Several studies focusing on elucidating the mechanism of NO (nitric oxide) signalling in plant cells have highlighted that its biological effects are partly mediated by protein kinases. The identity of these kinases and details of how NO modulates their activities, however, remain poorly investigated. In the present study, we have attempted to clarify the mechanisms underlying NO action in the regulation of NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SNF1 (sucrose non-fermenting 1)-related protein kinase 2 family. We found that in tobacco BY-2 (bright-yellow 2) cells exposed to salt stress, NtOSAK is rapidly activated, partly through a NO-dependent process. This activation, as well as the one observed following treatment of BY-2 cells with the NO donor DEA/NO (diethylamine-NONOate), involved the phosphorylation of two residues located in the kinase activation loop, one being identified as Ser158. Our results indicate that NtOSAK does not undergo the direct chemical modifications of its cysteine residues by S-nitrosylation. Using a co-immunoprecipitation-based strategy, we identified several proteins present in immunocomplex with NtOSAK in salt-treated cells including the glycolytic enzyme GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Our results indicate that NtOSAK directly interacts with GAPDH in planta. Furthermore, in response to salt, GAPDH showed a transient increase in its S-nitrosylation level which was correlated with the time course of NtOSAK activation. However, GADPH S-nitrosylation did not influence its interaction with NtOSAK and did not have an impact on the activity of the protein kinase. Taken together, the results support the hypothesis that NtOSAK and GAPDH form a cellular complex and that both proteins are regulated directly or indirectly by NO.

INTRODUCTION

Over the last few years, the free radical gas NO (nitric oxide) has become established as an intracellular messenger that affects signalling pathways involved in the plant cell response to various biotic and abiotic stimuli including hormones, pathogens and derived PAMPs (pathogen-associated molecular patterns), heavy metals or salt [13]. The emerging picture is that NO exerts part of its biological actions by modulating the activity of a wide array of proteins, some being involved in the production or in the mobilization of cellular messengers such as H2O2, Ca2+ and cGMP [46]. Ultimately, these processes might influence the expression of numerous NO target genes, thus leading to an adaptive response [7]. Mechanistically, a growing body of evidence indicates that NO has an impact on the activity of target proteins by the direct chemical modification of transition metals and critical residues, preferentially cysteine and tyrosine residues. In particular, proteins regulated by S-nitrosylation, which is the reversible coupling of an NO moiety to a critical cysteine residue (forming an S-nitrosothiol), have been identified in Arabidopsis thaliana. These include glycine decarboxylase [8], the glycolytic enzyme GAPDH (glyceraldehyde-3-phosphate dehydrogenase) [9,10], methionine adenosyltransferase 1 [11], metacaspase 9 [12], NPR1 [13], the salicylic-acid-binding protein 3 [14], peroxiredoxin II [15] and the transcription factor AtMYB2 [16]. These proteins are related to cell death, defence responses and metabolism. Depending on the protein, S-nitrosylation promotes or inhibits their activity.

Several studies have highlighted a role for protein kinases in conveying NO effects in plants. The bulk of the evidence for protein kinase regulation by NO has mainly relied on the ability of NO donors to trigger protein kinase activities in cell suspensions and/or tissues of various species [17]. Similarly, NO scavengers and inhibitors of NO synthesis were shown to impair the activation of protein kinases triggered by PAMPs [18], ABA (abscisic acid) [19], auxin [20] and sorbitol-induced hyperosmotic stress [21]. These protein kinases exhibited MAPK (mitogen-activated protein kinase) or CDPK (Ca2+-dependent protein kinase) properties [2224]. However, although the list of examples showing the involvement of protein kinases in mediating NO signalling in plants is increasing, only a few of them have been firmly identified. This includes the tobacco MAPK SIPK (salicylic-acid-induced protein kinase) [23,25] and the alfalfa p34cdc2 cyclin-dependent protein kinase [26].

Besides MAPK and CDPK, we recently investigated the possibility that NO also contributes to the activation of protein kinases belonging to the SnRK2 [SNF1 (sucrose non-fermenting 1)-related protein kinase 2]. Plant SnRKs include three subfamilies (SnRK1, SnRK2 and SnRK3), with the SnRK2 and SnRK3 subfamilies appearing to be specific to plants [2729]. The SnRK2 protein kinases have been described as key components of water stress and ABA signalling and were shown to contribute to the adaptive responses to drought tolerance [3034]. Work focused on several SnRK2 members has suggested that the activity of these protein kinases is regulated by phosphorylation [3537]. Accordingly, Burza et al. [38] provided evidence that in tobacco BY-2 (bright-yellow 2) cells exposed to salt, the SnRK2 protein kinase NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase) is reversibly activated via the phosphorylation on two serine residues (Ser154 and Ser158) located in the kinase activation loop. We also demonstrated that the exposure of Nicotiana plumbaginifolia cell suspensions to the NO donor DEA/NO (diethylamine-NONOate) led to rapid and transient activation of NtOSAK [21]. The NO-induced activation of NtOSAK appeared to be Ca2+-independent [25,39]. In further support of a key role for NO in the regulation of NtOSAK, we showed that NO, endogenously produced in N. plumbaginifolia cell suspensions exposed to a sorbitol-induced hyperosmotic stress, is required for the full activation of this SnRK2 protein kinase [21]. Taken together, these results suggest that SnRK2 protein kinases are NO signalling components.

In animals, NO modulates the activity of different classes of protein kinases including MAPKs, protein kinase C, Janus kinases and the tyrosine kinase Src, by direct chemical modification of the kinases themselves or by modulation of upstream factors such as small GTP-binding proteins or phosphatases [4046]. Direct chemical modifications include S-nitrosylation and tyrosine nitration [4]. In plants, the process by which NO regulates the activity of protein kinases remains enigmatic. In the present study, using BY-2 cell suspensions, we attempted to provide a first insight into the mechanisms underlying this regulation by performing a detailed analysis of NtOSAK post-translational modifications mediated by NO endogenously produced in response to salt treatment or exogenously released by the NO donor DEA/NO. Our results have shown that, in response to NO, NtOSAK is not regulated by S-nitrosylation but via the phosphorylation of two residues located within the kinase activation loop, one being identified as Ser158. Furthermore, we have identified a cellular partner of NtOSAK, the glycolytic enzyme GAPDH, and provided evidence that in BY-2 cells exposed to salt, GAPDH undergoes S-nitrosylation, but is not phosphorylated by NtOSAK. Functional analysis further indicated that GAPDH S-nitrosylation did not affect NtOSAK activation or the complex formation between both enzymes.

EXPERIMENTAL

Cell culture and treatments

BY-2 tobacco cells were cultured as described previously [38]. The cells were treated with NaCl (250 mM), cPTIO (carboxy PTIO; 500 μM) or DEA/NO (50 μM) for the time indicated, harvested by filtration, quickly frozen in liquid nitrogen, and stored at −80 °C until analysed. DEA/NO was prepared as described previously [21].

Immunoblotting

Western blot analysis was performed according to a standard procedure as described by Burza et al. [38]. An anti-NtOSAK-specific polyclonal antibody raised against the C-terminal peptide (KQVQQAHESGEVRLT) of the protein kinase, an anti-Ser158(P) phospho-specific polyclonal antibody raised against the phosphopeptide K157pSTVGT phosphorylated on residue Ser158 and anti-GAPDH-specific polyclonal antibodies raised against the peptide CYDDIKAAIKEESEG of GAPDH were obtained from Biogenes. Proteins from crude extracts were separated on SDS/PAGE (10% gels) and transferred on to nitrocellulose by electroblotting using transfer buffer (25 mM Tris base and 192 mM glycine) overnight at 15 V. The membrane was blocked overnight at 4 °C in TBST {TBS [Tris-buffered saline; 10 mM Tris/HCl (pH 7.5) and 100 mM NaCl] containing 0.1% Tween 20} buffer containing 2% (w/v) BSA, and then incubated for 1 h in TBST with the primary antibodies at a dilution of 1:1000. In the case of blots probed with the anti-Ser158(P) antibodies, in order to block non-specific binding, the membranes were incubated with 5% (w/v) BSA and 5% (w/v) skimmed milk powder in TBST overnight at room temperature (20 °C), and then for 2 h in the same solution with the antibodies at a 1:500 dilution at room temperature and then overnight at 4 °C. After removing unbound antibodies by extensive washing (five times, 5 min each) with TBST, the blots were incubated for 1 h with alkaline-phosphatase-conjugated secondary antibodies (anti-rabbit from Sigma) at a 1:5000 dilution, or with peroxidase-conjugated secondary antibodies (Bio-Rad; at a 1:50000 dilution). After washing (five times, 5 min each) with TBST, immunoreactive proteins were visualized using BCIP (5-bromo-4-chloroindol-3-yl phosphate)/NBT (Nitro Blue Tetrazolium) colour development substrate (Promega) or LumiGLO® reagent (Cell Signaling Technology) respectively.

Immunoprecipitation

Immunoprecipitation was performed as described previously [39] with some minor changes. Proteins from BY-2 crude extracts (4 mg) were incubated with an anti-NtOSAK antibody (120 μg) in immunoprecipitation buffer [20 mM Tris/HCl (pH 7.5), 2 mM EDTA, 2 mM EGTA, 50 mM 2-glycerophosphate, 100 μM sodium orthovanadate, 2 mM DTT (dithiothreitol), 500 μM PMSF, 1 μM pepstatin, 1 μM leupeptin, 1 μM aprotinin, 1% Triton X-100 and 150 mM NaCl] at 4 °C for 4 h on a rocker. Approx. 50 μl of packed volume of Protein A–agarose (Santa Cruz Biotechnology) was added, and the incubation was continued for a further 2 h. Agarose bead–protein complexes were pelleted by brief centrifugation and washed three times with immunoprecipitation buffer and two times with the following buffer: 20 mM Tris/HCl (pH 7.5), 2 mM EDTA, 2 mM EGTA, 50 mM 2-glycerophosphate, 100 μM sodium orthovanadate, 2 mM DTT, 500 μM PMSF, 1 μM pepstatin, 1 μM leupeptin and 1 μM aprotinin. After washing, the immunocomplexes were divided into two pools at a ratio of 1:10. The smaller portion was used directly for the immunocomplex kinase activity assay, whereas the major part was used for determination of phosphorylation sites by MS. In parallel, cell extracts were analysed by immunoblotting with anti-Ser158(P) antibodies.

Immunocomplex kinase activity assay

Sample buffer (50 μl) was added to the pelleted agarose bead–protein complex after immunoprecipitation, and the sample was heated at 95 °C for 3 min. After brief centrifugation, the supernatant was analysed by an in-gel kinase activity assay.

In-gel kinase activity assays

In-gel kinase activity assays were performed using a method described previously [47]. Protein samples were electrophoresed on SDS/PAGE (10% gels) with 0.5 mg/ml MBP (myelin basic protein) embedded in the separating gel as a substrate for the kinase. After electrophoresis, SDS was removed by washing the gel with washing buffer [25 mM Tris/HCl (pH 7.5), 5 mM sodium fluoride, 0.5 mg/ml BSA, 0.1% Triton X-100, 0.5 mM DTT and 0.1 mM sodium orthovanadate) three times each for 30 min at room temperature. Next, proteins were renaturated overnight in renaturating buffer [25 mM Tris/HCl (pH 7.5), 5 mM sodium fluoride, 0.1% Triton X-100, 1 mM DTT and 0.1 mM sodium orthovanadate) at 4 °C with three changes of buffer. The gel was then incubated for 1.5 h at room temperature in 10 ml of reaction buffer {10 mM Tris/HCl (pH 7.5), 2 mM DTT, 0.1 mM EGTA, 15 mM MgCl2 and 20 μM ATP, supplemented with 50 μCi of [γ-32P]ATP}. Unincorporated [γ-32P]ATP was removed by extensive washing of the gels in 5% trichloroacetic acid with 1% sodium phosphate. Finally, the gels were stained with Coomassie Brilliant Blue R250, dried and exposed to X-ray film.

Staining of NtOSAK with Pro-Q Diamond

Staining was performed as described previously [38].

MS

Protein samples were analysed by LC-ESI-MS-MS/MS (liquid chromatography-electrospray ionization MS with collisional fragmentation) as described previously [38].

Biotin-switch assay

The biotin-switch technique was performed according to a procedure described by Sell et al. [48] with some minor changes. After treatment, cells (0.25 g) were harvested by filtration, frozen in liquid nitrogen and ground in a mortar. Samples were mixed with 800 μl of HEN buffer [25 mM Hepes/NaOH (pH 7.7), 1 mM EDTA, 0.1 mM neucuproine, 2 mM PMSF, 10 μM antipain and 10 μM leupeptin) containing 0.5% Chaps and freshly prepared MMTS [methyl methanethiosulfonate; 2 M in DMF (dimethylformamide)] added to a final concentration of 5 mM. The mixture obtained was centrifuged at 15000 g for 20 min and the corresponding proteins were quantified. Then, 5 mg of the protein was adjusted to 0.8 μg/μl HEN buffer followed by the addition of freshly prepared MMTS (2 M in DMF) and SDS [25% (v/v)] to final concentrations of 20 mM and 2.5% respectively. Following frequent vortex mixing at 50 °C for 20 min, proteins were precipitated with 3 vol. of acetone at −20 °C for 20 min. The proteins were recovered by centrifugation at 3200 g for 12 min, followed by gentle rinsing of the pellet four times with 1 ml of acetone. The pellets were then suspended in 10 μl of HENS (HEN buffer containing 1% SDS) per 100 μg of starting material of proteins. The samples were divided into two equal pools and were mixed with 1 mM biotin-HPDP {N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide; Thermo Scentific; dissolved in DMF} and 1 mM ascorbate in HEN buffer. For the control samples, appropriate volumes of DMF and ascorbate were added. Labelling reactions were performed in the dark at room temperature for 1 h. Next, the labelling reaction was acetone-precipitated overnight at −20 °C. The washed pellet was resuspended in 240 μl of HENS buffer, followed by the addition of 750 μl of neutralization buffer [20 mM Hepes, 100 mM NaCl, 1 mM EDTA and 0.5% Triton X-100 (pH 7.7)]. This material was incubated for 1.5 h with 150 μl of 50% neutravidin–agarose slurry (previously equilibrated in neutralization buffer). The beads were washed four times with 750 μl of wash buffer (neutralization buffer supplemented with 500 mM NaCl). The beads were eluted with 70–90 μl of 2×Laemmli buffer with 100 mM DTT. The eluted mixture was then analysed by SDS/PAGE, followed by immunoblotting with anti-NtOSAK (1:1000) or anti-GAPDH (1:1000) antibodies.

Measurement of GAPDH activity

The activity of GAPDH was determined according to a method described by Lindermayr et al. [10] with slight modifications. Crude extracts of BY-2 cell suspension cultures [300 mg of protein in 50 mM Tris/HCl (pH 7.5)] were incubated with 4 mM arsenate and 100 μg/ml 3-phosphoglycerinaldehyde and were adjusted to 1900 μl with 50 mM Tris/HCl (pH 7.5). The reaction was performed at 30 °C and initiated by adding 100 μl of 100 mM NAD+. The reduction of NAD+ to NADH was monitored by spectrophotometry at 340 nm. For in vitro inhibition assays, protein extracts were incubated before initiation of the reaction with different concentrations of DEA/NO at room temperature for 2 min.

Cloning of cDNAs encoding NtGAPCa and NtGAPCb

cDNAs encoding NtGAPCa and NtGAPCb (N. tobacum cytosolic GAPDH a and b respectively) were obtained by RT (reverse transcription)–PCR with total RNA isolated from 3-week-old N. tabacum (LA Burley 21 line) plants. RNA was extracted using TRIzol® reagent (Invitrogen) according to the manufacturer's instructions. First-strand cDNA was synthesized using 5 μg of total RNA as a template with the Enhanced Avian HS RT–PCR kit (Sigma–Aldrich). The cDNA was used for PCR amplification of NtGAPCa and NtGAPCb with primer pairs (listed in Supplementary Table S1 at http://www.BiochemJ.org/bj/429/bj4290073add.htm) designed based on nucleotide sequences available in databases. PCR products were cloned into pCR II-TOPO vector (Invitrogen) and verified by DNA sequencing.

Expression of recombinant proteins in Escherichia coli

Expression and purification of GST (glutathione transferase)–NtOSAK was performed as described previously [38]. Full-length cDNAs encoding NtGAPCa and NtGAPCb were PCR-amplified using the appropriate primers listed in Supplementary Table S1 and EcoRI/SalI fragments of NtGAPCa and EcoRI/XhoI of NtGAPCb were cloned into the expression vector pET-28A (Novagen).

All PCRs were performed using a high-fidelity Pfu DNA polymerase (Stratagene) and verified by sequencing. Recombinant proteins were expressed overnight in E. coli BL21(DE3) cells at 18 °C and purified using glutathione–agarose beads (Sigma–Aldrich) as described previously [38] or Ni-NTA (Ni2+-nitrilotriacetate)–agarose beads (Qiagen) according to the manufacturer's instructions.

In vitro binding assay

Purified recombinant NtOSAK (5 μg) without a GST tag (the GST epitope was cleaved off with thrombin) was mixed with a similar amount of His6–NpGAPC1 or His6–NpGAPC2 attached to Ni-NTA–agarose beads (Qiagen), or as a control to Ni-NTA–agarose beads in binding buffer [50 mM Tris/HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole and 0.1% Triton X-100]. After gentle rotation (overnight at 4 °C) the beads were centrifuged (1 min at 1000 g) and washed six times with the binding buffer. Proteins attached to the resin were analysed by SDS/PAGE and then by Western blotting using anti-NtOSAK antibodies.

Protoplast transient expression assay and BiFC (bimolecular fluorescence complementation)

Protoplasts were isolated and transformed via PEG [poly(ethylene) glycol] treatment according to the protocol of He et al. [49] with minor modifications.

After transformation, Arabidopsis thaliana T87 protoplasts were suspended in WI incubation solution [0.5 M mannitol, 4 mM Mes (pH 5.7) and 20 mM KCl] and incubated at 25 °C in the dark for 2 days.

cDNAs encoding NtOSAK, NtGAPCa, NtGAPCb, NtGAPCa(C157S,C161S) and NtGAPCb(C154S,C158S) were PCR-amplified using Pfu DNA polymerase (the primers used are listed in Supplementary Table S1), cloned into pCRII-TOPO (Invitrogen) and verified by DNA sequencing. For analysis of the effect of NtGAPCs (wild-type and mutated forms) on NtOSAK activity, the XhoI/EcoRI fragment of cDNA encoding NtOSAK and XhoI/BamHI fragments of cDNAs encoding NtGAPCa, NtGAPCb, NtGAPCa(C157S,C161S) or NtGAPCb(C154S,C158S) were inserted into pSAT6-EGFP-C1. For BiFC, the cDNA encoding NtOSAK was inserted into pSAT4-nEYFP-C1 and the cDNA fragments for NtGAPCs into pSAT4-cEYFP-C1-B. The vectors were provided by Dr T. Tzfira (University of Michigan, Ann Arbor, MI, U.S.A.).

In each transformation approx. 2×105 protoplasts were transfected with approx. 40 μg of plasmid DNA; for BiFC experiments the plasmids were mixed in a 1:1 (w/w) ratio. The transfected protoplasts after incubation in the dark for 2 days were subjected to 250 mM NaCl treatment. In control experiments, water instead of NaCl was added to the transfected protoplasts. Protoplasts were carefully placed on to slides with home-made chambers preventing damage and drying. Fluorescent images was visualized with the Nikon EZ-C1 laser-scanning microscope, equipped with a 60× [NA (numerical aperture) 1.4] PlanApo oil-immersion objective mounted on an inverted epifluorescence microscope TE 2000E. The fluorescence of EYFP (enhanced yellow fluorescent protein) was excited with blue light at 488 nm emitted by a 40 mW argon-ion laser (Melles Griot). EYFP fluorescence was detected with a 535/30 nm band-pass filter and rendered in false green. Nuclei were stained with 0.3 mM Hoechst 33342 (H3570, Invitrogen) and excited with the 408 nm line from an MOD diode laser (Melles Griot). Hoechst fluorescence was detected with a 450/35 nm band-pass filter and rendered in false blue. Fluorescent images are single optical sections made with the standard EZ-C1 Nikon software. Brightness and contrast were adjusted with the Adobe Photoshop 6.0 program.

Site-directed mutagenesis

pSAT6-EGFP-NtGAPCa and pSAT6-EGFP-NtGAPCb constructs were used as a template for site-directed mutagenesis with the QuikChange® II site-directed mutagenesis kit (Stratagene). The primers used for substitution of Cys157 and Cys161 in NtGAPCa and Cys154 and Cys158 in NtGAPCb are listed in Supplementary Table S1. The constructs obtained were sequenced to verify the mutations introduced.

RESULTS

NO contributes to NaCl-induced NtOSAK activation

Protein kinase activities were assessed by in-gel kinase assays using MBP as a substrate. Exposure of BY-2 cells to 250 mM NaCl resulted in the rapid activation of two protein kinases with apparent molecular masses of 47 and 42 kDa previously identified as SIPK and NtOSAK respectively (Figure 1) [50]. To investigate the involvement of NO in mediating NaCl-induced SIPK and NtOSAK activation, cPTIO, an NO scavenger, was used. This compound has been shown to suppress the fast production of NO triggered by salt both in tobacco cell suspensions and plant tissues [51]. Pre-treatment of BY-2 cells with cPTIO affected the salt-induced activation of SIPK and NtOSAK: SIPK activation was almost completely suppressed, whereas NtOSAK activity was reduced by approx. 50%. As previously reported [21,25], the ability of NO to promote NtOSAK activation was confirmed by the demonstration that treatment of BY-2 cells with 50 μM of the NO donor DEA/NO promotes its transient activation within 30 min (Figure 2A). In contrast, SIPK activation by DEA/NO was detected only at millimolar concentrations of DEA/NO (results not shown; [25]). Taken together, these results indicate that the activation of NtOSAK triggered by salt depends, at least partially, on NO production. This result fits well with our previous study [21] showing that NO produced in response to sorbitol-induced hyperosmotic stress acts upstream of NtOSAK.

NO contributes to the activation of NtOSAK and SIPK induced in response to salt

Figure 1
NO contributes to the activation of NtOSAK and SIPK induced in response to salt

cPTIO (500 μM) was added to BY-2 cell suspensions 10 min prior to salt (250 mM NaCl). Aliquots of the culture were taken at the times indicated and analysed for protein kinase activity by in-gel kinase assay with MBP as substrate. Results shown are one from five representative experiments.

Figure 1
NO contributes to the activation of NtOSAK and SIPK induced in response to salt

cPTIO (500 μM) was added to BY-2 cell suspensions 10 min prior to salt (250 mM NaCl). Aliquots of the culture were taken at the times indicated and analysed for protein kinase activity by in-gel kinase assay with MBP as substrate. Results shown are one from five representative experiments.

NO induces NtOSAK phosphorylation

Figure 2
NO induces NtOSAK phosphorylation

(A) DEA/NO triggers NtOSAK activation in BY-2 cell suspensions. Cell suspensions were treated with DEA/NO (50 μM) for the time indicated or with NaCl (250 mM) for 5 min as a positive control. Protein extracts were immunoprecipitated with anti-NtOSAK antibodies. The resulting immunocomplexes were analysed by in-gel kinase assay with MBP as a substrate. Results shown are one from five representative experiments. (B) The immunocomplexes obtained in (A) were subjected to SDS/PAGE. Phosphorylation of NtOSAK was determined by Pro-Q Diamond stain. Results shown are one from three representative experiments. (C) Analysis of NtOSAK phosphorylation on residue Ser158. Crude extracts from BY-2 cells treated with DEA/NO (50 μM) for 60 min or NaCl (250 mM) for 5 min were subjected to Western blot analysis and probed with anti-Ser158(P) antibodies. Results shown are one from three representative experiments.

Figure 2
NO induces NtOSAK phosphorylation

(A) DEA/NO triggers NtOSAK activation in BY-2 cell suspensions. Cell suspensions were treated with DEA/NO (50 μM) for the time indicated or with NaCl (250 mM) for 5 min as a positive control. Protein extracts were immunoprecipitated with anti-NtOSAK antibodies. The resulting immunocomplexes were analysed by in-gel kinase assay with MBP as a substrate. Results shown are one from five representative experiments. (B) The immunocomplexes obtained in (A) were subjected to SDS/PAGE. Phosphorylation of NtOSAK was determined by Pro-Q Diamond stain. Results shown are one from three representative experiments. (C) Analysis of NtOSAK phosphorylation on residue Ser158. Crude extracts from BY-2 cells treated with DEA/NO (50 μM) for 60 min or NaCl (250 mM) for 5 min were subjected to Western blot analysis and probed with anti-Ser158(P) antibodies. Results shown are one from three representative experiments.

NO regulates NtOSAK by phosphorylation on residues Ser154 and Ser158, but not via S-nitrosylation

To decipher the mechanisms underlying NtOSAK activation by NO, we analysed its phosphorylation state in tobacco cells exposed to 50 μM DEA/NO for 30 min. For this purpose, proteins extracted from DEA/NO or salt-treated cells were immunoprecipitated with a specific anti-NtOSAK polyclonal antibody and the resulting immunocomplexes were analysed by an in-gel kinase assay (Figure 2A) and stained with the Pro-Q Diamond stain (Figure 2B) which recognizes phosphorylated serine, tyrosine and threonine residues. As shown in Figure 2(B), NtOSAK displays phosphorylation in BY-2 cells exposed to salt in accordance with previous results [38], but also in response to DEA/NO. The phosphorylation of NtOSAK was well correlated with its protein kinase activity (Figure 2A).

We next mapped the NtOSAK amino acid residues undergoing phosphorylation in tobacco cells exposed to DEA/NO. Particular attention was paid to residues Ser154 and Ser158 as active NtOSAK, isolated from NaCl-treated BY-2 cells, was shown to be phosphorylated on those residues [38]. First, protein extracts from DEA/NO or NaCl-treated BY-2 cells were subjected to Western blot analysis and probed with the phospho-specific antibody anti-Ser158(P). This antibody, raised against the phosphorylated peptide K157pSTVGT, specifically reacts with NtOSAK phosphorylated on Ser158 [38]. As expected, immunostaining revealed one band in the protein extracts corresponding to phosphorylated NtOSAK present in NaCl-treated cells (Figure 2C). Of interest, the antibodies recognized the phosphopeptide also in the protein extracts from cells exposed to DEA/NO for 30 min, indicating that NtOSAK is phosphorylated on Ser158 in response to NO. Secondly, in order to check whether NO promotes the phosphorylation of Ser154, NtOSAK was immunoprecipitated from BY-2 cells untreated or treated with DEA/NO for 30 min according to the procedure described by Burza et al. [38], and subjected to SDS/PAGE. Then the protein was excised from the gel and the corresponding tryptic fragments were analysed by LC-ESI-MS-MS/MS. In the samples corresponding to DEA/NO-treated cells, the MS analysis revealed the presence of two peptides (residues 149–157 and residues 158–173; Table 1) containing Ser154 and Ser158 respectively, and showing an m/z value increased by 40 as compared with the m/z values measured in the control samples. This difference in the m/z value, expected for doubly charged phosphorylated species, corresponds to a difference of 80 Da and highlights the presence of a phosphorylated residue in both peptides. Therefore, based on the immunoblot assay (Figure 2C) and the findings of Burza et al. [38] showing that NtOSAK is phosphorylated on Ser158 and Ser154 in response to salt treatment, our MS result strongly suggests that NO up-regulates NtOSAK activity by promoting the phosphorylation on both serine residues.

Table 1
Mass spectrometric identification of NtOSAK phosphorylation peptides

BY-2 cell suspensions were treated with DEA/NO (50 μM) for 30 min. Protein extracts were immunoprecipitated with anti-NtOSAK antibodies and separated by SDS/PAGE. Coomassie-Blue-stained protein bands were excised from the gel and analysed by LS-MS-MS/MS after in-gel digestion with trypsin. Protein sequence database searches were carried out by MASCOT (http://www.MatrixScience.com). MS/MS fragmentation ion scores (ion scores) indicate the presence or absence of a phosphorylated and unphosphorylated form of the peptide containing Ser154 and Ser158 (in bold where phosphorylated) in NtOSAK. For each peptide signal its m/z and charge is given. Results are one from three representative experiments. – indicates lack of the phosphorylated form of the peptide.

Peptides Control ion score (m/z, charge) DEA/NO treatment ion score (m/z, charge) 
Phosphorylated peptides: sequence   
 S149SLLHSRPK157 – 33 (552.77, 2+
S158TVGTPAYIAPEVLSR173 – 34 (870.93, 2+
Unphosphorylated peptides: sequence   
 S149SLLHSRPK157 42 (512.79, 2+40 (512.79, 2+
 S158TVGTPAYIAPEVLSR173 78 (830.94, 2+60 (830.94, 2+
Peptides Control ion score (m/z, charge) DEA/NO treatment ion score (m/z, charge) 
Phosphorylated peptides: sequence   
 S149SLLHSRPK157 – 33 (552.77, 2+
S158TVGTPAYIAPEVLSR173 – 34 (870.93, 2+
Unphosphorylated peptides: sequence   
 S149SLLHSRPK157 42 (512.79, 2+40 (512.79, 2+
 S158TVGTPAYIAPEVLSR173 78 (830.94, 2+60 (830.94, 2+

We also considered the possibility that NO might regulate NtOSAK through S-nitrosylation. To verify this assumption, NtOSAK putative S-nitrosylation was surveyed by the biotin-switch assay [52] after treatment of BY-2 cells with the NO donor DEA-NO or NaCl for various times (up to 1 h). In a first step, protein extracts were treated with MMTS in order to chemically block all free thiol groups without modifying nitrosothiols or disulfides. In a second step, nitrosothiols, but not disulfides, were reduced to free thiol groups by ascorbate. This step was followed by the biotinylation of the newly formed free thiols by biotin-HPDP. Next, the biotinylated proteins were concentrated by affinity purification with neutravidin–agarose, eluted under reducing conditions using a DTT-enriched buffer and then subjected to Western blot analysis using a specific anti-NtOSAK polyclonal antibody. As shown in Figure 3, immunostaining did not reveal the presence of NtOSAK in the samples corresponding to biotinylated proteins. In contrast, NtOSAK was clearly detected in the samples containing proteins that did not bind to neutravidin, that is those which were not biotinylated during the biotin-switch assay. The presence of NtOSAK in the samples corresponding to non-biotinylated proteins suggested that NtOSAK is not regulated by S-nitrosylation in response to salt or NO released by DEA/NO.

NtOSAK does not undergo S-nitrosylation

Figure 3
NtOSAK does not undergo S-nitrosylation

BY-2 cell suspensions were treated with DEA/NO (50 μM) or with NaCl (250 mM) for the time indicated. The corresponding crude extracts were subjected to the biotin-switch assay. Biotinylated proteins were purified with neutravidin–agarose and eluted under reducing conditions. Biotinylated proteins and non-biotinylated proteins (that is, proteins that did not bind to neutravidin) were analysed by Western blotting using anti-NtOSAK antibodies. Results shown are one from three representative experiments.

Figure 3
NtOSAK does not undergo S-nitrosylation

BY-2 cell suspensions were treated with DEA/NO (50 μM) or with NaCl (250 mM) for the time indicated. The corresponding crude extracts were subjected to the biotin-switch assay. Biotinylated proteins were purified with neutravidin–agarose and eluted under reducing conditions. Biotinylated proteins and non-biotinylated proteins (that is, proteins that did not bind to neutravidin) were analysed by Western blotting using anti-NtOSAK antibodies. Results shown are one from three representative experiments.

GAPDH is present in immunocomplex with NtOSAK and undergoes S-nitrosylation under salt treatment

To further explore NtOSAK regulation in the salt signalling pathway, a search for proteins which interact with NtOSAK was performed. For this purpose, we undertook the identification of proteins co-immunoprecipitating with NtOSAK in extracts prepared from BY-2 cells subjected to 250 mM NaCl for 5 min. Proteins pulled down together with NtOSAK in the immunocomplex or pre-immune serum (negative control) were separated by SDS/PAGE and the Coomassie-Blue-stained protein bands were excised from the gel (Supplementary Figure S1 at http://www.BiochemJ.org/bj/429/bj4290073add.htm). After in-gel digestion with trypsin, the corresponding peptides were analysed by ESI-MS-MS/MS and identified using the MASCOT search engine. The list of proteins selectively co-immunoprecipitated with NtOSAK, but not with the pre-immune serum, as well as their functional classification, is shown in Table 2. These proteins are related to N/C (nitrogen and carbon) metabolism, cellular architecture, protein synthesis and degradation, and ATP metabolism.

Table 2
Identification of proteins co-immunoprecipitating with NtOSAK in BY-2 cells exposed to salt

Cell suspensions were treated with NaCl (250 mM) for 5 min. Protein extracts were immunoprecipitated with anti-NtOSAK antibodies or pre-immune serum as a control. Proteins present in the immunocomplex were separated by SDS/PAGE. Coomassie-Blue-stained protein bands were excised from the gel and analysed by ESI-MS-MS/MS after in-gel digestion with trypsin. Results were used to search the NCBI (National Center for Biotechnology Information) database using the MASCOT search engine (http://www.MatrixScience.com) under the taxon restriction of Viridiplantae. The Table shows a list of proteins selectively co-immunoprecipitated with NtOSAK, but not with the pre-immune serum, in response to NaCl treatment. *Protein scores are derived from ion scores as a non-probabilistic basis for ranking protein hits; **number of peptides sequenced de novo found to be derived from a given protein; ***band numbers correspond to Supplementary Figure S1 (at http://www.BiochemJ.org/bj/429/bj4290073add.htm). Results are one from three representative experiments.

Accession number Name Protein score* Queries matched** Band number*** 
gi|19568098 OSAK 921 26 
gi|120676 GAPDH 325 12 
gi|1419092 Glutamine synthetase (cytosolic) 299 12 
gi|3021506 Isocitrate dehydrogenase (NAD+177 
gi|40036995 β-Tubulin 130 
gi|78191448 ADP/ATP translocator-like 122 
gi|50058115 Actin 84 
Accession number Name Protein score* Queries matched** Band number*** 
gi|19568098 OSAK 921 26 
gi|120676 GAPDH 325 12 
gi|1419092 Glutamine synthetase (cytosolic) 299 12 
gi|3021506 Isocitrate dehydrogenase (NAD+177 
gi|40036995 β-Tubulin 130 
gi|78191448 ADP/ATP translocator-like 122 
gi|50058115 Actin 84 

Interestingly, one of the potential partners of NtOSAK showing the highest score corresponds to the glycolytic enzyme GAPDH. In animals, NO was shown to elicit S-nitrosylation of GAPDH under several physiological contexts, including apoptosis [53,54]. In A. thaliana, Lindermayr et al. [10] demonstrated that GAPDH is S-nitrosylated in vitro by artificially released NO, this process leading to a reversible inhibition of the enzyme activity. Based on these results, we monitored the influence of NO on GAPDH by analysing its S-nitrosylation profile in BY2 cells exposed to salt or DEA/NO for up to 1 h. For this purpose, proteins extracted at different time points during both treatments were subjected to the biotin-switch assay. The resulting biotinylated proteins were purified by affinity chromatography using neutravidin–agarose and then subjected to Western blot analysis using a specific anti-GAPDH polyclonal antibody. As a negative control, the same procedure was performed without adding biotin-HPDP. The immunoblots in Figure 4 show the presence of a band only in the samples in which biotin-HPDP was added during the biotin-switch assay. Therefore the detection of the immunoreactive bands in those samples did not reflect a constitutive and/or inducible endogenous biotinylation of GAPDH in response to salt or DEA/NO treatment, but highlighted the S-nitrosylation of the enzyme. More precisely, S-nitrosylated GAPDH was already detected at zero time, suggesting that the enzyme is constitutively S-nitrosylated in our culture conditions. In response to DEA/NO, the level of S-nitrosylation increased after 30 min, a time at which NtOSAK showed its highest activity in NO-treated cells (Figure 2A). In cells challenged by salt, the increase in GAPDH S-nitrosylation was maximal at 5–15 min and returned to the basal level within 30 min (see also Supplementary Figure S2 at http://www.BiochemJ.org/bj/429/bj4290073add.htm for quantification). Here too, the increase in the GAPDH S-nitrosylation level was well correlated with NtOSAK kinetic activity (Figure 1). Taken together, these results indicate that in BY-2 cells exposed to salt, GAPDH is present in immunocomplex with NtOSAK and the induction of NtOSAK protein kinase activity and GAPDH S-nitrosylation occur in the same lapse of time. However, it should be specified that only a small proportion of GAPDH appeared to be regulated by S-nitrosylation in response to salt as most of the enzyme was clearly detected in the non S-nitrosylated fraction (Supplementary Figure S3 at http://www.BiochemJ.org/bj/429/bj4290073add.htm).

Salt-stress induced GAPDH S-nitrosylation

Figure 4
Salt-stress induced GAPDH S-nitrosylation

BY-2 cell suspensions were treated with DEA/NO (50 μM) or with NaCl (250 mM) for the time indicated. The corresponding crude extracts were subjected to the biotin-switch assay. As a negative control, the biotin-switch assay was performed without adding the sodium ascorbate. Biotinylated proteins were then purified with neutravidin–agarose, eluted under reducing conditions and analysed by Western blotting using anti-GAPDH antibodies. Results shown are one from three representative experiments.

Figure 4
Salt-stress induced GAPDH S-nitrosylation

BY-2 cell suspensions were treated with DEA/NO (50 μM) or with NaCl (250 mM) for the time indicated. The corresponding crude extracts were subjected to the biotin-switch assay. As a negative control, the biotin-switch assay was performed without adding the sodium ascorbate. Biotinylated proteins were then purified with neutravidin–agarose, eluted under reducing conditions and analysed by Western blotting using anti-GAPDH antibodies. Results shown are one from three representative experiments.

GAPDH activity is not impaired in BY2 cells exposed to salt or DEA/NO

As stated above, it was previously reported that the exposure of crude extracts of A. thaliana cell cultures to NO donors leads to the inhibition of GAPDH activity through direct S-nitrosylation [10]. Because GAPDH was found to be transiently S-nitrosylated in BY2 cells exposed to salt or DEA/NO (Figure 4), we investigated whether these treatments could affect the activity of the enzyme. Therefore BY2 cell suspensions were exposed to NaCl or DEA/NO for various times, and the total GAPDH activity was determined. As shown in Figure 5(A), treatment of cell suspensions with salt or DEA/NO did not affect the total GAPDH activity. To complete these data, we next analysed GAPDH activity in BY2 cell crude extracts exposed to DEA/NO at room temperature. Similarly to the Lindermayr et al. [10] study, under these in vitro conditions, the NO donor reduced the GAPDH activity by 25% and 85% when used at 50 μM or 500 μM respectively (Figure 5B). Taken together, these results indicate that, whereas an inhibition of GAPDH activity by NO occurred in vitro, the activity of the enzyme was not impaired in vivo following the exposure of BY2 cells to salt or DEA/NO. This suggests that the S-nitrosylation of GAPDH observed in vivo (Figure 4) had a negligible effect on the total GAPDH activity. This result seems plausible as only a small proportion of GAPDH undergoes S-nitrosylation under these conditions (Figure 4 and Supplementary Figure S3).

GAPDH activity is not inhibited in salt or DEA/NO treated cells

Figure 5
GAPDH activity is not inhibited in salt or DEA/NO treated cells

(A) In vivo activity. BY-2 cell suspensions were treated with DEA/NO (50 μM) for 30 min or with NaCl (250 mM) for 5 min. Then, GAPDH activity was measured by following the time course of NAD+ reduction to NADH by spectrophotometry at 340 nm (see the Experimental section). Results shown are one from three representative experiments. (B) In vitro activity. Proteins extracted from untreated BY-2 cells were exposed to NO artificially released by DEA/NO (50 or 500 μM) for 2 min and GAPDH activity was measured as specified in (A). Results shown are one from three representative experiments.

Figure 5
GAPDH activity is not inhibited in salt or DEA/NO treated cells

(A) In vivo activity. BY-2 cell suspensions were treated with DEA/NO (50 μM) for 30 min or with NaCl (250 mM) for 5 min. Then, GAPDH activity was measured by following the time course of NAD+ reduction to NADH by spectrophotometry at 340 nm (see the Experimental section). Results shown are one from three representative experiments. (B) In vitro activity. Proteins extracted from untreated BY-2 cells were exposed to NO artificially released by DEA/NO (50 or 500 μM) for 2 min and GAPDH activity was measured as specified in (A). Results shown are one from three representative experiments.

GAPDH interacts directly with NtOSAK

In order to establish whether GAPDH interacts directly or indirectly with NtOSAK, an in vitro pull-down assay was used. The A. thaliana genome encodes two cytosolic GAPDHs, GAPC1 (At3g04120) and GAPC2 (At1g13446). In the NCBI nucleotide database, two cDNA sequences for N. tabacum cytosolic GAPDHs were deposited [GenBank® accession numbers M14419 (partial cDNA sequence) and AJ133422]. We have cloned cDNAs encoding these GAPDHs (NtGAPCa and NtGAPCb) by RT–PCR using total RNA isolated from tobacco seedlings as a template and appropriate primers designed based on sequences available in nucleotide and EST (expressed sequence tag) databases. The sequences of the cDNA we cloned did not completely fit with those of M14419 and AJ133422 deposited in the NCBI database. The sequence identities between NtGAPCa and M14419, and between NtGAPCb and AJ133422 were approx. 99%. Analogous discrepancies were also observed for A. thaliana GAPCs [9]. Most probably, these variations are due to ecotype-specific GAPDHs in plants.

In order to produce the tobacco GAPDH proteins for a pull-down assay, the cloned cDNAs were introduced into the bacterial expression vector pET-28A and recombinant proteins His6–NtGAPCa and His6–NtGAPCb were produced in E. coli. NtOSAK was expressed in E. coli as a fusion protein with a GST tag, purified on glutathione–Sepharose beads, and the GST epitope was cleaved off with thrombin. The purified NtOSAK was incubated with His6–NtGAPCa or His6–NtGAPCb attached to Ni-NTA–agarose or with Ni-NTA–agarose beads, as a control. After incubation followed by extensive washing of the beads, the presence of NtOSAK bound to His6–NtGAPCa or His6–NtGAPCb or to free Ni-NTA–agarose (control) was analysed by SDS/PAGE and then by Western blotting using anti-NtOSAK antibodies (Figure 6). The results revealed that NtOSAK interacts directly with both NtGAPCs.

NtOSAK directly interacts with NtGAPCa and NtGAPCb

Figure 6
NtOSAK directly interacts with NtGAPCa and NtGAPCb

In vitro binding assay of His6–NtGAPCa and His6–NtGAPCb with NtOSAK. Ni-NTA–agarose with attached His6–NtGAPCa or His6–NtGAPCb was incubated with purified NtOSAK produced in E. coli., or as a control NtOSAK was incubated with Ni-NTA–agarose. In additional controls, NtOSAK was not added to His6–NtGAPCa or His6–NtGAPCb Ni-NTA–agarose. After washing, the presence of NtOSAK attached to the beads was analysed by Western blotting with anti-NtOSAK antibodies. Results shown are one from three representative experiments.

Figure 6
NtOSAK directly interacts with NtGAPCa and NtGAPCb

In vitro binding assay of His6–NtGAPCa and His6–NtGAPCb with NtOSAK. Ni-NTA–agarose with attached His6–NtGAPCa or His6–NtGAPCb was incubated with purified NtOSAK produced in E. coli., or as a control NtOSAK was incubated with Ni-NTA–agarose. In additional controls, NtOSAK was not added to His6–NtGAPCa or His6–NtGAPCb Ni-NTA–agarose. After washing, the presence of NtOSAK attached to the beads was analysed by Western blotting with anti-NtOSAK antibodies. Results shown are one from three representative experiments.

GAPDH interacts with NtOSAK in planta

To study the interaction between NtOSAK and GAPDH in plant cells we applied BiFC. Appropriate constructs for transient expression of NtOSAK and NtGAPCa or NtGAPCb fused to complementary non-fluorescent fragments of YFP (described in the Experimental section) were introduced into A. thaliana (T-87 cells) protoplasts. In the first approach, we have tried to transform tobacco BY-2 cells protoplasts for this purpose, however, in our hands this transformation was not efficient enough. Therefore we have chosen A. thaliana protoplasts for our studies. Interaction of the protein partners resulted in reconstruction of YFP and its fluorescence. For negative controls, each fusion protein was tested in the presence of the other half of YFP alone. We have observed NtOSAK–NtGAPCa and NtOSAK–NtGAPCb complex formation in protoplasts not treated and treated with 250 mM NaCl (Figure 7). In the absence of salt treatment, the NtOSAK–NtGAPCa complex formation occurred mainly in the cytoplasm and sporadically also in the nucleus, whereas the NtOSAK–NtGAPCb complex was localized more often to the nucleus (besides the cytoplasm), suggesting that both GADPHs may have a different function in plant cells. The salt treatment did not change significantly the complex localization. Moreover, NtGAPCb very often formed aggregates (when expressed in protoplasts with or without NtOSAK). Aggregate formation is a well-known feature of GAPDH, which can play a role in oxidative stress-induced cell death [55].

NtOSAK interacts with NtGAPCa and NtGAPCb in planta

Figure 7
NtOSAK interacts with NtGAPCa and NtGAPCb in planta

NtOSAK interacts with NtGAPCa and NtGAPCb in A. thaliana T87 protoplasts, as shown by BiFC. The physical interaction of NtOSAK and NtGAPCa or NtGAPCb leads to reconstitution of the EYFP molecule. The EYFP signal was localized mainly to the cytoplasm in the case of complex formation between NtOSAK and NtGAPCa before (A) and after (C) 1 h of treatment with 250 mM NaCl. EYFP was localized to the cytoplasm and nucleus in the case of interaction between NtOSAK and NtGAPCb before (B) and after (D) 1 h of treatment with 250 mM NaCl. Interaction between NtOSAK and GAPCa(C157S,C161S) or GAPCb(C154S,C158S) in protoplasts not treated (E and F respectively) and treated with NaCl (G and H respectively) gave the BiFC signal restricted only to the cytoplasm. False green colour represents EYFP (BiFC). False blue colour represents stained nuclei (Hoechst). Merge, overlay of the EYFP and Hoechst signals. Results shown are one from several independent experiments showing similar results.

Figure 7
NtOSAK interacts with NtGAPCa and NtGAPCb in planta

NtOSAK interacts with NtGAPCa and NtGAPCb in A. thaliana T87 protoplasts, as shown by BiFC. The physical interaction of NtOSAK and NtGAPCa or NtGAPCb leads to reconstitution of the EYFP molecule. The EYFP signal was localized mainly to the cytoplasm in the case of complex formation between NtOSAK and NtGAPCa before (A) and after (C) 1 h of treatment with 250 mM NaCl. EYFP was localized to the cytoplasm and nucleus in the case of interaction between NtOSAK and NtGAPCb before (B) and after (D) 1 h of treatment with 250 mM NaCl. Interaction between NtOSAK and GAPCa(C157S,C161S) or GAPCb(C154S,C158S) in protoplasts not treated (E and F respectively) and treated with NaCl (G and H respectively) gave the BiFC signal restricted only to the cytoplasm. False green colour represents EYFP (BiFC). False blue colour represents stained nuclei (Hoechst). Merge, overlay of the EYFP and Hoechst signals. Results shown are one from several independent experiments showing similar results.

We have also analysed the impact of NtGAPCa or NtGAPCb S-nitrosylation on their interaction with NtOSAK. It was shown previously that Cys155 and Cys159 of A. thaliana GAPC1 and GAPC2, corresponding to Cys157 and Cys161 or Cys154 and Cys158 of tobacco NtGAPCa or NtGAPCb respectively, undergo glutathionylation and S-nitrosylation [9]. Therefore we replaced these cysteine residues in NtGAPCa and NtGAPCb with serine residues and used constructs with cDNA encoding NtGAPCa(C157S,C161S) or GAPCb(C154S,C158S) and NtOSAK fused to complementary fragments of YFP for/in BiFC analysis. Mutated GAPCs interacted with NtOSAK, exclusively in the cytoplasm (Figure 7), since NtGAPCa(C157S,C161S) and GAPCb(C154S,C158S) were not found in the nucleus.

Taken together, our results indicate that NtOSAK interacts with GAPDH in living plant cells and this interaction is independent of stress application and S-nitrosylation. However, the absence of the complexes between NtOSAK and the mutated forms of NtGAPCa and NtGAPCb from the nucleus suggests that the corresponding cysteine residues in the native forms of NtGAPCs might play an important role in the cellular localization of the proteins.

NtOSAK does not phosphorylate GAPDH in vitro and under our experimental conditions GAPDH does not influence NtOSAK activity

The interaction between NtOSAK and NtGAPCa or NtGAPCb indicates that GAPDH can be a substrate of NtOSAK and/or one of its regulators in plant cells. To determine whether tobacco GAPDH could be phosphorylated by NtOSAK, an in vitro phosphorylation reaction was performed using purified proteins (NtOSAK and His6–NtGAPCa or His6–NtGAPCb) expressed in E. coli. Additionally, as a source of the active kinase, we used NtOSAK purified from tobacco BY-2 cells, as has been described previously [39]. The phosphorylation reaction was conducted under standard phosphorylation conditions elaborated for this kinase [39]. Our results indicate that GAPDH is not phosphorylated by NtOSAK in vitro (results not shown).

To corroborate the possible role of the S-nitrosylation of GAPDH in NtOSAK activation in response to salinity stress, we expressed EGFP–NtOSAK (EGFP is enhanced green fluorescent protein) with EGFP–NtGAPCa or EGFP–GAPCb [wild-types and their mutated forms, EGFP–NtGAPCa(C157S,C161S) or EGFP–GAPCb(C154S,C158S)] in A. thaliana protoplasts. At 2 days after transformation, protoplasts were treated with 250 mM NaCl and the activity of EGFP–NtOSAK expressed in protoplasts together with each of NtGAPCs (not mutated or mutated) or with EGFP (as control) was analysed. The kinase activity was monitored by an in-gel kinase activity assay using MBP as a substrate. In our experimental conditions we have not observed any significant changes of NtOSAK activity caused by the presence of NtGAPCs (wild-type or mutated forms) in plant protoplasts (Supplementary Figure S4 at http://www.BiochemJ.org/bj/429/bj4290073add.htm). However, it has to be stressed that in protoplasts the level of endogenous native GAPDH, was very high in most experiments, even higher than transiently expressed EGFP–NtGAPCa or EGFP–GAPCb (Supplementary Figure S4). Therefore the effect of artificially introduced NtGAPCs on NtOSAK activity is extremely difficult to estimate. Additionally, GAPDH exists in oligomeric forms; in the protoplasts analysed most probably the native A. thaliana GAPC1 or GAPC2 can form oligomers with expressed EGFP–NtGAPCs, and this makes the analysis even more complicated. In our experimental conditions we are simply unable to establish the role of GAPDH and its S-nitrosylation in NtOSAK signalling in response to salinity stress.

DISCUSSION

In previous studies, we have reported that NO promotes the activation of the SnRK2 protein kinase NtOSAK in tobacco cell suspensions subjected to a hyperosmotic stress triggered by sorbitol [21]. In the present study, we confirmed the key role of NO in regulating NtOSAK activity in vivo by showing that it also contributes to NtOSAK activation in BY-2 cells exposed to salt stress. The functional relationship between NO and NtOSAK is further strengthened by the demonstration that the protein kinase is transiently activated in response to NO artificially released by the NO donor DEA/NO (the present study and [21,25]). The observation that the NO scavenger cPTIO partly, but not completely, suppressed the salt-induced NtOSAK activation suggests that the up-regulation of the protein kinase may occur through both NO-dependent and NO-independent pathways. It is also plausible that the partial inhibition of NtOSAK might be related to the inability of cPTIO to fully scavenge NO produced in response to salt. Supporting this assumption, Gould et al. [51] reported that the scavenging efficiency of cPTIO varies according to the abiotic stress applied to plant cell suspensions. Notably, these authors observed that in tobacco cell suspensions, the addition of 250 mM of NaCl in the extracellular medium reduced by 40% the efficiency of cPTIO to scavenge NO released by the NO donor NOC-9. The mechanism underlying this observation remains to be investigated.

Previous studies have indicated that members of the SnRK2 subfamily including NtOSAK are activated by phosphorylation [35,38,50,56]. Accordingly, we found that the activation of NtOSAK observed in BY-2 cells exposed to DEA/NO is correlated with an increase in its phosphorylation status. Furthermore, MS analysis of NtOSAK purified from DEA/NO-treated cells revealed the presence of two phosphopeptides (S149SLLHSRPK157; S158TVGTPAYIAPEVLSR173) containing Ser154 and Ser158 respectively. Those residues are localized in the kinase activation loop (residues 143–169) and were previously identified as two key phosphorylation sites in active NtOSAK [38]. The NO-induced phosphorylation of Ser158 was further confirmed by its visualization by Western blotting with anti-Ser158(P) antibodies. Taken together, these results establish that in response to the NO donor, NtOSAK undergoes phosphorylation on Ser158 and on a second serine residue localized within the peptide S149SLLHSRPK157 and which might correspond to Ser154. Therefore the mechanisms by which NO activates NtOSAK resembles those observed in response to salt. This finding clearly reinforces the notion that NO is a key mediator of NtOSAK activation.

Beside phosphorylation, we also monitored whether NtOSAK could be regulated by S-nitrosylation. In animals, S-nitrosylation has been shown to have an impact on the activity of several protein kinases, including, for instance, Janus kinases and the apoptosis signalling kinase 1 [54]. It was therefore plausible that the S-nitrosylation of NtOSAK could promote conformational changes favouring its phosphorylation by an upstream kinase. Our finding indicates that in BY-2 cells exposed to salt or DEA/NO, NtOSAK did not undergo S-nitrosylation. Accordingly, in preliminary in vitro S-nitrosylation assays, we were not able to find NtOSAK among the S-nitrosylated proteins identified from BY-2 cell protein extracts exposed to the S-nitrosylating agent GSNO (S-nitrosoglutathione; results not shown). Therefore NtOSAK might not be regulated by direct S-nitrosylation of critical cysteine residue(s).

There are mounting data indicating that SnRKs link metabolism and stress signalling in plants [28]. Studies in rice and A. thaliana showed that ABF (ABA-response-element-binding factor) transcription factors, as well as proteins related to energy metabolism, are targets for SnRK2 protein kinases [5759]. Accordingly, we found that NtOSAK co-immunoprecipitated with proteins primarily involved in N/C metabolism, and ATP synthesis and transport. The other interacting proteins include the elongation factor-1α that binds aminoacyl-tRNAs to the acceptor site of ribosomes during peptide chain elongation [60], the 26S proteasome subunit 4-like and proteins related to the cytoskeleton. The possibility that elongation factors represent SnRK2-interacting proteins has been previously reported by Shin et al. [59] who identified several phosphorylated targets of the A. thaliana SnRK2.8 protein kinase.

Among the NtOSAK-interacting proteins, we preferentially focused our attention on GAPDH. Several arguments supported this choice. First, in animals, besides its glycolytic activity, GAPDH participates in several cellular events including gene transcription, RNA transport and DNA replication [61]. This multifunctional protein has been reported to be inhibited by S-nitrosylation of a conserved catalytic cysteine residue, both in vitro and in vivo. Interestingly, Hara et al. [53] reported that in macrophages exposed to endotoxin, as well as in neurons elicited by glutamate, the S-nitrosylation of GAPDH causes structural changes allowing GAPDH to interact with the E3 ubiquitin ligase Siah1. The complex is then translocated into the nucleus where Siah1 promotes cell death through the ubiquitin-mediated degradation of nuclear target proteins. Secondly, in A. thaliana, GAPDH from cell culture crude extracts as well as its recombinant purified cytosolic isoform were shown to be in vitro S-nitrosylated by the NO donors GSNO and sodium nitroprusside [9,10]. In those studies, S-nitrosylation led to the inhibition of its enzymatic activity. Finally, in various organisms, including plants, GAPDH was identified as a direct target of H2O2 [62,63]. Mechanistically, H2O2 triggers the inhibition of GAPDH through the oxidation of the catalytic cysteine residue also prone to S-nitrosylation. In yeast, the H2O2-induced oxidation of GAPDH was shown to promote its interaction with the response regulator Mcs4 which, in turn, activates the Spc1 MAPK cascade [63]. Taken together, these results indicate that GAPDH has roles outside of that of glycolysis, and modulates cellular signalling pathways once S-nitrosylated or oxidized on a critical cysteine residue.

Based on these studies, we investigated the degree of S-nitrosylation of GAPDH in salt-treated BY-2 cells and found that it undergoes an increased and transient S-nitrosylation. The kinetics of GAPDH S-nitrosylation in response to salt or DEA/NO was well correlated with the kinetics of NtOSAK activation, suggesting that the physical association between both proteins might have been related to their respective S-nitrosylated and phosphorylated status. However, our studies have not confirmed this hypothesis. The complex formation analysis in planta indicate that the proteins interact with each other before and after stress application, and their interaction does not depend on S-nitrosylation of GAPDH. The increased S-nitrosylation of GAPDH cells exposed to salt or DEA/NO was not accompanied by a reduction of the total GAPDH enzymatic activity. This observation resembles the situation encountered in stimulated macrophages in which only a small proportion of GAPDH is S-nitrosylated with a negligible effect on overall cellular glycolysis [53,64]. Consistently, after applying the biotin-switch assay to proteins extracted from salt and DEA/NO-treated BY-2 cells, GAPDH was also strongly detected in the samples corresponding to proteins that did not bind to neutravidin (Supplementary Figure S2).

In mammals the activity of GAPDH can be modulated by phosphorylation [65]. Therefore we have considered that in response to salinity stress, GAPDH can be regulated by phosphorylation catalysed by NtOSAK. However, we were unable to detect phosphorylation of tobacco GAPDH by NtOSAK (in vitro studies). Moreover, our MS data also did not show any phosphorylated peptides of GAPDH isolated from tobacco cells. To establish a possible role of GAPDH in NtOSAK signalling, we expressed NtOSAK in A. thaliana protoplasts together with tobacco GAPDH and monitored NtOSAK activity after salt stress application. Two different cytosolic GAPDHs were tested, that is wild-type and mutated forms in which the cysteine residues undergoing S-nitrosylation were replaced with serine residues. We did not observe any significant differences in the kinase activity due to GAPDH overexpression, suggesting that the S-nitrosylation state of GAPDH does not influence NtOSAK activity. These results do not exclude the possibility that interaction with GAPDH and its additional S-nitrosylation in response to salinity can influence NtOSAK substrate specificity and/or interaction with other NtOSAK signalling components.

In conclusion, the present study provides the first description of the mechanisms underlying NO-induced activation of protein kinases in a physiological context in plants. The finding that GAPDH associates with NtOSAK and following exposure to salt undergoes an increased S-nitrosylation suggests that, besides its glycolytic activity, GAPDH might act as a component of a signalling cascade involving phosphorylation-dependent events. Work is ongoing to address these issues.

Abbreviations

     
  • ABA

    abscisic acid

  •  
  • BiFC

    bimolecular fluorescence complementation

  •  
  • BY-2

    bright-yellow 2

  •  
  • CDPK

    Ca2+-dependent protein kinase

  •  
  • cPTIO

    carboxy PTIO

  •  
  • DEA/NO

    diethylamine-NONOate

  •  
  • DMF

    dimethylformamide

  •  
  • DTT

    dithiothreitol

  •  
  • EGFP

    enhanced green fluorescent protein

  •  
  • EYFP

    enhanced yellow fluorescent protein

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • GST

    glutathione transferase

  •  
  • LC-ESI-MS-MS/MS

    liquid chromatography-electrospray ionization MS with collisional fragmentation

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MBP

    myelin basic protein

  •  
  • MMTS

    methyl methanethiosulfonate

  •  
  • Ni-NTA

    Ni2+-nitrilotriacetate

  •  
  • NtGAPC

    Nicotiana tabacum cytosolic GAPDH

  •  
  • NtOSAK

    Nicotiana tabacum osmotic stress-activated protein kinase

  •  
  • PAMP

    pathogen-associated molecular pattern

  •  
  • RT

    reverse transcription

  •  
  • SIPK

    salicylic-acid-induced protein kinase

  •  
  • SnRK

    SNF1 (sucrose non-fermenting 1)-related protein kinase

AUTHOR CONTRIBUTION

Izabella Wawer was involved in the analysis of the NO-dependence of NtOSAK and of NtOSAK and GAPDH S-nitrosylation, identification of NtOSAK serine residues undergoing phosphorylation, immunoprecipitation assays and identification of GAPDH as the NtOSAK partner. Maria Bucholc was involved in cloning of NtGAPCa and NtGAPCb, preparation of the constructs for expression of NtOSAK and GAPDHs in the bacterial system and in plant protoplasts, site-directed mutagenesis of NtGAPCa and NtGAPCb and in vitro binding assays. Jeremy Astier was involved in setting up of the biotin-switch assay. Anna Anielska-Mazur was involved in confocal microscopy analysis. Jennifer Dahan helped in the measurement of NtOSAK activity. Anna Kulik analysed the influence of GAPDHs on NtOSAK activity, and measured NtOSAK activity in plant protoplasts transformed with NtOSAK and NtGAPCa or NtGAPCb. Aleksandra Wysłouch-Cieszynska and Monika Zaręba-Kozioł helped with the biotin-switch assay and with analysis of protein S-nitosylation. Ewa Krzywinska was involved in the expression and purification of NtOSAK and GAPDHs in the bacterial system and protoplast transformation. Michal Dadlez performed the MS analysis. Grazyna Dobrowolska and David Wendehenne supervised the project in their respective laboratories.

FUNDING

This work was supported by the Conseil Régional de Bourgogne [grant number 07 9201 CPER O2 S 5527]; the Agence Nationale de la Recherche [grant number BLAN07-2_184783]; the Ministère des Affaires Etrangères [grant number EGIDE Polonium, 11545WG]; and the Ministry of Science and Higher Education [grant numbers N N301 2540 and 500/N-COST/2009/0]. I.W. was supported by a fellowship from the Conseil Régional de Bourgogne [grant number 07 HCP 36].

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Author notes

1

These authors contributed equally to the present study

Supplementary data