Javier Narváez-Vásquez

Hydrogen Peroxide Acts as a Second Messenger for the Induction of Defense Genes in Tomato Plants in Response to Wounding, Systemin, and Methyl Jasmonate

The systemic accumulation of both hydrogen peroxide (H2O2) and proteinase inhibitor proteins in tomato leaves in response to wounding was inhibited by the NADPH oxidase inhibitors diphenylene iodonium (DPI), imidazole, and pyridine. The expression of several defense genes in response to wounding, systemin, oligosaccharides, and methyl jasmonate also was inhibited by DPI. These genes, including those of four proteinase inhibitors and polyphenol oxidase, are expressed within 4 to 12 hr after wounding. However, DPI did not inhibit the wound-inducible expression of genes encoding prosystemin, lipoxygenase, and allene oxide synthase, which are associated with the octadecanoid signaling pathway and are expressed 0.5 to 2 hr after wounding. Accordingly, treatment of plants with the H2O2-generating enzyme glucose oxidase plus glucose resulted in the induction of only the later-expressed defensive genes and not the early-expressed signaling-related genes. H2O2 was cytochemically detected in the cell walls of vascular parenchyma cells and spongy mesophyll cells within 4 hr after wounding of wild-type tomato leaves, but not earlier. The cumulative results suggest that active oxygen species are generated near cell walls of vascular bundle cells by oligogalacturonide fragments produced by wound-inducible polygalacturonase and that the resulting H2O2 acts as a second messenger for the activation of defense genes in mesophyll cells. These data provide a rationale for the sequential, coordinated, and functional roles of systemin, jasmonic acid, oligogalacturonides, and H2O2 signals for systemic signaling in tomato plants in response to wounding.

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 14C-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 14C-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 14C-lysophosphatidylcholine and the accumulation of defense proteins in response to systemin. 14C-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 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.

Autoradiographic and biochemical evidence for the systemic translocation of systemin in tomato plants

The movement of systemin, the 18-amino-acid polypeptide inducer of proteinase inhibitors in tomato (Lycopersicon esculentum L.) plants, was investigated in young tomato plants following the application of [14C]systemin to wounds on the surface of leaves. Wholeleaf autoradiographic analyses revealed that [14C]systemin was distributed throughout the wounded leaf within 30 min, and then during the next several hours was transported to the petiole, to the main stem, and to the upper leaves. The movement of [14C]systemin was similar to the movement of [14C]sucrose when applied to leaf wounds, except that sucrose was slightly more mobile than systemin. Analyses of the radioactivity in the petiole phloem exudates at intervals over a 5-h period following the application of [14C]systemin to a wound demonstrated that intact [14C]systemin was present in the phloem over the entire time, indicating that the polypeptide was either stable for long periods in the phloem or was being continually loaded into the phloem from the source leaf. The translocation pathway of systemin was also investigated at the cellular level, using light microscopy and autoradiography. Within 15 min after application of [3H]systemin to a wound on a terminal leaflet, it was found distributed throughout the wounded leaf and was primarily concentrated in the xylem and phloem tissues within the leaf veins. After 30 min, the radioactivity was found mainly associated with vascular strands of phloem tissue in the petiole and, at 90 min, label was found in the phloem of the main stem. Altogether, these and previous results support a role for systemin as a systemic wound signal in tomato plants.

Leucine Aminopeptidase Regulates Defense and Wound Signaling in Tomato Downstream of Jasmonic Acid

Leucine aminopeptidase A (LapA) is a late wound-response gene of tomato (Solanum lycopersicum). To elucidate the role of LapA, transgenic plants that overexpressed or abolished LapA gene expression were used. The early wound-response gene RNA levels were similar in wild-type and Lap-silenced (LapA-SI), -antisense (LapA-AS), and -overexpressing (LapA-OX) plants. By contrast, late wound-response gene RNA levels and protection against Manduca sexta damage were influenced by LapA RNA and protein levels. While LapA-OX plants had elevated levels of LapA RNAs and protein, ectopic expression of LapA was not sufficient to induce Pin (Ser proteinase inhibitor) or PPO (polyphenol oxidase) transcripts in nonwounded leaves. M. sexta larvae damaged less foliage and displayed delays in growth and development when feeding on LapA-OX plants. By contrast, LapA-SI and LapA-AS lines had lower levels of Pin and PPO RNAs than wild-type controls. Furthermore, larvae consumed more foliage and attained larger masses when feeding on LapA-SI plants. Jasmonic acid (JA) did not complement the wound-signaling phenotype of LapA-SI plants. Based on root elongation in the presence of JA, JA perception appeared to be intact in LapA-SI lines. Collectively, these data suggested that LAP-A has a role in modulating essential defenses against herbivores by promoting late wound responses and acting downstream of JA biosynthesis and perception.

Proteinase-inhibitor synthesis in tomato plants: Evidence for extracellular deposition in roots through the secretory pathway

The cellular and subcellular localization of proteinase Inhibitors I and II proteins, synthesized in transgenic tomato (Lycopersicon esculentum L.) plants from chimeric genes regulated by the 35S promoter, was investigated by immunocytochemical techniques. Newly synthesized inhibitor proteins were deposited in the cell vacuoles as in wild-type plants, but were also secreted into the cell walls of outer epidermal and secretory cells of the root cap. The Na ionophore monensin increased the levels of proteinase inhibitors found in rough endoplasmic reticulum, Golgi cisternae and in the cell walls of transgenic plants, supporting a role for the secretory pathway in the sorting and targeting of Inhibitor I and II proteins. The two inhibitor proteins were detected by Western-blot analysis in water-washes obtained from roots of transgenic tomato seedlings, confirming their extracellular presence. Wild-type tomato plants exhibited the presence of Inhibitor I and II proteins in the external cell walls, using silver-enhanced immunogold labelling, but not by Western-blot analysis. The extracellular Inhibitor I from transgenic plant roots migrated in electrophoretic gels with a slightly different apparent mass than the Inhibitor I isolated from tomato leaf vacuoles, indicating that specific structural features of this inhibitor protein have been altered during or after extracellular deposition. The presence of extracellular inhibitors in roots may help provide protection for the growing meristems against insects or microorganisms present in the soil.

Differential expression of a chimeric CaMV-tomato proteinase Inhibitor I gene in leaves of transformed nightshade, tobacco and alfalfa plants.

The open reading frame and terminator region of a wound-inducible tomato Inhibitor I gene, regulated by the CaMV 35S promoter, was stably integrated into the genomes of nightshade (Solanum nigrum), tobacco (Nicotiana tabacum), and alfalfa (Medicago sativa), using an Agrobacterium-mediated transformation system. The expression of the foreign Inhibitor I gene in leaves of each species was studied at the mRNA and protein levels. The levels of Inhibitor I protein present in leaves of each species correlated with the levels of mRNA. The average levels of both mRNA and Inhibitor I protein were highest in leaves of transgenic nightshade plants (over 125 μg of Inhibitor I per g tissue), less in tobacco plants (about 75 μg/g tissue), and lowest in leaves of transgenic alfalfa plants (below 20 μg/g tissue). Inhibitor I protein was observed in all tissues throughout transgenic plant species, but inhibitor concentration per gram of tissue was 2–3 times higher in young developing leaf tissues and floral organs. The differences in the expression of the CaMV-tomato Inhibitor I gene among the different plant genera suggests that either the rate of transcription of the foreign gene or the rate of degradation of the nascent Inhibitor I mRNA varies among genera. Using electron microscopy techniques, the newly synthesized pre-pro-Inhibitor I protein was shown to be correctly processed and stored as a mature Inhibitor I protein within the central vacuoles of leaves of transgenic nightshade and alfalfa. The results of these experiments suggest that maximal expression of foreign proteinase inhibitor genes, and perhaps other foreign defense genes, may require gene constructs that are fashioned with promoters and terminators that allow maximum expression in the selected plant species.

A Sulfhydryl Reagent Modulates Systemic Signaling for Wound-Induced and Systemin-Induced Proteinase Inhibitor Synthesis.

The sulfhydryl group reagent p-chloromecuribenzene sulfonic acid (PCMBS), an established inhibitor of active apoplastic phloem loading of sucrose in several plant species, is shown to be a powerful inhibitor of wound-induced and systemin-induced activation of proteinase inhibitor synthesis and accumulation in leaves of tomato plants (Lycopersicon esculentum cv Castlemart). PCMBS, supplied to young tomato plants through their cut stems, blocks accumulation of proteinase inhibitors in leaves in response to wounding. The application of systemin directly to fresh wounds enhances systemic accumulation of proteinase inhibitors to levels higher than wounding alone. Placed on fresh wounds, PCMBS severely inhibits systemic induction of proteinase inhibitors, in both the presence and absence of exogenous systemin. PCMBS inhibition can be reversed by cysteine, dithiothreitol, and glutathione. Radiolabeled systemin placed on fresh wounds is readily transported from the wounded leaves to upper leaves. However, in the presence of PCMBS, radiolabeled systemin is not transported away from wound sites. Induction of proteinase inhibitor I synthesis by oligouronides (degree of polymerization [almost equal to] 20), linolenic acid, or methyl jasmonate was not inhibited by PCMBS. The cumulative data support a possible role for sulfhydryl groups in mediating the translocation of systemin from wound sites to distal receptor sites in tomato plants and further support a role for systemin as a systemic wound signal.

The systemin precursor gene regulates both defensive and developmental genes in Solanum tuberosum

Transformation of Solanum tuberosum, cv. Desiree, with the tomato prosystemin gene, regulated by the 35S cauliflower mosaic virus promoter, resulted in constitutive increase in defensive proteins in potato leaves, similar to its effects in tomato plants, but also resulted in a dramatic increase in storage protein levels in potato tubers. Tubers from selected transformed lines contained 4- to 5-fold increases in proteinase inhibitor I and II proteins, >50% more soluble and dry weight protein, and >50% more total nitrogen and total free amino acids than found in wild-type tubers. These results suggest that the prosystemin gene plays a dual role in potato plants in regulating proteinase inhibitor synthesis in leaves in response to wounding and in regulating storage protein synthesis in potato tubers in response to developmental cues. The results indicated that components of the systemin signaling pathway normally found in leaves have been recruited by potato plants to be developmentally regulated to synthesize and accumulate large quantities of storage proteins in tubers.

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.