Jasmonate mediates salt induced nicotine biosynthesis in tobacco (Nicotiana tabacum L.)

Jasmonate (JA), as an important signal, plays a key role in multiple processes of plant growth, deve lopment and stress response. Nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L.) are essential secondary metabolites. Whether environmental factors control nicotine biosynthesis and the underlying mechanism remains previously unreported. Here, we applied physiological and biochemical approaches to investigate how salt stress affects nicotine biosynthesis in tobacco. We found that salt stress induced the biosynthesis of JA, which subsequently triggered the activation of JA responsive gene expression and, ultimately, nicotine synthesis. Bioinformatics analysis revealed the existence of many NtMYC2a recognized G box motifs in the promoter regions of NtLOX, NtAOS, NtAOC and NtOPR genes. Applying exogenous JA increased nicotine content, while suppressing JA biosynthesis reduced nicotine biosynthesis. Salt treatment could not efficiently induce nicotine biosynthesis in transgenic anti COI1 tobacco plants. These results demonstrate that JA acts as the essential signal which triggers nicotine biosynthesis in tobacco after salt stress.     

Jasmonate mediates salt-induced nicotine biosynthesis in tobacco(Nicotiana tabacum L.)

Jasmonate(JA),as an important signal,plays a key role in multiple processes of plant growth,development and stress response.Nicotine and related pyridine alkaloids in tobacco(Nicotiana tabacum L.) are essential secondary metabolites.Whether environmental factors control nicotine biosynthesis and the underlying mechanism remains previously unreported.Here,we applied physiological and biochemical approaches to investigate how salt stress affects nicotine biosynthesis in tobacco.We found that salt stress induced the biosynthesis of JA,which subsequently triggered the activation of JA-responsive gene expression and,ultimately,nicotine synthesis.Bioinformatics analysis revealed the existence of many Nt MYC2a-recognized G-box motifs in the promoter regions of Nt LOX,Nt AOS,Nt AOC and Nt OPR genes.Applying exogenous JA increased nicotine content,while suppressing JA biosynthesis reduced nicotine biosynthesis.Salt treatment could not efficiently induce nicotine biosynthesis in transgenic anti-COI1 tobacco plants.These results demonstrate that JA acts as the essential signal which triggers nicotine biosynthesis in tobacco after salt stress.     

Immunomodulation of jasmonate to manipulate the wound response

Jasmonates are signals in plant stress responses and development. The exact mode of their action is still controversial. To modulate jasmonate levels intracellularly as well as compartment-specifically, transgenic Nicotiana tabacum plants expressing single-chain antibodies selected against the naturally occurring (3R,7R)-enantiomer of jasmonic acid (JA) were created in the cytosol and the endoplasmic reticulum. Consequently, the expression of anti-JA antibodies in planta caused JA-deficient phenotypes such as insensitivity of germinating transgenic seedlings towards methyl jasmonate and the loss of wound-induced gene expression. Results presented here suggest an essential role for cytosolic JA in the wound response of tobacco plants. The findings support the view that substrate availability takes part in regulating JA biosynthesis upon wounding. Moreover, high JA levels observed in immunomodulated plants in response to wounding suggest that tobacco plants are able to perceive a reduced level of physiologically active JA and attempt to compensate for this by increased JA accumulation.     

A requirement for calcium and protein phosphatase in the jasmonate-induced increase in tobacco leaf acid phosphatase specific activity

Jasmonic acid (JA) is an established wound signal and also plays a role in plant-pathogen interactions. Application of JA to tobacco leaf explants, tobacco seedlings or to intact leaves via the petiole resulted in an increase in the specific activity of acid phosphatase (AP) and a reduction in overall protein content. Similar changes in AP activity were observed in wounded tissue and in tissue undergoing a hypersensitive response (HR) following infiltration with a virulent bacteria. The AP activity increase was restricted to wounded tissue and HR lesions and was absent from unwounded or uninfiltrated tissue on the same leaf. The JA response (AP increase and protein loss combined) was investigated pharmacologically. Co-incubation with EGTA, ruthenium red, LaCl3 and ()-verapamil blocked the JA response suggesting a requirement for Ca²⁺ mobilization. Similarly, okadaic acid, cantharidin and microcystin LR abrogated the response to JA implicating a protein phosphatase in the JA signal transduction mechanism(s). No evidence was found for kinase involvement as a mediator of JA signalling in this system.     

The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death

Salicylic acid (SA) has been proposed to antagonize jasmonic acid (JA) biosynthesis and signaling. We report, however, that in salicylate hydroxylase-expressing tobacco (Nicotiana tabacum) plants, where SA levels were reduced, JA levels were not elevated during a hypersensitive response elicited by Pseudomonas syringae pv phaseolicola. The effects of cotreatment with various concentrations of SA and JA were assessed in tobacco and Arabidopsis (Arabidopsis thaliana). These suggested that there was a transient synergistic enhancement in the expression of genes associated with either JA (PDF1.2 [defensin] and Thi1.2 [thionin]) or SA (PR1 [PR1a-beta-glucuronidase in tobacco]) signaling when both signals were applied at low (typically 10-100 microm) concentrations. Antagonism was observed at more prolonged treatment times or at higher concentrations. Similar results were also observed when adding the JA precursor, alpha-linolenic acid with SA. Synergic effects on gene expression and plant stress were NPR1- and COI1-dependent, SA- and JA-signaling components, respectively. Electrolyte leakage and Evans blue staining indicated that application of higher concentrations of SA + JA induced plant stress or death and elicited the generation of apoplastic reactive oxygen species. This was indicated by enhancement of hydrogen peroxide-responsive AoPR10-beta-glucuronidase expression, suppression of plant stress/death using catalase, and direct hydrogen peroxide measurements. Our data suggests that the outcomes of JA-SA interactions could be tailored to pathogen/pest attack by the relative concentration of each hormone.     

Kinetics of the Accumulation of Jasmonic Acid and Its Derivatives in Systemic Leaves of Tobacco (Nicotiana tabacum cv. Xanthi nc) and Translocation of Deuterium-Labeled Jasmonic Acid from the Wounding Site to the Systemic Site

In plants, the mobile signal needed for wound-induced systemic acquired resistance (WSR) has been elusive. The signal compound involved in WSR is supposed to be JA or its derivatives. On the basis of kinetic study of the accumulation of JA or its derivatives, it was discovered that JA, JA-Ile, tuberonic acid (TA, 12-OH epi-JA), and tuberonic acid glucoside (TAG) accumulated in systemic tissues in response to mechanical wounding stress in the tobacco plant (Nicotiana tabacum). Attempts to recover deuterium-labeled JA in systemic leaves after feeding the wounded leaves with deuterium-labeled JA were successfully done. It was also found that the translocated deuterium-labeled JA was metabolized to TA in systemic leaves under feeding of deuterium-labeled JA to the wounding leaves.     

Elevated CO2 differentially affects tobacco and rice defense against lepidopteran larvae via the jasmonic acid signaling pathway

Atmospheric CO_2 levels are rapidly increasing due to human activities. However, the effects of elevated CO_2(ECO_2) on plant defense against insects and the underlying mechanisms remain poorly understood. Here we show that ECO_2 increased the photosynthetic rates and the biomass of tobacco and rice plants, and the chewing lepidopteran insects Spodoptera litura and Mythimna separata gained less and more mass on tobacco and rice plants, respectively. Consistently, under ECO_2, the levels of jasmonic acid(JA), the main phytohormone controlling plant defense against these lepidopteran insects, as well as the main defense-related metabolites, were increased and decreased in insectdamaged tobacco and rice plants. Importantly, bioassaysand quantification of defense-related metabolites in tobacco and rice silenced in JA biosynthesis and perception indicate that ECO_2 changes plant resistance mainly by affecting the JA pathway. We further demonstrate that the defensive metabolites, but not total N or protein, are the main factors contributing to the altered defense levels under ECO_2. This study illustrates that ECO_2 changes the interplay between plants and insects, and we propose that crops should be studied for their resistance to the major pests under ECO_2 to predict the impact of ECO_2 on future agroecosystems.     

The Essential Role of Jasmonic Acid in Plant-Herbivore Interactions - Using the Wild Tobacco Nicotiana attenuata as a Model

The plant hormone jasmonic acid （JA） plays a central role in plant defense against herbivores. Herbivore damage elicits a rapid and transient JA burst in the wounded leaves and JA functions as a signal to mediate the accumulation of various secondary metabolites that confer resistance to herbivores. Nicotiana attenuata is a wild tobacco species that inhabits western North America. More than fifteen years of study and its unique interaction with the specialist herbivore insect Manduca sexta have made this plant one of the best models for studying plant-herbivore interactions. Here we review the recent progress in understanding the elicitation of JA accumulation by herbivore-specific elicitors, the regulation of JA biosynthesis, JA signaling, and the herbivore-defense traits in N. attenuata.     

A mitogen-activated protein kinase NtMPK4 activated by SIPKK is required for jasmonic acid signaling and involved in ozone tolerance via stomatal movement in tobacco

The mitogen-activated protein kinase (MAPK) cascade is involved in responses to biotic and abiotic stress in plants. In this study, we isolated a new MAPK, NtMPK4, which is a tobacco homolog of Arabidopsis MPK4 (AtMPK4). NtMPK4 was activated by wounding along with two other wound-responsive tobacco MAPKs, WIPK and SIPK. We found that NtMPK4 was activated by salicylic acid-induced protein kinase kinase (SIPKK), which has been isolated as an SIPK-interacting MAPK kinase. In NtMPK4 activity-suppressed tobacco, wound-induced expression of jasmonic acid (JA)-responsive genes was inhibited. NtMPK4-silenced plants showed enhanced sensitivity to ozone. Inversely, transgenic tobacco plants, in which SIPKK or the constitutively active type SIPKK(EE) was overexpressed, exhibited greater responsiveness to wounding with enhanced resistance to ozone. We further found that NtMPK4 was expressed preferentially in epidermis, and the enhanced sensitivity to ozone in NtMPK4-silenced plants was caused by an abnormal regulation of stomatal closure in an ABA-independent manner. These results suggest that NtMPK4 is involved in JA signaling and in stomatal movement.     

Tobacco mosaic virus inoculation inhibits wound-induced jasmonic acid-mediated responses within but not between plants

Jasmonic acid (JA) and salicylic acid (SA) have both been implicated as important signal molecules mediating induced defenses of Nicotiana tabacum L. against herbivores and pathogens. Since the application of SA to a wound site can inhibit both wound-induced JA and a defense response that it elicits, namely nicotine production, we determined if tobacco mosaic virus (TMV) inoculation, with its associated endogenous systemic increase in SA, reduces a plant's ability to increase JA and nicotine levels in response to mechanical damage, and evaluated the consequences of these interactions for the amount of tissue removed by a nicotine-tolerant herbivore, Manduca sexta. Additionally, we determined whether the release of volatile methyl salicylic acid (MeSA) from inoculated plants can reduce wound-induced JA and nicotine responses in uninoculated plants sharing the same chamber. The TMV-inoculated plants, though capable of inducing nicotine normally in response to methyl jasmonate applications, had attenuated wound-induced JA and nicotine responses. Moreover, larvae consumed 1.7- to 2.7-times more leaf tissue from TMV-inoculated plants than from mock-inoculated plants. Uninoculated plants growing in chambers downwind of either TMV-inoculated plants or vials releasing MeSA at 83- to 643-times the amount TMV-inoculated plants release, exhibited normal wound-induced responses. We conclude that tobacco plants, when inoculated with TMV, are unable to elicit normal wound responses, due likely to the inhibition of JA production by the systemic increase in SA induced by virus-inoculation. The release of volatile MeSA from inoculated plants is not sufficient to influence the wound-induced responses of neighboring plants. Received: 6 January 1999 / Accepted: 11 January 1999     

外源植物生长调节物质对烟叶中腐胺和烟碱含量的影响

水培的烟草打顶和打顶后喷施腐胺（Put）,烟叶中Put和烟碱含量均增加,钾含量下降,Put含量与烟碱含量之间呈显著正相关;打顶喷施吲哚乙酸（IAA）和赤霉素（GA3）的烟叶中Put和烟碱含量下降,钾含量上升;喷施茉莉酸（JA）的烟碱含量提高,而Put含量变化不大;喷施脱落酸（ABA）和6-BA的叶中Put含量下降。     

Simultaneous UPLC MS/MS analysis of endogenous jasmonic acid,salicylic acid,and their related compounds

Jasmonic acid (JA) and salicylic acid (SA) are plant hormones involved in plant growth and development. Recent studies demonstrated that presence of a complex interplay between JA and SA signaling pathways to response to pathogenesis attack and biotic stresses. To our best knowledge, no method has existed for simultaneous analyses of JA, SA, and their related compounds. Especially, the glucosides are thought to be the storages or the inactivated compounds, but their contribution should be considered for elucidating the amount of the aglycons. It is also valuable for measuring the endogenous amount of phenylalanine, cinnamic acid, and benzoic acid that are the biosynthetic intermediates of SA due to the existence of isochorismate pathway to synthesize SA. We established this method using deuterium labeled compounds as internal standards. This is the first report of simultaneous analysis of endogenous JA, SA, and their related compounds. Measuring the endogenous JA, SA, and their related compounds that had been accumulated in tobacco plants proved the practicality of the newly developed method. It was demonstrated that accumulation of JA, SA and their related compounds were induced in both case of TMV infection and abiotic stresses.     

Differential induction of tobacco MAP kinases by the defense signals nitric oxide, salicylic acid, ethylene, and jasmonic acid

In tobacco, two mitogen-activated protein (MAP) kinases, designated salicylic acid (SA)-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK) are activated in a disease resistance-specific manner following pathogen infection or elicitor treatment. To investigate whether nitric oxide (NO), SA, ethylene, or jasmonic acid (JA) are involved in this phenomenon, the ability of these defense signals to activate these kinases was assessed. Both NO and SA activated SIPK; however, they did not activate WIPK. Additional analyses with transgenic NahG tobacco revealed that SA is required for the NO-mediated induction of SIPK. Neither JA nor ethylene activated SIPK or WIPK. Thus, SIPK may function downstream of SA in the NO signaling pathway for defense responses, while the signals responsible for resistance-associated WIPK activation have yet to be determined.     

Distal transport of exogenously applied jasmonoyl-isoleucine with wounding stress

Determining the mobile signal used by plants to defend against biotic and abiotic stresses has proved elusive, but jasmonic acid (JA) and its derivatives appear to be involved. Using deuterium-labeled analogs, we investigated the distal transport of JA and jasmonoyl-isoleucine (JA-Ile) in response to leaf wounding in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum) plants. We recovered [(2)H(2)-2]JA ([(2)H(2)]JA) and [(2)H(3)-12]JA-Ile ([(2)H(3)]JA-Ile) in distal leaves of N. tabacum and S. lycopersicum after treating wounded leaves with [(2)H(2)]JA or [(2)H(3)]JA-Ile. We found that JA-Ile had a greater mobility than JA, despite its lower polarity, and that application of exogenous JA-Ile to wounded leaves of N. tabacum led to a higher accumulation of JA and JA-Ile in distal leaves compared with wounded control plants. We also found that exudates from the stem of S. lycopersicum plants with damaged leaflets contained JA and JA-Ile at higher levels than in an undamaged plant, and a significant difference in the levels of JA-Ile was observed 30 min after wounding. Based on these results, it was found that JA-Ile is a transportable compound, which suggests that JA-Ile is a signaling cue involved in the resistance to biotic and abiotic stresses in plants.     

Herbivory and caterpillar regurgitants amplify the wound-induced increases in jasmonic acid but not nicotine in Nicotiana sylvestris

Both herbivory and mechanical damage result in increases in the concentration of the wound-signal molecule, jasmonic acid (JA), and the defense metabolite, nicotine, in native tobacco plants, Nicotiana sylvestris Speg. et Comes (Solanaceae). We found that higher concentrations of JA resulted from herbivory by Manduca sexta (L.) larvae than from the mechanical damage designed to mimic the herbivory. While both herbivory and mechanical damage increased JA concentrations in roots of wounded plants, herbivory did not induce either higher root JA or nicotine responses than mechanical damage. In a separate experiment in which mechanical damage was not designed to mimic herbivory, JA responses to herbivory were higher than those to mechanical damage, but the whole-plant (WP) nicotine responses were smaller. Furthermore, when regurgitants from M. sexta larvae were applied to standardized mechanical leaf wounds, leaf JA responses were dramatically amplified. However, neither the root JA response nor the WP nicotine response was comparably amplified by application of regurgitants. Our findings demonstrate that the response of N. sylvestris to herbivory is different from its response to mechanical damage; moreover, oral secretions from larvae may be partly responsible for the difference. During feeding, M. sexta larvae appear to modify the plant's normal defensive response to leaf wounding by reducing the systemic increase in root JA after leaf damage and the subsequent WP nicotine response. Received: 28 February 1997 / Accepted: 9 June 1997     

一株内生砖红镰刀菌促进烟草生长和增强青枯病抗性

课题组前期报道了一株对马铃薯具有促生防病作用的内生砖红镰刀菌Fusarium lateritium (FL617)。为拓展该菌株的应用范围,本研究以同为茄科作物的烟草为研究对象,探究了砖红镰刀菌对其生长和抗病的影响。结果表明,与对照组相比,处理组叶表面积、主根数、叶片数、叶绿素a和叶绿素b含量分别提高了5.0、3.9、1.4、1.3和1.3倍;该结果表明砖红镰刀菌对烟草具有促生作用。生测结果表明,砖红镰刀菌增强了烟草对青枯病的抗病性,其青枯病病情指数下降约30%。植物激素合成相关基因表达模式分析,发现处理组植物激素合成相关基因表达显著上调(1.6-39.9倍);用青枯病菌Ralstonia solanacearum感染寄主植物后分析其水杨酸(SA)、茉莉酸(JA)和R基因信号相关基因的转录模式,发现与对照组相比,处理组SA、JA相关基因均显著上调(1.2-8.3倍),仅有一个R基因显著下调(50%)。进一步用GFP标记的菌株进行荧光定殖观察,发现植物根系周围簇生着带有绿色荧光信号的真菌菌丝,表明砖红镰刀菌可以定殖于烟草根系。综上所述,推测砖红镰刀菌F. lateritium能够通过定殖于烟草根系介导植物激素、免疫防御相关基因的表达从而影响植株的生长发育和抗病性。     

Pathogen-induced calmodulin isoforms in basal resistance against bacterial and fungal pathogens in tobacco

Thirteen tobacco calmodulin (CaM) genes fall into three distinct amino acid homology types. Wound-inducible type I isoforms NtCaM1 and 2 were moderately induced by tobacco mosaic virus (TMV)-mediated hypersensitive reaction, and the type III isoform NtCaM13 was highly induced, while the type II isoforms NtCaM3-NtCaM12 showed little response. Type I and III knockdown tobacco lines were generated using inverted repeat sequences from NtCaM1 and 13, respectively, to evaluate the contribution of pathogen-induced calmodulins (CaMs) to disease resistance. After specific reduction of type I and III CaM gene expression was confirmed in both transgenic lines, we analyzed the response to TMV infection, and found that TMV susceptibility was slightly enhanced in type III CaM knockdown lines compared with the control line. Resistance to a compatible strain of the bacterial pathogen Ralstonia solanacearum, and fungal pathogens Rhizoctonia solani and Pythium aphanidermatum was significantly lower in type III but not in type I CaM knockdown plants. Expression of jasmonic acid (JA)- and/or ethylene-inducible basic PR genes was not affected in these lines, suggesting that type III CaM isoforms are probably involved in basal defense against necrotrophic pathogens in a manner that is independent of JA and ethylene signaling.     

Differential Effect of Jasmonic Acid and Abscisic Acid on Cell Cycle Progression in Tobacco BY-2 Cells

Environmental stress affects plant growth and development. Several plant hormones, such as salicylic acid, abscisic acid (ABA), jasmonic acid (JA), and ethylene play a crucial role in altering plant morphology in response to stress. Developmental regulation often has the cell cycle machinery among its targets. We analyzed the effect of JA and ABA on cell cycle progression in synchronized tobacco (Nicotiana tabacum) BY-2 cells. Both compounds were found to prevent DNA replication, keeping the cells in the G1 stage, when applied just before the G1/S transition. However, ABA did not have any effect on subsequent phases of the cell cycle when applied at a later stage, whereas JA effectively prevented mitosis on application during DNA synthesis. This demonstrates that JA treatment can freeze synchronized BY-2 cells in both the G1 and G2 stages of the cell cycle. Jasmonate administered after the S-phase was less effective in decreasing the mitotic index, suggesting that cell sensitivity toward JA is dependent on the cell cycle phase. In cultures detained in the G2-phase, we observed a reduced histone H1 kinase activity of kinases associated with the p13(suc1) protein.