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First published online October 6, 2006; 10.1104/pp.106.088781 Plant Physiology 142:1621-1641 (2006) © 2006 American Society of Plant Biologists
Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata. VII. Changes in the Plant's Proteome1,[W] SvatoDepartment of Molecular Ecology (A.P.G., H.W., S.M., J.A.Z., I.T.B.) and Mass Spectrometry Research Group (A.M., A.S.), Max Planck Institute for Chemical Ecology, 07745 Jena, Germany; and Plant Molecular Biology Unit, Division of Biochemical Sciences, National Chemical Laboratory, Pune–411 008 (M.S.), India (A.P.G.)
When Manduca sexta attacks Nicotiana attenuata, fatty acid-amino acid conjugates (FACs) in the larvae's oral secretions (OS) are introduced into feeding wounds. These FACs trigger a transcriptional response that is similar to the response induced by insect damage. Using two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time of flight, and liquid chromatography-tandem mass spectrometry, we characterized the proteins in phenolic extracts and in a nuclear fraction of leaves elicited by larval attack, and/or in leaves wounded and treated with OS, FAC-free OS, and synthetic FACs. Phenolic extracts yielded approximately 600 protein spots, many of which were altered by elicitation, whereas nuclear protein fractions yielded approximately 100 spots, most of which were unchanged by elicitation. Reproducible elicitor-induced changes in 90 spots were characterized. In general, proteins that increased were involved in primary metabolism, defense, and transcriptional and translational regulation; those that decreased were involved in photosynthesis. Like the transcriptional defense responses, proteomic changes were strongly elicited by the FACs in OS. A semiquantitative reverse transcription-PCR approach based on peptide sequences was used to compare transcript and protein accumulation patterns for 17 candidate proteins. In six cases the patterns of elicited transcript accumulation were consistent with those of elicited protein accumulation. Functional analysis of one of the identified proteins involved in photosynthesis, RuBPCase activase, was accomplished by virus-induced gene silencing. Plants with decreased levels of RuBPCase activase protein had reduced photosynthetic rates and RuBPCase activity, and less biomass, responses consistent with those of herbivore-attacked plants. We conclude that the response of the plant's proteome to herbivore elicitation is complex, and integrated transcriptome-proteome-metabolome analysis is required to fully understand this ubiquitous ecological interaction.
The majority of studies examining the induced defense responses of plants after insect attack have focused on the dynamics of the specific genes, proteins, and metabolites that are thought to be responsible for changes in resistance elicited by attack. More recently, large-scale transcriptional analyses with microarrays have broadened the scope of the analysis and revealed coordinated changes in hundreds of transcripts, suggesting that large-scale shifts in metabolism accompany the activation of defense responses (Reymond et al., 2000  , 2004; Halitschke et al., 2001, 2003; Hermsmeier et al., 2001; Schittko et al., 2001; Kessler and Baldwin, 2002; Ralph et al., 2006a, 2006b). How the changes in the transcriptome translate into changes in the proteome and metabolome that eventually account for the phenotype of increased resistance is unknown, largely because the proteomic changes elicited by herbivore attack remain unstudied. It is becoming increasingly clear that proteomic changes cannot be directly predicted from changes in the transcriptome. Furthermore, in a given cellular microenvironment, both proteins and transcripts interact with other molecules in specific ways, and these interactions determine the regulation, expression, activity, and stability of specific mRNA and protein molecules. Parallel large-scale transcription and protein analyses promise to illuminate the function and regulation of genes and proteins (Bertone and Snyder, 2005; Lange and Ghassemian, 2005; Peck, 2005). Recent developments in mass spectrometric peptide sequencing now allow for protein identification on the proteomic scale (Aebersold and Mann, 2003; Glinski and Weckwerth, 2006). Information obtained with such tools will provide valuable insights into the mechanisms attacked plants use to shift their metabolic priorities from growth to defense (Walling, 2000; Hahlbrock et al., 2003).
Nicotiana attenuata Torr. Ex Wats. (Solanaceae), a post-fire annual inhabiting the Great Basin Desert, has a number of well-described herbivore-induced direct and indirect defenses (Baldwin, 2001
High-throughput transcriptional analysis of the interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host N. attenuata identified a large-scale transcriptional reconfiguration, which entailed decreases in photosynthetic-related processes and increases in defense-related processes (Walling, 2000 As a follow-up to transcriptional analysis, we undertook a comparative proteome analysis of the M. sexta-N. attenuata interaction. Proteomic analysis was carried out by comparing the patterns of leaf proteins in the leaves of undamaged plants to those in elicited and attacked plants by two-dimensional gel electrophoresis (2-DE). We performed two types of proteomic analysis, addressing two main questions. First, how does a plant respond to the different elicitors found in M. sexta OS? To answer this question, we compared the patterns of protein accumulation observed when punctured wounds were treated with OS to those observed when the punctured wounds were treated with water, FACs, OS that had their FACs removed by ion-exchange chromatography (OS-FAC-free), and feeding M. sexta larvae. Second, how do these responses change over the time when leaves are known to increase their resistance to insect attack? To answer this question, we measured the accumulation of identified proteins at 6, 12, 30, 48, and 72 h after OS treatment of puncture wounds. We used a reverse transcription (RT)-PCR approach to determine the association between candidate proteins showing differential accumulation patterns and the abundance of their encoding mRNAs. In addition, the functional analysis of one of the proteins identified as being involved in photosynthesis, RuBPCase activase (RCA), was accomplished by gene silencing. This study identifies several well-characterized proteins whose direct and indirect roles in insect-elicited responses were not previously known, as well as several proteins of unknown function.
Comparison of 2-DE Protein Profiles of Control and Elicited Leaves of N. attenuata Source leaves +1, +2, and +3 of rosette-stage plants of N. attenuata were punctured parallel to the midvein with a fabric pattern wheel six times at 30-min intervals (Fig. 1 ). To induce the plants with different elicitors, various solutions were applied to the punctured leaves (W + OS, W + FAC, and W + OS-FAC-free) or larvae were released on these leaves (Fig. 1). The leaf proteins were extracted using different methods and analyzed.
Phenolic extracts of N. attenuata leaf proteins yielded approximately 600 protein spots on a 2-DE. Protein spots exhibiting variations in extracts from control and elicited leaves were identified and compared among three to five biological replicates. One such biological replicate represents two to four plants and three elicited or adjacent unelicited (systemic) leaves on each plant. Representative 2-DE gel images of leaf proteins from the 30 h harvest of control and W + OS-elicited leaves are presented in Figure 2 . Each protein preparation was analyzed on at least three parallel 2-DE gels. In total, we generated and analyzed 72 gels for phenol extracts (three biological replicates, six treatments, and five time points) and 14 gels for nuclear extracts (three biological replicates and two time points). Analysis of the nuclear protein fractions revealed approximately 100 protein spots with no significant variation between control and OS-elicited leaf extracts (Supplemental Fig. S1). Although the total leaf protein profiles (phenolic extracts) responded to elicitation, the nuclear protein fraction did not, so we focused our efforts on characterizing the elicited changes in the total protein extracts. The similar protein patterns observed in control leaves from five harvests that were removed 6 to 72 h after elicitation indicated that the developmental or leaf maturation-related changes that occurred during this time did not significantly influence the part of the proteome that was detectable in our analysis (Fig. 2; Supplemental Figs. S2 and S3). In contrast, we identified 90 spots that exhibited differential accumulation patterns from 6 to 72 h after OS elicitation when wounded leaves were compared with the respective control leaves. These proteins were successfully identified using peptide mass fingerprints and/or peptide sequencing (Table I ). Among these 90 proteins, 35 showed consistent accumulation trends in induced leaves at 30 h after OS elicitation (Fig. 2; Table II ). Changes in these proteins were meticulously compared in four induction treatments (Table II). Fifty-one other proteins identified in this study, whose accumulation patterns consistently increased or decreased at specific harvest times in at least two replicates, were identified. The mass spectrometry (MS) data of four proteins did not yield information for the database search or de novo sequencing.
Identification of Proteins by MALDI-TOF and LC-MS/MS The tryptic peptide mass fingerprints of selected proteins were determined from matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) spectra using the green plant database of the National Center for Biotechnology Information (NCBI; http://www.ncim.nlm.nih.gov); protein spots showing identity with RuBPCase were omitted from further analysis. Peptide sequence data obtained from liquid chromatography (LC)-MS/MS were compared with green plant or nonredundant NCBI databases. Amino acid sequences from de novo data with unspecified protein hits were searched using MS-BLAST. Most of the 45 proteins identified by peptide sequences agree with the peptide mass fingerprints. An additional 42 proteins have been identified by their peptide mass fingerprints, which match at least four peptides. Table I presents detailed information about these 84 protein spots, such as identification scores, number of matching peptides, and peptide sequences, and identifies their putative function with accession numbers of database proteins, molecular masses, and isoelectric points. More than 20% of proteins remain unidentified due to the lack of a database match or a match only to proteins with unknown functions.
Accumulation patterns for 35 proteins were monitored in the four elicitation treatments and compared to patterns observed in control leaves. Differences among these treatments were seen in only a few spots. The larval feeding treatment was terminated after 48 h and the responses in this treatment were compared with those of the W + OS treatments. A similar but lower rate of protein accumulation was found in the larval treatment compared to the W + OS treatment, perhaps because the amount of damage and the amount of OS introduced into wounds after 48 h of feeding by five neonate larvae are likely less than those inflicted by the W + OS treatment. Of the 35 differentially regulated proteins in these two treatments, 37% (13 proteins) were commonly regulated (nine up- and four down-regulated; Fig. 3A ; Table II). Interestingly, spot 83, a chloroplast Gln synthetase, was up-regulated in caterpillar-attacked leaves but not regulated in the other elicitation treatments (Table II). However, a vacuolar H+-ATPase A2 subunit, TD, and one spot with glyceraldehyde-3-P dehydrogenase of caterpillar-attacked leaves share accumulation patterns similar to those of leaves treated with OS.
Because the amount of mechanical damage and elicitor solutions introduced represented parameters that did not differ among the W + OS, W + FAC, and W + OS-FAC-free treatments, the cause of the differences is more easily inferred. Of the 35 proteins that were differentially regulated by these three treatments, 12 (seven up- and five down-regulated) were commonly regulated among all three treatments (Fig. 3B; Table II). The seven commonly up-regulated protein spots were RNA-binding protein (spot 24), acyl-CoA synthetase 7 (ACS7; spot 32), S-adenosylmethionine synthetase (spot 34), O-acetylserine thiol lyase (spot 43), RCA (spot 46), spermidine synthase (spot 50), and potassium sodium symporter (spot 57). Interestingly, four of the five down-regulated spots identified as RCA consistently accumulated in lower amounts (spots 6, 9, 82, and 95); the fifth protein was the oxygen-evolving enhancer protein 1 (spot 75). To determine which proteins were regulated by the FACs in OS, we compared the regulation elicited by the W + OS and W + FAC treatments. Seven proteins were commonly regulated and no proteins were uniquely regulated by the FAC treatments that were not changed by the W + OS treatment. Only one spot (OSJNBa0039c07.4, spot 1) was uniquely regulated by W + OS-FAC-free treatment, and one spot (quinone oxidoreductase [QR]-like protein, spot 8) was commonly regulated by this treatment and by the W + FAC treatment, demonstrating that FACs account for the majority of the elicitor activity of OS at the protein level. However, OS clearly contain factors other than FACs that elicit differential accumulations of proteins, as can be seen by comparing the responses elicited by the W + OS and W + OS-FAC-free treatments (Fig. 3B). These treatments commonly regulated the following five proteins, none of which were regulated by the W + FAC treatment: RCA (spots 20 and 51), transketolase (TK; spot 2), the oxygen-evolving enhancer protein 1 (spot 12), and the Gly-rich RNA-binding protein (spot 72). These results demonstrate that although FACs are the main elicitors of specific changes in protein accumulation in OS, other factors are clearly also active.
The early (6, 12, and 30 h) and later (48–72 h) changes in the W + OS-elicited proteome were characterized by the relative densitometric quantitation of 90 regulated protein spots (Table I; Fig. 4 ; Supplemental Figs. S2 and S3). Of these 90 regulated protein spots, 17 are involved in photosynthesis and photorespiration—28 in primary metabolism, 13 in transcription and translation processes—and nine function in secondary metabolism pathways; one protein in each case was identified in signal transduction, secretory pathway, and cytoskeleton formation (Table I; Fig. 2). Twenty proteins were not identified or were identified but had unknown functions. To simplify the analysis, we categorized the protein accumulation patterns as: (I) consistently high accumulation, (II) consistently low accumulation, (III) early high accumulation (up to 30 h) and no change or low later (30 h onward), (IV) no change or low early on and high accumulation or no change later, and (V) no consistent pattern (Table I). Twenty-seven proteins exhibited a type I response (Table I). Of these, four proteins belong to the photosynthesis and photorespiration category and eight to the primary metabolism category; four proteins were categorized as involved in secondary metabolism, seven as unknown proteins; one is a translation elongation factor; and one protein contributes to the cytoskeleton and one to the secretory pathway. Fifteen proteins exhibited a type II response and are present in the following categories: eight are related to photosynthesis and photorespiration, four to primary metabolism, one to transcription and translation, one to signal transduction, and one is a putative protein. For type I and II responses, we also included proteins that showed consistent accumulation in four harvests. We categorized six proteins with type III responses. One is involved in photosynthesis, three in primary metabolism, one in transcription and translation, and one in secondary metabolism. Sixteen proteins exhibited type IV responses, seven in primary metabolism, four in transcription and translation, and five with no known function. We identified 26 proteins with type V responses, indicating they either have very specific kinetics or were not detected at other harvests of several replicates. In summary, we observed most proteins with similar accumulation patterns during the five time harvests after applying OS to N. attenuata leaves. However, few proteins specific to one or two harvest times and exhibiting increased or decreased accumulation patterns were also identified.
The Response of RCA Protein(s) to OS Elicitation
The changes in RCA protein spots were one of the most apparent OS-elicited responses, so much so that these spots reliably differentiated gels of control and elicited samples. We identified seven protein spots that have one or several peptides exhibiting homology with RCA (Tables I and II). In four spots, molecular weight and pI are very similar in terms of reported RCA proteins from several plant species. After W + OS in elicited leaves compared to controls, the levels of these proteins (spots 6, 9, 82, and 95) decreased (Fig. 5
), whereas levels of RCA (spots 20, 46, and 51) strongly increased. Since these changes were not significant in W + W or W + FAC treatments, we infer that factors other than FACs in OS are responsible (Fig. 5D). This inference is supported by the observation that the responses observed in the W + OS-FAC-free treatment were similar to those observed in the W + OS treatment (Fig. 5F). Peptide data obtained from spots 6 and 95 match data from the C-terminal region of RCA protein, whereas spots 9, 20, and 46 yielded peptide sequences spanning almost the entire length of RCA. Protein spot 51 generated peptides from the N-terminal region of RCA. These results suggest that massive changes are occurring in RCA proteins due to proteolytic cleavage, differential subunit formation, or gene expression. RCA is a member of the chaperonin protein family, which is involved in RuBPCase activation, and RuBPCase is known to be transcriptionally down-regulated after elicitation (Hermsmeier et al., 2001
To determine whether the changes observed in RCA proteins are due to the proteolytic activity of the OS fraction, we analyzed the 2-DE protein patterns of undamaged systemic leaves on elicited plants. Interestingly, proteins in spots 46 and 51 were detectable in OS-elicited systemic leaves, whereas decreasing amounts of protein in spots 6, 9, 82, and 95 were noticed in the systemic leaves compared to leaves growing at the same nodes on control plants (Fig. 5, G and H). Furthermore, to confirm that these changes in RCA spots are not due to a plant protein fraction present in OS, we performed a 2-DE analysis of OS. Of the nearly 100 proteins we identified by peptide mass fingerprints, no protein spots were detected with similarity to RCA (data not shown). These results demonstrate that even if proteolysis of RCA is responsible for these changes, it occurs via plant metabolism and is not due to the proteolytic activity of OS.
RT-PCR was used to quantify the transcripts of 17 genes coding for 17 differentially accumulated proteins to assess the correlations between protein and mRNA accumulation patterns (Figs. 6 and 7 ). Different peptide sequences obtained from protein spots were used to design primers to compare mRNA accumulation in W + OS-elicited leaves with that in control leaves 30 h after elicitation. The well-characterized insect-induced protein TPI was not identified in the protein gels; however, it was used as a positive control. TPI may not be detected in induced leaves in proteomic analysis due to its extractability and/or low levels. Patterns of mRNA accumulation were compared among 17 candidate genes 30 h after the different elicitation treatments (Fig. 6). Levels of mRNA increased after W + OS, W + FAC, W + OS-FAC-free, and larval treatments in seven transcripts, namely, tpi, td, pgam (phosphoglycerate mutase [PGM]), sams (S-adenosylmethionine synthetase), pme (pectin methyl esterase [PME]), gpdh (glyceraldehyde-3-P dehydrogenase), fdh (mitochondrial formate dehydrogenase), and atpe (vacuolar ATPase); however, treatment-specific increases were also observed (Fig. 6A). tpi, td, and sams transcripts responded maximally to larval feeding, whereas pgam and atpe accumulated maximally in response to the W + OS-FAC-free treatment. The W + FAC treatment elicited the largest response in pme mRNA. ald (aldolase [ALD]) transcripts decreased in response to W + OS and larval treatments, but increased after W + FAC and W + OS-FAC-free treatments. Levels of tef (translation elongation factor) increased after W + OS-FAC-free treatments compared to other elicitor treatments. Gln synthetase transcripts increased only after W + FAC and W + OS-FAC-free and larval treatments but not after W + OS treatment. Levels of grp (Gly-rich RNA-binding protein) and cpn (chaperonin) increased after W + FAC and W + OS-FAC-free treatments, but decreased in response to the presence of larvae. grp also showed increased accumulation after W + OS treatments. Levels of tk increased maximally after W + OS-FAC-free treatment. Transcripts of rca decreased after W + OS treatments and increased after larval and W + OS-FAC-free treatments, but remained unchanged after the W + FAC treatment (Fig. 6B). Transcript accumulation patterns of seven candidates correlated with their respective protein abundances. The specific mRNA accumulation patterns observed in OS-FAC-free-elicited N. attenuata leaves indicate that signaling molecules other than FACs exist in M. sexta OS and that these also elicit specific protein accumulation patterns.
The differential accumulation of mRNA was observed in all 17 genes. To simplify the data, we categorized accumulation patterns as we had done for the proteins (Fig. 7). In control plants, levels of tpi transcripts were barely detectable, but they were strongly elicited—a type I pattern—by W + OS treatment. This pattern was also found in td transcripts where both (tpi and td) had reached their peaks at 12 h and decreased slightly thereafter. pme transcripts were up-regulated at all time points (type I). mRNAs of pgam, sams, fdh, and atpe were consistently up-regulated up to 30 h, slightly down-regulated at 48 h, and unchanged at 72 h (type III; Fig. 7A). Transcripts of gpdh were slightly up-regulated at 6 h, down-regulated at 12 and 30 h, and up-regulated again at 48 and 72 h (type V). grp accumulated fewer transcripts in induced leaves at early compared to later time points (30–72 h; type IV). Levels of tk, rca, tef, mgpc (magnesium [Mg]-protoporphyrin IX chelatase), ald, and cpn (6-48 h) showed decreased mRNA accumulation at all time points (type II; Fig. 7B). Transcripts of gmps (Gln synthetase) and oep (oxygen-evolving protein) were down-regulated consistently up to 30 h, but at 48 h the levels of accumulation were the same as in control leaves; at 72 h after OS application (type V) the levels were down-regulated. In summary, mRNA accumulation patterns match protein accumulation patterns in several candidate proteins; however, cases in which protein and mRNA accumulation did not match need to be studied further. One possible explanation for such discrepancy might be that several isoforms of these proteins exist, which makes it difficult to identify specific ones.
Massive changes in RCA protein levels after elicitation were found in the proteomic analysis. Transcriptional analysis of induced tissues indicated that the Narca gene is down-regulated after herbivory, supporting our earlier large-scale transcriptional analysis (Hermsmeier et al., 2001
We conducted a proteomic analysis of a natural plant-herbivore interaction for which previous research had characterized the host plant's transcriptional responses and the larval elicitors that activate them. From this analysis of 90 herbivore-elicited protein spots in over 84 2-DE gels, we not only developed the techniques for extracting, separating, and analyzing complex protein mixtures reproducibly, but we also characterized proteomic responses to elicitation and learned that the larval elicitors responsible for the transcriptional response account for a majority of the specific changes in the proteome. Previous transcriptional analyses of this particular interaction, as well as of other plant-herbivore interactions, can be broadly seen as representing a shift in metabolism from growth- to defense-related processes (Walling, 2000 ; Reymond et al., 2004; Bostock, 2005; Leitner et al., 2005; Schmidt et al., 2005; Thompson and Goggin, 2006). We organize the discussion into five sections: the change in proteins associated with (1) photosynthesis; (2) primary and (3) secondary metabolism as these proteins relate to supporting known herbivore-elicited defense responses; (4) the kinetics and elicitors of the transcripts for a subset of 17 elicited proteins; and (5) the signals from the feeding larvae responsible for changes in the proteome.
We identified seven different functional photosynthesis-related proteins (from the analysis of 20 protein spots), which accumulate differentially in herbivore-induced leaf tissues. We excluded RuBPCase from the analysis because its transcripts are known to degrade in response to herbivore and pathogen attack (Hermsmeier et al., 2001
RCA belongs to the AAA+ protein super family, whose members generally function as chaperonins, and uses the Sensor-2 domain to recognize its substrate (Vargas-Suarez et al., 2004
RCA modulates the activity of RuBPCase, a key regulatory enzyme of photosynthetic carbon assimilation, by facilitating the removal of sugar phosphates (ribulose bisphosphate) that prevent substrate binding and carbamylation of the protein's active site. The VIGS experiment demonstrated that reducing RCA protein and transcript levels, as occurs in OS-elicited plants, reduced net photosynthetic rates of N. attenuata plants, which in turn was reflected in the reduced biomass of these plants. The role of RCA in regulating the activity of RuBPCase (Portis, 1995
In addition to RCA, five other photosynthetic proteins were found to be down-regulated during all harvests after elicitation: three oxygen-evolving enhancer proteins and two oxygen-evolving complex proteins. In contrast, PSI chain II D2 precursors and S2 hydroxy acid oxidase proteins increased after OS elicitation. These results confirm our earlier transcription analysis of N. attenuata-M. sexta interaction, which revealed that most photosynthesis-related genes are down-regulated after herbivore attack (Halitschke et al., 2001
Of the 28 primary metabolism proteins that were differentially regulated, eight are involved in amino acid metabolism and were up-regulated in response to OS elicitation: TD, S-adenosylmethionine synthetase, O-acetylserine thiol lyase, spermidine synthase, Gln synthetase, 3-dehydroquinate dehydratase, homo-Cys-S-methyltransferase, and hydroxymethyltransferase. Chen et al. (2005)
A protein spot identified as an ACS had a type III response. Several acs genes reportedly played diverse roles in plant metabolism (Hayashi et al., 2002
We found proteins similar to TK and plastidic ALD to be differentially regulated by OS elicitation. High levels of accumulation of the TK protein after OS elicitation might provide substrates for the synthesis of phenolic-based defenses. Proteomic analysis of germinating maize embryos infected by Fusarium verticilliodes showed the increased accumulation of ALD proteins (Campo et al., 2004
Seven out of eight proteins involved in the production of secondary metabolites showed increased accumulation levels at at least one time point after OS elicitation. Levels of a QR-like protein increased after OS elicitation. QRs are multisubunit enzymes that are involved in the generation of free radical semiquinones. Recently, QRs genes have been shown to be up-regulated in the epidermis during powdery mildew infection to wheat (Triticum aestivum; Greenshields et al., 2005
A protein spot showing similarity to spermidine synthase is up-regulated from 6 to 72 h after OS elicitation. Spermidine synthase is involved in polyamine metabolism and thereby the production of secondary metabolite defense compounds (Franceschetti et al., 2004
It is evident from several studies that the proteomic analysis of a plant's response does not necessarily match the transcriptional analysis of the same response. One of the major constraints to linking protein accumulation with mRNA in this study is the presence of several isoforms and/or subunits of certain proteins, especially when candidate proteins belong to a multigene family. The difficulty of differentiating between isoforms when using the RT-PCR approach remains a limitation for comparing protein and transcript accumulation. In the well-studied eukaryotic system of yeast, similar mRNA expression levels were accompanied by a wide range of protein abundance levels and vice versa (Gygi et al., 1999
The patterns of differential protein accumulation as well as the identity of the elicited proteins provide insights into the nature of the larval elicitors that are involved in regulating plant proteomes. Previous research using a detailed microarray and secondary metabolite analysis demonstrated that the two most abundant FACs in M. sexta OS can account for all measured direct (TPI and nicotine) and indirect (cis-
FACs are plant defense elicitors that are synthesized in the larval midgut and elicit defense responses in the plant that appear to benefit the plant. However, other elicitors are produced by the plant during caterpillar attack and some of these may benefit the caterpillar. Recent work from our group has shown that treatment of wounds with OS and larval attack dramatically increases methanol (MeOH) emissions from attacked plants. This MeOH release is elicited not by the FACs in OS but, rather, by the high pH of OS (pH 9.3), and is associated with increases in transcripts and activity of leaf PME and decreases in the degree of pectin methylation (von Dahl et al., 2006 In conclusion, FACs play a major role in organizing not only transcriptional responses but also proteomic responses. This dual role is not simply a result of an overlap of the transcriptional and proteomic responses to FACs because the responses of the transcriptome and proteome are clearly very different.
Plant Material and Preparation of Elicitors
A 17th-generation inbred line of Nicotiana attenuata (ecotype Utah), originally collected from a natural population in Washington County, Utah, was used for all experiments. Seed germination and plant growth were conducted as described earlier (Halitschke et al., 2003
The FAC solution was prepared as described earlier (Halitschke et al., 2001
Source leaves +1, +2, and +3 of rosette-stage plants of N. attenuata (Fig. 1) were punctured parallel to the central leaf vein with a fabric pattern wheel six times at 30-min intervals. Induction treatments were started between 9 and 10 AM and performed every 30 min for 3 h. Around 20 µL of 1:1 water-diluted OS or OS-FAC-free or FACs (in 0.0025% Triton X-100) or sterile water or 0.0025% Triton X-100 were applied to the punctured leaves (Halitschke et al., 2001
Total protein was extracted from elicited and control leaves with a modified phenolic extraction procedure (Schuster and Davies, 1983
Proteins of interest were processed on 96-well microtiter plates with an Ettan TA Digester running the Digester Version 1.10 software (both GE Healthcare Bio-Sciences AB) using the following protocol. The excised gel plugs were washed four times with 70 µL of acetonitrile/50 mM ammonium bicarbonate for 20 min each. The second wash was performed twice with 70 µL of 70% acetonitrile for 20 min and the gel plugs were air-dried for 1 h. Trypsin digestion was carried out overnight with 50 ng of trypsin (Porcine trypsin; Promega) in 15 µL of 50 mM ammonium bicarbonate at 37°C. The 15-µL solution from the preceding step was mixed with 25 µL of extraction buffer (50% acetonitrile and 0.1% trifluoroacetic acid), incubated for 20 min, and transferred to a 96-well plate. The gel plug was then incubated with 40 µL of extraction buffer for 20 min and transferred to the plate. The 60-µL solution containing the peptide mixture was then vacuum-dried for 1 h.
Dry peptides were dissolved in 15 µL of aqueous 0.1% trifluoroacetic acid. One µL aliquot was mixed with 1 µL of
The peptides, redissolved in aqueous 0.1% trifluoroacetic acid, were subsequently used for nanoLC-MS/MS analysis. The peptides were separated on a CapLC XE nanoLC system (Waters). A mobile phase flow of 0.1% aqueous formic acid (20 µL/min for 5 min) was used to concentrate and desalt the samples on a 5- x 0.35-mm Symmetry-300 C18 precolumn with 5-µm particle size. The samples were eluted and separated on a 150-mm x 75-µm NanoEase Atlantis C18 column, particle size 3 µm, using an increasing acetonitrile gradient (in 0.1% aqueous formic acid). Phases A (5% MeCN in 0.1% formic acid) and B (95% MeCN in 0.1% formic acid) were linearly mixed using a gradient program set to 5% phase B in A for 5 min, increased to 40% B in 25 min, and to 60% A in 10 min, and finally increased to 95% B for 4 min. The peptides were directly transferred to the NanoElectroSpray source of a Q-TOF Ultima tandem mass spectrometer through a Teflon capillary union and a metal-coated nanoelectrospray tip (Picotip; 50 x 0.36 mm; 10 µm i.d.; Waters). The source temperature was set to 40°C, cone gas flow 50 L/h, and the nanoelectrospray voltage was 1.6 kV. The TOF analyzer was used in reflectron mode. The MS/MS spectra were collected at 0.9-s intervals in the range of 50 to 1,700 m/z. A mixture of 100 fmol/µL human Glu-Fibrinopeptide B and 80 fmol/µL reserpine in 0.1% formic acid/acetonitrile (1:1 v/v) was infused at a flow rate of 0.9 µL/min through the reference NanoLockSpray source every fifth scan to compensate for mass shifts in the MS and MS/MS fragmentation mode due to temperature fluctuations. The data were collected by MassLynx Version 4.0 software. ProteinLynx Global Server Browser Version 2.1.5 (RC7) software (both from Waters) was used for further data processing (deconvolution, baseline subtraction, smoothing), de novo sequence identification, and database searches. The MS/MS data were searched against the NCBI Green Plant Version 1.0 database with the following parameters: peptide tolerance of 20 ppm, fragment tolerance of 0.05 D, estimated calibration error of 0.005 D, one missed cleavage, carbamidomethylation of cysteins, and the possible oxidation of Met. The amino acid sequences of peptides that did not provide conclusive results from the database searches were searched using MS-BLAST Internet (http://www.dove.embl-heidelberg.de) and an in-house-based search engine. Consolidated database (sp_nrdb) containing all nonredundant protein databases (Swiss-Prot, Swiss-ProtNew, SptremblNew, Sptrembl) and PAM30MS matrix with default settings were used for searches (Shevchenko et al., 2001
The total RNA was extracted from local leaves as described in the TRI reagent protocol (Sigma). From all the samples, 1 µg of total RNA was converted to cDNA using a Superscript first-strand synthesis system for RT-PCR according to the manufacturer's instructions (Invitrogen). Primers were designed from the peptide sequences obtained after LC-MS/MS analysis. Respective sites of the peptides were mapped on nucleotide regions for the respective database entries. Closely related species of N. attenuata were used for primer design when the sequence information of particular genes was unavailable. The primer design scheme is provided in Supplemental Table S1. The location of the peptides within the protein sequence is provided in Supplemental Figure S5. Two concentrations of cDNA (viz. 1x, 5x or 50x, 250x dilutions of original cDNA derived from 1 µg of RNA) derived from control and induced leaves were used as a template to amplify the respective cDNA fragment with the above primer combinations. PCR conditions were optimized for each primer set. PCR was carried out after denaturing cDNAs at 94°C for 5 min and then 30 cycles of 94°C for 60 s, annealing temperature (see Supplemental Table S1) for 30 s, and extension at 72°C for 60 s. The final extension step in PCR was at 72°C for 10 min. The amplified cDNA fragments were purified from agarose gel using GFX gel elution kit (Amersham) and cloned into PGMT-Easy vector (Promega). Cloned fragments were sequenced and confirmed with the NCBI database and novel sequences were deposited in the database.
The previously reported N. attenuata 268-nucleotide rca sequence (BU 494545) was used to make the Tobacco rattle virus-based construct for VIGS as described earlier (Saedler and Baldwin, 2004
Five-week-old plants were used for photosynthesis and nitrate measurements. Net photosynthetic rates and intercellular CO2 concentrations were measured on empty vector and Narca VIGS-silenced plants under saturating light (approximately 1,400 µM m–2 s–1) using a LI-COR 6400 portable photosynthesis system. Net photosynthesis was measured in at least five replicate plants each at six different CO2 concentrations, namely, 800, 600, 400, 200, 100, and 50 µmol/mol. Nitrate was measured spectrophotometrically according to the protocol described by Cataldo et al. (1975)
New sequences of putative partial genes of N. attenuata cloned and characterized in this study were deposited in the public database NCBI. Accession numbers allotted to these genes are as follows (in parentheses): N. attenuata (Na) ATPase, Naatpe (DQ682456); chaperonin, Nacpn (DQ682457); Gln synthase, Nagmps (DQ682458); glyceraldehydes-3-P dehydrogenase, Nagpdh (DQ682459); Gly-rich RNA-binding protein, Nagrp (DQ682460); Mg-protoporphyrin IX chelatase, NaMgpc (DQ682461); oxygen-evolving protein, Naoep (DQ682462); RCA, Narca (DQ682463); S-adenosylmethionine synthetase, Nasmas (DQ682464); translation elongation factor, Natef (DQ682465); PGM, Napgam (DQ682466); ALD, Naald (DQ682467); TK, Natk (DQ682468); mitochondrial formate dehydrogenase, Nafdh (DQ885565); and PME, Napme (DQ885566).
The following materials are available in the online version of this article.
We thank Klaus Gase, Thomas Hahn, and Albrecht Berg for DNA sequencing, providing chemically synthesized FACs, and HPLC analysis; Tamara Krügel and Andreas Weber for growing the plants; and Emily Wheeler for editorial assistance. We also thank Markus Hartl for critically reading the manuscript and Nan Qu for assistance in the preparation of the nuclear extracts and for helpful discussions. Received August 24, 2006; accepted September 27, 2006; published October 6, 2006.
1 This work was supported by the Max Planck Society. A.P.G. acknowledges the Alexander von Humboldt Foundation, Bonn, for a research fellowship. 
2 These authors contributed equally to the paper.
3 Present address: Institute of Genomic Biology, University of Illinois, Urbana, IL 61801. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ian T. Baldwin (baldwin{at}ice.mpg.de).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.088781 * Corresponding author; e-mail baldwin{at}ice.mpg.de; fax 49–(0)3641–571102.
Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422: 198–207[CrossRef][Medline] Anderson JP, Thatcher LF, Singh KB (2005) Plant defence responses: conservation between models and crops. Funct Plant Biol 32: 21–34[CrossRef] Baldwin IT (2001) An ecologically motivated analysis of plant-herbivore interactions in native tobacco. Plant Physiol 127: 1449–1458 Bertone P, Snyder M (2005) Prospects and challenges in proteomics. Plant Physiol 138: 560–562 Bischoff M, Schaller A, Bieri F, Kessler F, Amrhein N, Schmid J (2001) Molecular characterization of tomato 3-dehydroquinate dehydratase-shikimate:NADP oxidoreductase. Plant Physiol 125: 1891–1900 Bostock RM (2005) Signal crosstalk and induced resistance: straddling the line between cost and benefit. Annu Rev Phytopathol 43: 545–580[CrossRef][Web of Science][Medline] Bourgis F, Botha FC, Mani S, Hiten FN, Rigden DJ, Verbruggen N (2005) Characterization and functional investigation of an Arabidopsis cDNA encoding a homologue to the d-PGMase superfamily. J Exp Bot 56: 1129–1142 Campo S, Carrascal M, Coca M, Abian J, San Segundo B (2004) The defense response of germinating maize embryos against fungal infection: a proteomics approach. Proteomics 4: 383–396[CrossRef][Web of Science][Medline] Cataldo DA, Haroon M, Schrader LE, Youngs VL (1975) Rapid colorimetric determination of nitrate in plant-tissue by nitration of salicylic-acid. Commun Soil Sci Plant Anal 6: 71–80[CrossRef][Web of Science] Chen H, Wilkerson CG, Kuchar JA, Phinney BS, Howe GA (2005) Jasmonate-inducible plant enzymes degrade essential amino acids in the herbivore midgut. Proc Natl Acad Sci USA 102: 19237–19242 Chittoor JM, Leach JE, White FF (1997) Differential induction of a peroxidase gene family during infection of rice by Xanthomonas oryzae pv. oryzae. Mol Plant Microbe Interact 10: 861–871[Web of Science][Medline] Chivasa S, Simon WJ, Yu XL, Yalpani N, Slabas AR (2005) Pathogen elicitor-induced changes in the maize extracellular matrix proteome. Proteomics 5: 4894–4904[CrossRef][Web of Science][Medline] Franceschetti M, Fornale S, Tassoni A, Zuccherelli K, Mayer MJ, Bagni N (2004) Effects of spermidine synthase overexpression on polyamine biosynthetic pathway in tobacco plants. J Plant Physiol 161: 989–1001[CrossRef][Web of Science][Medline] Futcher B, Latter GI, Monardo P, McLaughlin CS, Garrels JI (1999) A sampling of the yeast proteome. Mol Cell Biol 19: 7357–7368 Glinski M, Weckwerth W (2006) The role of mass spectrometry in plant systems biology. Mass Spectrom Rev 25: 173–214[CrossRef][Web of Science][Medline] Greenshields DL, Liu GS, Selvaraj G, Wei YD (2005) Differential regulation of wheat quinone reductases in response to powdery mildew infection. Planta 222: 867–875[CrossRef][Web of Science][Medline] Gygi SP, Rochon Y, Franza BR, Aebersold R (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 19: 1720–1730 Hahlbrock K, Bednarek P, Ciolkowski I, Hamberger B, Heise A, Liedgens H, Logemann E, Nurnberger T, Schmelzer E, Somssich IE, et al (2003) Non-self recognition, transcriptional reprogramming, and secondary metabolite accumulation during plant/pathogen interactions. Proc Natl Acad Sci USA 100: 14569–14576 Halitschke R, Gase K, Hui DQ, Schmidt DD, Baldwin IT (2003) Molecular interactions between the specialist herbivore Manduca 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 conjugates. Plant Physiol 131: 1894–1902 Halitschke R, Schittko U, Pohnert G, Boland W, Baldwin IT (2001) 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. Plant Physiol 125: 711–717 Hancock JT, Henson D, Nyirenda M, Desikan R, Harrison J, Lewis M, Hughes J, Neill SJ (2005) Proteomic identification of glyceraldehyde 3-phosphate dehydrogenase as an inhibitory target of hydrogen peroxide in Arabidopsis. Plant Physiol Biochem 43: 828–835[CrossRef][Web of Science][Medline] Hayashi H, De Bellis L, Hayashi Y, Nito K, Kato A, Hayashi M, Hara-Nishimura I, Nishimura M (2002) Molecular characterization of an Arabidopsis acyl-coenzyme A synthetase localized on glyoxysomal membranes. Plant Physiol 130: 2019–2026 Hermsmeier D, Schittko U, Baldwin IT (2001) 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. Plant Physiol 125: 683–700 Hui DQ, Iqbal J, Lehmann K, Gase K, Saluz HP, Baldwin IT (2003) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. V. Microarray analysis and further characterization of large-scale changes in herbivore-induced mRNAs. Plant Physiol 131: 1877–1893 Jorgensen R, Merrill AR, Andersen GR (2006) The life and death of translation elongation factor 2. Biochem Soc Trans 34: 1–6[CrossRef][Web of Science][Medline] Kang J, Wang L, Giri AP, Baldwin IT (2006) Silencing threonine deaminase and the JAR1 homologue in Nicotiana attenuata impairs JA-isoleucine-mediated defenses against the specialist herbivore, Manduca sexta. Plant Cell (in press) Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular analysis. Annu Rev Plant Biol 53: 299–328[CrossRef][Medline] Kiddle G, Pastori GM, Bernard S, Pignocchi C, Antoniw J, Verrier PJ, Foyer CH (2003) Effects of leaf ascorbate content on defense and photosynthesis gene expression in Arabidopsis thaliana. Antioxid Redox Signal 5: 23–32[CrossRef][Web of Science][Medline] Kim ST, Kim SG, Hwang DH, Kang SY, Kim HJ, Lee BH, Lee JJ, Kang KY (2004) Proteomic analysis of pathogen-responsive proteins from rice leaves induced by rice blast fungus, Magnaporthe grisea. Proteomics 4: 3569–3578[CrossRef][Web of Science][Medline] Kirch HH, Schlingensiepen S, Kotchoni S, Sunkar R, Bartels D (2005) Detailed expression analysis of selected genes of the aldehyde dehydrogenase (ALDH) gene superfamily in Arabidopsis thaliana. Plant Mol Biol 57: 315–332[CrossRef][Web of Science][Medline] Koo AJK, Chung HS, Kobayashi Y, Howe GA (2006) Identification of a peroxisomal acyl-activating enzyme involved in the biosynthesis of jasmonic acid in Arabidopsis. J Biol Chem 281: 33511–33520 Lange BM, Ghassemian M (2005) Comprehensive post-genomic data analysis approaches integrating biochemical pathway maps. Phytochemistry 66: 413–451[CrossRef][Web of Science][Medline] Leitner M, Boland W, Mithofer A (2005) Direct and indirect defences induced by piercing-sucking and chewing herbivores in Medicago truncatula. New Phytol 167: 597–606[CrossRef][Web of Science][Medline] Li ZY, Zhang JS, Chen SY (1999) Molecular clotting, expression analysis and chromosomal mapping of salt-responsive cDNAs in rice (Oryza sativa L.). Sci China C Life Sci 42: 506–516[Medline] Lindermayr C, Saalbach G, Durner J (2005) Proteomic identification of S-nitrosylated proteins in Arabidopsis. Plant Physiol 137: 921–930 Manter DK, Kerrigan J (2004) A/Ci curve analysis across a range of woody plant species: influence of regression analysis parameters and mesophyll conductance. J Exp Bot 55: 2581–2588 Matt P, Krapp A, Haake V, Mock HP, Stitt M (2002) Decreased Rubisco activity leads to dramatic changes of nitrate metabolism, amino acid metabolism and the levels of phenylpropanoids and nicotine in tobacco antisense RBCS transformants. Plant J 30: 663–677[CrossRef][Web of Science][Medline] Mazarei M, Lennon KA, Puthoff DP, Rodermel SR, Baum TJ (2003) Expression of an Arabidopsis phosphoglycerate mutase homologue is localized to apical meristems, regulated by hormones, and induced by sedentary plant-parasitic nematodes. Plant Mol Biol 53: 513–530[CrossRef][Web of Science][Medline] Peck SC (2005) Update on proteomics in Arabidopsis. Where do we go from here? Plant Physiol 138: 591–599 Portis AR (1995) The regulation of Rubisco by Rubisco activase. J Exp Bot 46: 1285–1291[Abstract] Ralph S, Oddy C, Cooper D, Yueh H, Jancsik S, Kolosova N, Philippe RN, Aeschliman D, White R, Huber D, et al (2006a) Genomics of hybrid poplar (Populus trichocarpa x deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full-length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of insect-induced defences in poplar. Mol Ecol 15: 1275–1297[CrossRef][Medline] Ralph SG, Yueh H, Friedmann M, Aeschliman D, Zeznik JA, Nelson CC, Butterfield YSN, Kirkpatrick R, Liu J, Jones SJM, et al (2006b) Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome. Plant Cell Environ 29: 1545–1570[CrossRef][Medline] Rayapuram C, Baldwin IT (2006) Using nutritional indices to study LOX3-dependent insect resistance. Plant Cell Environ 29: 1585–1594[CrossRef][Medline] Reymond P, Bodenhausen N, Van Poecke RMP, Krishnamurthy V, Dicke M, Farmer EE (2004) A conserved transcript pattern in response to a specialist and a generalist herbivore. Plant Cell 16: 3132–3147 Reymond P, Weber H, Damond M, Farmer EE (2000) Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell 12: 707–719 Roda A, Halitschke R, Steppuhn A, Baldwin IT (2004) Individual variability in herbivore-specific elicitors from the plant's perspective. Mol Ecol 13: 2421–2433[CrossRef][Medline] Saedler R, Baldwin IT (2004) Virus-induced gene silencing of jasmonate-induced direct defences, nicotine and trypsin proteinase-inhibitors in Nicotiana attenuata. J Exp Bot 55: 151–157 Saravanan RS, Rose JKC (2004) A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4: 2522–2532[CrossRef][Web of Science][Medline] Schittko U, Hermsmeier D, Baldwin IT (2001) 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. Plant Physiol 125: 701–710 Schmidt DD, Voelckel C, Hartl M, Schmidt S, Baldwin IT (2005) Specificity in ecological interactions. Attack from the same lepidopteran herbivore results in species-specific transcriptional responses in two solanaceous host plants. Plant Physiol 138: 1763–1773 Schnurr J, Shockey J, Browse J (2004) The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis. Plant Cell 16: 629–642 Schuster AM, Davies E (1983) Ribonucleic acid and protein metabolism in pea epicotyls. I. The aging process. Plant Physiol 73: 809–816 Shevchenko A, Sunyaev S, Loboda A, Shevehenko A, Bork P, Ens W, Standing KG (2001) Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time of flight mass spectrometry and BLAST homology searching. Anal Chem 73: 1917–1926[Medline] Stenzel O, Teuber M, Drager B (2006) Putrescine N-methyltransferase in Solanum tuberosum L., a calystegine-forming plant. Planta 223: 200–212[CrossRef][Web of Science][Medline] Steppuhn A, Gase K, Krock B, Halitschke R, Baldwin IT (2004) Nicotine's defensive function in nature. PLoS Biol 2: 1074–1080[Web of Science] Thompson GA, Goggin FL (2006) Transcriptomics and functional genomics of plant defence induction by phloem-feeding insects. J Exp Bot 57: 755–766 Urao T, Yamaguchi-Shinozaki K, Shinozaki K (2000) Two-component systems in plant signal transduction. Trends Plant Sci 5: 67–74[CrossRef][Web of Science][Medline] Vargas-Suarez M, Ayala-Ochoa A, Lozano-Franco J, Garcia-Torres I, Diaz-Quinonez A, Ortiz-Navarrete VF, Sanchez-de-Jimenez E (2004) Rubisco activase chaperone activity is regulated by a post-translational mechanism in maize leaves. J Exp Bot 55: 2533–2539 von Dahl CC, Havecker M, Schlogl R, Baldwin IT (2006) Caterpillar-elicited methanol emission: a new signal in plant-herbivore interactions? Plant J 46: 948–960[CrossRef][Web of Science][Medline] Walling LL (2000) The myriad plant responses to herbivores. J Plant Growth Regul 19: 195–216[Medline] Yamada S, Komori T, Hashimoto A, Kuwata S, Imaseki H, Kubo T (2000) Differential expression of plastidic aldolase genes in Nicotiana plants under salt stress. Plant Sci 154: 61–69[Medline] Yan S-P, Zhang Q-Y, Tang Z-C, Su W-A, Sun W-N (2006) Comparative proteomic analysis provides new insights into chilling stress responses in rice. Mol Cell Proteomics 5: 484–496 Zangerl AR, Hamilton JG, Miller TJ, Crofts AR, Oxborough K, Berenbaum MR, de Lucia EH (2002) Impact of folivory on photosynthesis is greater than the sum of its holes. Proc Natl Acad Sci USA 99: 1088–1091 Zavala JA, Patankar AG, Gase K, Hui D, Baldwin IT (2004) Manipulation of endogenous trypsin proteinase inhibitor production in Nicotiana attenuata demonstrates their function as antiherbivore defenses. Plant Physiol 134: 1181–1190 This article has been cited by other articles:
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-bergamotene) defenses, the endogenous jasmonic acid burst that elicits them, and 65% to 86% of the induced transcriptional changes elicited by attack on N. attenuata leaves (Halitschke et al., 2001
