Presoaking with hemin improves salinity tolerance during wheat seed germination

The aim of this study was to investigate whether presoaking with hemin, an inducer of heme oxygenase-1 (HO-1), could alleviate salinity damage during wheat seed germination in comparison with the pretreatment of a well-known nitric oxide (NO) donor sodium nitroprusside (SNP). The results showed that, compared with the samples upon 150 mM NaCl salt stress alone, both 10 ??M hemin and 200 ??M SNP pretreatments could (1) significantly attenuate the inhibition of seed germination and thereafter seedling growth; (2) induce HO expression; (3) enhance amylase activity, thus accelerating the formation of reducing sugar and total soluble sugar; and (4) increase the potassium (K) to sodium (Na) ratio, particularly in the shoot parts. Hemin and SNP could also increase antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), and ascorbate peroxidase (APX), thus resulting in the alleviation of oxidative damage, as indicated by the decrease of thiobarbituric acid reactive substances (TBARS) content. Moreover, semi-quantitative RT-PCR and isozymatic analysis illustrated that hemin and SNP pretreatment were able to up-regulate the expression of Mn-SOD (especially) and Cu/Zn-SOD gene, and activate SOD isozymatic activities. Since the addition of the NO scavenger methylene blue (MB) differentially reversed the above effects, the protective roles of hemin might be related to the induction of endogenous NO signal. Meanwhile, hemin-driven NO production was confirmed. Together, these results indicated that hemin exerted an advantageous effect on enhancing salinity tolerance during wheat seed germination, which might interact with NO.     

外源一氧化氮对铅胁迫下小麦种子萌发及幼苗生理特性的影响

利用室内水培实验,研究了外源一氧化氮（NO）供体硝普钠（SNP）对Pb²⁺处理下小麦（Triticum aestivum L.）种子萌发、幼苗生长及相关生理指标变化的影响。结果表明,Pb²⁺处理使小麦种子发芽势、发芽率、幼苗根长和茎长均显著降低,诱导叶绿素a、叶绿素b含量减少及叶绿素荧光参数F_v/F_m和F_v/F_o的比值减小,25 μmol·L^-1 SNP明显缓解Pb²⁺胁迫对种子萌发及幼苗生长的抑制作用,提高Pb²⁺胁迫下叶绿素a、叶绿素b含量及F_v/F_m和F_v/F_o的比值,而100 μmol·L^-1SNP无明显缓解作用。此外,25和100 μmol·L^-1SNP诱导Pb²⁺胁迫下小麦幼苗叶片过氧化氢酶（CAT）活性增强和可溶性蛋白含量增多,但100 μmol·L^-1SNP处理降低了过氧化物酶（POD）活性。结果说明,外源NO促进Pb²⁺胁迫下小麦种子萌发及幼苗生长,提高叶绿素和可溶性蛋白含量,诱导CAT活性升高,从而增强小麦对Pb²⁺胁迫的适应性。     

Induction of chilling tolerance in wheat during germination by pre-soaking seed with nitric oxide and gibberellin

Chilling depresses seed germination and seedling establishment, and is one major constraint to grain yield formation in late sown winter wheat. Seeds of winter wheat (Triticum aestivum L.) were separately pre-soaked with sodium nitroprusside (SNP, as nitric oxide donor) and Gibberellic acid (GA₃) before germination and then germinated under low temperature. SNP and GA₃ pre-treatment increased seed germination rate, germination index, weights and lengths of coleoptile and radicle, while they decreased mean germination time and weight of seeds germinating under low temperature. Exogenous NO and GA₃ increased seed respiration rate and promoted starch degradation along with increased amylase activities. In addition, efficient antioxidant systems were activated by NO, and which effectively reduced concentrations of malondialdehyde and hydrogen peroxide (H₂O₂). Seedling growth was also enhanced by exogenous NO and GA₃ as a result of improved seed germination and maintenance of better reactive oxygen species homeostasis in seedling growing under chilling temperatures. It is indicated that exogenous NO was more effective than GA₃ in alleviating chilling stress during seed germination and seedling establishment in wheat.     

一氧化氮对水分胁迫下玉米种子萌发及幼苗生理特性的影响

以0.4mol/L的甘露醇(M)模拟水分胁迫状况,研究了外源一氧化氮(NO)供体硝普钠(SNP)对水分胁迫下玉米种子萌发、幼苗生长和生理特性的影响。结果表明:(1)水分胁迫下,玉米种子萌发和幼苗生长受到抑制,叶片丙二醛(MDA)含量、质膜相对透性、脯氨酸含量均显著增加;(2)SNP能显著提高水分胁迫下玉米种子的发芽率、发芽势、发芽指数和活力指数,增加玉米幼苗的根长、茎长、根重和整株干重,抑制水分胁迫下玉米幼苗叶片MDA含量的上升,降低叶片质膜相对透性,降低脯氨酸含量。其中以100μmol/L和200μmol/LSNP对水分胁迫的缓解效果最佳。     

Nitric oxide accelerates seed germination in warm-season grasses

The nitric oxide (NO) donor sodium nitroprusside (SNP) significantly promoted germination of switchgrass (Panicum virgatum L. cv Kanlow) in the light and in the dark at 25°C, across a broad range of concentrations. SNP also promoted seed germination in two other warm-season grasses. A chemical scavenger of NO inhibited germination and blocked SNP stimulation of seed germination. The phenolic (+)-catechin acted synergistically with SNP and nitrite in promoting seed germination. Acidified nitrite, an alternate NO donor also significantly stimulated seed germination. Interestingly, sodium cyanide, potassium ferricyanide and potassium ferrocyanide at 200 μM strongly enhanced seed germination as well, whereas potassium chloride was without effect. Ferrocyanide and cyanide stimulation of seed germination was blocked by an NO scavenger. Incubation of seeds with a fluorescent NO-specific probe provided evidence for NO production in germinating switchgrass seeds. Abscisic acid (ABA) at 10 μM depressed germination, inhibited root elongation and essentially abolished coleoptile emergence. SNP partially overcame ABA effects on radicle emergence but did not overcome the effects of ABA on coleoptile elongation. Light microscopy indicated extension of the radicle and coleoptiles in seeds maintained on water or on SNP after 2 days. In contrast, there was minimal growth of the radicle and coleoptile in ABA-treated seeds even after 3–4 days. These data indicate that seed germination of warm-season grasses is significantly influenced by NO signaling pathways and document that NO could be an endogenous trigger for release from dormancy in these species.     

Seed enhancement with cytokinins: changes in growth and grain yield in salt stressed wheat plants

Cytokinins are often considered abscisic acid (ABA) antagonists and auxins antagonists/synergists in various processes in plants. Seed enhancement (seed priming) with cytokinins is reported to increase plant salt tolerance. It was hypothesized that cytokinins could increase salt tolerance in wheat plants by interacting with other plant hormones, especially auxins and ABA. The present studies were therefore conducted to assess the effects of pre-sowing seed treatment with varying concentrations (100, 150 and 200 mg l⁻¹) of cytokinins (kinetin and benzylaminopurine (BAP)) on germination, growth, and concentrations of free endogenous auxins and ABA in two hexaploid spring wheat (Triticum aestivum L.) cultivars. The primed and non-primed seeds of MH-97 (salt-intolerant) and Inqlab-91 (salt-tolerant) were sown in both Petri dishes in a growth room and in the field after treatment with 15 dS m⁻¹ NaCl salinity. Both experiments were repeated during 2002 and 2003. Among priming agents, kinetin was effective in increasing germination rate in the salt-intolerant and early seedling growth in the salt-tolerant cultivar when compared with hydropriming under salt stress. Thus, during germination and early seedling growth, the cytokinin-priming induced effects were cultivar specific. In contrast, kinetin-priming showed a consistent promoting effect in the field and improved growth and grain yield in both cultivars under salt stress. The BAP-priming did not alleviate the inhibitory effects of salinity stress on the germination and early seedling growth in both cultivars. The increase in growth and grain yield in both cultivars was positively correlated with leaf indoleacetic acid concentration and negatively with ABA concentration under both saline and non-saline conditions. The decrease in ABA concentration in the plants raised from kinetin-primed seeds might reflect diminishing influence of salt stress. However, the possibility of involvement of other hormonal interactions is discussed.     

Effects of nitric oxide on the germination of cucumber seeds and antioxidant enzymes under salinity stress

Effects of exogenous nitric oxide (NO) on the germination and antioxidant enzyme during cucumber seed germination were investigated under salt stress. Seeds of cucumber (Cucumis sativus L. cv. Jinyou 1) were treated with distilled water or NaCl in the presence or absence of NO donor sodium nitroprusside (SNP) during germination. Excess 50 mM NaCl reduced significantly the seed germination rate in a short term and speed of germination. When salt concentration increased, germination of cucumber seed was reduced and the time needed to complete germination lengthened. Addition of exogenous SNP in salt solution attenuated the salt stress effects in a dose-dependent manner, as indicated by accelerating the seed germination, as well as weight increase of budding seeds, and 50 μM SNP was optimal concentration. At 150 mM NaCl, the 50 μM exogenous SNP significantly increased the activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and protein content, while decreased the contents of malondialdehyde (MDA). There were no obvious effects of exogenous NO on peroxidase (POD, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.6) activities under salt stress. Exogenous NO also increased the SOD and CAT isozyme expression under salt stress, which was in accordance with the improved antioxidant activities in the germinating seeds. The NO-induced salt stress resistance was associated with activated enzymes, and enhanced protein content, thus decreasing MDA content. It is concluded that exogenous NO treatment on cucumber seeds may be a good option to improve seed germination under saline conditions.     

Effects of the nitric oxide donor sodium nitroprusside on antioxidant enzymes in wheat seedling roots under nickel stress

The inhibitory effect of nickel on the growth of wheat (Triticum aestivum L.) seedlings and the alleviation of nickel toxicity by nitric oxide (NO) were investigated. Nickel (Ni) at 100 μM caused striking reduction in seedling growth and significant overproduction of MDA and H₂O₂ in the roots. Supplementation with NO donor sodium nitroprusside (SNP) could significantly reverse the inhibitory effect of nickel in a dose-dependent manner. K₃Fe(CN)₆, a SNP analogue, which does not release NO, had no ameliorative effect on Ni toxicity in wheat.. In addition, application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, could dramatically counteract the stimulatory effects of SNP on the growth of wheat seedling roots under Ni stress, confirming that NO rather than other compounds derived from SNP was responsible for the alleviating effect of Ni toxicity. Further results showed that SNP enhanced the activities of guaiacol peroxidase (POD, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase (SOD, EC 1..1..5.1..1), glutathione reductase (GR, EC 1.6.4.2), and glutathione S-transferase (GST, EC 2.5.1.18) in wheat seedling roots under nickel stress, while no significant difference in the activity of catalase (CAT, EC 1.11.1.6) in wheat roots supplemented with SNP or without it was observed. These results clearly indicate that NO has a protective role in Ni-induced oxidative damage through modulation of antioxidant enzymes.     

外源葡萄糖、果糖和NO供体(SNP)对盐胁迫下水稻种子萌发的影响

单独采用一氧化氮(nitric oxide,NO)供体硝普钠(sodiumnitroprusside,SNP)、葡萄糖和果糖浸种均不同程度地提高盐胁迫下水稻种子早期发芽率和发芽指数,SNP预处理可以不同程度地提高果糖和葡萄糖的含量;进一步采用葡萄糖和果糖分别与SNP混合后浸种,发现葡萄糖与SNP处理对盐胁迫下水稻种子的萌发有正协同效应,而果糖和SNP的组合处理对盐胁迫下水稻种子的萌发可能受到SNP一定程度的负调控.此外,SNP对盐胁迫下幼苗生长的促进效应可以被葡萄糖和果糖处理所加强,其中葡萄糖的效应更明显.     

Involvement of nitrate reductase-dependent nitric oxide production in magnetopriming-induced salt tolerance in soybean

In the present study, experiments were performed to investigate the role of nitric oxide (NO) in magnetopriming-induced seed germination and early growth characteristics of soybean (Glycine max) seedlings under salt stress. The NO donor (sodium nitroprusside, SNP), NO scavenger (2-[4-carboxyphenyl]-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, CPTIO), inhibitors of nitrate reductase (sodium tungstate, ST) or NO synthase (N-nitro-L-Arg-methyl ester, LNAME) and NADPH oxidase inhibitor (diphenylene iodonium, DPI) have been used to measure the role of NO in the alleviation of salinity stress by static magnetic field (SMF of 200 mT, 1 h). Salt stress (50 mM NaCl) significantly reduced germination and early growth of seedlings emerged from non-primed seeds. Pre-treatment of seeds with SMF positively stimulated the germination and consequently promoted the seedling growth. ST, LNAME, CPTIO and DPI significantly decreased the growth of seedling, activities of α-amylase, protease and nitrate reductase (NR), hydrogen peroxide (H₂O₂), superoxide (O₂^•−) and NO content in roots of seedlings emerged from non-primed and SMF-primed seeds. However, the extent of reduction was higher with ST in seedlings of SMF-primed seeds under both conditions, whereas SNP promoted all the studied parameters. Moreover, the generation of NO was also confirmed microscopically using a membrane permanent fluorochrome (4-5-diaminofluorescein diacetate [DAF-2 DA]). Further, analysis showed that SMF enhanced the NR activity and triggered the NO production and NR was maximally decreased by ST as compared to LNAME, CPTIO and DPI. Thus, in addition to ROS, NO might be one of the important signaling molecules in magnetopriming-induced salt tolerance in soybean and NR may be responsible for SMF-triggered NO generation in roots of soybean.     

Protective roles of nitric oxide on germination and antioxidant metabolism in wheat seeds under copper stress

Nitric oxide (NO) is a multifunctional gaseous signal in plant. In the present study, we found that pretreatment with NO could significantly improve wheat seeds germination and alleviate oxidative stress against copper toxicity. With the enhancement of copper stress, the germination percentage of wheat seeds decreased gradually. Pretreatment during wheat seed imbibition with sodium nitroprusside (SNP), an NO donor, could greatly reverse the inhibitory effect of the following copper stress to wheat seeds germination. SNP-pretreated seeds also tended to retain higher amylase activities than that of the control without SNP pretreatment. On the other hand, there was no apparent difference in the activities of esterase in wheat seeds pretreated with or without SNP. Further investigations showed that pretreatment with NO donor dramatically stimulated the activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6), decreased the activities of lipoxygenases, sustained a lower level of malondialdehyde, and interfered with hydrogen peroxide (H₂O₂) excessive accumulation compared with the control, thereby enhancing the antioxidative capacity in wheat seeds under copper stress. In addition, the seed copper contents were not significant different between those pretreated with SNP and the controls, inferring that protective roles of NO was not responsible for preventing Cu uptake. Kang-Di Hu and Lan-Ying Hu contributed equally to this paper.     

Salicylic acid and nitric oxide alleviate osmotic stress in wheat (Triticum aestivum L.) seedlings

Salicylic acid (SA) and nitric oxide (NO) are reported to alleviate the damaging effects of stress in plants rather similarly when applied at appropriate low concentrations. An experiment was therefore conducted to study the impact of SA, sodium nitroprusside (SNP; as NO donor), and methylene blue (MB; as a guanylate cyclase inhibitor) on wheat seedling performance under osmotic stress. Osmotic stress significantly reduced shoot fresh weight (SFW), chlorophyll contents (Chla, Chlb, total Chl), and membrane stability index (MSI) and also increased malondialdehyde (MDA) level, lipoxygenase (LOX) activity, and hydrogen peroxide production. Moreover, enzymatic antioxidant activities including superoxide dismutase, guaiacol peroxidase, and glutathione reductase activity were enhanced under osmotic stress. On the contrary, SA or SNP pretreatment reduced the damaging effects of osmotic stress by further enhancing the antioxidant activities that led to increased SFW, Chl, and MSI and reduced MDA level and LOX activity. However, pretreatment of plants with MB reversed or reduced the protective effects of SA and SNP suggesting that the protective effects were likely attributed to NO signaling. Therefore, NO may act as downstream of SA signaling in reduction of induced oxidative damage in wheat seedlings.     

外源CO和NO对水稻种子萌发过程中干旱胁迫损伤的缓解效应

选用水稻品种‘Ⅱ优128’种子为材料,以1.0μmol.L-1高铁血红素(Hematin,H)和200μmol.L-1硝普钠(sodium nitroprusside,SNP)分别作为CO和NO供体,采用PEG-6000模拟干旱胁迫,研究外源CO和NO对干旱胁迫下水稻种子萌发和萌发过程中抗氧化能力的影响。结果表明:高铁血红素和硝普钠处理可以显著提高干旱胁迫下水稻种子的发芽率、芽长和根长;同时显著提高种子淀粉酶活性,显著增加其可溶性糖、可溶性蛋白和脯氨酸含量;还不同程度地诱导增强超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)的活性,同时降低质膜相对透性和丙二醛(MDA)含量。研究证实,外源CO和NO可通过调节渗透调节物质含量和保护酶活性来有效缓解干旱胁迫对萌发水稻种子造成的氧化伤害,促进种子萌发生长。     

外源NO对缺铁豌豆幼苗生长以及光合作用的影响

以水培豌豆(Pisum sativum)品种‘陇碗一号’幼苗为材料,以硝普钠(SNP)为一氧化氮(NO)供体,研究外源NO对缺铁豌豆幼苗的生长及光合作用的影响。结果显示:0.6 mmol.L-1的外源SNP所产生的NO能够促进缺铁豌豆幼苗的生长,并促进叶绿素的合成,增强净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr),而使胞间CO2浓度(Ci)下降;同时,叶绿素荧光最大光化学量子产量(Fv/Fm)、实际光化学量子效率(Yield)和光化学淬灭系数(qP)均升高,在一定程度上缓解缺铁对豌豆幼苗叶片PSII反应中心的影响。外源NO对正常铁(100μmol.L-1Fe)处理下豌豆幼苗的生长和光合作用具有一定的抑制作用。研究表明,缺铁和正常铁处理的豌豆幼苗对NO的敏感性不同,适宜浓度的NO对缺铁下豌豆幼苗的生长和光合都具有一定的改善作用。     

一氧化氮供体硝普钠浸种缓解盐胁迫对小麦种子萌发的抑制作用

以小麦品种‘德抗961＇为材料,用NO供体硝普钠（SNP）浸种研究外源NO对盐胁迫下小麦种子萌发的影响.结果表明：0.06 mmol/L的SNP浸种24 h后对盐胁迫下小麦种子发芽率、发芽指数、活力指数和吸胀速率的下调都有显著缓解作用;SNP浸种对盐胁迫下α-淀粉酶的活性无明显影响,但能显著提高盐胁迫下β-淀粉酶的活性;进一步研究表明,SNP浸种预处理对盐胁迫下的α-淀粉酶同工酶变浅的条带有所恢复（尤其是条带3）,同时使盐胁迫下变浅的β-淀粉酶同工酶的条带有明显的恢复（尤其是d、e、f、g）.并且SNP能显著降低盐胁迫下小麦地上部分和根中的Na^＋含量,提高其K＋含量,从而使K^＋/Na^＋显著提高.以上结果表明：SNP浸种预处理提高盐胁迫下小麦种子的萌发,主要是通过提高β-淀粉酶的活性来实现的.     

Interactive Effect of Melatonin and Sodium Nitroprusside on Seed Germination and Seedling Growth of Catharanthus roseus under Cadmium Stress

Russian Journal of Plant Physiology - In this study, impact of seed presoaking with melatonin and sodium nitroprusside (SNP) (as a NO donor) was evaluated on seed germination and seedling growth of...     

Nitric oxide suppresses the inhibitory effect of abscisic acid on seed germination by S-nitrosylation of SnRK2 proteins

Nitric oxide (NO) plays important roles in plant development, and biotic and abiotic stress responses. In a recent study, we showed that endogenous NO negatively regulates abscisic acid (ABA) signaling in guard cells by inhibiting sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6)/open stomata 1(OST1) through S-nitrosylation. Application of NO breaks seed dormancy and alleviates the inhibitory effect of ABA on seed germination and early seedling growth, but it is unclear how NO functions at the stages of seed germination and early seedling development. Here, we show that like SnRK2.6, SnRK2.2 can be inactivated by S-nitrosoglutathione (GSNO) treatment through S-nitrosylation. SnRK2.2 and the closely related SnRK2.3 are known to play redundant roles in ABA inhibition of seed germination in Arabidopsis. We found that treatment with the NO donor SNP phenocopies the snrk2.2snrk2.3 double mutant in conferring ABA insensitivity at the stages of seed germination and early seedling growth. Our results suggest that NO negatively regulates ABA signaling in germination and early seedling growth through S-nitrosylation of SnRK2.2 and SnRK2.3.     

Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus

Nitric oxide (NO) is a bioactive molecule, which in plants was found to function as prooxidant as well as antioxidant. In the present study, we found that NO donor sodium nitroprusside (SNP) stimulates seed germination and root growth of lupin (Lupinus luteus L. cv. Ventus). Seed germination is promoted at concentrations between 0.1 and 800 μM SNP in a dose-dependent manner. The stimulation was most pronounced after 18 and 24 h and ceased after 48 h of imbibition. The promoting effect of NO on seed germination persisted even in the presence of heavy metals (Pb, Cd) and sodium chloride. Pretreatment of lupin seedlings for 24 h with 10 μM SNP resulted in efficient reduction of the detrimental effect of the abiotic stressors on root growth and morphology. The inhibitory effect of heavy metals on root growth was accompanied by increased activity of superoxide dismutase (SOD, EC 1.15.1.1.), which in roots preincubated with SNP was significantly higher. Some changes in the activity of other antioxidant enzymes, peroxidase (POX, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) were also detected. Using the superoxide anion (O₂^•–)-specific indicator dihydroethidium (DHE), we found intense DHE-derived fluorescence in heavy metal-stressed roots, whereas in those pretreated with SNP the fluorescence was very low, comparable to the level in unstressed roots. On the basis of the above data, we conclude that the protective effect of NO in stressed lupin roots may be at least partly due to the stimulation of SOD activity and/or direct scavenging of the superoxide anion.     

Arsenic toxicity: cell signalling and the attenuating effect of nitric oxide in Eichhornia crassipes

Nitric oxide (NO) is an important molecule involved in the perception of stress induced by toxic compounds such as arsenic (As). The present study investigated the role of NO applied as sodium nitroprusside (SNP) in cell signalling and the ability of NO to attenuate the toxic effects of As (in the form of sodium arsenate) in water hyacinth (Eichhornia crassipes). Water hyacinth plants were collected and assigned to one of the following treatments: control; 100 μM SNP; 20 μM As; or 20 μM As + 100 μM SNP. The plants remained under these conditions for 0, 4, 12, and 24 h. After each time interval, the plants were collected and As absorption, production of reactive oxygen species (ROS), integrity of membranes, and antioxidant enzyme activities were evaluated. The plants were able to absorb and accumulate large amounts of As, even after only four hours of exposure to the pollutant. The absorption and bioaccumulation factor of As was even greater when plants were exposed to both As and SNP. The accumulation of As triggered increases in ROS production and cell membrane damage. In the presence of SNP, the tolerance index to As increased and damage was mitigated. Therefore, from the present work, it was possible to conclude that exogenous NO influenced the ability of plants to tolerate As; this finding has implications for phytoremediation in areas contaminated by As.