Ethylene effect on extensin and peroxidase distribution in the subapical region of pea epicotyls

In dark grown pea (Pisum sativum) seedlings ethylene causes the triple response in which elongation growth is inhibited, radial growth is promoted, and orientation of shoots to gravity is altered. The distribution of extensin and peroxidase activity in pea epicotyls upon ethylene treatment was studied by tissue printing on nitrocellulose paper. It was found that the localization of extensin and peroxidase activity changes after 72 and 96 hours of ethylene treatment. In untreated plants, peroxidase activity is detected only in the vascular bundles. Nonetheless, after 72 and 96 hours of ethylene treatment peroxidase activity is hardly detected in the vascular system but present in the epidermal and cortical cells. Extensin increases in the epidermal and cortical cells upon ethylene treatment but it also appears in the vascular system when peroxidase activity is no longer detected.     

Autolysis of cell walls in pea epicotyls during growth. Enzymatic activities involved

Pisum sativum L. (cv. Lincoln) epicotyl cell walls show autohydrolysis and release into the incubation medium up to 120 μg of sugar per mg of cell wall dry weight in 30 h. Cell walls from younger epicotyls with high growth capacity showed higher auto-lytic capacity than older epicotyls. This suggests that both processes, growth and au-tolysis, are related. The proteins responsible for autolysis were extracted from the wall fraction with high saline solution (3 M LiCl) and enzymatic activities associated with the proteins were studied. The highest activity corresponded to α-galactosidase; lower activities were found for β-galactosidase, a-arabinosidase and exoglucanase. Changes in enzymatic activities and changes in the proportion of sugars released in autolysis by cell walls during the growth of epicotyls support the notion that α-galac-tosidase is one of the enzymes involved in the process of autolysis, and that the liberation of arabinose and galactose in this process occurs as arabinogalactan.     

Glucose as a growth medium factor regulating lipid composition of Yersinia pseudotuberculosis

Effects of glucose and growth temperature on Yersinia pseudotuberculosis O:1b serovar lipid composition have been studied. These growth parameters were shown to have drastic effects on biosynthetic processes in the pseudotuberculosis bacteria. The temperature effect is the most universal, extending to cell growth and to free lipid and lipopolysaccharide content and composition; it is most conspicuous in the bacteria cultivated on glucose-containing nutrient broth. The effect of glucose is selective, affecting only free lipids and depending on temperature (glucose favors phospholipid (PL) synthesis in the cold and inhibits it at 37°C); the effect of glucose is more evident in the cold. Determination of the contents of individual PL in percent dry bacterial weight indicates that the most obvious effect of glucose and/or growth temperature is on phosphatidylethanolamine (PE) content: on both media and at both temperatures an overall decrease in PL content stems from the inhibition of PE synthesis and is attended by decreasing ratio of neutral to acidic lipids.     

Effects of calcium and kinetin on growth and cell wall composition of pea epicotyls

Kinetin and CaCl₂, in the presence of indoleacetic acid, promoted lateral expansion of epicotyls of decapitated and derooted Alaska pea seedlings (Pisum sativum L.) and inhibited their elongation. This growth response was correlated with the development of cell walls unusually rich in pectic uronic acids. Epicotyls in calcium-auxin solutions continued to enlarge and to add new wall material long after tissues in auxin only had stopped. Longitudinal enlargement, associated with the development of walls poor in pectic uronic acids, was favored by KCl, MgCl₂, and ethylenediaminetetraacetate. The last of these agents promoted the loss of ⁴⁵Ca from the epicotyls. Seedings grown in vermiculite moistened with CaCl₂, KCl, or MgCl₂ solutions did not differ in appearance or in the composition of their walls. They responded similarly to experimental treatment except that the decapitated epicotyls of the MgCl₂-grown plants suffered an absolute loss of pectic uronate when incubated in that salt.     

Ethylene rapidly up-regulates the activities of both monomeric GTP-binding proteins and protein kinase(s) in epicotyls of pea

It is demonstrated that, in etiolated pea (Pisum sativum) epicotyls, ethylene affects the activation of both monomeric GTP-binding proteins (monomeric G-proteins) and protein kinases. For monomeric G-proteins, the effect may be a rapid (2 min) and bimodal up-regulation, a transiently unimodal activation, or a transient down-regulation. Pretreatment with 1-methylcyclopropene abolishes the response to ethylene overall. Immunoprecipitation studies indicate that some of the monomeric G-proteins affected may be of the Rab class. Protein kinase activity is rapidly up-regulated by ethylene, the effect is inhibited by 1-methylcyclopropene, and the activation is bimodal. Immunoprecipitation indicates that the kinase(s) are of the MAP kinase ERK1 group. It is proposed that the data support the hypothesis that a transduction chain exists that is separate and antagonistic to that currently revealed by studies on Arabidopsis mutants.     

Occurrence of diamine oxidase in the apoplast of pea epicotyls

Most of the diamine oxidase (EC 1.4.3.6) present in pea (Pisum sativum L. cv. Rondo) epicotyls is found in the fluid obtained by centrifuging pea epicotyl sections previously infiltrated under vacuum with a buffer solution. No detectable amount of the cytoplasmic enzyme glucose-6-phosphate dehydrogenase is present in this fluid, showing that there is very little contamination by cell contents. Polyacrylamide-gel electrophoresis and specific-activity data indicate that diamine oxidase is the most plentiful protein in the extracellular solution obtained from pea epicotyl sections and that an active process is involved in the selective transfer of the enzyme outside the cell. The possible involvement of diamine oxidase in the supply of H₂O₂ to peroxidase-catalyzed reactions occurring inside the cell wall is discussed.Abbreviations DAO diamine oxidase - Glc6P glucose-6-phosphate     

Auxin-induced extension of the isolated epidermis of light-grown pea epicotyls

1. The effect of IAA and FC on the extension of isolated epidermisof light-grown Alaska pea epicotyls was studied under differentconditions with an extension apparatus. The following resultswere obtained.
2. The epidermis extended in response to low pHbuffer solutionof 1–10 mM, maximum extension being achievedat pH below5.5.
3. IAA, 5 mg/liter, caused, although not consistently,an extensionof epidermal strips in 1 mM buffer, but not at10 mM.
4. Consistent extension of the isolated epidermis dueto IAA wasobtained by addition of GTP, ATP, ITP or UTP (sodiumsalts),but not nucleosides, nitrogen bases or sugars.
5. A fungaltoxin, FC, at 10^–5 M induced extension of theepidermiswithout addition of the nucleoside triphosphates.
6. IAA andFC caused H⁺ extrusion in peeled epicotyl segments bothin thepresence and absence of GTP. IAA caused appreciable H⁺extrusionin the isolated epidermis only in the presence ofGTP, whereasH⁺ extrusion by the epidermis was induced by FCeven in theabsence of GTP.

From these results, we concluded that IAA induces extensionof the isolated epidermis under the above conditions throughthe mediation of H⁺ ions. (Received July 12, 1976; )     

Asymbiotic association of Rhizobium with pea epicotyls treated with a plant hormone

Treatment of epicotyls of dark-grown pea (Pisum sativum L.) seedlings with indole-3-acetic acid causes swelling of the tissue. Application of Rhizobium to the cut surface of the swollen tissue results in the development of an infection. The infection spreads in the cortical cells and proceeds 2–3 mm deep into the stem within 3–4 days. An acetylene reduction assay used for detecting nitrogen-fixation capacity of the infected tissue was negative at 10% [O₂]; however, if [O₂] was reduced to below 1%, some activity could be detected. Ultrastructural observations indicate that the cytoplasmic contents of the infected cells are destroyed and no membrane structure around the bacteria is formed during this infection. Rhizobium does not appear to have developed any symbiotic relationship with the host. Failure to develop symbiosis appears to result in a parasitic or saprophytic association and the nitrogen fixed under such conditions may not be of any use to the plant.     

Lipoproteins as a factor regulating vascular tonus

Lipoproteins (very low, low and high density) purified from canine fresh plasma produced relaxation of isolated dog coronary artery and rabbit aortic ring segments precontracted with potassium, 30 mM or phenylephrine, 10(-6) M, respectively. No apparent species-specific effects of lipoproteins were revealed and they produced relaxation of other species vessels as well, which differed quantitatively. No apparent endothelium-dependent relaxation produced by animals' lipoproteins was evident. The interference of lipoproteins with some steps in electromechanical coupling is suggested which seemingly result to decrease in excitability of voltage and receptor operated calcium channels of smooth muscle cell membrane. The role of lipoproteins seems to be in maintenance of low level of smooth muscle cell membrane excitability.     

Activation of cell wall-associated peroxidase isoenzymes in pea epicotyls by a xyloglucan-derived nonasaccharide

The cell wall-derived xyloglucan nonasaccharide XXFG was foundto increase the extractable activity of distinct cationic cellwall-associated peroxidase isozyme groups isolated from etiolatedpea epicotyls. Peroxidase activation occurred in the first 10h of incubation with the nonasaccharide in the pea epicotylbioassay. At the same time varying concentrations of XXFG causedgrowth inhibition up to 35%. Neither the increase of peroxidaseactivity nor the growth inhibition was restricted to a certainXXFG concentration. The increase in peroxidase activity wasnot just an oligosaccharide effect in general. The correspondingheptasaccharide XXXG neither inhibited growth nor increasedperoxidase activity. The isozymes extracted from pea epicotylswere additionally separated by cation-exchange chromatographyand submitted to isoelectric focusing. With one exception, allof the ionically-bound, cell wall-associated peroxidases presentin pea epicotyls were cationic or slightly anionic. It is proposedthat the growth inhibition caused by XXFG is at least in partthe result of peroxidasecatalysed cell wall tightening inducedby the nonasaccharide. Key words: XXFG, growth inhibition, cell wall-associated peroxidases, cell wall tightening, pea epicotyls     

Temperature as a factor regulating growth and toxin content in the dinoflagellate Alexandrium catenella

Controlled laboratory culture of Alexandrium catenella was used to determine the effects of a range of temperatures between 10 and 16 °C on the growth and saxitoxin content of this dinoflagellate, using strain ACC02 isolated from seawater at Aysen, XI Region, Southern Chile. Cell cultures were made using L1 culture medium at 30‰ salinity, and a photon flux density of 59.53 μmol m² s⁻¹. The results showed that the duration of the exponential growth phase was determined by the experimental temperature, with maximum cell concentrations obtained at 12 °C; significantly lower cell concentrations and growth rates were obtained at 16 °C. Cell dry weight and chlorophyll a values followed cell growth trends. The toxicity of A. catenella was variable at all the experimental temperatures, with a tendency towards having an inverse relation to temperature, with the highest values occurring at 10 °C and the lowest at 16 °C. The optimal range of temperature for the growth of the Chilean strain of A. catenella differed from rates reported for this species isolated at other latitudes, and was correlated with natural temperature conditions predominant in the environment from which it was isolated. The inverse relation of toxicity with temperature in the laboratory was broadly reflected in observations on the toxicity of this dinoflagellate in the field, and coincided with results from the literature.     

Requirement for the gravity-controlled transport of auxin for a negative gravitropic response of epicotyls in the early growth stage of etiolated pea seedlings

Gravity-controlled transport of auxin was studied for a negative gravitropic response in the early growth stage of etiolated pea (Pisum sativum L. cv. Alaska) seedlings, in which epicotyl bending was observed near the cotyledon nodes of the seedlings grown continuously from seeds germinated in a horizontal or an inclined position. Increased expression of an auxin-inducible gene, PsIAA4/5, was observed in the elongated side of epicotyls grown in a horizontal or an inclined position. Regardless of the conditions of seed germination, polar auxin transport in the proximal side of the first internodes of the seedlings was significantly higher than in the distal side. Polar auxin transport in the proximal side of epicotyls grown in an inclined position was significantly lower than in those grown in a horizontal position. In contrast, lateral auxin distribution from the proximal to distal sides in epicotyls grown in an inclined position was significantly higher than in epicotyls grown in a horizontal position. Accumulation of PsPIN1 mRNA encoding a putative auxin efflux facilitator, which was observed in vascular tissue, cortex and epidermis in the proximal and distal sides of epicotyls, was markedly influenced by gravistimulation. These results strongly suggest that gravistimulation induces changeable polar auxin transport and one-way lateral auxin distribution in epicotyls as well as asymmetric auxin accumulation in the proximal and distal sides of epicotyls, resulting in a negative gravitropic response of epicotyls in the early growth stage of pea seedlings.     

Antioxidative responses and proline level in leaves and roots of pea plants subjected to nickel stress

The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione S-transferase (GST) as well as proline content were studied in leaves and roots of 14 day-old pea plants treated with NiSO₄ (10, 100, 200 μm) for 1, 3, 6 and 9 days. Exposure of pea plants to nickel (Ni) resulted in the decrease in CuZnSOD as well as total SOD activities in both leaves and roots. The activity of APX in leaves of plants treated with 100 and 200 μm Ni increased following the 3rd day after metal application, while in roots at the end of the experiment the activity of this enzyme was significantly reduced. In both organs CAT activity generally did not change in response to Ni treatment. The activity of GST in plants exposed to high concentrations of Ni increased, more markedly in roots. In both leaves and roots after Ni application accumulation of free proline was observed, but in the case of leaves concentration of this amino acid increased earlier and to a greater extent than in roots. The results indicate that stimulation of GST activity and accumulation of proline in the tissues rather than antioxidative enzymes are involved in response of pea plants to Ni stress.     

Ethylene as a factor limiting the legume-Rhizobium symbiosis in tube culture

Growth and nitrogen fixation by Trifolium subterraneum plants at 15 and 10 ^oC root temperatures is much less in test tubes plugged with cotton wool than in open pots of sand. Removing the ethylene produced by plants growing in cotton wool plugged flasks by adsorption onto activated charcoal, or by aerating with sterile air, increased nitrogenase activity and growth over the first 25 days, to levels approaching that of plants grown in open pots of sand.     

Effects of brefeldin A on the development of the Casparian strip in pea epicotyls

Summary The Casparian strip, a structure that is present in roots, is also present in epicotyls of dark-grown pea seedlings. In a dark-grown epicotyl, the cells in each stage of the development of the Casparian strip have been suggested to be lined up basipetally in the region 3 to 37 mm below the bending point of the hook, in order of the developmental stage. Brefeldin A (BFA), a specific inhibitor of secretory transport, was administrated at 200 M. to dark-grown pea epicotyls for 2 h via a thread passed through the epicotyl 40 mm below the bending point. The basipetal sequence of development of the modification of the cell wall at the Casparian strip, as judged by fluorescence microscopy, stopped 5 h after the start of 2 h treatment with BFA and resumed after 30 h. This basipetal sequence of development did not stop in control seedlings. Electron micrographs of endodermal cells in epicotyls treated with BFA showed striking morphological changes in the Golgi stacks and the ER. Histological examination made 20 h after the start of the experiment revealed that the basipetal sequence of development of the cell wall modification stopped at a point which was present at 25.2 ± 1.6 mm (mean with SD, n=5) from the bending point of the hook at the start while the basipetal sequence of development of the tight adhesion of the plasma membrane to the cell wall at the Casparian strip stopped 0.9 ± 0.5 mm (mean with SD, n=5) below this point. These results indicate the involvement of secretory transport not only in the introduction of the modification of the cell wall but also in the completion of the tight adhesion of the plasma membrane.Abbreviations BFA brefeldin A - PBS phosphate-buffered saline - ER endoplasmic reticulum     

Bradykinin as a factor regulating synaptic inflow to the neurons in Helix pomata]

The action of bradykinin on spontaneous electric activity of Helix pomata neurones from the subfaringeal complex was studied by microionophoresis. Bradykinin not only facilitates the neuronal responses to synaptic activation, but also prolongs the activation-induced generation of the potential action. Bradykinin effects were observed only in experiments conducted in spring.     

Expression and localization of a Rhizobium-derived cambialistic superoxide dismutase in pea (Pisum sativum) nodules subjected to oxidative stress

Two phylogenetically unrelated superoxide dismutase (SOD) families, i.e., CuZnSOD (copper and zinc SOD) and FeMn-CamSOD (iron, manganese, or cambialistic SOD), eliminate superoxide radicals in different locations within the plant cell. CuZnSOD are located within the cytosol and plastids, while the second family of SOD, which are considered to be of bacterial origin, are usually located within organelles, such as mitochondria. We have used the reactive oxygen species-producer methylviologen (MV) to study SOD isozymes in the indeterminate nodules on pea (Pisum sativum). MV caused severe effects on nodule physiology and structure and also resulted in an increase in SOD activity. Purification and N-terminal analysis identified CamSOD from the Rhizobium leguminosarum endosymbiont as one of the most active SOD in response to the oxidative stress. Fractionation of cell extracts and immunogold labeling confirmed that the CamSOD was present in both the bacteroids and the cytosol (including the nuclei, plastids, and mitochondria) of the N-fixing cells, and also within the uninfected cortical and interstitial cells. These findings, together with previous reports of the occurrence of FeSOD in determinate nodules, indicate that FeMnCamSOD have specific functions in legumes, some of which may be related to signaling between plant and bacterial symbionts, but the occurrence of one or more particular isozymes depends upon the nodule type.