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Tsubasa Shoji, Keiji Nakajima, Takashi Hashimoto, Ethylene Suppresses Jasmonate-Induced Gene Expression in Nicotine Biosynthesis, Plant and Cell Physiology, Volume 41, Issue 9, 15 September 2000, Pages 1072–1076, https://doi.org/10.1093/pcp/pcd027
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Abstract
In Nicotiana sylvestris , a set of nicotine biosynthesis genes were activated by exogenous application of methyl jasmonate, but the activation was effectively suppressed by simultaneous treatment with ethylene. When N. sylvestris transgenic hairy roots were treated with a natural ethylene precursor, the jasmonate-responsive expression of the promoter from a nicotine pathway enzyme gene was completely suppressed, and this suppressive effect was abolished when ethylene perception was blocked with silver cation. These and additional immunoblot results suggest that ethylene signal antagonizes jasmonate signal in nicotine biosynthesis.
(Received June 2, 2000; Accepted June 30, 2000).
Cross-talk between multiple signal transduction pathways appears to be prevalent and important in the regulation of many biological systems. In plant defense responses against a wide variety of biotic and abiotic agents, hormonal signals involving jasmonate, salicylate, and ethylene, constitute a complex signalling network leading to defense-related gene activation ( Maleck and Dietrich 1999 ). Cross-talk between these signalling pathways may enable plants to optimize their defense strategies by activating different sets of genes in different situations.
Jasmonic acid and its volatile methyl ester (MeJA) are fatty acid derivatives produced via the octadecanoid pathway ( Creelman and Mullet 1997 ). In response to insect herbivory and to assault by microbial pathogens, plants produce jasmonates and activate signal transduction cascades regulating many defense-related genes encoding, for example, proteinase inhibitors (PI), pathogenesis-related (PR) proteins, and antifungal proteins ( Penninckx et al. 1996 , Koiwa et al. 1997 , Thomma et al. 1998 ). The gaseous hormone ethylene controls a broad spectrum of physiological processes during development and in response to stress. Damage caused by pathogen infection and insect herbivory stimulates ethylene synthesis and activates a diverse array of genes, including genes for basic chitinase, β-1,3-glucanase and other basic-type PR proteins ( Ecker 1995 ).
As expected from their overlapping roles in wound response and disease resistance, several instances have recently been reported in which signal transduction cascades involving jasmonate and ethylene may interact. In tomato, jasmonate and ethylene stimulate the production of each other, and the inhibition of ethylene production or sensitivity suppresses the jasmonate-induced expression of the PI gene ( O’Donnell et al. 1996 ). Thus, ethylene is a necessary, but not sufficient, signal which acts cooperatively with jasmonates to induce the tomato PI gene. Cooperative cross-talk between jasmonate and ethylene has also been observed for disease resistance in plant ( Dong 1998 , Maleck and Dietrich 1999 ). In Arabidopsis , a gene for defensin, an antimicrobial peptide, is induced by either jasmonate or ethylene, and synergistically induced by the simultaneous application of both ( Penninckx et al. 1996 , Penninckx et al. 1998 ). In support of this synergy, defensin gene induction requires functional signalling pathways for both jasmonate and ethylene ( Penninckx et al. 1998 ). Hyperinduction by the combination of jasmonate and ethylene was also reported for the tobacco osmotin and PR-1b genes as well ( Xu et al. 1994 ).
In Nicotiana species, jasmonate acts as a signal molecule for the herbivore- and wound-induced accumulation of nicotine, an important defensive compound in these plants ( Baldwin 1999 ). After endogenous jasmonates increase in wounded leaves, a portion of these aerial jasmonates may be transported to the roots where nicotine biosynthesis is subsequently stimulated ( Zhang and Baldwin 1997 ). In accordance with this view, exogenous application of jasmonates induces the expression of several genes encoding nicotine biosynthetic enzymes ( Imanishi et al. 1998 , Shoji et al. 2000 ).
Nicotine is synthesized from putrescine, a diamine derived from ornithine and/or arginine (Fig. 1 ; Hashimoto and Yamada 1994 ). Ornithine is converted to putrescine by ornithine decarboxylase (ODC). Putrescine N -methyltransferase (PMT) catalyzes the N -methylation of putrescine at the first committed step in nicotine biosynthesis, and may represent a regulatory point controlling the metabolic flow into nicotine alkaloids ( Hibi et al. 1994 ). S -adenosylmethionine is supplied by S -adenosylmethionine synthase (SAMS) and is utilized as a cofactor by methyltransferases, including PMT. Tobacco ODC , PMT , and SAMS genes have been identified as jasmonate-inducible genes in tobacco BY-2 cell cultures ( Imanishi et al. 1998 ). Jasmonate-dependent induction of the ODC and PMT genes was shown to be under the control of their promoter regions ( Imanishi et al. 2000 , Shoji et al. 2000 ). The tobacco A622 gene encodes a reductase-like protein, and expression studies indicate that A622 may also be involved in nicotine metabolism ( Hibi et al. 1994 ; our unpublished data).
Effects of ethylene on expression of jasmonate-inducible genes
To examine whether ethylene affects the expression pattern of jasmonate-inducible genes, we treated N. sylvestris plantlets with MeJA, ethylene, or both (MeJA+ethylene). Eight-week-old plantlets were grown in Agripot container (Kirin, Tokyo), which had an air space of 250 cm 3 and a solid medium volume of 80 cm 3 . For MeJA treatment, a cotton ball soaked with 0.5 ml of 100 µM MeJA was placed on the gellam gum medium. Twenty microliters of ethylene gas was injected per pot by microsyringe. Twenty-four hours after treatment, leaves and roots were harvested for RNA gel blot analysis.
PI-II , PR-1b , and osmotin mRNAs were induced by either MeJA or ethylene treatment (Fig. 2 ), as reported previously ( Xu et al. 1994 , Balandin et al. 1995 ). Although the induction magnitude was smaller in our experiment than in a previous report ( Xu et al. 1994 ), transcript levels of these genes increased still further after simultaneous treatment with MeJA and ethylene. SAMS mRNA was detected in both leaves and roots, yet induction by MeJA was clearly observed only in roots. Ethylene, either in its own or in combination with MeJA, did not affect the level of SAMS mRNA. Expression patterns of PMT , A622 , and ODC genes were similar in roots. PMT , A622 , and ODC mRNAs increased after treatment with MeJA but not with ethylene. Remarkably, the simultaneous addition of MeJA and ethylene abolished the MeJA-induced increase of these transcripts. In leaves, ODC mRNA was almost undetectable in untreated and ethylene-treated plants and was induced by MeJA treatment but became very low in abundance after treatment with MeJA and ethylene together. These results indicate that ethylene suppresses the jasmonate-induced expression of the PMT , A622 , and ODC genes. Therefore, tobacco jasmonate-inducible genes can be classified into three groups according to their responsiveness to exogenously added ethylene: (i) ethylene-inducible genes which include PI-II , PR-1b , and osmotin , (ii) genes unresponsive to ethylene, e.g. SAMS , and (iii) genes which are not induced by ethylene and whose positive response to jasmonates is suppressed by ethylene; this group includes PMT , A622 , and ODC . The similar expression patterns of PMT , A622 , and ODC genes may reflect a common function in the same biosynthetic pathway. Although SAMS supplies the S -adenosylmethionine cofactor for the PMT reaction, thereby indirectly contributing to nicotine biosynthesis, this same cofactor is also used in many other methylation reactions. This may explain why SAMS is regulated differentially from bona fide nicotine biosynthetic genes.
Effects of jasmonate and ethylene on PMT and A622 protein accumulation
Next, we analyzed the effect of jasmonate and ethylene on PMT and A622 protein accumulation. Soluble protein extracts from N. sylvestris hairy roots were size-fractionated by SDS-PAGE (12.5%T), and transferred onto an Immobilon membrane (Millipore). The membrane was blocked and then incubated with primary and secondary antibodies as basically described in Nakajima and Hashimoto (1999) . The specificity of anti-PMT and anti-A622 antisera was confirmed when a protein extract from roots of the nic1nic2 low-nicotine tobacco mutant showed reduced signals of PMT and A622 in immunoblots (Fig. 3 A), reflecting a similar decrease in their transcript abundance ( Hibi et al. 1994 ). Both antisera detected proteins with the expected molecular masses. N. sylvestris hairy roots were subcultured at 2-week intervals in liquid MS medium ( Murashige and Skoog 1962 ) and treated for 24 h with the indicated combinations of MeJA (20 µM), 1-aminocyclopropane-1-carboxylic acid (ACC) (100 µM), and AgNO 3 (100 µM) by adding directly to 3-day-old hairy root cultures. ACC is known to be converted to ethylene in planta by the action of ACC oxidase ( Adams and Yang 1979 ).
Accumulation of PMT and A622 proteins increased gradually from 6 h until 24 h after MeJA treatment (Fig. 3 B), which followed the induction kinetics of these transcripts (data not shown). Addition of ACC alone did not induce protein accumulation. When MeJA and ACC were added together, PMT and A622 protein amounts remained at levels similar to untreated controls. This inhibitory effect of ACC clearly disappeared in the presence of AgNO 3 . Silver cation is known to inhibit ethylene perception by the ethylene receptor ( Beyer et al. 1976 ), thereby antagonizing the stimulatory effect of ACC on ethylene production. Therefore, in hairy root cultures, the ethylene signal acts negatively on the MeJA-mediated PMT and A622 protein accumulation.
Responses of PMT promoter to jasmonate and ethylene
Finally, we analyzed the response of a PMT promoter to jasmonate and ethylene. An N. sylvestris transgenic hairy root clone contained a transgene in which the 1.7 kb-promoter region of NsPMT2 was fused to the GUS coding sequence (Fig. 4 A; Shoji et al. 2000 ). We analyzed the effect of MeJA and ACC treatment on the GUS activity of the transgene in this clone. Treatment of the hairy root clone was done in the same manner as for the immunoblot analysis, and GUS activities were determined fluorometrically ( Shoji et al. 2000 ).
GUS activity increased to approximately 4-fold after 24 h MeJA treatment (Fig. 4 B), as reported previously ( Shoji et al. 2000 ). ACC treatment alone did not influence GUS activity, but ACC suppressed MeJA-induced GUS activity when added together with MeJA. The suppressive effect of ACC was negated when AgNO 3 was also included. The results were reproducible in another hairy root clone containing the same transgene (data not shown). Thus, the jasmonate-inducible expression of the NsPMT2 promoter was abolished by ethylene.
Biological relevance of antagonistic cross-talk between jasmonate and ethylene signals
In conclusion, jasmonate-inducible expression of structural genes involved in nicotine biosynthesis such as ODC , PMT , and possibly A622 , is suppressed by ethylene. This antagonistic effect of the ethylene signal is distinct from other well-characterized ethylene responses in which ethylene potentiates jasmonate signalling ( O’Donnell et al. 1996 , Penninckx et al. 1998 ), or acts synergistically with jasmonates ( Xu et al. 1994 ). Analogous antagonistic cross-talk between jasmonate and ethylene signals has been recently reported in Arabidopsis ( Rojo et al. 1999 ). When Arabidopsis leaves were damaged, several jasmonate-inducible genes were repressed in locally wounded tissue through the production and perception of ethylene, whereas these genes were induced in systemic tissues, probably because ethylene does not diffuse over long distances. In this case, the ethylene signalling pathway was used to execute distinct defense responses between local and remote tissues.
What may be the biological relevance of the antagonism between jasmonate and ethylene signals in nicotine biosynthesis? Jasmonate-induced nicotine production, although benefiting plants under insect attack, is costly and results in reduced seed yield ( Baldwin 1998 ), probably because a large proportion of the available total nitrogen is allocated for nicotine production ( Baldwin et al. 1994 ). To optimize the cost of defense, nicotine production should be strictly regulated, yet responsive to various forms of stress. One interesting case was found in the altered response of tobacco toward a herbivore which has evolved tolerance against nicotine ( Kahl et al. 2000 ). When the nicotine-tolerant larvae of Manduca sexta fed on leaves of Nicotiana attenuata , ethylene was released at the wound sites, and the subsequent expected increase in nicotine accumulation did not take place. Our results suggest that the herbivore-induced ethylene burst causes suppression of the jasmonate-mediated activation of nicotine biosynthesis genes in the root. Tobacco may repress nicotine biosynthesis against a nicotine-adapted herbivore so that resources will not be wasted on an ineffective nicotine defense, but rather allocated to other more effective defense mechanisms. Alternatively, a successful herbivore may have evolved to trigger an ethylene burst in damaged tobacco leaves, thus preventing excess nicotine accumulation. Whether it is the herbivores or the tobacco plants which are taking advantage, the ethylene signal is apparently being utilized here to suppress the nicotine defense by interfering with the jasmonate signal. Distinct cross-talk between jasmonate and ethylene signals might have evolved during the co-evolution of insect herbivores and nicotine synthesis in tobacco.
Acknowledgements
We thank K. Nakamura for tobacco ODC and SAMS cDNAs, H. Sano for tobacco PI-II and PR-1b cDNAs, J.-P. Nap for pLM9, R. Winz for the critical reading of the manuscript, and Y. Yamada for his continuous encouragement on this work. This research was supported by a grant (JSPS-RFTF L1605, in “Research for the Future” Program) from the Japan Society for the Promotion of Science to T.H. T.S. was supported by a Japan Society for the Promotion of Science Research Fellowship for Young Scientist (No. 6537).
Corresponding author: E-mail, hasimoto@bs.aist-nara.ac.jp; Fax, +81-743-72-5529.
Abbreviations
- ACC
1-aminocyclopropane-1-carboxylic acid
- GUS
β-glucuronidase
- MeJA
methyl jasmonate
- NsPMT
Nicotiana sylvestris putrescine N-methyltransferase
- ODC
ornithine decarboxylase
- PR-1b
pathogenesis-related protein I basic form
- PI-II
proteinase inhibitor II
- PMT
putrescine N-methyltransferase
- SAMS
S-adenosylmethionine synthase.
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