Cross-talk between calcium and reactive oxygen species originated from NADPH oxidase in abscisic acid-induced antioxidant defence in leaves of maize seedlings

The signal interactions between calcium (Ca2+) and reactive oxygen species (ROS) originated from plasma membrane NADPH oxidase in abscisic acid (ABA)-induced antioxidant defence were investigated in leaves of maize (Zea mays L.) seedlings. Treatment with ABA led to significant increases in the activity of plasma membrane NADPH oxidase, the production of leaf O2-, and the activities of several antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). However, such increases were blocked by the pretreatment with Ca2+ chelator EGTA or Ca2+ channel blockers La3+ and verapamil, and NADPH oxidase inhibitors such as diphenylene iodonium (DPI), imidazole and pyridine. Treatment with Ca2+ also significantly induced the increases in NADPH oxidase activity, O2- production and the activities of antioxidant enzymes, and the increases were arrested by pretreatment with the NADPH oxidase inhibitors. Treatment with oxidative stress induced by paraquat, which generates O2-, led to the induction of antioxidant defence enzymes, and the up-regulation was suppressed by the pretreatment of Ca2+ chelator and Ca2+ channel blockers. Our data suggest that a cross-talk between Ca2+ and ROS originated from plasma membrane-bound NADPH oxidase is involved in the ABA signal transduction pathway leading to the induction of antioxidant enzyme activity, and Ca2+ functions upstream as well as downstream of ROS production in the signal transduction event in plants.     

Effect of short-term heat stress on growth, physiology and antioxidative defence system in wheat seedlings

An experiment was conducted to find out the effect of short-term heat stress on morpho-physiological characters and antioxidants in 10 diverse wheat genotypes. Seed were aseptically grown in test tubes containing filter paper whose lower half was dipped in one-fourth MS media. Heat stress conditions were created by exposing the seedlings at 45 °C for 2 h after 7 days of their germination. Measurements were taken after 3 days of treatment. Heat stress significantly reduced the shoot dry mass, root dry mass, shoot length and root length in all the genotypes. The chlorophyll content and membrane stability index decreased, whereas proline content increased in heat-treated plants. There was significant increase in the activity of catalase, guaiacol peroxidase and superoxide dismutase under stress conditions. The genotypic variations were also significant. On the basis of a coordinated simulation of all these parameters, wheat genotypes Raj 4037 and PBW 373 were identified as tolerant to high temperature stress. The study provides evidence that the tolerant genotypes were equipped with better management of physiological processes along with an efficient antioxidative defence system, sensitivity of which can be evaluated to a sufficient level of certainty at seedling stage.     

渗透胁迫对杧果叶片活性氧伤害的影响

杧果叶片经渗透胁迫处理后,叶水势Ψ_L下降,O₂^·产生速率和MDA含量增加,SOD、POD和CAT的活性水平与O₂^·和MDA的变化相一致。结果表明,杧果叶片的渗透胁迫损伤,是由O₂^·引发的膜脂过氧化,致使MDA含量增加,破坏细胞膜系统所致。渗透胁迫处理过程中,GSH和AsA含量下降。     

Role of active oxygen species and NO in plant defence responses.

Research in the area of active oxygen species is going through a reflective stage. There is controversy whether multiple mechanisms for active oxygen species generation exist and some data may need reassessing since the discovery of a role for NO in defence responses. Important work concerning upstream and downsteam signalling in this area is emerging, and the stage is set for approaches utilising transgenic knockouts and mutants to resolve many questions.     

Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves

The interrelationship among water-stress-induced abscisic acid (ABA) accumulation, the generation of reactive oxygen species (ROS), and the activities of several antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) was investigated in leaves of detached maize (Zea mays L.) plants exposed to -0.7 MPa water stress induced by polyethylene glycol (PEG 6000). Time-course analyses of ABA content, the production of ROS, and the activities of antioxidant enzymes in water-stressed leaves showed that a significant increase in the content of ABA preceded that of ROS, which was followed by a marked increase in the activities of these antioxidant enzymes. Pretreatment with an ABA biosynthesis inhibitor, tungstate, significantly suppressed the accumulation of ABA, and also reduced the increased generation of ROS and the up-regulation of these antioxidant enzymes in water-stressed leaves. A mild oxidative stress induced by paraquat, which generates O(2)(-) and then H(2)O(2), resulted in a significant enhancement in the activities of antioxidant enzymes in non-water-stressed leaves. Pretreatment with some ROS scavengers, such as Tiron and dimethylthiourea (DMTU), and an inhibitor of NAD(P)H oxidase, diphenyleneiodonium (DPI), almost completely arrested the increase in ROS and the activities of these antioxidant enzymes induced by water stress or ABA treatment. These data suggest that water stress-induced ABA accumulation triggers the increased generation of ROS, which, in turn, leads to the up-regulation of the antioxidant defence system.     

Effect of high temperature on active oxygen species,senescence and photosynthetic properties in cucumber leaves

To gain a physiological understanding of the effects of high temperatures on cucumber (Cucumis sativus L.), we subjected seedlings to heat treatment at daytime temperatures of 28 °C, 32 °C, and 36 °C for 7 h a day for 30 days. The amount of active oxygen species, indicators of senescence, and photosynthetic properties in the second and third leaves were determined at the start of temperature treatment and on the 15th and 30th days of treatment. The amount of active oxygen species superoxide in leaves was greatest in the high temperature zones on the 15th day of treatment, and the amount of hydrogen peroxide was greatest in the high temperature zones on both the 15th and 30th days of treatment. The reduction in the amount of protein and the increase in the amount of malondialdehyde, both indicators of senescence, were greatest in the high temperature zones on both the 15th and 30th days of treatment, and the amount of chlorophyll was lowest in the 36 °C zone on the 15th day, and lower in the high temperature zones on the 30th day. It is clear from these results that a large amount of active oxygen species is generated and accumulated in the leaves at high temperatures, and senescence is significantly accelerated. The photosynthetic properties of stomatal conductance, sub-stomatal CO₂ concentration, and transpiration rate were at the same level on both the 15th and 30th days of treatment in all three temperature treatment zones. No significant difference was seen in the net photosynthesis rate between the 28 °C and 32 °C zones, was lower in the 36 °C zone than the 32 °C zone on the 15th day, and lowest in the 36 °C zone on the 30th day. CO₂ intake and water absorption are only mildly affected by high temperatures, and the reduction in net photosynthesis rate due to the 36 °C high temperature stress suggests that the large amount of active oxygen species induces inhibition of photosynthesis and damage to the mechanism of photosynthesis.     

Effect of paclobutrazol on abscisic acid levels in wheat seedlings

Contradictory results have been reported for the effects of triazoles on abscisic acid (ABA) levels in plants. Paclobutrazol reduced the height, fresh weight, and ABA levels of wheat seedlings. The magnitude of the inhibitory effect of paclobutrazol on ABA levels was dependent on the length of time after application. ABA levels in plants as determined by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS) were reduced by 50–60% 2 days after soil application. A further 15% reduction occurred by the seventh day after treatment. ABA level analyses began at the time of treatment with 4-day-old seedlings and continued until 21 days after treatment when only a 20% reduction was detected. These determinations using GC-SIM-MS should increase the understanding of triazole effects on ABA levels.     

Effect of paclobutrazol on abscisic acid levels in wheat seedlings

Contradictory results have been reported for the effects of triazoles on abscisic acid (ABA) levels in plants. Paclobutrazol reduced the height, fresh weight, and ABA levels of wheat seedlings. The magnitude of the inhibitory effect of paclobutrazol on ABA levels was dependent on the length of time after application. ABA levels in plants as determined by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS) were reduced by 50–60% 2 days after soil application. A further 15% reduction occurred by the seventh day after treatment. ABA level analyses began at the time of treatment with 4-day-old seedlings and continued until 21 days after treatment when only a 20% reduction was detected. These determinations using GC-SIM-MS should increase the understanding of triazole effects on ABA levels.     

Triticale and barley microspore embryogenesis induction requires both reactive oxygen species generation and efficient system of antioxidative defence

Plant Cell, Tissue and Organ Culture (PCTOC) - The effectiveness of microspore embryogenesis (ME) is determined by a complex network of internal and environmental factors. In the present study on...     

Girdling induces oxidative damage and triggers enzymatic and non-enzymatic antioxidative defences in Citrus leaves

The effects of girdling on oxidative damage, antioxidant enzyme activity, antioxidant metabolites and proline (Pro) were studied in leaves arising from different shoot types of potted 2-year-old ‘Loretina’ mandarin (Citrus reticulata Blanco) trees during the spring flush period. Girdling increased malonyldialdehyde (MDA) and basal chlorophyll (Chl) a fluorescence (F_o) in young leaves 30 days after girdling but not in the mature leaves (ML) suggesting a disruption of photosynthetic apparatus and oxidative damage in young leaves. This phenomenon was accompanied by increasing levels of Pro. Paralleling these changes, an increase of all antioxidant enzyme activities occurred in leaves from vegetative (VG) and multiflowered leafy shoots (MLY) of girdled trees. Similarly, in ML of girdled trees, ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) activity also increased. However, dehydroascorbate reductase (DHAR) activity decreased and superoxide dismutase (SOD) activity remained unchanged. Total leaf carbohydrate content and starch also increased as a result of girdling in all shoot types. Whilst soluble sugars increased markedly in young leaves, they increased only slightly in ML. In conclusion, this study provides evidence that girdling gives rise to oxidative damage in Citrus during carbohydrate accumulation, triggering enzymatic and non-enzymatic defence mechanisms.     

Girdling induces oxidative damage and triggers enzymatic and non-enzymatic antioxidative defences in Citrus leaves

The effects of girdling on oxidative damage, antioxidant enzyme activity, antioxidant metabolites and proline (Pro) were studied in leaves arising from different shoot types of potted 2-year-old ‘Loretina’ mandarin (Citrus reticulata Blanco) trees during the spring flush period. Girdling increased malonyldialdehyde (MDA) and basal chlorophyll (Chl) a fluorescence (F_o) in young leaves 30 days after girdling but not in the mature leaves (ML) suggesting a disruption of photosynthetic apparatus and oxidative damage in young leaves. This phenomenon was accompanied by increasing levels of Pro. Paralleling these changes, an increase of all antioxidant enzyme activities occurred in leaves from vegetative (VG) and multiflowered leafy shoots (MLY) of girdled trees. Similarly, in ML of girdled trees, ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) activity also increased. However, dehydroascorbate reductase (DHAR) activity decreased and superoxide dismutase (SOD) activity remained unchanged. Total leaf carbohydrate content and starch also increased as a result of girdling in all shoot types. Whilst soluble sugars increased markedly in young leaves, they increased only slightly in ML. In conclusion, this study provides evidence that girdling gives rise to oxidative damage in Citrus during carbohydrate accumulation, triggering enzymatic and non-enzymatic defence mechanisms.     

Phosphorus toxicity disrupts Rubisco activation and reactive oxygen species defence systems by phytic acid accumulation in leaves

Phosphorus (P) is an essential mineral nutrient for plants. Nevertheless, excessive P accumulation in leaf mesophyll cells causes necrotic symptoms in land plants; this phenomenon is termed P toxicity. However, the detailed mechanisms underlying P toxicity in plants have not yet been elucidated. This study aimed to investigate the molecular mechanism of P toxicity in rice. We found that under excessive inorganic P (Pi) application, Rubisco activation decreased and photosynthesis was inhibited, leading to lipid peroxidation. Although the defence systems against reactive oxygen species accumulation were activated under excessive Pi application conditions, the Cu/Zn-type superoxide dismutase activities were inhibited. A metabolic analysis revealed that excessive Pi application led to an increase in the cytosolic sugar phosphate concentration and the activation of phytic acid synthesis. These conditions induced mRNA expression of genes that are activated under metal-deficient conditions, although metals did accumulate. These results suggest that P toxicity is triggered by the attenuation of both photosynthesis and metal availability within cells mediated by phytic acid accumulation. Here, we discuss the whole phenomenon of P toxicity, beginning from the accumulation of Pi within cells to death in land plants.     

Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid

Plants, in common with all organisms, have evolved mechanisms to cope with the problems caused by high temperatures. We examined specifically the involvement of calcium, abscisic acid (ABA), ethylene, and salicylic acid (SA) in the protection against heat-induced oxidative damage in Arabidopsis. Heat caused increased thiobarbituric acid reactive substance levels (an indicator of oxidative damage to membranes) and reduced survival. Both effects required light and were reduced in plants that had acquired thermotolerance through a mild heat pretreatment. Calcium channel blockers and calmodulin inhibitors increased these effects of heating and added calcium reversed them, implying that protection against heat-induced oxidative damage in Arabidopsis requires calcium and calmodulin. Similar to calcium, SA, 1-aminocyclopropane-1-carboxylic acid (a precursor to ethylene), and ABA added to plants protected them from heat-induced oxidative damage. In addition, the ethylene-insensitive mutant etr-1, the ABA-insensitive mutant abi-1, and a transgenic line expressing nahG (consequently inhibited in SA production) showed increased susceptibility to heat. These data suggest that protection against heat-induced oxidative damage in Arabidopsis also involves ethylene, ABA, and SA. Real time measurements of cytosolic calcium levels during heating in Arabidopsis detected no increases in response to heat per se, but showed transient elevations in response to recovery from heating. The magnitude of these calcium peaks was greater in thermotolerant plants, implying that these calcium signals might play a role in mediating the effects of acquired thermotolerance. Calcium channel blockers and calmodulin inhibitors added solely during the recovery phase suggest that this role for calcium is in protecting against oxidative damage specifically during/after recovery.     

Contrasting photo- and thermoperiod-induced changes in abscisic acid and lipid contents in leaves of mungbean seedlings

Seedlings of Phaseolus aureus ROXB were grown under 12/12 h light/dark cycles with the light period at 32.5°C and darkness at 10°C (normal conditions N) or with light at 10°C and darkness at 32.5°C (inverse conditions, I). I-conditions affected the level of chlorophyll and carotenoids (very low), monogalactosyldiacylgycerol (low) and phosphatidylinositol (high) in the leaves. Leaves of I-seedlings showed a sharp and durable decline of relative water content during the low temperature phase. For the N-seedlings, loss of water was restricted to the end of this period. The loss of water was accompanied by visible symptoms of wilting at specific times of day. Although the pigment content remained nearly unchanged, ABA content of leaves of both N-and I-seedlings increased during water stress. Upon return to the warm period, ABA level continued to increase after the leaves had regained turgor, this 'after stress'increase being more pronounced in the leaves of I-seedlings. Exogenous application of ABA induced a slight increase in the content of phospholipids in N- and I-leaves and a decrease in free fatty acids, whereas monogalactosyldiacylglycerol content was significantly reduced in N-leaves after application of ABA. Upon transfer of I-plants to 20°C for 12 h during the light period, pigment and chloroplastic lipid content increased rapidly whereas upon a further exposure to 10°C in light, pigments and especially monogalactosyldiacylglycerol were lost. The control of pigment and lipid metabolism and the role of ABA during chilling stress are discussed.     

Cross-talk between calcium-calmodulin and nitric oxide in abscisic acid signaling in leaves of maize plants

Using pharmacological and biochemical approaches, the signaling pathways between hydrogen peroxide （H2O2）, calcium （Ca^2＋）-calmodulin （CAM）, and nitric oxide （NO） in abscisic acid （ABA）-induced antioxidant defense were investigated in leaves of maize （Zea mays L.） plants. Treatments with ABA, H2O2, and CaCl2 induced increases in the generation of NO in maize mesophyll cells and the activity of nitric oxide synthase （NOS） in the cytosolic and microsomal fractions of maize leaves. However, such increases were blocked by the pretreatments with Ca^2＋ inhibitors and CaM antagonists. Meanwhile, pretreatments with two NOS inhibitors also suppressed the Ca^2＋-induced increase in the production of NO. On the other hand, treatments with ABA and the NO donor sodium nitroprusside （SNP） also led to increases in the concentration of cytosolic Ca^2＋ in protoplasts of mesophyll cells and in the expression of calmodulin 1 （CaM1） gene and the contents of CaM in leaves of maize plants, and the increases induced by ABA were reduced by the pretreatments with a NO scavenger and a NOS inhibitor. Moreover, SNP-induced increases in the expression of the antioxidant genes superoxide dismutase 4 （SOD4）, cytosolic ascorbate peroxidase （cAPX）, and glutathione reductase 1 （GR1） and the activities of the chloroplastic and cytosolic antioxidant enzymes were arrested by the pretreatments with Ca^2＋ inhibitors and CaM antagonists. Our results suggest that Ca^2＋-CaM functions both upstream and downstream of NO production, which is mainly from NOS, in ABA- and H2O2-induced antioxidant defense in leaves of maize plants.     

Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings

The roles of the plasma-membrane (PM) NADPH oxidase in abscisic acid (ABA)- and water stress-induced antioxidant defense were investigated in leaves of maize ( Zea mays L.) seedlings. Treatment by exogenous ABA (100 micro M ABA) or osmotic stress (-0.7 MPa induced by polyethylene glycol) significantly increased the activity of the PM NADPH oxidase, the production of leaf O(2)(-), the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), and the contents of antioxidant metabolites (ascorbate and reduced glutathione). Pretreatment with three different inhibitors of NADPH oxidase (diphenylene iodonium, imidazole and pyridine) or an inhibitor of ABA biosynthesis (tungstate) reduced the increase in the activity of the PM NADPH oxidase and the production of leaf O(2)(-), and the capacity of antioxidant defense systems mediated by ABA. The inhibitory effects above caused by tungstate were reversed by exogenous ABA. These data indicate that NADPH oxidase is involved in the ABA-induced production of active oxygen species (AOS), and our results depict a minimal chain of events initiated by water stress-induced ABA accumulation, which then triggers the production of AOS by membrane-bound NADPH oxidase, resulting in the induction of antioxidant defense systems against oxidative damage in plants.     

Mitogen-activated protein kinase is involved in abscisic acid-induced antioxidant defense and acts downstream of reactive oxygen species production in leaves of maize plants

The role of mitogen-activated protein kinase (MAPK) in abscisic acid (ABA)-induced antioxidant defense was investigated in leaves of maize (Zea mays) plants. Treatments with ABA or H(2)O(2) induced the activation of a 46-kD MAPK and enhanced the expression of the antioxidant genes CAT1, cAPX, and GR1 and the total activities of the antioxidant enzymes catalase, ascorbate peroxidase, glutathione reductase, and superoxide dismutase. Such enhancements were blocked by pretreatment with several MAPK kinase inhibitors and reactive oxygen species inhibitors or scavengers. Pretreatment with MAPK kinase inhibitors also substantially arrested the ABA-induced H(2)O(2) production after 2 h of ABA treatment, but did not affect the levels of H(2)O(2) within 1 h of ABA treatment. Pretreatment with several inhibitors of protein tyrosine phosphatase, which is believed to be a negative regulator of MAPK, only slightly prevented the ABA-induced H(2)O(2) production, but did not affect the ABA-induced MAPK activation and ABA-enhanced antioxidant defense systems. These results clearly suggest that MAPK but not protein tyrosine phosphatase is involved in the ABA-induced antioxidant defense, and a cross talk between H(2)O(2) production and MAPK activation plays a pivotal role in the ABA signaling. ABA-induced H(2)O(2) production activates MAPK, which in turn induces the expression and the activities of antioxidant enzymes. The activation of MAPK also enhances the H(2)O(2) production, forming a positive feedback loop.     

Effect of auxin and abscisic acid on cell wall extensibility in maize coleoptiles

Plastic and elastic in-vitro extensibilities (E _pland E _el ) of cell walls from growing maize (Zea mays L.) coleoptile segments were measured by stretching frozen-thawed tissue, pre-extended to its in-vivo length, at constant force (creep test) in a custom-buildt extensiometer, equipped with a linear-displacement transducer. The indole-3-acetic acid (IAA)-induced change of E _pl (E _pl ) is strictly correlated with the growth rate for a period of 3–4 h. Subsequently, E _plremains constant while the growth rate is slowing down. Since this discrepancy can be accounted for by a growth-dependent reduction of osmotic pressure, it is concluded that E _plrepresents quantitatively the relative increase of in-vivo extensibility (cell wall loosening) involved in IAA-mediated cell growth over a much longer time. On the other side it is argued that the growth rate may not be strictly correlated with wall extensibility during long-term growth. Abscisic acid (ABA) inhibits segment growth induced by auxin, fusicoccin, or exogenous acid, and this effect can be quantitatively attributed to an ABA-mediated reduction of cell wall extensibility as determined by the E _plmeasurement. Both, IAA and ABA have no effect on total protein synthesis, RNA synthesis, and amount of osmotic solutes. Fusicoccin-induced proton excretion is only slightly inhibited by ABA. In contrast to ABA, growth inhibition by cycloheximide (CHI) is always much larger than the concomitant reduction of E _pl , indicating that a further growth parameter is also involved in the inhibition of cell growth by CHI. E _el is not affected by either IAA, ABA, or CHI. It is concluded that E _pl as determined by the applied method, represents a relative measure of the actual in-vivo extensibility of the growing cell wall at the very moment when the tissue is killed, rather than an average extensibility accumulated over some immediate-past period of time as suggested by Cleland (1984, Planta 160, 514–520). Hence, we further draw the conclusion that IAA and ABA control of cell growth can entirely be attributed to a modulation of cell wall extensibility by these hormones in maize coleoptiles.Abbreviations ABA ±abscisic acid - CHI cycloheximide - E _el , E_pl elastic and plastic in vitro extensibilities, respectively (E _el+E_pl=E_tot>) - FC fusicoccin - IAA indole-3-acetic acid