Silencing an N-Acyltransferase-Like Involved in Lignin Biosynthesis in Nicotiana attenuata Dramatically Alters Herbivory-Induced Phenolamide Metabolism

Baldwin IT (2013) Silencing an N-Acyltransferase-Like Involved in Lignin Biosynthesis in Nicotiana attenuata Dramatically Alters Herbivory-Induced Phenolamide Metabolism. PLoS ONE 8(5): e62336. doi:10.1371/journal.pone.0062336 Silencing an N -Acyltransferase-Like Involved in Lignin Biosynthesis in Nicotiana attenuata Dramatically Alters Herbivory-Induced Phenolamide Metabolism Emmanuel Gaquerel 0 Hemlata Kotkar 0 Nawaporn Onkokesung 0 Ivan Galis 0 Ian T. Baldwin 0 Frederik Bo rnke, Friedrich-Alexander-University Erlangen-Nurenberg, Germany 0 1 Department of Molecular Ecology, Max Planck Institute for Chemical Ecology , Jena, Germany, 2 Plant Molecular Biology Unit, Division of Biochemical Sciences, National Chemical Laboratory (CSIR) , Pune , India , 3 Institute of Plant Science and Resources, Okayama University , Kurashiki , Japan In a transcriptomic screen of Manduca sexta-induced N-acyltransferases in leaves of Nicotiana attenuata, we identified an Nacyltransferase gene sharing a high similarity with the tobacco lignin-biosynthetic hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) gene whose expression is controlled by MYB8, a transcription factor that regulates the production of phenylpropanoid polyamine conjugates (phenolamides, PAs). To evaluate the involvement of this HCT-like gene in lignin production as well as the resulting crosstalk with PA metabolism during insect herbivory, we transiently silenced (by VIGs) the expression of this gene and performed non-targeted (UHPLC-ESI/TOF-MS) metabolomics analyses. In agreement with a conserved function of N. attenuata HCT-like in lignin biogenesis, HCT-silenced plants developed weak, soft stems with greatly reduced lignin contents. Metabolic profiling demonstrated large shifts (up to 12% deregulation in total extracted ions in insect-attacked leaves) due to a large diversion of activated coumaric acid units into the production of developmentally and herbivory-induced coumaroyl-containing PAs (N9,N99-dicoumaroylspermidine, N9,N99-coumaroylputrescine, etc) and to minor increases in the most abundant free phenolics (chlorogenic and cryptochlorogenic acids), all without altering the production of well characterized herbivory-responsive caffeoyl- and feruloyl-based putrescine and spermidine PAs. These data are consistent with a strong metabolic tension, exacerbated during herbivory, over the allocation of coumaroyl-CoA units among lignin and unusual coumaroyl-containing PAs, and rule out a role for HCT-LIKE in tuning the herbivory-induced accumulation of other PAs. Additionally, these results are consistent with a role for lignification as an induced anti-herbivore defense. - Funding: This research was supported by the Max Planck Society. The Max Planck Society also provided a Max Planck-India Visiting Fellowship for H. Kotkar to conduct experiments at MPI-CE in Jena. The work of Emmanuel Gaquerel is funded by Advanced Grant No 293926 of the European Research Council to Ian T. Baldwin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Plants as sessile organisms are exposed during their development to variable stress conditions which have shaped, from a macro-evolutionary point of view, the emergence of highly adaptive tolerance and resistance traits. The feeding damage of herbivores has provided a major evolutionary selective pressure that has sculpted all aspects of plant metabolism including the composition and size of their pools and the regulatory networks that determine fluxes [13]. When chewing leaves, insects elicit a burst of the plant hormone, jasmonic acid (JA); this signaling molecule mediates rapid changes in secondary metabolic pathways [4,5]. Native tobacco plants, Nicotiana attenuata, accumulate, as a consequence of JA signaling, large amounts of nicotine, a neurotoxic compound and a spectrum of phenolic amide conjugates (phenolamides, PAs), derived from the phenylpropanoid pathway, to defend against specialist and generalist chewing herbivores [6,7]. As is frequently observed in transcriptomic analyses, herbivoreinduced changes in a plants metabolome result from specific reorganizations of metabolic pathways. However, how attacked cells rechannel metabolic fluxes towards the production of a specific spectrum of metabolites is frequently unknown. The underlying mechanisms by which plants perceive insect herbivory are known to be controlled by elicitors present in the oral secretions (OS) of feeding larvae [8]. As an essential part of the signal transduction mechanisms, plants employ specific transcription factors (TFs) to synchronize the expression of relevant gene networks. For example, genes controlling PA biogenesis in N. attenuata are tightly regulated by MYB8, an herbivory-inducible TF of the MYB family [6]. Figure 1 summarizes major steps in the formation of PA and connections with the lignin pathway. MYB8 activates the core phenylpropanoid genes and specific Nacyltransferases that redirect the flux of phenylpropanoids towards PA production during insect herbivory. Using microarray analysis and metabolic profiling of MYB8-silenced plants (irMYB8), we recently identified several N-acyltranferase enzymes that conjugate activated phenolic acids (p-coumaroyl-, caffeoyl-, feruloyl) with polyamines (putrescine and spermidine) [9]: AT1 is a hydroxycinnamoyl-CoA: putrescine acyltransferase responsible for caffeoyl- and coumaroyputrescine accumulation during insect herbivory. Another gene (DH29), specific for spermidine conjugation, mediates the initial acylation step in the formation of caffeoyl-, coumaroyl and feruloyl-containing di-acylated spermidine structures. Although this enzyme was not able to perform the second acylation towards diacylated spermidine biosynthesis, another acyltransferase gene, called CV86, proposed to act on mono-acylated spermidines was isolated and partially characterized [9]. Among the herbivory-regulated genes screened in the aforementioned study, we identified a candidate N-acyltransferase which shared a high sequence similarity with previously characterized hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) genes from N. tabacum (Acc. No. CAD47830) and N. benthamiana (Acc. No. CAD88491) (Figure S1). HCT enzymes have previously been shown to catalyze developmentally-regulated lignin biosynthesis in tobacco [10,11]. Interestingly, the herbivory-induced expression of this HCT-like gene is dependent on and is temporally synchronized by MYB8 transcriptional activity. This suggests that MYB8 controls, via an HCT-like conserved activity, certain aspects of lignin production in herbivore-attacked leaf tissues. The possibility of cross-talk between HCT activity and the PA pathway (Figure 1), especially when the PA pathway is m (...truncated)