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     

Changes in extracellular pH are neither required nor sufficient for activation of mitogen-activated protein kinases (MAPKs) in response to systemin and fusicoccin in tomato

Leaf wounding and the wound signaling peptide systemin induce expression of wound response genes while the fungal toxin fusicoccin (FC) induces expression of pathogenesis-related genes. Consistent with their functional differences, FC and systemin regulate the extracellular pH in opposite ways, with systemin inducing an alkalinization and FC an acidification response. Here we show that systemin, wounding and FC activate the same mitogen-activated protein kinases (MAPKs; MPKs) MPK1 and 2 in tomato (Lycopersicon esculentum) leaves and L. peruvianum suspension-cultured cells. Wounding and FC activated an additional MAPK, MPK3. Pronounced differences were observed with regard to MAPK activation kinetics. FC induced prolonged, and systemin transient activity of the MAPKs. This shows that functionally different elicitors engage the same signaling components, yet induce signal-specific activation dynamics. A comparative analysis of pH effects and MAPK activity in response to specific treatments revealed that the kinetics of pH changes and MAPK activation did not correlate. Simultaneous application of FC and systemin did not lead to immediate pH changes but resulted in rapid increases in MAPK activity. Furthermore, changes in extracellular pH could be induced without concomitant MAPK activation by exchanging conditioned medium with fresh medium. This shows that changes in the extracellular pH are neither required nor sufficient for MAPK activation, suggesting that signaling pathways involving MAPKs and extracellular pH changes operate in parallel and are not part of the same linear pathway.     

Tomato BRASSINOSTEROID INSENSITIVE1 is required for systemin-induced root elongation in Solanum pimpinellifolium but is not essential for wound signaling

The tomato Leu-rich repeat receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) has been implicated in both peptide (systemin) and steroid (brassinosteroid [BR]) hormone perception. In an attempt to dissect these signaling pathways, we show that transgenic expression of BRI1 can restore the dwarf phenotype of the tomato curl3 (cu3) mutation. Confirmation that BRI1 is involved in BR signaling is highlighted by the lack of BR binding to microsomal fractions made from cu3 mutants and the restoration of BR responsiveness following transformation with BRI1. In addition, wound and systemin responses in the cu3 mutants are functional, as assayed by proteinase inhibitor gene induction and rapid alkalinization of culture medium. However, we observed BRI1-dependent root elongation in response to systemin in Solanum pimpinellifolium. In addition, ethylene perception is required for normal systemin responses in roots. These data taken together suggest that cu3 is not defective in systemin-induced wound signaling and that systemin perception can occur via a non-BRI1 mechanism.     

Enhancement of wound-induced ethylene synthesis by ethylene in preclimacteric cantaloupe

Although intact fruits of unripe cantaloupe (Cucumis melo L.) produce very little ethylene, a massive increase in ethylene production occurred in response to excision. The evidence indicates that this wound ethylene is produced from methionine via 1-aminocyclopropanecarboxylic acid (ACC) as in ripening fruits. Excision induced an increase in both ACC synthase and the enzyme converting ACC to ethylene. Ethylene further increased the activity of the enzyme system converting ACC to ethylene. The induction by ethylene required a minimum exposure of 1 hour; longer exposure had increasingly larger effect. The response was saturated at approximately 3 microliters per liter ethylene and was inhibited by Ag⁺. Neither ethylene nor ACC had a promotive or inhibitory effect on ACC synthase beyond the effect attributable to wounding.     

Tissue-type specific systemin perception and the elusive systemin receptor

Systemin is a wound signaling peptide from tomato that is important for plant defenses against herbivory. The systemin receptor was initially identified as the tomato homolog of the brassinosteroid receptor BRI1, but genetic evidence argued against this finding. However, we found that BRI1 may function as an inappropriate systemin binding protein that does not activate the systemin signaling pathway. Here we provide evidence that systemin perception is localized in a tissue-type specific manner. Mesophyll protoplasts were not sensitive to systemin, while they responded to other elicitors. We hypothesize that the elusive systemin receptor is a protein with high similarity to BRI1 which is specifically localized in vascular tissue like the systemin precursor prosystemin. Binding of systemin to BRI1 may be an artifact of transgenic BRI1-overexpressing plants, but does not take place in wild type tomato cells.Key words: systemin, systemin receptor, brassinosteroids, BRI1, BRL, protoplastsSystemin is thought to be processed from its precursor prosystemin upon insect attack and wounding of tomato leaves. Strong evidence has been gathered for an important role of (pro-)systemin in the activation of defenses against insects, and the underlying signaling pathway has been studied in detail.¹ However, the perception of systemin is controversial. Meindl et al.² and Scheer and Ryan³ identified high affinity, saturable, reversible and specific cell surface binding sites on Solanum peruvianum suspension-cultured cells which are known to be highly sensitive to systemin.⁴ A purification approach using a photoaffinity systemin analog identified a 160 kDa protein as the systemin receptor (SR160).⁵ Follow-up studies showed that overexpression of tomato 35S::SR160 in systemin-insensitive tobacco plants conferred systemin sensitivity to tobacco.⁶ Surprisingly, SR160 turned out to be the tomato homolog of the brassinosteroid receptor BRI1,⁷ which raised many questions as to the functionality of a receptor for two structurally and functionally diverse ligands. It was then shown in two independent papers that a null mutant for tomato BRI1, cu-3, exhibited a normal response to systemin.^8,9 This was strong evidence that SR160/BRI1 does not represent the functional systemin receptor. Our recent data added a peculiar twist to this story. We found that overexpression of tomato BRI1 in tobacco suspension-cultured cells resulted in binding of a fluorescently labeled systemin to the plasma membranes of the transgenic tobacco cells, but not to wild type cells. Surprisingly, this did not result in BRI1-dependent signal transduction and activation of a defense response, although we detected weak BRI1-independent signaling responses to systemin.¹⁰ Together with the identification of BRI1 as the systemin receptor by Scheer and Ryan,⁵ the simplest explanation for this phenomenon is that BRI1 is a systemin binding protein, but not the physiological systemin receptor.Therefore and for other reasons, we suggested that the true systemin receptor may be a protein with very similar properties as BRI1, e.g., a homolog of the BRI1-like (BRL) proteins. The purification strategy employed by Scheer and Ryan5 may have resulted in binding of a photoaffinity-systemin derivative to BRI1 and one or more BRL proteins. Since BRLs and BRI1 have a very similar MW, multiple bands on a SDS-PAGE would not be detectable.Here, we would like to add another aspect of systemin perception. We provide evidence for tissue-specific systemin sensitivity and discuss how this may affect systemin binding to BRI1 and the elusive systemin receptor. Prosystemin is only present in phloem parenchyma cells.¹¹ It can be surmised that the systemin receptor is located close to these cells. Systemin perception results in JA synthesis in companion cells of vascular bundles.¹² Since JA or a JA derivative is the most likely phloem-mobile candidate for a systemic long-distance wound signal, it is thought that JA is moving from companion cells into sieve cells to reach distant parts of the plant for upregulation of wound response genes in leaf cells, including mesophyll cells.^13–15Here, we tested the hypothesis that mesophyll cells lack systemin perception. We generated mesophyll protoplasts from tomato leaf material as well as protoplasts from S. peruvianum suspension-cultured cells, the same cell line that had been used for the purification of SR160/BRI1 and is known to be highly sensitive to systemin. Mesophyll protoplasts showed increased phosphorylation of MAP kinases (MPKs) in response to the elicitors flg22 and chitosan, bacterial and fungal MAMPs, respectively. However, they did not respond to systemin. In contrast, the S. peruvianum protoplasts did respond to systemin and to flg22, demonstrating that the protoplasting procedure did not compromise the systemin perception mechanism (Fig. 1). Immunocomplex kinase assays with specific antibodies against tomato MPK2 produced similar results (data not shown). Since flg22, chitosan and systemin activated the same MPKs (Fig. 1), our data indicate that systemin perception is absent in mesophyll protoplasts. Our leaf protoplasting protocol is a modification of the protocol by Yoo et al. which results in the generation of mesophyll protoplasts.¹⁶ In contrast, suspension-cultured cells do not normally represent specific cell types and it is not known why the S. peruvianum cells are highly sensitive to systemin.Open in a separate windowFigure 1Absence of systemin-induced MPK phosphorylation in mesophyll cells. Protoplasts were generated (protocol available upon request) from S. peruvianum suspension-cultured cells and from S. lycopersicum cv. MicroTom leaves. After a 1.5 hour recovery phase on ice, protoplasts were resuspended in WI medium (0.5 M mannitol, 5 mM ME S pH 5.7, 20 mM KCl), recovered for 1 hour in non-stick tubes with constant rotation on a rotary shaker at room temperature, and then treated with either water (con), 10 nM systemin (sys), 100 nM flg22, or 2.5 µg/ml chitosan (from crab shells—chi) for 10 min at room temperature. Protoplasts were analyzed for MPK phosphorylation by immunoblotting using an anti-phospho-ER K antibody (phospho-p44/42 MA PK (Erk1/2) (Thr202/Tyr204); D13.14.4E; Cell Signaling Technology) at a dilution of 1:2,000. This antibody recognizes MPKs that are phosphorylated on either the Thr and Tyr or on only the Thr within the TE Y phosphorylation motif which is conserved among plant and metazoan MPKs. It is known to recognize the tobacco MPKs SIPK and WIPK²¹ and Arabidopsis MPK6 and MPK3,²² the orthologs of tomato MPK1/2 and MPK3.²³ Bands were visualized as described.¹⁰ Proteins on membranes were stained with Ponceau S to demonstrate equal loading.Intriguingly, BRL1, BRL2 and BRL3 are expressed in the vasculature and function in vascular pattern formation in Arabidopsis, while BRI1 is ubiquitously expressed in dividing and elongating cells. BRL3 is even specifically expressed in phloem cells.¹⁷ This matches the highly specific localization of prosystemin in the phloem parenchyma cells.^11,18 The highest BRI1 expression is found in growing parts of young leaves^17,19 while prosystemin is specifically present in the phloem parenchyma cells throughout all developmental stages.¹¹ In this context, it is also interesting to note that application of systemin to tomato plants via the cut stem results in rapid and strong MPK activation. In this assay, systemin is delivered to leaf cells via the transpiration stream and therefore present in vascular tissue.²⁰Based on the combined evidence, we propose that the true systemin receptor is a BRL or similar protein which is expressed in phloem cells in the vicinity of the parenchyma cells that express prosystemin, but not in mesophyll cells. Because of the similarity between BRLs and BRI1, BRI1 was erroneously identified as the systemin receptor. Inappropriate binding of systemin to BRI1 is consistent with the high similarity between BRI1 and BRLs. However, because of the tissue-specificity of the systemin signaling pathway, inappropriate binding of systemin to BRI1 may rarely occur in wild type plants and may not pose an interference problem for either systemin or brassinosteroid signaling.     

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.The authors acknowledge the Washington State University Electron Microscope Center and staff for their technical advice and collaboration. We also thank Greg Wichelns for growing our plants and Dr. Steven Doares for providing [³H]systemin. This research was supported in part by the Washington State College of Agriculture and Home Economics Project No. 1791 and National Science Foundation grants IBN 9117795 and IBN 9104542     

No difference in the regulation pattern of calcium on ethylene biosynthesis between wild-type and never-ripe tomato fruit at mature green stage

This work investigated how calcium regulates the ethylene biosynthesis in the fruits of wild-type tomato (Lycopersicon esculentum L.) and their ethylene receptor never-ripe (Nr) mutants. In Nr tomato, the ethylene perception was blocked. When both materials were treated with calcium, the content of 1-aminocyclopropane-1-carboxylic acid (ACC)/malonyl-ACC and the activity of ACC oxidase (ACO) in tomato fruit discs increased, whereas the production of ethylene, content of malondialdehyde, and membrane permeability decreased. Calcium treatment did not affect the activity of ACC synthase, which is the first committed step in the ethylene biosynthesis pathway. The expression of LeACO1 in mature green fruit was inhibited significantly by calcium treatment in wild-type and Nr tomatoes, but the expression of LeACS2, the key ACC synthase gene in ethylene synthesis during tomato fruit maturing, was not affected. These results revealed that the effect of calcium on ethylene biosynthesis in tomato mature green fruit was independent of ethylene perception. The results also revealed that the targeting step of calcium preventing ethylene production was located at the ACC conversion to ethylene, by means of inhibiting ACC availability for ACO through enhancing cell membrane integrity and by means of preventing LeACO1 gene expression. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 1, pp. 60–67. The text was submitted by the authors in English.     

Proteinase inhibitors I and II in fruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal

The juice of unripe fruit from a wild species of tomato, Lycopersicon peruvianum (L.) Mill., LA 107, contains over 50% of its soluble proteins as the sum of two proteinase inhibitors. These are the highest levels of proteinase inhibitors and highest percentage of soluble proteins as proteinase inhibitors of any plant or animal tissue found to date. Fruit of the modern tomato, L. esculentum Mill., contains only negligible quantities of the two inhibitors. The two proteinase inhibitors in the fruit of L. peruvianum are members of the Inhibitor I and II families previously found in potato tubers and in leaves of wounded potato and tomato plants. The levels of the two inhibitors in the unripe fruit decrease significantly during ripening. Unripe fruit from other wild Lycopersicon species such as L. parviflorum Rick, Kesicki, Fobes et Holle, L. hirsutum Humb. et Bonpe., L. pimpinellifolium Mill., and other lines of L. peruvianum contain moderate levels of the inhibitors that also decrease during ripening. Another wild tomato species, L. pennellii Corr., is similar to L. esculentum in not containing the two proteinase inhibitors in either unripe or ripe fruit. The transient levels of the inhibitors in fruit of wild species indicate that they are present in unripe fruit as defensive chemicals against insects, birds or small mammals and their disappearance during ripening may render them edible to facilitate seed dispersal. High levels of mRNAs coding for Inhibitors I and II in unripe fruit of L. peruvianum, LA 107, indicate that strong promoters may regulate the developmentally expressed proteinase-inhibitor genes in tomato fruit that may have a substantial potential for use in genetic-engineering experiments to enhance the production of large quantities of proteinase inhibitors or other proteins in field tomatoes.Abbreviations poly(A)⁺ mRNA polyadenylated mRNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis Project 1791, College of Agriculture and Home Economics Research Center, Washington, State University     

Tomato BRI1 and systemin wound signalling

Brassinosteroids (BRs) are perceived by Brassinosteroid Insensitive 1 (BRI1), that encodes a leucine-rich repeat receptor kinase. Tomato BRI1 has previously been implicated in both systemin and BR signalling. The role of tomato BRI1 in BR signalling was confirmed, however it was found not to be essential for systemin/wound signalling. Tomato roots were shown to respond to systemin but this response varied according to the species and growth conditions. Overall the data indicates that mutants defective in tomato BRI1 are not defective in systemin-induced wound signalling and that systemin perception can occur via a non-BRI1 mechanism.Key words: tomato BRI1, brassinosteroids, systemin, wound signallingBrassinosteroids (BRs) are steroid hormones that are essential for normal plant growth. The most important BR receptor in Arabidopsis is BRASSINOSTERIOD INSENSITIVE 1 (BRI1), a serine/threonine kinase with a predicted extracellular domain of ∼24 leucine-rich repeats (LRRs).^1,2 BRs bind to BRI1 via a steroid-binding domain that includes LRR 21 and a so-called “island” domain.^2,3 In tomato a BRI1 orthologue has been identified that when mutated, as in the curl3 (cu3) mutation, results in BR-insensitive dwarf plants.⁴ Tomato BRI1 has also been purified as a systemin-binding protein.⁵ Systemin is an eighteen amino acid peptide, which is produced by post-translational cleavage of prosystemin. Systemin has been implicated in wound signalling and is able to induce the production of jasmonate, protease inhibitors (PIN) and rapid alkalinization of cell suspensions (reviewed in ref. ⁶).To clarify whether tomato BRI1 was indeed a dual receptor it was important to first confirm its role in BR signalling. Initially this was carried out by genetic complementation of the cu3 mutant phenotype.⁷ Overexpression of tomato BRI1 restored the dwarf phenotype and BR sensitivity and normalized BR levels (

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

Ca2+ effects on ethylene, carbon dioxide and 1-aminocyclopropane-1-carboxylic acid synthase activity

The response of pericarp disks from ripening tomato (Lycopersicon esculentum Mill. cv. Traveler‘76) to CaCl₂, additions was studied to determine the effect of Ca²⁺ on ethylene and CO₂ production. Application of 5 mM CaCl₂ resulted in a 2, 20, 33, 39, and 50% increase in ethylene production in disks obtained from preclimacteric minimum, climacteric rise, climacteric peak, one, and two days postclimacteric fruit, respectively. CaCl₂ concentrations of 10 and 50 mM gave no additional stimulation of ethylene production; CO₂ production at 5 mM CaCl₂ was not different from controls, but is increased at 10 and 50mM CaCl₂. CaCl₂ also increased ethylene production in disks treated with 1-aminocyclopropane-1-carboxylic acid (ACC) or aminoethoxy-vinylglycine. Chloride salts of K⁺, Na⁺, Mg²⁺, Sr²⁺ and La³⁺ did not stimulate ethylene production. SrCl₂ stimulated ethylene production to a lesser degree than CaCl₂. Disks from potato (Solanum tuberosum L. cv. Katahdin) tubers produced greater quantities of ethylene and ACC when 5 mM CaCl₂ was included in the incubation medium (K. B. Evensen, 1983. Physiol. Plant. 60:125–128). Ca²⁺-treated disks had more than three times as much ACC synthase activity as control disks after 18 to 24 h incubation, when ethylene and ACC were maximal. The apparent K_m for S-adenosylmethionine was 13 μM at 29°C, pH 8.0 in extracts from both Ca²⁺-treated and control disks. Inclusion of 1 to 50 mM CaCl₂ in the assay medium did not significantly affect enzyme activity. ACC synthase extracted from control and Ca²⁺-treated disks had a pH optimum of 8.5 and an apparent molecular weight of 72 kdalton, estimated by gel filtration. It is likely that the presence of Ca²⁺ in the buffer allows greater synthesis of ACC synthase as part of the wound-healing response in potato, while in tomato the predominant effect is on membrane stabilization.     

Ultraviolet-B activates components of the systemin signaling pathway in Lycopersicon peruvianum suspension-cultured cells

Among the early responses of Lycopersicon peruvianum suspension-cultured cells to the polypeptide wound signal systemin are the alkalinization of the culture medium and the activation of a 48-kDa mitogen-activated protein kinase (MAPK). Here, we report that both responses are induced in the cells by exposure to ultraviolet-B (UV-B) radiation. Suramin, an inhibitor of systemin receptor function, strongly inhibited the UV-B-induced medium alkalinization and MAPK activity. The UV-B response was also reduced when cells were initially treated with systemin or the systemin antagonist Ala-17-systemin, which competitively inhibits binding of systemin to the systemin receptor. Cells that were initially treated with either UV-B or systemin exhibited a strongly reduced response to a subsequent elicitation with systemin. The desensitization was transient, reaching a maximum at 30-60 min after the initial treatment. Several hours later, depending on the initial UV-B dose or systemin concentration, the cells regained their initial responsiveness. When cells were irradiated with low doses of UV-B and subsequently treated with systemin, the UV-B response reached levels higher than the response without UV-B treatment. The data provide evidence for an involvement of the systemin receptor and/or systemin-responsive signaling elements in the UV-B response.     

Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants.

Systemin is an important mediator of wound-induced defense gene activation in tomato plants, and it elicits a rapid alkalinization of the growth medium of cultured Lycopersicon peruvianum cells. A possible mechanistic link between proton fluxes across the plasma membrane and the induction of defense genes was investigated by modulating plasma membrane H+-ATPase activity. Inhibitors of H+-ATPase (erythrosin B, diethyl stilbestrol, and vanadate) were found to alkalinize the growth medium of L. peruvianum cell cultures and to induce wound response genes in whole tomato plants. Conversely, an activator of the H+-ATPase (fusicoccin) acidified the growth medium of L. peruvianum cell cultures and suppressed systemin-induced medium alkalinization. Likewise, in fusicoccin-treated tomato plants, the wound- and systemin-triggered accumulation of wound-responsive mRNAs was found to be suppressed. However, fusicoccin treatment of tomato plants led to the accumulation of salicylic acid and the expression of pathogenesis-related genes. Apparently, the wound and pathogen defense signaling pathways are differentially regulated by changes in the proton electrochemical gradient across the plasma membrane. In addition, alkalinization of the L. peruvianum cell culture medium was found to depend on the influx of Ca2+ and the activity of a protein kinase. Reversible protein phosphorylation was also shown to be involved in the induction of wound response genes. The plasma membrane H+-ATPase as a possible target of a Ca2+-activated protein kinase and its role in defense signaling are discussed.     

Systemin transiently depolarizes the tomato mesophyll cell membrane and antagonizes fusicoccin-induced extracellular acidification of mesophyll tissue

The plant polypeptide signal systemin induces proteinase inhibitor synthesis in tomato leaves. We show here that systemin elicits a transient depolarization of the tomato mesophyll cell membrane. Furthermore it triggers a transient decrease in the external pH of the mesophyll tissue which is followed by a sustained pH increase. In the presence of fusicoccin (which has been shown to antagonize the synthesis of proteinase inhibitors) the depolarization and transient H⁺ efflux are attenuated whereas the slower phase of the sustained electroneutral H⁺ influx persists. These results suggest that systemin-induced changes in ion transport play a role in the early phases of systemin signal transduction.     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Ethylene regulation of fruit ripening: Molecular aspects

Progress in ethylene regulating fruit ripening concerning itsperception and signal transduction and expression of ACC synthaseand ACC oxidase genes is reviewed. ACC synthase and ACC oxidasehave been characterized and their genes cloned from various fruittissues. Both ACC synthase and ACC oxidase are encoded bymultigene families, and their activities are associated withfruit ripening. In climacteric fruit, the transition toautocatalytic ethylene production appears to be due to a seriesof events in which ACC sythase and ACC oxidase genes have beenexpressed developmentally. Differential expression of ACCsynthase and ACC oxidase gene family members is probably involvedin such a transition that ultimately controls the onset of fruitripening.In comparison to ACC synthase and ACC oxidase, less is knownabout ethylene perception and signal transduction because of thedifficulties in isolating and purifying ethylene receptors orethylene-binding proteins using biochemical methods. However, theidentification of the Nr tomato ripening mutant as anethylene receptor, the applications of new potent anti-ethylenecompounds and the generation of transgenic fruits with reducedethylene production have provided evidence that ethylenereceptors regulate a defined set of genes which are expressedduring fruit ripening. The properties and functions of ethylenereceptors, such as ETR1, are being elucidated.Application of molecular genetics, in combination withbiochemical approaches, will enable us to better understand theindividual steps leading from ethylene perception and signaltransduction and expression of ACC synthase and ACC oxidase genefamily member to the physiological responses.     

Properties and Partial Purification of 1-Aminocyclopropane-1-carboxylate Synthase

We studied the regulation of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in tomato (Lycopersicon esculentum Mill.) fruit tissue and attempted the purification of this enzyme. The increase of ACC synthase activity in wounded tomato pericarp was inhibited by cordycepin and cycloheximide. Density labeling studies showed a 0.75% increase in the buoyant density of ACC synthase isolated from tomato pericarp tissue that had been incubated on ²H₂O as compared to ACC synthase from H₂O-treated tissue. These data are consistent with the hypothesis that ACC synthase is synthesized de novo following wounding of tomato pericarp tissue. SDS-gel electrophoresis and fluorography showed that the pattern of incorporation of l-[³⁵S]methionine into protein changed with time after wounding of the tissue. Radioactive protein bands that were not detected 1 hour after wounding, became apparent 2 to 3 hours after wounding.