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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.35 n.3-4 Santiago  2002 

Biol Res 35: 373-383, 2002


Signal transduction in lemon seedlings in the
hypersensitive response against Alternaria alternata:
participation of calmodulin, G-protein and protein


1Laboratorio de Bioquímica, Facultad Ciencias de la Salud, Universidad Nacional Andrés Bello
2Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas,
Universidad de Chile
3Departamento de Biología, Facultad de Ciencias Básicas, Universidad Metropolitana de Ciencias de la


The development of an effective hypersensitive response (HR) in any plant system relies, not only in their gene composition and expression, but also on an effective and rapid signal transduction system. Lemon seedlings induce the phenylpropanoid pathway, which results in the de novo biosynthesis of the phytoalexin scoparone, as part of the hypersensitive response against Alternaria alternata. In order to elucidate some of the signaling elements that participate in the development of HR in lemon seedlings, we used several compounds that are known as activators or inhibitors of signal transduction elements in plants or in animal cells.

Lemon seedlings treated either with cholera toxin or with phorbol 12-myristate 13-acetate (PMA), in the absence of A. alternata induced phenylalanine ammonia-lyase (PAL, E. C. and the synthesis of scoparone, suggesting the participation of a G-protein and of a serine/threonine kinase, respectively, in signal transduction.

The use of trifluoperazine (TFP), W-7, staurosporine, lavendustin A or 2,5dihydroximethyl cinnamate (DHMC) prevented PAL induction as well as scoparone biosynthesis in response to the fungal inoculation, thus allowing us to infer the participation of Calmodulin (CaM), of serine/threonine and of tyrosine protein kinases (TPK) for signal transduction in Citrus limon in response to A. alternata.

Key terms: Citrus limon, Alternaria alternata, hypersensitive response, signal transduction.


The hypersensitive response (HR) is one of the effective mechanisms that plants use for their defense against phytopathogens (Agrios, 1997). Development of HR depends not only in an early recognition of the pathogen or in the expression of defense genes, but also in an effective signaling to induce gene expression at an appropriate time period to control pathogen development and penetration to the host tissue. The plant defense becomes effective once phytoalexins (de novo synthesized antimicrobial compounds) and pathogenesis-related proteins are formed.

Their synthesis is triggered after several reactions, which include an initial and rapid oxidative burst at the plasma membrane, followed by ion fluxes, cross-linking of proteins from the cell wall and synthesis of phenolics. These early stages of plant defense are followed by several signaling reactions, which trigger the expression of defence genes (Chrispeels et al, 1999). Classical signaling components such as G-proteins and protein phosphorylation, have been identified in plant systems (Blumwald et al, 1998; Chrispeels et al, 1999). Studies on the signaling components involved in the development of different physiological responses of plant systems have used different compounds whose targets are well-known components of animal signal transduction pathways. For example, calcium ionophore A23187 has been used to establish the participation of calcium in the abscisic acid (ABA) transduction system (Chrispeels et al, 1999) as well as in the HR developed in lemon seedlings (Castañeda and Pérez, 1996). The participation of G-protein has been analyzed using cholera toxin in french beans (Bolwell et al, 1991). This same toxin, as well as mastoparan and of PMA, were tested in Sanguinaria canadiensis (Mahady et al, 1998) to establish the participation of G-proteins and protein kinases, respectively, in the signal transduction for the synthesis of alkaloids induced by fungal elicitors and ABA. Calmodulin (CaM) participation has been inferred after trifluoperazine (TFP) treatment of Phaseolus radiatus (Long et al, 1998), or after treatment of detached wheat leaves with the W-7 (Noel et al, 2001), where staurosporine was also tested to get some information on the participation of protein kinases in the synthesis of fructans. Staurosporine has also been used to establish the participation of PKC-like proteins in potato (Yoshioka et al, 2001). Nevertheless, most of these findings have been obtained using herbaceous plants, as signaling systems are almost unknown in woody plants with the exception of Larix decidua (Bach and Seitz, 1997) and Santalum album (Anil and Sankara, 2001).

Lemon seedlings develop a hypersensitive response (HR) after inoculation with Alternaria alternata or with elicitors derived from the fungus. This HR includes the induction of the phenylpropanoid pathway, which results in the de novo synthesis of the phytoalexin scoparone (Roco et al, 1993; Pérez et al, 1994a). In addition, an early maceration of plant tissue at the site of inoculation with the fungus can be observed (Pérez et al, 1994b). This maceration is produced as a consequence of the action of an A. alternata-derived pectinase on lemon tissues (Fanta et al, 1992), which provides oligogalacturonides that act as the true elicitors of the HR (Roco et al, 1993). Therefore, the secretion of pectinolytic enzymes by A. alternata for the degradation of the plant cell wall appears to be the initial step that triggers the development of the HR in lemon seedlings, thus supporting the role of the extracellular matrix as an important source of these elicitors (Chrispeels et al., 1999). The participation of calcium ions and of calcium channels (Castañeda and Pérez, 1996), as well as of IP3 (Ortega and Pérez, 2001), have been established in the defensive response of this woody plant system. As calcium ions are involved in the activation of several proteins, such as calmodulin and protein kinases, and IP3 has been involved in calcium release from different compartments (vacuole and endoplasmic reticulum), we decided to explore whether CaM and/or protein kinases were components of the signal transduction pathway involved in the HR elicited by A. alternata. On the other hand, a G-protein or protein kinases could be participating in the activation of a phospholipase C for the production of IP3, whose increases are related to HR (Ortega and Pérez, 2001). We therefore decided to explore the participation of these specific signaling components using an indirect approach through the use of activators or inhibitors. This information is important for developing a more detailed study of the components of signaling and of their organization for HR in lemon seedlings, in order to understand the way in which the plant prevents fungal infection.



All reagents were analytical grade and were obtained from Sigma or Merck. Antibody to phosphotyrosine (monoclonal, mAbaPY) and peroxidase-labeled goat anti-rat antibodies (clone 4G10) were purchased from Amersham.


Lemon seedlings were obtained from seeds as described (Roco et al., 1993), and A. alternata was isolated from Citrus trees infected with sooty molds (Pérez et al, 1991).

Cell free extracts, measurement of PAL
activity and quantitation of proteins.

Cell free extracts were prepared from intact or wounded lemon seedlings using 1 ml of 100 mM pH 8.8 sodium borate containing 1 µM PMSF and 10 mM ß-mercaptoethanol per g of fresh tissue as described (Roco et al, 1993). Treatments with inhibitors or activators of different components of signal transduction are specified below. Controls were performed in the same conditions in the absence of any elicitor. PAL activity was measured by the method of Zucker (1965), four hours after elicitation with 1 x 106 fungal conidia per g fresh tissue. Results are expressed as PAL ratio (treated/control seedlings) or as PAL activity (pkat/mg protein) and correspond to the mean of at least three different experiments run in triplicates where S. D. did not exceed 10%. Proteins were quantified by the method of Bradford (1976).

Formation of the phytoalexin scoparone

The presence of scoparone was established by TLC (Pérez et al., 1994a) 42 hours after PAL induction. Quantitation was made at 268 nm using an e of 2,085 cm-1 M-1 for scoparone in ethyl acetate (Roco et al., 1993). Three different experiments, run in duplicates, were performed for each treatment.

Proposed elements involved in signal

Groups of five wounded seedlings were treated with 2 mL of 1 µg/mL cholera toxin in the absence of fungal conidia. Samples were taken at different time intervals to test PAL induction and formation of scoparone.


Groups of five wounded seedlings were treated with 1 µM TFP or 1 µM W-7 for 5 minutes before elicitation with the fungus. PAL activity and scoparone synthesis were analyzed as above. The direct effect of TFP and of W-7 on PAL activity was tested including these chemicals in the assay medium.

Serine/threonine protein kinases.

Groups of five wounded seedlings were treated with 1 mL of 1 µM staurosporine for 5 minutes before elicitation with the fungus. PAL activity and scoparone synthesis was analyzed as below. The direct effect of staurosporine on PAL activity was tested including this chemical in the assay medium.

Time course experiments also were performed with different concentrations of phorbol 12myristate 13-acetate (0 _ 100 µM). Wounded seedlings were elicited with the phorbol ester in the absence of fungal conidia; samples taken at different time periods were analyzed for PAL activity and scoparone synthesis as above.

Tyrosine protein kinases

Groups of five wounded seedlings were treated with 2 mL of 7.5 nM lavendustin A or with 0.5µM dihydroxymethyl cinnamate (DHMC) for 5 minutes before elicitation with the fungus. PAL activity and scoparone synthesis were analyzed as above. The direct effect of lavendustin A and of DHMC on PAL activity was tested including these chemicals in the assay medium.

Protein phosphorylation in tyrosine.

Cell free extracts from control, lavendustin A, and DHMC-treated lemon seedlings were prepared 1, 5, 10, 15, 20, 30, 60 and 240 min after elicitation with fungal conidia. Seedlings were frozen in liquid nitrogen, powdered in a mortar, and homogenized by means of an Ultraturrex (20,500 rpm, three cycles of 30 sec) using 2 volumes of buffer (2 mM sodium orthovanadate, 50 mM HEPES pH 7.4, 100 mM NaCl, 4 mM sodium pyrophosphate, 10 mM sodium EDTA, 10 mM sodium fluoride, 1% Triton X-100, 2 µg/mL leupeptin, 2 µg/mL aprotinin and 1 mM PMSF) per g of powdered tissue. Homogenates were stirred three times for 10 minutes each on a vortex mixer before centrifugation at 12,100 x g for 20 minutes. Proteins from supernatants (70 µg per lane) were separated by PAGE-SDS (Weber and Osborne, 1969) using the buffer system of Laemmli (1970). Proteins were either stained with Coomasie blue R-250 or electrotransferred for one hour at 100 V to a nitro-cellulose membrane. After blocking with 3% BSA in PBS buffer, the membrane was treated with the monoclonal antibody against phosphotyrosine for three hours, washed with PBS-1% Tween 20 and treated with a peroxidase conjugated antibody for one hour. A final wash with PBS-1% Tween 20 was made before submerging the membrane into ECL reagent for one minute.

After drying and wrapping with plastic film, the membrane was placed in contact with HyperfilTMECL inside an exposure cassette. Controls were performed incubating the membrane immediately after electrotransference with the peroxidase conjugate antibody.


Participation of a G-protein.

A six-fold transient induction of PAL activity was observed after a one-hour treatment of wounded lemon seedlings with 1 µg/mL cholera toxin (Fig 1). Toxin concentrations higher or lower than 1 µg/mL induced PAL to a lesser extent (Fig 2). As is observed in this plant system in response to fungal conidia, 42 hours after PAL induction, scoparone synthesis was detected (Fig 3). Cholera toxin acts through the ADP ribosylation of the G protein a subunit (Ga); this blocks the GTP hydrolysis associated to this G-protein subunit thus maintaining the protein in its activated form. This permanent activation results in the stimulation of down stream signal transduction elements involved in a determinate response. A G-protein therefore appears to be involved in PAL induction as well as in scoparone synthesis in this plant system; which agrees with the results obtained with cholera toxin or with Pertussis toxin in bean cell cultures, where PAL induction was observed after treatment with these compounds (Bolwell et al, 1991),or in cell suspensions of Sanguinaria canadiensis, where the synthesis of the phytoalexin benzophenanthridine was observed in response to cholera toxin, or to a fungal elicitor, or to abscisic acid (Mahady et al, 1998).

Figure 1. Kinetics of PAL induction after elicitation of wounded lemon seedlings with 1 µg/mL cholera toxin. PAL activity was measured at 290 nm using L-Phe as substrate, in samples taken every 30 minutes. Results are the mean of at least three different experiments run in triplicates, where SD did not exceed 10%.

Figure 2. Induction of PAL at different concentrations of cholera toxin. PAL activity was measured at 290 nm using L-Phe as substrate, after one hour elicitation with the toxin. Results are the mean of at least three different experiments run in triplicates, where SD did not exceed 10%.

Figure 3. Synthesis of scoparone in lemon seedlings in response to inoculation with Alternaria alternata, with the G-protein activator cholera toxin, with the PKC-activator PMA, and with Alternaria alternata after pre-incubation with the CaM antagonists TFP and W-7, or with the Ser/Treo kinase inhibitor Staurosporine, or with the Tyr protein kinase inhibitors lavendustin A and DHMC.

Participation of Calmodulin. Effect of
trifluoperazine and W-7 on the induction of

The 24-fold increase of PAL activity observed after four hours of treatment of wounded lemon seedlings with fungal conidia (Castañeda and Pérez, 1996) was completely suppressed by 1 µM TFP or by 1 µM W-7. Basal PAL activity of lemon seedlings (5 - 8 pkat/mg proteins) did not increase at four hours as a consequence of wounding or treatment with TFP or W-7, in the absence of A. alternata. TFP is a CaM antagonist, early described as an inhibitor of mammalian CaM-dependent stimulation of 3': 5'- cyclic nucleotide phosphodiesterase (Huang et al, 1981). W-7 is also a CaM antagonist that inhibits the same phosphodiesterase with an IC50 of 28µM and the myosin light chain kinase with an IC50 of 51 µM (Hidaka and Tanaka, 1983), as well as DcCPK1 from Daucus carota which contains a calmodulin-like domain (Farmer and Choi, 1999). The same inhibitory effect observed for TFP and W-7 could be explained through their similar mechanisms of action on enzyme systems. As cAMP has not been described in plants, the action of these antagonists might be on a cyclic guanosine monophosphate (cGMP) phosphodiesterase, which would affect cGMP levels inside plant cells (Chrispeels et al, 1999) or on CaM-dependent protein kinases (CDPK), whose presence has been described in soybean (Lee et al, 1998). Furthermore, we cannot discard an effect of these CaM antagonists on plant potassium channels (Chrispeels et al, 1999) that share large similitude to animal channels, which may include the presence of CaM-binding regions to confer Ca-sensitivity to the channels (Orrego, 2000).

As external or cell wall calcium participates in early events of this signal transduction process in our plant system (Castañeda and Pérez, 1996), it may be suggested that its interaction with CaM might be produced down stream in the transduction pathway. Nevertheless, an interaction between calcium from internal compartments, released to the cytoplasm as a consequence of IP3 interaction with its receptors cannot be ruled out (Ortega and Pérez, 2001).

In order to compare the behavior of C. limon with animal systems, IC50 for TFP was determined to be 1.2 + 0.1 µM for this plant system after elicitation with fungal conidia. This IC50 value is one order of magnitude lower than the IC50 of 13 µM, described for cAMP-gated cationic channels (Kleene, 1994), suggesting that CaM-dependent signaling component(s) in C. limon appears to be more sensitive to this antagonist than the ones involved in animals.

Additionally, scoparone was not formed in lemon seedlings inoculated with A. alternata where PAL induction had been suppressed by TFP or W-7 (Fig 3), suggesting the direct involvement of CaM in signal transduction for the synthesis of this phytoalexin.

Participation of protein kinases.
Serine/threonine protein kinases

The effect of staurosporine was initially studied in lemon seedlings using a concentration of 1 µM as described for other plant systems (Felix et al, 1991). This compound suppressed PAL induction in response to fungal conidia (Fig 4), suggesting that a Ser/Treo protein kinase could be involved in this signaling system. These enzymes could correspond to the PKC-type or CDPK-type protein kinases, which could be activated by calcium ions proceeding from external or internal compartments or by CaM, respectively (Castañeda and Pérez, 1996; Lee et al, 1998). In fact, the DcCPK1 from carrot contains a CaM-like domain in its structure and is inhibited both by staurosporine and by W-7 (Farmer and Choi, 1999). These results agree with those obtained by Grosskopf et al (1990), who blocked PAL induction and ethylene biosynthesis in tomato cell cultures, using K-252a a known inhibitor of mammalian protein kinases, and with those obtained by Bach and Seitz (1997), where PAL and peroxidase activities were blocked by staurosporine. It also agrees with the finding in potato of a protein kinase similar to PKC involved in defense against different elicitors (Subramaniam et al, 1997). In addition, PAL has been described as a substrate of AtCPK1, a PAL kinase activity (calcium dependent PK) in maize protoplasts (Cheng et al, 2001).

A concentration-dependent suppression of PAL induction was observed when different concentrations of staurosporine were used, allowing us to determine an IC50 of 0.52 + 0.07 µM. IC50 values between 3 nM and 70 nM, depending on the protein kinase analyzed, have been described for purified enzymes (Nishimura and Simpson, 1994). Thus, the IC50 value obtained for staurosporine in the lemon system could represent a mean value for one or more kinases involved in signal transduction for PAL induction and synthesis of scoparone. In fact, staurosporine also suppressed the synthesis of scoparone in response to fungal elicitation (Fig 3).

The participation of PKC has been also inferred after use of phorbol esters (Hortelano et al., 1992). PAL activity was induced six-fold in lemon seedlings after one hour of treatment with 100 µM phorbol 12-miristate 13-acetate (PMA) (Fig 5), a known activator of PKC (Schenk and Snaar-Jagalska, 1999). When PMA was reduced to 1 µM, PAL induction was observed after two hours of treatment (Fig 5), suggesting not only the participation of a PKC-like protein, but also of more than one kinase, differing in their sensitivity to different concentrations of the activator. In addition, the formation of scoparone was also observed after treatment with PMA (Fig 3).

Tyrosine protein kinases

The effect of DHMC, an erbstatin analogue and a potent inhibitor of tyrosine protein kinases (PTK), was analyzed. This compound penetrates the cell easily and acts as a competitive inhibitor for the binding of the protein kinase substrate, without altering ATP binding (Umezawa et al, 1990). PAL induction in response to A. alternata was reduced 80% when the lemon seedlings were pre-treated with 0.5 µM DHMC (Fig. 4), a concentration described as inhibitory for animal systems. Experiments run with different concentrations of DHMC showed a concentration dependent reduction of PAL induction and allowed us to estimate an IC50 of 0.3 + 0.03 µM for this compound.

An IC50 of 0,77 µM was described for the EGF receptor tyrosine kinase activity (Umezawa et al, 1990), which agrees with the value obtained in the lemon system.

In addition, Lavendustin A, described as a competitive inhibitor for ATP binding to PTK (Umezawa et al, 1990), reduced PAL induction observed in response to fungal inoculation. A 55% reduction of PAL induction was observed in seedlings treated with 7.5 nM Lavendustin A (Fig. 4). The IC50 determined for this compound was of 8.0 + 1.3 nM. An IC50 of 11,5 nM was described for the EGF receptor associated tyrosine kinase (Umezawa et al, 1990) that is in the same order of magnitude than the one obtained in C. limon. Therefore, the suppressive effect of DHMC and of Lavendustin A on PAL induction, strongly suggests that a PTK could be involved in signal transduction in the lemon system. The synthesis of scoparone was also suppressed in the presence of DHMC or Lavendustin A (Fig. 3) confirming the relationship between PAL induction and phytoalexin synthesis in response to inoculation with A. alternata.

Figure 4. PAL activity in lemon seedlings treated with 1 µM Staurosporine, with 0.5 µM DHMC, or with 7.5 nM lavendustin A for five minutes before inoculation with A. alternata. PAL activity was measured four hours after fungal inoculation at 290 nm using L-Phe as substrate. Results are the mean of at least three different experiments run in triplicates, where SD did not exceed 10%.

Figure 5. Kinetics of PAL induction after elicitation of wounded lemon seedlings with 100 µM or with 1 µM phorbol 12-myristate 13-acetate (PMA). PAL activity was measured at 290 nm using L-Phe as substrate, in samples taken every 30 minutes. Results are the mean of at least three different experiments run in triplicates, where SD did not exceed 10%.

Protein phosphorylation in tyrosine.

Control lemon seedlings showed several tyrosine-phosphorylated proteins whose phosphorylation level increased after inoculation with A. alternata (Fig 6), while the Coomasie blue stained proteins did not change their pattern (Fig 7). A group of phosphorylated proteins, which migrated around 84 kDa, began to appear after one minute of inoculation with the fungus, as well as several peptides with MM less than 20 kDa. Pre-phosphorylated proteins with MM between ~48.5 and ~23.5 kDa reached a maximum phosphorylation at 15 minutes after fungal inoculation. Among these, a band with a MW of ~37 kDa was observed (Figs 6 and 7). The size of this protein resembles that of MAPK, whose activation results in transcriptional activation of several plant defence genes (Treisman, 1996). MAPK cascades are considered among components downstream of receptors or sensors that participate in signal transduction for cellular responses in yeast and animal systems (Davis, 2000). It has been described that the Arabidopsis thaliana genome encodes aproximately 20 different MAPKs, where several of them appear to be activated by pathogen infection (Zhang and Klessig, 2001). Also, NtMEK2, a MAP kinase of tobacco, is activated by various pathogens or pathogen-derived elicitors (Yang et al, 2001). Therefore, the activation of protein tyrosine phosphorylation as an early response of lemon seedlings to inoculation with A. alternata, suggest that these tyrosine kinases and tyrosine phosphorylated proteins are involved in the HR development.

The A. alternata induced phosphorylation pattern changed when seedlings were treated with 7.5 nM lavendustin A or with 0,5 µM DHMC and to a lesser extent, after treatment with staurosporine (Fig 7). In all cases, the phosphorylation of the ~37 kDa protein was prevented. Staurosporine and Gd3+, a calcium ion channel blocker, suppresses activation of an MAPK in tobacco (Susuki and Shinshi, 1995), which agrees with our results and suggests that in lemon seedlings upstream kinases might be involved in phosphorylation of this specific protein.

In conclusion, the blockade of PAL induction and of the de novo biosynthesis of the phytoalexin scoparone in lemon seedlings inoculated with A. alternata, by TFP or W-7, or by staurosporine, lavendustin A, or DHCM, suggests that CaM and protein phosphorylation through the action of PKC-like proteins and protein tyrosine kinases might be involved in the development of HR in this woody plant system. The increase in PAL activity and biosynthesis of scoparone in response to PMA reinforces the participation of PKC-like proteins. Additionally, obtaining similar results, i.e. PAL induction and synthesis of scoparone as a consequence of the use of cholera toxin, allows us to suggest the participation of a G-protein in this defense process. The sequence of these elements within the signal transduction pathway remains to be elucidated.

Figure 6. Kinetics of tyrosine phosphorylation of proteins from wounded lemon seedlings inoculated with A. alternata. Proteins were separated by SDS-PAGE, electrotransferred to a nitro-cellulose membrane and treated with a monoclonal antibody against phosphotyrosine. MM markers (lane 1), wounded seedlings (lane 2), wounded seedlings after fungal inoculation: one minute (lane 3), five minutes (lane 4), 10 minutes (lane 5), 15 minutes (lane 6), 20 minutes (lane 7), 30 minutes (lane 8), 60 minutes (lane 9), 240 minutes (lane 10).

Figure 7.

A. Effect of Ser/Treo and tyrosine protein kinase inhibitors on tyrosine phosphorylation of proteins from wounded lemon seedlings after 15 minutes inoculation with A. alternata. Proteins were separated by SDS-PAGE, electrotransferred to a nitro-cellulose membrane and treated with a monoclonal antibody against phosphotyrosine. MM markers (lane 1), A. alternata (lane 2), A. alternata plus 7.5 nM lavendustin A (lane 3) or plus 0.5 µM DHMC (lane 4) or plus 1 µM Staurosporine (lane 5).

B. Effect of protein kinase inhibitors on the protein pattern of extracts from lemon seedlings. Proteins were separated by SDS-PAGE and stained with Coomasie blue R-250. MM markers (lane 1), intact seedlings (lane 2), wounded seedlings (lane 3), wounded seedlings inoculated with A. alternata (lane 4), or plus 7.5 nM lavendustin A (lane 5) or plus 0.5 µM DHMC (lane 6).


This work was funded by FONDECYT grants Nº 1970532 and 2990090.


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**Corresponding author: Luz M. Pérez, Laboratorio de Bioquímica, Facultad Ciencias de la Salud, Universidad Andrés Bello. Sazie 2325. Telephone: (56-2) 661-8400. Fax: (56-2) 671-1936. e-mail:

Received: March 11, 2002. In revised form: August 2, 2002. Accepted: September 3, 2002

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