Cloning the tomato curl3 gene highlights the putative dual role of the leucine-rich repeat receptor kinase tBRI1/SR160 in plant steroid hormone and peptide hormone signaling

Brassinosteroids (BRs) are plant steroid hormones that are essential for normal plant development. To gain better understanding of the conservation of BR signaling, the partially BR-insensitive tomato mutant altered brassinolide sensitivity1 (abs1) was identified and found to be a weak allele at the curl3 (cu3) locus. BR content is increased in both of these mutants and is associated with increased expression of DWARF: The tomato homolog of the Arabidopsis Brassinosteroid Insensitive1 Leu-rich repeat (LRR) receptor-like kinase, named tBri1, was isolated using degenerate primers. Sequence analysis of tBRI1 in the mutants cu3 and abs1 revealed that cu3 is a nonsense mutant and that abs1 is a missense mutant. A comparison of BRI1 homolog sequences highlights conserved features of BRI1 sequences, with the LRRs in close proximity to the island domain showing more conservation than N-terminal LRRs. The most homologous sequences were found in the kinase and transmembrane regions. tBRI1 (SR160) also has been isolated as the putative receptor for systemin, a plant peptide hormone. This finding suggests a possible dual role for tBRI1 in steroid hormone and peptide hormone signaling.     

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 (

Brassinosteroid signal transduction--choices of signals and receptors

Small signaling molecules that mediate cell-cell communication are essential for developmental regulation in multicellular organisms. Among them are the steroids and peptide hormones that regulate growth in both plants and animals. In plants, brassinosteroids (BRs) are perceived by the cell surface receptor kinase BRI1, which is distinct from the animal steroid receptors. Identification of components of the BR signaling pathway has revealed similarities to other animal and plant signal transduction pathways. Recent studies demonstrated that tomato BRI1 (tBRI1) perceives both BR and the peptide hormone systemin, raising new questions about the molecular mechanism and evolution of receptor-ligand specificity.     

The tomato mutant spr1 is defective in systemin perception and the production of a systemic wound signal for defense gene expression

Wound-induced systemic expression of defensive proteinase inhibitor (PI) genes in tomato plants requires the action of systemin and its precursor protein prosystemin. Although it is well established that systemin induces PI expression through the octadecanoid pathway for jasmonic acid (JA) biosynthesis, relatively little is known about how systemin and JA interact to promote long-distance signaling between damaged and undamaged leaves. Here, this question was addressed by characterizing a systemin-insensitive mutant (spr1) that was previously identified as a suppressor of prosystemin-mediated responses. In contrast to JA biosynthetic or JA signaling mutants that lack both local and systemic PI expression in response to wounding, spr1 plants were deficient mainly in the systemic response. Consistent with this phenotype, spr1 plants exhibited normal PI induction in response to oligosaccharide signals that are thought to play a role in the local wound response. Moreover, spr1 abolished JA accumulation in response to exogenous systemin, and reduced JA accumulation in wounded leaves to approximately 57% of wild-type (WT) levels. Analysis of reciprocal grafts between spr1 and WT plants showed that spr1 impedes systemic PI expression by blocking the production of the long-distance wound signal in damaged leaves, rather than inhibiting the recognition of that signal in systemic undamaged leaves. These experiments suggest that Spr1 is involved in a signaling step that couples systemin perception to activation of the octadecanoid pathway, and that systemin acts at or near the site of wounding (i.e. in rootstock tissues) to increase JA synthesis to a level that is required for the systemic response. It was also demonstrated that spr1 plants are not affected in the local or systemic expression of a subset of rapidly induced wound-response genes, indicating the existence of a systemin-independent pathway for wound signaling.     

Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase

Brassinosteroid (BR) mutants of Arabidopsis have pleiotropic phenotypes and provide evidence that BRs function throughout the life of the plant from seedling development to senescence. Screens for BR signaling mutants identified one locus, BRI1, which encodes a protein with homology to leucine-rich repeat receptor serine (Ser)/threonine (Thr) kinases. Twenty-seven alleles of this putative BR receptor have been isolated to date, and we present here the identification of the molecular lesions of 14 recessive alleles that represent five new mutations. BR-insensitive-1 (BRI1) is expressed at high levels in the meristem, root, shoot, and hypocotyl of seedlings and at lower levels later in development. Confocal microscopy analysis of full-length BRI1 fused to green fluorescent protein indicates that BRI1 is localized in the plasma membrane, and an in vitro kinase assay indicates that BRI1 is a functional Ser/Thr kinase. Among the bri1 mutants identified are mutants in the kinase domain, and we demonstrate that one of these mutations severely impairs BRI1 kinase activity. Therefore, we conclude that BRI1 is a ubiquitously expressed leucine-rich repeat receptor that plays a role in BR signaling through Ser/Thr phosphorylation.     

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).     

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.     

Brassinosteroid and systemin: two hormones perceived by the same receptor

Brassinosteroids, coordinating developmental events, and systemin, inducing systemic wound responses to attacks by insect pests, are newly recognized plant hormones that are perceived by plasma membrane-localized leucine-rich repeat receptor kinases. The recent characterization of the brassinosteroid receptor BRI1 from tomato revealed that this protein is identical to the previously isolated SR160 systemin receptor, strongly suggesting that both brassinosteroid and systemin signalling use the same surface receptor.     

Specifying the role of BAK1‐interacting receptor‐like kinase 3 in brassinosteroid signaling

Brassinosteroids (BR) are involved in the control of several developmental processes ranging from root elongation to senescence and adaptation to environmental cues. Thus, BR perception and signaling have to be precisely regulated. One regulator is BRI1‐associated kinase 1 (BAK1)‐interacting receptor‐like kinase 3 (BIR3). In the absence of BR, BIR3 forms complexes with BR insensitive 1 (BRI1) and BAK1. However, the biophysical and energetic requirements for complex formation in the absence of the ligand have yet to be determined. Using computational modeling, we simulated the potential complexes between the cytoplasmic domains of BAK1, BRI1 and BIR3. Our calculations and experimental data confirm the interaction of BIR3 with BAK1 and BRI1, with the BAK1 BIR3 interaction clearly favored. Furthermore, we demonstrate that BIR3 and BRI1 share the same interaction site with BAK1. This suggests a competition between BIR3 and BRI1 for binding to BAK1, which results in preferential binding of BIR3 to BAK1 in the absence of the ligand thereby preventing the active participation of BAK1 in BR signaling. Our model also suggests that BAK1 and BRI1 can interact even while BAK1 is in complex with BIR3 at an additional binding site of BAK1 that does not allow active BR signaling.     

BRL1, a leucine-rich repeat receptor-like protein kinase, is functionally redundant with BRI1 in regulating Arabidopsis brassinosteroid signaling

BRI1-like receptor kinase (BRL1) was identified as an extragenic suppressor of a weak bri1 allele, bri1-5, in an activation-tagging genetic screen for novel brassinosteroid (BR) signal transduction regulators. BRL1 encodes a leucine-rich repeat receptor-like protein kinase (LRR-RLK). Sequence alignment revealed that BRL1 is closely related to BRI1, which is involved in BR perception. Overexpression of a BRL1 cDNA, driven by a constitutive CaMV 35S promoter, recapitulates the bri1-5 suppression phenotypes, and partially complements the phenotypes of a null bri1 allele, bri1-4. Analysis of a BR-specific feedback response gene, CPD, indicates that BRL1 functions in BR signaling. BRL1 expression pattern overlaps with, but is distinct from, that of BRI1. In addition, both the expression level and in vitro kinase autophosphorylation activity of BRL1 are significantly lower than those of BRI1. bri1-5 brl1-1 double mutant plants have enhanced developmental defects relative to bri1-5 mutant plants, revealing that BRL1 plays a partially redundant role with BRI1 in controlling Arabidopsis growth and development. These findings enhance our understanding of functional redundancy and add an additional layer of complexity to RLK-mediated BR signaling transduction in Arabidopsis.     

The wound response mutant suppressor of prosystemin-mediated responses6 (spr6) is a weak allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1)

The systemic defense response of tomato plant in response to insect attack and wounding is regulated by the 18 amino acid peptide systemin and the phytohormone jasmonic acid (JA). Recent genetic analyses based mainly on spr (suppressors of prosystemin-mediated responses) mutant screens have led to the hypothesis that systemin acts at, or near, the site of wounding to amplify the production of JA, which in turn functions as a mobile signal to promote the systemic defense response. In order to identify more components involved in the systemin/JA-signaled defense response, we carried out a larger scale screen for new spr mutants in tomato. Here we describe the characterization of spr6, a mutant impaired in wound- and systemin-induced defense gene expression. Using a candidate gene approach based on genetic linkage, we demonstrate that spr6 is allelic to jai1-1, which is a loss-of-function allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. We show several aspects of the spr6 mutant phenotype distinct from that of jai1-1. First, the responsiveness of spr6 plants to exogenous JA shows a dosage dependency, i.e. it is more sensitive to JA than jai1-1 while less sensitive to JA than the wild-type. Secondly, unlike the sterile jai1-1, the spr6 plant displays normal fertility and seed set and thus can be maintained as a pure line and does not require selection. Therefore, spr6 provides a valuable tool, which can complement the limitations of jai1-1, to study JA signaling in tomato. The gene identification process of Spr6 we described herein represents an example showing the convenience of a candidate gene approach, based on genetic linkage, to identify gene functions of genetic loci defined by tomato wound response mutants.     

Genetic evidence for an indispensable role of somatic embryogenesis receptor kinases in brassinosteroid signaling

The Arabidopsis thaliana somatic embryogenesis receptor kinases (SERKs) consist of five members, SERK1 to SERK5, of the leucine-rich repeat receptor-like kinase subfamily II (LRR-RLK II). SERK3 was named BRI1-Associated Receptor Kinase 1 (BAK1) due to its direct interaction with the brassinosteroid (BR) receptor BRI1 in vivo, while SERK4 has also been designated as BAK1-Like 1 (BKK1) for its functionally redundant role with BAK1. Here we provide genetic and biochemical evidence to demonstrate that SERKs are absolutely required for early steps in BR signaling. Overexpression of four of the five SERKs-SERK1, SERK2, SERK3/BAK1, and SERK4/BKK1-suppressed the phenotypes of an intermediate BRI1 mutant, bri1-5. Overexpression of the kinase-dead versions of these four genes in the bri1-5 background, on the other hand, resulted in typical dominant negative phenotypes, resembling those of null BRI1 mutants. We isolated and generated single, double, triple, and quadruple mutants and analyzed their phenotypes in detail. While the quadruple mutant is embryo-lethal, the serk1 bak1 bkk1 triple null mutant exhibits an extreme de-etiolated phenotype similar to a null bri1 mutant. While overexpression of BRI1 can drastically increase hypocotyl growth of wild-type plants, overexpression of BRI1 does not alter hypocotyl growth of the serk1 bak1 bkk1 triple mutant. Biochemical analysis indicated that the phosphorylation level of BRI1 in serk1 bak1 bkk1 is incapable of sensing exogenously applied BR. As a result, the unphosphorylated level of BES1 has lost its sensitivity to the BR treatment in the triple mutant, indicating that the BR signaling pathway has been completely abolished in the triple mutant. These data clearly demonstrate that SERKs are essential to the early events of BR signaling.     

Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3beta-like kinase

Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3beta family. Unlike human glycogen synthase kinase 3beta, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways.     

Wound-induced RNaseLE expression is jasmonate and systemin independent and occurs only locally in tomato (Lycopersicon esculentum cv. Lukullus)

Tomato RNaseLE is induced by phosphate deficiency and wounding and may play a role in macromolecular recycling as well as wound healing. Here, we analyzed the role of jasmonate and systemin in the wound-induced RNaseLE activation. The rapid expression of RNaseLE upon wounding of leaves leading to maximal RNase activity within 10 h, appeared only locally. Jasmonic acid (JA) or its molecular mimic ethyl indanoyl isoleucine conjugate did not induce RNaseLE expression. Correspondingly, RNaseLE was expressed upon wounding of 35S::allene oxide cyclase antisense plants known to be JA deficient. RNaseLE was not expressed upon systemin treatment, but was locally expressed in the spr1 mutant which is affected in systemin perception. In tomato plants carrying a PromLE::uidA construct, GUS activity could be detected upon wounding, but not following treatment with JA or systemin. The data indicate a locally acting wound-inducible systemin- and JA-independent signaling pathway for RNaseLE expression.     

Micro-electrode flux estimation confirms that the Solanum pimpinellifolium cu3 mutant still responds to systemin

In this study, we introduce the Micro-Electrode Ion Flux Estimation technique as a sensitive and accurate technique to study systemin-induced changes in ion fluxes from isolated nearly intact plant tissues. Our results demonstrate the effectiveness and value of the Micro-Electrode Ion Flux Estimation technique to monitor and characterize those elicitor-induced ion flux changes from intact tissues. We used the method to monitor the systemin-induced changes in ion fluxes from leaf tissue of various plant species, including wild-type and cu3 mutant tomato (Solanum pimpinellifolium) plants, and confirm previous observations, but now in intact leaf tissue. Upon exposure of leaf tissue of plant species from the subtribe solaneae to systemin, the H(+) influx and K(+) efflux were transiently strongly increased. Plant species of other clades did not show a response upon systemin exposure. Although it has been reported that the gene containing the cu3 null mutation is identical to the SR160/tBRI1 gene, which encodes the systemin/brassinosteroid receptor and is essential in systemin and brassinosteroid perception, we observed no differences in the response of H(+) and K(+) fluxes from both wild-type and mutant leaf tissue to systemin. Also, the effects of various pharmacological effectors on systemin-induced flux changes were similar. Moreover, a SR160/tBRI1 transgene-containing tobacco (Nicotiana tabacum) line was insensitive to systemin, whereas both this line and its wild-type predecessor were responsive to the elicitor flg22. Our results support the conclusion that the Cu3 receptor of tomato is not the systemin receptor, and, hence, another receptor is the principal systemin receptor.     

A Mathematical Model for BRASSINOSTEROID INSENSITIVE1-Mediated Signaling in Root Growth and Hypocotyl Elongation

Brassinosteroid (BR) signaling is essential for plant growth and development. In Arabidopsis (Arabidopsis thaliana), BRs are perceived by the BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor. Root growth and hypocotyl elongation are convenient downstream physiological outputs of BR signaling. A computational approach was employed to predict root growth solely on the basis of BRI1 receptor activity. The developed mathematical model predicts that during normal root growth, few receptors are occupied with ligand. The model faithfully predicts root growth, as observed in bri1 loss-of-function mutants. For roots, it incorporates one stimulatory and two inhibitory modules, while for hypocotyls, a single inhibitory module is sufficient. Root growth as observed when BRI1 is overexpressed can only be predicted assuming that a decrease occurred in the BRI1 half-maximum response values. Root growth appears highly sensitive to variation in BR concentration and much less to reduction in BRI1 receptor level, suggesting that regulation occurs primarily by ligand availability and biochemical activity.     

Jasmonates as Signals in the Wound Response

Plant responses to wounding and herbivore attack are orchestrated by complex signaling pathways that link the production of chemical and physical signals at the wound site to activation of gene expression and other cellular processes. The systemic nature of many wound-induced responses provides an attractive opportunity to study intercellular signaling pathways that operate over long distances within the plant. Genetic dissection of the wound-response pathway in tomato indicates that (1) systemin and its precursor protein, prosystemin, are upstream components of an intercellular signaling cascade that requires the biosynthesis and action of jasmonic acid (JA); and (2) physiological processes regulated by this pathway confer host resistance to a broad spectrum of plant invaders. Grafting experiments conducted with mutants defective in systemic wound signaling indicate that systemin functions at or near the wound site to trigger the production of JA, which in turn acts non-cell autonomously to promote systemic defense responses. The location of JA biosynthetic enzymes within the companion cell-sieve element complex of vascular bundles, together with the accumulation of JA in vascular tissues, support a role for jasmonates as phloem-mobile signals. The recent discovery of enzymes involved in the metabolism of JA to volatile methyl-JA and bioactive JA-amino acid conjugates has potential implications for the mechanism by which JA promotes wound signaling. Species-specific differences in the mechanism of wound signaling appear to reflect the way in which the wound-induced jasmonate pathway is regulated by other signals including systemin, cell wall-derived oligosaccharides, ethylene, and insect-derived elicitors. Adding to the complexity of the wound-induced jasmonate cascade are wound-signaling pathways that operate independently of JA.