外源一氧化氮对镉胁迫下玉米幼苗根生长及氧化伤害的影响

以玉米幼苗为材料,通过在镉处理的同时补充外源一氧化氮(NO)供体硝普钠(SNP)及其类似物[K3Fe(CN)6]、以及NO消除剂,分析NO对植物耐镉性的影响,探讨NO在植物逆境胁迫响应中的作用及其机理。结果显示:添加20μmol·L-1 SNP能显著降低镉引发的玉米幼苗根生长抑制及根尖内源镉的积累,减少电解质的渗漏以及超氧化物自由基(O2.-)和过氧化氢(H2O2)的上升幅度,抑制超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)活性的增加,进一步提高镉胁迫下谷胱甘肽还原酶(GR)的活性。SNP的上述效应可被NO消除剂2-(4-羧基-2-苯基)-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)所逆转,而SNP类似物K3Fe(CN)6的应用对上述反应几乎无影响,说明该反应具有NO特异性。研究表明,外源NO能够显著缓解镉胁迫对玉米幼苗生长造成的伤害,该缓解作用主要是通过降低植株体内内源镉积累和减轻镉诱发的氧化伤害来实现的。     

NO参与玉米幼苗对盐胁迫的应答

以玉米幼苗为材料,研究盐胁迫下其內源NO含量、NR和NOS活性的变化;NOS专一性抑制剂L-NAME和NR非专一性抑制剂NaN3对玉米幼苗內源NO含量的影响;利用激光共聚焦显微技术观测盐胁迫下玉米幼苗根部NO含量的变化及其分布特点。结果表明,盐胁迫下玉米幼苗根尖和叶片中NO含量有猝发现象,NOS活性也随之显著提高,NR活性则显著降低;L-NAME或NaN3均可降低盐胁迫所引起的玉米幼苗NO水平的增加,L-NAME对NO含量的影响比NaN3更显著。推测,NO参与玉米幼苗对盐胁迫的应答,NOS途径是盐胁迫下玉米幼苗內源NO合成的主要途径。     

Calcium is involved in nitric oxide- and auxin-induced lateral root formation in rice

In the present study, the role of nitric oxide (NO) in the regulation of lateral root (LR) formation in rice was examined. Application of sodium nitroprusside (SNP; a NO donor) and indole-3-butyric acid (IBA; a naturally occurring auxin) to rice seedlings induced LR formation. The effect is specific for NO because the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (cPTIO) blocked the action of SNP and IBA. Endogenous NO was detected by the specific fluorescence probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. SNP- and IBA-induced NO fluorescence was specifically suppressed by cPTIO. Nitrate reductase (NR) inhibitor sodium tungstate completely inhibited IBA-induced LR formation and NO fluorescence. However, nitric oxide synthase inhibitor N ^G-nitro-l-arginine methyl ester hydrochloride slightly reduced IBA-induced LR formation and NO generation. It appears that NO generation that occurs in response to IBA might primarily involve NR activity. Moreover, NO production caused by SNP and IBA was localized in root area corresponding to LR emergence. The effects of Ca²⁺ chelators, Ca²⁺-channel inhibitors, and calmodulin antagonists on LR formation induced by SNP and IBA were also examined. All these inhibitors were effective in reducing the action of SNP and IBA. However, Ca²⁺ chelators and Ca²⁺-channel inhibitors had no effect on SNP- and IBA-induced NO generation. It is concluded that cytosolic levels of Ca²⁺ may regulate SNP and IBA action through calmodulin-dependent mechanism.     

Inhibition of nitric oxide synthase (NOS) underlies aluminum-induced inhibition of root elongation in Hibiscus moscheutos

Aluminum (Al) is toxic to plants when solubilized into Al(3+) in acidic soils, and becomes a major factor limiting plant growth. However, the primary cause for Al toxicity remains unknown. Nitric oxide (NO) is an important signaling molecule modulating numerous physiological processes in plants. Here, we investigated the role of NO in Al toxicity to Hibiscus moscheutos. Exposure of H. moscheutos to Al(3+) led to a rapid inhibition of root elongation, and the inhibitory effect was alleviated by NO donor sodium nitroprusside (SNP). NO scavenger and inhibitors of NO synthase (NOS) and nitrate reductase had a similar inhibitory effect on root elongation. The inhibition of root elongation by these treatments was ameliorated by SNP. Aluminum inhibited activity of NOS and reduced endogenous NO concentrations. The alleviation of inhibition of root elongation induced by Al, NO scavenger and NOS inhibitor was correlated with endogenous NO concentrations in root apical cells, suggesting that reduction of endogenous NO concentrations resulting from inhibition of NOS activity could underpin Al-induced arrest of root elongation in H. moscheutos.     

Nitric oxide plays a central role in determining lateral root development in tomato

Nitric oxide (NO) is a bioactive molecule that functions in numerous physiological processes in plants, most of them involving cross-talk with traditional phytohormones. Auxin is the main hormone that regulates root system architecture. In this communication we report that NO promotes lateral root (LR) development, an auxin-dependent process. Application of the NO donor sodium nitroprusside (SNP) to tomato (Lycopersicon esculentum Mill.) seedlings induced LR emergence and elongation in a dose-dependent manner, while primary root (PR) growth was diminished. The effect is specific for NO since the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) blocked the action of SNP. Depletion of endogenous NO with CPTIO resulted in the complete abolition of LR emergence and a 40% increase in PR length, confirming a physiological role for NO in the regulation of root system growth and development. Detection of endogenous NO by the specific probe 4,5-diaminofluorescein diacetate (DAF-2 DA) revealed that the NO signal was specifically located in LR primordia during all stages of their development. In another set of experiments, SNP was able to promote LR development in auxin-depleted seedlings treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Moreover, it was found that LR formation induced by the synthetic auxin 1-naphthylacetic acid (NAA) was prevented by CPTIO in a dose-dependent manner. All together, these results suggest a novel role for NO in the regulation of LR development, probably operating in the auxin signaling transduction pathway.Abbreviations CPTIO 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide - DAF-2 DA 4,5-Diaminofluorescein diacetate - LR Lateral root - NAA 1-Naphthylacetic acid - NO Nitric oxide - NPA N-1-Naphthylphthalamic acid - PR Primary root - SNP Sodium nitroprusside     

镉胁迫下紫花苜蓿幼苗内源一氧化氮和活性氧的生成

以"甘农三号"紫花苜蓿幼苗为材料,在水培条件下,研究了不同浓度镉(Cd)胁迫下紫花苜蓿根、茎和叶内源一氧化氮(NO)和活性氧(ROS)的生成机制以及根系活力的变化.结果表明:在0~2.0 mmol·L-1范围内,随着Cd浓度的增加,幼苗内NO含量呈现先升高后降低的趋势,最后可维持在略高或持平于对照的水平.幼苗内一氧化氮合成酶(NOS)活性、硝酸还原酶(NR)活性、亚硝酸根离子(NO2-)含量和类胡萝卜素(Car)含量的变化与NO含量变化规律相似却又不全相同.NOS和NR是影响幼苗茎中NO含量的主要因素,NOS、NO2-和NR则是影响叶中NO含量的主要因素,而根中NO含量主要与NOS活性和NO2-含量有较大相关性.随着Cd浓度的增加,幼苗内过氧化氢(H2 O2)含量、丙二醛(MDA)含量、超氧阴离子(O-2·)含量和相对电导率(REC)呈现显著升高趋势,说明高浓度的Cd处理会使ROS大量积累,细胞膜遭破坏,细胞质外流,进而引发膜脂过氧化.随着Cd浓度的增加,紫花苜蓿根系活力的变化为先升高后降低,指示了低浓度Cd处理会促进植物代谢,增强其生命力;而高浓度Cd会致使植株代谢受抑制,细胞受损害.NO和ROS的相关性不大,说明二者虽同为自由基,但它们产生和变化方式大有差别.     

NO对He-Ne激光和增强UV-B辐射小麦幼苗气孔运动的作用机理研究

为探讨NO对He-Ne激光和增强UV-B辐射小麦（Triticum aestivuml）气孔运动的作用机理,采用低剂量（5 mW.mm-2）He-Ne激光和增强（10.08 kJ.m-2.d-1）UV-B辐射并结合药理学实验和激光共聚焦显微技术,对ML7113小麦的叶片及表皮条进行不同的处理,结果显示：（1）UV-B辐射既可诱导小麦叶片气孔关闭,又能够明显增加气孔保卫细胞和叶片的NO水平,且NO清除剂明显抑制了UV-B辐射诱导的小麦叶片气孔关闭,同时气孔保卫细胞和叶片内的NO含量明显减少。（2）一氧化氮合酶（NOS）抑制剂L-NAME对经UV-B辐射诱导的小麦幼苗气孔开度及保卫细胞和叶片内NO含量的抑制程度明显大于硝酸还原酶（NR）抑制剂NaN3对其的抑制程度,说明一氧化氮合酶（NOS）合成途径是小麦叶片经UV-B辐射后NO的主要产生途径。（3）就气孔开度而言,L〉CK〉BL〉B。就小麦叶片及保卫细胞内NO含量而言,B〉BL〉CK〉L。就硝酸还原酶（NR）和一氧化氮合酶（NOS）的活性而言,B组NR活性最低,NOS活性最高,L组NR活性最高,NOS活性最低。表明经He-Ne激光和增强UV-B辐射诱导的小麦气孔开度的变化确实与保卫细胞及叶片中NO含量的多少有关,气孔开度的减小及增大对应于NO含量的增多或减少,同时进一步证实了小麦叶片经He-Ne激光单独辐照后,NO的主要合成途径也来源于NOS途径。     

Constitutive arginine-dependent nitric oxide synthase activity in different organs of pea seedlings during plant development

Nitric oxide (NO) is an important signalling molecule in different animal and plant physiological processes. Little is known about its biological function in plants and on the enzymatic source or site of NO production during plant development. The endogenous NO production from l-arginine (NO synthase activity) was analyzed in leaves, stems and roots during plant development, using pea seedlings as a model. NOS activity was analyzed using a novel chemiluminescence-based assay which is more sensitive and specific than previous methods used in plant tissues. In parallel, NO accumulation was analyzed by confocal laser scanning microscopy using as fluorescent probes either DAF-2 DA or DAF-FM DA. A strong increase in NOS activity was detected in stems after 11 days growth, coinciding with the maximum stem elongation. The arginine-dependent NOS activity was constitutive and sensitive to aminoguanidine, a well-known irreversible inhibitor of animal NOS, and this NOS activity was differentially modulated depending on the plant organ and seedling developmental stage. In all tissues studied, NO was localized mainly in the vascular tissue (xylem) and epidermal cells and in root hairs. These loci of NO generation and accumulation suggest novel functions for NO in these cell types.     

Exogenous nitric oxide enhances cadmium tolerance of rice by increasing pectin and hemicellulose contents in root cell wall

To study the mechanisms of exogenous NO contribution to alleviate the cadmium (Cd) toxicity in rice (Oryza sativa), rice plantlets subjected to 0.2-mM CdCl₂ exposure were treated with different concentrations of sodium nitroprusside (SNP, a NO donor), and Cd toxicity was evaluated by the decreases in plant length, biomass production and chlorophyll content. The results indicated that 0.1 mM SNP alleviated Cd toxicity most obviously. Atomic absorption spectrometry and fluorescence localization showed that treatment with 0.1 mM SNP decreased Cd accumulation in both cell walls and soluble fraction of leaves, although treatment with 0.1 mM SNP increased Cd accumulation in the cell wall of rice roots obviously. Treatment with 0.1 mM SNP in nutrient solution had little effect on the transpiration rate of rice leaves, but this treatment increased pectin and hemicellulose content and decreased cellulose content significantly in the cell walls of rice roots. Based on these results, we conclude that decreased distribution of Cd in the soluble fraction of leaves and roots and increased distribution of Cd in the cell walls of roots are responsible for the NO-induced increase of Cd tolerance in rice. It seems that exogenous NO enhances Cd tolerance of rice by increasing pectin and hemicellulose content in the cell wall of roots, increasing Cd accumulation in root cell wall and decreasing Cd accumulation in soluble fraction of leaves.     

Carbon Monoxide Promotes Lateral Root Formation in Rapeseed

Carbon monoxide (CO), an odorless, tasteless and colorless gas, has recently proved to be an important bioactive or signalmolecule in mammalian cells, with its effects mediated mainly by nitric oxide (NO). In the present report, we show thatexogenous CO induces lateral root (LR) formation, an NO-dependent process. Administration of the CO donor hematin torapeseed (Brassica napus L. Yangyou 6) seedlings for 3 days, dose-dependently promoted the total length and number ofLRs. These responses were also seen following the application of gaseous CO aqueous solutions of different saturatedconcentrations. Furthermore, the actions of CO on seedlings were fully reversed when the CO scavenger hemoglobin (Hb)or the CO-specific synthetic inhibitor zinc protoporphyrin-IX (ZnPPIX) were added. Interestingly, depletion of endogenousNO using its specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO)or the nitric oxide synthase (NOS) inhibitor N~G-nitro-L-arginine methyl ester (L-NAME),led to the complete abolition ofLR development, illustrating an important role for endogenous NO in the action of CO on LR formation. However, theinduction of LR development by 200 umol/L sodium nitroprusside (SNP),an NO donor, was not affected by the presenceor absence of ZnPPIX. Furthermore, using an anatomical approach combined with laser scanning confocal microscopywith the NO-specific fluorophore 4,5-diaminofluorescein diacetate, we observed that both hematin and SNP increased NOrelease compared with control samples and that the NO signal was mainly distributed in the LR primordia (LRP), especiallyafter 36 h treatment. The LRP were found to have similar morphology in control, SNP-and hematin-treated seedlings.Similarly, the enhancement of the NO signal by CO at 36 h was differentially quenched by the addition of cPTIO, L-NAME,ZnPPIX and Hb. In contrast, the induction of NO caused by SNP was not affected by the application of ZnPPIX. Therefore,we further deduced that CO induces LR formation probably mediated by the NO/NOS pathway and NO may act downstreamof CO signaling, which has also been shown to occur in animals.     

铅胁迫下三叶鬼针草内源一氧化氮的生成及其对氧化损伤的缓解效应

对不同浓度铅(Pb)胁迫下三叶鬼针草(Bidens pilosa L.)叶、茎和根中内源一氧化氮(NO)和活性氧(ROS)的生成机制及根系活力的变化,内源NO对Pb胁迫下三叶鬼针草幼苗氧化损伤的缓解效应进行了研究。结果显示,在0~1000 mg/L范围内,随着Pb浓度的增加,叶片中NO含量呈升高趋势,根中NO含量呈先升高后降低的趋势,但仍高于对照,Pb浓度在0~400 mg/L范围内,茎中NO含量与对照持平,Pb浓度大于600 mg/L时,茎中NO含量低于对照;600 mg/L Pb处理能显著增强叶、茎和根中一氧化氮合成酶(NOS)和硝酸还原酶(NR)活性,显著增加叶和茎中亚硝酸根离子(NO_2~-)和类胡萝卜素(Car)含量,NOS、NR、NO_2~-和Car均能促进叶片中内源NO的生成,NOS是根中内源NO生成的主要途径。Pb胁迫使超氧阴离子(O_2~(·-))产生速率、过氧化氢(H_2O_2)含量、丙二醛(MDA)含量和相对电导率(REC)显著升高,从而造成幼苗严重的膜脂过氧化损伤,而胁迫诱发产生的NO能降低根中ROS的产生,促进幼苗根系活力,进而缓解胁迫造成的膜脂过氧化损伤。     

Carbon Monoxide Promotes Lateral Root Formation in Rapeseed

Carbon monoxide (CO), an odorless, tasteless and colorless gas, has recently proved to be an important bioactive or signal molecule in mammalian cells, with its effects mediated mainly by nitric oxide (NO). In the present report, we show that exogenous CO induces lateral root (LR) formation, an NO-dependent process. Administration of the CO donor hematin to rapeseed (Brassica napus L. Yangyou 6) seedlings for 3 days, dose-dependently promoted the total length and number of LRs. These responses were also seen following the application of gaseous CO aqueous solutions of different saturated concentrations. Furthermore, the actions of CO on seedlings were fully reversed when the CO scavenger hemoglobin (Hb)or the CO-specific synthetic inhibitor zinc protoporphyrin-Ⅸ (ZnPPIX) were added. Interestingly, depletion of endogenous NO using its specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO)or the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME), led to the complete abolition of LR development, illustrating an important role for endogenous NO in the action of CO on LR formation. However, the or absence of ZnPPIX. Furthermore, using an anatomical approach combined with laser scanning confocal microscopy with the NO-specific fluorophore 4,5-diaminofluorescein diacetate, we observed that both hematin and SNP increased NO release compared with control samples and that the NO signal was mainly distributed in the LR primordia (LRP), especially after 36 h treatment. The LRP were found to have similar morphology in control, SNP- and hematin-treated seedlings.Similarly, the enhancement of the NO signal by CO at 36 h was differentially quenched by the addition of cPTIO, L-NAME,ZnPPIX and Hb. In contrast, the induction of NO caused by SNP was not affected by the application of ZnPPIX. Therefore,we further deduced that CO induces LR formation probably mediated by the NO/NOS pathway and NO may act downstream of CO signaling, which has also been shown to occur in animals.     

Cascade transduction and production of the light-nitric oxide signal from shoots to roots in maize seedlings exposed to enhanced UV-B radiation

The biomasses, rate of apparent nitric oxide (NO)-release, nitric oxide synthase (NOS) activity as well as β-d-endo and exo-glucanase activity of the cell wall were analyzed and determined in the roots of maize seedlings. It was found that rhizospheric treatments of 2-phenyl-4,4,5,5-tetramethlimida-zoline-l-oxyl-3-oxide (PTIO), a NO scavenger, and radiation of enhanced ultraviolet-B (UV-B) to aerial parts of the seedling markedly inhibited the rate of NO release in roots, raised the activity of β-d-endo and exo-glucanase, and increased the biomasses of roots. The patent inhibitor, N-nitro-l-arginine (LNNA), of NOS was unable to inhibit NOS activity and NO generation. Inversely, reactive oxygen species (ROS) eliminator, N-acetyl-cysteine (NAC), stimulated the rate of NO release. There is no relationship between NOS activity and the rate of NO release. The latter showed a positive correlation with nitrate reductase (NR) activity, whereas it showed a negative correlation with the bio-masses and the activity of β-d-endo and exo-glucanase. All results implicated that NO was a by-product generated by NR catalysis, whereas NR activity was sensitively repressed by the systemic signal network (involved in ROS) induced by enhanced UV-B. It indicated that the downstream signal molecule of enhanced UV-B light is probably ROS which decreased NO generation through inhibiting NR activity. The endogenous NO generated by NR catalysis is perhaps such a messenger for restraining β-d-endo and exo-glucanase activity that the root growth was retarded.     

The dynamic distribution of NO and NADPH–diaphorase activity during IBA-induced adventitious root formation

Nitric oxide (NO) is a multifunctional molecule involved in numerous physiological processes in plants. In this study, we investigate the spatiotemporal changes in NO levels and endogenous NO‐generating system in auxin‐induced adventitious root formation. We demonstrate that NO mediates the auxin response, leading to adventitious root formation. Treatment of explants with the auxin indole‐3‐butyric acid (IBA) plus the NO donor sodium nitroprusside (SNP) together resulted in an increased number of adventitious roots compared with explants treated with SNP or IBA alone. The action of IBA was significantly reduced by the specific NO scavenger, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (c‐PTIO), and the nitric oxide synthase (NOS, enzyme commission 1.14.13.39) inhibitor, N^G‐nitro‐l ‐arg‐methyl ester (l ‐NAME). Detection of endogenous NO by the specific probe 4,5‐diaminofluorescein diacetate and survey of NADPH–diaphorase activity (commonly employed as a marker for NOS activity) by histochemical staining revealed that during adventitious root formation, NO and NADPH–diaphorase signals were specifically located in the adventitious root primordia in the basal 2‐mm region (as zone I) of both control and IBA‐treated explants. With the development of root primordia, NO and NADPH–diaphorase signals increased gradually and were mainly distributed in the root meristem. Endogenous NO and NADPH–diaphorase activity showed overall similarities in their tissue localization. Distribution of NO and NADPH–diaphorase activity similar to that in zone I were also observed in the basal 2–4‐mm region (zone II) of IBA‐treated explants, but neither NO nor NADPH–diaphorase signals were detected in this region of the control explants. l ‐NAME and c‐PTIO inhibited the formation of adventitious roots induced by IBA and reduced both NADPH–diaphorase staining and NO fluorescence. These results show the dynamic distribution of endogenous NO in the developing root primordia and demonstrate that NO plays a vital role in IBA‐induced adventitious rooting. Also, the production of NO in this process may be catalyzed by a NOS‐like enzyme.     

NOS-like-mediated nitric oxide is involved in Pinus thunbergii response to the invasion of Bursaphelenchus xylophilus

The content of NO and H(2)O(2) as well as the activities of nitric oxide synthase (NOS)-like and nitrate reductase (NR) were monitored in the needles of Pinus thunbergii infected by Bursaphelenchus xylophilus. The results showed that the content of NO increased significantly only 8?h after the invasion of B. xylophilus, while H(2)O(2) increased 12?h after invasion. NO donor SNP could promote and NO scavenger cPTIO could prevent the production of NO and H(2)O(2). The content of NO changed earlier than that of H(2)O(2). In addition, the symptoms appeared 9, 5 and 12?days, respectively, after the inoculation with B. xylophilus, SNP pre-treatment and cPTIO pre-treatment followed by B. xylophilus infection. After B. xylophilus infection, the content of NO in P. thunbergii changed fiercely more earlier than the appearance of external symptoms, which indicated that the content of NO was related with the appearance and the development of the symptoms. The treatment with L-NNA (NOS inhibitor) inhibited the content of NO significantly, whereas, Na(2)WO(4) (NR inhibitor) had no effect. The further analysis of NOS revealed that NO changed in consistent with cNOS activity. To sum up, NO, as the upstream signal molecule of H(2)O(2), was involved in the pine early response to the invasion of B. xylophilus and influenced the accumulation of the content of H(2)O(2). Moreover, NOS-like rather than NR was responsible for the endogenous NO generation, which was modulated by cNOS during the interaction between P. thunbergii and B. xylophilus. Key message NO is involved in early response of P. thunbergii to the invasion of B. xylophilus and NOS is the key enzyme responsible for NO generation in P. thunbergii.     

Nitric oxide is involved in nitrate-induced inhibition of root elongation in Zea mays

BACKGROUND AND AIMS: Root growth and development are closely dependent upon nitrate supply in the growth medium. To unravel the mechanism underlying dependence of root growth on nitrate, an examination was made of whether endogenous nitric oxide (NO) is involved in nitrate-dependent growth of primary roots in maize. METHODS: Maize seedlings grown in varying concentrations of nitrate for 7 d were used to evaluate the effects on root elongation of a nitric oxide (NO) donor (sodium nitroprusside, SNP), a NO scavenger (methylene blue, MB), a nitric oxide synthase inhibitor (N(omega)-nitro-L-arginine, L-NNA), H(2)O(2), indole-3-acetic acid (IAA) and a nitric reducatse inhibitor (tungstate). The effects of these treatments on endogenous NO levels in maize root apical cells were investigated using a NO-specific fluorescent probe, 4, 5-diaminofluorescein diacetate (DAF-2DA) in association with a confocal microscopy. KEY RESULTS: Elongation of primary roots was negatively dependent on external concentrations of nitrate, and inhibition by high external nitrate was diminished when roots were treated with SNP and IAA. MB and L-NNA inhibited root elongation of plants grown in low-nitrate solution, but they had no effect on elongation of roots grown in high-nitrate solution. Tungstate inhibited root elongation grown in both low- and high-nitrate solutions. Endogenous NO levels in root apices grown in high-nitrate solution were lower than those grown in low-nitrate solution. IAA and SNP markedly enhanced endogenous NO levels in root apices grown in high nitrate, but they had no effect on endogenous NO levels in root apical cells grown in low-nitrate solution. Tungstate induced a greater increase in the endogenous NO levels in root apical cells grown in low-nitrate solution than those grown in high-nitrate solution. CONCLUSIONS: Inhibition of root elongation in maize by high external nitrate is likely to result from a reduction of nitric oxide synthase-dependent endogenous NO levels in maize root apical cells.     

Involvement of nitric oxide-induced NADPH oxidase in adventitious root growth and antioxidant defense in Panax ginseng

Nitric oxide (NO) affects the growth and development of plants and also affects plant responses to various stresses. Because NO induces root differentiation, we examined whether or not it is involved in increased ROS generation. Treatments with sodium nitroprusside (SNP), an NO donor, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger, and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), an NO synthase (NOS) inhibitor, revealed that NO is involved in the adventitious root growth of mountain ginseng. Supply of an NO donor, SNP, activates NADPH oxidase activity, resulting in increased generation of O₂ ^·−, which subsequently induces growth of adventitious roots. Moreover, treatment with diphenyliodonium chloride (DPI), an NADPH oxidase inhibitor, individually or with SNP, inhibited root growth, NADPH oxidase activity, and O₂ ^·− anion generation. Supply of the NO donor, SNP, did not induce any notable isoforms of enzymes; it did, however, increase the activity of pre-existing bands of NADPH oxidase, superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase. Enhanced activity of antioxidant enzymes induced by SNP supply seems to be responsible for a low level of H₂O₂ in the adventitious roots of mountain ginseng. It was therefore concluded that NO-induced generation of O₂ ^·− by NADPH oxidase seems to have a role in adventitious root growth of mountain ginseng. The possible mechanism of NO involvement in O₂ ^·− generation through NADPH oxidase and subsequent root growth is discussed.     

Involvement of nitric oxide in water stress-induced responses of cucumber roots

The effect of water deficit on nitric oxide (NO) generation was investigated in cucumber (Cucumis sativus cv. Dar) seedling roots using bio-imaging with an NO-selective fluorophor, diaminofluorescein-2-diacetate (DAF-2DA). Roots subjected to mild (5 and 10 h) water deficit showed slightly enhanced NO synthesis in cells of root tips and in the surrounding elongation zone. However, severe (17 h) stress resulted in an intensive NO production localized mainly in and above the elongation zone. Water stress-induced NO generation was blocked by a specific NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) as well as nitrate reductase (NR) and partially by nitric oxide synthase (NOS-like) inhibitors.A pharmacological approach was used in order to verify the capacity of NO to induce adaptive responses of cucumber roots to water deficit. A positive correlation was found between NO donor (SNP 100 μM and GSNO 100 μM) pretreatment and plant hydration status, measured as relative water content (RWC) during progressive dehydration. At an early stage (5 h) of stress duration NO caused a periodical increase in lipoxygenase (LOX) activity, correlated with time-dependent enhancement of lipid peroxidation. Beginning from 10 h up to severe stress (17 h) exogenous NO was able to diminish LOX activity and alleviate water deficit-induced membrane permeability and lipid peroxidation, measured as TBARS content and visualised by histochemical staining in situ. Observed changes via NO were accompanied by a significant reduction of proline level, suggesting that the accumulation of this osmolyte might not be essential in water stress tolerance. Obtained results clearly indicate that NO augmentation is likely to help the plant at the initial stage of tissue dehydration to trigger efficient mechanisms, mitigating severe water deficit effects in roots of cucumber seedlings.     

Regulation of nitrate reductase by nitric oxide in Chinese cabbage pakchoi (Brassica chinensis L.)

Nitrate reductase (NR), a committed enzyme in nitrate assimilation, involves generation of nitric oxide (NO) in plants. Here we show that the NR activity was significantly enhanced by the addition of NO donors sodium nitroprusside (SNP) and NONOate (diethylamine NONOate sodium) to the culturing solution, whereas it was decreased by NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, both NO gas and SNP directly enhanced but cPTIO inhibited the NR activities of crude enzyme extracts and purified NR enzyme. The cPTIO terminated the interaction between NR-generated NO and the NR itself. Furthermore, the NR protein content was not affected by the SNP treatment. The investigation of the partial reactions catalysed by purified NR using various electron donors and acceptors indicated that the haem and molybdenum centres in NR were the two sites activated by NO. The results suggest that the activation of NR activity by NO is regulated at the post-translational level, probably via a direct interaction mechanism. Accordingly, the concentration of nitrate both in leaves and roots was decreased after 2 weeks of cultivation with SNP. The present study identifies a new mechanism of NR regulation and nitrate assimilation, which provides important new insights into the complex regulation of N-metabolism in plants.     

Nitric Oxide Functions as a Positive Regulator of Root Hair Development

The root epidermis is composed of two cell types: trichoblasts (or hair cells) and atrichoblasts (or non-hair cells). In lettuce (Lactuca sativa cv. Grand Rapids var. Rapidmor oscura) plants grown hydroponically in water, the root epidermis did not form root hairs. The addition of 10 µM sodium nitroprusside (SNP), a nitric oxide (NO) donor, resulted in almost all rhizodermal cells differentiated into root hairs. Treatment with the synthetic auxin 1-naphthyl acetic acid (NAA) displayed a significant increase of root hair formation (RHF) that was prevented by the specific NO scavenger carboxy-PTIO (cPTIO). In Arabidopsis, two mutants have been shown to be defective in NO production and to display altered phenotypes in which NO is implicated. Arabidopsis nos1 has a mutation in an NO synthase structural gene (NOS1), and the nia1 nia2 double mutant is null for nitrate reductase (NR) activity. We observed that both mutants were affected in their capacity of developing root hairs. Root hair elongation was significantly reduced in nos1 and nia1 nia2 mutants as well as in cPTIO-treated wild type plants. A correlation was found between endogenous NO level in roots detected by the fluorescent probe DAF-FM DA and RHF. In Arabidopsis, as well as in lettuce, cPTIO blocked the NAA-induced root hair elongation. Taken together, these results indicate that: (1) NO is a critical molecule in the process leading to RHF and (2) NO is involved in the auxin-signaling cascade leading to RHF.Key Words: auxin, nitric oxide, root hair, lettuce, arabidopsis, nos1 mutant, nia1, nia2 mutant