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Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase
Plant Physiology and Biochemistry 60 (2012) 157e164
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Research article
Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase Pooja Saigal Talwar, Ravi Gupta, Arun Kumar Maurya 1, Renu Deswal* Molecular Plant Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, Delhi 110007, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 June 2012 Accepted 8 August 2012 Available online 21 August 2012
Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4 mM) and 40% by calcium (0.4 mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300 mM) and L-NAME (5 mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased Km (w13%) and almost two fold increase in Vmax. PMA (438 nM), a kinase activator, activated BjNOSla w1.9 fold while its inactive analog 4aPDD was ineffective. Calcium and PMA activated the enzyme to w3 folds. Interestingly, 1,2-DG6 (2.5 mM) and PS (1 mM) with calcium activated the enzyme activity to w7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (w90%) and calphostin-C (w40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7e46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: BjNOSla L-NAME NBT Nitric oxide Nitric oxide synthase Phosphorylation Protein kinase-C
1. Introduction NO is involved as a signaling and regulatory molecule in an array of physiological processes including seed germination, root development, stomatal movements, flowering, senescence and programmed cell death [1]. Besides, NO evolution during different abiotic and biotic stress conditions (such as temperature, salinity, drought, heavy metal stress and pathogen attack) is well established [1]. Furthermore, NO also functions as a protective molecule against reactive oxygen species (ROS) [1].
Abbreviations: 1,2-DG6, 1,2-dihexanoylglycerol; 4aPDD, 4a-phorbol 12,13didecanoate; BH4, tetrahydrobiopterin; BjNOSla, Brassica juncea nitric oxide synthase like activity; CaM, calmodulin; EGTA, ethylene glycol tetraacetic acid; LNAME, N-nitro-L-Arg-methyl ester; NBT, nitro blue tetrazolium; NO, nitric oxide; PKC, protein kinase-C; PMA, phorbol 12-myristate acetate; PS, phosphatidylserine. * Corresponding author. Tel.: þ91 11 27667573; fax: þ91 11 27667829. E-mail address: [email protected] (R. Deswal). 1 Present address: Department of Botany, Hans Raj College, University of Delhi, Delhi 110007, India. 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2012.08.005
NO is produced by both enzymatic and non-enzymatic reactions. In animals, enzymatic NO production is mediated by NADPH dependent Nitric Oxide synthase (NOS), which catalyzes the oxidation of L-arginine to L-citrulline and NO is evolved as a byproduct. Animal NOS occur in three homodimeric isoformsendothelial NOS, neuronal NOS and inducible NOS [2]. NO production in plants was first observed in herbicide treated soybean by Klepper in 1979 [3] but its role in plant defence was established by Durner [4] and Delledonne [5] in 1998. The fact that NO production in animals is mediated by NOS led to the search of similar enzyme in plants. NOS like activity has been reported in bacteria [6], slime mold (Physarum polycephalum) [7], algae (Ostreococcus tauri, Ostreococcus lucimarinus) [8] gymnosperms (Taxus brevifolia) and higher plants like Nicotiana tabacum, Glycine max, Pisum sativum, Mucuna hasszoo, Lupinus albus, Arabidopsis thaliana, Zea mays [9] and Chorispora bungeana [10]. NO generation in plants have been detected either by in planta (citrulline assay, oxyhemoglobin assay, Griess reaction, DAF dye, ESR, NO electrode and Mass spectrometry) or ex planta (chemiluminescence and laser based infrared spectroscopy) assays [11].
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The combination of biochemical and molecular approach along with mutant analysis led to the identification of NO generating enzyme AtNOS1 in A. thaliana [12]. Later, it was found to be a circularly permuted plastid GTPase and was renamed as NO associated protein (AtNOA1) as it did not exhibit NOS like activity [13]. Recently, an L-arginine dependent NOS with 45% sequence homology with human endothelial NOS was purified from an alga, O. tauri [8]. In spite of all these studies, purification of NOS from any plant is still eluding researchers; therefore it is important to understand its biochemical properties including its regulation to device a successful purification strategy. Phosphorylation regulates NO mediated calcium release in guard cells of Arabidopsis [14] and Vicia faba [15]. The calcium release was reversibly suppressed by protein kinase antagonist staurosporine and K252A but not by tyrosine kinase antagonist genistein. Although, the role of protein phosphorylation in calcium release and regulating Kþ and Cl channel is shown in Arabidopsis guard cells, but whether phosphorylation modulates NOS like activity is not known. There are few reports suggesting the involvement of calcium and protein kinases as upstream signal for NO synthesis in plants [16]. The aim of this study was to detect and characterize the NOS like activity in Brassica juncea and its regulation.
w57% activation (Fig. 1C) while CaCl2 showed 40% increase in the BjNOSla at 0.4 mM (Fig. 1D). Respective optimized concentrations of the assay components (3 mM L-arginine, 10 mL oxyhemoglobin, 10 mM MgCl2 and 0.4 mM CaCl2) were used for further experimentation. The specific activity of rat neuronal NOS, used as positive control, was calculated as w1.7 nmol min1 mg1 using this assay indicating good efficacy/efficiency of the oxyhemoglobin assay. The BjNOSla was completely abolished by 2 mM EGTA, suggesting that calcium is essential for the activity. Cofactors NADPH (0.4 mM) and BH4 (4 mM) led to 25% and 10% activation respectively (data not shown). However, FMN (1e40 mM), FAD (1e40 mM) and CaM (0.5e1 mM) were ineffective (data not shown). 2.2. Mammalian NOS inhibitors inhibit NOS like activity Inhibitors of mammalian NOS, NBT (300 mM) and L-NAME (5 mM) led to 100% and 67% inhibition of NOS like activity in B. juncea, suggesting similarity between mammalian NOS and BjNOSla (Fig. 2A and B). NBT was able to inhibit 100% activity even at 1/10th concentration of L-NAME suggesting it to be a more potent inhibitor of BjNOSla. 2.3. BjNOSla is predominantly localized in the guard cells
2. Results 2.1. Optimization of NOS like activity by the oxyhemoglobin assay B. juncea seedlings were homogenized in the extraction buffer as described in methods. For optimization of BjNOSla activity assay, freshly prepared oxyhemoglobin was used. BjNOSla activity was assayed in 65 mM Hepes buffer, pH 7.4 and other reaction components were varied independently to optimize the reaction conditions (Fig. 1AeD). Maximum activity was observed at 3 mM L-arginine, the substrate (Fig. 1A). Oxyhemoglobin (10 mL) showed 40% higher activity than control (Fig. 1B). To know the divalent cation requirement of BjNOSla, the assay was performed with varying concentration of MgCl2 and CaCl2. MgCl2 (10 mM) showed
To observe the effect of light on BjNOSla and its distribution in different parts, the activity was checked in etiolated and light grown seedlings in shoot and root. NOS like activity was predominantly (about 80%) localized in shoots (Fig. 3A). It was 15% and 20% higher in light grown shoots and roots respectively in comparison to respective etiolated control (Fig. 3A). NADPH-diaphorase co-localizes with the NOS [16], therefore, an attempt was made to localize the BjNOSla in the epidermal peel. Maximum NADPH-diaphorase staining was detected in the guard cells (Fig. 3B and C). To further confirm the localization of BjNOSla in the guard cells, DAF-2DA, a NO sensitive fluorophore was used. DAF-2DA showed strongest intensity with the substrate, L-arginine (Fig. 3I), further supporting the involvement of an arginine dependent enzymatic source in NO biosynthesis.
Fig. 1. Optimization of oxyhemoglobin assay for analyzing NOS like activity in B. juncea seedlings. The assay was performed by varying only one component at a time in 65 mM Hepes buffer pH 7.4. NOS activity with (A) substrate L-arginine (0e4 mM), (B) oxyhemoglobin (0e20 mL), (C) magnesium chloride (0e40 mM) and (D) calcium chloride (0e0.6 mM). Error bars represents standard deviation from ten biological replicates. Asterisk showed significant difference in comparison with control (Student’s t-test, p < 0.05).
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suggest the NOS modulation by phosphorylation in plants [17]. In order to evaluate the modulation of BjNOSla by PKC/PKC-like kinase, PKC activators (PMA, 1,2-DG6, a synthetic diacylglycerol and PS) were used. With calcium (0.4 mM) and PMA (438 nM), the stimulation of BjNOSla was 2.8 fold, whereas, 4aPDD, an inactive analog of PKC, was found to be ineffective (Fig. 4A). The stimulation of BjNOSla with 1,2-DG6 (1,2-dihexanoylglycerol, 2.5 mM), PS (phosphatidylserine, 1 mM) and calcium was w7 fold higher than control (Fig. 4A). PMA, 1,2-DG6 and PS stimulated BjNOSla even without calcium probably due to the residual calcium. Kinase inhibitor staurosporine (20 nM) inhibited the activity by 90% (Fig. 4B) while calphostin-C (0.5 mM) inhibited it by 40% (Fig. 4C). 2.6. Abiotic stress conditions stimulate BjNOSla
Fig. 2. Effect of NOS inhibitors on BjNOSla. The activity was assayed with NOS inhibitors (A) NBT (1e300 mM) and (B) L-NAME (1e0.5 mM). Error bars represents standard deviation from four biological replicates with each assay performed in duplicates. Asterisk showed significant difference in comparison with control (Student’s t-test, p < 0.05).
L-NAME brought back the DAF-2DA fluorescence to the control level while NBT inhibited it further (Fig. 3J and K). These results were in consonance with the results of in vitro inhibition studies as described in Fig. 2A and B.
2.4. BjNOSla shows positive cooperativity with calcium The kinetic properties of BjNOSla were analyzed using both oxyhemoglobin and citrulline assays. With oxyhemoglobin assay, the Km was 9.43 mM and Vmax was 2.43 pmol min1 mg1 without calcium. However, Km decreased to 7.407 mM while Vmax increased by 2 folds to 4.76 pmol min1 mg1 with calcium. Using radioactive citrulline assay, MichaeliseMenten curve of L-[U-14C] arginine versus NOS like activity showed Km 14.28 mM and Vmax 3 pmol min1 mg1 without calcium whereas, a decrease in the Km to 12.5 mM and an increase of 1.6 folds in Vmax to 5.55 pmol min1 mg1 was observed with calcium (Table 1). Results suggest that calcium not only increased the affinity of the substrate for the enzyme as indicated by a decreased Km, but also enhanced the rate of the reaction by two fold. To further confirm this positive cooperativity, Hill plots were plotted. The Hill coefficient without calcium was calculated as 1 and 0.38 by oxyhemoglobin and citrulline assay respectively. However, the Hill coefficient increased to 5.2 and 1.65 with calcium for oxyhemoglobin and citrulline assay respectively. The increase in cooperativity with calcium was almost 5 fold by both the assays. 2.5. PKC activators and calcium act synergistically to stimulate the NOS like activity Earlier, NO mediated calcium release in the guard cell was shown to be modulated by phosphorylation and only few reports
To know whether NOS like enzyme contributes to NO production in response to environmental stress conditions, the BjNOSla activity was analyzed in different abiotic stress such as low and high temperature, salinity, drought and injury. LT (4 C, 30 min) stimulated the activity by w20% (Fig. 5A) while heat treatment (50 C, 5 h) showed 46% increase (Fig. 5B). The increase was relatively less (w7%) by salinity stress (0.5 M NaCl, 1 h, Fig. 5C). Drought stress showed 35% and 22% higher activity at 30 min and 1 h respectively (Fig. 5D). The injured seedlings showed 20% higher activity after 30 min which remained elevated up to 1 h (Fig. 5E). Immediate effect of stress was a decline in NOS activity but activation was observed after 30 min which continued to increase up to 1 h except for HT where significant activation was observed after 5 h. These results suggest existence of NOS like activity in B. juncea which seems somewhat similar to mammalian NOS. Furthermore, the BjNOSla showed positive cooperativity with calcium and is probably regulated by phosphorylation. 3. Discussion In animal system, it is a well established that NOS is mainly responsible for NO synthesis [18]. NO synthesis in plants is also known since long [3] and reports suggest involvement of diverse enzymes such as putative NOS like enzyme, NR, Ni-NOR and xanthine oxidoreductase [18]. Search for animal like NOS led to the identification of a unique AtNOS1 gene in Arabidopsis which did not exhibit NOS activity in vitro [13] and therefore was renamed as AtNOA1. Investigation revealed it to be a plastid GTPase, which is mainly responsible for the post-transcriptional regulation of plastid-localized methylerythritol phosphate pathway enzyme [13] and ribosome assembly [18]. Recently, AtNOA1 has been shown to regulate physiological processes such as chlorophyll biosynthesis and Rubisco formation in rice [19]. AtNOA1 indeed modulates NO accumulation in plants, possibly indirectly [20]. To date, no NOS has been purified from higher plants except from some lower organisms such as slime mold (P. polycephalum) [7] and alga (O. tauri) [8] that have raised some hope for existence of NOS like activity/ enzyme in plants. The aim of this work was to characterize the arginine dependent NOS like activity in B. juncea, its modulation by various pharmacological agents and to analyze its functional role in abiotic stress. BjNOSla was detected by oxyhemoglobin and citrulline assays which was further confirmed using the mammalian NOS inhibitors. Complete inhibition of BjNOSla by EGTA indicates that calcium plays critical role in regulation of its activity. The BjNOSla showed dependence on calcium as observed in Physarum [7], AtNOA1 [13], Lupinus [21], Pisum [22], maize [23], soybean [24], and Staphylococcus [25]. However, the NOS like activity from Flammulina
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velutipes [26] and O. tauri [7] was calcium independent. In contrast to NOS like activity reported in P. sativum, Arabidopsis, Z. mays [9] and C. bungeana [10], the BjNOSla was found to be independent of CaM, FAD and FMN. NOS like activity purified from F. velutipes [26] and O. tauri [8] was also CaM independent, supporting the CaM independency of BjNOSla. Moreover, the NOS like activity analyzed in Brassica seedlings was comparable to the activity reported in other plants except Pisum, which showed almost 1000 times higher activity (Table 2). BjNOSla was significantly inhibited by mammalian NOS inhibitors. Nitro group (-NO2) of NBT and L-NAME competitively interacts with the active site of the NOS and therefore probably does not allow the binding of the substrate, L-arginine, leading to inhibition of NOS activity. Inhibition of BjNOSla by mammalian NOS inhibitors also indicates oxidative nature of the enzyme. After ascertaining the existence of NOS like activity in B. juncea, its localization was investigated in shoot and root which showed higher activity in shoots than roots. As the activity was higher in shoot, its localization was analyzed in the epidermal peels using NADPH-diaphorase staining. NO levels were higher in the guard cells, supporting the earlier observations of NO being a key regulator of stomatal closure in Arabidopsis [17] and V. faba [15]. It was reconfirmed using the fluorescent probe DAF-2DA which also showed NOS localization in guard cells confirming that guard cells are the major sites of NO production.
Table 1 Showing Km, Vmax and Hill coefficient for BjNOSla, by oxyhemoglobin and citrulline assays. Assay conditions
Km (mM)
Vmax (pmol min1 mg1)
Hill coefficient
Hb assay (Ca) Hb assay (þCa) Citrulline assay (Ca) Citrulline assay (þCa)
9.43 103 7.4 103 14.28 12.5
2.43 4.76 3 5.55
1 5.2 0.38 1.65
However, detection of BjNOSla in roots could indicate the involvement of NO in signal transduction pathways. Previously, the role of NO during root initiation and development has been reported in pea [27], soybean [28], tomato [29], Arabidopsis [12], Cucumis sativus [30], maize [31] and Lupinus [21]. Kinetic analysis of BjNOSla using citrulline assay showed a Km of 12.5 mM with calcium which is comparable to the mammalian (rat nNOS) NOS Km (8.4 mM) [32], Arabidopsis (12.5 mM) [12] and alga (O. tauri) Km (12.6 5 mM) [8]. The Hill coefficient for oxyhemoglobin and citrulline assay was 5.2 and 1.65 respectively. The binding of a ligand to a macromolecule is often enhanced if there are already other ligands present on the same macromolecule. A value higher than 1 indicates that there is positive cooperativity for the substrate resulting in higher affinity for the other ligand
Fig. 3. Localization studies of BjNOSla in Brassica seedlings. (A) The activity was tested in shoot and root grown in light and dark. (BeC) NADPH-diaphorase activity staining of epidermal peel showing localization of BjNOSla in the guard cells. (DeG) bright field microscopic images and (HeK) fluorescent microscopic images of epidermal peel in absence of L-arginine (control), presence of L-arginine, L-NAME and NBT respectively.
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Fig. 4. Effect of PKC activators (PMA, 1,2-DG6 and PS) and kinase inhibitors (staurosporine, calphostin-C) on BjNOSla in vitro. (A) Assays were done in the presence of different additives i.e. Ca2þ (0.4 mM), EGTA (2 mM), PMA (438 nM), 4aPDD (438 nM), PS (1 mM) and/or 1,2-DG6 (2.5 mM). The activity was maximal in Ca2þ, PS and 1,2-DG6 followed by Ca2þ and PMA. (B) Effect of staurosporine (1.5e20 nM) and (C) calphostin-c (0.1e1 mM) on BjNOSla. Error bars represents standard deviation from four biological replicates with each assay performed in duplicates. Multiple comparisons were carried out with one-way ANOVA followed by TukeyeKramer test. Values with same letters are not significantly different (p < 0.05).
molecules. Hill coefficient without calcium was equal to one indicating no cooperativity. Higher Hill coefficient 1.65 (hemoglobin assay) and 5.2 (citrulline assay) with calcium suggest positive cooperativity leading to conformational change in the enzyme which in turn leads to its activation by making it more receptive as evident by a decreased Km and two fold increase in the rate of the reaction. To provide an insight into the regulation of NOS, the effect of kinase activators and inhibitors were tested on BjNOSla. PMA is a NO promoter and promotes the expression of inducible NOS as reported in murine microglial BV2 cells [33]. Activation of BjNOSla by PMA, 1,2-DG6 and PS with calcium suggests involvement of PKC in modulating its activity by phosphorylation. Although the existence of PKC in plants has recently been questioned [34], existence of some other PKC-like kinases in plants cannot be ruled out. Some of the other members of AGC kinase family (family of kinases to which PKC belongs) contain a PKC-like kinase extension domain in S6 kinase, nuclear Dbf2-related kinase (NDR) and incomplete root hair elongation (IRE) genes. It has also been proposed that phosphorylation recognition sequence of plant calcium dependent protein kinase (CDPKs) is similar to the animal PKCs and therefore it is more likely that CDPKs would be performing the role of PKCs in plants [35]. In addition, PKC-like
activity has been reported and extensively characterized in many plants including B. juncea, Zea mays, V. faba and Arabidopsis, supporting existence of PKC-like kinase in plants [35]. Identification of the PKC-like kinase involved and its mode of action, culminating in the induction of NOS like activity needs further investigation. Higher stimulation of BjNOSla activity with PMA, 1,2-DG6 and PS in the presence of calcium in comparison with these components alone suggests that a calcium stimulated PKC-like kinase isoform could be involved. Almost complete inhibition of the BjNOSla activity by PKC inhibitors suggests that phosphorylated form is the active form of the enzyme. To understand the functional significance of BjNOSla, the effect of abiotic stress conditions (LT, HT, salinity, drought and injury) was analyzed. All stress conditions (except HT) stimulated BjNOSla within first few hours of the stress treatment followed by a decline later. Collectively, the data indicated an increase in the BjNOSla in all abiotic conditions. NOS mediated NO accumulation could be an early event. This result is consistent with other reports on abiotic stresses [1]. The present study showed the existence of NOS like activity in B. juncea. Strong inhibition of BjNOSla by mammalian NOS inhibitors and its activation by calcium and NADPH suggest its similarity with
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Table 2 A comparative account of specific activity of NOS in different organisms. Species
Specific activity (pmol min1 mg1 crude protein)
Rat nNOS Angiosperms Nicotiana tobaccum Lupinus albus Pisum sativum Zea mays Glycine max Arabidopsis Brassica juncea
0.5 150 5610 3.36 (Leaves) 3.88 (Root tips) 4.5 3.6 3e13
Algae Ostreococcus tauri
Km value (mM)
Reference
8.4
[32]
N.A. N.A. N.A. N.A.
[4] [21] [22] [23]
N.A. 12.5 12.5
[24] [12,38]
12 5
[8]
4. Methods 4.1. Plant material and growth conditions B. juncea var. varuna seeds were obtained from National Seed Corporation, Indian Agricultural Research Institute, New Delhi, India. Seeds were treated with ethanol for 20 s followed by washing thrice with distilled water and soaked overnight. These were plated in wet germination paper rolls and transferred to B.O.D incubator (25 2 C with 260 mmol m2 s1 light for 16 h/8 h light/dark photoperiod) after keeping for 1 d in dark. 4.2. Protein extraction and estimation
Bacteria Staphylococcus aureus Nocardia
25.2 103 150
13.4 5.7
[25] [39]
Fungi Flammulina velutipes Phycomyces blakesleeanus
1000e10,000
0.33 N.A.
[26] [40]
66.7 14.5
[7]
Slime mold Physarum polycephalum
regulation of NOS like activity in plants is reported. Furthermore, the activation of BjNOSla by different abiotic stress indicates that NOS like activity could be responsible for NO evolution during stress conditions (Fig. 6).
244 47 103 (Isoform A) 253 47 103 (Isoform B)
26.8 9.7
N.A. e not available.
mammalian NOS. Its activation by PMA, 1,2-DG6 and PS (PKC activators), and inhibition by staurosporine and calphostin (PKC inhibitors) indicate that the phosphorylated form is the active form of the enzyme. Further, for the first time role of phosphorylation in
Seven days old B. juncea seedlings were homogenized in Hepes buffer (65 mM, pH 7.4) containing sucrose (514 mM), EDTA (0.16 mM), DTT (1.6 mM), PMSF (0.5 mM) and protease inhibitor cocktail (Sigma, 5 mL g1). The extract was centrifuged at 12,500 rpm for 25 min at 4 C. Supernatant was passed through two layered muslin cloth and was incubated with AG50-WX8 resin (Naþ form, BioRad) for 1 h with intermittent shaking. Resin was allowed to settle and the extract was used for activity assay. Proteins were quantified using Bradford’s method [36]. 4.3. Nitric oxide synthase activity assay Both oxyhemoglobin and citrulline assays were employed to analyze the NOS activity in B. juncea. For oxyhemoglobin assay, fresh oxyhemoglobin was prepared every day [28]. Hemoglobin
Fig. 5. Effect of abiotic stress conditions on BjNOSla. Treatments were given for 0.25e24 h. (A) Low temperature stress: seedlings were incubated in ice cold buffer at 4 C. (B) Heat stress: seedlings were placed in a vial containing buffer in a water bath maintained at 50 C. (C) Salinity stress was provided by submerging the roots of the seedlings in 0.5 M NaCl. (D) Dehydration: induction of water deficit was achieved by drying the roots of the seedling using a tissue paper and incubating them in B.O.D. at 25 2 C. (E) Injury: the seedling was pricked by a needle at 4e5 places and then placed the seedlings in B.O.D. at 25 2 C in buffer solution. Error bars represent standard deviation from three biological replicates. Asterisk showed significant difference in comparison with control (Student’s t-test, p < 0.05).
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Fig. 6. BjNOSla is phosphorylated. Calcium, NADPH, light, PMA, PS, 1,2-DG6 and abiotic stress activate BjNOSla while the PKC inhibitors-staurosporine and calphostin inhibit it.
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5 mL of 10 mM Tris pH 7.2 and 10 mM DAF-2DA. Epidermal peels were maintained in dark for 1 h. The peels were removed and washed with buffer (10 mM Tris, pH 7.2) for 15 min and then with 1 mM L-arginine for 30 min for 25 C. L-NAME (5 mM, 30 min, 25 C) and NBT (300 mM), were added to the buffer after washing the probe. Fluorescent microscope (Zeiss, Oberkochen, Germany) was used for analysis and sections were excited with UV light. Dye emissions were recorded using a 505e530 nm band pass filter. Autofluorescence of chloroplasts was captured using a 585 nm long pass filter. Images were processed and captured by Zeiss Microsystems camera and analyzed using Zeiss LSM510 software. Experiments were carried out three times in duplicates. 4.5. NOS inhibitor, PKC inhibitors and PKC activators treatments
(25 mg) was suspended in 1 mL of TriseHCl buffer (10 mM, pH 7.0) and incubated with sodium dithionite (20 mg mL1) for 30 min. The solution was passed through sephadex G-25 (10 mL) column for desalting. Aliquots (1 mL) were eluted using 10 mM Tris containing 0.1 M NaCl, at a flow rate of 1 mL min1 and the fraction having maximum oxyhemoglobin was used for the assay [21]. For optimization, the assay was performed in 65 mM Hepes with varying concentrations of the substrate (L-arginine, 1e4 mM) and other reaction mixture components like oxyhemoglobin (2.5e20 mL), CaCl2 (0.1e0.6 mM) and MgCl2 (5e40 mM). BjNOSla was also monitored in presence of NADPH (0.1e1 mM), calmodulin (CaM, 0.5e1 mM), FAD (1e40 mM), FMN (1e40 mM) and BH4 (4e12 mM). All experiments were performed with ten biological replicates, with each experiment carried out in duplicates. NOS like activity was expressed as pmol min1 mg1 protein and the results are expressed as percent activity. Km, Vmax and Hill coefficient were calculated by plotting MichaeliseMenten curve (v versus s), LineweavereBurk plot (1/v versus 1/s) and Hill plot [log v/ (Vmax v) versus log s]. Rat neuronal NOS was used as positive control. Citrulline assay was performed according to Cueto et al. with slight modifications [21]. The assay was performed using 20 mL of the crude extract, 0.2 mM L-arginine, 0.3 mM CaCl2 and 0.05e 0.45 mCi of L-[U-14C] arginine. The reaction was terminated using AG50WX-8, suspended in Milli Q water (1:1 ratio). The supernatant was added to 3 mL scintillation cocktail-W and quantified by liquid scintillation counting (Beckman Coulter, Model LS 6000). Four biological replicates were performed with each assay performed in duplicates.
Effect of mammalian NOS inhibitors, NBT (10e300 mM) and LNAME (1e5 mM) was observed on BjNOSla. EGTA (1e2 mM), PKC inhibitors, staurosporine (1.5e20 nM), calphostin-C (0.1e1 mM) were incubated with the extract for 5 min before measuring the activity. Each experiment was carried out in duplicates with four biological replicates. NOS like activity was analyzed with PMA (438 nM), 4aPDD (438 nM), 1,2-DG6 (2.5 mM) and PS (1 mM) in vitro.
4.4. Nitric oxide detection by fluorescent microscopy
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In vivo detection of BjNOSla was performed in epidermal peel using NADPH-diaphorase [21] and DAF-2DA staining [37]. Epidermal peels were gently removed from the abaxial surface of the 4 weeks old Brassica leaves. For NADPH-diaphorase staining, epidermal peels were fixed with 4% p-formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffered saline pH 7.4 and then incubated with 4% p-formaldehyde for 2 h. These peels were mounted in 20% glycerol followed by washed thrice for 10 min each in 10 mM Tris buffer pH 7.6 and was suspended in 5% Triton X-100 in Tris buffer pH 7.6. The peels were incubated in dark for 2 h at 37 C in a solution containing NBT (0.25 mg mL1), NADPH (1 mg mL1) and Triton X-100 (0.5%) in 0.1 M Tris pH 7.6 [21]. Peels were mounted on slides using glycerine and were observed under light microscope (40 magnification). In the control set, NADPH was omitted. Experiments were repeated thrice in duplicates. DAF-2DA staining was performed according to Foissner et al. [37]. In brief, epidermal peels were placed in a petri dish containing
4.6. Abiotic stress treatments The seedlings were subjected to different abiotic stress conditions for 0.25e24 h. Seedlings were incubated in ice cold Hepes buffer at 4 C for low temperature and 50 C for high temperature. Dehydration was done by keeping seedlings on a tissue paper at 25 C and for salinity in 0.5 M NaCl. Injury stress was given to the seedlings by pricking at 4e5 places by sterilized needle. Experiments were carried out in three biological replicates with two technical replicates. Acknowledgments The research work was partially supported by SAP program from University Grants Commission and Council of Scientific and Industrial Research (CSIR), Government of India. PST acknowledges CSIR for fellowship. We thank Prof. S. K. Bansal (Department of Biochemistry, V. P. Patel Chest Institute, Delhi) for providing the liquid scintillation counting facility. References
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[26] N. Song, C. Jeong, H. Choi, Identification of nitric oxide synthase in Flammulina velutipes, Mycologia 92 (2000) 1027e1032. [27] F.J. Corpas, J.B. Barroso, A. Carreras, Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants, Plant Physiol. 136 (2004b) 2722e2733. [28] X. Hu, S.J. Neill, Z. Tang, W. Cai, Nitric oxide mediates gravitropic bending in soybean roots, Plant Physiol. 137 (2005) 663e670. [29] N. Correa-Aragunde, M. Graziano, C. Chevalier, L. Lamattina, Nitric oxide modulates the expression of cell cycle regulatory genes during lateral root formation in tomato, J. Exp. Bot. 57 (2006) 581e588. [30] G.C. Pagnussat, M.L. Lanteri, M.C. Lombardo, L. Lamattina, Nitric oxide mediates the indole acetic acid induction activation of a mitogen-activated protein kinase cascade involved in adventitious root development, Plant Physiol. 135 (2004) 279e286. [31] C.M.C.P. Gouvea, J.F. Souza, A.C.N. Magalhães, I.S. Martins, NO-releasing substances that induce growth elongation in maize root segments, Plant Growth Regul. 21 (1997) 183e187. [32] R.G. Knowles, M. Palacios, R.M.J. Palmer, S. Monacada, Kinetic characteristics of nitric oxide synthase from rat brain, Biochem. J. 269 (1990) 207e210. [33] I.O. Han, H.S. Kim, H.C. Kim, E.H. Joe, W.K. Kim, Synergistic expression of inducible nitric oxide synthase by phorbol ester and interferon g is mediated through NF-kB and ERK in microglial cells, J. Neurosci. Res. 73 (2003) 659e669. [34] T. Munnik, C. Testerink, Plant phospholipid signaling: “in a nutshell”, J. Lipid Res. (Suppl.) (April 2009) S260eS265. [35] A. Sato, F. Gambale, I. Dreyer, N. Uozumi, Modulation of the Arabidopsis KAT1 channel by an activator of protein kinase C in Xenopus laevis oocytes, FEBS J. 277 (2010) 2318e2328. [36] M.M. Bradford, A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 42 (1976) 248e254. [37] I. Foissner, D. Wendehenne, C. Langebartels, J. Durner, In vivo imaging of an elicitor-induced nitric oxide burst in tobacco, Plant J. 23 (2000) 817e824. [38] D. Zeidler, U. Zahringer, I. Gerber, I. Dubery, T. Hartung, W. Bors, P. Hutzler, J. Durner, Innate immunity in Arabidopsis thaliana: lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 15811e15816. [39] Y.J. Chen, J.P.N. Rosazza, A bacterial nitric oxide synthase from Nocardia species, Biochem. Biophys. Res. 203 (1994) 1251e1258. [40] J. Maier, R. Hacker, P. Rockel, H. Ninnemann, Role of nitric oxide synthase in the light-induced development of sporangiophore in Phycomyces blakesleeanus, Plant Physiol. 126 (2001) 1323e1330.
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Research article
Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase Pooja Saigal Talwar, Ravi Gupta, Arun Kumar Maurya 1, Renu Deswal* Molecular Plant Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, Delhi 110007, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 June 2012 Accepted 8 August 2012 Available online 21 August 2012
Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4 mM) and 40% by calcium (0.4 mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300 mM) and L-NAME (5 mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased Km (w13%) and almost two fold increase in Vmax. PMA (438 nM), a kinase activator, activated BjNOSla w1.9 fold while its inactive analog 4aPDD was ineffective. Calcium and PMA activated the enzyme to w3 folds. Interestingly, 1,2-DG6 (2.5 mM) and PS (1 mM) with calcium activated the enzyme activity to w7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (w90%) and calphostin-C (w40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7e46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: BjNOSla L-NAME NBT Nitric oxide Nitric oxide synthase Phosphorylation Protein kinase-C
1. Introduction NO is involved as a signaling and regulatory molecule in an array of physiological processes including seed germination, root development, stomatal movements, flowering, senescence and programmed cell death [1]. Besides, NO evolution during different abiotic and biotic stress conditions (such as temperature, salinity, drought, heavy metal stress and pathogen attack) is well established [1]. Furthermore, NO also functions as a protective molecule against reactive oxygen species (ROS) [1].
Abbreviations: 1,2-DG6, 1,2-dihexanoylglycerol; 4aPDD, 4a-phorbol 12,13didecanoate; BH4, tetrahydrobiopterin; BjNOSla, Brassica juncea nitric oxide synthase like activity; CaM, calmodulin; EGTA, ethylene glycol tetraacetic acid; LNAME, N-nitro-L-Arg-methyl ester; NBT, nitro blue tetrazolium; NO, nitric oxide; PKC, protein kinase-C; PMA, phorbol 12-myristate acetate; PS, phosphatidylserine. * Corresponding author. Tel.: þ91 11 27667573; fax: þ91 11 27667829. E-mail address: [email protected] (R. Deswal). 1 Present address: Department of Botany, Hans Raj College, University of Delhi, Delhi 110007, India. 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2012.08.005
NO is produced by both enzymatic and non-enzymatic reactions. In animals, enzymatic NO production is mediated by NADPH dependent Nitric Oxide synthase (NOS), which catalyzes the oxidation of L-arginine to L-citrulline and NO is evolved as a byproduct. Animal NOS occur in three homodimeric isoformsendothelial NOS, neuronal NOS and inducible NOS [2]. NO production in plants was first observed in herbicide treated soybean by Klepper in 1979 [3] but its role in plant defence was established by Durner [4] and Delledonne [5] in 1998. The fact that NO production in animals is mediated by NOS led to the search of similar enzyme in plants. NOS like activity has been reported in bacteria [6], slime mold (Physarum polycephalum) [7], algae (Ostreococcus tauri, Ostreococcus lucimarinus) [8] gymnosperms (Taxus brevifolia) and higher plants like Nicotiana tabacum, Glycine max, Pisum sativum, Mucuna hasszoo, Lupinus albus, Arabidopsis thaliana, Zea mays [9] and Chorispora bungeana [10]. NO generation in plants have been detected either by in planta (citrulline assay, oxyhemoglobin assay, Griess reaction, DAF dye, ESR, NO electrode and Mass spectrometry) or ex planta (chemiluminescence and laser based infrared spectroscopy) assays [11].
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The combination of biochemical and molecular approach along with mutant analysis led to the identification of NO generating enzyme AtNOS1 in A. thaliana [12]. Later, it was found to be a circularly permuted plastid GTPase and was renamed as NO associated protein (AtNOA1) as it did not exhibit NOS like activity [13]. Recently, an L-arginine dependent NOS with 45% sequence homology with human endothelial NOS was purified from an alga, O. tauri [8]. In spite of all these studies, purification of NOS from any plant is still eluding researchers; therefore it is important to understand its biochemical properties including its regulation to device a successful purification strategy. Phosphorylation regulates NO mediated calcium release in guard cells of Arabidopsis [14] and Vicia faba [15]. The calcium release was reversibly suppressed by protein kinase antagonist staurosporine and K252A but not by tyrosine kinase antagonist genistein. Although, the role of protein phosphorylation in calcium release and regulating Kþ and Cl channel is shown in Arabidopsis guard cells, but whether phosphorylation modulates NOS like activity is not known. There are few reports suggesting the involvement of calcium and protein kinases as upstream signal for NO synthesis in plants [16]. The aim of this study was to detect and characterize the NOS like activity in Brassica juncea and its regulation.
w57% activation (Fig. 1C) while CaCl2 showed 40% increase in the BjNOSla at 0.4 mM (Fig. 1D). Respective optimized concentrations of the assay components (3 mM L-arginine, 10 mL oxyhemoglobin, 10 mM MgCl2 and 0.4 mM CaCl2) were used for further experimentation. The specific activity of rat neuronal NOS, used as positive control, was calculated as w1.7 nmol min1 mg1 using this assay indicating good efficacy/efficiency of the oxyhemoglobin assay. The BjNOSla was completely abolished by 2 mM EGTA, suggesting that calcium is essential for the activity. Cofactors NADPH (0.4 mM) and BH4 (4 mM) led to 25% and 10% activation respectively (data not shown). However, FMN (1e40 mM), FAD (1e40 mM) and CaM (0.5e1 mM) were ineffective (data not shown). 2.2. Mammalian NOS inhibitors inhibit NOS like activity Inhibitors of mammalian NOS, NBT (300 mM) and L-NAME (5 mM) led to 100% and 67% inhibition of NOS like activity in B. juncea, suggesting similarity between mammalian NOS and BjNOSla (Fig. 2A and B). NBT was able to inhibit 100% activity even at 1/10th concentration of L-NAME suggesting it to be a more potent inhibitor of BjNOSla. 2.3. BjNOSla is predominantly localized in the guard cells
2. Results 2.1. Optimization of NOS like activity by the oxyhemoglobin assay B. juncea seedlings were homogenized in the extraction buffer as described in methods. For optimization of BjNOSla activity assay, freshly prepared oxyhemoglobin was used. BjNOSla activity was assayed in 65 mM Hepes buffer, pH 7.4 and other reaction components were varied independently to optimize the reaction conditions (Fig. 1AeD). Maximum activity was observed at 3 mM L-arginine, the substrate (Fig. 1A). Oxyhemoglobin (10 mL) showed 40% higher activity than control (Fig. 1B). To know the divalent cation requirement of BjNOSla, the assay was performed with varying concentration of MgCl2 and CaCl2. MgCl2 (10 mM) showed
To observe the effect of light on BjNOSla and its distribution in different parts, the activity was checked in etiolated and light grown seedlings in shoot and root. NOS like activity was predominantly (about 80%) localized in shoots (Fig. 3A). It was 15% and 20% higher in light grown shoots and roots respectively in comparison to respective etiolated control (Fig. 3A). NADPH-diaphorase co-localizes with the NOS [16], therefore, an attempt was made to localize the BjNOSla in the epidermal peel. Maximum NADPH-diaphorase staining was detected in the guard cells (Fig. 3B and C). To further confirm the localization of BjNOSla in the guard cells, DAF-2DA, a NO sensitive fluorophore was used. DAF-2DA showed strongest intensity with the substrate, L-arginine (Fig. 3I), further supporting the involvement of an arginine dependent enzymatic source in NO biosynthesis.
Fig. 1. Optimization of oxyhemoglobin assay for analyzing NOS like activity in B. juncea seedlings. The assay was performed by varying only one component at a time in 65 mM Hepes buffer pH 7.4. NOS activity with (A) substrate L-arginine (0e4 mM), (B) oxyhemoglobin (0e20 mL), (C) magnesium chloride (0e40 mM) and (D) calcium chloride (0e0.6 mM). Error bars represents standard deviation from ten biological replicates. Asterisk showed significant difference in comparison with control (Student’s t-test, p < 0.05).
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suggest the NOS modulation by phosphorylation in plants [17]. In order to evaluate the modulation of BjNOSla by PKC/PKC-like kinase, PKC activators (PMA, 1,2-DG6, a synthetic diacylglycerol and PS) were used. With calcium (0.4 mM) and PMA (438 nM), the stimulation of BjNOSla was 2.8 fold, whereas, 4aPDD, an inactive analog of PKC, was found to be ineffective (Fig. 4A). The stimulation of BjNOSla with 1,2-DG6 (1,2-dihexanoylglycerol, 2.5 mM), PS (phosphatidylserine, 1 mM) and calcium was w7 fold higher than control (Fig. 4A). PMA, 1,2-DG6 and PS stimulated BjNOSla even without calcium probably due to the residual calcium. Kinase inhibitor staurosporine (20 nM) inhibited the activity by 90% (Fig. 4B) while calphostin-C (0.5 mM) inhibited it by 40% (Fig. 4C). 2.6. Abiotic stress conditions stimulate BjNOSla
Fig. 2. Effect of NOS inhibitors on BjNOSla. The activity was assayed with NOS inhibitors (A) NBT (1e300 mM) and (B) L-NAME (1e0.5 mM). Error bars represents standard deviation from four biological replicates with each assay performed in duplicates. Asterisk showed significant difference in comparison with control (Student’s t-test, p < 0.05).
L-NAME brought back the DAF-2DA fluorescence to the control level while NBT inhibited it further (Fig. 3J and K). These results were in consonance with the results of in vitro inhibition studies as described in Fig. 2A and B.
2.4. BjNOSla shows positive cooperativity with calcium The kinetic properties of BjNOSla were analyzed using both oxyhemoglobin and citrulline assays. With oxyhemoglobin assay, the Km was 9.43 mM and Vmax was 2.43 pmol min1 mg1 without calcium. However, Km decreased to 7.407 mM while Vmax increased by 2 folds to 4.76 pmol min1 mg1 with calcium. Using radioactive citrulline assay, MichaeliseMenten curve of L-[U-14C] arginine versus NOS like activity showed Km 14.28 mM and Vmax 3 pmol min1 mg1 without calcium whereas, a decrease in the Km to 12.5 mM and an increase of 1.6 folds in Vmax to 5.55 pmol min1 mg1 was observed with calcium (Table 1). Results suggest that calcium not only increased the affinity of the substrate for the enzyme as indicated by a decreased Km, but also enhanced the rate of the reaction by two fold. To further confirm this positive cooperativity, Hill plots were plotted. The Hill coefficient without calcium was calculated as 1 and 0.38 by oxyhemoglobin and citrulline assay respectively. However, the Hill coefficient increased to 5.2 and 1.65 with calcium for oxyhemoglobin and citrulline assay respectively. The increase in cooperativity with calcium was almost 5 fold by both the assays. 2.5. PKC activators and calcium act synergistically to stimulate the NOS like activity Earlier, NO mediated calcium release in the guard cell was shown to be modulated by phosphorylation and only few reports
To know whether NOS like enzyme contributes to NO production in response to environmental stress conditions, the BjNOSla activity was analyzed in different abiotic stress such as low and high temperature, salinity, drought and injury. LT (4 C, 30 min) stimulated the activity by w20% (Fig. 5A) while heat treatment (50 C, 5 h) showed 46% increase (Fig. 5B). The increase was relatively less (w7%) by salinity stress (0.5 M NaCl, 1 h, Fig. 5C). Drought stress showed 35% and 22% higher activity at 30 min and 1 h respectively (Fig. 5D). The injured seedlings showed 20% higher activity after 30 min which remained elevated up to 1 h (Fig. 5E). Immediate effect of stress was a decline in NOS activity but activation was observed after 30 min which continued to increase up to 1 h except for HT where significant activation was observed after 5 h. These results suggest existence of NOS like activity in B. juncea which seems somewhat similar to mammalian NOS. Furthermore, the BjNOSla showed positive cooperativity with calcium and is probably regulated by phosphorylation. 3. Discussion In animal system, it is a well established that NOS is mainly responsible for NO synthesis [18]. NO synthesis in plants is also known since long [3] and reports suggest involvement of diverse enzymes such as putative NOS like enzyme, NR, Ni-NOR and xanthine oxidoreductase [18]. Search for animal like NOS led to the identification of a unique AtNOS1 gene in Arabidopsis which did not exhibit NOS activity in vitro [13] and therefore was renamed as AtNOA1. Investigation revealed it to be a plastid GTPase, which is mainly responsible for the post-transcriptional regulation of plastid-localized methylerythritol phosphate pathway enzyme [13] and ribosome assembly [18]. Recently, AtNOA1 has been shown to regulate physiological processes such as chlorophyll biosynthesis and Rubisco formation in rice [19]. AtNOA1 indeed modulates NO accumulation in plants, possibly indirectly [20]. To date, no NOS has been purified from higher plants except from some lower organisms such as slime mold (P. polycephalum) [7] and alga (O. tauri) [8] that have raised some hope for existence of NOS like activity/ enzyme in plants. The aim of this work was to characterize the arginine dependent NOS like activity in B. juncea, its modulation by various pharmacological agents and to analyze its functional role in abiotic stress. BjNOSla was detected by oxyhemoglobin and citrulline assays which was further confirmed using the mammalian NOS inhibitors. Complete inhibition of BjNOSla by EGTA indicates that calcium plays critical role in regulation of its activity. The BjNOSla showed dependence on calcium as observed in Physarum [7], AtNOA1 [13], Lupinus [21], Pisum [22], maize [23], soybean [24], and Staphylococcus [25]. However, the NOS like activity from Flammulina
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velutipes [26] and O. tauri [7] was calcium independent. In contrast to NOS like activity reported in P. sativum, Arabidopsis, Z. mays [9] and C. bungeana [10], the BjNOSla was found to be independent of CaM, FAD and FMN. NOS like activity purified from F. velutipes [26] and O. tauri [8] was also CaM independent, supporting the CaM independency of BjNOSla. Moreover, the NOS like activity analyzed in Brassica seedlings was comparable to the activity reported in other plants except Pisum, which showed almost 1000 times higher activity (Table 2). BjNOSla was significantly inhibited by mammalian NOS inhibitors. Nitro group (-NO2) of NBT and L-NAME competitively interacts with the active site of the NOS and therefore probably does not allow the binding of the substrate, L-arginine, leading to inhibition of NOS activity. Inhibition of BjNOSla by mammalian NOS inhibitors also indicates oxidative nature of the enzyme. After ascertaining the existence of NOS like activity in B. juncea, its localization was investigated in shoot and root which showed higher activity in shoots than roots. As the activity was higher in shoot, its localization was analyzed in the epidermal peels using NADPH-diaphorase staining. NO levels were higher in the guard cells, supporting the earlier observations of NO being a key regulator of stomatal closure in Arabidopsis [17] and V. faba [15]. It was reconfirmed using the fluorescent probe DAF-2DA which also showed NOS localization in guard cells confirming that guard cells are the major sites of NO production.
Table 1 Showing Km, Vmax and Hill coefficient for BjNOSla, by oxyhemoglobin and citrulline assays. Assay conditions
Km (mM)
Vmax (pmol min1 mg1)
Hill coefficient
Hb assay (Ca) Hb assay (þCa) Citrulline assay (Ca) Citrulline assay (þCa)
9.43 103 7.4 103 14.28 12.5
2.43 4.76 3 5.55
1 5.2 0.38 1.65
However, detection of BjNOSla in roots could indicate the involvement of NO in signal transduction pathways. Previously, the role of NO during root initiation and development has been reported in pea [27], soybean [28], tomato [29], Arabidopsis [12], Cucumis sativus [30], maize [31] and Lupinus [21]. Kinetic analysis of BjNOSla using citrulline assay showed a Km of 12.5 mM with calcium which is comparable to the mammalian (rat nNOS) NOS Km (8.4 mM) [32], Arabidopsis (12.5 mM) [12] and alga (O. tauri) Km (12.6 5 mM) [8]. The Hill coefficient for oxyhemoglobin and citrulline assay was 5.2 and 1.65 respectively. The binding of a ligand to a macromolecule is often enhanced if there are already other ligands present on the same macromolecule. A value higher than 1 indicates that there is positive cooperativity for the substrate resulting in higher affinity for the other ligand
Fig. 3. Localization studies of BjNOSla in Brassica seedlings. (A) The activity was tested in shoot and root grown in light and dark. (BeC) NADPH-diaphorase activity staining of epidermal peel showing localization of BjNOSla in the guard cells. (DeG) bright field microscopic images and (HeK) fluorescent microscopic images of epidermal peel in absence of L-arginine (control), presence of L-arginine, L-NAME and NBT respectively.
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Fig. 4. Effect of PKC activators (PMA, 1,2-DG6 and PS) and kinase inhibitors (staurosporine, calphostin-C) on BjNOSla in vitro. (A) Assays were done in the presence of different additives i.e. Ca2þ (0.4 mM), EGTA (2 mM), PMA (438 nM), 4aPDD (438 nM), PS (1 mM) and/or 1,2-DG6 (2.5 mM). The activity was maximal in Ca2þ, PS and 1,2-DG6 followed by Ca2þ and PMA. (B) Effect of staurosporine (1.5e20 nM) and (C) calphostin-c (0.1e1 mM) on BjNOSla. Error bars represents standard deviation from four biological replicates with each assay performed in duplicates. Multiple comparisons were carried out with one-way ANOVA followed by TukeyeKramer test. Values with same letters are not significantly different (p < 0.05).
molecules. Hill coefficient without calcium was equal to one indicating no cooperativity. Higher Hill coefficient 1.65 (hemoglobin assay) and 5.2 (citrulline assay) with calcium suggest positive cooperativity leading to conformational change in the enzyme which in turn leads to its activation by making it more receptive as evident by a decreased Km and two fold increase in the rate of the reaction. To provide an insight into the regulation of NOS, the effect of kinase activators and inhibitors were tested on BjNOSla. PMA is a NO promoter and promotes the expression of inducible NOS as reported in murine microglial BV2 cells [33]. Activation of BjNOSla by PMA, 1,2-DG6 and PS with calcium suggests involvement of PKC in modulating its activity by phosphorylation. Although the existence of PKC in plants has recently been questioned [34], existence of some other PKC-like kinases in plants cannot be ruled out. Some of the other members of AGC kinase family (family of kinases to which PKC belongs) contain a PKC-like kinase extension domain in S6 kinase, nuclear Dbf2-related kinase (NDR) and incomplete root hair elongation (IRE) genes. It has also been proposed that phosphorylation recognition sequence of plant calcium dependent protein kinase (CDPKs) is similar to the animal PKCs and therefore it is more likely that CDPKs would be performing the role of PKCs in plants [35]. In addition, PKC-like
activity has been reported and extensively characterized in many plants including B. juncea, Zea mays, V. faba and Arabidopsis, supporting existence of PKC-like kinase in plants [35]. Identification of the PKC-like kinase involved and its mode of action, culminating in the induction of NOS like activity needs further investigation. Higher stimulation of BjNOSla activity with PMA, 1,2-DG6 and PS in the presence of calcium in comparison with these components alone suggests that a calcium stimulated PKC-like kinase isoform could be involved. Almost complete inhibition of the BjNOSla activity by PKC inhibitors suggests that phosphorylated form is the active form of the enzyme. To understand the functional significance of BjNOSla, the effect of abiotic stress conditions (LT, HT, salinity, drought and injury) was analyzed. All stress conditions (except HT) stimulated BjNOSla within first few hours of the stress treatment followed by a decline later. Collectively, the data indicated an increase in the BjNOSla in all abiotic conditions. NOS mediated NO accumulation could be an early event. This result is consistent with other reports on abiotic stresses [1]. The present study showed the existence of NOS like activity in B. juncea. Strong inhibition of BjNOSla by mammalian NOS inhibitors and its activation by calcium and NADPH suggest its similarity with
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Table 2 A comparative account of specific activity of NOS in different organisms. Species
Specific activity (pmol min1 mg1 crude protein)
Rat nNOS Angiosperms Nicotiana tobaccum Lupinus albus Pisum sativum Zea mays Glycine max Arabidopsis Brassica juncea
0.5 150 5610 3.36 (Leaves) 3.88 (Root tips) 4.5 3.6 3e13
Algae Ostreococcus tauri
Km value (mM)
Reference
8.4
[32]
N.A. N.A. N.A. N.A.
[4] [21] [22] [23]
N.A. 12.5 12.5
[24] [12,38]
12 5
[8]
4. Methods 4.1. Plant material and growth conditions B. juncea var. varuna seeds were obtained from National Seed Corporation, Indian Agricultural Research Institute, New Delhi, India. Seeds were treated with ethanol for 20 s followed by washing thrice with distilled water and soaked overnight. These were plated in wet germination paper rolls and transferred to B.O.D incubator (25 2 C with 260 mmol m2 s1 light for 16 h/8 h light/dark photoperiod) after keeping for 1 d in dark. 4.2. Protein extraction and estimation
Bacteria Staphylococcus aureus Nocardia
25.2 103 150
13.4 5.7
[25] [39]
Fungi Flammulina velutipes Phycomyces blakesleeanus
1000e10,000
0.33 N.A.
[26] [40]
66.7 14.5
[7]
Slime mold Physarum polycephalum
regulation of NOS like activity in plants is reported. Furthermore, the activation of BjNOSla by different abiotic stress indicates that NOS like activity could be responsible for NO evolution during stress conditions (Fig. 6).
244 47 103 (Isoform A) 253 47 103 (Isoform B)
26.8 9.7
N.A. e not available.
mammalian NOS. Its activation by PMA, 1,2-DG6 and PS (PKC activators), and inhibition by staurosporine and calphostin (PKC inhibitors) indicate that the phosphorylated form is the active form of the enzyme. Further, for the first time role of phosphorylation in
Seven days old B. juncea seedlings were homogenized in Hepes buffer (65 mM, pH 7.4) containing sucrose (514 mM), EDTA (0.16 mM), DTT (1.6 mM), PMSF (0.5 mM) and protease inhibitor cocktail (Sigma, 5 mL g1). The extract was centrifuged at 12,500 rpm for 25 min at 4 C. Supernatant was passed through two layered muslin cloth and was incubated with AG50-WX8 resin (Naþ form, BioRad) for 1 h with intermittent shaking. Resin was allowed to settle and the extract was used for activity assay. Proteins were quantified using Bradford’s method [36]. 4.3. Nitric oxide synthase activity assay Both oxyhemoglobin and citrulline assays were employed to analyze the NOS activity in B. juncea. For oxyhemoglobin assay, fresh oxyhemoglobin was prepared every day [28]. Hemoglobin
Fig. 5. Effect of abiotic stress conditions on BjNOSla. Treatments were given for 0.25e24 h. (A) Low temperature stress: seedlings were incubated in ice cold buffer at 4 C. (B) Heat stress: seedlings were placed in a vial containing buffer in a water bath maintained at 50 C. (C) Salinity stress was provided by submerging the roots of the seedlings in 0.5 M NaCl. (D) Dehydration: induction of water deficit was achieved by drying the roots of the seedling using a tissue paper and incubating them in B.O.D. at 25 2 C. (E) Injury: the seedling was pricked by a needle at 4e5 places and then placed the seedlings in B.O.D. at 25 2 C in buffer solution. Error bars represent standard deviation from three biological replicates. Asterisk showed significant difference in comparison with control (Student’s t-test, p < 0.05).
P.S. Talwar et al. / Plant Physiology and Biochemistry 60 (2012) 157e164
Fig. 6. BjNOSla is phosphorylated. Calcium, NADPH, light, PMA, PS, 1,2-DG6 and abiotic stress activate BjNOSla while the PKC inhibitors-staurosporine and calphostin inhibit it.
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5 mL of 10 mM Tris pH 7.2 and 10 mM DAF-2DA. Epidermal peels were maintained in dark for 1 h. The peels were removed and washed with buffer (10 mM Tris, pH 7.2) for 15 min and then with 1 mM L-arginine for 30 min for 25 C. L-NAME (5 mM, 30 min, 25 C) and NBT (300 mM), were added to the buffer after washing the probe. Fluorescent microscope (Zeiss, Oberkochen, Germany) was used for analysis and sections were excited with UV light. Dye emissions were recorded using a 505e530 nm band pass filter. Autofluorescence of chloroplasts was captured using a 585 nm long pass filter. Images were processed and captured by Zeiss Microsystems camera and analyzed using Zeiss LSM510 software. Experiments were carried out three times in duplicates. 4.5. NOS inhibitor, PKC inhibitors and PKC activators treatments
(25 mg) was suspended in 1 mL of TriseHCl buffer (10 mM, pH 7.0) and incubated with sodium dithionite (20 mg mL1) for 30 min. The solution was passed through sephadex G-25 (10 mL) column for desalting. Aliquots (1 mL) were eluted using 10 mM Tris containing 0.1 M NaCl, at a flow rate of 1 mL min1 and the fraction having maximum oxyhemoglobin was used for the assay [21]. For optimization, the assay was performed in 65 mM Hepes with varying concentrations of the substrate (L-arginine, 1e4 mM) and other reaction mixture components like oxyhemoglobin (2.5e20 mL), CaCl2 (0.1e0.6 mM) and MgCl2 (5e40 mM). BjNOSla was also monitored in presence of NADPH (0.1e1 mM), calmodulin (CaM, 0.5e1 mM), FAD (1e40 mM), FMN (1e40 mM) and BH4 (4e12 mM). All experiments were performed with ten biological replicates, with each experiment carried out in duplicates. NOS like activity was expressed as pmol min1 mg1 protein and the results are expressed as percent activity. Km, Vmax and Hill coefficient were calculated by plotting MichaeliseMenten curve (v versus s), LineweavereBurk plot (1/v versus 1/s) and Hill plot [log v/ (Vmax v) versus log s]. Rat neuronal NOS was used as positive control. Citrulline assay was performed according to Cueto et al. with slight modifications [21]. The assay was performed using 20 mL of the crude extract, 0.2 mM L-arginine, 0.3 mM CaCl2 and 0.05e 0.45 mCi of L-[U-14C] arginine. The reaction was terminated using AG50WX-8, suspended in Milli Q water (1:1 ratio). The supernatant was added to 3 mL scintillation cocktail-W and quantified by liquid scintillation counting (Beckman Coulter, Model LS 6000). Four biological replicates were performed with each assay performed in duplicates.
Effect of mammalian NOS inhibitors, NBT (10e300 mM) and LNAME (1e5 mM) was observed on BjNOSla. EGTA (1e2 mM), PKC inhibitors, staurosporine (1.5e20 nM), calphostin-C (0.1e1 mM) were incubated with the extract for 5 min before measuring the activity. Each experiment was carried out in duplicates with four biological replicates. NOS like activity was analyzed with PMA (438 nM), 4aPDD (438 nM), 1,2-DG6 (2.5 mM) and PS (1 mM) in vitro.
4.4. Nitric oxide detection by fluorescent microscopy
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In vivo detection of BjNOSla was performed in epidermal peel using NADPH-diaphorase [21] and DAF-2DA staining [37]. Epidermal peels were gently removed from the abaxial surface of the 4 weeks old Brassica leaves. For NADPH-diaphorase staining, epidermal peels were fixed with 4% p-formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffered saline pH 7.4 and then incubated with 4% p-formaldehyde for 2 h. These peels were mounted in 20% glycerol followed by washed thrice for 10 min each in 10 mM Tris buffer pH 7.6 and was suspended in 5% Triton X-100 in Tris buffer pH 7.6. The peels were incubated in dark for 2 h at 37 C in a solution containing NBT (0.25 mg mL1), NADPH (1 mg mL1) and Triton X-100 (0.5%) in 0.1 M Tris pH 7.6 [21]. Peels were mounted on slides using glycerine and were observed under light microscope (40 magnification). In the control set, NADPH was omitted. Experiments were repeated thrice in duplicates. DAF-2DA staining was performed according to Foissner et al. [37]. In brief, epidermal peels were placed in a petri dish containing
4.6. Abiotic stress treatments The seedlings were subjected to different abiotic stress conditions for 0.25e24 h. Seedlings were incubated in ice cold Hepes buffer at 4 C for low temperature and 50 C for high temperature. Dehydration was done by keeping seedlings on a tissue paper at 25 C and for salinity in 0.5 M NaCl. Injury stress was given to the seedlings by pricking at 4e5 places by sterilized needle. Experiments were carried out in three biological replicates with two technical replicates. Acknowledgments The research work was partially supported by SAP program from University Grants Commission and Council of Scientific and Industrial Research (CSIR), Government of India. PST acknowledges CSIR for fellowship. We thank Prof. S. K. Bansal (Department of Biochemistry, V. P. Patel Chest Institute, Delhi) for providing the liquid scintillation counting facility. References
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