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The cytochemical localization of nucleotide pyrophosphatase activity in plant tissues using naphthyl esters of thymidine-5'-phosphate.

A cytochemical method for the localization of nucleotide pyrophosphatase activity in plants employing naphthol AS-BI thymidine 5'-monophosphate and alpha-naphthyl thymidine 5'-monophosphate as specific substrates is reported. Biochemical evidence for the validity of this method is presented and the synthesis of the naphthol AS-BI ester is described. The application of this cytochemical technique to shoots of Triticum sp. and roots of Vicia faba has shown nucleotide pyrophosphatase to be ubiquitous in its distribution in these organs and to occur in a structurally-bound form in the cytoplasm. The highest activity was detected in developing fibres adjacent to the leaf vascular bundles, in the coleoptile epidermal and hypodermal cells and in the coleoptile and leaf xylem.

Chemical Phenomena↗

On the alignment of cellulose microfibrils by cortical microtubules: a review and a model.

The hypothesis that microtubules align microfibrils, termed the alignment hypothesis, states that there is a causal link between the orientation of cortical microtubules and the orientation of nascent microfibrils. I have assessed the generality of this hypothesis by reviewing what is known about the relation between microtubules and microfibrils in a wide group of examples: in algae of the family Characeae, Closterium acerosum, Oocystis solitaria, and certain genera of green coenocytes and in land plant tip-growing cells, xylem, diffusely growing cells, and protoplasts. The salient features about microfibril alignment to emerge are as follows. Cellulose microfibrils can be aligned by cortical microtubules, thus supporting the alignment hypothesis. Alignment of microfibrils can occur independently of microtubules, showing that an alternative to the alignment hypothesis must exist. Microfibril organization is often random, suggesting that self-assembly is insufficient. Microfibril organization differs on different faces of the same cell, suggesting that microfibrils are aligned locally, not with respect to the entire cell. Nascent microfibrils appear to associate tightly with the plasma membrane. To account for these observations, I present a model that posits alignment to be mediated through binding the nascent microfibril. The model, termed templated incorporation, postulates that the nascent microfibril is incorporated into the cell wall by binding to a scaffold that is oriented; further, the scaffold is built and oriented around either already incorporated microfibrils or plasma membrane proteins, or both. The role of cortical microtubules is to bind and orient components of the scaffold at the plasma membrane. In this way, spatial information to align the microfibrils may come from either the cell wall or the cell interior, and microfibril alignment with and without microtubules are subsets of a single mechanism.

Cell Polarity↗

Tissue print-immunoblotting reveals an uneven distribution of beet necrotic yellow vein and beet soil-borne viruses in sugarbeets.

An uneven distribution of the coat protein antigens of beet necrotic yellow vein (BNYVV) and beet soil-borne (BSBV) viruses in tap roots of naturally infected sugarbeets and of BNYVV coat protein antigen in leaves and petioles of mechanically inoculated sugarbeet seedlings was detected by means of tissue print-immunoblotting. BNYVV antigen-containing areas in the tap roots were usually found underneath a root beard. Occasionally BNYVV antigen was detected predominantly, but not exclusively in the xylem vessels.

Antigens, Viral↗

Properties of a previously undescribed supercoiled filamentous virus infecting papaya in Venezuela.

Supercoiled filamentous virus particles with lengths of 400 to 700 nm and 3 nm wide were isolated from leaves of Carica papaya L. plants showing a mild yellowing between the veins. The morphological properties of this virus resemble those of tenuiviruses. However, it was serologically unrelated to three of the five definitive members of this group of plant viruses and had biochemical features quite different from tenuiviruses. Therefore, the virus described here is possible an unreported new virus infecting papaya for which the name of papaya mild yellow leaf virus (PMYLV) is proposed. PMYLV was mechanically transmitted to papaya and to several Cucurbitaceae species. Virus particles sedimented as one component in sucrose density gradients, containing one molecule of ssRNA with an apparent size of 6400 nucleotides which constitutes 5% of the particle weight. The buoyant density of PMYLV was 1.26 g/cm3 in cesium chloride equilibrium gradients, and the virus coat protein consisted of a single polypeptide with mol. wt. of c. 39 kDa. Estimated virus yield in purified preparations was 2.6 g/kg leaf tissues. An antiserum was produced with a titer of 1:1500. Ultrastructural observations of PMYLV-infected leaf tissues showed crystalline aggregates of virus particles, closely associated with electron dense amorphous inclusion bodies only within xylem cells.

Animals↗

Mineral composition of some traditional Mexican teas.

Teas of plant origin traditionally consumed by the Mountain Pima of Chihuahua, Mexico, were analyzed for mineral nutritional content. Fe, Cu, Zn, Ca, and Mg composition was determined for native teas made from shoots of Tagetes lucida, T. filifolia, Elytraria imbricata, and Holodiscus dumosus, and from root xylem of Ceanothus depressus and Phaseolus ritensis. Native uses of these teas are also described.

Beverages↗

Localization of cell wall polysaccharides in nonarticulated laticifers of Asclepias speciosa Torr.

Asclepias speciosa Torr. has latex-containing cells known as nonarticulated laticifers. In stem sections of this species, we have analyzed the cell walls of nonarticulated laticifers and surrounding cells with various stains, lectins, and monoclonal antibodies. These analyses revealed that laticifer walls are rich in (1-->4) beta-D-glucans and pectin polymers. Immunolocalization of pectic epitopes with the antihomogalacturonan antibodies JIM5 and JIM7 produced distinct labeling patterns. JIM7 labeled all cells including laticifers, while JIM5 only labeled mature epidermal cells and xylem elements. Two antibodies, LM5 and LM6, which recognize rhamnogalacturonan I epitopes distinctly labeled laticifer walls. LM6, which binds to a (1-->5) alpha-arabinan epitope, labeled laticifer walls more intensely than walls of other cells. LM5, which recognizes a (1-->4) beta-D-galactan epitope, did not label laticifer segments at the shoot apex but labeled more mature portions of laticifers. Also the LM5 antibody did not label cells at the shoot apical meristem, but as cells grew and matured the LM5 epitope was expressed in all cells. LM2, a monoclonal antibody that binds to beta-D-glucuronic acid residues in arabinogalactan proteins, did not label laticifers but specifically labeled sieve tubes. Sieve tubes were also specifically labeled by Ricinus communis agglutinin, a lectin that binds to terminal beta-D-galactosyl residues. Taken together, the analyses conducted showed that laticifer walls have distinctive cytochemical properties and that these properties change along the length of laticifers. In addition, this study revealed differences in the expression of pectin and arabinogalactan protein epitopes during shoot development or among different cell types.

Apocynaceae↗

Structure and function of metal chelators produced by plants: the case for organic acids, amino acids, phytin, and metallothioneins.

Plants produce a range of ligands for cadmium (Cd), copper (Cu), nickel (Ni), and zinc (Zn). Cd- and Zn-citrate complexes are prevalent in leaves, even though malate is more abundant. In the xylem sap moving from roots to leaves, citrate and histidine are the principal ligands for Cu, Ni, and Zn. Phosphorus-rich globular bodies in young roots are probably Zn-phytate. Metallothioneins (MTs) are cysteine (Cys)-rich ligands. Plants produce class II MTs (MT-IIs) which differ from the archetypal mammalian MT-I in the location and number of Cys. The Ec protein from wheat embryos has Cys in three domains, binds Zn, and disappears with seedling development. The first 59 amino acids have been sequenced for the protein. Fifty-eight genes for MT-IIs, from a range of plants and tissues, predict proteins with Cys in two domains. Most of the predicted proteins have not been isolated, and their metal binding is poorly documented. Three protein bands, corresponding to six MT genes, have been isolated from Arabidopsis, and the amino acids sequenced for nine fragments. The MT-IIIs are atypical, nontranslationally synthesized polypeptides with variously repeating gamma-glutamylcysteine units. Of the five families known, those with carboxy-terminal glycine are the most widespread among plants, algae, and certain yeasts. A heterogeneous grouping of these molecules form Cd-binding complexes with tetrahedral coordination and a Cd-sulfur interatomic distance of 2.52 A. One complex is cytosolic, the dominant one is vacuolar. Together, they can bind a large proportion of cellular Cd; other ligands may also function. Little is known about the counterpart situation for Cu and Zn.

Cadmium↗

Detection of cross-reactive antigens shared by Fusarium oxysporum and Glycine max by indirect ELISA and their cellular location in root tissues.

Pathogenicity test of Fusarium oxysporum on ten cultivars of soybean revealed Soymax and Punjab-1 to be most resistant while JS-2 and UPSM-19 were most susceptible. Antigens were prepared from the roots of all the ten varieties of soybean and the mycelium of F. oxysporum. Polyclonal antisera were raised against the mycelial suspension of F. oxysporum and the root antigen of the susceptible cultivar UPSM-19. Cross reactive antigens shared by the host and the pathogen were detected first by immunodiffusion. The immunoglobulin fraction of the antiserum was purified by ammonium sulfate precipitation and DEAE-Sephadex column chromatography. The immunoglobulin fractions were used for detection of cross-reactive antigens by enzyme-linked immunosorbent assay. In enzyme-linked immunosorbent assay, antigens of susceptible cultivars showed higher absorbance values when tested against the purified anti-F. oxysporum antiserum. Antiserum produced against UPSM-19 showed cross-reactivity with the antigens of other cultivars. Indirect staining of antibodies using fluorescein isothiocyanate indicated that in cross-sections of roots of susceptible cultivar (UPSM-19) cross-reactive antigens were concentrated around xylem elements, endodermis and epidermal cells, while in the resistant variety, fluorescence was concentrated mainly around epidermal cells and distributed in the cortical tissues. CRAs were also present in microconidia, macroconidia and chlamydospores of the fungus.

Antigens↗

Uptake of tributyltin into willow trees.

BACKGROUND: Organotins have been used world-wide as antifoulants in ship paints. Repeatedly, severe effects on aquatic species have resulted. The use of organotins for this purpose was ruled out, and dumping of contaminated harbor sludge into the sea was prohibited. Land-based dumping is seen as an alternative. OBJECTIVE: This study investigates sorption, uptake and translocation of tributyltin (TBT) to willow trees in order to evaluate phytoremediation as treatment option. The study considers the influence of pH on the plant uptake of organotins. EXPERIMENTAL SET-UP: Chemicals investigated were the weak base tributyltin chloride (TBTCl) and the neutral tributyltin hydride (TBTH). Organotins were extracted from solution and plant material with toluene, and analyzed as tin by AAS with graphite oven. The pH in solution varied from pH 4 to pH 7. The sorption to living and dead roots, stems and leaves was measured in shaking experiments. The uptake into intact trees was measured at nominal levels of 1 and 10 mg TBT/l for TBTH and TBTCl at low and high pH. RESULTS: The sorption to roots and leaves dropped for dead tissue, but did not vary much with pH. The sorption to stems increased for dead stems and with pH. The solubility of TBTCl in water was below 10 mg/l and lowest at pH 4. Concentrations of TBTCl and TBTH in solutions with trees dropped rapidly to low values. Highest TBT contents in trees were found in roots and lower stems. The concentrations followed the concentrations in solution. The pH had only a small effect on the plant uptake of TBTCl, and no effect on the uptake of TBTH. No effective translocation to higher stems or leaves was found. DISCUSSION: An ion trap mechanism that accumulates the weak base TBTCl in the xylem sap of plants and leads to upward translocation could not be detected. Neither TBTCl at low or high pH, nor the neutral lipophilic chemical TBTH, were translocated effectively to leaves. The TBT+ cation sorbed strongly to plant tissue. The exact mechanism for the strong sorption of the cation is unknown, but similar effects have been observed for algae, liposomes and isolated biomembranes. CONCLUSIONS: Both the uptake of the neutral TBTH and the uptake of the neutral molecule form of TBTCl into willows was as is to be expected from theory. The cation TBT+ showed an unexpected behavior which has been observed before. No ion trap occurs, and the phytoextraction of TBT is not feasible. OUTLOOK: Planting trees, or other appropriate vegetation, could have a beneficial remediation effect by aeration of the TBT-contaminated soil or sludge. In a follow-up paper, the toxicity of TBT to willow trees will be described.

Biodegradation, Environmental↗

Hybrid Weakness in Phaseolus vulgaris L. II. Disruption of Root-Shoot Integration.

We have been examining the importance of the root system on shoot growth and development using a developmentally disabled hybrid of the common bean Phaseolus vulgaris L. Parental cultivars (P. Vulgaris cv. Redkloud of Mesoamerican origin, and P. vulgaris cv. Batt of Andean origin) grow normally, but crosses produce F1 hybrids exhibiting hybrid weakness associated with reduced root and shoot growth. In this study, applications of benzylaminopurine (BAP) to roots of F1 hybrids increased the number of root tips and leaves. Reciprocal grafting was used to study the effects of the root system on shoots. Grafting of roots of the Mesoamerican cultivar onto shoots of F1 hybrids increased the cytokinin concentrations in leaves of F1 hybrids and removed the characteristics associated with hybrid weakness. To determine whether factors in the xylem sap enhanced leaf growth, leaf discs were incubated on sap collected from Mesoamerican and Andean cultivars. Sap from Mesoamerican plants enhanced the growth of leaf discs excised from F1 hybrids more than sap collected from Andean cultivars. Estimates of the transport of zeatin riboside (ZR)-type cytokinins from roots of F1 hybrids indicated that transport out of hybrid roots was reduced compared with those transported out of Mesoamerican or Andean roots. Results suggest that ZR-type cytokinins are involved in hormonal integration between roots and shoots of P. vulgaris and that one of the barriers to hybridization between Andean and Mesoamerican landraces is related to hormone transport.

Journal Article↗

New Plant Growth Regulators Protect Photosynthesis and Enhance Growth Under Drought of Jack Pine Seedlings.

To determine whether natural plant growth regulators (PGRs) can enhance drought tolerance and the competitive ability of transplanted seedlings, 1.5-year-old jack pine (Pinus banksana Lamb.) seedlings were treated with homobrassinolide, salicylic acid, and two polyamines, spermine and spermidine, triacontanol, abscisic acid (ABA), and the synthetic antioxidant, Ambiol. PGRs were fed into the xylem for 7 days and plants were droughted by withholding water for 12 days. ABA, Ambiol, spermidine, and spermine at a concentration of 10 µg L(-1) stimulated elongation growth under drought, whereas ABA, Ambiol, and spermidine maintained higher photosynthetic rates, higher water use efficiency, and lower Ci/Ca ratio under drought compared with control plants. The damaging effects of drought on membrane leakage was reversed by Ambiol, ABA, triacontanol, spermidine, and spermine. Because ABA, Ambiol, and both polyamines enhanced elongation growth and also reduced membrane damage in jack pine under drought, they show promise as treatments to harden seedlings against environmental stress. The protective action of these compounds on membrane integrity was associated with an inhibition of ethylene evolution, with a reduction in transpiration rate and an enhancement of photosynthesis, which together increased water use efficiency under drought. Although most of the tested compounds acted as antitranspirants, the inhibition in membrane leakage in ABA-, Ambiol-, and polyamine-treated plants appeared more closely related to the antiethylene action.

Journal Article↗

Graviresponses in herb and trees: a major role for the redistribution of tissue and growth stresses.

Tissue stresses, which occur in turgid herbaceous stems, both elongating and non-elongating. and tree growth stresses (TGSs) which occur in woody stems, are similar in that (i) they form self-equilibrating patterns of stresses (tensile and compressive) in stems, and (ii) the asymmetric, graviresponsive change in the pattern tends to bend the stem. The longitudinal tensile tissue stress (TS) which occurs in the outer layers of turgid stems is a few times higher than the osmotic pressure of cell sap in such a layer. Usually it is considered that TSs originate from the differential growth of tissues in a stem; however, physical analysis of a turgid stem model has shown that TSs are an unavoidable physical consequence of the variation in structural characteristics of cell layers or vascular strands in turgid stems. The model applied to the sunflower hypocotyl gives forces which fit well to those measured. The structural characteristics are sufficient to explain fully the TSs which exist in turgid stems. Differential growth is not necessary in this respect. Examination of the model shows also that the longitudinal elastic strain of all cell walls in a turgid stem is the same at a given stem level regardless of wall thickness, i.e. the structure-based TSs compensate for the variation in turgor-induced wall stress in single cells with variable diameter and wall thickness. The importance of this compensation for the anisotropy of wall stresses is presented. The forces which generate TSs exert bending moments which are high but they sum mutually to zero in a vertical stem. In gravistimulated turgid stems of Reynoutria, the TSs decrease considerably on the lower side while those on the upper side remain unaltered. The consequences of this asymmetric change for gravitropic bending are analysed. Tree growth stresses arise in a process by which new cells added by the cambium to the secondary xylem tend to shrink longitudinally (except compression wood) during maturation of the cell walls. The pattern of TGSs is characterized by tensile or compressive stress in the peripheral or the core wood, respectively. An asymmetrical pattern of TGSs due to asymmetrical deposition of wood results in a bending moment which equilibrates the bending moment caused by the weight of a lateral branch. In response to a gravity-derived stimulus the TGS pattern may be modified by asymmetric formation of reaction wood which differs histologically from normal wood: tension wood in many arborescent angiosperms, and compression wood in conifers. The formation and functioning of the reaction wood is discussed.

Cell Wall↗

The kinetics of calcium and magnesium entry into mycorrhizal spruce roots.

The entry of calcium and magnesium from external sources into mycorrhizal roots of 3-year-old Norway spruce trees (Picea abies [L.] Karst.) was monitored. Roots of intact plants were exposed for various periods of time, ranging from 2 min to 48 h, to nutrient solutions which contained the stable-isotope tracers 25Mg and 44Ca. After labelling, samples of roots were excised from the plants, shock-frozen, cryosubstituted and embedded. The resulting isotope composition in this material was analysed by a laser-microprobe-mass-analyser (LAMMA) at relevant positions within cross-sections of the roots. For both elements, we determined (i) the fractions of the isotopes originating from the plant prior to labelling, and (ii) the fraction of isotopes originating from the corresponding tracer that penetrated into the root. Both divalent cations rapidly penetrated across the cortical apoplast and reached the endodermis. After 2 min of exposure to the labelling solution, an initial transient gradient of the tracers could be observed within the root cortex. Subsequently, calcium as well as magnesium equilibrated between the apoplast of the entire cortex and the external tracer with a half-time, t1/2, of about 3 min. In contrast, the kinetics of radial movement into the vascular stele showed a delay with a t1/2 of 100-120 min. We take this as strong evidence that there exists a free apoplastic path for divalent cations in the cortex and that the endodermis is a major barrier to the further passage of Mg and Ca into the xylem. While 25Mg in the labelling solution exchanged rapidly with Mg in the cortical apoplast, the exchange across the plasma membrane with Mg present in the protoplasm of the same cortical cells was almost 2 orders of magnitude slower. The kinetics of Ca and Mg entry at + 6 degrees C were similar to those obtained at a root temperature of +22 degrees C.

Calcium↗

Speciation of iron coordinated by phytosiderophores by use of HPLC with pulsed amperometric detection and AAS.

Phytosiderophores of the mugineic acid family, and the respective iron species, have been separated by anion-exchange chromatography with NaOH gradient elution. Two different detection methods were used in parallel, pulsed amperometry (PAD) for phytosiderophores and atomic absorption spectrometry (AAS) for iron. This combination enables identification of separated iron species. Up to five different iron species were separated and detected within 30 min - two different phytosiderophore species, two amino acid species, and one species which has not yet been identified but which is most probably a decomposition product of phytosiderophores. The detection limit was in the low micro mol L(-1) concentration range, which is sufficiently low for determination in real plant samples, even after dilution. The method has been applied to root washings of iron-deficient wheat and barley plants and to a xylem exudate of non-deficient maize.

Chromatography, High Pressure Liquid↗

Biological upgrading of wheat straw through solid-state fermentation with Streptomyces cyaneus.

The biological upgrading of wheat straw with Streptomyces cyaneus was examined through the analysis of chemical and structural changes of the transformed substrate during solid-state fermentation. Analysis of enzymes produced during the growth of S. cyaneus showed that phenol oxidase was the predominant enzyme. The reduction in Klason lignin content (16.4%) in the transformed substrate indicated the ability of this strain to delignify lignocellulose residues and suggests a role for phenol oxidase in the bacterial delignification process. Microscopic examination of the transformed substrate showed that the initial attack occurred at the less lignified cell walls (phloem and parenchyma), while xylem and sclerenchyma were slowly degraded. The pattern of degradation of sclerenchymatic tissues by S. cyaneus showed delamination between primary and secondary walls and between S1 and S2 due to partial removal of lignin. In the later stages of the decay a disorganization of the secondary walls was detected on account of fibrillation of this layer. A comparison of the properties of the pulp from wheat straw transformed by S. cyaneus with untreated wheat straw showed that pretreatment improved the characteristics that determine the quality of pulp. This was indicated by an increase in pulp brightness and by a decrease in the kappa number. These changes occurred without significantly affecting the viscosity, a measure of the quality of the cellulose fibres. These results support the potential application of this organism or its oxidative enzymes in biopulping.

Culture Media↗

Developing an ecosystem perspective from experimental monitoring programs: II. Ecophysiological responses of a rare geothermal grass to soil water.

Measurements of xylem water potential, leaf conductance, and leaf pressure-volume characteristics on the geothermal endemic Dichanthelium lanuginosum var. thermale (DILA) were used to delineate operational ranges during wet and dry years and among several microsites at Little Geysers, Sonoma County, California, USA. Plants seldom experienced water potentials more negative that -1.5 MPa. Other nongeothermal, widespread species experienced the lower water potentials typical of chaparral and woodland plants. DILA was able to effectively utilize geothermal water while the widespread species could not and was able to keep stomata open during most of the year. There was evidence to suggest that DILA had some ability to acclimate with significant shifts in Pio and psio during the dry 1994 summer, especially in the upland microhabitat. Nevertheless, minimum leaf turgor values in the upland came very close to, or dropped below, the 0.2-0.3 MPa threshold thought necessary to maintain stomatal opening and photosynthesis. DILA thus depends upon the unique water status of fumarole soils in the vicinity of the Little Geysers to persist in an otherwise lethal regional mosaic of climate, soil, and vegetation. The physiological data were used to derive reference ranges for subsequent monitoring of DILA at Little Geysers. Such ranges are required to determine the future impact, if any, of geothermal development on the persistence of this rare grass and its complex ecosystem.

Conservation of Natural Resources↗

Establishment of a genetically marked insect-derived symbiont in multiple host plants.

Alcaligenes xylosoxidans subsp. denitrificans, originally isolated from the cibarial region of the foregut of the glassy-winged sharpshooter (Homalodisca coagulata), was transformed using the Himar1 transposition system to express EGFP. Seedlings of six potential host plants were inoculated with transformed bacteria and 2 weeks later samples were taken 5 cm away and analyzed by quantitative real-time PCR using primers designed to amplify the gene insert. The largest colony of 3,591,427 cells/2 cm of A. xylosoxidans subsp. denitrificans was found in Citrus limon, with almost all plants testing positive in both trials. The amount of colonization decreased in the other plants tested in the following order: orange (Citrus sinensis "sweet orange") > chrysanthemum (Chrysanthemum grandiflora cv. "White Diamond") > periwinkle (Vinca rosea) > crepe myrtle (Lagerstroemia indica) > grapevine (Vitis vinifera cv. Chardonnay). The bacterium's preference for citrus paralleled the host insect's preference for this same plant. Additional tests determined that A. xylosoxidans subsp. denitrificans thrives as a nonpathogenic, xylem-associated endophyte.

Alcaligenes↗

Expression of a salt-induced protein (SALT) in suspension-cultured cells and leaves of rice following exposure to fungal elicitor and phytohormones.

Phytohormones are essential signal compounds in the regulation of stress-related and defense-related genes. However, there is no clear evidence for any effect of these signal molecules and biotic elicitors on the regulation of the SALT gene in suspension-cultured rice cells. We characterized the expression of a SALT gene following treatment with fungal elicitor, phytohormones, cycloheximide, and inhibitors of protein kinase/phosphatases. SALT expression was up-regulated following treatment with a fungal elicitor, jasmonic acid (JA), abscisic acid (ABA), and NaCl. However, salicylic acid (SA) alone or in combination with one of the other elicitors not only strongly inhibited SALT gene expression but also exhibited an antagonistic effect in suspension cells and leaves. Cycloheximide inhibited SALT accumulation in suspension cells and in leaves, but the inhibitors of protein kinase/phosphatase did not. Immunolocalization revealed that SALT protein was present in xylem parenchyma cells of vascular bundles in the major and minor leaf veins.

Abscisic Acid↗