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.     

Function of nitric oxide and superoxide anion in the adventitious root development and antioxidant defence in <Emphasis Type="Italic">Panax ginseng</Emphasis>

The involvement of NO in O₂ ^·− generation, rootlet development and antioxidant defence were investigated in the adventitious root cultures of mountain ginseng. Treatments of NO producers (SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetylpenicillamine; and sodium nitrite with ascorbic acid), and NO scavenger (PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide) revealed that NO is involved in the induction of new rootlets. Severe decline in number of new rootlets compared to the control under PTIO treatment indicates that NO acts downstream of auxin action in the process. NO producers (SNP, SNAP and sodium nitrite with ascorbic acid) activated NADPH oxidase activity, resulting in greater O₂ ^·− generation and higher number of new rootlets in the adventitious root explants. Moreover, treatment of diphenyliodonium chloride, a NADPH oxidase inhibitor, individually or along with SNP, inhibited root growth, NADPH oxidase activity and O₂ ^·− anion generation. NO supply also enhanced the activities of antioxidant enzymes that are likely to be responsible for reducing H₂O₂ levels and lipid peroxidation as well as modulation of ascorbate and non-protein thiol concentrations in the adventitious roots. Our results suggest that NO-induced generation of O₂ ^·− by activating NADPH oxidase activity is related to adventitious root formation in mountain ginseng.     

Modulation of copper toxicity-induced oxidative damage by nitric oxide supply in the adventitious roots of <Emphasis Type="Italic">Panax ginseng</Emphasis>

Nitric oxide (NO) is a highly reactive, membrane-permeable free radical, which has recently emerged as an important signalling molecule and antioxidant. Here we investigated the protective effect of NO against the toxicity caused by excess CuSO₄ (50 μM) in the adventitious roots of mountain ginseng. It was found that NO donor, sodium nitroprusside (SNP), was effective in reducing Cu-induced toxicity in the mountain ginseng adventitious roots. Protective effect of SNP, as indicated by extent of lipid peroxidation, was reversed by incorporation of 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (CPTIO), a NO scavenger, in the medium suggesting that the protective effect of SNP is attributable to NO released, which was revealed from in situ confocal laser scanning microscopic localization of NO in the adventitious roots of mountain ginseng. Results obtained in the present study suggest that reduction of excess Cu-induced toxicity by SNP is most likely mediated through the modulation in the activities of antioxidant enzymes involved in H₂O₂ detoxification (catalase, peroxidase, ascorbate peroxidase) and in the maintenance of cellular redox couples (glutathione reductase), and contents of molecular antioxidants (particularly non-protein thiol, ascorbate and its redox status). Exogenous NO supply also improved the activity of superoxide dismutase, an enzyme responsible for O₂ ^·− dismutation, and NADPH oxidase, an enzyme responsible for O₂ ^·− generation, in excess Cu supplied adventitious roots of mountain ginseng.     

Salicylic Acid-induced Nitric Oxide and ROS Generation Stimulate Ginsenoside Accumulation in Panax ginseng Roots

We evaluated the involvement of nitric oxide (NO) in salicylic acid (SA)-induced accumulation of ginsenoside in adventitious roots of Panax ginseng and its mediation by reactive oxygen species (ROS). Related effects of SA on components of the antioxidant system were also sought. Adventitious roots of P. ginseng were grown in suspension culture for 3 weeks in MS medium and treated over 5 days with SA (100 μM) alone, SA in combination with the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), or PTIO alone. Nitric oxide, the superoxide anion (O₂^·−), H₂O₂, nitrite, nonprotein thiol, and ascorbate were monitored together with ginsenoside, NADPH oxidase activity, and several antioxidant enzymes. Salicylic acid did not inhibit root growth but induced accumulation of ginsenoside, lipid peroxidation, and generation of NO and O₂^·−. It also enhanced activities of NADPH oxidase, superoxide dismutase, catalase, and peroxidase, including ascorbate peroxidase. These effects were suppressed by PTIO. Salicylic acid also decreased glutathione reductase activity. Inclusion of PTIO with SA decreased the activity of glutathione reductase further. Treatment with SA plus PTIO also decreased nonprotein thiol and ascorbate contents but caused nitrite to overaccumulate. Salicylic acid applied to adventitious roots in culture induced accumulation of ginsenoside in an NO-dependent manner that was mediated by the associated increases in O₂^·−, which gave other antioxidant responses that were dependent on NO.     

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.     

Nitric oxide affecting root growth,lignification and related enzymes in soybean seedlings

This study analyzed the involvement of nitric oxide (NO) in the root lignification of soybean seedlings. To this end, changes in root cell viability; phenylalanine ammonia-lyase (PAL) and soluble and cell wall bound peroxidase (POD) activities and lignin and hydrogen peroxide (H₂O₂) contents of soybean roots treated with the NO-donor sodium nitroprusside (SNP) and its relationships with root growth were evaluated. Seedlings were cultivated in a nutrient solution supplemented with 5 to 1,000 μM SNP for 24 h. At an extremely low concentration (5 μM), SNP induced root growth and increased lignification and activities of related enzymes (PAL and cell wall-bound POD). At a high concentration (1,000 μM), SNP reduced root growth and lignification (PAL activity and H₂O₂ and lignin contents) and caused a loss of cell viability. Application of potassium ferrocyanide (an analog of SNP that cannot release NO) and PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoleline-1-oxyl-3-oxide, a scavenger of NO) revealed that the inhibitory/stimulatory effects on root lignification may be due to NO itself. These results indicate that NO, depending on its concentration, may act as a stress factor, due to its toxic action, or as a signal molecule, inducing soybean root growth and lignification.     

Effects of root and foliar applications of exogenous NO on alleviating cadmium toxicity in lettuce seedlings

The effects of sodium nitroprusside (SNP, a donor of NO) on cadmium (Cd) toxicity in lettuce seedlings were studied. SNP was added into hydroponic systems or sprayed directly on the leaves of plants grown with and without Cd. Excess supply of Cd (100 μM) caused growth inhibition, dramatically increased Cd accumulation in both leaves and roots, and inhibited the absorption of Ca, Mg, Fe and Cu. Excess Cd also decreased activities of superoxide dismutase peroxidase and catalase in leaves and roots, and increased the accumulation of superoxide anion (O ₂ ^·? ), hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). Root or foliar applications of exogenous NO alleviated Cd-induced growth suppression, especially root application of 250 μM SNP and foliar addition of 500 μM SNP. Addition of SNP promoted the chlorophyll synthesis suggesting that the photosynthesis was up-regulated. Exogenous NO increased Cd-decreased activities of antioxidant enzymes and markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation. Moreover, the absorption of Ca, Mg, Fe and Cu was increased, indicating that exogenous NO stimulated H⁺-ATPase activity to promote sequestration or uptake of ions. In addition, exogenous NO also inhibited Cd transfer from roots to shoots, which may indicate that Cd retention in roots induced by NO plays a significant role in Cd tolerance in lettuce seedlings. These data suggest that under Cd stress, exogenous NO improves photosynthesis by increasing chlorophyll synthesis, protects lettuce seedlings against oxidative damage by scavenging ROS, helps to maintain the uptake of nutrient elements, and inhibits Cd transferred to shoots effectively.     

Role and relationship of nitric oxide and hydrogen peroxide in adventitious root development of marigold

Several lines of evidence suggest that nitric oxide (NO) and hydrogen peroxide (H₂O₂) are important signal molecules involved in plant development and other physiological processes. Marigold (Tagetes erecta L. ‘Marvel’) was used to understand the role and relationship of NO and H₂O₂ in adventitious root development of plants. The results showed that the effects of H₂O₂ or NO on adventitious root organogenesis of explants were dose dependent, with maximal biological responses at 200 μM H₂O₂ or 50 μM NO donor sodium nitroprusside (SNP). The results also indicated the importance of both putative NO synthase (NOS)-like and nitrate reductase (NR) enzymes, which might be responsible for the production of NO in explants during rooting. Additionally, guanosine 3′, 5′ -cyclic monophosphate (cGMP) was involved in NO- induced root formation of marigold, but it was not involved in H₂O₂- mediated rooting process. The root number and length of explants treated with NO and H₂O₂ simultaneously were significantly higher than those of explants treated with H₂O₂ or NO alone. Moreover, NO treatments enhanced endogenous H₂O₂ levels in hypocotyls. Together, these results indicate that NO and H₂O₂ play crucial roles in the adventitious root development of marigold explants both synergistically and independently.     

Arsenic-induced oxidative stress in the common bean legume,Phaseolus vulgaris L. seedlings and its amelioration by exogenous nitric oxide

The adverse effects of arsenic (As) toxicity on seedling growth, root and shoot anatomy, chlorophyll and carotenoid contents, root oxidizability (RO), antioxidant enzyme activities, H₂O₂ content, lipid peroxidation and electrolyte leakage (EL%) in common bean (Phaseolus vulgaris L.) were investigated. The role of exogenous nitric oxide (NO) in amelioration of As-induced inhibitory effect was also evaluated using sodium nitroprusside (100 μM SNP) as NO donor and 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (200 μM PTIO) as NO scavenger in different combinations with 50 μM As. As-induced growth inhibition was associated with marked anomalies in anatomical features, reduction in pigment composition, increased RO and severe perturbations in antioxidant enzyme activities. While activity of superoxide dismutase and catalase increased, levels of ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase decreased significantly and guaiacol peroxidase remained normal. The over-accumulation of H₂O₂ content along with high level of lipid peroxidation and electrolyte leakage indicates As-induced oxidative damage in P. vulgaris seedlings with more pronounced effect on the roots than the shoots. Exogenous addition of NO significantly reversed the As-induced oxidative stress, maintaining H₂O₂ in a certain level through balanced alterations of antioxidant enzyme activities. The role of NO in the process of amelioration has ultimately been manifested by significant reduction of membrane damage and improvement of growth performance in plants grown on As + SNP media. Onset of oxidative stress was more severe after addition of PTIO, which confirms the protective role of NO against As-induced oxidative damage in P. vulgaris seedlings.     

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.     

The interaction between H<Subscript>2</Subscript>O<Subscript>2</Subscript> and NO,Ca<Superscript>2+</Superscript>, cGMP,and MAPKs during adventitious rooting in mung bean seedlings

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In the present study, we present a signaling network involving H₂O₂, nitric oxide (NO), calcium (Ca²⁺), cyclic guanosine monophosphate (cGMP), and the mitogen-activated protein kinase (MAPK) cascade during adventitious rooting in mung bean seedlings. Both exogenous H₂O₂ and the NO donor sodium nitroprussiate were capable of promoting the formation and development of adventitious roots. H₂O₂ and NO signaling pathways were elicited in parallel in auxin-induced adventitious rooting. Cytosolic Ca²⁺ was required for adventitious rooting, and Ca²⁺ served as a downstream component of H₂O₂, as well as cGMP or MAPK, signaling cascades. cGMP and MAPK cascades function downstream of H₂O₂ signaling and depend on auxin responses in adventitious root signaling processes.     

Regulation of defence responses in avocado roots infected with Phytophthora cinnamomi (Rands)

Phytophthora cinnamomi occurs worldwide and has a host range in excess of 1,000 plant species. Avocados (Persea americana Mill) have been described as highly susceptible to this soil-borne pathogen. Here, the regulation of defence responses in avocado root seedlings inoculated with P. cinnamomi mycelia is described. A burst of reactive oxygen species (ROS) was observed 4 days after inoculation. The higher physiological concentration of H₂O₂ induced by P. cinnamomi on avocado roots had no effect on in vitro growth of the oomycete. Total phenols and epicathecin content showed a significant decrease, but lignin and pyocianidins exhibited no changes after inoculation. Also, increased nitric oxide (NO) production was observed 72 h after treatment. We studied the effects of one NO donor [sodium nitroprusside (SNP)], and one NO scavenger [2- to 4-carboxyphenyl-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (CPTIO)] to determine the role of NO during root colonisation by P. cinnamomi mycelia. Pretreatment of the roots with CPTIO, but not with SNP, inhibited root colonisation suggesting an important role for NO production during the avocado–P. cinnamomi interaction. Our data suggest that although defence responses are activated in avocado roots in response to P. cinnamomi infection, these are not sufficient to avoid pathogen invasion.     

Nitric oxide (NO) counteracts cadmium induced cytotoxic processes mediated by reactive oxygen species (ROS) in Brassica juncea: cross-talk between ROS, NO and antioxidant responses

Research on NO in plants has achieved huge attention in recent years mainly due to its function in plant growth and development under biotic and abiotic stresses. In the present study, we investigated Cd induced NO generation and its relationship to ROS and antioxidant regulation in Brassica juncea. Cd accumulated rapidly in roots and caused oxidative stress as indicated by increased level of lipid peroxidation and H₂O₂ thus, inhibiting the overall plant growth. It significantly decreased the root length, leaf water content and photosynthetic pigments. A rapid induction in intracellular NO was observed at initial exposures and low concentrations of Cd. A 2.74-fold increase in intracellular NO was recorded in roots treated with 25 μM Cd than control. NO effects on Malondialdehyde (MDA) content and on antioxidant system was investigated by using sodium nitroprusside (SNP), a NO donor and a scavenger, [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide] (cPTIO). Roots pretreated with 5 mM SNP for 6 h when exposed to 25 μM Cd for 24 h reduced the level of proline, non-protein thiols, SOD, APX and CAT in comparison to only Cd treatments. However, this effect was almost blocked by 100 μM cPTIO pretreatment to roots for 1 h. This ameliorating effect of NO was specific because cPTIO completely reversed the effect in the presence of Cd. Thus, the present study report that NO strongly counteracts Cd induced ROS mediated cytotoxicity in B. juncea by controlling antioxidant metabolism as the related studies are not well reported in this species.     

Chromium Differentially Affects Hydrogen Peroxide Distribution in Primary and Adventitious Roots of <i>Arabidopsis thaliana</i> L.

The post-embryonic growth of the Arabidopsis thaliana root system can be modified by different types of stress, such as sublethal concentrations of metals, which may induce the production of reactive oxygen species (ROS). In this study, the effects of different concentrations of potassium chromate (K_₂CrO₄) on the distribution and relative quantity of hydrogen peroxide (H₂O₂) were determined in primary and adventitious roots in A. thaliana HyPer line seedlings. This line has a biosensor that specifically reports H₂O₂ levels within tissues as fluorescence. Primary root growth was inhibited at 100 μM Cr (VI); in contrast, adventitious root formation was induced over the main root growth axis. These structures proliferated from 100-160 μM Cr (VI), and much higher concentrations (180-200 μM) of K_₂CrO₄ were required to affect their growth. The H₂O₂ distributions were observed in the columella and lateral root cap of primary roots of plants grown in medium lacking dichromate, but following the development of toxicity symptoms, H₂O₂ changed its distribution to the meristem and differentiation zones. Conversely, adventitious roots had comparable H₂O₂ distribution patterns in untreated plants and those exposed to Cr (VI) supplementation. Thus, differential H₂O₂ distribution correlates with the resistance of adventitious roots, but not primary roots, to dichromate and underlies cell reorganization at the apex to support growth.     

Hydrogen peroxide acts as a signal molecule in the adventitious root formation of mung bean seedlings

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In this study we demonstrated that the exogenous H₂O₂ was able to promote the formation and development of adventitious roots in mung bean seedlings. Treatments with 1–100 mM H₂O₂ for 8–18 h significantly induced the formation and development of adventitious roots. Catalase (CAT) and ascorbic acid, which are H₂O₂ scavengers or inhibitors, eliminated the adventitious root-promoting effects of exogenous H₂O₂. H₂O₂ may have a downstream signaling function in the auxin signaling pathway and be involved in auxin-induced adventitious root formation. 2,3,5-Triiodobenzoic acid (TIBA), an inhibitor of auxin polar transport, strongly inhibited adventitious rooting of mung bean seedlings; however, the inhibiting effects of TIBA on adventitious rooting can be partially reversed by the exogenous IBA or H₂O₂. Diphenylene iodonium (DPI) strongly inhibits the activity of NADPH oxidase, which is one of the main sources of H₂O₂ formation in plant cells. DPI treatment strongly inhibited the formation of adventitious roots in mung bean, but the inhibitory effects of DPI on rooting can be partially reversed by the exogenous H₂O₂ or IBA. This indicates that the formation of adventitious roots was blocked once the generation of H₂O₂ through NADPH oxidase was inhibited, and H₂O₂ mediated the IBA-induced adventitious root formation. Furthermore, a rapid increase in the endogenous level of H₂O₂ was detected during incubation with water 12–36 h after the primary root removal in mung bean seedlings. Three hours after the primary root removal, the generation of endogenous H₂O₂ was markedly induced in IBA-treated seedlings in comparison with water-treated seedlings. This implies that IBA induced overproduction of H₂O₂ in mung bean seedlings, and that IBA promoted adventitious root formation via a pathway involving H₂O₂. Results obtained suggest that H₂O₂ may function as a signaling molecule involved in the formation and development of adventitious roots in mung bean seedlings.     

Nitric oxide improves thermotolerance in spring maize by inducing varied genotypic defense mechanisms

The investigation aimed at determining the effect of nitric oxide on antioxidant defense system of spring maize (Zea mays L.) genotypes namely, LM 11 (stress susceptible) and CML 32 (stress tolerant), that showed differential tolerance towards heat stress. Seed priming with a NO donor, sodium nitroprusside (SNP) improved seedling growth and induced varied defense mechanisms, under stress conditions. 75 μM SNP improved seedling lengths and their biomasses. It specifically enhanced catalase (CAT) activity in the roots of stressed seedlings of the two genotypes. However, it could induce CAT activity only in LM 11 shoots, under heat stress. It also enhanced peroxidase (POX) activity in CML 32 roots. However, such induction of POX activity with SNP treatment was not observed in LM 11 roots. This showed that NO increased the H₂O₂ scavenging efficiency of CML 32 genotype by enhancing the cumulative activation of CAT and POX in its roots. However, it did not induce activation of any of the H₂O₂ detoxifying enzymes in CML 32 shoots which showed that ascorbate–glutathione cycle remained non-operational in shoots of SNP-treated seedlings of the tolerant genotype, under high temperature stress. With seed priming, superoxide dismutase (SOD) activity increased in both the tissues of LM 11 seedlings. The shoots of SNP primed CML 32 seedlings, however, did not show any effect on SOD activity which illustrated that nitric oxide might act as a direct scavenger of superoxide radicals in CML 32 seedlings. SNP decreased the contents of H₂O₂ and MDA and increased proline content in seedlings of both the genotypes indicating reduced oxidative damage. The results thus showed that nitric oxide might induce different mechanisms of stress tolerance in these maize genotypes.     

Reactive oxygen species and nitric oxide are involved in polyamine-induced growth inhibition in wheat plants

Polyamines (PAs) produce H₂O₂ and nitric oxide (NO) during their normal catabolism and modulate plant growth and development. To explore the biochemical basis of PAs-induced growth inhibition in Triticum aestivum L seedlings, we examined the role of O₂^·-, H₂O₂ or NO in shoot and root development. Although all PA treatments resulted in a variable reduction of root and shoot elongation, spermine (Spm) caused the greater inhibition in a similar way to that observed with the NO donor, sodium nitroprusside (SNP). In both cases, O₂^·- production was completely blocked whereas H₂O₂ formation was high in the root apex under SNP or Spm treatments. Catalase recovered root and shoot growth in SNP but not in Spm-treated plants, revealing the involvement of H₂O₂ in SNP-root length reduction. The addition of the NO scavenger, cPTIO, restored root length in SNP- or Spm-treated plants, respectively, and partially recovered O₂^·- levels, compared to the plants exposed to PAs or SNP without cPTIO. A strong correlation was observed between root growth restoration and O₂^·- accumulation after treating roots with SNP + aminoguanidine, a diamine oxidase inhibitor, and with SNP + 1,8-diaminoctane, a polyamine oxidase inhibitor, confirming the essential role of O₂^·- formation for root growth and the importance of the origin and level of H₂O₂. The differential modulation of wheat growth by PAs through reactive oxygen species or NO is discussed.

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Graphical abstract Polyamines, nitric oxide and ROS interaction in plants during plant growth

     

Hydrogen Peroxide Is a Second Messenger in the Salicylic Acid-Triggered Adventitious Rooting Process in Mung Bean Seedlings

In plants, salicylic acid (SA) is a signaling molecule that regulates disease resistance responses, such as systemic acquired resistance (SAR) and hypertensive response (HR). SA has been implicated as participating in various biotic and abiotic stresses. This study was conducted to investigate the role of SA in adventitious root formation (ARF) in mung bean (Phaseolus radiatus L) hypocotyl cuttings. We observed that hypocotyl treatment with SA could significantly promote the adventitious root formation, and its effects were dose and time dependent. Explants treated with SA displayed a 130% increase in adventitious root number compared with control seedlings. The role of SA in mung bean hypocotyl ARF as well as its interaction with hydrogen peroxide (H₂O₂) were also elucidated. Pretreatment of mung bean explants with N, N’-dimethylthiourea (DMTU), a scavenger for H₂O₂, resulted in a significant reduction of SA-induced ARF. Diphenyleneiodonium (DPI), a specific inhibitor of membrane-linked NADPH oxidase, also inhibited the effect of adventitious rooting triggered by SA treatment. The determination of the endogenous H₂O₂ level indicated that the seedlings treated with SA could induce H₂O₂ accumulation compared with the control treatment. Our results revealed a distinctive role of SA in the promotion of adventitious rooting via the process of H₂O₂ accumulation. This conclusion was further supported by antioxidant enzyme activity assays. Based on these results, we conclude that the accumulation of free H₂O₂ might be a downstream event in response to SA-triggered adventitious root formation in mung bean seedlings.     

Ethylene-Induced Activation of Xylanase in Adventitious Roots of Maize as a Response to the Stress Effect of Root Submersion

Submersion of roots of ten-day-old maize (Zea maysL.) seedlings was accompanied by a decrease in pO₂and an increase in pCO₂of the medium adjacent to the roots. These changes stimulated ethylene evolution in intact plants. Enhanced biosynthesis of ethylene was accompanied by xylanase activation in adventitious roots. As a result, an enhanced formation of aerenchyma was observed in the cortex of adventitious roots. Therefore, these processes resulted in the development of a ventilation system by which O₂can reach the root system exposed to hypoxia. The volume of aerenchyma was assessed by the volume of gas cavities (porosity). In contrast to the main root, the growth of adventitious roots was not inhibited under these conditions. Enlargement of the stem base and increase in the number of aerenchymatous adventitious roots facilitated the oxygen supply to the submerged organs of the plants.     

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.