This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 138/3/1763    most recent pp.105.061192v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (11) Citing Articles via Google Scholar Google Scholar Articles by Schmidt, D. D. Articles by Baldwin, I. T. Search for Related Content PubMed PubMed Citation Articles by Schmidt, D. D. Articles by Baldwin, I. T. Agricola Articles by Schmidt, D. D. Articles by Baldwin, I. T.

PLANTS INTERACTING WITH OTHER ORGANISMS

Specificity in Ecological Interactions. Attack from the Same Lepidopteran Herbivore Results in Species-Specific Transcriptional Responses in Two Solanaceous Host Plants^1,[w]

Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Beutenberg Campus, 07745 Jena, Germany

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Model systems have proven enormously useful in elucidating the biochemical function of plant genes. However their ecological function, having been sculpted by evolutionary forces specific to a species, may be less conserved across taxa. Responses to wounding and herbivore attack differ among plant families and are known to be mediated by oxylipin, ethylene, and systemin-signaling networks. We analyzed transcriptional responses of two native Solanaceous species to the attack of an herbivore whose elicitors are known not to be influenced by diet. With The Institute for Genomic Research 10k-cDNA potato (Solanum tuberosum) microarray, we compared the transcriptional responses of Nicotiana attenuata with those of black nightshade (Solanum nigrum) when both were attacked by the Solanaceous generalist herbivore, Manduca sexta. Based on an NADH dehydrogenase subunit F phylogeny, S. nigrum is more closely related to potato than N. attenuata but responded significantly less to M. sexta attack. Apart from transcriptional differences anticipated from their differences in secondary metabolism, both species showed distinct transcriptional patterns (with only 10% overlap in significantly regulated genes), which point to fundamental differences in the signaling cascades and downstream genes mediating herbivore resistance. The lackluster transcriptional response of S. nigrum could not be attributed to its inability to respond to elicitation, because methyl jasmonate elicitation of S. nigrum resulted in a strong transcriptional response. Given that attack from the same herbivore elicits profoundly different responses in two Solanaceaous taxa, we conclude that blueprints for commonly regulated responses to plant-herbivore interactions appear unlikely.

Three plant hormones, jasmonic acid (JA), ethylene (ET), and salicylic acid, mediate responses to wounding and attack from herbivores and pathogens in most taxa studied, but research on crosstalk among these signals has identified important differences at the family level. For example in Arabidopsis, wounding leads to the activation of two pathways: an oligosaccharide-dependent and a JA-dependent pathway in the damaged leaves (Walling, 2000; Leon et al., 2001). The former produces ET, which antagonizes the elicitation of JA-responsive genes in the wounded leaves but not in unwounded leaves from the same plant. In contrast, both ET and JA are required for the elicitation of wound-inducible genes in tomato (Walling, 2000; Leon et al., 2001). In addition to differences in crosstalk among the same signal molecules, Brassicaceous and Solanaceous plants differ in the signal molecules that are recruited. Systemin, a polypeptide hormone, which mediates local and systemic responses to wounding, has been found only in Solanaceous species to date (Ryan, 2000). In addition to these family-level differences, differences have also been found between taxa within the same family.

Within-family differences have been observed in the crosstalk between JA and ET signaling in the elicitation of defense responses. While JA and ET interact synergistically to elicit proteinase inhibitor expression in tomato, attack from Manduca sexta larvae in Nicotiana attenuata elicits an ET burst, which antagonizes the JA-mediated increase in nicotine (Kahl et al., 2000; Winz and Baldwin, 2001). Within-family differences in signals have also been found. Systemins of potato (Solanum tuberosum), bell pepper (Capsicum annuum), and black nightshade (Solanum nigrum) are structurally similar and differ each in three (of 18) amino acids from the tomato systemin and their ability to elicit tomato proteinase inhibitors (Constabel et al., 1998). Two tobacco systemins that elicit the accumulation of proteinase inhibitors have been identified, but these are structurally dissimilar to the tomato systemin (Pearce et al., 2001). Tobacco does not produce peptide signals that activate the tomato systemin receptor and does not react to tomato systemin (Scheer et al., 2003).

Given this evidence for differences in defense signaling among several Solanaceous taxa, we were interested in a more thorough characterization of the potential differences in responses to herbivore attack within a plant family. For this analysis, we compared the herbivore-induced transcriptome of two native Solanaceous species, N. attenuata and S. nigrum, to attack from the same native herbivore, M. sexta larvae. Three aspects of this analysis make this a valuable comparison. First, we analyze the responses of two native plants in which the responses observed to herbivore attack are not confounded by a history of artificial selection for yield-associated traits. Second, we measure the responses to attack by a shared native herbivore (Fraenkel, 1959), which is particularly well-studied with regard to how it elicits responses in its host plants. M. sexta larvae produce a suite of eight fatty acid-amino acid conjugates (FAC), which are thought to be introduced into wounds during feeding and are necessary and sufficient to account for all of the observed changes in the plant's wound response, including defense metabolites, signals, and transcriptional responses, that are elicited by larval feeding (Halitschke et al., 2001, 2003; Roda et al., 2004). Simply adding these FACs to plant wounds simulates the plant's responses to herbivore attack. Moreover, M. sexta's FAC profile is not substantially altered when it feeds on different host plants (Alborn et al., 2003) and specifically does not quantitatively or qualitatively change when it feeds on N. attenuata or S. nigrum (D.D. Schmidt, A. Steppuhn, and R. Halitschke, unpublished data). Third, the secondary metabolites produced by both species are well studied and provide a valuable backdrop against which to compare their herbivore-elicited transcriptomes. N. attenuata and S. nigrum constitutively produce nicotine and glycoalkaloids (Baldwin, 1999; Dopke et al., 1987; Ridout et al., 1989), respectively, and both species are capable of producing proteinase inhibitors in response to wounding and release monoterpenoid and sesquiterpenoid volatiles in response to herbivore feeding (Van Dam et al., 2001; Kessler and Baldwin, 2001; Schmidt et al., 2004). In summary, for this pair of host plants, attack by M sexta larvae is likely to provide a standardized elicitation of the responses to herbivore attack, which, in turn, are likely sculpted by natural selection.

In order to provide an unbiased comparison of the herbivore-regulated transcriptome in both species, we used a microarray with over 10,000 potato cDNAs (representing approximately a third of the potato genome). We were particularly interested in the scope of the response (How many genes are involved?) and the specificity of the response (How many transcripts are commonly and specifically regulated?). The potato microarray was established through the National Science Foundation Potato Functional Genomics project (http://www.tigr.org/tdb/potato) and is available from The Institute of Genomic Research (TIGR, Rockville, MD). The scope and specificity of the response could be influenced by the taxonomic similarity between the source of the genes on the array (potato) and the samples used in the hybridizations (S. nigrum and N. attenuata). To clarify the phylogentic relationship among the three species, we analyzed sequence similarities among the plastidial NADH dehydrogenase subunit F (ndhF) gene of several Solanacaeous species. Based on this analysis, S. nigrum is more closely related to potato than N. attenuata, which generated the expectation that the array analysis would likely reveal a stronger response from S. nigrum than from N. attenuata. However, we found the opposite to be true: S. nigrum showed a weaker response to M. sexta than N. attenuata. To test the hypothesis whether S. nigrum is generally less responsive, we examined the transcriptional responses of S. nigrum after elicitation by methyl jasmonate (MeJA), the volatile derivative of JA. MeJA is known to be both a product and elicitor of the oxilipin signaling pathway, the signal cascade that mediates many defense responses to herbivore and pathogen attack (Walling, 2000).

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Phylogenetic Analysis

To clarify the phylogenetic relationship between N. attenuata and S. nigrum, we analyzed sequence similarities among the plastidial ndhF gene of several Solanaceous species. Based on this comparison, S. nigrum is more closely related to potato than N. attenuata, as demonstrated by a 97% homology between S. nigrum and potato and 94% between N. attenuata and potato (Fig. 1).

View larger version (18K): [in this window] [in a new window]   Figure 1. The single most parsimonious tree recovered using sequences of ndhF (length = 372, CI = 0.949, RI = 0.835). The two species of interest (N. attenuata and S. nigrum) are in bold as is the source species for the cDNA clones spotted onto the TIGR potato array. Branch lengths correspond to parsimony steps and the numbers above branches indicate parsimony bootstrap support values for that clade.

Three replicate TIGR chips were hybridized with RNA from thrice replicated M. sexta-infested N. attenuata plants and M. sexta-infested S. nigrum plants. Of 11,243 cDNAs, a total of 754 were regulated (mean ratio >1.5 or <0.67 for up- and down-regulated genes, respectively) in either N. attenuata, S. nigrum, or both species in response to M. sexta attack (Fig. 2

Figure 3. Categories of genes up-regulated in response to M. sexta herbivory with examples from both plant species (Both: clear boxes), N. attenuata (NA: darkly shaded boxes), and S. nigrum (SN: lightly shaded boxes; multiple clones for the same gene are indicated in parentheses).

). Interestingly, there were more genes up-regulated (561) than down-regulated (203) and only 75 cDNAs (10%) were equally regulated in both species (Fig. 2). When the expression ratios (ERs) of the 754 responsive cDNAs were subjected to a cluster analysis, the three N. attenuata arrays were clearly separated from the three S. nigrum arrays and patterns of commonly and specifically regulated genes were discernable (Supplemental Fig. 1). Based on the annotations of the 754 regulated cDNAs, we grouped the genes into functional categories (Supplemental Table I; Fig. 3) and, in the following, use this classification to discuss some striking differences in gene up-regulation that occur between these two Solanaceous species. For the details (ERs, annotations, categories) of gene down-regulation, see Supplemental Table I.

View larger version (21K): [in this window] [in a new window]   Figure 2. Venn diagram of the numbers of overlapping and non-overlapping significantly up-regulated (; ER > 1.5) or down-regulated (; ER < 0.67) genes in N. attenuata and S. nigrum that are elicited by M. sexta herbivory. In summary, 561 and 203 genes were found to be up- and down-regulated, respectively, but only 10% had the same regulation in both plant species. (S. nigrum from http://www.rmc.sierraclub.org/outings/images/weeds_blacknightshade_drawing.jpg.)

Proteolysis
Among the genes coding for proteolytic enzymes, a Leu aminopeptidase (LAP), which catalyzes the release of N-terminal residues from proteins and peptides, was up-regulated up to 20-fold in S. nigrum. In contrast, increases in ubiquitin-mediated proteolysis (polyubiquitin, ubiquitin-conjugating enzyme) are specific to N. attenuata. An up-regulation of protein disulfide isomerase and peptidylprolyl isomerase was found in both species, but the response of the latter was stronger in S. nigrum.

Secondary Metabolism
Little overlap between N. attenuata and S. nigrum was found in the expression of genes involved in secondary metabolism (Fig. 4), including up-regulation of hydroperoxide lyase (HPL), an important gene in the synthesis of C6 volatile organic compounds (VOCs), spermidine synthase, which is involved in polyamine synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR2), the key enzyme in the cytosolic route to the terpenoid precursor isopentenyl pyrophosphate (IPP). In S. nigrum, HMGR1 is also up-regulated, and it has been shown for potato that HMGR2 is mainly involved in the biosynthesis of sesquiterpenes, whereas HMGR1 produces precursors for sterol biosynthesis (Choi et al., 1994). Moreover, enzymes involved in the biosynthesis of sterols, such as cholesterol (7-dehydrocholesterol reductase), were up-regulated in S. nigrum. Sterol contents in Solanum species are tightly linked to levels of glycoalkaloids, and cholesterol is thought to be a precursor of steroidal alkaloids (Bergenstrahle et al., 1996), which in turn suggests increased production of glycoalkaloids in S. nigrum in response to M. sexta feeding. Further evidence for the deployment of alkaloidal defenses in S. nigrum is the up-regulation of a 2-oxoglutarate-dependent dioxygenase, which is homologous to hyoscyamine 6-hydroxylase of Hyoscyamus niger (Lantin et al., 1999). Up-regulated genes of terpenoid biosynthesis include farnesyl pyrophosphate synthase and several sesquiterpene synthases, which are among the most strongly up-regulated genes in S. nigrum. It is known that S. nigrum produces a rich bouquet of VOCs in response to herbivory from flea beetles or the moth Acherontia atropos (Schmidt et al., 2004).

View larger version (49K): [in this window] [in a new window]   Figure 4. Simplified overview of major pathways of secondary-metabolite biosynthesis and their interrelationships with primary metabolism (modified from Gershenzon, 2002). Genes found to be up-regulated are italicized and examples of Solanaceous secondary compounds are listed for some classes of secondary metabolites. Genes up-regulated in N. attenuata (#), S. nigrum (), or both (#,) are indicated. Abbreviations: 4CL, 4-coumarate coenzyme A ligase; 7DCR, 7-dehydrocholesterol reductase; ADC, Arg decarboxylase; C4H, cinnamate-4-hydrogenase; CCR, cinnamoyl-CoA reductase; CM, chorismate mutase; CS chorismate synthase; CS3'H, p-coumaroyl shikimate 3'-hydroxylase; DHFR, dihydroflavonol reductase; DHQS, 3-dehydroquinate synthase; DXR, 1-deoxy-D-xylulose-5-P reductoisomerase; DXS, 1-deoxy-D-xylulose-5-P synthase; EPSP, 5-enolpyruvylshikimate-3-P synthase; G3P, glycerol-3-P; HMGS, 3-hydroxy-3-methylglutaryl coenzyme A synthase; LDOX, leucanthocyanidin dioxygenase; LIS, linalool synthase; ODC, Orn decarboxylase; OPR, oxophytodienoate reductase; PAL, Phe ammonia lyase; PLC, phospholipase C; SabSYN, sabinene synthase; SAMDC, S-adenosyl Met decarboxylase; SAMS, S-adenosyl Met synthase; SHKK, shikimate kinase; SPDSYN, spermidine synthase; STS, sesquiterpene synthase(s).

Also in contrast to the response in S. nigrum, N. attenuata plants elicit a strong transcriptional commitment to the production of phenol-based secondary compounds. Starting with the synthesis of shikimate (dehydroquinate synthase, shikimate kinase, 3-phosphoshikimate 1-carboxyvinyltransferase, chorismate synthase), proceeding to the synthesis of prephenate (the committed step in Phe and Tyr synthesis catalyzed by chorismate mutase), continuing with the synthesis of cinnamic acid (Phe ammonia lyase), p-coumaric acid (cinnamate-4-hydroxylase), and p-coumaroyl-CoA (coumaric acid-CoA ligase), the genes providing the precursors for flavonoid and phenylpropanoid biosynthesis are induced. Transcripts related to flavonoid metabolism (UDP rhamnose-anthocyanidin-3-glucoside rhamnosyltransferase, dihydroflavonol reductase) and phenylpropanoid metabolism (p-coumaroyl shikimate 3'-hydroxylases, cinnamoyl-CoA reductase) were up-regulated as well. These results are consistent with the following earlier findings in the M. sexta-N. attenuata interaction: (1) The cloning of a UDP rhamnose-anthocyanidin-3-glucoside rhamnosyltransferase by differential display reverse transcription-PCR from M. sexta-induced N. attenuata plants (Voelckel and Baldwin, 2003), the production of phenylpropanoid-derived compounds (caffeoyl-putrescine, chlorogenic acid) in M. sexta-attacked N. attenuata plants (Kessler and Baldwin, 2004), and the production of flavonoids (quercetin, rutin) in N. attenuata leaf trichomes (Roda et al., 2003). Several transcripts for polyphenyl oxidases, which catalyze the production of o-quinones, which, in turn react with insect dietary proteins and impair their digestion (e.g. Constabel et al., 2000), increase in abundance specifically in M. sexta-attacked N. attenuata.

Proteinase inhibitor (PI) induction, an induced-defense response well characterized in Manduca-attacked N. attenuata (Zavala et al., 2004), was seen only in S. nigrum, probably because of the lack of tobacco-specific probes on the array.

Primary Metabolism
Manduca attack up-regulated in both plant species genes involved in cell wall biosynthesis, such as pectin methyl esterase and extensin, genes related to lipid metabolism (acyl-CoA synthetase), and genes that play a central role in carbohydrate metabolism (e.g. glyceraldehyde 3-phosphate dehydrogenase). Interestingly, transcripts for acetyl-CoA carboxylase which catalyzes the formation of malonyl-CoA, an essential precursor of flavonoid biosynthesis, increased specifically in N. attenuata. Other precursors of secondary metabolism whose production is elicited in N. attenuata include acetyl-CoA, S-adenosylmethionine, and putrescine. Putrescine may be needed for nicotine and polyamine synthesis, and S-adenosylmethionine may be used in ET and polyamine synthesis. The up-regulation of two different isoforms of S-adenosylmethionine decarboxylase (secondary metabolism) suggests that a supply of decarboxlated S-adenosylmethionine is required to complement the supply of putrescine for higher polyamine synthesis. Additional evidence of tobacco-specific regulation in primary metabolism includes increases in xyloglucan endotransglycosylase (XTH), starch synthase, ATP citrate lyase, adenine phosphoribosyl transferase, and nucleotide sugar epimerase. S. nigrum up-regulates genes involved in cell wall biosynthesis (cellulose synthase), amino acid metabolism, and lipid synthesis. The most striking difference with the responses of primary metabolism in N. attenuata was the up-regulation of photosynthesis-related genes (e.g. Rubisco, light-harvesting complex b6) in caterpillar-infested S. nigrum plants. In general, herbivory is thought to down-regulate photosynthesis genes and, thereby, divert resources to secondary metabolism (e.g. Hermsmeier et al., 2001).

Stress Responses and Selected Genes
The stressed metabolic state of herbivore-attacked plants is reflected in the up-regulation of chaperones (HSP90, luminal binding protein, endoplasmin, etc.), dehydration induced proteins (with distinct genes elicited in both species, e.g. dehydrin in S. nigrum), and several cytochrome P450 monooxygenases. Among the most strongly up-regulated genes in both species were a putative acid phosphatase and the EEF53 gene. N. attenuata induces a vegetative storage protein reported to be herbivory induced in Arabidopsis (Berger et al., 2002), a salt stress-related phospholipid-hydroperoxide glutathione peroxidase (Mittova et al., 2002), a tobacco mosaic virus response-related gene, an iron-stress related protein, a superoxid dismutase, and a metal-transporting ATPase. A remarkably large number of chaperones are induced only in S. nigrum. Further stress-related responses in S. nigrum include the up-regulation of enzymes involved in oxidative stress, such as an herbivory-induced NADPH oxidase subunit (whitefly-induced gp91-phox), a peroxidase, and a glutathione transferase.

Verification of Microarray Data by TaqMan Real-Time PCR
The results of the quantitative PCR analysis for the genes -DOX, HPL, lipoxygenase (LOX), and XTH confirmed the ERs obtained with microarrays in N. attenuata (Supplemental Table II). The relative ERs of -DOX, LOX, and XTH were significantly higher (approximately 2-fold) than the microarray ERs. Hence, the microarray data seem to even underestimate the magnitude of the gene expression differences.

Microarray Analysis of MeJA-Induced Responses of S. nigrum

The weaker response of S. nigrum to M. sexta herbivory in comparison to the response of N. attenuata raised the question whether S. nigrum was a nonresponding species. To test this hypothesis, we hybridized three additional microarrays with RNA from three biological replicates of MeJA-treated plants. Of 11,243 analyzed clones on the microarray, 339 were differentially regulated (263 > 1.5; 76 < 0.67) in S. nigrum in response to MeJA elicitation (about 3% of the total number of spotted clones; Supplemental Table III). In accordance with the ubiquitous role of oxilipins (JA, MeJA) in mediating plants' responses to herbivory, 72 genes are commonly up-regulated in S. nigrum in response to MeJA or to M. sexta (Fig. 5). Again LAP was among the highest up-regulated genes in MeJA-elicited plants and further up-regulated genes are involved in oxylipin biosynthesis (LOX), secondary metabolism (HMGR, farnesyl pyrophosphate synthase, PIs), synthesis of chaperones (luminal binding protein), to name just a few (Supplemental Table III). A majority of the up-regulated genes (191) were up-regulated only in MeJA-elicited plants clearly demonstrating that S. nigrum is fully capable of responding to elicitation. These genes included genes involved in secondary metabolism (e.g. shikimate 5-dehydrogenase), transcription factor genes (e.g. C2H2 zinc finger protein), genes related to ubiquitin-mediated proteolysis (clone nos. STMDG12, STMHH55), and genes coding for several cytochrome P450s. A subset of 33 genes were commonly up-regulated in MeJA-elicited S. nigrum and M. sexta-infested N. attenuata plants (Fig. 5B) and were dominated by genes involved in primary metabolism (carbohydrates) and secondary metabolism (phenylpropanoids, terpenoids). A similar-sized (33) subset of genes were commonly up-regulated among all three treatments and were dominated by genes related to oxylipin signaling and chaperones.

View larger version (30K): [in this window] [in a new window]   Figure 5. Transcriptional responses of S. nigrum to MeJA and M. sexta. A, Venn diagram of the numbers of overlapping and nonoverlapping significantly up-regulated (; ER > 1.5) or down-regulated (; ER < 0.67) genes in S. nigrum that are elicited by MeJA treatment or M. sexta herbivory. B, Overlapping up-regulated genes of M. sexta-infested N. attenuata and MeJA-elicited S. nigrum. S. nigrum's weaker response to M. sexta is not due to a basic unresponsiveness, because MeJA induced the expression of many genes that are up-regulated in N. attenuata in response to M. sexta herbivory, such as genes related to carbohydrate or secondary metabolism (multiple clones for the same gene are indicated in parentheses).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The TIGR array analysis confirmed previously measured increases in the expression of jasmonate cascade genes and of genes known to be positively regulated by jasmonate signaling, such as genes involved in green leaf volatile, polyamine, or phenylpropanoid synthesis. Additionally, the TIGR array analysis found many more genes representing the above-mentioned branches of metabolism, including p-coumaroyl shikimate 3'-hydroxylase and cinnamoyl-CoA reductase (phenylpropanoids and their conjugates, Gang et al., 2002), S-adenosyl Met (SAM) synthetase and spermidine synthase (polyamines), Arg and Orn decarboxylase (precursors for polyamines and nicotine), and several polyphenol oxidases to be induced. The TIGR array analysis also revealed the activation of almost every gene in the shikimate pathway, the plastidic route to terpenoid synthesis and additional cell wall related genes (pectin methyl esterase, extensin), to name just a few of the genes not monitored with the oligonucleotide array. Disagreements between the two analyses were mainly due to four reasons: (1) Some genes were not present on the TIGR array (e.g. thionins); (2) differential regulation was observed in less than three biological replicates (e.g. proteinase inhibitors), and hence the gene was automatically excluded from further analysis; (3) some genes were not regulated in the TIGR array analysis (e.g. Rubisco); and (4) the raw signals of the hybridized TIGR arrays were not interpretable (e.g. Thr deaminase). In general, the large-scale transcriptional analysis with the TIGR array has substantially extended our understanding of the plastic responses of N. attenuata to M. sexta attack, and additionally allowed for a detailed comparison of these responses with those elicited in S. nigrum by this Solanaceous generalist.

Apart from the anticipated differences in alkaloid (steroidal alkaloids in S. nigrum) and alkaloid/polyamine precursor (putrescine in N. attenuata) formation, our analysis revealed the production of different defense metabolites in both species. N. attenuata predominantly elicited genes for the production of antinutritive polyphenol oxidases, phenylpropanoids and their precursors, and plastidic IPP, which is primarily channeled into mono- and diterpene synthesis (Lichtenthaler, 1999). In S. nigrum, the transcriptional emphasis was on sesquiterpene synthesis. Both species likely increase flavonoids, polyamines, and green leaf volatiles in response to attack. Analyses of the metabolome are needed to test the predictions emerging from this transcriptional analysis.

Contrary to our assumption that the two species would activate a similar suite of signaling genes, N. attenuata and S. nigrum activated different sets of signaling genes and in N. attenuata more signaling cascades appeared to be up-regulated. JA signaling was regulated in both species; calcium-based signaling, inositol-phospholipid signaling, and G-protein-mediated signaling were found to be specific to N. attenuata, and cytokinin signaling was detected only in S. nigrum. While calcium-signaling has been implicated in systemin-mediated activation of phospholipase A2 and subsequent JA synthesis (Ryan, 2000), the role of phospholipase C and heterotrimeric G proteins in defense signaling in Solanaceous plants is largely unexplored. The only prosystemin gene on the array was up-regulated in two of three replicates of S. nigrum but did not yield interpretable results in N. attenuata, consistent with the structural dissimilarities of potato and tobacco systemins (Scheer et al., 2003; Ryan, 2000).

Our analysis also provided a deeper insight into the changes that accompany alterations in signaling and secondary metabolism, namely changes in carbohydrate and amino acid metabolism, nucleic acid and protein metabolism, or changes associated with other biotic or abiotic stressors. For example, both plants increased similar and dissimilar transcripts for chaperones, dehydration-related genes, and oxidative stress genes. Transcript levels of enzymes involved in protein degradation/processing were increased in both species but with distinct differences. Ubiquitin-related proteolysis, which plays an important role in eliminating misfolded or abnormal proteins that probably accumulate in stressed plants, is activated only in N. attenuata. Furthermore, ubiquitin-dependent protein turnover influences many cellular processes by modulating levels of regulatory proteins (Hare et al., 2003) and there is increasing evidence that ubiquitin-dependent proteolysis is essential in regulating oxilipin-mediated plant responses. An important element in JA signal transduction, CORONATINE INSENSITIVE 1, is part of an E3-type ubiquitin ligase complex, and tomato mutants expressing nonfunctional CORONATINE INSENSITIVE 1 are compromised in their resistance to two-spotted spider mites (Xie et al., 1998; Devoto et al., 2002). Hence, ubiquitin-dependent proteolysis may play an important role in regulatory networks that mediate defense responses of N. attenuata.

In S. nigrum other genes involved in protein metabolism are induced by M. sexta herbivory, with the most dramatic example being a LAP gene whose expression was up-regulated 20-fold. LAPs are present in pro- and eukaryotes, and plant LAPs have been intensively studied in tomato, where they are elicited by JA, wounding, and pathogens and are present in floral tissues (Tu et al., 2003, and references therein). Although tomato LAPs are biochemically and physiologically well characterized, their function in plant defense responses remains elusive. Animal aminopeptidases are involved in modulation of peptide and protein activities (Barr, 1991) and therefore it is tempting to speculate that a LAP cleaves precursors of polypeptide hormones that are known especially among Solanaceous plants (Ryan, 2000). Alternatively, wound-induced LAPs may have a more general role in the turnover of proteins that are essential components of plant defense responses (Walling et al., 1995). The fact that LAP was also highly up-regulated on the arrays of MeJA-elicited plants emphasized the importance of this gene in defense responses of S. nigrum.

When S. nigrum was treated with MeJA, a derivative of the jasmonate cascade that is largely responsible for mediating herbivore resistance, branches of secondary metabolism and signaling were activated that the previous microarray experiment had shown only for N. attenuata's responses to M. sexta attack. These included genes involved in phenylpropanoid and shikimate biosynthesis, the plastidic pathway of terpenoid production, genes involved in ubiquitin-mediated proteolysis, and a gene related to G protein signaling. Hence, the genuinely weaker transcriptional response of S. nigrum to M. sexta herbivory cannot be explained by S. nigrum's inability to respond to elicitation. N. attenuata has likely had a longer evolutionary association with M. sexta and hence has had more time to evolve a strong response to M. sexta attack. S. nigrum appears not to have evolved strong responses to this herbivore, but has to others, e.g. flea beetles (Schmidt et al., 2004).

In summary, the transcriptional comparison of M. sexta-attacked N. attenuata and S. nigrum plants did not support the existence of a Solanaceous blueprint for herbivore defense. Most strikingly, the differences extended beyond the activation of different alkaloid pathways and included a profound divergence in signaling pathways mediating the elicited responses. The likely difference in the length of their evolutionary associations with M. sexta may account for the differences in their responses.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant and Insect Growth and Experimental Setup

Seeds of a Nicotiana attenuata inbred line were smoke germinated on Phytagel as described by Krugel et al. (2002). Twelve days after germination seedlings were planted in soil into Teku pots (Waalwijk, The Netherlands), and after 12 additional days, transferred to 0.5-L pots with a peat-based substrate (Klasmann Tonsubstrat, Geeste-Gro Hesepe, Germany). Seeds of a Solanum nigrum inbred line were germinated as described in Schmidt et al. (2004) and 10-d-old seedlings were transferred to 0.5-L pots with the same substrate as for N. attenuata. Both plant species were grown in the glasshouse of the Max Planck Institute for Chemical Ecology (Jena, Germany) at 24°C to 26°C (16 h light; supplemental lighting by Philips Sun-T Agro 400 and 600 W sodium lights [Eindhoven, The Netherlands]; 65% humidity).

Insect Treatment
One day prior to the herbivore treatment, 24 randomly selected plants of each species were placed in glass insect cages (30 cm x 30 cm x 60 cm, each cage accommodating four plants). Twelve plants were used in both herbivore and control treatment; plants were harvested individually, and later RNA from the four plants of a single cage were pooled to provide one biological replicate of the experiment. Eggs of Manduca sexta were obtained from a laboratory colony. On each plant of the herbivore treatment (n = 12 plants for each species) 10 freshly hatched caterpillars were placed on two different leaves and allowed to feed freely. After 24 h of feeding, herbivores and their frass were removed, and shoots and leaves of the herbivore-damaged and of nonattacked control plants were harvested, flash-frozen in liquid nitrogen, and stored at –80°C until microarray analysis.

To determine whether first instar M. sexta larvae consume different amounts of leaf area from the two host plant species, we placed two freshly hatched caterpillars on each of another five plants per species and measured the amount of leaf material consumed after 24 h. After 24 h, the damaged leaves were harvested, scanned (HP Scanjet 8200, 300 dpi; Hewlett-Packard, Palo Alto, CA), the leaf area consumed was determined by counting pixels with image analysis software (Sigma Scan Pro 5, Point Richmond, CA), and the larvae were weighed. Five leaves were not included in the analysis because the caterpillars died or walked off the leaf during the experiment leaving n = 8 for N. attenuata and n = 7 for S. nigrum. The amount of leaf area consumed did not differ significantly between the two host species (N. attenuata 17.0 ± 2.9 mm²; S. nigrum 23.6 ± 3.0 mm²; t_{0.025, 13} = –1.375, P = 0.192). Lavae feeding on N. attenuata (2.7 ± 0.2 mg) tended to be slightly heavier than those feeding on S. nigrum (2.0 ± 0.1 mg).

MeJA Treatment
Twelve S. nigrum plants were used in both control and MeJA treatment. Ten microliters lanolin containing 75 µg MeJA (both Sigma, St. Louis) were applied to one leaf (seventh leaf above cotyledons) of each plant; control plants were treated with 10 µL pure lanolin. After 24 h, the treated leaves were harvested, flash-frozen in liquid nitrogen, and stored at –80°C until RNA extraction. RNA of four individual plants was pooled to provide one biological replicate.

Microarray Analysis

For gene expression analysis, we used two versions of the TIGR potato 10,000-clone cDNA microarray that contain 11,243 (version 10Kv1) and 11,512 (10Kv2) annotated cDNA clones spotted as duplicates on the array. Detailed information about this microarray can be found under http://www.tigr.org/tdb/potato/microarray_comp.shtml. For the transcriptional analysis of M. sexta-induced responses of N. attenuata and S. nigrum, we used array version 10Kv1; for the MeJA-elicited responses of S. nigrum we used array version 10Kv2. For comparisons of the datasets produced from the two versions of the microarray, we analyzed only the clones present on both versions of the array (11,243 clones). Gene expression data obtained from hybridizations of this potato microarray with a variety of Solanaceous species can be accessed through a database maintained at the TIGR Web site (http://www.tigr.org/tigr-scripts/sgedb/studies_SGED.pl) and thus compared and shared across different laboratories. Although Solanaceous species clearly differ in morphology, life cycle, secondary metabolism, tuber and fruit formation, etc., they have similar genomes with respect to gene content and genome organization (see transcriptional analysis of several Solanaceous plants by Robin Buell, http://www.tigr.org/tigr-scripts/sgedb/search2_std.pl?study_id=35).

Plant material was ground under liquid nitrogen and total RNA was extracted with TRI Reagent (Sigma) according to the manufacturer's instructions. All steps of microarray processing (cDNA production, cDNA labeling, microarray hybridization, data quantification, data normalization using LOWESS) were carried out by the TIGR Expression Profiling Service according to published methods (http://www.tigr.org/tdb/potato/microarray_SOPs.shtml). The cDNAs hybridized to an individual array was produced from RNA extracted individually from the four plants of a control cage (Cy5 labeled) and the four plants of the treatment cage (Cy3 labeled). The three biological replicates of the M. sexta-N. attenuata elicitation experiment are named NA1, NA2, and NA3. SN1, SN2, and SN3 designated the corresponding replicate microarrays of the M. sexta-S. nigrum elicitation experiment; the arrays of the S. nigrum MeJA experiment are named SNmj1, SNmj2, and SNmj3. The raw data from the nine hybridizations including all details of the experiment are available from the TIGR Solanaceae Gene Expression Database (http://www.tigr.org/tigr-scripts/sgedb/search2_std.pl?study_id=53, experiment IDs 2450, 2455, 2460, 2465, 2470, 2475, 2928, 2929, 2930).

We analyzed the normalized data of the six microarrays with GeneSpring 6.1 (Silicon Genetics, Redwood City, CA) using Hierarchical Cluster Analysis. For further analysis we calculated the mean ER from the biological replicates and defined a transcript as being differentially regulated when the following criteria were fullfilled: (1) The ER was significantly different from 1 as determined by a Student's t test (P < 0.05); and (2) the ER exceeded the thresholds of 0.67 and 1.5 for down- and up-regulation, respectively. These criteria had been previously tested and found to give reproducible results (Heidel and Baldwin, 2004; Halitschke et al., 2003).

We confirmed the microarray expression data using TaqMan real-time PCR (ABI PRISM 7700 Sequence Detection System; Applied Biosystems, Foster City, CA). Additionally, to test the suitability of this potato microarray for S. nigrum and N. attenuata, we chose to assay the species that is more distantly related to potato, namely N. attenuata (Fig. 1). We analyzed the three biological replicates of M. sexta-challenged N. attenuata using TaqMan probes for N. attenuata-specific genes that are homologous to genes present on the array, namely -DOX, LOX, HPL, and XTH, formerly XET; Supplemental Table II). We calculated the relative gene expression of each sample using the comparative 2^–Ct method (Livak and Schmittgen, 2001) with sulfite reductase as endogenous control gene, which under our experimental conditions is not regulated (B. Bubner and I.T. Baldwin, unpublished data) and the noninfested control plants as the calibrator.

	ACKNOWLEDGMENTS

 
We thank TIGR for generously providing the microarrays and the data acquisition, Dr. Ryan Oyama for invaluable support with the phylogenetic analysis, Jens Wurlitzer for help with the experiments, and Ben Bubner for TaqMan support.

Received February 12, 2005; returned for revision April 5, 2005; accepted April 5, 2005.

	FOOTNOTES

 
¹ This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (FOR 456; BA2138/1–1).

² These authors contributed equally to the paper.

³ Present address: John Innes Centre, Department of Disease and Stress Biology, Colney, Norwich NR4 7UH, UK.

^[w] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.061192.

^* Corresponding author; e-mail baldwin{at}ice.mpg.de; fax: 49–3641–571102.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Alborn HT, Brennan MM, Tumlinson JH (2003) Differential activity and degradation of plant volatile elicitors in regurgitant of tobacco hornworm (Manduca sexta) larvae. J Chem Ecol 29: 1357–1372[CrossRef][ISI][Medline]

Baldwin IT (1999) Inducible nicotine production in native Nicotiana as an example of adaptive phenotypic plasticity. J Chem Ecol 25: 3–30

Barr PJ (1991) Mammalian subtilisins—the long-sought dibasic processing endoproteases. Cell 66: 1–3[CrossRef][ISI][Medline]

Bergenstrahle A, Borga P, Jonsson MV (1996) Sterol composition and synthesis in potato tuber discs in relation to glycoalkaloid synthesis. Phytochemistry 41: 155–161

Berger S, Mitchell-Olds T, Stotz HU (2002) Local and differential control of vegetative storage protein expression in response to herbivore damage in Arabidopsis thaliana. Physiol Plant 114: 85–91[CrossRef][Medline]

Choi D, Bostock RM, Avdiushko S, Hildebrand DF (1994) Lipid-derived signals that discriminate wound-responsive and pathogen-responsive isoprenoid pathways in plants: methyl jasmonate and the fungal elicitor arachidonic acid induce different 3-hydroxy-3-methylglutaryl-coenzyme A reductase genes and antimicrobial isoprenoids in Solanum tuberosum L. Proc Natl Acad Sci USA 91: 2329–2333[Abstract/Free Full Text]

Constabel CP, Yip L, Patton JJ, Christopher ME (2000) Polyphenol oxidase from hybrid poplar: cloning and expression in response to wounding and herbivory. Plant Physiol 124: 285–295[Abstract/Free Full Text]

Constabel CP, Yip L, Ryan CA (1998) Prosystemin from potato, black nightshade, and bell pepper: primary structure and biological activity of predicted systemin polypeptides. Plant Mol Biol 36: 55–62[CrossRef][ISI][Medline]

Devoto A, Nieto-Rostro M, Xie DX, Ellis C, Harmston R, Patrick E, Davis J, Sherratt L, Coleman M, Turner JG (2002) COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis. Plant J 32: 457–466[CrossRef][ISI][Medline]

Dopke W, Duday S, Matos N (1987) The isolation and identification of alkaloids and steroids from Solanum nigrum. Z Chem 27: 64

Ehrlich PR, Raven PH (1964) Butterflies and plants: a study in coevolution. Evolution Int J Org Evolution 18: 586–608

Fraenkel GS (1959) Raison d'etre of secondary plant substances. Science 129: 1466–1470[Free Full Text]

Frohne D, Jensen U (1992) Systematik des Pflanzenreichs. Urban & Fischer, Munich

Gang DR, Beuerle T, Ullmann P, Werck-Reichhart D, Pichersky E (2002) Differential production of meta hydroxylated phenylpropanoids in sweet basil peltate glandular trichomes and leaves is controlled by the activities of specific acyltransferases and hydroxylases. Plant Physiol 130: 1536–1544[Abstract/Free Full Text]

Gershenzon J (2002) Secondary metabolism and plant defense. In L Taiz, E Zeiger, eds, Plant Physiology, Ed 3. Sinauer Associates, Sunderland, MA, pp 283–308

Halitschke R, Gase K, Hui D, Schmidt DD, Baldwin IT (2003) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata: microarray analysis reveals that most herbivore-specific transcriptional changes are mediated by fatty acid-amino acid conjugates. Plant Physiol 131: 1894–1902[Abstract/Free Full Text]

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[Abstract/Free Full Text]

Hare PD, Seo HS, Yang JY, Chua NH (2003) Modulation of sensitivity and selectivity in plant signaling by proteasomal destabilization. Curr Opin Plant Biol 6: 453–462[CrossRef][Medline]

Heidel A, Baldwin IT (2004) Microarray analysis of salicylic acid- and jasmonic acid-signalling in responses of Nicotiana attenuata to attack by insects from multiple feeding guilds. Plant Cell Environ 27: 1362–1373[CrossRef]

Hermsmeier D, Schittko U, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata: large-scale changes in the accumulation of growth- and defense-related plant mRNAs. Plant Physiol 125: 683–700[Abstract/Free Full Text]

Jones CG, Firn RD (1991) On the evolution of plant secondary chemical diversity. Philos Trans R Soc Lond B Biol Sci 333: 273–280

Kahl J, Siemens DH, Aerts RJ, Gabler R, Kuhnemann F, Preston CA, Baldwin IT (2000) Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore. Planta 210: 336–342[CrossRef][ISI][Medline]

Kessler A, Baldwin IT (2001) Defensive function of herbivore-induced plant volatile emissions in nature. Science 291: 2141–2144[Abstract/Free Full Text]

Kessler A, Baldwin IT (2004) Herbivore-induced plant vaccination: the orchestration of plant defenses in nature and their fitness consequences in the wild tobacco Nicotiana attenuata. Plant J 38: 639–649[CrossRef][ISI][Medline]

Krugel T, Lim M, Gase K, Halitschke R, Baldwin IT (2002) Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system. Chemoecology 12: 177–183

Kutchan TM (2001) Ecological arsenal and developmental dispatcher: the paradigm of secondary metabolism. Plant Physiol 125: 58–60[Free Full Text]

Lantin S, O'Brien M, Matton DP (1999) Pollination, wounding and jasmonate treatments induce the expression of a developmentally regulated pistil dioxygenase at a distance, in the ovary, in the wild potato Solanum chacoense Bitt. Plant Mol Biol 41: 371–386[Medline]

Leon J, Rojo E, Sanchez-Serrano JJ (2001) Wound signalling in plants. J Exp Bot 52: 1–9[Abstract/Free Full Text]

Lichtenthaler HK (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 50: 47–65[CrossRef][ISI]

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2^–Ct method. Methods 25: 402–408[CrossRef][ISI][Medline]

Mitchell-Olds T (2001) Arabidopsis thaliana and its wild relatives: a model system for ecology and evolution. Trends Ecol Evol 16: 693–700[CrossRef]

Mittova V, Tal M, Volokita M, Guy M (2002) Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species. Physiol Plant 115: 393–400[CrossRef][Medline]

Pearce G, Moura DS, Stratmann J, Ryan CA (2001) Production of multiple plant hormones from a single polyprotein precursor. Nature 411: 817–820[CrossRef][Medline]

Ridout CL, Price KR, Coxon DT, Fenwick GR (1989) Glycoalkaloids from Solanum nigrum L., alpha-solamargine and alpha-solasonine. Pharmazie 44: 732–733

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]

Roda AL, Oldham NJ, Svatos A, Baldwin IT (2003) Allometric analysis of the induced flavonols on the leaf surface of wild tobacco (Nicotiana attenuata). Phytochemistry 62: 527–536[Medline]

Ryan CA (2000) The systemin signaling pathway: differential activation of plant defensive genes. Biochim Biophys Acta 1477: 112–121[CrossRef][Medline]

Scheer JM, Pearce G, Ryan CA (2003) Generation of systemin signaling in tobacco by transformation with the tomato systemin receptor kinase gene. Proc Natl Acad Sci USA 100: 10114–10117[Abstract/Free Full Text]

Schmidt DD, Kessler A, Kessler D, Schmidt S, Lim M, Gase K, Baldwin IT (2004) Solanum nigrum: A model ecological expression system. Mol Ecol 13: 981–995[CrossRef][Medline]

Tu CJ, Park SY, Walling LL (2003) Isolation and characterization of the neutral leucine aminopeptidase (LapN) of tomato. Plant Physiol 132: 243–255[Abstract/Free Full Text]

Van Dam NM, Horn M, Mares M, Baldwin IT (2001) Ontogeny constrains systemic protease inhibitor response in Nicotiana attenuata. J Chem Ecol 27: 547–568[CrossRef][ISI][Medline]

Voelckel C, Baldwin IT (2003) Detecting herbivore-specific transcriptional responses in plants with multiple DDRT-PCR and subtractive library procedures. Physiol Plant 118: 240–252[CrossRef]

Voelckel C, Baldwin IT (2004) Herbivore-induced plant vaccination: array-studies reveal the transience of herbivore-specific transcriptional imprints and a distinct imprint from stress combinations. Plant J 38: 650–663[CrossRef][ISI][Medline]

Walling LL (2000) The myriad plant responses to herbivores. J Plant Growth Regul 19: 195–216[Medline]

Walling LL, Pautot V, Gu YQ, Chao WS, Holzer FM (1995) Leucine aminopeptidase: a complex array of proteins induced during the plant defense response. J Cell Biochem (Suppl) 19A: 137[CrossRef]

Winz RA, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata: insect-induced ethylene reduces jasmonate-induced nicotine accumulation by regulating putrescine N-methyltransferase transcripts. Plant Physiol 125: 2189–2202[Abstract/Free Full Text]

Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG (1998) COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280: 1091–1094[Abstract/Free Full Text]

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 anti-herbivore defenses. Plant Physiol 134: 1181–1190[Abstract/Free Full Text]

This article has been cited by other articles:

				A. Singh, I. K. Singh, and P. K. Verma Differential transcript accumulation in Cicer arietinum L. in response to a chewing insect Helicoverpa armigera and defence regulators correlate with reduced insect performance J. Exp. Bot., June 1, 2008; 59(9): 2379 - 2392. [Abstract] [Full Text] [PDF]

				S. P. Pandey, P. Shahi, K. Gase, and I. T. Baldwin Chemical Ecology Special Feature: Herbivory-induced changes in the small-RNA transcriptome and phytohormone signaling in Nicotiana attenuata PNAS, March 25, 2008; 105(12): 4559 - 4564. [Abstract] [Full Text] [PDF]

				K. Zhu-Salzman, D. S. Luthe, and G. W. Felton Arthropod-Inducible Proteins: Broad Spectrum Defenses against Multiple Herbivores Plant Physiology, March 1, 2008; 146(3): 852 - 858. [Full Text] [PDF]

				O. Barazani, C. C. von Dahl, and I. T. Baldwin Sebacina vermifera Promotes the Growth and Fitness of Nicotiana attenuata by Inhibiting Ethylene Signaling Plant Physiology, June 1, 2007; 144(2): 1223 - 1232. [Abstract] [Full Text] [PDF]

				A. P. Giri, H. Wunsche, S. Mitra, J. A. Zavala, A. Muck, A. Svatos, and I. T. Baldwin Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata. VII. Changes in the Plant's Proteome Plant Physiology, December 1, 2006; 142(4): 1621 - 1641. [Abstract] [Full Text] [PDF]

				S. Schmidt and I. T. Baldwin Systemin in Solanum nigrum. The Tomato-Homologous Polypeptide Does Not Mediate Direct Defense Responses Plant Physiology, December 1, 2006; 142(4): 1751 - 1758. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF)