Journal of Experimental Botany

JXB Advance Access originally published online on March 10, 2006
Journal of Experimental Botany 2006 57(6):1341-1351; doi:10.1093/jxb/erj109

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RESEARCH PAPER

Calcium and calcium-dependent protein kinases are involved in nitric oxide- and auxin-induced adventitious root formation in cucumber

María Luciana Lanteri, Gabriela Carolina Pagnussat ^* and Lorenzo Lamattina

Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina

To whom correspondence should be addressed. E-mail: lolama{at}mdp.edu.ar

Received 11 November 2005; Accepted 9 January 2006

	Abstract

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A few years ago it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during adventitious root (AR) formation in cucumber (Cucumis sativus). More recently, a mitogen-activated protein kinase cascade was shown to be induced by IAA in a NO-dependent, but cGMP-independent, pathway. In the present study, the involvement of Ca²⁺ and the regulation of Ca²⁺-dependent protein kinase (CDPK) activity during IAA- and NO-induced AR formation was evaluated in cucumber explants. The effectiveness of several broad-spectrum Ca²⁺ channel inhibitors and Ca²⁺ chelators in affecting AR formation induced by IAA or NO was also examined. Results indicate that the explants response to IAA and NO depends on the availability of both intracellular and extracellular Ca²⁺ pools. Protein extracts from cucumber hypocotyls were assayed for CDPK activity by using histone IIIS or syntide 2 as substrates for in-gel or in vitro assays, respectively. The activity of a 50 kDa CDPK was detected after 1 d of either NO or IAA treatments and it extended up to the third day of treatment. This CDPK activity was affected in both extracts from NO- and IAA-treated explants in the presence of the specific NO-scavenger cPTIO, suggesting that NO is required for its maximal and sustained activity. The in-gel and the in vitro CDPK activity, as well as the NO- or IAA-induced AR formation, were inhibited by calmodulin antagonists. Furthermore, the induction of CDPK activity by NO and IAA was shown to be reliant on the activity of the enzyme guanylate cyclase.

Key words: Adventitious roots, auxin, Ca²⁺, Ca²⁺-dependent protein kinase, cGMP, Cucumis sativus, guanylate cyclase, nitric oxide

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
The auxin indole acetic acid (IAA) regulates many aspects of plant growth and development from seed germination to fruit ripening, mediating cell division, expansion, and differentiation (Davies, 1995). However, despite the critical role of auxins throughout the plant life cycle, the molecular mechanisms underlying their action are still poorly understood. Adventitious root (AR) formation involves the development of a meristematic tissue after removal of the primary root system. The auxin IAA promotes AR formation by inducing the dedifferentiation of cells to develop new meristems. This process is essential for the propagation of woody species and for adaptation to particular environmental conditions (Moncousin, 1992). It was recently demonstrated that nitric oxide (NO) is involved in the auxin response during AR formation in cucumber (Cucumis sativus; Pagnussat et al., 2002) and that convergent and complex cGMP-dependent and -independent signalling pathways are orchestrating the formation of a new root system when the primary root is removed (Pagnussat et al., 2003, 2004).

Although first described as a signal molecule in animals, new and exiting roles for NO as a regulator of plant growth and developmental processes have been reported during the past few years (Beligni and Lamattina, 2001; Lamattina et al., 2003; Neill et al., 2003; Wendehenne et al., 2004). A connection among NO, cGMP, Ca²⁺, and calmodulin (CaM) pathways was suggested in previous studies that showed the participation of NO in light-mediated processes in plants (Beligni and Lamattina, 2000). In tobacco (Nicotiana tabacum) cells, cADPR was able to mimic the NO-mediated induction of defence gene expression. This cADPR effect was inhibited by ruthenium red, an inhibitor of Ca²⁺ release via cADPR/ryanodine-sensitive Ca²⁺ channels (Durner et al., 1998). Furthermore, 8-Br-cADPR, an antagonist of cADPR, blocked gene expression mediated by recombinant neuronal nitric oxide synthase in tobacco (Klessig et al., 2000). More recently, it was demonstrated that NO triggered an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) by promoting Ca²⁺ release from intracellular stores through the activation of cADPR/ryanodine-sensitive Ca²⁺ channels in fava bean (Vicia faba) and tobacco cells (García-Mata et al., 2003; Lamotte et al., 2004).

The second messenger inositol 1,4,5-trisphosphate (IP₃) diffuses within the cell where it encounters specific receptors allowing the Ca²⁺ stored in organelles to enter the cytosol (Bootman et al., 2001). There is evidence that a rise in IP₃ concentration follows auxin signalling and activates Ca²⁺ channels located in intracellular compartments such as the vacuole (Ettlinger and Lehle, 1988; Zbell and Walter-Back, 1988). Moreover, it was reported that both the auxin-induced cytosolic acidification and elongation growth were blocked by inhibiting the Ca²⁺ influx into the cytosol (Poovaiah and Reddy, 1987; Cho and Hong, 1996; Shishova and Lindberg, 1999), which indicate that some cell responses to auxins depend on activity of Ca²⁺ channels.

Available evidence supports the idea that both cADPR/ryanodine-sensitive Ca²⁺ channels and IP₃-regulated Ca²⁺ channels are functionally active in plant cells (Muir and Sanders, 1996, 1997). However, their biochemical and pharmacological properties cannot be extrapolated from experiments done with animal Ca²⁺ channel inhibitors since there is little evidence for direct effects and specificity on plant Ca²⁺ channels. Thus, an understanding of the participation and occurrence of these signalling pathways is not yet complete, since no orthologues of both types of Ca²⁺ channels were found in plants at the molecular level (Nagata et al., 2004).

In plants, the elevations in [Ca²⁺]_cyt induced by a particular stimulus are perceived and transduced by specific effectors involving Ca²⁺-binding proteins and protein kinases that initiate downstream events leading to changes in gene expression, metabolism, and cell division and elongation (Sanders et al., 2002; White and Broadley, 2003; Reddy and Reddy, 2004). These proteins are also referred to as Ca²⁺ sensors and include (i) CaM, (ii) Ca²⁺-dependent protein kinases (CDPKs), (iii) Ca²⁺-regulated phosphatases, and (iv) annexins and integrins. CDPKs (EC 2.7.1.37 [EC] ) are a family of serine/threonine protein kinases that are unique to plants and protists (for reviews see Roberts and Harmon, 1992; Roberts, 1993; Harmon et al., 2000, 2001; Cheng et al., 2002; Harper et al., 2004; Ludwig et al., 2004). These enzymes contain four domains: (i) an amino terminal domain of variable length and sequence, (ii) a protein kinase catalytic domain, (iii) an autoinhibitory junction domain, and (iv) a CaM-like Ca²⁺-binding domain in their carboxyl terminal (Harmon et al., 2001). CDPKs are activated by the binding of Ca²⁺ to their CaM-like domain. In resting conditions, [Ca²⁺]_cyt is maintained at low levels assuring a basal CDPK activity. Different biotic and abiotic stimuli trigger increases in [Ca²⁺]_cyt, resulting in the activation of certain CDPKs and in the subsequent initiation of signalling events (Webb et al., 1996; Trewavas and Malhó, 1998; Hrabak, 2000). In many plant species, CDPKs are encoded by a multigene family. In cucumber, there have been isolated and characterized four partial cDNAs (Ullanat and Jayabaskaran, 2002) and one full-length cDNA (Kumar et al., 2004) encoding putative CDPK isoforms. Activity of CDPKs was found in various organs of cucumber seedlings and the proteins responsible for that activity were immunolocalized in the phloem sieve elements. A role for CDPKs in the Ca²⁺-regulated phloem transport of assimilates from leaves to sink organs was suggested (Kumar and Jayabaskaran, 2004).

In the present study, the involvement of Ca²⁺ and the regulation of CDPK activity was evaluated during the IAA- and NO-induced AR formation in cucumber explants. Evidence is provided that Ca²⁺ and CDPK activity are downstream messengers in the signalling pathway triggered by auxins and NO to promote AR formation.

	Materials and methods

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Plant material and experimental design
Cucumber seeds (Cucumis sativus cv. Poinsett 76) were germinated in Petri dishes on filter papers soaked in distilled H₂O and maintained at 25 °C for 7 d with a 14 h photoperiod (photosynthetically active radiation=200 µmol m^–2 s^–1). Primary roots of 7-d-old seedlings were removed and cucumber explants were maintained under the same conditions of temperature and photoperiod for up to 5 d in the presence of H₂O (control) or 10 µM of the auxin IAA (Fluka Buch, Switzerland) or 10 µM of the NO-donor sodium nitroprusside (SNP; Merck, Darmstadt, Germany). As a control, 200 µM of the specific NO-scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, potassium salt (cPTIO; Molecular Probes Eugene, Oregon, USA) was added together with SNP or IAA.

Treatments with Ca²⁺ chelators, Ca²⁺ channel inhibitors, and calmodulin antagonists
All the chemicals except for 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM; Calbiochem, San Diego, USA) were purchased from Sigma-Aldrich (St Louis, USA). Before the removal of the primary root, cucumber seedlings were pretreated in Petri dishes for 2 h in the presence of 100 µM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) or 100 µM BAPTA/AM or 500 µM lanthanum chloride (LaCl₃) or 150 µM methoxyverapamil hydrochloride (MV) or 5 mM lithium chloride (LiCl) or 50 µM neomycin sulphate (NEO) or 50 µM ruthenium red (RR) or 100 µM nicotinamide (NA). Primary roots were then removed and whole explants were treated for 5 d in Petri dishes containing filter papers imbibed in solutions with the above compounds in the presence or absence of 10 µM IAA, or 10 µM SNP. As a control, cucumber explants were maintained in H₂O, IAA or SNP. The same experimental procedure was used to evaluate the effect of the membrane-permeable CaM antagonists chlorpromazine hydrochloride (CPZ), trifluoperazine dihydrochloride (TFP), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W-7) on the AR formation. To establish the inhibitor assay conditions, dose–response analysis was performed with different pretreatment times. All the inhibitors except for LiCl were assayed between 10 µM and 1 mM. In the case of LiCl, the dose–response ranged from 1 mM to 10 mM. The time of pretreatment was assayed from 2 h to 24 h. The concentrations of the inhibitors and the time of pretreatments were chosen as the minor ones that were effective. Until the 5th day no particular phenotype concerning explants growth or development could be observed and ascribed to the effect of the inhibitors. By contrast, all the explants treated with the inhibitors tested in this study looked healthy.

Protein extracts and protein determination
All the extraction procedures were performed on ice. Hypocotyls from cucumber explants were ground in a mortar with liquid nitrogen and extracted with 3 vols (3 ml g^–1 FW) of extraction buffer (100 mM TRIS pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM Na₃VO₄, 10 mM NaF, 50 mM ß-glycerophosphate, 1 mM PMSF, 5 µg ml^–1 aprotinin, and 5 µg ml^–1 leupeptin). After centrifugation at 20 000 g, 4 °C for 15 min, supernatants were transferred into clean tubes and immediately used for analysis. The quantification of proteins was performed according to Bradford (1976) using bovine serum albumin as standard.

In-gel protein kinase activity assay
In-gel protein kinase activity assays were performed as described previously with some minor modifications (Zhang et al., 2000). Extracts containing 10 µg of protein were electrophoresed on 12% SDS-polyacrylamide gels imbibed in 0.25 mg ml^–1 of histone IIIS (Sigma-Aldrich, St Louis, USA) in the separating gel as substrate for the protein kinases. After electrophoresis, SDS was removed by washing the gel with washing buffer (25 mM TRIS, pH 7.5, 0.5 mM DTT, 0.1 mM Na₃VO₄, 5 mM NaF, 0.5 mg ml^–1 BSA, and 0.1% Triton X-100 [v/v]) three times for 30 min each at room temperature. The protein kinases were allowed to renature in 25 mM TRIS, pH 7.5, 1 mM DTT, 0.1 mM Na₃VO₄, and 5 mM NaF at 4 °C overnight with three changes of buffer. The gel was then incubated at room temperature in 30 ml of reaction buffer (25 mM TRIS, pH 7.5, 12 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄, and 2 mM CaCl₂ or 2 mM EGTA) with 200 nM cold ATP plus 50 µCi -³²P-ATP (6000 Ci mmol^–1) for 60 min. The reaction was stopped by transferring the gel into 5% TCA (w/v)/1% NaPPi (w/v). The unincorporated -³²P ATP was removed by washing five times with the same solution for at least 6 h. The gel was dried at 80 °C for 60 min and exposed to Kodak X-OMAT UV film. Prestained size markers (Bio-Rad, Hercules, California, USA) were used to estimate the size of the protein kinases.

In vitro protein kinase activity assay
The protein kinase activity was determined in vitro by measuring phosphate incorporation into syntide 2 (Sigma-Aldrich, St Louis, USA) following Roskoski's procedure (Roskoski, 1983) with some minor modifications. The reaction was carried out in a final volume of 30 µl. Fifteen µl of sample was assayed in a reaction mixture containing 25 µM syntide 2, 20 mM TRIS pH 7.5, 10 mM MgCl₂, 1 mM DTT, and 5 µM CaCl₂ or 500 µM EGTA. The reactions were started by the addition of 100 µM cold ATP and 0.7 µCi -³²P-ATP (6000 Ci mmol^–1). After incubating at room temperature for 15 min, 25 µl of the mixture were spotted onto P81 phosphocellulose squares (Whatman, Springfield Mill, Maidstone, UK) and washed extensively by shaking in 75 mM H₃PO₄. The paper squares were dried and counted with a Liquid Scintillation Counter Beckman LS 7000 as described by Ulloa et al. (1991). The protein kinase activity was measured in the absence (EGTA) or presence (CaCl₂) of Ca²⁺. The Ca²⁺-dependent protein kinase (CDPK) activity was expressed as pmol ³²P min^–1 mg^–1 protein after subtracting the Ca²⁺-independent protein kinase activity. Where indicated, 100 µM of the CaM antagonists TFP and W-7, or 50 µM of the guanylate cyclase inhibitor 6-anilino-5, 8-quinilinedione (LY83583; Sigma-Aldrich, St Louis, USA) were administered to explants. To determine the effect of the CaM antagonists, a combination of both TFP and W-7 was added to the reaction mixture and the CDPK activity was measured as described above.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Ca²⁺ channel inhibitors and Ca²⁺ chelators prevent the IAA- and nitric oxide-induced adventitious root formation
Available evidence shows that generation of cGMP, cADPR, and elevations in [Ca²⁺]_cyt are involved in plant responses to NO (Wendehenne et al., 2001; García-Mata et al., 2003; Neill et al., 2003). It was previously reported that in cucumber explants, NO operates downstream of IAA promoting AR formation through the guanylate cyclase (GC)-catalysed synthesis of cGMP (Pagnussat et al., 2003). Taking advantage of the knowledge on Ca²⁺ participation in some NO-regulated signalling pathways, the requirement of Ca²⁺ for the IAA- and NO-induced AR formation was examined. The effectiveness was tested of several broad-spectrum Ca²⁺ channel inhibitors and Ca²⁺ chelators in reducing AR formation. The inhibitors of Ca²⁺ release from intracellular stores via cADPR/ryanodine-sensitive channels, methoxyverapamil hydrochloride (MV; Moyen et al., 1998; Shishova and Lindberg, 2004) and ruthenium red (RR; Price et al., 1994; Trewavas and Knight, 1994; Allen et al., 1995), were assayed. As expected, the treatment with 10 µM of the NO-donor sodium nitroprusside (SNP) or with 10 µM of the auxin IAA promoted AR formation in cucumber explants (Fig. 1). As shown in Fig. 1, the application of the inhibitors prevented the induction of AR formation by either NO or IAA (t test, P <0.05). Since externally provided RR and verapamil has also been shown to inhibit plasma membrane (PM) Ca²⁺ channels (White, 2000; White et al., 2002), the effect of nicotinamide (NA), which as a product of the activity of the enzyme ADPR cyclase inhibits cADPR synthesis (Wu et al., 1997) was tested. Treatment of cucumber explants with NA resulted in a significant reduction in both IAA- and NO-induced AR formation (Fig. 1; t test, P <0.05).

View larger version (22K): [in this window] [in a new window]   Fig. 1. Ca2+ is required for adventitious root formation induced by indole acetic acid (IAA) and nitric oxide (NO). Cucumber explants were incubated for 5 d with different Ca2+ chelators or Ca2+ channel inhibitors, in the presence or absence of 10 µM of the NO-donor SNP or 10 µM of the auxin IAA. Root number values are expressed as mean ±SE (n=10 explants from at least three independent experiments). Bars with asterisk are significantly different (t test, P <0.05) from their control without SNP or IAA. See Materials and methods section for the full description of the experimental procedure. MV, methoxyverapamil hydrochloride (150 µM); RR, ruthenium red (50 µM); NA, nicotinamide (100 µM); LiCl, lithium chloride (5 mM); NEO, neomycin sulphate (50 µM); LaCl3, lanthanum chloride (500 µM); EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (100 µM); BAPTA/AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (100 µM).

 
Ca²⁺ efflux from intracellular stores can also be mediated by the well-described ligand IP₃ (Allen et al., 1995). Therefore, the blockers of IP₃-regulated Ca²⁺ channels lithium chloride (LiCl, which suppress IP₃ cycling and signalling by inhibiting inositol-1-phosphatase; Gillaspy et al., 1995; Liang et al., 1996) and neomycin sulphate [NEO, which inhibits IP₃ formation by avoiding the binding of the enzyme phospholipase C (PLC) to its substrate phosphatidylinositol 4,5-bisphosphate; Munnik et al., 1998] were administered to cucumber explants in the presence or absence of NO or IAA. Again, these inhibitors significantly suppressed the AR formation induced by IAA or NO (Fig. 1, t test, P <0.05).

Since Ca²⁺-mediated signalling pathways are thought to depend on the activation of PM Ca²⁺ channels and the consequent influx of extracellular Ca²⁺ into cells for their function (Sanders et al., 2002), the effect of the Ca²⁺ channel inhibitor lanthanum chloride (LaCl₃; Tlalka and Gabrys, 1993; Huang et al., 1994; Bush, 1995; van der Meulen et al., 1996; Clayton et al., 1999) on the induction of AR formation was also analysed. The results presented in Fig. 1 indicate that LaCl₃ was able to prevent the stimulating effect of both NO and IAA on AR formation (t test, P <0.05). However, these results should be interpreted with some caution. Even though lanthanum is used as a blocker of PM Ca²⁺ channels, it can also block intracellular Ca²⁺ channels. It has been observed that lanthanum enters the cells when provided in milimolar concentrations for periods greater than 1 min (Peeters et al., 1989; Quiquampoix et al., 1990; Zha and Morrison, 1995). Nevertheless, the involvement of extracellular and intracellular Ca²⁺ was further confirmed by the use of the membrane-impermeable Ca²⁺ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and the membrane-permeable Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM; van der Luit et al., 1999; Cousson, 2003), respectively. Figure 1 shows that these compounds significantly blocked both the auxin- and NO-induced AR formation (t test, P <0.05).

CDPK activity is induced by IAA in a NO-mediated pathway
In order to study a possible link between Ca²⁺ and downstream signalling sensors, the Ca²⁺-dependent protein kinase (CDPK) activity in cucumber explants was measured. To characterize the CDPK activity present in soluble extracts of cucumber hypocotyls, in-gel protein kinase assays were performed in the presence of an exogenous substrate histone IIIS (Roberts and Harmon, 1992). Only one activity band that was Ca²⁺-dependent was detected in the in-gel analysis. This band corresponded to a protein kinase with an apparent molecular mass of approximately 50 kDa (Fig. 2A, B). Since no signal was detected in the absence of Ca²⁺ (SNP+EGTA and IAA+EGTA; Fig. 2A, B, respectively), the 50 kDa phosphorylating signal was attributed to a CDPK activity. This activity was detected after the first day of both SNP and IAA treatments and it extended up to the third and the fourth days of treatment, respectively (Fig. 2A, B). When analysed, the activation of this 50 kDa CDPK was delayed in explants treated with SNP plus the specific NO-scavenger cPTIO (Fig. 2A). Furthermore, this activity band was faint in both the third and the fourth day of IAA+cPTIO treatment, while it was clearly visible in the case of the IAA treatment (Fig. 2B). Under these experimental conditions, no activity could be detected in both control H₂O- and cPTIO-treated explants (Fig. 2C).

View larger version (37K): [in this window] [in a new window]   Fig. 2. In-gel CDPK activity is induced by IAA and NO during adventitious root formation. (A) Cucumber explants were incubated for different times with 10 µM of the NO-donor SNP or with SNP plus 200 µM of the specific NO-scavenger cPTIO. In-gel protein kinase activity assays were assayed in total soluble extracts from the hypocotyls of those explants in the presence (SNP) or absence of Ca2+ (SNP+EGTA). The arrow indicates an apparent molecular mass of approximately 50 kDa. (B) Cucumber explants were incubated for different times either with 10 µM IAA or with IAA plus 200 µM cPTIO. Protein kinase activity was measured in the presence (IAA) or absence of Ca2+ (IAA+EGTA). The arrow indicates an apparent molecular mass of approximately 50 kDa. (C) Cucumber explants were incubated for different times with either H2O or 200 µM of cPTIO and protein extracts from the hypocotyls of those explants were used for in-gel protein kinase activity assays. (D) Cucumber explants were treated with 10 µM SNP or 10 µM IAA in the presence or absence of 200 µM cPTIO. Root number was measured during 4 d of treatment every 24 h and the values are expressed as mean ±SE (n=10 explants from at least three independent experiments). Bars with asterisk are significantly different (t test, P <0.05) from the H2O-treatment. Protein kinase activity was measured using histone IIIS as substrate copolymerised in the gels.

 
A previous study showed that within 3 d after removal of the primary root system, adventitious root primordia formation was detected in explants treated with IAA or NO, while at that time cell proliferation was barely detected and no root primordia could be observed in H₂O-treated explants (Pagnussat et al., 2004). Figure 2D displays the kinetics of AR formation in cucumber explants treated with H₂O, NO or IAA in the presence or absence of cPTIO. After 4 d of treatment, the number of adventitious roots was 400% higher in NO- and IAA-treated explants than in the control ones. As was already demonstrated, the scavenging of NO with cPTIO results in a decrease in adventitious root number (Fig. 2D; Pagnussat et al., 2002).

To further confirm that the 50 kDa CDPK activity detected during the IAA- and NO-induced AR formation was univocally a member of the cucumber CDPK family, in-gel protein kinase experiments were performed in extracts from explants treated with SNP or IAA in the presence of a combination of both CaM antagonists trifluoperazine dihydrochloride (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W-7). TFP was reported to inhibit plant CDPKs by competing with the binding of Ca²⁺ to their CaM-like domain (Harmon et al., 1987; Roberts and Harmon, 1992; Li et al., 1998; Chico et al., 2002). W-7 is widely used to study the role of CaM in different signalling pathways and it was also shown to be a rather non-specific inhibitor of Ca²⁺-dependent enzymes (Lam et al., 1989; Anil and Rao, 2000). No phosphorylating signal could be detected when in-gel protein kinase activity assays were carried out with extracts from inhibitor-treated explants (data not shown), indicating that the 50 kDa CDPK activity might posses CaM-like Ca²⁺-binding domains.

The results obtained from in-gel protein kinase activity assays might be conditioned by the ability of each CDPK to resist denaturalization and subsequent renaturalization. Thereby, in vitro protein kinase activity assays were used to confirm and quantify the effect of the CaM antagonists TFP and W-7. Table 1 shows that, in the protein extracts from explants treated with SNP plus a combination of TFP and W-7 (SNP+INH), the CDPK activity was totally inhibited at the first day and more than 40% between the second and the fourth days. Furthermore, the in vitro CDPK activity was strongly inhibited in extracts from IAA+INH-treated cucumber explants (Table 1).

View this table: [in this window] [in a new window]   Table 1. Effect of calmodulin antagonists on the CDPK activity induced by NO or IAA in cucumber explants

 
CDPK activity is required for adventitious root formation
It was also studied how the inhibition of the CDPK activity affects both the IAA- and NO-induced AR formation. Cucumber explants were incubated with H₂O, IAA, or the NO-donor SNP either in the presence or absence of 100 µM of the CaM antagonists TFP or W-7. The CaM antagonist chlorpromazine hydrochloride (CPZ) was also assayed. As shown in Fig. 3A and B, all the CaM antagonists assayed were able to prevent the promotion of ARs by IAA and NO (t test, P <0.05). The inhibitory effect displayed by TFP was stronger than those of W-7 and CPZ (Fig. 3A). The potency of these drugs is in accordance with previously published reports in which the efficacy of TFP, W-7, and CPZ in inhibiting the activity of CDPKs was evaluated (Abo-El-Saad and Wu, 1995; MacIntosh et al., 1996). Figure 3A also shows that the inhibitor TFP strongly decreased the AR formation compared with the H₂O-treated explants, suggesting that the signalling pathways under the control of endogenous pools of auxins and NO have been affected. Figure 3B displays representative photographs of AR formation in cucumber explants treated for 5 d as described above.

View larger version (46K): [in this window] [in a new window]   Fig. 3. Inhibition of CDPK activity blocks the NO- and IAA-induced adventitious root formation. (A) Cucumber explants were treated with 10 µM SNP or 10 µM IAA in the presence or absence of 100 µM of trifluoperazine dihydrochloride (TFP), 100 µM of N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W-7) or 100 µM of chlorpromazine hydrochloride (CPZ) for 5 d as indicated. Root number were quantified and expressed as mean ±SE (n=10 explants from at least three independent experiments). Bars with asterisk are significantly different (t test, P <0.05) from their control without SNP or IAA. (B) Photographs are representative for each treatment and were taken after 5 d of treatment. Bar indicates 5 mm.

 
The guanylate cyclase inhibitor LY83583 prevents the NO- and IAA-induced CDPK activity
According to a previous report, the IAA-induced AR formation is mediated by NO and requires the activity of the enzyme GC (Pagnussat et al., 2003). GC catalyses the synthesis of the second messenger cGMP and was shown to be positively regulated by NO in plants (Pfeiffer et al., 1994; Durner et al., 1998). To study the dependence on cGMP for the activation of CDPKs, the in vitro and in-gel CDPK activity was measured in soluble protein extracts of explants treated with the auxin IAA or with the NO-donor SNP in the presence or absence of 50 µM of the GC inhibitor 6-anilino-5, 8-quinilinedione (LY83583). It was previously shown that, at this concentration, LY83583 was able to block the effect of both IAA and NO in promoting AR formation in cucumber explants (Pagnussat et al., 2003). As shown in Fig. 4, the in vitro CDPK activity was strongly inhibited by the presence of LY83583. Furthermore, the in-gel protein kinase activity assays performed using extracts from SNP+LY83583- or IAA+LY83583-treated explants showed a total inhibition of the 50 kDa CDPK activity (data not shown). Finally, the LY83583-mediated inhibition of the CDPK activity was transiently reversed by the addition of the membrane-permeable cGMP analogue 8-Br-cGMP (data not shown). This evidence suggests that the CDPK activity is induced by IAA and NO in a cGMP-dependent pathway.

View larger version (18K): [in this window] [in a new window]   Fig. 4. The guanylate cyclase inhibitor LY83583 prevents the NO- and IAA-mediated induction of CDPK activity. Cucumber explants were incubated for different times with 10 µM SNP or 10 µM IAA in the presence or absence of 50 µM of the guanylate cyclase inhibitor 6-anilino-5,8-quinilinedione (LY83583). In vitro CDPK activity was measured in total soluble extracts from the hypocotyls of those explants. Values are expressed as pmol 32P min–1 mg–1 protein. Data are from two independent experiments with two repetitions for each point.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
Activation of Ca²⁺-mediated signalling pathways during adventitious root formation
cADPR and increases in [Ca²⁺]_cyt have been described as messengers in the cGMP-dependent signalling pathways induced by NO in both animals and plants (Lamattina et al., 2003; Neill et al., 2003; Stamler, 1994). In the present study, it was analysed whether the NO-induced and cGMP-mediated AR formation (Pagnussat et al., 2003) involves a cADPR/Ca²⁺-dependent pathway. Data obtained from treating cucumber explants with compounds that are known to block cADPR/ryanodine-sensitive Ca²⁺ channels (methoxyverapamil hydrochloride and ruthenium red) or cADPR synthesis (nicotinamide) indicate that these inhibitors provoked a significant reduction in both IAA- and NO-induced AR formation. These results are consistent with a report that showed that the stimulation of de novo root formation by auxins in the monocotyledonous plant Commelina communis was suppressed by treatment with either (i) a GC inhibitor, (ii) a Ca²⁺ chelator, (iii) an inhibitor of Ca²⁺ release from intracellular stores, or (iv) an inhibitor of cADPR synthesis (Cousson, 2004). These findings suggest that cGMP could be implicated in Ca²⁺ mobilization by stimulating cADPR synthesis and subsequent Ca²⁺ release from cADPR/ryanodine-sensitive Ca²⁺ channels. Results indicate that this signalling pathway is also operative in a dicotyledonous plant during AR formation and that it relies on NO. Although the possibility cannot be excluded that some other Ca²⁺ channels might be affected due to the low specificity of the inhibitors methoxyverapamil hydrochloride and ruthenium red, the results presented in this work collectively argue that cADPR/ryanodine-sensitive Ca²⁺ channels are involved in the signalling pathways triggered by IAA and NO to induce AR formation. A similar process occurs in animals in which NO was shown to activate cADPR/ryanodine-sensitive Ca²⁺ channels indirectly via a cGMP/cADPR-dependent pathway (Willmott et al., 1996) or directly through S-nitrosylation (Xu et al., 1998). Besides the function of cGMP in mediating a cADPR/Ca²⁺-dependent pathway, cGMP has also been shown to activate PM Ca²⁺ channels. These channels are called cyclic nucleotide-gated ion channels (CNGCs) and were identified throughout the plant kingdom. CNGCs are permeable to both monovalent and divalent cations (typically K⁺, Na⁺, and Ca²⁺) and are directly activated by cGMP and/or cAMP (Lemtiri-Chlieh et al., 2004; Bridges et al., 2005).

Another pathway in which Ca²⁺ is released from intracellular stores is through IP₃-regulated channels (Alexandre et al., 1991; Allen et al., 1995). These data indicate that inhibitors of IP₃-regulated Ca²⁺ channels promoted a significant reduction in AR formation in both NO- and IAA-treated cucumber explants. In accordance, there is evidence that auxins could induce an increase in IP₃ concentration (Ettlinger and Lehle, 1988; Zbell and Walter-Back, 1988). It was also proposed that IP₃-regulated Ca²⁺ channels at the tonoplast might be involved in auxin-triggered increases in [Ca²⁺]_cyt (Shishova and Lindberg, 2004). In animals, it has been reported that NO could activate the enzyme PLC, which catalyses the formation of IP_3. Thus, PLC activity has been proposed to be part of the NO-dependent pathway that controls [Ca²⁺]_cyt via IP₃ (Clementi et al., 1995). The data presented in this study together with results obtained in this laboratory showing NO-dependent PLC activation in xylanase-elicited tomato (Solanum lycopersicum) cells (AM Laxalt et al., unpublished results), suggest that auxins and NO effects might also be accomplished through IP₃-regulated Ca²⁺ channels.

A recent report investigated whether cADPR/ryanodine- and IP₃-regulated Ca²⁺ channels exist in plants like the ones described in animals. On the basis of sequence analysis the authors found no homologous proteins in Arabidopsis thaliana and rice (Oryza sativa; Nagata et al., 2004). However, both types of channels have been implicated in different plant processes by biochemical, electrophysiological, and pharmacological studies. Similarly, neither nitric oxide synthase (NOS; an enzyme that catalyses NO formation) gene nor protein with sequence similarities to mammalian-type NOS has been found in the A. thaliana genome. However, pharmacological and biochemical studies demonstrated that NOS-like activities as well as inhibition of NO synthesis by inhibitors of mammalian NOS occurred in plants (Wendehenne et al., 2001). More recently, Guo et al. (2003) identified in A. thaliana a functional NOS gene that encodes a protein with sequence similarity to a protein that has been implicated in NO synthesis in the snail Helix pomatia.

Results indicate that the PM Ca²⁺ channel blocker lanthanum chloride significantly compromised the IAA- and NO-induced AR formation. Accordingly, in a recent study it was reported that NO can regulate changes in [Ca²⁺]_cyt through the control of Ca²⁺ influx across the PM in tobacco cells (Lamotte et al., 2004). In the same direction, auxin might activate Ca²⁺ transport from the extracellular space through PM Ca²⁺ channels as shown in wheat (Triticum aestivum) leaf protoplasts (Shishova and Lindberg, 2004). The requirement of extracellular Ca²⁺ to promote AR formation in cucumber explants was corroborated via the use of the membrane-impermeable Ca²⁺ chelator EGTA. Notwithstanding the limitations of the pharmacological approach used in this study, these data collectively indicate that both intracellular and extracellular Ca²⁺ pools are required for the action of IAA and NO in triggering AR formation. Thus, in addition to the function of Ca²⁺ as a mineral nutrient modulating the root growth (Druart, 1997; Bellamine et al., 1998), these results provide evidence to support the involvement of Ca²⁺ as a second messenger linking both auxins and NO to the activation of processes leading to AR formation.

CDPK activity is induced by IAA and NO during adventitious root formation
CDPKs are involved in signalling pathways that utilize changes in [Ca²⁺]_cyt to couple cellular responses to extracellular stimuli (Roberts and Harmon, 1992). An increase in a 50 kDa CDPK activity is reported here after 1 d of exposure of the cucumber explants to either the NO-donor SNP or the auxin IAA. The specific NO-scavenger cPTIO delayed the CDPK activity in SNP-treated explants and diminished it in IAA-treated ones. These results suggest that the 50 kDa CDPK detected by in-gel assays in both NO and IAA treatments is the same and requires the presence of NO for its maximal and sustained activity. The induction of CDPK gene expression in response to auxins was previously reported in alfalfa (Medicago sativa; Davletova et al., 2001). In the present study auxins and NO are put together in the Ca²⁺-mediated signalling pathway that regulates CDPK activity leading to AR formation. The rate of NO release from the NO-donor SNP displays a peak around the second day in aqueous solutions (MV Beligni and L Lamattina, unpublished results). This fact might explain the partial inactivation of the 50 kDa CDPK in SNP+cPTIO treatment at the second and third days of treatment. With regard to the auxin action, a transient increase in the endogenous NO level was reported to occur during the first day of IAA-treated cucumber explants (Pagnussat et al., 2002). Thus, the amount of cPTIO could be insufficient to scavenge the NO entirely and therefore to inhibit the CDPK activity completely during the first and second days of IAA+cPTIO treatment. The different dynamics of CDPK activity measured by in vitro and in-gel assays could be explained by the nature of the experiments. While in the in vitro assays the total CDPK activity is measured, in the in-gel assays only the CDPKs that are able to renature under the experimental conditions are detected. In addition, syntide 2 is used as substrate for in vitro assays and histone IIIS for in-gel analysis.

Overall, Fig. 5 summarizes the available data that suggest a scenario in which basipetal transport of auxins induces an NO burst in the basal region of the cucumber hypocotyl, where the AR primordia develop (Pagnussat et al., 2002). Then, NO triggers a bifurcated signalling pathway that includes: (i) increases in the levels of cGMP, cADPR, and IP₃ that results in elevations in both [Ca²⁺]_cyt and CDPK activity; and (ii) the induction of a cGMP-independent MAPK cascade (Fig. 5; Pagnussat et al., 2003, 2004). It would be noteworthy that the compounds TFP, W-7, and CPZ are not specific for inhibiting CDPKs. They also affect the activity of other Ca²⁺-binding proteins such as CaM and calcineurin B-like proteins (Anil and Rao, 2000). However, the model presented in Fig. 5 is in accordance with these results showing an inhibition of the CDPK activity by those compounds in both in vitro and in-gel protein kinase assays. Nevertheless, this does not preclude the possibility that inhibition of other proteins by TFP, W-7, and CPZ could also be affecting AR formation. The activation of all the pathways triggered by NO seems to be required for AR formation since the stimulatory effect of auxins and NO is abolished when one of the pathways is compromised. It remains to be determined whether co-ordination and/or synchronization between MAPK- and Ca²⁺/CDPK-dependent signalling pathways occur during AR formation.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Schematic representation of the signalling pathways induced by IAA and NO during adventitious root formation in cucumber. NO accumulates in the basal region of the hypocotyls in response to basipetal transport of IAA (Pagnussat et al., 2002). NO induces an increase in [Ca2+]cyt through the regulation of both the release of Ca2+ from intracellular stores (via cADPR/ryanodine- and IP3-regulated Ca2+ channels) and the influx of Ca2+ from the extracellular space (via PM Ca2+ channels). As a consequence, certain CDPKs become activated. These protein kinases could probably contribute to the downstream responses that result in AR formation. cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, potassium salt; GC, guanylate cyclase; ADPRC, ADP-ribosyl cyclase; cADPR, cyclic adenosine diphosphoribose; PLC, phospholipase C; IP3, inositol 1,4,5-trisphosphate; PM, plasma membrane; CDPKs, Ca2+-dependent protein kinases; , inhibition. Dashed lines denote indirect or still undescribed pathways, or uncharacterised proteins. All the inhibitors assayed in this study are boxed.

 
The isolation of five cDNAs encoding cucumber CDPKs provided evidence for the existence of multiple isoforms and raises the question of the physiological function of each one (Ullanat and Jayabaskaran, 2002; Kumar et al., 2004). In this study, the activity of the 50 kDa cucumber CDPK was detected at earlier stages of AR formation, suggesting that this protein kinase could be associated with cell dedifferentiation, division and/or differentiation. These results fit with those previously reported by Kobayashi and Fukuda (1994) and by Komatsu et al. (1996), in which Ca²⁺-binding proteins and protein kinases were found to be involved in plant differentiation processes. Future studies including cell type-, tissue- and organ-specific expression profiles, identification of specific substrates, and subcellular localization will be necessary to elucidate the biological function of each cucumber CDPK isoform.

Most eukaryotic signalling pathways involve spatially and temporally specific elevations in [Ca²⁺]_cyt, either via release from intracellular stores and/or via influx from the extracellular space, being both processes closely related. The other major mechanism in cell signalling is the reversible protein phosphorylation, including protein kinases and phosphatases (Neill et al., 2003). It seems that these two signalling events are activated during the NO-induced AR formation and may represent key components of the NO responses in plants.

	Acknowledgements

 
This work was financially supported by grants to LL from CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas), ANPCyT (Agencia Nacional de Promoción Científica y Tecnológica), Fundación Antorchas, and UNMdP (Universidad Nacional de Mar del Plata), Argentina. LL is a career member and MLL is a PhD fellow from CONICET, Argentina. LL is a fellow from the JS Guggenheim Foundation.

	Footnotes

 
^* Present address: University of California, Davis, Section of Plant Biology, CA 95616, USA.

Abbreviations: AR, adventitious root; CaM, calmodulin; [Ca²⁺]_cyt, cytosolic Ca²⁺ concentration; CDPK, Ca²⁺-dependent protein kinase; IAA, indole acetic acid; GC, guanylate cyclase; NO, nitric oxide; PLC, phospholipase C; PM, plasma membrane.

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