The action of gravity in agravitropic Zea primary roots: Effect of gravistimulation on the extracellular free-Ca2+ content in the 1-mm apical root tip in the dark

The action of gravity stimulation in darkness was examined in agravitropic primary roots of Zea mays L. (cv. Golden Cross Bantam 70). Contents of diffusible and nitric-acid-extractable Ca²⁺ in 1-mm apical tips of roots gravistimulated in the dark were measured by flowinjection analysis as free Ca²⁺ and bound Ca²⁺, respectively. The free-Ca²⁺ content increased transiently, reaching a maximum 0.5 h after gravistimulation. This transient increase was also observed when gravistimulation was applied by changing the orientation of the roots back from horizontal to vertical again. On the other hand, the bound-Ca²⁺ content decreased transiently following gravistimulation. Furthermore, when the root caps were treated with 10 mM 2-(N-morpholino) ethanesulfonic acid buffer, the elevation of free Ca²⁺ following gravistimulation was prevented. These results indicate that gravity perception and the initial transduction steps proceed in the dark, and moreover that the elevation of free Ca²⁺ brought about by the interaction of Ca²⁺/H ⁺ in the apoplast of root tips may be involved in transmission of the gravity signal.Abbreviations FIA flow-injection analysis - Mes 2-(N-morpholino) ethanesulfonic acid - Pipes 1,4-piperazinediethanesulfonic acid - Quin 2 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis(carboxymethyl) aminoquinoline     

Isolation and characterization of a rice WUSCHEL-type homeobox gene that is specifically expressed in the central cells of a quiescent center in the root apical meristem

The Arabidopsis WUSCHEL (WUS) protein, which plays an important role in the specification of the stem cells in the shoot apical meristem (SAM), contains an 'atypical' homeodomain (HD) with extra residues in its loop and turn regions. We speculated that a WUS-type atypical HD protein might also be involved in the specification and maintenance of the root apical meristem (RAM) stem cells of rice. To investigate this possibility, we isolated and characterized a rice WUS-type homeobox gene designated quiescent-center-specific homeobox (QHB) gene. Using transformants carrying the QHB promoter-GUS and in situ hybridization, we found that QHB was specifically expressed in the central cells of a quiescent center (QC) of the root. During embryogenesis and crown root formation, QHB expression was observed prior to the morphological differentiation of the root. However, we detected different QHB expression patterns in the process of the RAM development, specifically between radicle and crown root formation, suggesting that the cell-fate determination of the QC may be controlled by different mechanisms. We also produced transformants that overexpress QHB or Arabidopsis WUS. These transformants did not form crown roots, but developed multiple shoots from ectopic SAMs with malformed leaves. On the basis of these observations, we propose that the WUS-type homeobox gene is involved in the specification and maintenance of the stem cells (QC cells) in the RAM, by a mechanism similar to that for WUS in the SAM.     

Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation

Post-translational hydroxylation of peptide-bound proline residues, catalyzed by peptidyl-prolyl-4 hydroxylase (EC 1.14.11.2) using ascorbate as co-substrate, is a key event in the maturation of a number of cell wall-associated hydroxyproline-rich glycoproteins (HRGPs), including extensins and arabinogalactan-proteins, which are involved in the processes of wall stiffening, signalling and cell proliferation. Allium cepa L. roots treated with 3,4-DL-dehydroproline (DP), a specific inhibitor of peptidyl-prolyl hydroxylase, showed a 56% decrease in the hydroxyproline content of HRGP. Administration of DP strongly affected the organization of specialized zones of root development, with a marked reduction of the post-mitotic isodiametric growth zone, early extension of cells leaving the meristematic zone and a huge increase in cell size. Electron-microscopy analysis showed dramatic alterations both to the organization of newly formed cell walls and to the adhesion of the plasma membranes to the cell walls. Moreover, DP administration inhibited cell cycle progression. Root tips grown in the presence of DP also showed an increase both in ascorbate content (+53%) and ascorbate-specific peroxidase activity in the cytosol (+72%), and a decrease in extracellular “secretory” peroxidase activity (−73%). The possible interaction between HRGPs and the ascorbate system in the regulation of both cell division and extension is discussed. Received: 14 October 1998 / Accepted: 31 May 1999     

Structural differentiation of membranes involved in the secretion of polysaccharide slime by root cap cells of cress (Lepidium sativum L.)

The peripheral secretion tissue of the root cap of Lepidium sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins — recognizable as IMPs — are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face - IMP intramembranous particle     

Overexpression of OsKTN80a,a katanin P80ortholog,caused the repressed cell elongation and stalled cell division mediated by microtubule apparatus defects in primary root in Oryza sativa

Katanin, a microtubule‐severing enzyme, consists of two subunits: the catalytic subunit P60, and the regulatory subunit P80. In several species, P80 functions in meiotic spindle organization, the flagella biogenesis, the neuronal development, and the male gamete production. However,the P80 function in higher plants remains elusive. In this study, we found that there are three katanin P80 orthologs(OsKTN80a, OsKTN80b, and OsKTN80c) in Oryza sativa L.Overexpression of OsKTN80a caused the retarded root growth of rice seedlings. Further investigation indicates that the retained root growth was caused by the repressed cell elongation in the elongation zone and the stalled cytokinesis in the division zone in the root tip. The in vivo examination suggests that OsKTN80a acts as a microtubule stabilizer. We prove that OsKTN80a, possibly associated with OsKTN60, is involved in root growth via regulating the cell elongation and division.     

Regulation of cell length in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid: a matter of apoplastic reactions

Treatment of the Arabidopsis thaliana root with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) immediately imposes a reduced maximal cell length beyond which further elongation is blocked. Here, we investigated possible apoplastic reactions involved in the inhibition of cell elongation. Five-day-old Arabidopsis seedlings were transferred to a growth medium supplemented with ACC and the effect on root cell length was recorded after 3 h of treatment. Altered characteristics in the apoplast of the nonelongating cells in the ACC-treated root, such as 'reactive oxygen species' (ROS) production and callose deposition, were detected using specific fluorochromes. The presence of functional hydroxyproline-rich glycoproteins (HRGPs) and the crosslinking of these cell-wall proteins are essential in limiting cell elongation. The ROS that drive the oxidative crosslinking of HRGPs, accumulate in the apoplast of cells in the zone where cell elongation stops. In the same cells, callose is deposited in the cell wall. The final cell length in the Arabidopsis root treated for a short period with ACC is determined in the zone of fast elongation. Both HRGPs crosslinking by ROS and callose deposition in the cell wall of this zone are suggested as causes for the reduced cell elongation.     

Particle density and protein composition of the peribacteroid membrane from soybean root nodules is affected by mutation in the microsymbiont Bradyrhizobium japonicum

Particle frequency of the peribacteroid membrane (PBM) from nodules of Glycine max (L.) Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101 (wild-type strain) was determined by freeze-fracturing to be about 2200·m^-2 in the protoplasmic fracture face and 700·m^-2 in the exoplasmic fracture face. In membranes isolated from nodules infected with the mutant RH 31-Marburg of B. japonicum, the particle frequency was similar in both fracture faces with 1200–1300 particles·m^-2. Analysis of particlesize distribution on peribacteroid membranes showed a loss, especially of particle sizes larger than 11 nm, in the mutant-infected nodules. Two-dimensional gel electrophoresis (isoelectric focussing and sodium dodecyl sulfate-polyacrylamide) showed 27 different polypeptides in the PBM from nodules infected with the wild-type strain, four of which were absent from the PBM of nodules infected with the mutant RH 31-Marburg, which also exhibited one extra small-molecular-weight polypeptide. At least 14 of the 27 polypeptides in the PBM from the wild-type-infected nodule were glycoproteins. In three of these glycoproteins, post-translational modifications were either lacking or different when the membrane was derived from mutant-infected nodules.Abbreviations EF exoplasmatic fracture face - HRPO horse radish peroxidase - IEF Isoelectric focussing - PBM peribacteroid membrane - PF protoplasmatic fracture face - PNA peanut agglutinin - PSA Pisum sativum agglutinin - SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Xylem transport and shoot accumulation of lumichrome, a newly recognized rhizobial signal, alters root respiration, stomatal conductance, leaf transpiration and photosynthetic rates in legumes and cereals

* Root respiration, stomatal conductance, leaf transpiration and photosynthetic rates were measured in phytotron and field-grown plants following the application of 5 or 10 nM lumichrome, 10 nM ABA (abscisic acid) and 10 ml of 0.2 OD600 infective rhizobial cells. * Providing soybean and cowpea roots with their respective homologous rhizobia and/or purified lumichrome increased the concentration of this molecule in xylem sap and leaf extracts. Relative to control, rhizobial inoculation and lumichrome application significantly increased root respiration in maize, decreased it in lupin, but had no effect on the other test species. * Applying either lumichrome (10 nM), infective rhizobial cells or ABA to roots of plants for 44 h in growth chambers altered leaf stomatal conductance and transpiration in cowpea, lupin, soybean, Bambara groundnut and maize, but not in pea or sorghum. Where stomatal conductance was increased by lumichrome application or rhizobial inoculation, it resulted in increased leaf transpiration relative to control plants. Treating roots of field plants of cowpea with this metabolite up to 63 d after planting showed decreased stomatal conductance, which affected CO2 intake and reduction by Rubisco. * The effect of rhizobial inoculation closely mirrored that of lumichrome application to roots, indicating that rhizobial effects on these physiological activities were most likely due to lumichrome released into the rhizosphere.     

Red light causes a reduction in IAA levels at the apical tip by inhibiting de novo biosynthesis from tryptophan in maize coleoptiles

When maize coleoptiles were unilaterally exposed to red light (7.9 μmol m⁻²s⁻¹ for 5 min), 3 h after treatment IAA levels in coleoptiles decreased in all regions, from top to basal, with levels about 60% of dark controls. Localized irradiation in the 5 mm top zone was sufficient to cause the same extent of IAA reduction in the tips to that in the tips of whole irradiated shoots. When coleoptiles were treated with N-1-naphthylphthalamic acid (NPA), an accumulation of IAA in the tip and a decrease of diffusible IAA from tips were simultaneously detected. IAA accumulation in red-light treated coleoptiles by NPA was much lower than that of dark controls. NPA treatment did not affect the content of conjugated IAA in either dark or light treated coleoptile tips. When ¹³C₁₁ ¹⁵N₂-tryptophan (Trp) was applied to the top of coleoptiles, substantial amounts of stable isotope were incorporated into free IAA in dark and red-light treated coleoptile tips. The ratio of incorporation was slightly lower in red-light treated coleoptile tips than that in dark controls. The label could not be detected in conjugated IAA. The rate of basipetal transport of IAA was about 10 mm h⁻¹ and the velocity was not affected by red light. These results strongly suggest that red light does not affect the rates of conversion of free IAA to the conjugate form or of the basipetal transport, but just reduces the IAA level in the tips, probably inhibited by IAA biosynthesis from Trp in this region.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Fibronectin is elevated in the cerebrospinal fluid of patients suffering from bacterial meningitis and enhances inflammation caused by bacterial products in primary mouse microglial cell cultures

Toll-like receptors (TLR) play a key role in the recognition of pathogenic organisms. Fibronectin, an extracellular matrix protein, is considered a potent stimulator of the innate immune system through TLR4. In bacterial meningitis, several extracellular matrix proteins and bacterial compounds are elevated in the CSF. For this reason, we hypothesized that these molecules may jointly stimulate the innate immune system and increase neuronal damage in bacterial meningitis. Concentrations of fibronectin were elevated in the CSF of patients suffering from bacterial meningitis, but not in patients with multiple sclerosis, when compared with control patients without CSF abnormalities. In primary cultures of mouse microglial cells, co-administration of fibronectin at concentrations occurring in the CSF in bacterial meningitis (10 microg/mL) with defined TLR agonists [lipopolysaccharide (TLR4), the synthetic lipopeptide tripalmytoyl-cysteinyl-seryl-(lysyl)3-lysine (TLR2) and single-stranded unmethylated cytosine-guanosine oligodesoxynucleotide (TLR9)] led to an additive release of nitric oxide and tumor necrosis factor-alpha when compared with the release elicited by either compound alone. In conclusion, the inflammatory reaction to bacterial compounds can be aggravated by endogenous fibronectin at elevated levels during bacterial CNS infections. This additive or synergistic effect may contribute to neuronal damage during bacterial meningitis.     

Properties of the leak permeability induced by a cytotoxic protein from Pseudomonas aeruginosa (PACT) in rat erythrocytes and black lipid membranes

A cytotoxic protein, isolated from Pseudomonas aeruginosa (PACT), was tested on red blood cells of rats and on black lipid membranes for changes of membrane permeability. In rat erythrocytes PACT induces lysis indicative of the formation of a leak permeable to monovalent ions. The dose response curve for the PACT-induced hemolysis demonstrates that the rate of lysis as well as the fraction of lytic cells increases with increasing toxin concentration. Furthermore, the leak pathway discriminates hydrophilic non-electrolytes according to their molecular weight. The findings indicate formation by PACT of a pore with an apparent radius of about 1.2 nm. In pure lipid membranes PACT forms hydrophilic pathways with moderate selectivity for small cations over small anions. The presence of cholesterol is a prerequisite for the occurrence of these PACT-induced permeability changes.     

Production of root hair deformation factors by Rhizobium meliloti nodulation genes in Escherichia coli: HsnD (NodH) is involved in the plant host-specific modification of the NodABC factor

The role of the hsnD (nodH) gene in the determination of the host-specific nodulation ability of Rhizobium meliloti was studied by expressing the common nodulation genes (nodABC) with or without the hsnD gene in Escherichia coli and testing for biological activity on various leguminous plants. In this way, four categories of plants were established. Upon infection with E. coli carrying the nodABC construct, root hair deformation (Had) was detected on clovers while the hsnD gene was additionally needed for the elicitation of the same response on alfalfa and sweet clover. A weak root hair deformation was seen on siratro by inoculation with E. coli harbouring the nodABC genes and was highly increased when hsnD was also introduced. Cowpea and Desmodium did not respond to any of the E. coli strains constructed. Exudates or cytosolicfractions of the respective E. coli derivatives elicited the same root hair deformation as the intact bacteria. These data indicate that not only the nodABC gene products but also the hsnD product are involved in the synthesis of Had factors. Subclones expressing only the nodA, nodB, or nodC genes or the same genes in pairs (nodAB, nodBC, nodAC) did not provide a compound with activity comparable to the NodABC factor, suggesting that all three genes are required for the production of the Had factor which is active on clover. Coinoculation of alfalfa plants with two strains of E. coli, one carrying the nodABC genes and the other expressing only hsnD, or combining exudates or cytosolic fractions from these strains did not result in root hair deformation on alfalfa. These data indicate that the HsnD protein itself or its product is not an additional alfalfa-specific extracellular signal but more likely is enzymatically involved in the modification of the basic compound determined by the nodABC genes.     

The 2013 SFRBM discovery award: Selected discoveries from the butterfield laboratory of oxidative stress and its sequela in brain in cognitive disorders exemplified by Alzheimer disease and chemotherapy induced cognitive impairment

This retrospective review on discoveries of the roles of oxidative stress in brain of subjects with Alzheimer disease (AD) and animal models thereof as well as brain from animal models of chemotherapy-induced cognitive impairment (CICI) results from the author receiving the 2013 Discovery Award from the Society for Free Radical Biology and Medicine. The paper reviews our laboratory’s discovery of protein oxidation and lipid peroxidation in AD brain regions rich in amyloid β-peptide (Aβ) but not in Aβ-poor cerebellum; redox proteomics as a means to identify oxidatively modified brain proteins in AD and its earlier forms that are consistent with the pathology, biochemistry, and clinical presentation of these disorders; how Aβ in in vivo, ex vivo, and in vitro studies can lead to oxidative modification of key proteins that also are oxidatively modified in AD brain; the role of the single methionine residue of Aβ(1–42) in these processes; and some of the potential mechanisms in the pathogenesis and progression of AD.CICI affects a significant fraction of the 14 million American cancer survivors, and due to diminished cognitive function, reduced quality of life of the persons with CICI (called “chemobrain” by patients) often results. A proposed mechanism for CICI employed the prototypical ROS-generating and non-blood brain barrier (BBB)-penetrating chemotherapeutic agent doxorubicin (Dox, also called adriamycin, ADR). Because of the quinone moiety within the structure of Dox, this agent undergoes redox cycling to produce superoxide free radical peripherally. This, in turn, leads to oxidative modification of the key plasma protein, apolipoprotein A1 (ApoA1). Oxidized ApoA1 leads to elevated peripheral TNFα, a proinflammatory cytokine that crosses the BBB to induce oxidative stress in brain parenchyma that affects negatively brain mitochondria. This subsequently leads to apoptotic cell death resulting in CICI. This review outlines aspects of CICI consistent with the clinical presentation, biochemistry, and pathology of this disorder. To the author’s knowledge this is the only plausible and self-consistent mechanism to explain CICI.These two different disorders of the CNS affect millions of persons worldwide. Both AD and CICI share free radical-mediated oxidative stress in brain, but the source of oxidative stress is not the same.Continued research is necessary to better understand both AD and CICI. The discoveries about these disorders from the Butterfield Laboratory that led to the 2013 Discovery Award from the Society of Free Radical and Medicine provide a significant foundation from which this future research can be launched.     

Functional characterisation of LKT1, a K+ uptake channel from tomato root hairs, and comparison with the closely related potato inwardly rectifying K+ channel SKT1 after expression in Xenopus oocytes

A cDNA encoding a novel inwardly rectifying potassium (K⁺ _in) channel, LKT1, was cloned from a root-hair-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The LKT1 mRNA was shown to be most strongly expressed in root hairs by Northern blot analysis. The LKT1 channel is a member of the AKT family of K⁺ _in channels previously identified in Arabidopsis thaliana (L.) Heynh. and potato (Solanum tuberosum L.). Moreover, LKT1 is closely related (97% identical amino acids) to potato SKT1. An electrophysiological comparison of the two channels should therefore assist the identification of possible molecular bases for functional differences. For this comparison, both channels were functionally expressed and electrophysiologically characterised within the same expression system, i.e. Xenopus laevis oocytes. Voltage-clamp measurements identified LKT1 as a K⁺-selective inward rectifier which activates with slow kinetics upon hyperpolarising voltage pulses to potentials more negative than −50 mV. The activation potential of LKT1 is shifted towards positive potentials with respect to SKT1 which might be due to single amino acid exchanges in the rim of the channel's pore region or in the S4 domain. Like SKT1, LKT1 reversibly activated upon shifting the external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K⁺ _in channels. The pharmacological inhibitor Cs⁺, applied externally, inhibited K⁺ _in currents mediated by LKT1 and SKT1 half-maximally with a concentration (IC₅₀) of 21 μM and 17 μM, respectively. In conclusion, LKT1 may serve as a low-affinity influx pathway for K⁺ into root hair cells. Comparison of homologous K⁺ _in rectifiers from different plant species expressed in the same heterologous system allows conclusions to be drawn in respect to structure-function relationships. Received: 3 August 1999 / Accepted: 2 November 1999     

Behavioral expression of cocaine sensitization in rats is accompanied by a distinct pattern of modifications in the PKA/DARPP-32 signaling pathway

Repeated cocaine administration induces behavioral sensitization and modifications in the phosphorylation pattern of dopamine and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32), characterized by a tonic increase in the Thr75 phosphorylated form, and a decrease in the Thr34 phosphorylated form. This study further investigated the correlations between cocaine sensitization and modifications in the DARPP-32 phosphorylation pattern, cAMP-dependent protein kinase (PKA) activity, and mGluR5 tone in the medial prefrontal cortex and nucleus accumbens. Behavioral sensitization and modifications in these neurochemical markers followed a similar temporal pattern. Moreover, in sensitized rats acute cocaine administration modified phosphorylation levels of Thr75- and Thr34-DARPP-32, GluR1, and NR1 subunits in the nucleus accumbens only at a dose double the efficacious dose in control rats. These results suggest that the high levels of phospho-Thr75 DARPP-32 maintain PKA in a prevalent inhibited state. Furthermore, in sensitized rats the acute administration of 6-methyl-2-(phenylethynyl)-pyridine, a mGluR5 antagonist, reinstated the phosphorylation levels of Thr75- and Thr34-DARPP-32, GluR1, and NR1 to control values, and a subsequent cocaine challenge did not elicit a sensitized response. These data suggest that a tonic increase in mGluR5 transmission in cocaine-sensitized rats sustains both the increase in phospho-Thr75 DARPP-32 levels and the expression of behavioral sensitization.