the presence of several Z. punctata structures, such as multicellular trichomes, leaflets, stems and young leaves. Remarkable was the richness of the propolis in two bioactive chalcones, also present in Z. punctata resin; these compounds can be regarded as possible markers for propolis identification and justify its use as a dietary supplement, functional food and medicinal product. This study indicates that the source of resin used by honey bees to produce propolis in the Monte region of Argentina is only Z. punctata, a native shrub widespread in this phytogeographical region, while other more abundant species (L. divaricata and L. cuneifolia) in the region were not found, indicating that this propolis could be defined as a mono-resin, type-Zuccagnia.

[Clonal micropropagation of a rare species Hedysarum theinum Krasnob (Fabaceae) and assessment of the genetic stability of regenerated plants using ISSR markers].

In the present study, a protocol was developed for the in vitro propagation of a rare medicinal plant, Hedysarum theinum (tea sweetvetch), from axillary buds, and identification of the regenerants was performed with the use of ISSR markers. It was demonstrated that Gamborg and Eveleigh medium supplemented with 5 µM 6-benzylaminopurine was the best for H. theinum for initial multiplication. On the other hand, half-strength Murashige and Skoog (MS) basal medium supplemented with 7 µM α-naphthaleneacetic acid proved to be the best for explant rooting. Molecular genetic analysis of the H. theinum mother plants and the obtained regenerants was performed with six ISSR markers. Depending on the primer, four to ten amplified fragments with sizes ranging from 250 to 3000 bp were identified. Our results confirmed the genetic stability of regenerants obtained in five passages and their identity to the mother plant.

Phytoremediation of heavy and transition metals aided by legume-rhizobia symbiosis.

Legumes are important for nitrogen cycling in the environment and agriculture due to the ability of nitrogen fixation by rhizobia. In this review, we introduce an important and potential role of legume-rhizobia symbiosis in aiding phytoremediation of some metal contaminated soils as various legumes have been found to be the dominant plant species in metal contaminated areas. Resistant rhizobia used for phytoremediation could act on metals directly by chelation, precipitation, transformation, biosorption and accumulation. Moreover, the plant growth promoting (PGP) traits of rhizobia including nitrogen fixation, phosphorus solubilization, phytohormone synthesis, siderophore release, and production of ACC deaminase and the volatile compounds of acetoin and 2, 3-butanediol may facilitate legume growth while lessening metal toxicity. The benefits of using legumes inoculated with naturally resistant rhizobia or recombinant rhizobia with enhanced resistance, as well as co-inoculation with other plant growth promoting bacteria (PGPB) are discussed. However, the legume-rhizobia symbiosis appears to be sensitive to metals, and the effect of metal toxicity on the interaction between legumes and rhizobia is not clear. Therefore, to obtain the maximum benefits from legumes assisted by rhizobia for phytoremediation of metals, it is critical to have a good understanding of interactions between PGP traits, the symbiotic plant-rhizobia relationship and metals.

Using the pollen viability and morphology for fluoride pollution biomonitoring.

The methods using plants for biomonitoring of air and soil quality are simple, cheap, and fast and can supplement the classical physicochemical methods. In this study, biological pollen characterization of some collected legume species from an aluminum smelter area in Iran (IRALCO) was carried out to determine the actual value of pollen as a bioindicator of the effects of soil and atmospheric pollution. Young buds and flowers of six legumes (Cercis siliquastrum L., Medicago sativa L., Robinia pseudoacacia L., Melilotus officinalis (L.) lam, Trifolium repens L., and Sophora alopecuroides L.) in polluted and control plants were removed and compared. Studies of light and electron microscopic preparation showed some abnormalities during pollen development in affect of fluoride pollution. The viability of pollen grains estimated by staining with acetocarmine shows sharp differences in smearing advanced pollen grains from abnormal ones. Except M. officinalis, the pollen grains of C. siliquastrum, M. sativa, R. pseudoacacia, T. repens, and S. alopecuroides in polluted areas showed light, partial, or no staining with acetocarmine, whereas almost all of the control ones clearly stained. Observation of the pollen grains by light microscopy and scanning electron microscopy showed the significant effect of fluoride on shapes and sizes of pollen grains. The stimulation and inhibition of these pollen characteristics depend on the pollen species as well as on the pollutant and its concentration. Therefore, pollen grains provide essential information on biological impact of pollutants and they are good candidates for biomonitoring the atmospheric and edaphic pollutions.

Pyrazinecarboxamides as potential elicitors of flavonolignan and flavonoid production in Silybum marianum and Ononis arvensis cultures in vitro.

The effect of new synthetic pyrazinecarboxamide derivatives as potential elicitors of flavonolignan and flavonoid production in Silybum marianum and Ononis arvensis cultures in vitro was investigated. Both tested elicitors increased the production of flavonolignans in S. marianum callus and suspension cultures and flavonoids in O. arvensis callus and suspension cultures. Compound I, 5-(2-hydroxybenzoyl)-pyrazine-2-carboxamide, has shown to be an effective elicitor of flavonolignans and taxifoline production in Silybum marianum culture in vitro. The maximum content of silydianin (0.11%) in S. marianum suspension culture was induced by 24 h elicitor application in concentration of 1.159 × 10⁻³ mol/L. The maximum content of silymarin complex (0.08%) in callus culture of S. marianum was induced by 168 h elicitor application of a concentration 1.159 × 10⁻4 mol/L, which represents contents of silydianin (0.03%), silychristin (0.01%) and isosilybin A (0.04%) compared with control. All three tested concentrations of compound II, N-(2-bromo-3-methylphenyl)-5-tert-butylpyrazin-2-carboxamide increased the flavonoid production in callus culture of O. arvensis in a statistically significant way. The best elicitation effect of all elicitor concentrations had the weakest c3 concentration (8.36 × 10⁻6 mol/L) after 168 h time of duration. The maximum content of flavonoids (about 5,900%) in suspension culture of O. arvensis was induced by 48 h application of c3 concentration (8.36 × 10⁻6 mol/L).

Abiotic stress enhances androgenesis from isolated microspores of some legume species (Fabaceae).

To induce androgenesis in field pea, grass pea and the model legume species Medicago truncatula, isolated microspores of various genotypes of these three species were submitted to a range of abiotic stresses prior to and during their initial culture, in order to stimulate them to divide and form embryos. Some stress agents had a positive effect on androgenesis from the treated microspores. Submission of flower buds to a cold period prior to anther excision or microspore isolation, modifying the osmotic pressure of the medium during initial culture and electroporation of isolated microspores were the three major individual stress agents to have an impact on the efficiency of androgenetic proliferation and subsequent differentiation from the microspores of pea, grass pea and M. truncatula genotypes. A combination of osmotic and electric shocks significantly improved responses from isolated microspores and yielded microcalluses and then calluses, but only few underwent morphogenesis. Further work is under way to improve responses and extend them to other genotypes. The results reported here are, to the best of our knowledge, the first successful results from isolated microspores of these species.

[Study on pharmacognostic identifcation of Cassia siamea].

OBJECTIVE: To establish methods for identification of Dai medicine, the heartwoods and leaves of Cassia siamea. METHODS: Macroscopic, microscopic observation and TLC technique were used to anthenticate this crude drug, and the identification characteristics were studied. RESULTS: Macroscopic and microscopic identification methods and TLC characters of the ethnomedicine were re-ported, and the simple and detailed drawings of the transections of the heartwoods and leaves, as well as microscopic drawings of their powders were drawn in this paper. CONCLUSION: The results can serve as evidence for identification of the ethnomedicine in the utilization.

[Pharmacognostic studies on Desmodium gyrans].

OBJECTIVE: To study the characteristic features of Desmodium gyrans in order to provide a basis for rational exploitation and utilization of the herb. METHOD: Samples of the title plant were collected, the microscopic features of cross sections and powders were studied. TLC profiles and UV absorption of the plant extract were examined. RESULT: Calcium oxalate crystals were found in cells of transverse sections. Nonglandular hairs were observed on leaf surfaces. Characteristic peaks in the UV spectrum were identified. CONCLUSION: The distinct characteristic features revealed in this studies can serve as evidence for the identification of D. gyrans.

Characterization of DNA end-binding activities in higher plants.

DNA double-strand-breaks (DSB) are the most severe lesion in cells exposing to ionizing radiation and many other stress environments. Repair of DNA DSB is therefore critical to cellular survival. In this work, we observed the double-stranded DNA end-binding (DEB) like activities in rice (Oryza sativa L. cv. TN5) suspension cells and hypocotyls from etiolated mung bean (Vigna radiata L. TN5) seedlings. Higher plant DEB-like protein binds primarily to linearized double-stranded DNA ends. Competition of unlabeled probe was examined in double-stranded DEB assay of cell extracts from rice and mung bean. DEB-like activities of higher plants did not depend on sequence and types of double-stranded DNA ends. Distinct electrophoretic mobility shift patterns and binding features further indicate that DEB-like factors from various sources might not share identical structure and function, and probably belong to different types of DEB proteins from higher plants. Our evidence suggests that DEB proteins are certainly ubiquitous in all organisms probably for repairing and processing double-stranded DNA breaks from formidable lethal lesion.

Primary sites of ozone-induced perturbations of photosynthesis in leaves: identification and characterization in Phaseolus vulgaris using high resolution chlorophyll fluorescence imaging.

High resolution imaging of chlorophyll a fluorescence was used to identify the sites at which ozone initially induces perturbations of photosynthesis in leaves of Phaseolus vulgaris. Leaves were exposed to 250 and 500 nmol mol(-1) ozone at a photosynthetically active photon flux density of 300 micromol m(-2) s(-1) for 3 h. Images of fluorescence parameters indicated that large decreases in both the maximum and operating quantum efficiencies of photosystem II had occurred in cells adjacent to stomata in the upper, but not lower, leaf surfaces. However, this treatment did not produce any significant changes in the maximum or operating quantum efficiencies of photosystem II in the leaves when estimated from fluorescence parameters measured with a conventional, integrating fluorometer. The localized decreases in photosystem II photochemical efficiencies were accompanied by an increase in the minimal fluorescence level, which is indicative of photoinactivation of photosystem II complexes and a decrease in stomatal conductance. Perturbations of photochemical efficiencies were not observed in cells associated with all of the stomata on the upper leaf surface or within cells distant from the upper leaf surface. It is concluded that ozone penetrates the leaf through stomata and initially damages only cells close to stomatal pores.

The shoot apical meristem restores its symplasmic organization during chilling-induced release from dormancy.

The shoot apex of overwintering perennials ceases its morphogenetic activity at the end of the growing season and transforms into a bud which is dormant and freezing-tolerant. In birch (Betula pubescens) these events are triggered by short photoperiod, and involve the production of 1,3-beta-D-glucan containing sphincters on the plasmodesmata. As a result, all symplasmic pathways shut down. Here we show that breakage of bud dormancy by chilling involves restoration of the symplasmic organization of the meristem. This restoration is likely to be mediated by 1,3-beta-D-glucanase, which was present in small spherosome-like vacuoles that arose de novo during dormancy induction. During chilling these vacuoles were displaced from the bulk cytoplasm to the cortical cytoplasm where they became aligned with the plasma membrane, often associated with plasmodesmata. At this stage the enzyme also appeared outside the vacuoles. During chilling, 1,3-beta-D-glucan disappeared from the plasmodesmal channels and wall sleeves, and the plasmodesmata regained the capacity for cell-cell transport, as demonstrated by microinjection of Lucifer Yellow CH and Fluorescein-tagged gibberellic acid. Collectively, the present experiments demonstrate that restoration of the symplasmic organization of the meristem is indispensable for the release of buds from dormancy and the assumption of a proliferation-competent state, and implicate 1,3-beta-D-glucanase action at the plasmodesmata. Based on these findings we propose a model for 'dormancy cycling' which depicts the meristem as passing through three sequential states of cellular communication with characteristic sensitivities to distinct environmental cues.

Transmembrane topography of plasma membrane constituents in mung bean (Vigna radiata L.) hypocotyl cells. I. Transmembrane distribution of phospholipids.

The transmembrane distribution of phospholipids (PLs) in the plasma membrane (PM) of mung bean (Vigna radiata L.) hypocotyl cells was investigated using annexin V-fluorescein isothiocyanate, porcine pancreas phospholipase A(2), and (31)P-nuclear magnetic resonance (NMR) spectroscopy. Phosphatidylserine was not located on the cell surface of mung bean protoplasts. However, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid were found to be almost symmetrically distributed across right-side-out PM vesicles obtained by aqueous two-phase partitioning by porcine pancreas phospholipase A(2) assay. (31)P-NMR assay showed that the amount of PLs is about equal in the outer and the inner leaflets of the right-side-out PM vesicles. These results suggest that the topography of PM PLs might not contribute to well-known asymmetrical properties of the outer and inner surfaces of higher plant PMs. It is also indicated that inside-out PM vesicles created by Brij 58-treatment do not retain the native PL topography on dithionate reduction of 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled PLs incorporated in the PM vesicles.

Hydrogen peroxide-induced changes in intracellular pH of guard cells precede stomatal closure.

Epidermal bioassay demonstrated that benzylamine, a membrane-permeable weak base, can mimick hydrogen peroxide (H2O2) to induce stomatal closure, and butyric acid, a membrane-permeable weak acid, can partly abolish the H2O2-induced stomatal closure. Confocal pH mapping with the probe 5-(and-6)-carboxy seminaphthorhodafluor-1-acetoxymethylester (SNARF-1-AM) revealed that H2O2 leads to rapid changes in cytoplasmic and vacuolar pH in guard cells of Vicia faba L, i. e. alkalinization of cytoplasmic areas occur red in parallel with a decrease of the vacuolar pH, and that butyric acid pretreatment can abolish alkalinization of cytoplasmic areas and acidification of vacuolar areas of guard cells challenged with H2O2. These results imply that the alkalinization of cytoplasm via efflux of cytosol protons into the vacuole in guard cells challenged with H2O2 is important at an early stage in the signal cascade leading to stomatal closure.

Early elicitor-induced events in chickpea cells: functional links between oxidative burst, sequential occurrence of extracellular alkalinisation and acidification, K+/H+ exchange and defence-related gene activation.

Elicitation of cultured chickpea (Cicer arietinum L.) cells stimulates a signal transduction pathway leading to several rapid responses: (1) oxidative burst, (2) extracellular alkalinisation, (3) extracellular acidification, (4) transient K+ efflux, and (5) activation of defence related genes all within 2 hours. Induced genes are encoding acidic and basic chitinases, a thaumatin-like protein and isoflavone reductase. All these elicitor-induced responses are inhibited by the Ser/Thr protein kinase inhibitor staurosporine and the anion channel blocker anthracene-9-carboxylic acid but stimulated by the Ser/Thr protein phosphatase 2A inhibitor cantharidin. The oxidative burst leads to a transient extracellular H2O2 accumulation which seems to be preceded by O2- production, indicating dismutation of O2- to H2O2. The oxidative burst is accompanied by transient alkalinisation of the culture medium which is followed by long-lasting extracellular acidification. An 80 percent inhibition of the alkalinisation after complete inhibition of the H2O2 burst with diphenylene iodonium indicates that the elicitor induced increase of extracellular pH is mainly based on a proton consumption for O2-dismutation. A simultaneous deactivation of the plasma membrane H+-ATPase during oxidative burst and extracellular alkalinisation is also suggested. The elicitor-stimulated extracellular acidification is inhibited by the plasma membrane H+-ATPase inhibitor N, N'-dicyclohexylcarbodiimide assuming a reactivation of the H+-ATPase 25 min after elicitation. Extracellular acidification seems not to be necessary for elicitor-induced activation of defence related genes. Opposite modulation of K+ and proton fluxes after elicitation and/or treatment with the H+-ATPase effectors fusicoccin or N, N'-dicyclohexylcarbodiimide indicate that the elicitor induced transient K+ efflux is regulated by a K+/H+ exchange reaction.

Guard cell volume and pressure measured concurrently by confocal microscopy and the cell pressure probe.

Guard cell turgor pressures in epidermal peels of broad bean (Vicia faba) were measured and controlled with a pressure probe. At the same time, images of the guard cell were acquired using confocal microscopy. To obtain a clear image of guard cell volume, a fluorescent dye that labels the plasma membrane was added to the solution bathing the epidermal peel. At each pressure, 17 to 20 optical sections (each 2 microm thick) were acquired. Out-of-focus light in these images was removed using blind deconvolution, and volume was estimated using direct linear integration. As pressure was increased from as low as 0.3 MPa to as high as 5.0 MPa, guard cell volume increased in a saturating fashion. The elastic modulus was calculated from these data and was found to range from approximately 2 to 40 MPa. The data allow inference of guard cell osmotic content from stomatal aperture and facilitate accurate mechanistic modeling of epidermal water relations and stomatal functioning.

Possible role of root border cells in detection and avoidance of aluminum toxicity.

Root border cells are living cells that surround root apices of most plant species and are involved in production of root exudates. We tested predictions of the hypothesis that they participate in detection and avoidance of aluminum (Al) toxicity by comparing responses of two snapbean (Phaseolus vulgaris) cultivars (cv Dade and cv Romano) known to differ in Al resistance at the whole-root level. Root border cells of these cultivars were killed by excess Al in agarose gels or in simple salt solutions. Percent viability of Al-sensitive cv Romano border cells exposed in situ for 96 h to 200 microM total Al in an agarose gel was significantly less than that of cv Dade border cells; similarly, relative viability of harvested cv Romano border cells was significantly less than that of cv Dade cells after 24 h in 25 microM total Al in a simple salt solution. These results indicate that Al-resistance mechanisms that operate at the level of whole roots also operate at the cellular level in border cells. Al induced a thicker mucilage layer around detached border cells of both cultivars. Cultivar Dade border cells produced a thicker mucilage layer in response to 25 microM Al compared with that of cv Romano cells after 8 h of treatment and this phenomenon preceded that of observed cultivar differences in relative cell viability. Release of an Al-binding mucilage by border cells could play a role in protecting root tips from Al-induced cellular damage.

Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules.

Biological nitrogen fixation involves the reduction of atmospheric N2 to ammonia by the bacterial enzyme nitrogenase. In legume-rhizobium symbioses, the nitrogenase-producing bacteria (bacteroids) are contained in the infected cells of root nodules within which they are enclosed by a plant membrane to form a structure known as the symbiosome. The plant provides reduced carbon to the bacteroids in exchange for fixed nitrogen, which is exported to the rest of the plant. This exchange is controlled by plant-synthesised transport proteins on the symbiosome membranes. This review summarises our current understanding of these transport processes, focusing on ammonia and amino acid transport.

Recovery of gravitropic response during regeneration of root caps does not require developed columella cells and sedimentation of amyloplasts.

Correlations between regeneration of the root cap and recovery of a gravitropic response were studied using primary roots of Phaseolus vulgaris. After removal of various lengths of the root tip a gravistimulus was continuously given to the root. The statistical analysis of data showed that recovery of the gravitropic response was gradually delayed as the length of the tips removed increased. This suggested that the columella cells of the root cap were involved in gravitropism. When the root cap was completely removed, the roots did not respond to gravistimuli for the first 15 h and began to reorient their growth direction at 20 h. At this time, the columella cells had just begun to regenerate and had immature amyloplasts which did not sufficiently form a sediment. These results suggest that other systems of perception exist in plant cells in addition to the amyloplast-based model of graviperception.

Identification of a membrane-associated cysteine protease with possible dual roles in the endoplasmic reticulum and protein storage vacuole.

SH-EP is a vacuolar cysteine proteinase from germinated seeds of Vigna mungo. The enzyme has a C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum (ER) retention signal, KDEL. The KDEL-tail has been suggested to function to store SH-EP as a transient zymogen in the lumen of the ER, and the C-terminal propeptide was thought to be removed within the ER or immediately after exit from the ER. In the present study, a protease that may be involved in the post-translational processing of the C-terminal propeptide of SH-EP was isolated from the microsomes of cotyledons of V. muno seedlings. cDNA sequence for the protease indicated that the enzyme is a member of the papain superfamily. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form. In addition, protein fractionations of the cotyledonary microsome and Sf9 cells expressing the recombinant protease indicated that the enzyme associates with the microsomal membrane on the luminal side. The protease was named membrane-associated cysteine protease, MCP. The possibility that a papain-type enzyme, MCP, exists as mature enzyme in both ER and protein storage vacuoles will be discussed.