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1.
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 相似文献
2.
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. 相似文献
3.
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. 相似文献
4.
5.
Enhancement of wound-induced ethylene synthesis by ethylene in preclimacteric cantaloupe 总被引:10,自引:4,他引:6
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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. 相似文献
6.
Sarah R Hind Robert Malinowski Roopa Yalamanchili Johannes W Stratmann 《Plant signaling & behavior》2010,5(1):42-44
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 leaves17,19 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. 相似文献
7.
Autoradiographic and biochemical evidence for the systemic translocation of systemin in tomato plants 总被引:9,自引:0,他引:9
Javier Narváez-Vásquez Gregory Pearce Martha L. Orozco-Cardenas Vincent R. Franceschi Clarence A. Ryan 《Planta》1995,195(4):593-600
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 相似文献
8.
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. 相似文献
9.
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 相似文献
10.
Nicholas Holton Kate Harrison Takao Yokota Gerard J Bishop 《Plant signaling & behavior》2008,3(1):54-55
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 (35S:TomatoBRI1 complemented line Wt* cu3* 6-deoxocathasterone 566 964 676 6-deoxoteasterone nd 47 48 3-dehydro-6-deoxoteasterone 87 62 69 6-deoxotyphasterol nd 588 422 6-deoxocastasterone 1,755 6,247 26,210 castasterone 255 637 17,428 brassinolide nd nd nd