Peroxidase-mediated conjugation of glutathione to unsaturated phenylpropanoids. Evidence against glutathione S-transferase involvement

A number of plant species are thought to possess a glutathione S-transferase enzyme (GST: EC 2.5.1.18) that will conjugate glutathione (GSH) to trans -cinnamic acid (CA) and para -coumaric acid (4-CA). However, we present evidence that this activity is mediated by peroxidase enzymes and not GSTs. The N-terminal amino acid sequence of the GSH-conjugating enzyme purified from etiolated corn shoots exhibited a strong degree of homology to cytosolic ascorbate peroxidase enzymes (APX: EC 1.11.1.11) from a number of plant species. The GSH-conjugating and APX activities of corn could not be separated during chromatography on hydrophobic-interaction. anion-exchange, and gel filtration columns. Spectral analysis of the enzyme revealed that the protein had a Soret band at 405 nm. When the enzyme was reduced with dithionite, the peak was shifted to 423 nm with an additional peak at 554 nm. The spectrum of the dithionite-reduced enzyme in the presence of 0.1 m M KCN exhibited peaks at 430, 534 and 563 nm. These spectra are consistent with the presence of a heme moiety. The GSH-conjugating and APX activities of the enzyme were both inhibited by KCN. NaN₃, p -chloromercuribenzoate ( p CMB), and iodoacetate. The APX specific activity of the enzyme was 1.5-fold greater than the GSH-conjugating specific activity with 4-CA. In addition to the corn enzyme, a pea recombinant APX (rAPX) and horseradish peroxidase (HRP; EC 1.11.1.7) were also able to conjugate GSH to CA and 4-CA. The peroxidase enzymes may generate thiyl free radicals of GSH that react with the alkyl double bond of CA and 4-CA resulting in the formation of a GSH conjugate.     

Characterization of protochlorophyllide and protochlorophyllide esters in roots of dark-grown plants

The protochlorophyll pools of roots of dark-grown wheat ( Triticum aestivum L. cv. Walde), maize ( Zea mays L. cv. Goldcrest) and wrinkledseeded pea ( Pisum sativum L. ssp. sativurh cv. Kelvedon Wonder) were investigated by high performance liquid chromatography (HPLC) and low temperature fluorescence spectroscopy. All roots contained protochlorophyllide and esterified protochlorophyllides (protochlorophylls) but with considerably larger relative amounts of the latter compared with etiolated leaves. The alcohol moieties of the 4 detected protochlorophylls were geranylgeraniol (GG), dihydrogeranylgeraniol (DHGG), tetrahydrogeranylgeraniol (THGG) and phytol. The relative amounts of the different protochlorophylls varied between the species. Protochlorophyllide and the 4 protochlorophylls all contained monovinyl forms. The divinyl forms could not be detected by our instruments. Wrinkledseeded pea contained in addition chlorophyll a , some unidentified chlorophylls and negligible amounts of chlorophyllide. Small amounts of carotenoids were found in roots of all investigated species. The carotenoids were the same as those found in green or etiolated leaves, but present in different relative amounts.     

Molecular characterization of corn glutathione S-transferase isozymes involved in herbicide detoxication

Corn ( Zea mays L.) glutathione S-transferases (EC 2.5.1.18) have attracted interest, in part, due to their involvement in the metabolism of several herbicides, including atrazine and alachlor. Three corn, glutathione S-transferases have been purified, and cDNA clones have been isolated and sequenced for two of these, GST I and GST III. In addition to showing some amino acid sequence similarity to each other, the two sequenced corn glutathione S-transferases also show some similarity to rat and human enzymes. The corn glutathione S-transferases responsible for atrazine tolerance have not yet been purified or cloned, but purification attempts indicate that corn has two glutathione S-transferases with activity towards atrazine. While many glutathione S-transferases from various organisms have been detected by using 1-chloro-2,4-dinitrobenzene as a substrate, the atrazine-specific glutathione S-transferases have very little or no activity with 1-chloro-2,4-dinitrobenzene. This shows the importance of assaying with a variety of substrates when characterizing glutathione S-transferases.     

Influence of selenium on uptake and toxicity of copper and cadmium in pea (Pisum sativum) and wheat (Triticum aestivum)

The detoxifying effect of selenium on animals toxicated with heavy metals is well known. In this study we examine if there is a similar effect in plants. Wheat ( Triticum aestivum L. cv. Sunny) and pea ( Pisum sativum L. cv. Fenomen) were grown for 21 days on a nutrient solution based on the nutrient proportions in healthy plants. Nutrients along with cadmium, copper, selenite, selenate or selenite and selenate in combinations with copper or cadmium were supplied in small amounts with a daily incremental increase of 0.12 (wheat) and 0.20 (pea). The metal and selenium uptake and distribution in the plants as well as the effects on growth were investigated.
The results show that selenium does not reduce the toxicity of heavy metals to plants. Instead, selenium enhances metal uptake and toxicity, especially in peas grown in the presence of metal and selenate. Selenite increased cadmium concentrations of pea roots up to 300% and selenate that of wheat shoots up to 50%.     

Tribology of the root cap in maize (Zea mays) and peas (Pisum sativum)

Frictional resistance to a penetrating body can account for more than 80% of the total resistance to penetration of soil. We measured the frictional resistance between growing root caps of maize and pea and ground and smooth glass surfaces, which was linearly correlated to load, allowing calculation of the coefficient of kinetic friction and adhesion. Coefficients of kinetic friction between the root caps and the ground and smooth glass surfaces were approximately 0.04 and 0.02, respectively, the first measurements of the frictional properties of root tips at rates approaching those of root elongation, and an order of magnitude smaller than those previously reported. Results suggest that roots are well designed for penetrating soil, and encounter only small frictional resistance on the root cap. These data provide important parameters for modelling soil stresses and deformation around growing root tips.     

Cold-Shock 310-kD Protein Uncouples Oxidative Phosphorylation in Plant Mitochondria

We studied the effects of cold-shock 310-kD protein (CSP310) isolated from winter rye seedlings on the energetic activity of plant mitochondria. CSP310 was shown to enhance nonphosphorylating respiration and uncoupled oxidative phosphorylation in isolated mitochondria. The uncoupling effect was enhanced with increasing protein concentration. An antibody against CSP310 interfered with the uncoupling effect of CSP310. Free fatty acids were not evidently involved in uncoupling. The physiological role of uncoupling between oxidation and phosphorylation during plant adaptation to low temperatures is discussed.     

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.     

Methionyl sulfoxide content and protein-methionine-S-oxide reductase activity in response to water deficits or high temperature

Cellular injury resulting from partially reduced oxygen species (superoxide, peroxides and/or hydroxyl radicals) or singlet oxygen frequently increases during environmental stress. Because protein methionine residues are susceptible to oxidation, we investigated the effects of water-deficit stress and high temperature stress on the content of oxidized methionyl residues [Met(O)] in leaves. Leaf proteins from water-deficit-stressed cotton ( Gossypium hirsutum L. cv. Paymaster HS-26). pea ( Pisum sativum L. cv. Progress No. 9). wheat ( Triticum aestivum L. em. Thell. cv. Len) and potato ( Solanum tuberosum L. cv. Norgold M) and from the leaves of high-temperature-stressed pea seedlings were evaluated. The activity of protein methionine-S-oxide reductase (PrMSR). an enzyme responsible for re-reducing oxidized methionyl residues, was also determined. Protein Met(O) content did not change in response to either water-deficit or high temperature stress. PrMSR activity decreased in pea and cotton leaves, remained unchanged in potato leaves and significantly increased in leaves of water-deficit-stressed wheat. The findings demonstrate that these plants have developed protection systems that effectively maintain stable levels of oxidized methionyl residues in leaf proteins despite exposure to severe water and high temperature stress. The findings also suggest that changes in PrMSR activity do not fully account for the observed maintenance of protein methionyl sulfoxide content at constant levels.     

Relationships between shoot to root ratio, growth and leaf soluble protein concentration of Pisum sativum, Phaseolus vulgaris and Triticum aestivum under different nutrient deficiencies

Relations between shoot to root dry weight ratio (S : R), total plant dry weight (DW), shoot and plant N concentration and leaf soluble protein concentration were examined for pea ( Pisum sativum L.), common bean ( Phaseolus vulgaris L.) and wheat ( Triticum aestivum L.) under different nutrient deficiencies. A regression model incorporating leaf soluble protein concentration and plant DW could explain greater than 80% of the variation in S : R within and between treatments for pea supplied different concentrations of NO₃^– or NH₄⁺ in solid substrate; pea and bean supplied different concentrations of N, P, K and Mg in liquid culture; and wheat supplied different concentrations of N, P, K, Mg, Ca and S in liquid culture. Addition of shoot or plant N concentration to the model explained little more of the variation in S : R. It is concluded that results are consistent with the proposal that macronutrient effects on S : R are primarily mediated through their effects on protein synthesis and growth.     

How can the electrical polarity of axial organs regulate plant growth and IAA transport?

Alteration of the electrical polarity in sections of maize ( Zea mays L. cv. Odesskaya-80) coleoptiles and pea ( Pisum sativum L. cv. Cubanecz) internodes by passing a weak electric current longitudinally (6 μA, anode placed at the apical end of the section) increased their elongation rate 2–3 fold with a lag period of 2.5 min. Inhibitors of polar auxin transport, e.g. N-1-naphthylphtalamic and 2,3,5-triiodobenzoic acids and also ethylene glycol-bis(β-aminoethylether)-N,N,N'N'-tetraacetic acid (EGTA), a chelator of divalent cations, decreased the growth rate of the sections and inhibited the growth-stimulating effect of the electric current. The observed acceleration of growth of axial plant organs under the action of a weak electric current is suggested to be connected with changes in the mode of action of the basipetal auxin transport system.     

昆虫谷胱甘肽S-转移酶的基因结构及其表达调控

谷胱甘肽S-转移酶（glutathione S-transferases, GSTs）属于一个超家族,目前已从20多种昆虫中克隆得到了近百个GSTs基因序列。这些基因分属于至少3个类别,Ⅰ（Delta）类,Ⅱ类和Ⅲ（Epsilon）类,其中Ⅰ类和Ⅲ类是昆虫特异性的类别。昆虫Ⅰ类GSTs基因通常由多基因家族编码,基因多态性在不同昆虫种类中差异很大。Ⅱ类基因的种类较少,基因的结构较简单,通常是单拷贝基因。Ⅲ类基因是最近才鉴定出来的新类别,目前仅在黑腹果蝇和冈比亚按蚊中明确了其在染色体上的定位。基因簇、可变剪接和基因融合等机制是导致昆虫GSTs基因多态性的主要原因。在抗性昆虫种群中,GSTs表达量的增加有mRNA水平的提高和基因扩增两种机制,但后一种机制的报道很少。GSTs活性的增加是由于属于一类或多类的多个同工酶的增量调控,也有少数是由于单个同工酶的增量调控。GSTs的表达受反式调控元件和顺式调控元件的调控。目前仅有少数含有调节基因的染色体大致位点和可能的调控元件得到鉴定。     

Glycine oxidation in mitochondria isolated from light grown and etiolated plant tissue

Mitochondria were isolated from light grown and dark grown monocotyledonous (wheat- Triticum aestivum and barley- Hordeum vulgare ) and dicotyledonous (pea- Pisum sativum ) plants and their capacity to oxidize glycine was measured. In all of the studied plant species the rate of mitochondrial glycine oxidation was high in light grown leaves. Glycine oxidation in mitochondria from etiolated leaves was also very substantial; the rate of glycine oxidation relative to the oxidation of other substrates was about half as compared to green tissue. In etiolated non-photosynthetic tissues the relative glycine oxidation was only ca 20% of that measured in green leaves. The effect of light on the development of glycine oxidation capacity was studied using etiolated barley which was transferred to light for 6 to 24 h. During this time the rate of glycine oxidation as compared to the oxidation of NADH and malate increased, approaching the ratio observed in light grown leaves. It is concluded that the synthesis of proteins involved in glycine oxidation is regulated both in a light dependent and in a tissue specific manner. Monocotyledonous plants should be very useful for further studies of this aspect due to the relatively small developmental difference between etiolated and light grown leaf tissue.     

Time course effects of vanadium supplement on cytosolic reduced glutathione level and glutathione S-transferase activity

The influence of vanadium, an important dietary micronutrient, was evaluated on the cytosolic reduced glutathione (GSH) content and glutathione S-transferase (GST) activity in several rat target tissues. Supplementation of drinking water with vanadium at the level of 0.2 or 0.5 ppm for 4, 8, or 12 wk was found to increase the GSH level with a concomitant elevation in GST activity in the liver followed by small intestine mucosa, large intestine mucosa, and kidney. The results were almost dose-dependent and mostly pronounced with 0.5 ppm vanadium after 12 wk of its continuous supplementation. Neither the GSH level nor GST activity was significantly altered in forestomach and lung following vanadium supplementation throughout the study. The levels of vanadium that were found to increase the content of GSH and activity of GST in the liver, intestine, and kidney did not exert any toxic manifestation was evidenced from water and food consumption as well as the growth responses of the experimental animals. Moreover, these doses of vanadium did not impair either hepatic or renal functions as they did not alter the serum activities of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), sorbitol dehydrogenase (SDH), as well as serum urea and creatinine levels. All these results clearly indicate that vanadium under the doses employed in our study has a significant inducing role on GSH content with a concurrent elevation in GST activity in the liver and specific extrahepatic tissues without any apparent sign of cytotoxicity. This attribute of vanadium may have a greater importance in terms of biotransformation and detoxification of xenobiotics, including carcinogens. In addition, since the ability to afford an increment in the endogenous GSH-GST pool by anticarcinogenic natural substances has been found to correlate with their activity to inhibit neoplastic transformation, the trace element vanadium may be considered as a novel anticancer agent.     

Effect of apex excision and replacement by 1-naphthylacetic acid on cytokinin concentration and apical dominance in pea plants

As known from literature lateral buds from pea ( Pisum sativum ) plants are released from apical dominance when repeatedly treated with exogenous cytokinins. Little is known, however, about the endogenous role of cytokinins in this process and whether they interact with basipolar transported IAA, generally regarded as the main signal controlling apical dominance. This paper presents evidence that such an interaction exists.
The excision of the apex of pea plants resulted in the release of inhibited lateral buds from apical dominance (AD). This could be entirely prevented by applying 1-naphthylacetic acid (NAA) to the cut end of the shoot. Removal of the apex also resulted in a rapid and rather large increase in the endogenous concentrations of zeatin riboside (ZR), isopentenyladenosine (iAdo) and an as yet unidentified polar zeatin derivative in the node and internode below the point of decapitation. This accumulation of ZR and iAdo, was strongly reduced by the application of NAA. The observed increase in cytokinin concentration preceded the elongation of the lateral buds, suggesting that endogenous cytokinins play a significant role in the release of lateral buds from AD. However, the effect of NAA on the concentration of cytokinins clearly demonstrated the dominant role of the polar basipetally transported auxin in AD. The results suggest a mutual interaction between the basipolar IAA transport system and cytokinins obviously produced in the roots and transported via the xylem into the stem of the pea plants.     

Polyamine concentrations and arginine decarboxylase activity in wheat exposed to osmotic stress

The activity of L-arginine decarboxylase (ADC: EC 4.1.1.19)and polyamine content were examine in intact wheat plants ( Triticum aestivum L. cv. Sappo) exposed to osmotic stress (0.4 M mannitol) for 5 days. ADC activity was increased in first and second leaves and in roots of mannitol-stressed plants. Concentrations of putrescine, cadaverine and spermine were generally increased in leaves and roots of plants exposed to mannitol, whereas spermidine was reduced in first leaves and roots of these plants. In an attempt to determine the localization of mannitol in stressed wheat. ¹⁴C-mannitol was fed to plants grown in liquid culture. Most of the mannitol was detected in roots (84%), while small amounts were found in first (9%) and second (7%) leves.
Since it seemed possible that some of the effects on polyamine metabolism caused by exposure to mannitol could have been the result of water stress. polyamine metabolism was also studied in plants water stressed by exposure to 2% polyethylene glycol (PEG) 4000. ADC activity was not altered by exposure to PEG. but concentrations of putrescine, spermidine and spermine were generally reduced in leaves and roots of stressed plants. Cadaverine concentrations were not significantly affected by exposure to PEG. Spermidine and spermine concentrations were reduced in first and second leaves but remained unchanged in roots of plants exposed to PEG.     

Characterisation of glutathione transferases and glutathione peroxidases in pea (Pisum sativum)

Glutathione peroxidases (GPOXs) and glutathione transferases, also termed glutathione S-transferases (GST, EC 2.5.1.18), with activities toward a range of xenobiotic substrates including herbicides, have been characterized in etiolated pea (Pisum sativum L. cv. Feltham's First) seedlings. Crude extracts showed high activity toward a range of GST substrates including 1-chloro-2,4-dinitrobenzene (GSTC activity) and the herbicide fluorodifen (GSTF) but low activities toward chloroacetanilides and atrazine. Treatment of the pea seedlings with the herbicide safener dichlormid selectively increased the activity of GSTC and the GST which detoxified atrazine. This induction was restricted to the roots and was not observed with any of the other GST or GPOX activities. In contrast, treatment with CuCl₂ increased GPOX activity in the root but had no effect on any GST activity, while treatment of epicotyls with elicitors of the phytoalexin response increased GST activity toward ethacrynic acid, but had no effect on other GST or GPOX activities. The major enzymes with GSTC, GSTF and GPOX activities were purified from pea epicotyls 3609-fold, 1431-fold and 1554-fold, respectively. During purification by hydrophobic interaction chromatography and affinity chromatography using S-hexyl-glutathione as ligand all three activities co-eluted but could be partially resolved by anion exchange chromatography and gel filtration chromatography. Both GSTC and GPOX had a molecular mass of 48 kDa and their activities were associated with a similar 27.5-kDa subunit but distinct 29-kDa subunits. GSTF could be resolved into two isoenzymes with molecular masses of 49.5 and 54 kDa. GSTF activity was associated with a unique 30-kDa subunit in addition to 27.5- and 29-kDa peptides, suggesting that the two isoenzymes were composed of differing subunits. These results demonstrate that peas contain multiple GST isoenzymes some of which have GPOX activity and that the various activities are differentially responsive to biotic and abiotic stress.     

Elevation of glutathione levels and glutathione S-transferase activity in arsenic-resistant chinese hamster ovary cells

Summary Arsenic-resistant Chinese hamster ovary (CHO) cells were established by progressively increasing the concentration of sodium arsenite in culture medium. One of the resistant clones, SA7, was also cross-resistant to As(V), Zn, Fe(II), Co, and Hg. The susceptibilities to sodium arsenite in parental CHO cells, revertant SA7N cells, and resistant SA7 cells were correlated with their intracellular glutathione (GSH) levels and glutathione S-transferase (GST) activity. The resistance in SA7 cells was diminished by depletion of GSH in cells after treatment with buthionine sulfoximine. Furthermore, after reexposure of revertant SA7N cells to sodium arsenite, the intracellular GSH levels, GST activity, and resistance to sodium arsenite were raised to the same levels as SA7 cells. These data indicate that the elevation of intracellular GSH levels and GST activity in SA7 cells may be responsible for the resistance to arsenite. A p25 protein, which could be a monomer subunit of GST, accumulated in SA7 cells. In addition, an outward transport inhibitor, verapamil, indiscriminately increased the arsenite toxicity in resistant and parental cells. This work was supported in part by grant NSC77-0201-B001-31 from the National Science Council, Republic of China.     

Intensity of hydrostimulation for the induction of root hydrotropism and its sensing by the root cap

Roots of Pisum sativum L. and Zea mays L. were exposed to different moisture gradients established by placing both wet cheesecloth (hydrostimulant) and saturated aqueous solutions of various salts in a closed chamber. Atmospheric conditions with different relative humidity (RH) in a range between 98 and 86% RH were obtained at root level, 2 to 3mm from the water-saturated hydrostimulant. Roots of Silver Queen corn placed vertically with the tips down curved sideways toward the hydrostimulant in response to approximately 94% RH but did not respond positively to RH higher than approximately 95%. The positive hydrotropic response increased linearly as RH was lowered from 95 to 90%. A maximum response was observed at RH between 90 and 86%. However, RH required for the induction of hydrotropism as well as the responsiveness differed among plant species used; gravitropically sensitive roots appeared to require a somewhat greater moisture gradient for the induction of hydrotropism. Decapped roots of corn failed to curve hydrotropically, suggesting the root cap as a major site of hydrosensing.     

The effects of O3 fumigation during leaf development on photosynthesis of wheat and pea: An in vivo analysis

Previous studies have shown that short exposure of plants to high doses of ozone decreases subsequent photosynthesis; initially by reducing carboxylation capacity. This study tests the hypothesis that this is also the primary cause of loss of photosynthetic capacity in leaves affected by development under a low level of ozone. Triticum aestivum and Pisum sativum plants were exposed from germination to ozone in air (80 nmol mol^-1 for 7 hours per day, for 18 days. Leaves that had completed lamina expansion at this time were free of visible injury and light absorptance was unaffected. However, some significant changes in photosynthetic gas exchange were evident. Photosynthetic CO2 uptake at light saturation was decreased significantly by 35% in T. aestivum but was unchanged in P. sativum. The reduction in photosynthesis of T. aestivum was accompanied by a 31% decline in the maximum velocity of carboxylation measured in vivo. Decreased stomatal conductance did not contribute to this reduction of photosynthesis because there was no significant change in the stomatal limitation to CO2. Processes directly dependent upon photochemical reactions; that is, the quantum yield of CO2 uptake and capacity for regeneration of ribulose 1,5-bisphosphate were not affected by O3 fumigation in either species. This suggests that for wheat, the quantitative cause of decreased photosynthetic rate in vivo is a decrease in the quantity of active ribulose-1,5- bisphosphate carboxylase-oxygenase.