壳聚糖对镉胁迫下玉米幼苗叶片AsA-GSH循环的调控效应

以玉米（Zea mays L.）品种‘郑单958’为实验材料,分析外施壳聚糖对镉胁迫下玉米幼苗生物量、叶片镉含量、叶片超氧阴离子（O₂^·-）产生速率和过氧化氢（H₂O₂）的含量,以及抗坏血酸-谷胱甘肽（AsA-GSH）循环中抗氧化酶的活性及抗氧化物含量的影响。结果显示,随着镉胁迫时间的延长,玉米幼苗发生氧化胁迫,叶片抗氧化酶（APX、GR、DHAR、MDHAR）活性和抗氧化物（AsA、GSH）的含量降低,镉积累过量会抑制玉米幼苗的生长。施加壳聚糖可以降低镉胁迫下玉米幼苗叶片O₂^·-的产生速率和H₂O₂含量,提高上述抗氧化酶活性和抗氧化物的含量,促进AsA和GSH的再生,维持细胞的氧化还原状态,促进玉米幼苗的生长。研究结果表明壳聚糖处理后玉米幼苗保持了较高的AsA-GSH循环运作效率,提高了抗氧化能力,可有效缓解镉胁迫对玉米幼苗生长的抑制。     

硫化氢对盐碱胁迫下裸燕麦叶片抗坏血酸-谷胱甘肽循环的调控效应

盐碱胁迫是植物遭受的常见非生物胁迫之一,气体信号硫化氢(H₂S)在植物响应盐碱胁迫中发挥着重要作用。为探讨H₂S对盐碱胁迫下裸燕麦抗坏血酸(AsA)-谷胱甘肽(GSH)循环的调控效应,以品种‘定莜9号’为材料,研究了喷施H₂S供体硫氢化钠(NaHS)或H₂S合成抑制剂羟胺(HA)对盐碱混合胁迫下植株生长、叶片活性氧、膜脂过氧化和AsA-GSH循环中抗氧化物质和关键酶的影响。结果表明: 喷施50 μmol·L^-1 NaHS可缓解50 mmol·L^-1盐碱混合胁迫对裸燕麦生长的抑制,降低超氧阴离子、H₂O₂、丙二醛、氧化型抗坏血酸(DHA)、还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量,提高AsA/DHA和GSH/GSSG,而对还原型抗坏血酸(AsA)含量无显著影响。喷施NaHS还提高了盐碱混合胁迫下裸燕麦叶片AsA合成关键酶L-半乳糖脱氢酶(GalDH)和L-半乳糖-1,4-内酯脱氢酶(GalLDH)及AsA-GSH循环中单脱氢抗坏血酸还原酶(MDHAR)活性,降低了抗坏血酸过氧化物酶(APX)和脱氢抗坏血酸还原酶(DHAR)活性,而对抗坏血酸氧化酶(AO)和谷胱甘肽还原酶(GR)活性的影响不大。增添HA后部分或完全解除了喷施NaHS的上述作用。这说明H₂S可通过促进AsA合成和增强MDHAR活性提高AsA-GSH循环效率,降低盐碱胁迫对裸燕麦的氧化伤害。     

Photosynthetic gas exchange characteristics in three different almond species during drought stress and subsequent recovery

Three different drought stress levels (water potential of the nutrient solution, Ψ_s = −0.6, −1.2 and −1.8 MPa, respectively), and a control treatment (Ψ_s = −0.1 MPa), were applied during 2 weeks to three almond species, followed by 3 weeks of recovery. The selected test species were Prunus dulcis (Miller) D.Webb (bitter almond) and two wild almond species, P. lycioides (Spach) C.K. Schneider and P. scoparia (Spach) C.K. Schneider. All three are species native to Iran, and can be used as rootstock, but only P. dulcis is actually used for commercial almond production.In the absence of drought stress, maximum net assimilation rate (A_max) is highest for P. scoparia and lowest for P. dulcis. For all species A_max was above 16 μmol CO₂ m⁻² s⁻¹. A similar relationship between A_max and dark respiration rate (R_d), was observed for all species. This relationship suggests that optimisation of the carbon budget is independent of species.The three investigated species seem to have a different reaction to a similar stress, indicating different drought stress coping strategies. P. scoparia lost all its leaves during the experiment, while P. lycioides only kept some leaves, however, the remaining leaves were almost totally wilted and did not allow for any photosynthesis measurement. P. scoparia did not recover during the experiment, as no new leaves were developed once Ψ_s was restored to pre-drought stress levels. However, this species has green stems, indicating that stem photosynthesis might play an important role in the plants’ overall carbon balance. This species is an opportunistic one (sensu [Higgins, S.S., Larsen, F.E., Bendel, R.B., Radamaker, G.K., Bassman, J.H., Bidlake, W.R., Alwir, A., 1992. Comparative gas-exchange characteristics of potted, glasshouse-grown almond, apple, fig, grape, olive, peach and Asian pear. Sci. Hortic. 52 (4), 313–329]), where assimilation is seriously limited by non-stomatal processes as evidenced by measurements of intercellular CO₂ concentration, eventually resulting in total leaf loss. All P. lycioides leaves almost completely wilted during the experiment, but this species recovered rather quickly. Leaves, newly formed at the end of the experiment, obtained maximal assimilation rates under control Ψ_s levels, equivalent to those measured in the control treatment. Finally, P. dulcis did keep at least part of its leaves during drought stress. However, assimilation rates after 2 weeks of drought treatment and 3 weeks of recovery were only about half of those measured in the control treatment. Of the three investigated species, non-stomatal limitation of assimilation seems to be the least important in P. dulcis.Intrinsic water use efficiency, defined as the ratio of assimilation rate over stomatal conductance, increased for P. dulcis with increasing drought stress, while a different pattern was observed for P. lycioides and P. scoparia, indicating non-stomatal processes prevail over stomatal limitations of the assimilation process. It was concluded that P. dulcis is the species most tolerant to drought. P. scoparia tries to avoid drought, whereas P. lycioides has an intermediate behaviour. Besides P. dulcis, also P. lycioides seems to have some potential for use as rootstock for commercial almond production.     

Ultraviolet-B-induced oxidative stress and responses of the ascorbate-glutathione cycle in a marine macroalga Ulva fasciata

The regulation of the antioxidant defence system by ultraviolet-B (UV-B) was determined in a marine macroalga Ulva fasciata Delile exposed to low (0.5, 1 W m(-2)), medium (2.5, 5 W m(-2)), and high (10, 20 W m(-2)) UV-B irradiance. UV-B > or =2.5 W m(-2) increased H2O2 contents that are positively correlated with lipid peroxidation and total peroxide contents. Inhibition of the UV-B-induced H2O2 increase by a specific O2.- scavenger, 1,2-dihydroxy-benzene-3,5-disulphonic acid, shows that O2.- is the primary source of H2O2. Superoxide dismutase activity was increased by UV-B with a peak at 2.5 W m(-2), which did not match the H2O2 pattern. Alleviation of UV-B-induced oxidative damage by a H2O2 scavenger, dimethylthiourea, and a free radical scavenger, sodium benzoate, which inhibited UV-B-induced H2O2 accumulation, suggests that oxidative damage caused by UV-B > or = 2.5 W m(-2) is ascribed to accumulated H2O2. However, a decrease in growth rate and TTC reduction ability only at high UV-B doses indicates that the defence and repairing systems operate at low and medium UV-B doses. H2O2 not only can be excreted but can also be detoxified via the ascorbate-glutathione cycle. Increases in catalase, peroxidase, ascorbate peroxidase, and glutathione reductase activities and ascorbate (AsA) and glutathione pools, as well as AsA regeneration ability, function to keep the balance of cellular H2O2 under low UV-B doses. Dehydroascorbate reductase and monodehydroascorbate reductase are responsible for AsA regeneration under low and medium UV-B radiation, respectively. The appearance of oxidative damage in medium and high UV-B flux is attributable to a lower induction of the ascorbate-glutathione cycle as an antioxidant defence system. Overall, the availability of antioxidants and the induction of antioxidant enzyme activities for detoxifying reactive oxygen species (ROS) are regulated in U. fasciata against UV-B-induced oxidative stress, and experiments using ROS scavengers demonstrate that the antioxidant defence system is modulated by O2.- or H2O2.     

Function of the ascorbate-glutathione cycle in aged sunflower seeds

The function of the ascorbate-glutathione (AsA/GSH) cycle was analyzed in seeds of sunflower ( Helianthus annuus L. cv. Peredovik) subjected to accelerated ageing at 43°C and 75% relative humidity for 1 to 11 days. The study was performed using dry seeds and seeds hydrated by imbibition in distilled water for 4 h at 25 °C. Lipid peroxidation was also determined by measuring the malondialdehyde (MDA) level. As the ageing period increased, a progressive loss of seed viability became increasingly evident. Even though high levels of MDA were delected, the MDA level did not change during accelerated ageing, suggesting that lipid peroxidation might occur to some extent. The study of the ascorbate/glutathione (AsA/GSH) cycle revealed that the GSH system is the major detoxifying mechanism in both dry and imbibed sunflower seeds. The GSH system is mainly located in the embryo, and its protective role is mediated by reactions that consume the GSH pool and, thereby, minimize the increase of the oxidized form (GSSG). Seed imbibition activates cellular metabolism and allows some antioxidant enzymes like glutathione reductase (EC 1,6,4,2) to act upon toxic agents. These reactions provide a reducing status, so that repair of damage becomes possible. However, prolonged ageing conditions (11 days) result in an irreversible damage, as evidenced by the appearance of dead seeds when the germination period ended. Multiple regression analysis revealed the effectiveness of the GSH system in aged seeds, especially upon imbibition and until the AsA/GSH cycle became completely functional.     

海藻酸铈配合物对毒死蜱胁迫下菠菜叶片抗坏血酸-谷胱甘肽循环的影响

以菠菜(Spinacia oleracea L.)为材料,研究了毒死蜱胁迫下海藻酸铈配合物对菠菜叶片抗坏血酸-谷胱甘肽循环的影响。结果表明,在毒死蜱胁迫下,菠菜叶片中H2O2积累量比对照明显增加,非酶促抗氧化物质-抗坏血酸(AsA)和还原型谷胱甘肽(GSH)含量明显降低,抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)、脱氢抗坏血酸还原酶(DHAR)和单脱氢抗坏血酸还原酶(MDAR)的活性明显升高。在毒死蜱胁迫下,喷施不同浓度的海藻酸铈配合物使菠菜叶片中的H2O2积累量减少,AsA和GSH含量升高,APX、GR、DHAR和MDAR等抗氧化酶活性也有所提高,缓解了毒死蜱胁迫。试验表明,适宜浓度的海藻酸铈配合物处理可使菠菜叶片对毒死蜱胁迫有一定的缓解作用。     

外源一氧化氮对低温胁迫下枇杷叶片AsA-GSH循环的影响

采用0.2、0.5、1.0和1.5 mmol·L^-1的硝普钠(SNP)处理3年生‘早钟6号’枇杷幼苗,以喷清水为对照(CK),于-3 ℃低温胁迫处理6 h后在25 ℃下培养4 d,测定恢复0、1和4 d时叶片非酶抗氧化物质和抗氧化酶活性的变化.结果表明：与CK相比,经SNP处理的枇杷叶片过氧化氢(H₂O₂)含量降低,还原型谷胱甘肽(GSH)和抗坏血酸(AsA)含量及抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)、脱氢抗坏血酸还原酶(DHAR)和单脱氢抗坏血酸还原酶(MDAR)活性升高.低温处理后恢复4 d时,经0.5 mmol·L^-1 SNP处理的枇杷叶片H₂O₂含量下降至CK的75.3%,而GSH和AsA含量分别比CK增加了29.12%和23.40%,APX、GR、DHAR和MDAR活性分别比CK增加了50.0%、44.4%、49.53%和62.68%.适当的外源NO处理可提高枇杷叶片的抗氧化系统活性,减轻细胞在低温胁迫下的损伤,其中以0.5 mmol·L^-1的SNP处理效果较理想.     

Microplate quantification of enzymes of the plant ascorbate-glutathione cycle

Here, we describe microplate assays for determining the specific activities of four enzymes that constitute the ascorbate-glutathione cycle: APX, MDHAR, DHAR, and GR. In plants, these enzymes play a major role in detoxifying reactive oxygen species produced in cells under environmental stress. This work presents the development of plate reader assays to allow rapid analysis of the ascorbate-glutathione cycle activity using tomato fruits subjected to salt stress as a model. With this method, it is possible to analyze easily in one day the activities of the four enzymes for 30 experimental samples, all in triplicate and with blanks.     

外源NO对铜胁迫下番茄幼苗根系抗坏血酸-谷胱甘肽循环的影响

采用营养液培养方法,研究外源NO对铜胁迫下番茄(Lycopersicon esculentum Mill.)幼苗根系抗坏血酸(AsA)-谷胱甘肽(GSH)循环中抗氧化物质和抗氧化酶系的影响.结果表明:外施适量NO(硝普钠)可提高铜胁迫下番茄幼苗根系AsA、GSH含量和AsA/DHA(氧化型抗坏血酸)、GSH/GSSG(氧化型谷胱甘肽),降低DHA和GSSG含量.添加100 μmol·L-1 BSO(谷胱甘肽合成酶抑制剂)处理下,外源NO可提高铜胁迫下番茄幼苗根系的AsA含量、AsA/DHA及抗坏血酸酶(AAO)、单脱氢抗坏血酸还原酶(MDHAR)和脱氢抗坏血酸还原酶(DHAR)比活性,降低DHA、GSH、GSSG含量及抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)比活性;添加250 μmol·L-1 BSO处理下,外源NO提高了铜胁迫下番茄幼苗根系的AsA、GSH、GSSG含量、AsA/DHA及APX和GR比活性,降低了DHA含量及AAO、DHAR和MDHAR比活性.说明外源NO影响了铜胁迫下番茄根系的AsA-GSH代谢循环,并通过调节AsA/DHA、GSH/GSSG的变化来减轻氧化胁迫,从而缓解铜胁迫对番茄根系的伤害.     

Calcium and L-histidine effects on ascorbate-glutathione cycle components under nickel-induced oxidative stress in tomato plants

The effects of NiSO₄, calcium, and L-histidine (His) on the components of ascorbate-glutathione cycle, antioxidant enzymes and lipid peroxidation in a tomato cultivar Early Urbana Y was investigated. The activities of enzymes including catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione reductase (GR), lipoxygenase (LOX), and phenylalanine ammonia lyase (PAL) were measured. In addition, the content of H₂O₂, ascorbate (ASC), dehydroascorbate (DHA), reduced glutathione (GSH), chlorophyll (Chl) a+b, carotenoids, proteins, malondialdehyde (MDA), membrane aldehydes, and electrolyte leakage (EL) were determined. Results suggest that the excess of Ni increased the content of H₂O₂, MDA, membrane aldehydes and proteins in roots as well as GPX, LOX, APX activities, and EL in leaves, whereas Ca and His ameliorated these effects. Moreover, decreasing leaf GSH and DHA content and GR activity were observed under the Ni stress, but these parameters were raised by Ca plus His treatment. However, no improvement in leaf protein, ASC, root GSH content, and activities of PAL and CAT were observed by using Ca or His under Ni stress.     

Cadmium causes oxidative stress in mung bean by affecting the antioxidant enzyme system and ascorbate-glutathione cycle metabolism

Ascorbate (AsA)-glutathione (GSH) cycle metabolism is an essential mechanism for the resistance of plants under stress conditions. In a greenhouse pot experiment, the influence of cadmium (Cd) (25, 50, and 100 mg/kg soil) on plant dry weight and leaf area, photosynthetic parameters (net photosynthetic rate (P_N) and chlorophyll (Chl) content) and oxidative stress, and the possible protective role of AsA-GSH cycle metabolism was studied in two mung bean (Vigna radiata (L.) Wilczek.) cvs. Pusa 9531 (Cd-tolerant) and PS 16 (Cd-susceptible) at 30 days after sowing. The contents of thiobarbituric acid-reactive substances (TBARS), H₂O₂, and the leakage of ions were the highest at 100 mg Cd/kg soil, and the effect was more pronounced in cv. PS 16 than in cv. Pusa 9531. This was concomitant with the strongest decreases in P_N, plant dry weight, and leaf area. The changes in the AsA-GSH redox state and an increase in AsA-GSH-regenerating enzymes, such as glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, and other antioxidant enzymes, such as superoxide dismutase and ascorbate peroxidase, strongly supported over-utilization of AsA-GSH in Cd-treated plants. However, the oxidative stress caused by Cd toxicity was partially overcome by AsA-GSH-based detoxification mechanism in the two genotypes studied because an increases in lipid peroxidation (TBARS, ion leakage) and H₂O₂ content were accompanied by a corresponding decrease in reduced AsA and GSH pools. Thus, changes in AsA-GSH pools and the coordination between AsA-GSH-regenerating enzymes and other enzymatic antioxidants of the leaves suggest their relevance to the defense against Cd stress.     

外源NO对铜胁迫下番茄幼苗根系抗坏血酸谷胱甘肽循环的影响

采用营养液培养方法,研究外源NO对铜胁迫下番茄(Lycopersicon esculentum Mill.)幼苗根系抗坏血酸(AsA)-谷胱甘肽(GSH)循环中抗氧化物质和抗氧化酶系的影响.结果表明：外施适量NO(硝普钠)可提高铜胁迫下番茄幼苗根系AsA、GSH含量和AsA/DHA(氧化型抗坏血酸)、GSH/GSSG(氧化型谷胱甘肽),降低DHA和GSSG含量.添加100 μmol·L^-1 BSO(谷胱甘肽合成酶抑制剂)处理下,外源NO可提高铜胁迫下番茄幼苗根系的AsA含量、AsA/DHA及抗坏血酸酶(AAO)、单脱氢抗坏血酸还原酶(MDHAR)和脱氢抗坏血酸还原酶(DHAR)比活性,降低DHA、GSH、GSSG含量及抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)比活性;添加250 μmol·L^-1 BSO处理下,外源NO提高了铜胁迫下番茄幼苗根系的AsA、GSH、GSSG含量、AsA/DHA及APX和GR比活性,降低了DHA含量及AAO、DHAR和MDHAR比活性.说明外源NO影响了铜胁迫下番茄根系的AsA-GSH代谢循环,并通过调节AsA/DHA、GSH/GSSG的变化来减轻氧化胁迫,从而缓解铜胁迫对番茄根系的伤害.     

Modulation of cadmium-induced oxidative stress in Ceratophyllum demersum by zinc involves ascorbate-glutathione cycle and glutathione metabolism.

To understand the interaction between Zn, an essential micronutrient and Cd, a non-essential element, Cd-10 microM and Zn supplemented (10, 50, 100, and 200 microM) Cd 10 microM treated Ceratophyllum demersum L. (Coontail), a free floating freshwater macrophyte was chosen for the study. Cadmium at 10 microM concentration decreased thiol content, enhanced oxidation of ascorbate (AsA) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively, a clear indication of oxidative stress. Zinc supplementation to Cd (10 microM) treated plants effectively restored thiols, inhibited oxidation of AsA and GSH maintaining the redox molecules in reduced form. Cd-10 microM slightly induced ascorbate peroxidase (APX, E.C. 1.11.1.11) but inhibited monodehydroascorbate reductase (MDHAR, E.C. 1.6.5.4), dehydroascorbate reductase (DHAR, E.C. 1.8.5.1) and glutathione reductase (GR, E.C. 1.6.4.2), enzymes of ascorbate-glutathione cycle (AGC). Zn supplementation restored and enhanced the functional activity of all the AGC enzymes (APX, MDHAR, DHAR and GR). Gamma-glutamylcysteine synthetase (gamma-GCS, E.C. 6.3.2.2) was not affected by Cd as well as Zn, but Zn supplements increased glutathione-S-transferase (GST, E.C. 2.5.1.18) activity to a greater extent than Cd and simultaneously restored glutathione peroxidase (GSH-PX, E.C. 1.11.1.9) activity impaired by Cd toxicity. Zn-alone treatments did not change above investigated parameters. These results clearly indicate the protective role of Zn in modulating the redox status of the plant system through the antioxidant pathway AGC and GSH metabolic enzymes for combating Cd induced oxidative stress.     

Exogenous proline alleviates the effects of H2O2-induced oxidative stress in wild almond species

The effect of proline on the antioxidant system in the leaves of eight species of wild almond (Prunus spp.) exposed to H₂O₂-mediated oxidative stress was studied. The levels of endogenous proline (Pro) and hydrogen peroxide, and the activities of total superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and guaiacol peroxidase (POD) were measured. The degradation of chlorophyll but not carotenoids occurred in leaves in the solution of 5 mM H₂O₂. An increase in membrane lipid peroxidation was observed in H₂O₂ treatment, as assessed by MDA level and percentage of membrane electrolyte leakage (EL). Significant increases in total SOD and CAT activities, as well as decreases in APX and POD activities, were detected in H₂O₂-treated leaves. The three SOD isoforms showed different behavior, as Mn-SOD activity was enhanced by H₂O₂, whereas Fe-SOD and Cu/Zn-SOD activities were inhibited. In addition, Pro accumulation up to 0.1 ??mol/g fr wt, accompanied by significant decreases in ascorbate and glutathione levels, was observed in H₂O₂-treated leaves. After two different treatments with 10 mM Pro + 5 mM H₂O₂, total SOD and CAT activities were similar to the levels in control plants, while POD and APX activities were higher if compared to the leaves exposed only to H₂O₂. Pro + H₂O₂ treatments also caused a strong reduction in the cellular H₂O₂ and MDA contents and EL. The results showed that Pro could have a key role in protecting against oxidative stress injury of wild almond species by decreasing membrane oxidative damage.     

Activation of ascorbate-glutathione cycle inArabidopsis leaves in response to aminotriazole

Aminotriazole(AT)-induced changes in growth, hydrogen peroxide content and activities of H₂O₂-scavenging antioxidant enzymes were investigated in the growing leaves ofArabidopsis plants (Arabidopsis thaliana cv Columbia). Catalase activity of rosette leaves was reduced by 65% with an application of 0.1 mM AT (a herbicide known as a catalase inhibitor), whereas the leaf growth and H₂O₂ content were almost unaffected. However, an approximate 1.6 to 2-fold increase in cytosolic ascorbate peroxidase (APX) activity concomitant with a substantial activation of glutathione reductase (GR) (approx. 22% increase) was observed during leaf growth in the presence of 0.1 mM AT. The activity of cytosolic APX in leaves was also increased by 1.8-fold with an application of exogenous 2 mM paraquat (an inducer of H₂O₂ production in plant cells) in the absence of AT. These results collectively suggest that (a) cytosolic APX and GR operate to activate an ascorbate-glutathione cycle for the removal of H₂O₂ under severe catalase deactivation, and (b) the expression of APX seems to be regulated by a change of the endogenous H₂O₂ level in leaf cells.     

Arabidopsis dehydroascorbate reductase 1 and 2 modulate redox states of ascorbate-glutathione cycle in the cytosol in response to photooxidative stress

Ascorbate and glutathione are indispensable cellular redox buffers and allow plants to acclimate stressful conditions. Arabidopsis contains three functional dehydroascorbate reductases (DHAR1-3), which catalyzes the conversion of dehydroascorbate into its reduced form using glutathione as a reductant. We herein attempted to elucidate the physiological role in DHAR1 and DHAR2 in stress responses. The total DHAR activities in DHAR knockout Arabidopsis plants, dhar1 and dhar2, were 22 and 92%, respectively, that in wild-type leaves. Under high light (HL), the levels of total ascorbate and dehydroascorbate were only reduced and increased, respectively, in dhar1. The oxidation of glutathione under HL was significantly inhibited in both dhar1 and dhar2, while glutathione contents were only enhanced in dhar1. The dhar1 showed stronger visible symptoms than the dhar2 under photooxidative stress conditions. Our results demonstrated a pivotal role of DHAR1 in the modulation of cellular redox states under photooxidative stress.