Cell Wall Accumulation of Cu Ions and Modulation of Lignifying Enzymes in Primary Leaves of Bean Seedlings Exposed to Excess Copper

Copper is both a nutrient and an environmental toxin that is taken up by plants. In order to determine the subcellular localization of copper and to assess the resulting metabolic changes, we exposed 14-day-old bean seedlings to nutrient solutions containing varying concentrations of Cu²⁺ ions for 3 days. Biochemical analyses revealed that the cell wall was the major site of Cu²⁺ accumulation in the leaves of treated plants. Excess copper modified the activity of lignifying peroxidases in both soluble and ionic cell wall-bound fraction. The activity of ionic GPX (guaiacol peroxidase, EC 1.11.1.7) was increased by 50 and 75 μM CuSO_4. The activities of both ionic CAPX (coniferyl alcohol peroxidase, EC 1.11.1.4) and NADH oxidase were increased by both copper concentrations tested. While soluble CAPX activity decreased in leaves treated by all copper concentrations tested, the activity of soluble NADH oxidase remained unchanged at 50 μM and was enhanced at 75 μM. Treatment with CuSO₄ also increased the abundance of total phenol compounds and induced stimulation in the activity of PAL (phenylalanine ammonia lyase, EC. 4.3.1.5). Using histochemistry in combination with fluorescence microscopy we show that bean leaves from copper-exposed plants displayed biochemical and structural modifications reinforcing the cell walls of their xylem tissues. On the other hand, the perivascular fiber sclerenchyma appeared to be less developed in treated leaves.     

New Insights into Regulation of Proteome and Polysaccharide in Cell Wall of Elsholtzia splendens in Response to Copper Stress

Background and Aims

Copper (Cu) is an essential micronutrient for plants. However, excess amounts of Cu are toxic and result in a wide range of harmful effects on the physiological and biochemical processes of plants. Cell wall has a crucial role in plant defense response to toxic metals. To date, the process of cell wall response to Cu and the detoxification mechanism have not been well documented at the proteomic level.

Methods

An recently developed 6-plex Tandem Mass Tag was used for relative and absolute quantitation methods to achieve a comprehensive understanding of Cu tolerance/detoxification molecular mechanisms in the cell wall. LC–MS/MS approach was performed to analyze the Cu-responsive cell wall proteins and polysaccharides.

Key Results

The majority of the 22 up-regulated proteins were involved in the antioxidant defense pathway, cell wall polysaccharide remodeling, and cell metabolism process. Changes in polysaccharide amount, composition, and distribution could offer more binding sites for Cu ions. The 33 down-regulated proteins were involved in the signal pathway, energy, and protein synthesis.

Conclusions

Based on the abundant changes in proteins and polysaccharides, and their putative functions, a possible protein interaction network can provide new insights into Cu stress response in root cell wall. Cu can facilitate further functional research on target proteins associated with metal response in the cell wall.     

Changes in Thiol Content in Roots of Wheat Cultivars Exposed to Copper Stress

Wheat (Triticum aestivum L.) cultivars GK Tiszatáj, Yubileinaya, GK Öthalom and a landrace Kobomugi were grown for 18 d in hydroponic cultures containing 0 (control), 0.1, 1.0 or 10.0 µM Cu²⁺. On a dry mass basis, cvs. Tiszatáj and Kobomugi accumulated slightly more Cu²⁺ in the root tissues than did cvs. Yubileinaya and Öthalom, but their controls also contained higher amounts of Cu²⁺. As a result of perturbation in the plasma membrane functions the K⁺ content of roots was reduced at 10 µM Cu²⁺ in all cultivars, whereas the K⁺/Na⁺ ratio decreased significantly only in the roots of cv. Öthalom. In the sensitive cultivar, Öthalom, the dry mass of the roots decreased while the cysteine content, which is a limiting factor for glutathione synthesis, did not satisfactorily increase with increasing tissue Cu²⁺ content. This suggests that in cv. Öthalom the membrane damage of the root cells at 10 µM Cu²⁺ concentration may affect the sulphur availability or metabolism. Concentrations of glutathione and hydroxymethyl-glutathione, a tripeptide which may play a similar biochemical role to glutathione, were also lower in the sensitive cultivar. In the absence of glutathione the root tissues failed to cope with the oxidative stress caused by the excessive amount of Cu²⁺. A significant accumulation of iron in the roots of the sensitive cultivar at 10 µM Cu²⁺ supply enhanced the oxidative damage.     

Changes in Cell Wall Polysaccharides of Green Bean Pods during Development

The changes in cell wall polysaccharides and selected cell wall-modifying enzymes were studied during the development of green bean (Phaseolus vulgaris L.) pods. An overall increase of cell wall material on a dry-weight basis was observed during pod development. Major changes were detected in the pectic polymers. Young, exponentially growing cell walls contained large amounts of neutral, sugar-rich pectic polymers (rhamnogalacturonan), which were water insoluble and relatively tightly connected to the cell wall. During elongation, more galactose-rich pectic polymers were deposited into the cell wall. In addition, the level of branched rhamnogalacturonan remained constant, while the level of linear homogalacturonan steadily increased. During maturation of the pods, galactose-rich pectic polymers were degraded, while the accumulation of soluble homogalacturonan continued. During senescence there was an increase in the amount of ionically complexed pectins, mainly at the expense of freely soluble pectins. The most abundant of the enzymes tested for was pectin methylesterase. Peroxidase, beta-galactosidase, and alpha-arabinosidase were also detected in appreciable amounts. Polygalacturonase was detected only in very small amounts throughout development. The relationship between endogenous enzyme levels and the properties of cell wall polymers is discussed with respect to cell wall synthesis and degradation.     

Responses of Proteolytic Enzymes in Embryonic Axes of Germinating Bean Seeds under Copper Stress

The changes in protease activities in embryonic axes during the first days of bean (Phaseolus vulgaris L.) seed germination were investigated in response to copper stress. Synthetic substrates and specific protease inhibitors have been used to define qualitatively and quantitatively different catalytic classes, particularly endoproteases (EP), carboxypeptidases (CP) and aminopeptidases (AP), then identify which ones were affected in the presence of copper. In fact, a failure in storage proteins mobilization and a disorder of nitrogen supply at enzymatic level occurred in Cu. In fact, Cu inhibited azocaseinolytic activity (ACA) and cysteine-, aspartic-, serine-, and metallo-endopeptidases activities (Cys-EP, Asp-EP, Ser-Ep, and Met-EP, respectively). Besides, Cu affected leucine- and proline-aminopeptidases (LAP and PAP, respectively) and glycine-carboxypeptidases (Gly-CP). The proteolytic responses might also be associated with the decrease in defense capacity in the Cu-treated embryos.     

果实软化的胞壁物质和水解酶变化

果实软化通常被认为是由于胞壁水解酶如多聚半乳糖醛酸酶,果胶酯酶,纤维素酶降解胞壁物质导致。本文概述了这三种酶分子与果实软化关系的研究进展。反义基因证明,这三种酶基因的任一种表达被报制,果实能够正常软化,暗示果实的软化有其它因子的参与。其中由细胞内的淀粉酶和蔗糖酶引起的细胞膨压的变化及果胶的溶解可能是引起果肉软化的重要原因。     

Response to Phosphate Deficiency in Bean (Phaseolus vulgarisL.) Roots. Respiratory Metabolism, Sugar Localization and Changes in Ultrastructure of Bean Root Cells

Bean plants (Phaseolus vulgarisL. ‘Zlota Saxa’)were cultured on complete (+P) or phosphate-deficient (-P) nutrientmedium. A large increase in glucose concentration was foundin the meristematic zone of -P roots compared to control roots.The increased glucose concentration in the meristematic zonedid not influence total respiration rate. Glucose or uncoupler(carbonyl cyanide m-chlorophenylhydrazone) failed to increasethe respiration rate in -P root segments, but stimulated respirationin +P roots. The ultrastructure of cortical cells from the meristematicroot zone showed marked differences between +P and -P plants.Large vacuoles, invaginations of the plasmalemma and condensedforms of mitochondria were dominating features in cortical cellsof -P roots. Analysis of extracts after treating roots withdimethylsulfoxide (DMSO) indicated different localization ofsugars in the cell compartments. In roots of -P plants, mostof the reducing sugars were detected in the cytoplasm fractionwhile most sucrose was in the vacuole. Observations of the effectof 10% DMSO on cell ultrastructure indicated partial destructionof the plasmalemma but not the tonoplast. The localization ofreducing sugars in secondary vacuoles or plasmalemma invaginationsin the cells from the meristematic region of -P roots is discussed.Copyright1998 Annals of Botany Company. Bean (Phaseolus vulgarisL.), roots, Pi deficiency, respiration, meristematic zone, ultrastructure, sugar efflux, reducing sugars and sucrose localization.     

Surface Stress Induces a Conserved Cell Wall Stress Response in the Pathogenic Fungus Candida albicans

The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using ¹⁵N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the β-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.     

Changes in Stomatal Behavior and Guard Cell Cytosolic Free Calcium in Response to Oxidative Stress

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicals, and (b) H2O2. Both methyl viologen and H2O2 inhibited stomatal opening and promoted stomatal closure. At concentrations [less than or equal to]10-5 M, the effects of methyl viologen and H2O2 on stomatal behavior were reversible and were abolished by 2 mM EGTA or 10 [mu]M verapamil. In addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by EGTA or verapamil, methyl viologen and H2O2 caused an increase in guard cell cytosolic free Ca2+ ([Ca2+]i), which was abolished in the presence of EGTA. Therefore, at low concentrations of methyl viologen and H2O2, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatal aperture and the associated increases in [Ca2+]i. This suggests that in this concentration range the effects of the treatments are Ca2+-dependent and are mediated by changes in [Ca2+]i. In contrast, at concentrations of methyl viologan and H2O2 > 10-5 M, EGTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H2O2 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+]i and the possible disruption of guard-cell Ca2+ homeostasis are discussed in relation to the processes of Ca2+-based signal transduction in stomatal guard cells and the control of stomatal aperture.     

低温胁迫下西番莲叶片的生理反应及超微结构变化

以西番莲扦插苗为实验材料,比较预冷处理和常温处理(对照)情况下,西番莲叶片对-6℃低温胁迫的生理反应及叶肉细胞的超微结构变化,以探讨预冷处理对西番莲的抗寒作用及其机理。结果显示:(1)常温处理组在-6℃低温胁迫处理后,西番莲叶片的质膜相对透性、MDA含量随着低温处理时间的延长而逐渐增加,而预冷处理组这两个指标在处理期间都比常温处理组低;(2)常温处理组叶片H2O2含量在48h时达到峰值后出现下降,而预冷处理组的H2O2含量都低于对照,在48h时对照的H2O2含量比预冷锻炼的高1.14倍;(3)常温处理组的脯氨酸含量在24h内增加以后出现下降,而预冷处理组在处理期间逐渐增加且高于对照;(4)常温处理组的POD活性在12h时最高,CAT、SOD活性在24h时最高,以后出现下降。预冷处理组提高了3个酶的活性,其中POD在12h时是对照的2.73倍,CAT、SOD活性在24h时比对照分别提高18.7%和42.7%。(5)超微结构研究显示,预冷处理组在低温胁迫72h时虽出现叶绿体片层肿胀、核膜消失、质膜内陷等症状,但叶绿体伤害症状轻于常温处理。研究表明,低温预冷处理能够提高西番莲的抗寒性。     

Effects of Periodic- and Transient-Osmotic Stress on Electric Potentials in Bean Roots

Intact bean (Phaseolus mungo) roots were subjected to periodic-and transient-osmotic stress by treatment with a solution oflow water potential °. Both the intracellular potential,E, and the extracellular potential, V, were measured with amultimicrochamber system that combined intracellular microelectrodesand external electrodes. In elongating regions, ° simultaneouslyaffected potentials E and V. The response of E to ° couldbe presented by a transfer function that included dead timeand first order elements. These elements were consistent withthe emergence of an electromotive force, produced by respiration,between the cortex and the stele in the elongating region. (Received January 14, 1983; Accepted June 23, 1983)     

Changes of Cell Wall Composition and Polymer Size in Primary Roots of Cotton Seedlings Under High Salinity

The aim of this study was to investigate changes in cell wallchemical composition and polymer size in the root tip of intactcotton seedlings (Gossypium hirsutum L. cv. Acala SJ-2) grownin saline environments, in order to relate the interaction betweenhigh salinity and root growth to possible changes in cell wallmetabolism. Cotton seedlings were grown in modified Hoagland nutrient solutionwith various combinations of NaCl and CaCl₂. Cell walls werefractionated into four fractions (pectin, hemicellulose 1 and2, cellulose), and analysed for their total sugar content, neutralsugar composition and size of polysaccharides. At 1 mol m^–3Ca, 150 mol m^–3 NaCl resulted in a significant increasein the cell wall uronic acid content, but a reduction in cellulosecontent on a per unit dry weight basis. Supplemental Ca overcamethe inhibitory effect of high Na on cellulose content. The neutralsugar composition of the cell wall fractions showed no majorchanges caused by varied Na/Ca ratios. Determinations of polysaccharidepolymer size showed that high Na at 1 mol m^–3 Ca led toan increase in the amount of polysaccharides of intermediatemolecular size and a decrease in that of small size in the hemicellulose1 fraction, indicating a possible inhibition of polysaccharidedegradation by high Na. This change was not observed in the10 mol m^–3 Ca treatments. The results reveal a relationshipbetween the effects of high salinity on root growth and cellwall metabolism, particularly in regard to cellulose biosynthesis Key words: Gossypium hirsutum, salinity, root, cell wall     

Cytochemical Localization of Hydrogen Peroxide in Lignifying Cell Walls

The presence of endogenous H₂O₂ was demonstrated at the electronmicroscope level with the CeCl₃ technique in lignifying cellwalls of poplar. Reaction product was precisely located in thevery same walls which could oxidize hydrogen donors in the absenceof exogeneous H₂O₂. As evidenced by these results, peroxidasesare truly involved in the polymerisation of lignin monomerseven if other oxidases may participate in this biosyntheticpathway.Copyright 1993, 1999 Academic Press Cytochemistry, hydrogen peroxide, lignification, peroxidase, polyphenoloxidase, Populus x euramericana, poplar     

Response to Water Stress and Recovery of Nitrate-Fed and Nitrogen-Fixing Faba Bean

Comparative studies of response mechanisms to progressive waterstress were carried out in vegetative faba bean plants. Thesewere grown under controlled environmental conditions and eitherreceived nitrate-N (+ N) or were dependent on N₂-fixation (N₂).N₂-fixing plants reacted faster to water stress by increasedroot growth but were unable to maintain this response. +N plantsshowed a slower response but were able to maintain root andshoot growth throughout the treatment period. Leaf expansionwas similarly affected in +N and N₂ plants but there was anincrease of specific leaf weight in the former. In both +N andN₂ plants response mechanisms to water stress were aimed atpostponing dehydration: they tolerated stress at high waterpotentials by maintaining water absorption with increased rootgrowth and early stomatal closure. Recovery after watering thestressed plants was faster in +N plants. Stomatal resistancein leaves of N₂ plants attained similar values to those of control,non-stressed, plants after 68–82 h compared with only20 h in + N plants. Key words: Faba bean, water stress, nitrogen nutrition     

Effect of Auxin on Cell Wall Degrading Enzymes

The effect of auxin on the activities of amylase, cellulase, β-1, 3- and/or β-l, 6-glucanase and hemieellulase were observed using etiolated barley coleoptile and pea epicotyl internode segments. The activities of β-1, 3- and/or β-l, 6-glueanase and hemicellulase of barley were increased by indole-3-acetic acid in a 3 hours' treatment. Amylase activity was not influenced by the auxin. Cellulase activity was not detected under the experimental conditions. 2, 4-Dichlorophenoxyacetic acid increased hemicellulase activity, but not cellulase and amylase activities, in pea epicotyl segments in 3 hours. Fungal β-1, 3-glucanase exogenously applied induced the elongation of barley coleoptile segments. The elongation induced by the enzyme was as high as that induced by indole-3-acetic acid at least for the first 1 to 3 hours.     

Cell Wall Degrading Enzymes in Cuscuta reflexa and Its Hosts

Pectin degrading enzymes, hemicellulose degrading enzyme andcellulose degrading enzymes were studied in Cuscuta reflexaRoxb., its susceptible hosts, Brassica campestris L., Cocciniaindica W. & A. Datura innoxia Mill, Helianthus annuus L.,Holoptelea indica Planch, Lantana camara L., Medicago sativaL., Manihot utilissima Pohl, Petunia hybrida X Hort exvilm,Pisum sativum L., Phaseolus vulgaris L. and Solanum nigrum L.and non-susceptible plants Ipomoea batata Lam. and Solanum tuberosumL. Pectin esterase and polygalacturonase were present in higheramounts in Cuscuta parasitic on P. vulgaris and S. nigrum, whichneeded more time for haustorial establishment. Exo-l, 4-ß-D-glucosidaseactivity was found in Cuscuta but could not be detected in itshosts. Xylanase and cellulase activity of host plants increasedwhile cellobiase activity decreased as a result of infectionby the parasite. Higher pectin esterase, polygalacturonase,xylanase and exo-l, 4-ß-D-glucosidase activities inthe haustorial region of the parasite is likely to bring aboutthe lysis of the cell wall of the host plant and thus facilitatethe penetration of the parasite haustoria into the host sieveelement, which is necessary for the transport of nutrients betweenthe host and the parasite. Key words: Cell wall degrading enzymes, Cuscuta reflexa     

Alternaria Brassicae Induced Changes in the Activity of Cell Wall Degrading Enzymes in Leaves of Brassica Juncea

After inoculation of Brassica juncea leaves with Alternaria brassicae, activities of the cell wall degrading enzymes, polygalacturonase (EC 3.2.1.15) and cellulase (EC 3.2.1.4) decreased in leaf blight resistant cultivar RC-781 and increased in the susceptible cultivar Varuna upto 3 d. In the leaves of both the cultivars 11 poly-peptides were observed in the absence of A. brassicae inoculation. After inoculation in the resistant cultivar RC-781 there was no change in the polypeptide pattern, while in the susceptible cultivar Varuna, four polypeptides (43.7 to 58.8 kDa) disappeared only at 3^rd day after inoculation. This revised version was published online in June 2006 with corrections to the Cover Date.