Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata.

Inhibition of jasmonic acid (JA) signaling has been shown to decrease herbivore resistance, but the responsible mechanisms are largely unknown because insect resistance is poorly understood in most model plant systems. We characterize three members of the lipoxygenase (LOX) gene family in the native tobacco plant Nicotiana attenuata and manipulate, by antisense expression, a specific, wound- and herbivory-induced isoform (LOX3) involved in JA biosynthesis. In three independent lines, antisense expression reduced wound-induced JA accumulation but not the release of green leaf volatiles (GLVs). The impaired JA signaling reduced two herbivore-induced direct defenses, nicotine and trypsin protease inhibitors (TPI), as well as the potent indirect defense, the release of volatile terpenes that attract generalist predators to feeding herbivores. All these defenses could be fully restored by methyl-JA (MeJA) treatment, with the exception of the increase in TPI activity, which was partially restored, suggesting the involvement of additional signals. The impaired ability to produce chemical defenses resulted in lower resistance to Manduca sexta attack, which could also be restored by MeJA treatment. Expression analysis using a cDNA microarray, specifically designed to analyze M. sexta-induced gene expression in N. attenuata, revealed a pivotal role for LOX3-produced oxylipins in upregulating defense genes (protease inhibitor, PI; xyloglucan endotransglucosylase/hydrolase, XTH; threonine deaminase, TD; hydroperoxide lyase, HPL), suppressing both downregulated growth genes (RUBISCO and photosystem II, PSII) and upregulated oxylipin genes (alpha-dioxygenase, alpha-DOX). By genetically manipulating signaling in a plant with a well-characterized ecology, we demonstrate that the complex phenotypic changes that mediate herbivore resistance are controlled by a specific part of the oxylipin cascade.

[1]  F. Schaller.  Enzymes of the biosynthesis of octadecanoid-derived signalling molecules. , 2001, Journal of experimental botany.

[2]  I. Baldwin,et al.  Herbivore-induced allene oxide synthase transcripts and jasmonic acid in Nicotiana attenuata. , 2001, Phytochemistry.

[3]  C. Preston,et al.  Eating the evidence? Manduca sexta larvae can not disrupt specific jasmonate induction in Nicotiana attenuata by rapid consumption , 2000, Planta.

[4]  H. Leyser,et al.  Ethylene as a Signal Mediating the Wound Response of Tomato Plants , 1996, Science.

[5]  R. Creelman,et al.  Jasmonate is essential for insect defense in Arabidopsis. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[6]  M. Hamberg,et al.  Involvement of the Arabidopsis alpha-DOX1 fatty acid dioxygenase in protection against oxidative stress and cell death. , 2002, The Plant journal : for cell and molecular biology.

[7]  I. Baldwin,et al.  Defensive function of herbivore-induced plant volatile emissions in nature. , 2001, Science.

[8]  Ho Bang Kim,et al.  A knock-out mutation in allene oxide synthase results in male sterility and defective wound signal transduction in Arabidopsis due to a block in jasmonic acid biosynthesis. , 2002, The Plant journal : for cell and molecular biology.

[9]  G. Griffiths,et al.  Lipoxygenase H1 Gene Silencing Reveals a Specific Role in Supplying Fatty Acid Hydroperoxides for Aliphatic Aldehyde Production* , 2002, The Journal of Biological Chemistry.

[10]  J. Tumlinson,et al.  Quantitative relationships between induced jasmonic acid levels and volatile emission in Zea mays during Spodoptera exigua herbivory , 2003, Planta.

[11]  I. Baldwin,et al.  Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. IV. Insect-Induced ethylene reduces jasmonate-induced nicotine accumulation by regulating putrescine N-methyltransferase transcripts. , 2001, Plant physiology.

[12]  I. Baldwin,et al.  Molecular Interactions between the Specialist HerbivoreManduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata: V. Microarray Analysis and Further Characterization of Large-Scale Changes in Herbivore-Induced mRNAs1 , 2003, Plant Physiology.

[13]  M. Farag,et al.  Jasmonate‐deficient plants have reduced direct and indirect defences against herbivores , 2002 .

[14]  I. Baldwin,et al.  Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris , 1997, Planta.

[15]  S. Rosahl,et al.  Expression of Lipoxygenase in Wounded Tubers of Solanum tuberosum L , 1994, Plant physiology.

[16]  C. Preston,et al.  Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore , 2000, Planta.

[17]  G. Howe,et al.  Resistance of Cultivated Tomato to Cell Content-Feeding Herbivores Is Regulated by the Octadecanoid-Signaling Pathway1 , 2002, Plant Physiology.

[18]  I. Baldwin,et al.  Ecophysiological comparison of direct and indirect defenses in Nicotiana attenuata , 2000, Oecologia.

[19]  D. Hannapel,et al.  Lipoxygenase Is Involved in the Control of Potato Tuber Development , 2001, Plant Cell.

[20]  J. Albar,et al.  Antisense-mediated depletion of a potato lipoxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pests. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[21]  I. Baldwin.  An ecologically motivated analysis of plant-herbivore interactions in native tobacco. , 2001, Plant physiology.

[22]  C. Ryan,et al.  A Gene Encoding a Chloroplast-Targeted Lipoxygenase in Tomato Leaves Is Transiently Induced by Wounding, Systemin, and Methyl Jasmonate , 1997, Plant physiology.

[23]  I. Baldwin,et al.  Herbivory and caterpillar regurgitants amplify the wound-induced increases in jasmonic acid but not nicotine in Nicotiana sylvestris , 1997, Planta.

[24]  M. Hamberg,et al.  alpha-oxidation of fatty acids in higher plants. Identification of a pathogen-inducible oxygenase (piox) as an alpha-dioxygenase and biosynthesis of 2-hydroperoxylinolenic acid. , 1999, The Journal of biological chemistry.

[25]  I. Baldwin,et al.  Molecular Interactions between the Specialist HerbivoreManduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata. VI. Microarray Analysis Reveals That Most Herbivore-Specific Transcriptional Changes Are Mediated by Fatty Acid-Amino Acid Conjugates1,212 , 2003, Plant Physiology.

[26]  G. Howe,et al.  An octadecanoid pathway mutant (JL5) of tomato is compromised in signaling for defense against insect attack. , 1996, The Plant cell.

[27]  I. Baldwin,et al.  Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses. , 2001, Plant physiology.

[28]  S. Berger.  Jasmonate-related mutants of Arabidopsis as tools for studying stress signaling , 2002, Planta.

[29]  I. Baldwin,et al.  Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system , 2002, CHEMOECOLOGY.

[30]  J. Browse,et al.  Plant defense in the absence of jasmonic acid: The role of cyclopentenones , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Sánchez-Serrano,et al.  Characterization of Three Potato Lipoxygenases with Distinct Enzymatic Activities and Different Organ-specific and Wound-regulated Expression Patterns* , 1996, The Journal of Biological Chemistry.

[32]  I. Baldwin,et al.  Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. I. Large-scale changes in the accumulation of growth- and defense-related plant mRNAs. , 2001, Plant physiology.

[33]  E. Blée,et al.  Impact of phyto-oxylipins in plant defense. , 2002, Trends in plant science.

[34]  I. Baldwin,et al.  Rapid HPLC screening of jasmonate-induced increases in tobacco alkaloids, phenolics, and diterpene glycosides in Nicotiana attenuata , 2001 .

[35]  Andrea Pitzschke,et al.  Allene oxide cyclase dependence of the wound response and vascular bundle-specific generation of jasmonates in tomato - amplification in wound signalling. , 2003, The Plant journal : for cell and molecular biology.

[36]  S. Rothstein,et al.  The Cloning of Two Tomato Lipoxygenase Genes and Their Differential Expression during Fruit Ripening , 1994, Plant physiology.

[37]  Alessandra Devoto,et al.  The Jasmonate Signal Pathway Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.000679. , 2002, The Plant Cell Online.

[38]  P. M. Sanders,et al.  The Arabidopsis DELAYED DEHISCENCE1 Gene Encodes an Enzyme in the Jasmonic Acid Synthesis Pathway , 2000, Plant Cell.

[39]  P. Reymond,et al.  Jasmonate and salicylate as global signals for defense gene expression. , 1998, Current opinion in plant biology.

[40]  J. Browse,et al.  The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[41]  J. Tumlinson,et al.  Synergistic interactions between volicitin, jasmonic acid and ethylene mediate insect-induced volatile emission in Zea mays. , 2003, Physiologia plantarum.

[42]  I. Baldwin,et al.  Ontogeny Constrains Systemic Protease Inhibitor Response in Nicotiana attenuata , 2001, Journal of Chemical Ecology.

[43]  I. Baldwin,et al.  ECOLOGICAL COSTS AND BENEFITS CORRELATED WITH TRYPSIN PROTEASE INHIBITOR PRODUCTION IN NICOTIANA ATTENUATA , 2003 .

[44]  Ivo Feussner,et al.  The lipoxygenase pathway. , 2003, Annual review of plant biology.

[45]  I. Baldwin,et al.  Anti-sense expression of putrescine N-methyltransferase confirms defensive role of nicotine in Nicotiana sylvestris against Manduca sexta , 2001, CHEMOECOLOGY.

[46]  J. Browse,et al.  The Critical Requirement for Linolenic Acid Is Pollen Development, Not Photosynthesis, in an Arabidopsis Mutant. , 1996, The Plant cell.

[47]  I. Baldwin,et al.  Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. II. Accumulation of plant mRNAs in response to insect-derived cues. , 2001, Plant physiology.

[48]  E. Weiler,et al.  Transgenic Nicotiana tabacum and Arabidopsis thaliana plants overexpressing allene oxide synthase , 2000, Planta.

[49]  J E Mullet,et al.  A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[50]  R. Fall,et al.  Volatile organic compounds emitted after leaf wounding: On‐line analysis by proton‐transfer‐reaction mass spectrometry , 1999 .