Characterization of prosystemin expressed in the baculovirus/insect cell system reveals biological activity of the systemin precursor

 Tomato (Lycopersicon esculentum Mill.) prosystemin in fusion with a viral signal peptide was expressed in Sf21 insect cell cultures after infection with recombinant baculoviruses. Prosystemin was purified from culture supernatants and its identity was confirmed by N-terminal sequence and mass-spectral analyses. Recombinant prosystemin was found to be equally active as compared to systemin in inducing the expression of wound-response genes in tomato plants. In cultured cells of L. peruvianum, prosystemin elicited a rapid alkalinization of the growth medium. The timing and dose-dependence of the alkalinization response were found to be identical for prosystemin and systemin, respectively. Prosystemin-triggered defense responses were inhibited by a competitive antagonist of systemin activity, indicating that the systemin sequence within the primary structure of prosystemin determines its activity. Received: 30 August 1999 / Accepted: 6 December 1999     

Systemic wound signaling in tomato leaves is cooperatively regulated by systemin and hydroxyproline-rich glycopeptide signals

Hydroxyproline-rich glycopeptides (HypSys peptides) have been isolated recently from tobacco and tomato leaves that are powerful activators of protease inhibitor synthesis. The peptides are processed from polyprotein precursors, two from a single tobacco precursor and three from a single tomato precursor. The precursor genes are expressed in response to wounding and methyl jasmonate, similar to the expression of the systemin precursor prosystemin in tomato leaves. Here we investigate the relationships between systemin and the tomato HypSys peptides in regulating wound signaling in tomato plants. Analysis of transgenic tomato plants over-expressing sense and antisense constructs of the tomato HypSys precursor under the 35S CaMV promoter show that the transgenic plants regulate protease inhibitor gene expression in response to wounding in a manner similar to prosystemin. The evidence indicates that the expression of both the tomato HypSys precursor gene and the prosystemin gene in response to wounding are necessary for strong systemic signaling. The data supports a role for both genes in an amplification loop that up-regulates the octadecanoid pathway and the synthesis of jasmonates to effect strong systemic signaling of defense genes. This report provides the first demonstration of the involvement of two plant peptides derived from two unrelated genes in regulating long distance wound signaling in plants. 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 () is Clarence A. Ryan.     

An insect peptide engineered into the tomato prosystemin gene is released in transgenic tobacco plants and exerts biological activity

Tomato systemin is a signalling peptide produced in response to wounding that locally and systemically activates several defence genes. The peptide is released from the C-terminus of prosystemin, the 200 amino acid precursor, following post-translational modifications involving unknown events and enzymes. In tobacco, two systemin molecules have been recently isolated, neither sharing any sequence homologies with the tomato prosystemin gene/protein, but performing similar functions. We modified the tomato prosystemin gene by replacing the systemin-encoding region with a synthetic sequence encoding TMOF (trypsin-modulating oostatic factor), a 10 amino acid insect peptide hormone toxic to Heliothis virescens larvae, and expressed the chimeric gene in tobacco. The results reported here show that transformed leaves contain the TMOF peptide and exert toxic activity against insect larvae reared on them. In addition, subcellular localization studies showed the cytoplasmic location of the released TMOF, suggesting that in tobacco the enzymes responsible for the post-translational modifications of the tomato precursor protein are present and act in the cytoplasm to recognise the modified prohormone. The molecular engineering of the precursor, beside supplying new clues towards the understanding of prosystemin processing, constitutes an useful tool for plant genetic manipulation, by enabling the delivery of short biological active peptides.     

Wound- and systemin-inducible calmodulin gene expression in tomato leaves

Using a calmodulin (CaM) cDNA as a probe in northern analyses, transgenic tomato plants that overexpress the prosystemin gene were found to express increased levels of CaM mRNA and protein in leaves compared to wild-type plants. These transgenic plants have been reported previously to express several wound-inducible defense-related genes in the absence of wounding. Calmodulin mRNA and protein levels were found to increase in leaves of young wild-type tomato plants after wounding, or treatment with systemin, methyl jasmonate, or linolenic acid. CaM mRNA appeared within 0.5 h after wounding or supplying young tomato plants with systemin, and peaked at 1 h. The timing of CaM gene expression is similar to the expression of the wound- or systemin-induced lipoxygenase and prosystemin genes, signal pathway genes whose expression have been reported to begin at 0.5–1 h after wounding and 1–2 h earlier than the genes coding for defensive proteinase inhibitor genes. The similarities in timing between the synthesis of CaM mRNA and the mRNAs for signal pathway components suggests that CaM gene expression may be associated with the signaling cascade that activates defensive genes in response to wounding.     

The expression of tomato prosystemin gene in tobacco plants highly affects host proteomic repertoire

Systemin, an octadecapeptide isolated from tomato, is a primary signal molecule involved in the local and systemic responses to pest attack, elicited by activation of a set of defence genes. It derives from processing of prosystemin, a prohormone of almost 200 amino acids. Prosystemin orthologues have been found in other Solanaceae species but not in tobacco, where are present hydroxyproline-rich peptides functionally but not structurally related to tomato systemin. Molecular events leading to the release of signalling peptides from protein precursors are unknown in plants; the occurrence of a family of signal molecules suggests that initiation of wound response may involve different processing mechanisms. It has been previously shown that the protein product from an engineered tomato prosystemin gene is processed in tobacco, thus suggesting that the components responsible for its post-translational modifications are present in this species. By analyzing analysing the proteome repertoire of transformed tobacco plant leaves with 2-DE, here we demonstrate that the constitutive expression of the tomato prosystemin gene highly affected host protein synthesis. In particular, engineered plants showed a number of differentially synthesized proteins that were identified by PMF MALDI-TOF and microLC-ESI-IT-MS/MS experiments as polypeptide species involved in protection from pathogens and oxidative stress, or in carbon/energy metabolism. Significant differences in over-produced proteins were observed with respect to previous data reported on systemin-engineered tomato plants. Our results strongly support the need of using proteomic approaches during systematic analysis of plant tissues to investigate the principle of substantial equivalence in transgenic plants expressing a transgene coding for a signalling molecule.     

Systemin-inducible defence against pests is costly in tomato

The possible costs of inducible defences against pests were evaluated in tomato. To activate inducible resistance traits, we used transgenic plants that over-expressed the systemin precursor (prosystemin). The constitutive expression of the prosystemin, which is normally induced by herbivores in tomato, allowed the measurement of the impact of induced defences in a pest-free environment. The results showed that the continuous activation of traits that are normally induced by pests should be costly, affecting the growth, physiology and reproductive success of tomato plants.     

The organization of the prosystemin gene

The organization of the gene encoding tomato prosystemin, a 200 amino acid protein precursor of the 18 amino acid polypeptide inducer of proteinase inhibitor synthesis in tomato and potato plants, is reported. The prosystemin sequence reveals that the gene, which is composed of five homologous pairs of exons plus a non-homologous exon at the C-terminus containing the systemin sequence, has evolved by several gene duplication-elongation events from a much smaller ancestral gene. The nucleotide and amino acid sequence homologies among the exons suggest that a small ancestral gene was duplicated to form at least two tandem repeats, followed by subsequent duplication-elongation events that resulted in five tandemly repeated nucleotide sequences and three duplicated amino acid sequence elements. Since the systemin nucleotide or amino acid sequence was not duplicated, it was either not part of the gene duplication-elongation events or its coding region evolved separately and may even have been added to the tandemly repeated part of the gene at a later time.     

Tomato prosystemin promoter confers wound-inducible, vascular bundle-specific expression of the β-glucuronidase gene in transgenic tomato plants

Tomato (Lycopersicon esculentum Mill. cv. Better Boy) plants were transformed with a fused gene containing a 2.2-kb promoter fragment of the tomato prosystemin gene and the coding region of the β-glucuronidase (GUS) reporter gene. The transgenic plants exhibited a low constitutive level of prosystemin-β-glucuronidase gene expression, assayed by histochemical staining and GUS enzyme activity, that was associated in the vascular bundles of leaf main veins, petiolules, petioles and stems. The GUS activity in the vascular bundles in each tissue was increased by wounding and by treatment of the plants with methyl jasmonate, similar to the induction of prosystemin in wild-type plants. The increase in GUS activity in the vascular bundles of leaves in response to wounding correlated with the wound-inducible increase in prosystemin mRNA. Tissue printing, using rabbit anti-serum prepared against prosystemin, confirmed that inducible prosystemin protein was localized in vascular bundles of petiolules, petioles and stems of wild-type tomato plants. The evidence indicates that the 2.2-kb promoter region of the tomato prosystemin gene contains elements conferring its correct temporal and spatial expression in the vascular bundles of transgenic tomato plants. Received: 7 January 1997 / Accepted: 2 April 1997     

Positional specificity of a phospholipase A activity induced by wounding, systemin, and oligosaccharide elicitors in tomato leaves

Phospholipase A (PLA) activity, as measured by the accumulation of (14)C-lysophosphatidylcholine in leaves of tomato plants, increased rapidly and systemically in response to wounding. The increase in PLA activity in the systemic unwounded leaves was biphasic in wild-type tomato plants, peaking at 15 min and again at 60 min, but the second peak of activity was absent in transgenic prosystemin antisense plants. Supplying young excised tomato plants with the polypeptide hormone systemin also caused (14)C-lysophosphatidylcholine to increase to levels similar to those induced by wounding, but the increase in activity persisted for >2 hr. Antagonists of systemin blocked both the release of (14)C-lysophosphatidylcholine and the accumulation of defense proteins in response to systemin. (14)C-lysophosphatidylcholine levels did not increase in response to jasmonic acid. Chemical acylation of the lysophosphatidylcholine produced by wounding, systemin, and oligosaccharide elicitors followed by enzymatic hydrolysis with lipases of known specificities demostrated that the lysophosphatidylcholine is generated by a PLA with specificity for the sn-2 position.     

The expression of tomato prosystemin in Escherichia coli: A structural challenge.

Prosystemin is the 200-amino-acid prohormone of the 18-amino-acid polypeptide called systemin, a systemic mobile signal that activates the synthesis of defense genes in solanaceous plants in response to herbivore attacks. The unusual primary structural features of the tomato prosystemin cDNA and protein provided an extraordinary challenge in devising an expression system to obtain the full-length protein. Prosystemin expression inhibited the growth of a eukaryotic and several prokaryotic hosts used. Prosystemin was initially synthesized as a truncated protein of 185 amino acids in length using a T7 RNA polymerase expression system in E. coli strain BL21[DE3]. The truncation was found to be due to two factors: (1) the intramolecular associations of the 5' coding region of the prosystemin sequence with the expression vector's ribosome binding site and (2) the presence of a translation start site just prior to the amino acid methionine at position 15. Mutations that permitted the synthesis of the full-length prosystemin protein were introduced into the amino-terminal 5' coding region of the prosystemin cDNA. A 199-amino-acid recombinant prosystemin lacking the N-terminal methionine was purified from lysates and confirmed by N-terminal amino acid sequence and immunoblot analysis.     

The cellular localization of prosystemin: a functional role for phloem parenchyma in systemic wound signaling

The systemin precursor, prosystemin, has been previously shown to be sequestered in vascular bundles of tomato (Lycopersicon esculentum Mill.) plants, but its subcellular compartmentalization and association with a specific cell type has not been established. We present in situ hybridization and immunocytochemical evidence at the light, confocal, and transmission electron microscopy levels that wound-induced and methyl jasmonate-induced prosystemin mRNA and protein are exclusively found in vascular phloem parenchyma cells of minor veins and midribs of leaves, and in the bicollateral phloem bundles of petioles and stems of tomato. Prosystemin protein was also found constitutively in parenchyma cells of various floral organs, including sepals, petals and anthers. At the subcellular level, prosystemin was found compartmentalized in the cytosol and the nucleus of vascular parenchyma cells. The cumulative data indicate that vascular phloem parenchyma cells are the sites for the synthesis and processing of prosystemin as a first line of defense signaling in response to herbivore and pathogen attacks.Abbreviations IgG immunoglobulin - TEM transmission electron microscope     

Systemin-dependent salinity tolerance in tomato: evidence of specific convergence of abiotic and biotic stress responses

Plants have evolved complex mechanisms to perceive environmental cues and develop appropriate and coordinated responses to abiotic and biotic stresses. Considerable progress has been made towards a better understanding of the molecular mechanisms of plant response to a single stress. However, the existence of cross-tolerance to different stressors has proved to have great relevance in the control and regulation of organismal adaptation. Evidence for the involvement of the signal peptide systemin and jasmonic acid in wound-induced salt stress adaptation in tomato has been provided. To further unravel the functional link between plant responses to salt stress and mechanical damage, transgenic tomato ( Lycopersicon esculentum Mill.) plants constitutively expressing the prosystemin cDNA have been exposed to a moderate salt stress. Prosystemin over-expression caused a reduction in stomatal conductance. However, in response to salt stress, prosystemin transgenic plants maintained a higher stomatal conductance compared with the wild-type control. Leaf concentrations of abscissic acid (ABA) and proline were lower in stressed transgenic plants compared with their wild-type control, implying that either the former perceived a less stressful environment or they adapted more efficiently to it. Consistently, under salt stress, transgenic plants produced a higher biomass, indicating that a constitutive activation of wound responses is advantageous in saline environment. Comparative gene expression profiling of stress-induced genes suggested that the partial stomatal closure was not mediated by ABA and/or components of the ABA signal transduction pathway. Possible cross-talks between genes involved in wounding and osmotic stress adaptation pathways in tomato are discussed.     

Suppressors of systemin signaling identify genes in the tomato wound response pathway.

In tomato plants, systemic induction of defense genes in response to herbivory or mechanical wounding is regulated by an 18-amino-acid peptide signal called systemin. Transgenic plants that overexpress prosystemin, the systemin precursor, from a 35S::prosystemin (35S::prosys) transgene exhibit constitutive expression of wound-inducible defense proteins including proteinase inhibitors and polyphenol oxidase. To study further the role of (pro)systemin in the wound response pathway, we isolated and characterized mutations that suppress 35S::prosys-mediated phenotypes. Ten recessive, extragenic suppressors were identified. Two of these define new alleles of def-1, a previously identified mutation that blocks both wound- and systemin-induced gene expression and renders plants susceptible to herbivory. The remaining mutants defined four loci designated Spr-1, Spr-2, Spr-3, and Spr-4 (for Suppressed in 35S::prosystemin-mediated responses). spr-3 and spr-4 mutants were not significantly affected in their response to either systemin or mechanical wounding. In contrast, spr-1 and spr-2 plants lacked systemic wound responses and were insensitive to systemin. These results confirm the function of (pro)systemin in the transduction of systemic wound signals and further establish that wounding, systemin, and 35S::prosys induce defensive gene expression through a common signaling pathway defined by at least three genes (Def-1, Spr-1, and Spr-2).     

Systemic signaling in tomato plants for defense against herbivores. Isolation and characterization of three novel defense-signaling glycopeptide hormones coded in a single precursor gene

An 18-amino acid peptide in tomato leaves called systemin is a primary signal released at wound sites in response to herbivory that systemically signals the activation of defense genes throughout the plants. We report here the isolation of three hydroxyproline-rich glycopeptides from tomato leaves, of 20, 18, and 15 amino acids in length, that signal the activation of defense genes, similar to the activity of the systemin peptide. The three new peptides cause an alkalinization of suspension-cultured cells and induce the synthesis of defensive proteinase inhibitor proteins when supplied at fmol levels to young tomato plants through their cut stems. This suggests that they are part of the wound signaling of tomato plants that activates defense against herbivores and pathogens. Isolation of cDNAs coding for the tomato peptides revealed that they are all derived from the same pre-proprotein precursor that is systemically wound-inducible. The peptides are considered members of the functionally characterized systemin family of defense signals from plants that are synthesized both in wounded leaves and in distal, unwounded leaves in response to herbivory or other mechanical wounding. The precursor deduced from the cDNA exhibits a leader sequence, indicating that it is synthesized through the secretory pathway, where it is hydroxylated and glycosylated. The amino acid sequence of the precursor exhibited weak identity to the precursor of two hydroxyproline-rich defense signals recently found in tobacco, suggesting that the two pre-protein precursors have evolved from a common ancestral protein. The identification of hydroxyproline-rich glycoprotein systemins in tomato indicates that the initiation of wound signaling is more complex than previously thought and appears to involve multiple peptide signals.