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First published online October 27, 2006; 10.1104/pp.106.089755 Plant Physiology 142:1751-1758 (2006) © 2006 American Society of Plant Biologists
Systemin in Solanum nigrum. The Tomato-Homologous Polypeptide Does Not Mediate Direct Defense Responses1,[W]Max Planck Institute for Chemical Ecology, Department of Molecular Ecology, Beutenberg Campus, 07745 Jena, Germany
We extend Ryan's seminal work on the 18-amino acid polypeptide systemin in tomato's (Solanum lycopersicum) systemic wound response to the closely related solanaceous species Solanum nigrum. We compared wild-type plants to plants transformed with an inverted repeat prosystemin construct (IRSys) to silence the expression of the endogenous S. nigrum prosystemin gene. In wild-type plants elicited with wounding + oral secretions from Manduca sexta larvae, trypsin-proteinase inhibitors (TPIs) accumulated even though prosystemin transcripts were down-regulated. Neither reducing the endogenous systemin levels by RNAi nor complementing the plants with systemin by exogenously supplying the polypeptide through excised stems significantly increased TPI activity, indicating that systemin and TPIs are not correlated in S. nigrum. The performance of two herbivore species from two feeding guilds, M. sexta larvae and Myzus persicae nicotianae, did not differ between wild-type and IRSys plants, demonstrating that varying endogenous systemin levels do not alter the direct defenses of S. nigrum. Field experiments with wild-type and IRSys plants and the flea beetle Epitrix pubescens supported these glasshouse data. That levels of oral secretion-elicited jasmonic acid did not differ between wild-type and IRSys plants suggests that systemin is unlikely to mediate jasmonate signaling in S. nigrum as it does in tomato. We conclude that the tomato-homologous polypeptide does not mediate direct defense responses in S. nigrum.
Plants not only respond locally to leaf damage caused by wounding, herbivory, or pathogen attack, but also induce defenses in distal, unwounded leaves. These systemic defense responses have been extensively studied in tomato (Solanum lycopersicum), where an 18-amino acid polypeptide called systemin is known to play an essential role in generating the mobile wound signal. Systemin is processed from its larger precursor, prosystemin, which is synthesized and processed in the vascular phloem parenchyma cells (Narvaez-Vasquez and Ryan, 2004  ). Constitutive prosystemin mRNA expression has been found throughout the plant except for the roots (McGurl et al., 1992). After leaf wounding, prosystemin mRNA is induced systemically; the highest accumulation is seen after 3 to 4 h (McGurl et al., 1992). Like the prosystemin mRNA, proteinase inhibitor (PI) I mRNA, which encodes for a protein with antinutritional effects against several lepidopteran herbivores (Johnson et al., 1989; Ryan, 1990), accumulates systemically after wounding; it is most abundant 8 to 10 h after wounding (McGurl et al., 1992). Young tomato plants supplied with low concentrations of systemin through their cut stems accumulated PI I and II (Pearce et al., 1991). This positive correlation between systemin and PIs is supported by the work of McGurl et al. (1992), who transformed tomato plants with an antisense prosystemin construct. A transgenic line lacking prosystemin was almost completely suppressed in its systemic induction of PIs I and II. Furthermore, Manduca sexta larvae that fed on another transgenic plant silenced in its prosystemin expression consumed more leaf material and became 3 times heavier than those that fed on wild-type plants (Orozco-Cardenas et al., 1993). After wounding, transgenic plants transformed to overexpress the prosystemin gene constitutively produce PI I and II proteins and accumulate more PIs in local and systemic leaves than do wild-type plants (McGurl et al., 1994). Grafting experiments using these overexpressers as root stocks revealed the constitutive production of PIs in wild-type scions (McGurl et al., 1994), indicating the central role of systemin in generating the systemic wound signal in tomato.
More recent grafting experiments between jasmonic acid (JA) biosynthesis mutants (called spr2 mutants) and wild-type plants, or between systemin signaling mutants (called spr1 mutants) and wild-type plants, showed that both JA biosynthesis and the presence of systemin are required in the local, wounded leaf to produce the systemic signal and hence to induce PIs systemically. On the other hand, neither JA nor systemin is needed in the systemic, undamaged leaves of tomato plants (Howe, 2004
Using the tomato cDNA as a probe, systemin homologs have been found in three other solanaceous species (Constabel et al., 1998
S. nigrum systemin, which has 83% amino acid identity to the tomato systemin with the respective prosystemins being 81% identical, induced 10 times less PI I when supplied to excised tomato plants than did tomato systemin itself or any of the other systemin homologs (Constabel et al., 1998
Spatial and Temporal Prosystemin Transcript Patterns Prosystemin, of which at least three genes are present in S. nigrum (Supplemental Fig. S2A), was constitutively expressed in all reproductive and vegetative wild-type tissues except for the roots. The sites with the highest expression were the flower buds and the leaves, respectively (Fig. 1, A and B ). Interestingly, low prosystemin mRNA levels were detected in black berries and stems (Fig. 1, A and B). After a wounding + OS treatment, the expression of prosystemin decreased rapidly in leaves of wild-type plants and was lowest 30 min after elicitation, whereas, in both lines transformed with an inverted repeat prosystemin construct (IRSys lines), the expression of prosystemin remained very low (Fig. 2, A and B ).
TPI Accumulation in Wild-Type and IRSys Plants To test whether the accumulation of TPI depends on the (pro)systemin level of a plant, the amount of TPIs was quantified in uninduced and induced wild-type and IRSys plants. Although constitutive levels in wild-type and transgenic plants were below the detection limit of the assay, levels increased dramatically after induction (Fig. 3 ). No significant difference was detected between wild-type and IRSys plants, either in local or in systemic leaves.
Influence of Exogenously Applied Systemin on TPI Levels As reducing prosystemin mRNA levels in IRSys plants did not reduce TPI accumulation, we tried to enrich plants with systemin by applying the polypeptide through their cut stems. Applying S. nigrum systemin or tomato systemin to wild-type S. nigrum plants did not increase TPI levels compared to those of controls (Fig. 4A ). However, the application of MeJA was clearly capable of inducing TPIs (Fig. 4A). In tomato plants, the application of tomato systemin significantly increased the level of TPIs compared to control levels, whereas S. nigrum systemin did not (Fig. 4B). Using a MeJA dilution series, we demonstrated that S. nigrum wild-type plants are able to respond to an exogenously applied elicitor in a dose-dependent manner (Fig. 4A, insert). The patterns resulting from treatment of the IRSys lines did not differ significantly from those of treated wild-type plants (Supplemental Fig. S3).
Influence of Systemin Levels on Herbivores of Different Feeding Guilds To evaluate the influence of systemin on direct defense mechanisms, we compared the performance of herbivores on wild-type plants and IRSys lines. Herbivores from two different feeding guilds, namely, leaf chewers (i.e. caterpillars of the tobacco hornworm M. sexta [Sphingidae] and the flea beetle E. pubescens [Chrysomelidae]) and phloem sap suckers (i.e. Myzus persicae nicotianae [Aphididae]), were chosen. As measures of the leaf quality of the different genotypes, we quantified the mass gain of M. sexta, the leaf damage caused by E. pubescens, and the population growth of M. persicae nicotianae, respectively. The measures of M. sexta larval mass and the E. pubescens assay were repeated three times. The data shown in Figure 5 are representative for all three experiments. In none of the three herbivore species was a significant difference between wild-type and IRSys lines detected (Fig. 5; all Ps > 0.1240).
Influence of Systemin on JA Levels To test whether systemin acts at the top of the octadecanoid pathway upstream of JA, the level of the plant hormone was quantified in the leaves of wild-type plants and IRSys lines treated with wounding + OS. The time series of wild-type plants were characterized by two peaks 30 min and 3 h after elicitation (Fig. 6 , insert). The pattern was similar for IRSys lines, with the second peak more prominent than the first. Neither in uninduced nor in induced leaves were significant differences between wild-type and transgenic lines detected (Fig. 6). The wild-type time series as well as the comparison between wild-type plants and IRSys lines were repeated twice with similar results (only one graph is shown).
The aim of this study was to determine whether systemin's role in tomato also applies to a solanaceous species that is closely related to tomato. The question was addressed by extending Ryan's work to S. nigrum and testing whether systemin mediates direct systemic defense responses in this species. To compare transgenic S. nigrum plants silenced in their prosystemin expression to wild-type plants, we measured TPI and JA accumulation after wounding + OS treatment. In addition, we compared the performance of three different herbivore species on wild-type and IRSys plants and observed TPI levels in plants supplied with systemin.
S. nigrum harbors at least three prosystemin genes (Supplemental Fig. S2A) that are effectively silenced in both IRSys lines (Fig. 2, A and B). The tissue-specific expression pattern of prosystemin in S. nigrum (Fig. 1, A and B) seems to reflect that observed in tomato (McGurl et al., 1992
The ability of a transgenic tomato line, silenced in its prosystemin expression, to systemically increase PI I and II (McGurl et al., 1992
To increase endogenous systemin levels, plants were supplied with additional systemin by applying the polypeptide through the cut stems or petioles. In S. nigrum wild-type plants, neither S. nigrum systemin nor tomato systemin was able to elicit higher TPI levels compared to those of water-treated controls (Fig. 4A). The former phenomenon was reported previously by Constabel et al. (1998)
To test the influence of endogenous systemin levels on S. nigrum's resistance to insect attack, the performance of different herbivores on wild-type and IRSys plants was evaluated. Larvae of the tobacco hornworm M. sexta, a Solanaceae specialist, gained the same mass when reared on the different genotypes (Fig. 5A). Dramatic differences in weight gain have been reported for tomato plants overexpressing the prosystemin gene compared to wild-type tomato plants (Orozco-Cardenas et al., 1993
To test whether the endogenous systemin levels influence a plant's ability to produce JA, the plant hormone was quantified in both uninduced and OS-elicited wild-type and IRSys plants. Wild-type plants clearly responded to the treatment (Fig. 6, insert), showing a two-peaked pattern of JA accumulation. The amount of JA did not differ significantly between wild-type and IRSys plants (Fig. 6), indicating little or no correlation between systemin and JA in S. nigrum. Given that prosystemin transcripts are down-regulated after induction (Fig. 2), systemin and JA may be negatively correlated in S. nigrum, but the low constitutive JA levels in both IRSys lines argue against this. These data suggest that systemin in S. nigrum does not act at the top of the octadecanoid-based signaling pathway upstream of JA as has been proposed for tomato (Farmer and Ryan, 1992
The possibility that systemin might play completely different roles even in closely related species is supported by Boller's (2005)
Plant Growth
The Solanum nigrum inbred line Sn30 (Schmidt et al., 2004 The procedure was the same for tomato (Solanum lycopersicum cv Castlemart), except that the seeds were soaked in water at 4°C overnight. Plants used in the two field experiments in July and August 2005 (flea beetle herbivory and prosystemin expression after wounding + OS treatment) were planted at the field site in Dornburg (north of Jena, Germany) 24 or 21 d postsowing after being acclimatized to outdoor conditions for 3 to 5 d. The release of transformed plants at the Dornburg field site was conducted in compliance with EU and German regulations (release application nos. 6786–01–0156 [IRSys line 1] and 6786–01–0165 [IRSys line 2]) as administered by the Thüringer Landesverwaltungsamt and the Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz. All experiments were conducted with 4- to 5-week-old plants except those involving the harvesting of reproductive tissues.
Transgenic S. nigrum lines silenced in their prosystemin expression were constructed using the silencing vector pSOL3SYS1 (Supplemental Fig. S1), which is based on the pSOL3RCA silencing vector described in detail by Bubner et al. (2006)
Genomic DNA of wild-type and IRSys plants was isolated from S. nigrum leaves using a modified cetyl trimethyl ammonium bromide method (Rogers and Bendich, 1994
To mimic herbivore feeding, one leaf was wounded with a fabric pattern wheel, causing three rows of punctured wounds on each side of the midrib. The wounds were immediately supplied with OS of Manduca sexta larvae. OS were collected from third- to fourth-instar larvae hatched from eggs (Carolina Biological Supply) and reared on S. nigrum wild-type plants. OS were diluted 1:1 (v/v) with deionized water prior usage. For the prosystemin expression time series, the PI assay, and the JA measurements, a fully expanded leaf of the main axis (normally one leaf at nodes six to eight) was induced as described above. After the respective time points (for the PI assay, after 72 h), the locally treated leaf and in the case of the prosystemin time series the uninduced leaf one node above the treated leaf were harvested, flash frozen in liquid nitrogen, and stored at –80°C until further processing. As the plants grew slower in the field than in the glasshouse, in the field experiment the leaf at the fourth node was treated and harvested as described above. To measure the constitutive prosystemin expression in different plant tissues, the whole plant remained untreated and RNA was extracted from old (third node) leaf blades, petioles, and midribs; young (seventh node) leaf blades, petioles, and midribs; and stems, roots, buds, flowers, and green and black berries.
The PI-inducing effect of S. nigrum and tomato systemin was tested by excising S. nigrum and tomato wild-type plants at the base of the stem according to Pearce et al. (1993) All experiments described above were based on four to six individual plants for each treatment and/or harvest time point. The individual reproductive tissues samples were pooled out of 15 buds, five flowers, three green berries, and two black berries per plant, respectively.
M. sexta larvae were reared from eggs (obtained from the M. sexta colony at the Max Planck Institute for Chemical Ecology in Jena, Germany) and one neonate was placed on each of the 25 individual plants per genotype (wild type and both IRSys lines). The caterpillars were weighed after 3, 5, 9, and 11 d. Naturally occurring adult flea beetles (Epitrix pubescens) were allowed to feed on 45 field-grown plants planted as triplicates of the three genotypes over a period of 10 d. The damage done to the plant was recorded on days 2, 4, and 10. To quantify the damage, each individual leaf was categorized according to the following damage classes: 0 = 0% damage, 1 = 1% to 5% damage, 2 = 6% to 10% damage, 3 = 11% to 25% damage, 4 = 26% to 50% damage, and 5 = 51% to 100% damage. Based on the damage level estimated for each individual leaf, a mean value was calculated for the entire plant. As these mean plant data are noncontinuous percentage values and the damage experienced by wild-type plants was compared singly to the damage done to each IRSys line, thus having two analyses per recording day, Bonferroni-corrected Wilcoxon signed rank tests were performed. Myzus persicae nicotianae aphids were collected on Nicotiana attenuata plants in our greenhouse and transferred to S. nigrum, where they were allowed to establish a population for about 2 weeks. Single females of this population were used to infest 15 S. nigrum plants of each genotype (wild type and both IRSys lines), and after 10 d the population size on each plant was counted.
Harvested tissues were ground individually in liquid nitrogen and total RNA was isolated following a modified TRI Reagent procedure for polysaccharide- and proteoglycan-rich sources (The Institute for Genomic Research, 2003
Harvested leaves were ground in liquid nitrogen individually and total protein was extracted using 2 mL of protein extraction buffer (Jongsma et al., 1994
Approximately 300 mg of harvested leaf tissue were homogenized in 1 mL of ethyl acetate spiked with 200 ng mL–1 methanolic [13C2]JA as an internal standard. After centrifugation at 13,000 rpm for 20 min at 4°C, the supernatant was transferred to another tube and the extract was redissolved in 1 mL of ethyl acetate. Following another centrifugation step, the combined supernatants were evaporated and the dried sample was redissolved in 500 µL of 70% (v/v) methanol. After vortexing for 5 min, the sample was centrifuged for 10 min at 13,000 rpm and 15 µL of the supernatant was analyzed using a Varian 1200 L triple quadrupole mass spectrometer. For the HPLC, a Pursuit C8 column (150 mm x 2.0 mm, 3-µm particle size) was used and a gradient of water and methanol, both including 0.05% (v/v) formic acid, was run as the mobile phase with a flow rate of 0.2 mL/min. The mass spectrometer was run in negative electrospray ionization mode with an argon pressure of 0.279972 Pa (=2.1 mTorr) in the collision cell. The mass spectrometer was set up with a capillary voltage of –3,200 V, a shield voltage of 600 V, and a detector voltage of 1,800 V. The pressure of the drying gas was 131,005 Pa (=19 psi) at 300°C; that of the nebulizing gas was 379,225 Pa (=55 psi). The most abundant and characteristic fragment ion was chosen for quantification. The amount of JA per sample was calculated with the following formula [(peak area endogenous JA x 200 ng mL–1) x peak area ISTD–1] and related to 1 mg leaf tissue.
Data were analyzed by ANOVA followed by an LSD post-hoc test. To ensure homogeneity of variances data were transformed if necessary (square root: Figs. 1A and 2A; LG10: Figs. 3, left, 4A, 7C, and, partly, 8B; reciprocal: Figs. 3, right, and 6). In cases in which variances could not be homogenized by transformation, a Welch test followed by a Dunnett T3 test was performed (Figs. 1B, 2B, and 5A). The M. sexta caterpillar mass data were analyzed using repeated-measures ANOVA. All analyses were done using the software package SPSS.
The following materials are available in the online version of this article.
We dedicate this paper to Clarence "Bud" Ryan, whose research program inspired more than three decades of research into herbivore-induced defenses. We thank K. Gase, W. Kröber, A. Wissgott, and S. Kutschbach for the transformation construct and for plant transformation, A. Berg and E. Rothe for assistance with the JA analysis, J. Kellmann for managing the onerous legal obstacles to conducting field work with genetically modified plants in Germany, G. Howe for kindly providing us S. lycopersicum seeds, and C. Kost, M. Hartl, C. Voelckel, T. Kaiser, and two anonymous reviewers for critically commenting on earlier versions of the manuscript. Received September 12, 2006; accepted October 22, 2006; published October 27, 2006.
1 This work was supported by the Max Planck Society.  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.106.089755 * Corresponding author; e-mail baldwin{at}ice.mpg.de; fax 49–(0)3641–571102.
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