The AtrbohD-mediated oxidative burst elicited by oligogalacturonides in Arabidopsis is dispensable for the activation of defense responses effective against Botrytis cinerea

Oligogalacturonides (OGs) are endogenous elicitors of defense responses released after partial degradation of pectin in the plant cell wall. We have previously shown that, in Arabidopsis (Arabidopsis thaliana), OGs induce the expression of PHYTOALEXIN DEFICIENT3 (PAD3) and increase resistance to the necrotrophic fungal pathogen Botrytis cinerea independently of signaling pathways mediated by jasmonate, salicylic acid, and ethylene. Here, we illustrate that the rapid induction of the expression of a variety of genes by OGs is also independent of salicylic acid, ethylene, and jasmonate. OGs elicit a robust extracellular oxidative burst that is generated by the NADPH oxidase AtrbohD. This burst is not required for the expression of OG-responsive genes or for OG-induced resistance to B. cinerea, whereas callose accumulation requires a functional AtrbohD. OG-induced resistance to B. cinerea is also unaffected in powdery mildew resistant4, despite the fact that callose accumulation was almost abolished in this mutant. These results indicate that the OG-induced oxidative burst is not required for the activation of defense responses effective against B. cinerea, leaving open the question of the role of reactive oxygen species in elicitor-mediated defense.     

Involvement of nitrate reductase in auxin-induced NO synthesis

It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.¹ Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (N^G-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.^2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.^4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation.Key words: atnoa1, indole-3-butyric acid, nia1, nia2 double mutant, nitric oxideLateral roots are formed from root pericycle cells postembryonically which process is promoted by indole-acetic acid (IAA). It was recognized that IAA share common steps with NO in the signal transduction cascade towards the auxin induced adventitious and lateral root formation.^6–8 Previously it was suggested that besides IAA, indol-3-butyric (IBA) is a true endogenous auxin in Arabidopsis, which acts in adventious and lateral root development.^9,10 Our results showed that IBA induced LR initials emitted intensive NO fluorescence in Arabidopsis. This increased level of NO was present only in the LR initials in contrast to primary root (PR) sections where it remained at the control level.In plants NO can be produced by a number of enzyme systems and non-enzymatic ways. In roots, the most likely candidates of NO synthesis are NR enzymes (cytoplasmic and plasma membrane-bounded isoenzymes, cNR and PM-NR). Recently a new type of enzyme, the PM-bounded nitrite:NO reductase (Ni:NOR) was identified as a possible source of NO in roots.¹¹ Because of the several formation potentials of NO, the identification of its source in plant tissues under different conditions is complicated. Using diverse mutants proved to be a good opportunity to investigate the possible sources of NO. In our experiments wild-type (Col-1), Atnoa1 (nitric oxide synthase associated 1 deficient) and nia1, nia2 (NR deficient) seedlings were applied in order to determine the enzymatic source of NO induced by auxin. In roots of these plants, different NO levels were measured in their control state (i.e., without IBA treatment). The NO content in Atnoa1 roots was similar to that of wild-type, while nia1, nia2 showed lower NO fluorescence than the other groups of plants. This result suggests that NR activity is needed to NO synthesis in roots. Further on, it was demonstrated that IBA induced NO generation in both the wild type and Atnoa1 root primordia, but this induction failed in the NR-deficient mutant. This reveals that the NO accumulation in root primordia induced by auxin requires NR activity. These observations were evidenced also by biochemical manner. On the one part, we applied L-NMMA, which is a specific inhibitor of mammalian NOS, on the other part, the inhibitor of NR enzyme tungstate was used and we monitored NO fluorescence in wild-type roots. The NOS inhibitor displayed no effect on NO levels neither at control state nor during auxin treatment, while tungstate inhibited NO synthesis in lateral roots and primary roots of control plants. The effect of tungstate was similar in auxin-treated roots, since application of this NR enzyme inhibitor decreased NO levels in PRs and LRs (Fig. 1).Open in a separate windowFigure 1NO fluorescence in lateral roots (white columns) and primary roots (grey columns) of control, control + 1 mM tungstate, IBA and IBA + 1 mM tungstate-treated wild-type Arabidopsis thaliana. Vertical bars are standard errors.Some speculations can be made on these results. Although more efforts are needed to make the scene clear, now we can predict that auxin somehow may induce NR isoenzymes, which produce nitrite in root cells. From this point, two further scenarios are possible: as the result of accumulated nitrite, either the NO-producing activity of NR or Ni:NOR activity are promoted, hereby NO is generated from nitrite reduction. NO formed in these two possible ways modulates the expression of certain cell cycle regulatory genes contributing to division of pericycle cells in LR primordia, as was published in tomato.¹²Nowadays research in the “NO-world” of plants is running very actively. Nevertheless, lot of more work is needed to reveal all the unknown faces of this novel multipurpose signaling molecule.     

Verticillium dahliae toxins-induced nitric oxide production in Arabidopsis is major dependent on nitrate reductase

The source of nitric oxide (NO) in plants is unclear and it has been reported NO can be produced by nitric oxide synthase (NOS) like enzymes and by nitrate reductase (NR). Here we used wild-type, Atnos1 mutant and nia1, nia2 NR-deficient mutant plants of Arabidopsis thaliana to investigate the potential source of NO production in response to Verticillium dahliae toxins (VD-toxins). The results revealed that NO production is much higher in wild-type and Atnos1 mutant than in nia1, nia2 NR-deficient mutants. The NR inhibitor had a significant effect on VD-toxins-induced NO production; whereas NOS inhibitor had a slight effect. NR activity was significantly implicated in NO production. The results indicated that as NO was induced in response to VD-toxins in Arabidopsis, the major source was the NR pathway. The production of NOS-system appeared to be secondary.     

Exogenous auxin-induced NO synthesis is nitrate reductase-associated in Arabidopsis thaliana root primordia

Nitric oxide (NO) functions in various physiological and developmental processes in plants. However, the source of this signaling molecule in the diversity of plant responses is not well understood. It is known that NO mediates auxin-induced adventitious and lateral root (LR) formation. In this paper, we provide genetic and pharmacological evidence that the production of NO is associated with the nitrate reductase (NR) enzyme during indole-3-butyric acid (IBA)-induced lateral root development in Arabidopsis thaliana L. NO production was detected using 4,5-diaminofluorescein diacetate (DAF-2DA) in the NR-deficient nia1, nia2 and Atnoa1 (former Atnos1) mutants of A. thaliana. An inhibitor for nitric oxide synthase (NOS) N(G)-monomethyl-l-arginine (l-NMMA) was applied. Our data clearly show that IBA increased LR frequency in the wild-type plant and the LR initials emitted intensive NO-dependent fluorescence of the triazol product of NO and DAF-2DA. Increased levels of NO were restricted only to the LR initials in contrast to primary root (PR) sections, where NO remained at the control level. The mutants had different NO levels in their control state (i.e. without IBA treatment): nia1, nia2 showed lower NO fluorescence than Atnoa1 or the wild-type plant. The role of NR in IBA-induced NO formation in the wild type was shown by the zero effects of the NOS inhibitors l-NMMA. Finally, it was clearly demonstrated that IBA was able to induce NO generation in both the wild-type and Atnoa1 plants, but failed to induce NO in the NR-deficient mutant. It is concluded that the IBA-induced NO production is nitrate reductase-associated during lateral root development in A. thaliana.     

Dependence of UV-B-induced camptothecin production on nitrate reductase-mediated nitric oxide signaling in Camptotheca acuminata suspension cell cultures

UV-B irradiation induced production of secondary metabolites in plant cells. However, the mechanisms of UV-B-induced secondary metabolite production remained largely unknown. Here we report that UV-B treatment stimulated nitric oxide (NO) generation and camptothecin (CPT) production in Camptotheca acuminata cells. To investigate the origin of the UV-B-triggered NO and the role of NO in UV-B-induced CPT production, we assayed the responses of nitrate reductase (NR) and NO synthase (NOS) activities of the cells to UV-B exposure and examined the effects of NR and NOS inhibitors on CPT production in UV-B-treated cells. The data showed that UV-B irradiation enhanced NR activities in the cells. Pretreatment with NR inhibitors tungstate and okadaic acid not only suppressed the UV-B-triggered NR activities but also inhibited the UV-B-induced NO generation and CPT production in the cells. In contrast, UV-B irradiation had no effects on NOS activity of the cells and treatment of NOS inhibitor did not suppress UV-B-induced CAT production. Together, the results demonstrated that NR activity was essential for UV-B-triggered NO generation and that NR-mediated NO signaling was involved in UV-B-induced CPT production in C. acuminata cells.     

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     

Nia1 and Nia2 are Involved in Exogenous Salicylic Acid-induced Nitric Oxide Generation and Stomatal Closure in Arabidopsis

Phytohormone salicylic acid (SA) plays important roles in plant responses to environmental stress. However, knowledge about the molecular mechanisms for SA affecting the stomatal movements is limited. In this paper, we demonstrated that exogenous SA significantly induced stomatal closure and nitric oxide (NO) generation in Arabidopsis guard cells based on genetic and physiological data. These effects were significantly inhibited by the NO scavenger c-PTIO, NO synthase (NOS) inhibitor L-NAME or nitrate reductase suppressor tungstate respectively, implying that NOS and nitrate reductase (NR) participate in SA-evoked stomatal closing. Furthermore, the effects of SA promotion of stomatal closure and NO synthesis are significantly suppressed in NR single mutants of nia1, nia2 or double mutant nia1/nia2, compared with the wild type plants. This suggests that both Nia1 and Nia2 are involved in SA-stimulated stomatal closure. In addition, pharmacological experiments showed that protein kinases, cGMP and cADPR are involved in SA-mediated NO accumulation and stomatal closure induced by SA in Arabidopsis.     

ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H(2)O(2) have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H(2)O(2) in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H(2)O(2) production. Stomatal closure induced by H(2)O(2) is inhibited by the removal of NO with NO scavenger, and both ABA and H(2)O(2) stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H(2)O(2) synthesis nor does NO treatment induce H(2)O(2) production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H(2)O(2) and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H(2)O(2) synthesis. In the NR double mutant nia1, nia2 both ABA and H(2)O(2) fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H(2)O(2) are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H(2)O(2) production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H(2)O(2) and NO-induced stomatal closure.     

Mechanical Stress Elicits Nitric Oxide Formation and DNA Fragmentation in Arabidopsis thaliana

The effect of mechanical stress (centrifugation) on the inductionof nitric oxide (NO) formation and DNA fragmentation was investigatedin leaf cells of Arabidopsis thaliana. Centrifuged and non-centrifugedleaves from wild-type and nitrate reductase (NR)nia1, nia2 doublemutant, defective in the assimilation of nitrate, were labelledwith 4,5-diaminofluorescein diacetate (DAF-2 DA) to visualizein vivo NO production. After these treatments, DNA fragmentationwas detected by the terminal deoxynucleotidyl transferase-mediateddUTP nick end in situ labelling (TUNEL) method. Exposure toan NO-releasing compound, sodium nitroprusside (SNP) mimickedthe cell response to centrifugation (20 g). The involvementof endogenous NO as a signal in mechanical stress and in DNAfragmentation was confirmed by inhibition of NO production usinga nitric oxide synthase (NOS) inhibitor viz. N^G-monomethyl-L -arginine (L -NMMA). These results indicate that NOS-likeactivity was present in A. thaliana leaves and was increasedby mechanical stress. The effect of leaf-wounding on nitricoxide production was identical to that of centrifugation. Experimentswith A. thaliana NR mutant also showed that NO bursts were inducedby mechanical and wounding stresses and that NO was not a by-productof NR activity. A positive and significant correlation betweenNO production and DNA fragmentation was recorded for both centrifugedand non-centrifuged cells. Our results suggest that factorsother than NO contribute to DNA damage and cell death, and furthermore,that an inducible form of NOS is present in A. thaliana. Copyright2001 Annals of Botany Company Arabidopsis thaliana, cell death, DNA fragmentation, NO, plant stress, wounding     

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。     

Zeatin-induced nitric oxide (NO) biosynthesis in Arabidopsis thaliana mutants of NO biosynthesis and of two-component signaling genes

* Here, cytokinin-induced nitric oxide (NO) biosynthesis and cytokinin responses were investigated in Arabidopsis thaliana wild type and mutants defective in NO biosynthesis or cytokinin signaling components. * NO release from seedlings was quantified by a fluorometric method and, by microscopy, observed NO biosynthesis as fluorescence increase of DAR-4M AM (diaminorhodamine 4M acetoxymethyl ester) in different tissues. * Atnoa1 seedlings were indistinguishable in NO tissue distribution pattern and morphological responses, induced by zeatin, from wild-type seedlings. Wild-type and nia1,2 seedlings, lacking nitrate reductase (NR), responded to zeatin with an increase within 3 min in NO biosynthesis so that NR does not seem relevant for rapid NO induction, which was mediated by an unknown 2-(2-aminoethyl)2-thiopseudourea (AET)-sensitive enzyme and was quenched by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide (PTIO). Long-term morphological responses to zeatin were severely altered and NO biosynthesis was increased in nia1,2 seedlings. As cytokinin signaling mutants we used the single-receptor knockout cre1/ahk4, three double-receptor knockouts (ahk2,3, ahk2,4, ahk3,4) and triple-knockout ahp1,2,3 plants. All cytokinin-signaling mutants showed aberrant tissue patterns of NO accumulation in response to zeatin and altered morphological responses to zeatin. * Because aberrant NO biosynthesis correlated with aberrant morphological responses to zeatin the hypothesis was put forward that NO is an intermediate in cytokinin signaling.     

Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.     

Modulation of nitrate reductase activity by photosynthetic electron transport chain and nitric oxide balance in the red macroalga Gracilaria chilensis (Gracilariales, Rhodophyta)

Nitrate reductase (NR), a key enzyme in nitrogen metabolism, has been implicated in the production of nitric oxide (NO) in plants. The effect of photosynthetic electron transport chain inhibitors and NO scavengers or donors on NR activity of Gracilaria chilensis was studied under experimental laboratory conditions. Effective quantum yield (Φ _PSII) and NR activity were significantly diminished by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, two photosynthetic electron flux inhibitors of photosystem (PS) II and PSI, respectively, but not by diphenyleneiodonium, a NADPH oxidase inhibitor, indicating a direct dependence of NR activity on the PSII and PSI electron flux. Nitrate reductase activity was sensitive to a decrease or increase of NO levels when NO scavenger (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and NO donor (sodium nitroprusside) were added. Moreover, the addition of 8Br-cGMP, a secondary signal molecule, stimulated NR activity. These results evidence a modulation of the photosynthetic electron transport chain and NO balance on G. chilensis NR activity. This association could be linked to the crucial tight modulation of nitrogen assimilation and carbon metabolism to guarantee nitrite incorporation into organic compounds and to avoid toxicity by nitrite, reactive oxygen species, or nitric oxide in the cells. Nitric oxide showed to be an important signaling molecule regulating NR activity and cGMP could participate as secondary messenger on this regulation by phosphorylation and desphosphorylation processes.     

Nitrate reductase-mediated nitric oxide generation is essential for fungal elicitor-induced camptothecin accumulation of Camptotheca acuminata suspension cell cultures

Secondary metabolite accumulation and nitric oxide (NO) generation are two common responses of plant cells to fungal elicitors, and NO has been reported to play important roles in elicitor-induced secondary metabolite production. However, the source of elicitor-triggered NO generation in plant cells remains largely unknown. To investigate the origin of elicitor-triggered NO, we examined nitrate reductase (NR) activities and the expression levels of NIA1 and NIA2 genes of Camptotheca acuminata cells treated with PB90, a protein elicitor from Phytophthora boehmeriae. The data show that PB90 treatment stimulates NR activity and induces upregulation of NIA1 but does not affect NIA2 expression in the cells. Pretreatment of the cells with NR inhibitors tungstate and Gln abolishes not only the fungal elicitor-triggered NR activities but also the PB90-induced NO generation. Treatment of PB90 enhances camptothecin contents of the cells, suggesting that the fungal elicitor might stimulate camptothecin biosynthesis. Furthermore, application of tungstate and Gln suppresses the fungal elicitor-induced camptothecin accumulation of the cells and the suppression of NR inhibitors on PB90-induced camptothecin production can be reversed by NO via its donor sodium nitroprusside. Together, the results suggest that NIA1 is sensitive to PB90 and the fungal elicitor-induced upregulation of NIA1 may lead to higher NR activity. Furthermore, our data demonstrate that NR is involved in the fungal elicitor-triggered NO generation and the fungal elicitor induces camptothecin production of C. acuminata cells dependently on NR-mediated NO generation.     

Exogenous nitric oxide improves antioxidative capacity and reduces auxin degradation in roots of Medicago truncatula seedlings under cadmium stress

The effects of nitric oxide (NO) on cadmium toxicity in Medicago truncatula seedlings were studied by investigating root growth and uptake of antioxidants, IAA and ions. Exposure to cadmium reduced root growth and NO accumulation, and increased the production of reactive oxygen species (ROS) in roots. Supplementation with NO improved root growth and reduced ROS accumulation in roots. The NO-scavenger cPTIO, the nitrate reductase (NR) inhibitor tungstate, and the NO synthase (NOS) inhibitor L-NAME all inhibited the accumulation of NO in roots and reversed the effects of NO in promoting the root growth and accumulation of proline and glutathione. Application of NO reduced auxin degradation by inhibiting the activity of IAA oxidase. Exogenous NO also enhanced the uptake of K⁺ and Ca²⁺. These results suggest that NO improves cadmium tolerance in plants by reducing oxidative damage, maintaining the auxin equilibrium and enhancing ion absorption.