Nitrate efflux is an essential component of the cryptogein signaling pathway leading to defense responses and hypersensitive cell death in tobacco

There is much interest in the transduction pathways by which avirulent pathogens or derived elicitors activate plant defense responses. However, little is known about anion channel functions in this process. The aim of this study was to reveal the contribution of anion channels in the defense response triggered in tobacco by the elicitor cryptogein. Cryptogein induced a fast nitrate (NO(3)(-)) efflux that was sensitive to anion channel blockers and regulated by phosphorylation events and Ca(2+) influx. Using a pharmacological approach, we provide evidence that NO(3)(-) efflux acts upstream of the cryptogein-induced oxidative burst and a 40-kD protein kinase whose activation seems to be controlled by the duration and intensity of anion efflux. Moreover, NO(3)(-) efflux inhibitors reduced and delayed the hypersensitive cell death triggered by cryptogein in tobacco plants. This was accompanied by a delay or a complete suppression of the induction of several defense-related genes, including hsr203J, a gene whose expression is correlated strongly with programmed cell death in plants. Our results indicate that anion channels are involved intimately in mediating defense responses and hypersensitive cell death.     

Involvement of Free Calcium in Action of Cryptogein,a Proteinaceous Elicitor of Hypersensitive Reaction in Tobacco Cells

Treatment of suspension-cultured tobacco (Nicotiana tabacum var Xanthi) cells with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea, induced a great stimulation of Ca2+ influx within the first minutes. Ca2+ influx is essential for the initiation of cryptogein-induced responses, since ethyleneglycol-bis([beta]-amino-ethyl ether)-N,N[prime]-tetraacetic acid or La3+, which block Ca2+ entrance, suppress cryptogein-induced responses such as extracellular alkalinization, active oxygen species, and phytoalexin production. Moreover, once initiated, these responses require sustained Ca2+ influx within the 1st h. A Ca2+ ionophore (A23187) was able to trigger an extracellular alkalinization but not the formation of active oxygen species and phytoalexins, even in the presence of cryptogein. Staurosporine, a protein kinase inhibitor that was recently reported to suppress cryptogein-induced responses (M.-P. Viard, F. Martin, A. Pugin, P. Ricci, J.-P. Blein [1994] Plant Physiol 104: 1245-1249), inhibited Ca2+ influx induced by cryptogein in a dose-dependent manner. These results suggest that protein phosphorylation followed by Ca2+ influx might be involved in the initial steps of cryptogein signal transduction.     

Integrated signaling network involving calcium, nitric oxide, and active oxygen species but not mitogen-activated protein kinases in BcPG1-elicited grapevine defenses

We have already reported the identification of the endopolygalacturonase 1 (BcPG1) from Botrytis cinerea as a potent elicitor of defense responses in grapevine, independently of its enzymatic activity. The aim of the present study is the analysis of the signaling pathways triggered by BcPG1 in grapevine cells. Our data indicate that BcPG1 induces a Ca2+ entry from the apoplasm, which triggers a phosphorylation-dependent nitric oxide (NO) production via an enzyme probably related to a NO synthase. Then NO is involved in (i) cytosolic calcium homeostasis, by activating Ca2+ release from internal stores and regulating Ca2+ fluxes across the plasma membrane, (ii) plasma membrane potential variation, (iii) the activation of active oxygen species (AOS) production, and (iv) defense gene expression, including phenylalanine ammonia lyase and stilbene synthase, which encode enzymes responsible for phytoalexin biosynthesis. Interestingly enough, mitogen-activated protein kinase (MAPK) activation is independent of this regulation pathway that closely connects Ca2+, NO, and AOS.     

Identification of putative voltage-dependent Ca2+-permeable channels involved in cryptogein-induced Ca2+ transients and defense responses in tobacco BY-2 cells

Ca(2+) is the pivotal second messenger for induction of defense responses induced by treatment of pathogen-derived elicitor or microbial infection in plants. However, molecular bases for elicitor-induced generation of Ca(2+) signals (Ca(2+) transients) are largely unknown. We here identified cDNAs for putative voltage-dependent Ca(2+)-permeable channels, NtTPC1A and NtTPC1B, that are homologous to TPC1 (two pore channel) from suspension-cultured tobacco BY-2 cells. NtTPC1s complemented the growth of a Saccharomyces cerevisiae mutant defective in CCH1, a putative Ca(2+) channel, in a low Ca(2+) medium, suggesting that both products permeate Ca(2+) through the plasma membrane. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of rise in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in response to sucrose and a fungal elicitor cryptogein, while it did not affect hypoosmotic shock-induced [Ca(2+)](cyt) increase. Cosuppression of NtTPC1s also caused suppression of cryptogein-induced programmed cell death and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca(2+) mobilization induced by the cryptogein and sucrose, and have crucial roles in cryptogein-induced signal transduction pathway.     

机械刺激诱导的烟草悬浮细胞一氧化氮产生途径及其与C矿和钙调素的关系

通过提高摇床转速对烟草细胞施加机械刺激（Ms）可诱导其胞内一氧化氮（No）的快速产生和一氧化氮合酶（Nos）活性的提高,这种MS诱导的NO产生可被N0清除剂cPTIO和NOS抑制剂L-NMMA显著抑制。此外,Ca2＋螯合剂EGTA、质膜Ca＋通道阻断剂La3＋、胞内Ca2＋通道拮抗剂钌红,以及钙调素抑制剂CPZ和TFP预处理均不同程度地抑制了机械刺激诱导的烟草细胞NO的产生,而机械刺激过程中钙调素活性显著上升并与NOS活性和NO含量的变化相一致。这些结果暗示着（类）Nos酶催化的精氨酸依赖途径可能是机械刺激诱发烟草细胞NO产生的主要途径,Ca2＋／CAM可能通过调节（类）NOS活性来调控No的产生。     

An S-Type Anion Channel SLAC1 Is Involved in Cryptogein-Induced Ion Fluxes and Modulates Hypersensitive Responses in Tobacco BY-2 Cells

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl⁻ and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl⁻ efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl⁻ efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.     

Nitric oxide (NO) as an intermediate in the cryptogein-induced hypersensitive response--a critical re-evaluation

A hypersensitive response (HR) was induced in tobacco leaves and cell suspensions by the fungal elicitor cryptogein, and NO production was followed by chemiluminescence and occasionally by diaminofluorescein (DAF)-fluorescence. Results from both methods were at least partly consistent, but kinetics was different. NO emission was not induced by cryptogein in leaves, whereas in cell suspensions some weak NO emission was observed, which was nitrate reductase (NR)-dependent, but not required for cell death. Nitric oxide synthase (NOS) inhibitors did not prevent cell death, but PR-1 expression was weakened. In conclusion, neither NR nor NOS appear obligatory for the cryptogein-induced HR. However, a role for NO was still suggested by the fact that the NO scavenger cPTIO prevented the HR. Unexpectedly, cPTI, the reaction product of cPTIO and NO, also impaired the HR but without scavenging NO. Thus, prevention of the HR by cPTIO is not necessarily indicative for a role of NO. Further, even a 100-fold NO overproduction (over wild type) by a nitrite reductase-deficient mutant did not interfere with the cryptogein-induced HR. Accordingly, the role of NO in the HR should be reconsidered.     

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

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

Real-time electrochemical detection of extracellular nitric oxide in tobacco cells exposed to cryptogein, an elicitor of defence responses

It was previously reported that cryptogein, an elicitor of defence responses, induces an intracellular production of nitric oxide (NO) in tobacco. Here, the possibility was explored that cryptogein might also trigger an increase of NO extracellular content through two distinct approaches, an indirect method using the NO probe 4,5-diaminofluorescein (DAF-2) and an electrochemical method involving a chemically modified microelectrode probing free NO in biological media. While the chemical nature of DAF-2-reactive compound(s) is still uncertain, the electrochemical modified microelectrodes provide real-time evidence that cryptogein induces an increase of extracellular NO. Direct measurement of free extracellular NO might offer important new insights into its role in plants challenged by biotic stresses.     

Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway.

Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L. cv. Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores. We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea. Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors. Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions. However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC). The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC). On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein. A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein. These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DeltapH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC.     

Phosphoproteins involved in the signal transduction of cryptogein, an elicitor of defense reactions in tobacco

We previously reported that the signal transduction of cryptogein, an elicitor of defense reactions in Nicotiana tabacum cells, involves upstream protein phosphorylation. In the present study, induction of these early physiological events was further investigated with inhibitors of protein phosphatase (PP), okadaic acid, and calyculin A. Calyculin A mimicked the effects of cryptogein, inducing an influx of calcium, an extracellular alkalinization, and the production of active oxygen species (AOS), suggesting that during cryptogein signal transduction the balance between specific protein kinase (PK) and PP activities was modified. To identify the phosphorylated proteins that could be involved early in the elicitor signaling pathway, we analyzed by 2-D electrophoresis the in vivo phosphorylation status of proteins after cryptogein, staurosporine, and calyculin A treatments of tobacco cells (5 min). Of about 100 phospho-labeled polypeptides, 19 showed increased 32P incorporation after 5 min of cryptogein treatment. Phosphorylation of 12 of the 19 polypeptides depended upon calcium influx. Staurosporine inhibited the phosphorylations induced by cryptogein whereas calyculin A activated the phosphorylation of 18 of these polypeptides. This study highlighted the role of PKs and/or constitutive active PPs whose activation and inhibition, respectively, resulted in an increased phosphorylation of proteins that may be involved in cryptogein signal transduction. Identification of the phosphoproteins is in progress and will increase our knowledge of signal transduction pathways implicated in plant defense responses.     

植物一氧化氮生物学的研究进展

一氧化氮 (NO) 是植物中的一种关键的信号分子。在植物中, NO的潜在来源包括一氧化氮合成酶、硝酸还原酶、黄嘌呤氧化还原酶和非酶促途径。NO能促进植物生长, 延缓叶片、花和果实衰老, 促进休眠和需光种子的萌发, 能与植物激素相互作用调节气孔运动, 诱导程序性细胞死亡和防御相关基因的表达, 并在逆境中作为一种抗氧化剂起作用。 NO的细胞内信号反应包括环鸟苷酸、环腺苷二磷酸核糖的产生和细胞质Ca2+浓度的增加, 其信号转导途径及其生物化学和细胞学本质还不十分清楚。     

植物一氧化氮生物学的研究进展

一氧化氮(NO)是植物中的一种关键的信号分子.在植物中,NO的潜在来源包括一氧化氮合成酶、硝酸还原酶、黄嘌呤氧化还原酶和非酶促途径.NO能促进植物生长,延缓叶片、花和果实衰老,促进休眠和需光种子的萌发,能与植物激素相互作用调节气孔运动,诱导程序性细胞死亡和防御相关基因的表达,并在逆境中作为一种抗氧化剂起作用.NO的细胞内信号反应包括环鸟苷酸、环腺苷二磷酸核糖的产生和细胞质Ca2 浓度的增加,其信号转导途径及其生物化学和细胞学本质还不十分清楚.     

In vivo imaging of an elicitor-induced nitric oxide burst in tobacco

A growing body of evidence suggests that nitric oxide (NO), an important signalling and defence molecule in mammals, plays a key role in activating disease resistance in plants, acting as signalling molecule and possibly as direct anti-microbial agent. Recently, a novel fluorophore (diaminofluorescein diacetate, DAF-2 DA) has been developed which allows bio-imaging of NO in vivo. Here we use the cell-permeable DAF-2 DA, in conjunction with confocal laser scanning microscopy, for real-time imaging of NO in living plant cells. Epidermal tobacco cells treated with cryptogein, a fungal elicitor from Phytophthora cryptogea, respond to the elicitor with a strong increase of intracellular NO. NO-induced fluorescence was found in several cellular compartments, and could be inhibited by a NO scavenger and an inhibitor of nitric oxide synthase. The NO burst was triggered within minutes, reminiscent of the oxidative burst during hypersensitive response reactions. These results reveal additional similarities between plant and animal host responses to infection.     

Calcium signatures and signaling in cytosol and organelles of tobacco cells induced by plant defense elicitors

Calcium signatures induced by two elicitors of plant defense reactions, namely cryptogein and oligogalacturonides, were monitored at the subcellular level, using apoaequorin-transformed Nicotiana tabacum var Xanthi cells, in which the apoaequorin calcium sensor was targeted either to cytosol, mitochondria or chloroplasts. Our study showed that both elicitors induced specific Ca(2+) signatures in each compartment, with the most striking difference relying on duration. Common properties also emerged from the analysis of Ca(2+) signatures: both elicitors induced a biphasic cytosolic [Ca(2+)] elevation together with a single mitochondrial [Ca(2+)] elevation concomitant with the first cytosolic [Ca(2+)] peak. In addition, both elicitors induced a chloroplastic [Ca(2+)] elevation peaking later in comparison to cytosolic [Ca(2+)] elevation. In cryptogein-treated cells, pharmacological studies indicated that IP(3) should play an important role in Ca(2+) signaling contrarily to cADPR or nitric oxide, which have limited or no effect on [Ca(2+)] variations. Our data also showed that, depending on [Ca(2+)] fluxes at the plasma membrane, cryptogein triggered a mitochondrial respiration increase and affected excess energy dissipation mechanisms in chloroplasts. Altogether the results indicate that cryptogein profoundly impacted cell functions at many levels, including organelles.     

Mathematical modeling of the nitric oxide/cGMP pathway in the vascular smooth muscle cell

The nitric oxide (NO)/cGMP pathway in the vascular smooth muscle cell (VSMC) is an important cellular signaling system for the regulation of VSMC relaxation. We present a mathematical model to investigate the underlying mechanisms of this pathway. The model describes the flow of NO-driven signal transduction: NO activation of soluble guanylate cyclase (sGC), sGC- and phosphodiesterase-catalyzed cGMP production and degradation, cGMP-mediated regulation of protein targets including the Ca2+-activated K+ (KCa) channel, and the myosin contractile system. Model simulations reproduce major NO/cGMP-induced VSMC relaxation effects, including intracellular Ca2+ concentration reduction and Ca2+ desensitization of myosin phosphorylation and force generation. Using the model, we examine several testable principles. 1) Rapid sGC desensitization is caused by end-product cGMP feedback inhibition; a large fraction of the steady-state sGC population is in an inactivated intermediate state, and cGMP production is limited well below maximum. 2) NO activates the K(Ca) channel with both cGMP-dependent and -independent mechanisms; moderate NO concentration affects the K(Ca) via the cGMP-dependent pathway, whereas higher NO concentration is accommodated by a cGMP-independent mechanism. 3) Chronic NO synthase inhibition may cause underexpressions of K+ channels including inward rectifier and K(Ca) channels. 4) Ca2+ desensitization of the contractile system is distinguished from Ca2+ sensitivity of myosin phosphorylation. The model integrates these interactions among the heterogeneous components of the NO signaling system and can serve as a general modeling framework for studying NO-mediated VSMC relaxation under various physiological and pathological conditions. New data can be readily incorporated into this framework for interpretation and possible modification and improvement of the model.     

Disruption of microtubular cytoskeleton induced by cryptogein, an elicitor of hypersensitive response in tobacco cells

The dynamics of microtubular cytoskeleton were studied in tobacco (Nicotiana tabacum cv Xanthi) cells in response to two different plant defense elicitors: cryptogein, a protein secreted by Phytophthora cryptogea and oligogalacturonides (OGs), derived from the plant cell wall. In tobacco plants cryptogein triggers a hypersensitive-like response and induces systemic resistance against a broad spectrum of pathogens, whereas OGs induce defense responses, but fail to trigger cell death. The comparison of the microtubule (MT) dynamics in response to cryptogein and OGs in tobacco cells indicates that MTs appear unaffected in OG-treated cells, whereas cryptogein treatment caused a rapid and severe disruption of microtubular network. When hyperstabilized by the MT depolymerization inhibitor, taxol, the MT network was still disrupted by cryptogein treatment. On the other hand, the MT-depolymerizing agent oryzalin and cryptogein had different and complementary effects. In addition to MT destabilization, cryptogein induced the death of tobacco cells, whereas OG-treated cells did not die. We demonstrated that MT destabilization and cell death induced by cryptogein depend on calcium influx and that MT destabilization occurs independently of active oxygen species production. The molecular basis of cryptogein-induced MT disruption and its potential significance with respect to cell death are discussed.     

L-arginine stimulates an endogenous ADP-ribosyltransferase

An ubiquitous biochemical pathway known to synthesize nitric oxide (NO) from L-arginine has been identified in many cell types. Recent studies indicate that besides activating soluble guanylate cyclase NO is likely to have effects unrelated to the known signal transduction pathway. Activation of the soluble NO synthase stimulates an endogenous ADP-ribosylation of a predominant 39 kDa protein, known to be activated by NO releasing agents. This is demonstrated using the cytosolic fraction of rat cerebellum and HL-60 cells. The ADP-ribosylation is suppressed by the known NO synthase inhibitors N-nitro-L-arginine and N-methyl-L-arginine. These observations indicate that NO derived from its physiological precursor L-arginine activates an endogenous ADP-ribosyltransferase.