Gating of aquaporins by low temperature in roots of chilling-sensitive cucumber and chilling-tolerant figleaf gourd

Effects of low temperature (8 degrees C) on the hydraulic conductivity of young roots of a chilling-sensitive (cucumber, Cucumis sativus L.) and a chilling-resistant (figleaf gourd, Cucurbita ficifolia Bouche) crop have been measured at the levels of whole root systems (root hydraulic conductivity, Lp(r)) and of individual cortical cells (cell hydraulic conductivity, Lp). Exposure of roots to low temperature (LRT) for up to 6 d caused a stronger suberization of the endodermis in cucumber compared with figleaf gourd, but no development of exodermal Casparian bands in either species. Changes in anatomy after 6 d of LRT treatment corresponded with a reduction in hydrostatic root Lp(r) of cucumber roots by a factor of 24, and by a factor of 2 in figleaf gourd. In figleaf gourd, there was a reduction only in hydrostatic Lp(r) but not in osmotic Lp(r) suggesting that the activity of water channels was not much affected by LRT treatment in this species. Changes in cell Lp in response to chilling and recovery were similar to the root levels, although they were more intense at the root level. Activation energies (E(a)) and Q10 of water flow as measured at the cell level were high in cucumber (E(a)=109+/-13 kJ mol(-1); Q(10)=4.8+/-0.7; n=6-10 cells), but small in figleaf gourd (E(a)=11+/-2 kJ mol(-1); Q10=1.2+/-0.1; n=6-10 cells). Roots of figleaf gourd recovered better from LRT treatment than those of cucumber. In figleaf gourd, recovery (at both the root and cell level) often resulted in Lp and Lp(r) values which were even bigger than the original, i.e. there was an overshoot in hydraulic conductivity. These effects were larger for osmotic (representing the cell-to-cell passage of water) than for hydrostatic Lp(r). After a short-term (1 d) exposure to 8 degrees C followed by 1 d at 20 degrees C, hydrostatic Lp(r) of cucumber nearly recovered and that of figleaf gourd still remained higher due to the overshoot. By contrast, osmotic Lp(r) and cell Lp in both species remained high by a factor of 3 compared with the control, possibly due to an increased activity of water channels. After preconditioning of roots at LRT, increased hydraulic conductivity was completely inhibited by HgCl2 at both the root and cell levels. Different from figleaf gourd, recovery from chilling was not complete in cucumber after longer exposure to LRT. It is concluded that at LRT, both changes in the activity of aquaporins (AQPs) and alterations of root anatomy determine the water uptake in both species. The high temperature dependence of cell Lp in cucumber suggests conformational changes of AQPs during LRT treatment which result in channel closure and in a strong gating of AQP activity by low temperature. This mechanism is thought to be different from that in figleaf gourd where AQPs reacted in the conventional way, i.e. low temperature affected the mobility of water molecules in AQPs rather than their open/closed state, and Q(10) was low.     

A cohesion/tension mechanism explains the gating of water channels (aquaporins) in Chara internodes by high concentration

Isolated internodes of Chara corallina have been used to study the gating of aquaporins (water channels) in the presence of high concentrations of osmotic solutes of different size (molecular weight). Osmolytes were acetone and three glycol ethers: ethylene glycol monomethyl ether (EGMME), diethylene glycol monomethyl ether (DEGMME), and triethylene glycol monoethyl ether (TEGMEE). The 'osmotic efficiency' of osmolytes was quite different. Their reflection coefficients ranged between 0.15 (acetone), 0.59 (EGMME), 0.78 (DEGMME), and 0.80 (TEGMEE). Bulk water permeability (Lp) and diffusive permeabilities (Ps) of heavy water (HDO), hydrogen peroxide (H2O2), acetone, and glycol ethers (EGMME, DEGMME, and TEGMEE) were measured using a cell pressure probe. Cells were treated with different concentrations of osmotic solutes of up to 800 mM ( approximately 2.0 MPa of osmotic pressure). Inhibition of aquaporin activity increased with both increasing concentration and size of solutes (reflection coefficients). As cell Lp decreased, Ps increased, indicating that water and solutes used different passages across the plasma membrane. Similar to earlier findings of an osmotic gating of ion channels, a cohesion/tension model of the gating of water channels in Chara internodes by high concentration is proposed. According to the model, tensions (negative pressures) within water channels affected the open/closed state by changing the free energy between states and favoured a distorted/collapsed rather than the open state. They should have differed depending on the concentration and size of solutes that are more or less excluded from aquaporins. The bigger the solute, the lower was the concentration required to induce a reversible closure of aquaporins, as predicted by the model.     

Stimulus-induced downregulation of root water transport involves reactive oxygen species-activated cell signalling and plasma membrane intrinsic protein internalization

The water uptake capacity of plant roots (i.e. their hydraulic conductivity, Lp(r)) is determined in large part by aquaporins of the plasma membrane intrinsic protein (PIP) subfamily. In the present work, we investigated two stimuli, salicylic acid (SA) and salt, because of their ability to induce an accumulation of reactive oxygen species (ROS) and an inhibition of Lp(r) concomitantly in the roots of Arabidopsis plants. The inhibition of Lp(r) by SA was partially counteracted by preventing the accumulation of hydrogen peroxide (H(2)O(2)) with exogenous catalase. In addition, exogenous H(2)O(2) was able to reduce Lp(r) by up to 90% in <15 min. Based on the lack of effects of H(2)O(2) on the activity of individual aquaporins in Xenopus oocytes, and on a pharmacological dissection of the action of H(2)O(2) on Lp(r), we propose that ROS do not gate Arabidopsis root aquaporins through a direct oxidative mechanism, but rather act through cell signalling mechanisms. Expression in transgenic roots of PIP-GFP fusions and immunogold labelling indicated that external H(2)O(2) enhanced, in <15 min, the accumulation of PIPs in intracellular structures tentatively identified as vesicles and small vacuoles. Exposure of roots to SA or salt also induced an intracellular accumulation of the PIP-GFP fusion proteins, and these effects were fully counteracted by co-treatment with exogenous catalase. In conclusion, the present work identifies SA as a novel regulator of aquaporins, and delineates an ROS-dependent signalling pathway in the roots of Arabidopsis. Several abiotic and biotic stress-related stimuli potentially share this path, which involves an H(2)O(2)-induced internalization of PIPs, to downregulate root water transport.     

A re-examination of the minor role of unstirred layers during the measurement of transport coefficients of Chara corallina internodes with the cell pressure probe

The impact of unstirred layers (USLs) during cell pressure probe experiments with Chara corallina internodes has been quantified. The results show that the hydraulic conductivity (Lp) measured in hydrostatic relaxations was not significantly affected by USLs even in the presence of high water flow intensities ('sweep-away effect'). During pressure clamp, there was a reversible reduction in Lp by 20%, which was explained by the constriction of water to aquaporins (AQPs) in the C. corallina membrane and a rapid diffusional equilibration of solutes in arrays where water protruded across AQPs. In osmotic experiments, Lp, and permeability (Ps) and reflection (sigma s) coefficients increased as external flow rate of medium increased, indicating some effects of external USLs. However, the effect was levelling off at 'usual' flow rates of 0.20-0.30 m s(-1) and in the presence of vigorous stirring by air bubbles, suggesting a maximum thickness of external USLs of around 30 microm including the cell wall. Because the diameters of internodes were around 1 mm, internal USLs could have played a significant or even a dominating role, at least in the presence of the rapidly permeating solutes used [acetone, 2-propanol and dimethylformamide (DMF)]. A comparison of calculated (diffusion kinetics) and of measured permeabilities indicated an upper limit of the contribution of USLs for the rapidly moving solute acetone of 29%, and of 15% for the less rapidly permeating DME The results throw some doubt on recent claims that in C. corallina, USLs rather than the cell membrane dominate solute uptake, at least for the most rapidly moving solute acetone.     

氮磷亏缺对玉米根系水流导度的影响

在人工气候室水培条件下,从单根和整株根系两个层次研究了N、P营养与玉米(Zea mays L.)根系水流导度(root hydraulic conductivity,Lpr)间的关系。结果表明：表型抗旱的杂交种F1代户单4号和母本天四的单根水导和整株根系水导均高于不抗旱的父本478,其中天四的单根水导最高,而户单4号的整株根系水导最高。N、P亏缺均使玉米单根水导和整株根系水导降低,但与N亏块相比,P亏缺的植株具有较高的整株根系水导和较低的单根水导。整株根系的水导更能反映植物根系的输水性能。     

Effect of root anaerobiosis on the water relations of several Pyrus species

Solution culture experiments were designed to investigate the plant water relations of 3 Pyrus species subjected to root anaerobiosis. Root anaerobiosis induced partial stomatal closure prior to alterations in leaf water potential (ΨLW) or root osmotic potential (ΨRπ). In contrast, stomatal closure was accompanied by a decline in root hydraulic conductivity (Lp). Anoxia markedly reduced ΨLW for Pyrus communis L. and eventually led to wilting and defoliation. Pyrus betulaefolia Bunge and Pyrus calleryana Decne, however, were less affected by root anaerobiosis. To delineate if the increased root resistance was in the radial or longitudinal direction, 10⁻⁴ M cistrans abscisic acid (ABA) was added to detopped root systems of P. communis in solution culture after steady-state rates of Lp were established. A consistent 25 to 30% promotion of Lp was observed 1.5 h after the addition of ABA for aerobically treated plants. ABA did not influence Lp when applied to roots previously deprived of O₂ for 4 days. Additional evidence against the limiting resistance being in the radial direction was obtained when water fluxes were compared through intact P. communis roots, roots with all feeder roots detached, and stems without root systems. Severing feeder roots from anaerobically treated plants did not increase water flux to rates observed for aerobically treated plants. Resistance progressed basipetally to eventually encompass the stem itself. These results can only be explained by occlusion of the xylem vessels.     

Apoplastic barriers,aquaporin gene expression and root and cell hydraulic conductivity in phosphate-limited sheepgrass plants

Mineral nutrient supply can affect the hydraulic property of roots. The aim of the present work on sheepgrass (Leymus chinensis L.) plants was to test whether any changes in root hydraulic conductivity (Lp; exudation analyses) in response to a growth-limiting supply of phosphate (P) are accompanied by changes in (1) cell Lp via measuring the cell pressure, (2) the aquaporin (AQP) gene expression by performing qPCR and (3) the formation of apoplastic barriers, by analyzing suberin lamella and Casparian bands via cross-sectional analyses in roots. Plants were grown hydroponically on complete nutrient solution, containing 250 µM P, until they were 31–36 days old, and then kept for 2–3 weeks on either complete solution, or transferred on solution containing 2.5 µM (low-P) or no added P (no-P). Phosphate treatments caused significant decreases in root and cell-Lp and AQP gene expression, while the formation of apoplastic barriers increased, particularly in lateral roots. Experiments using the AQP inhibitor mercury (Hg) suggested that a significant portion of radial root water uptake in sheepgrass occurs along a path involving AQPs, and that the Lp of this path is reduced under low- and no-P. It is concluded that a growth-limiting supply of phosphate causes parallel changes in (1) cell Lp and aquaporin gene expression (decrease) and (2) apoplastic barrier formation (increase), and that the two may combine to reduce root Lp. The reduction in root Lp, in turn, facilitates an increased root-to-shoot surface area ratio, which allocates resources to the root, sourcing the limiting nutrient.     

Evidence that hydroxyl radicals mediate auxin-induced extension growth

Reactive oxygen intermediates, i.e. the superoxide radical (O*-)(2), hydrogen peroxide (H2O2) and the hydroxyl radical (*OH), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry may also play a useful role in polymer breakdown leading to wall loosening during extension growth of plant cells controlled by the phytohormone auxin. Backbone cleavage of cell wall polysaccharides can be accomplished in vitro by (*OH) produced from H2O2 in a Fenton reaction or in a reaction catalyzed by peroxidase supplied with O2 and NADH. Here, we show that coleoptile growth of maize seedlings is accompanied by the release of reactive oxygen intermediates in the cell wall. Auxin promotes release of (O*-)(2) and subsequent generation of (*OH)when inducing elongation growth. Experimental generation of (*OH) in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of (O*-)(2), H2O2 or (*OH), or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which (*OH), produced from (O*-)(2) and H2O2 by cell wall peroxidase, acts as a wall-loosening agent.     

Zinc treatment of hydroponically grown barley plants causes a reduction in root and cell hydraulic conductivity and isoform‐dependent decrease in aquaporin gene expression

The cellular and molecular basis of a reduction in root water uptake in plants exposed to heavy metals such as zinc (Zn) is poorly studied. The aim of the present study on hydroponically grown barley (Hordeum vulgare) was to test whether any reduction in root hydraulic conductivity (Lp) in response to Zn treatment is accompanied by a reduction in cell Lp and gene expression level of aquaporin (AQP) isoforms. Plants were grown in the presence of 0.25 μM, (control), 0.1 and 1 mM Zn in the root medium and analysed when they were 16–18 days old. Root and cell Lp was determined through exudation and cell pressure probe analyses, respectively, and gene expression of five candidate AQPs was analysed [real time quantitative polymerase chain reaction (PCR)]. Zinc treatments caused significant reductions (25–83%) in transpiration rate, root and shoot fresh weight, surface area and stomatal conductance. Zinc concentrations in tissues increased more than 100‐fold. Root Lp decreased by 24% (0.1 mM Zn) and 58% (1 mM Zn), while cell Lp decreased by 45 and 71%, respectively. Gene expression of AQPs decreased by 14–80%; decreases were statistically significant for HvPIP1;3, HvPIP2;4 and HvPIP2;5. Turgor in root cortex cells was not reduced by Zn treatments. It is concluded that reductions in plant water flow in response to Zn treatment are facilitated through decreases in root (Lp) and shoot (stomata) hydraulics. The decrease in root Lp is facilitated through reductions in cell Lp and AQP gene expression and may also reflect increased suberization in the endodermis.     

Production of reactive oxygen intermediates (O(2)(.-), H(2)O(2), and (.)OH) by maize roots and their role in wall loosening and elongation growth

Cell extension in the growing zone of plant roots typically takes place with a maximum local growth rate of 50% length increase per hour. The biochemical mechanism of this dramatic growth process is still poorly understood. Here we test the hypothesis that the wall-loosening reaction controlling root elongation is effected by the production of reactive oxygen intermediates, initiated by a NAD(P)H oxidase-catalyzed formation of superoxide radicals (O(2)(.-)) at the plasma membrane and culminating in the generation of polysaccharide-cleaving hydroxyl radicals ((.)OH) by cell wall peroxidase. The following results were obtained using primary roots of maize (Zea mays) seedlings as experimental material. (1) Production of O(2)(.-), H(2)O(2), and (.)OH can be demonstrated in the growing zone using specific histochemical assays and electron paramagnetic resonance spectroscopy. (2) Auxin-induced inhibition of growth is accompanied by a reduction of O(2)(.-) production. (3) Experimental generation of (.)OH in the cell walls with the Fenton reaction causes wall loosening (cell wall creep), specifically in the growing zone. Alternatively, wall loosening can be induced by (.)OH produced by endogenous cell wall peroxidase in the presence of NADH and H(2)O(2). (4) Inhibition of endogenous (.)OH formation by O(2)(.-) or (.)OH scavengers, or inhibitors of NAD(P)H oxidase or peroxidase activity, suppress elongation growth. These results show that juvenile root cells transiently express the ability to generate (.)OH, and to respond to (.)OH by wall loosening, in passing through the growing zone. Moreover, inhibitor studies indicate that (.)OH formation is essential for normal root growth.     

Allelochemical stress causes inhibition of growth and oxidative damage in Lycopersicon esculentum Mill

The aim of this study was to analyse the effect of allelochemical stress on Lycopersicon esculentum growth. Our results showed that allelochemical stress caused by Sicyos deppei aqueous leachate inhibited root growth but not germination, and produced an imbalance in the oxidative status of cells in both ungerminated seeds and in primary roots. We observed changes in activity of catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione reductase (GR) and the plasma membrane NADPH oxidase, as well as in the levels of H(2)O(2) and O(2) (*-) in seeds at 12 and 24 h, and in primary roots at 48 and 72 h of treatment, which could account for the oxidative imbalance. There were changes in levels of expression of the mentioned enzymes, but without a correlation with their respective activities. Higher levels of membrane lipid peroxidation were observed in primary roots at 48 and 72 h of treatment. No effect on the expression of metacaspase and the PR1 was observed as indicators of cell death or induction of plant defence. This paper contributes to the understanding of plant-plant interactions through the phytotoxic allelochemicals released in an aqueous leachate of the weed S. deppei, which cause a negative effect on other plants.     

Rapid accumulation of hydrogen peroxide in cucumber roots due to exposure to low temperature appears to mediate decreases in water transport

Water transport across root systems of young cucumber (Cucumis sativus L.) seedlings was measured following exposure to low temperature (LT, 8-13 degrees C) for varying periods of time. In addition, the amount of water transported through the stems was evaluated using a heat-balance sap-flow gauge. Following LT treatment, hydrogen peroxide was localized cytochemically in root tissue by the oxidation of cerium (III) chloride. The effects of hydrogen peroxide on the hydraulic conductivity of single cells (Lp) in root tissues, and on the H+-ATPase activity of isolated root plasma membrane, have been worked out. Cytochemical evidence suggested that exposure of roots to LT stress caused a release of hydrogen peroxide in the millimolar range in the vicinity of plasma membranes. In response to a low root temperature (8 degrees C), the hydraulic conductivity of the root (Lp(r)) decreased by a factor of 4, and the half-times of water exchange increased by a factor of 5-6. Decreasing root temperatures from 25-13 degrees C increased the half-times of water exchange in a cell by a factor of 6-9. The measurement of axial water transport with a heat-balance sap-flow gauge showed that only a small amount of water was transported when 8 degrees C was imposed on cucumber roots. Lp and the H+-ATPase activity of the isolated root plasma membrane were very sensitive to externally applied hydrogen peroxide at a concentration of 1-16 mM. These observations suggest that the accumulation of hydrogen peroxide appears to mediate decreases in water transport in cucumber roots under low temperature.     

Redox-related peroxidative responses evoked by methyl-jasmonate in axenically cultured aeroponic sunflower (Helianthus annuus L.) seedling roots

Methyl-jasmonate (MeJA) has been proposed to be involved in the evocation of defense reactions, as the oxidative burst in plants, substituting the elicitors or enhancing their effect. 48 h dark- and sterilely cultured (axenic) aeroponic sunflower seedling roots excised and treated with different concentrations of MeJA showed a strong and quick depression of the H(+) efflux rate, 1.80 microM MeJA totally stopping it for approximately 90 min and then reinitiating it again at a lower rate than controls. These results were wholly similar to those obtained with nonsterilely cultured roots and have been interpreted as mainly based on H(+) consumption for O(2)(*-) dismutation to H(2)O(2). Also K(+) influx was strongly depressed by MeJA, even transitorily reverting to K(+) efflux. These results were consistent with those associated to the oxidative burst in plants. MeJA induced massive H(2)O(2) accumulation in the middle lamella and intercellular spaces of both the root cap cells and the inside tissues of the roots. The native acidic extracellular peroxidase activity of the intact (nonexcised) seedling roots showed a sudden enhancement (by about 52%) after 5 min of MeJA addition, maintained for approximately 15 min and then decaying again to control rates. O(2) uptake by roots gave similar results. These and other results for additions of H(2)O(2) or horseradish peroxidase, diphenylene iodonium, and sodium diethyldithiocarbamate trihydrate to the reaction mixture with roots were all consistent with the hypothesis that MeJA induced an oxidative burst, with the generation of H(2)O(2) being necessary for peroxidase activity. Results with peroxidase activity of the apoplastic fluid were in accordance with those of the whole root. Finally, MeJA enhanced NADH oxidation and inhibited hexacyanoferrate(III) reduction by axenic roots, and diphenylene iodonium cancelled out these effects. Redox activities by CN(-)- preincubated roots were also studied. All these results are consistent with the hypothesis that MeJA enhanced the NAD(P)H oxidase of a redox chain linked to the oxidative burst, so enhancing the generation of O(2)(*-) and H(2)O(2), O(2) uptake, and peroxidase activity by roots.     

The ApoCorrect assay: a novel, rapid method to determine the biological functionality of radiolabeled and fluorescent Annexin A5

N-[4-(3)H]Benzoylglycylglycylglycine ([(3)H]BzG(3)) was tested as a probe for detecting hydroxyl radicals (*OH). Aerated solutions of l-ascorbate generated *OH, which oxidized [(3)H]BzG(3), yielding hydrophilic (probably hydroxylated) derivatives plus tritiated water. The (3)H(2)O was separated from organic products and remaining [(3)H]BzG(3) on Dowex-1. (3)H(2)O production was much greater with *OH than with other reactive oxygen species (ROS) (e.g., H(2)O(2), superoxide). The slight (3)H(2)O production in the presence of H(2)O(2) or superoxide was blocked by *OH scavengers (e.g., glycerol, mannitol, butan-1-ol) that do not scavenge H(2)O(2) or superoxide. This indicates that (3)H(2)O production was caused by *OH and that other ROS only generated any (3)H(2)O by forming traces of *OH. Doses of *OH that caused detectable nonenzymic polysaccharide scission also caused (3)H(2)O production, indicating that [(3)H]BzG(3) is a sensitive *OH probe in studies of polymer scission. The ability of scavengers and chelators to protect against ascorbate-mediated polysaccharide scission paralleled their ability to inhibit concurrent (3)H(2)O production, indicating that both processes were due to *OH. Thus, [(3)H]BzG(3) is a simple, specific, sensitive, and robust probe for detecting *OH production in vitro. It may have applications for in vivo detection of extracellular *OH in arthritic joints and of apoplastic *OH in plant cell walls.     

A Negative Hydraulic Message from Oxygen-Deficient Roots of Tomato Plants? (Influence of Soil Flooding on Leaf Water Potential,Leaf Expansion,and Synchrony between Stomatal Conductance and Root Hydraulic Conductivity)

Four to 10 h of soil flooding delayed and suppressed the normal daily increase in root hydraulic conductance (Lp) in tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) plants. The resulting short-term loss of synchrony between Lp and stomatal conductance decreased leaf water potential ([psi]L) relative to well-drained plants within 2 h. A decrease in [psi]L persisted for 8 h and was mirrored by decreased leaf thickness measured using linear displacement transducers. After 10 h of flooding, further closing of stomata and re-convergence of Lp in flooded and well-drained roots returned [psi]L to control values. In the second photoperiod, Lp in flooded plants exceeded that in well-drained plants in association with much increased Lp and decreased stomatal conductance. Pneumatic balancing pressure applied to roots of intact flooded plants to prevent temporary loss of [psi]L in the 1st d did not modify the patterns of stomatal closure or leaf expansion. Thus, the magnitude of the early negative hydraulic message was neither sufficient nor necessary to promote stomatal closure and inhibit leaf growth in flooded tomato plants. Chemical messages are presumed to be responsible for these early responses to soil flooding.     

Structural insights into the Aedes aegypti aquaporins and aquaglyceroporins – an in silico study

There has been a fair bit of understanding on the structure–function relationship of Aquaporins (AQPs) from plants and vertebrates obtained from available X-ray crystallography data. However, there is a lacuna in understanding the structure of AQPs from sanguinivorous insects like the mosquito where it plays a crucial role in survival. In this study, we have built homology models for the Aedes aegypti AQPs, identified key channel lining residues and compared the structure and sequence with orthodox AQPs. Although Ar/R filter residues of AaAQP1 were exactly similar to orthodox AQPs, AaAQP2 has a substitution at LE1position possibly making it less efficient in high capacity water transport. The huge difference in the selectivity filter region of AaAQP3 suggests a different transport property for this channel. The changes observed in the H5 position of the filter of AaAQP4 and AaAQP5 may explain the presence of a larger pore aperture to permit the passage of larger solute molecules. AaAQP6 possesses a completely hydrophobic filter like that in mammalian super aquaporins. The identified key residues are pivotal in understanding the mechanism of action and gating of these channels.     

Exogenous cadaverine induces oxidative burst and reduces cadaverine conjugate content in the common ice plant

The effect of free cadaverine (Cad) on its conjugates formation was analyzed in roots of the common ice plants (Mesembryanthemum crystallinum L.). It was found for the first time that Cad could induce oxidative burst in the roots of adult plants, as was evident from the sharp decrease in the content of Cad soluble or insoluble conjugates. This unusual effect was associated with the increased oxidative degradation of exogenous Cad (1mM, 1.5h) and intense H(2)O(2) production in the root cells of adult plants. Root treatment of both juvenile and adult plants with H(2)O(2) (1mM, 1.5h) reduced the content of soluble Cad conjugates and increased the content of their components, free Cad and phenols. We also found that one of the possible reasons of the negative effect of exogenous diamine on the formation of conjugated forms in adult roots was alkalization of the root apoplast at Cad addition to nutrient medium and the unusual O(2)(-) synthase function as a pH-dependent guaiacol peroxidase in the presence of a high content of H(2)O(2). This was confirmed by the data on the accumulation of O(2)(-) and enhanced superoxide dismutase activity in adult roots under treatment with Cad. It is possible that the accumulation of O(2)(-) together with H(2)O(2) was also responsible for oxidative burst, which induced a decrease in the content of Cad conjugates in adult roots of the common ice plants.     

Biochemistry of the oxidation of lignin by Phanerochaete chrysosporium

The objective of this research was to identify the biochemical agents responsible for the oxidative degradation of lignin by the white-rot fungus Phanerochaete chrysosporium. We examined the hypothesis that activated oxygen species are involved, and we also sought the agent in ligninolytic cultures responsible for a specific oxidative degradative reaction in substructure model compounds. Results of studies of the production of activated oxygen species by cultures, of the effect of their removal on ligninolytic activity, and of their action on substructure model compounds support a role for hydrogen peroxide (H(2)O(2)) and possibly superoxide (O(2)(*)(-)) in lignin degradation. Involvement of hydroxyl radical (*OH) or singlet oxygen (1O(2)) is not supported by our data. The actual biochemical agent responsible for one important oxidative C-C bond cleavage reaction in non-phenolic lignin substructure model compounds, and in lignin itself, was found to be an enzyme. The enzyme is extracellular, has a molecular weight of 42,000 daltons, is azide-sensitive, and requires H(2)O(2) for activity.     

Comparison between gradient-dependent hydraulic conductivities of roots using the root pressure probe: the role of pressure propagations and implications for the relative roles of parallel radial pathways

Hydrostatic pressure relaxations with the root pressure probe are commonly used for measuring the hydraulic conductivity (Lp(r)) of roots. We compared the Lp(r) of roots from species with different root hydraulic properties (Lupinus angustifolius L. 'Merrit', Lupinus luteus L. 'Wodjil', Triticum aestivum L. 'Kulin' and Zea mays L. 'Pacific DK 477') using pressure relaxations, a pressure clamp and osmotic gradients to induce water flow across the root. Only the pressure clamp measures water flow under steady-state conditions. Lp(r) determined by pressure relaxations was two- to threefold greater than Lp(r) from pressure clamps and was independent of the direction of water flow. Lp(r) (pressure clamp) was two- to fourfold higher than for Lp(r) (osmotic) for all species except Triticum aestivum where Lp(r) (pressure clamp) and Lp(r) (osmotic) were not significantly different. A novel technique was developed to measure the propagation of pressure through roots to investigate the cause of the differences in Lp(r). Root segments were connected between two pressure probes so that when root pressure (P(r)) was manipulated by one probe, the other probe recorded changes in P(r). Pressure relaxations did not induce the expected kinetics in pressure in the probe at the other end of the root when axial hydraulic conductance, and probe and root capacitances were accounted for. An electric circuit model of the root was constructed that included an additional capacitance in the root loaded by a series of resistances. This accounted for the double exponential kinetics for intact roots in pressure relaxation experiments as well as the reduced response observed with the double probe experiments. Although there were potential errors with all the techniques, we considered that the measurement of Lp(r) using the pressure clamp was the most unambiguous for small pressure changes, and provided that sufficient time was allowed for pressure propagation through the root. The differences in Lp(r) from different methods of measurement have implications for the models describing water transport through roots and the potential role of aquaporins.     

Impact of the auxin signaling inhibitor p-chlorophenoxyisobutyric acid on short-term Cd-induced hydrogen peroxide production and growth response in barley root tip

Short-term treatment (30min) of barley roots with a low 10μM Cd concentration induced significant H(2)O(2) production in the elongation and differentiation zone of the root tip 3h after treatment. This elevated H(2)O(2) production was accompanied by root growth inhibition and probably invoked root swelling in the elongation zone of the root tip. By contrast, a high 60μM Cd concentration induced robust H(2)O(2) production in the elongation zone of the root tip already 1h after short-term treatment. This robust H(2)O(2) generation caused extensive cell death 6h after short-term treatment. Similarly to low Cd concentration, exogenously applied H(2)O(2) caused marked root growth inhibition, which at lower H(2)O(2) concentration was accompanied by root swelling. The auxin signaling inhibitor p-chlorophenoxyisobutyric acid effectively inhibited 10μM Cd-induced root growth inhibition, H(2)O(2) production and root swelling, but was ineffective in the alleviation of 60μM Cd-induced root growth inhibition and H(2)O(2) production. Our results demonstrated that Cd-induced mild oxidative stress caused root growth inhibition, likely trough the rapid reorientation of cell growth in which a crucial role was played by IAA signaling in the root tip. Strong oxidative stress induced by high Cd concentration caused extensive cell death in the elongation zone of the root tip, resulting in the cessation of root growth or even in root death.