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Effect of short-term salinity on the nitrate reductase activity in cucumber roots
Plant Science 180 (2011) 783–788
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Effect of short-term salinity on the nitrate reductase activity in cucumber roots ˙ Małgorzata Reda ∗ , Magdalena Migocka, Grazyna Kłobus Department of Plant Physiology, Institute of Plant Biology, University of Wrocław, Kanonia 6/8, 50-328 Wrocław, Poland
a r t i c l e
i n f o
Article history: Received 4 October 2010 Received in revised form 10 February 2011 Accepted 14 February 2011 Available online 21 February 2011 Keywords: Cucumber Nitrate reductase Posttranslational regulation Salt stress
a b s t r a c t In short-term experiments, the effect of high salinity on cucumber (Cucumis sativus) nitrate reductase activity was studied. The 60-min exposure of cucumber roots to 200 mM NaCl resulted in significant increase of the actual NR activity (measured in the presence of Mg2+ ), whereas the total enzyme activity (measured with EDTA) was not affected. NaCl-induced stimulation of the actual NR activity was rapidly reversed upon transfer of roots to salt-free solution. The increase in actual activity was completely prevented by microcystin-LR and cantharidin, protein phosphatases inhibitors. In addition, a significant decrease in ATP level was also observed in roots incubated with NaCl. These data suggest that the reversible protein phosphorylation is involved in the induction of NR activity during the first hour of salt stress. The effect of short-term salinity on the expression of genes encoding for nitrate reductase in cucumber roots was also studied. 200 mM NaCl diminished the increase in CsNR1 expression observed in control roots. During the same time period, the expression of CsNR2 was not affected, whereas the expression of CsNR3 decreased significantly after 1 h incubation of the excised roots in both, control and salt-containing nutrient solutions. Incubation of roots in the presence of iso-osmotic concentration of PEG had no effect on both, NR activity and expression. This indicates that only the ionic component of salt stress was involved in the salt-induced modifications of nitrate reductase activity. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Nitrate reductase (NR – EC 1.6.6.1.) is the first and key enzyme of the nitrate assimilation pathway in higher plants. Its catalytic activity in tissue is subjected to complex regulation in response to different environmental stimuli. The modulation of NR activity by a number of external factors is very rapid and occurs due to the posttranslational modifications of nitrate reductase protein [reviewed by [1,2]]. The best known is reversible phosphorylation catalyzed by specific protein kinase followed by association with 14-3-3 protein in the presence of divalent cations (Mg2+ ) and/or polyamines [3], which leads to an inactive complex formation [4]. Reactivation of NR occurs after dephosphorylation of the enzyme protein mediated by protein phosphatase type 1 and 2A and subsequent dissociation of the inhibitor protein [5]. Such post-translational modifications of NR in response to changes in light conditions, carbon dioxide availability or low temperature have been well described for green tissue [6]. Also in roots, NR activity has been shown to be modified through reversible phosphorylation of the protein due to differ-
Abbreviations: NR, nitrate reductase; PEG, polyethyleneglycol; MC, microcystinLR. ∗ Corresponding author. Tel.: +48 71 3754113; fax: +48 71 3754118. E-mail addresses: [email protected] (M. Reda), [email protected] (M. Migocka), [email protected] (G. Kłobus). 0168-9452/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2011.02.006
ent external and internal stimuli. It has been previously reported that NR activity in root extracts changed rapidly in the presence of adenine nucleotides and Mg2+ [7]. Different aeration and uncoupler treatment of pea [8], barley [9] and cucumber roots [10,11] resulted in post-translational modifications of NR in root tissue. In recent years, excessive salinity became a common environmental factor limiting plant growth and development [12]. Since sodium chloride constitutes the majority of the salts in soil solution, it is considered as the main reason for the increased salinization of the environment. It is well known that excessive salinity affects a number of metabolic processes in plants, including nitrogen assimilation. It has been also reported that the negative effect of NaCl on plant nitrogen assimilation is strictly related to salt-induced modifications of the enzymes involved in NO3 − assimilation pathway [13–15]. Namely, the activity of nitrate reductase has been shown to be modified by salinity. The salt-induced modification of NR activity depends on many factors, such as plant species, nitrogen source availability, salt concentration and the time of plant exposure to stress conditions. A significant decrease of NR activity was observed in roots and leaves of bean [16], leaves of maize [17], sugar beet [18] and tomato [15] after 5–10 days of the exposure of plants to high concentrations of NaCl in the environment. On the contrary, a stimulatory effect of salt stress on the NR activity was reported in sprouting bean seeds [19], soybean roots [20] and tomato roots [15]. Conversely, the enzyme activity remained unaffected in poplar plants treated with NaCl for 1 or 2 weeks [12]. Nevertheless, the
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available reports do not describe the molecular mechanism underlying modifications of NR activity under salt stress conditions. It has been suggested that a decrease in nitrate reductase activity observed during long-term (above 24 h) salinity could be the result of the reduced nitrate uptake [13], lower nitrate content in tissues [15] and a decreased expression of Nia genes under salt stress [16]. Indeed, a high concentration of NaCl in the environment led to the significant decrease of NO3 − uptake by wheat [21], tobacco [22], bean [16] and tomato [13]. However, the information explaining molecular alterations in the process of nitrogen assimilation during the first hours of salt stress is still lacking. Furthermore, the available data concerning the salinity-induced regulation of the nitrate assimilation pathway predominantly focus on leaves. Although nitrate assimilation occurs mainly in green tissue, the activities of nitrate reductase and other nitrate assimilating enzymes have been also detected in roots. Moreover, root is the part of plant, which is directly exposed to the soil solution and thus first affected by high concentrations of salt. Therefore, the initial plant response to salt stress is expected to occur in root cells. In this work we have investigated nitrate reductase activity in cucumber roots subjected to short-term sodium chloride treatment. We present evidence that the rapid modifications of NR activity in response to salinity occur at the post-translational (phosphorylation/dephosphorylation) level. 2. Materials and methods 2.1. Plant material Cucumber plants were grown hydroponically in growth chamber under a 16-h photoperiod (180 mmol m−2 s−1 ) at 25 ◦ C during the day and 22 ◦ C during the night. For the first 6 days plants were grown in N-free solution, pH 6.0, containing 1 mM K2 SO4 , 0.17 mM Ca(H2 PO4 ), 1.5 mM CaSO4 , 0.33 mM MgSO4 and the following micronutrients: 25 M Fe-citrate, 3.3 M MnSO4 , 1.7 M H3 BO4 , 0.3 M CuSO4 , 0.003 M ZnSO4 , 0.017 M Na2 MoO4 . After that, plants were transferred for 1 day into the fresh nutrient solution, pH 6.0, containing 1.7 mM Ca(NO3 )2 , 1.7 mM KNO3 , 0.33 mM KH2 PO4 , 0.33 mM MgSO4 and micronutrients [7]. The roots of 7day-old seedlings were excised from plants after 6 h of illumination period and incubated up to 60 min in 10 mM Mes-KOH, pH 5.5, containing 5 mM KNO3 (incubation buffer) with or without addition of 200 mM NaCl or 20% PEG. In the experiments where the inhibitors of protein phosphatases were used, 1 M microcystin-LR or 50 M cantharidin were added to the incubation solutions and the equal amount of their solvent, DMSO was applied to control assays. After the incubation for the times indicated in the figures, the roots were blotted with paper, frozen in liquid nitrogen and used for the enzymatic assays. 2.2. Nitrate reductase assay For the extraction of NR, 1 g of frozen roots was ground in a chilled mortar with 1.5 ml of 50 mM Hepes-KOH (pH 7.5) containing 1 mM DTT, 1 mM PMSF, 1% BSA and 1 mM PVPP. The homogenate was filtered through 4 layers of cheesecloth and centrifuged at 15,000 × g for 15 min at 4 ◦ C. The obtained supernatant was desalted on Sephadex G-25 Fine. Nitrate reductase activity was measured in the desalted supernatant in the absence of MgCl2 (−Mg2+ , total activity) and/or in the presence of MgCl2 (+Mg2+ , actual activity), according to Kaiser and Huber [23] with some modifications. The reaction medium contained 50 mM Hepes-KOH, pH 7.5, 5 mM EDTA (−Mg2+ ) or 5 mM MgCl2 (+Mg2+ ), 10 mM KNO3 and crude extract. Following the addition of 0.2 mM NADH, the reaction was carried out for 5 min at 27 ◦ C and stopped with 0.066 ml of 1 mM zinc acetate. The mixture was centrifuged
and the amount of nitrite was determined colorimetrically at 540 nm [24]. 2.3. Extraction and determination of ATP Adenosine triphosphate (ATP) was determined according to Glaab and Kaiser [25] with some modifications. Following incubation of roots in 10 mM Mes-KOH, pH 5.5, with or without NaCl, 1 g of the fresh tissue was immediately frozen and ground in liquid nitrogen. Afterward, 5 ml of 4.5% HClO4 was added to the frozen powder and mixed until complete thawing. The mixture was then supplemented with 0.125 ml of 2 mM tris(hydroxymethyl)aminomethane and centrifuged for 5 min at 5000 × g at 4 ◦ C. The pH of the supernatant was adjusted to 7.4 with 5 mM K2 CO3 and samples were centrifuged once again (5 min, 5000 × g, 4 ◦ C). The ATP level in the supernatant was determined luminometrically in a TD-20/20 Luminometer (Turner Designs, USA) using Firefly luciferin-luciferase (Sigma). 2.4. Expression of CsNR genes To evaluate the expression of the genes encoding for nitrate reductase CsNR1 (HM755943), CsNR2 (HM755944) and CsNR3 (HO116134) the Real Time PCR was performed using LightCycler (2.0) system from Roche Diagnostics. For the normalization of each CsNR gene expression, a gene encoding TIP41-like protein (GW881871) was used as the internal standard. Total RNA was isolated from 50 mg of frozen roots tissue with Tri Reagent (Sigma) according to manufacturer’s instruction. Total RNA yield was determined using NanoDrop Spectrophotometer ND-1000 (Thermo Scientific) and the 260/280 nm ratio showed expected values between 1.8 and 2.0. To avoid any DNA contaminations, the RNA samples were treated with RNAse-free DNAseI (Fermentas) and then reversed transcribed into first-strand cDNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following manufacturer’s instruction. The cDNA was then used as the template for PCR amplification with RealTime 2× PCRMaster Mix SYBR® (A&A Biotechnology) kit. Gene specific primers used for PCR were carefully designed using Lightcycler Probe Design program. The CsNRs expression was analyzed with the following primer pairs: 5 -GGACGGTAGAGTAAAGAAGGC-3 (forward) and 5 -TATCCCTTTTACTCCATTCA-3 (reverse) for CsNR1, 5 CGACTCCTCCTCCAACTCC-3 (forward) and 5 -CCACTTCCATGTTGTCCAA-3 (reverse) for CsNR2, 5 -TCCAATGGCGACTGCTG-3 (forward) and 5 -CATCATCATCAATAAGGAGCGG-3 (reverse) for CsNR3 and 5 -CAACAGGTGATATTGGATTATGATTATAC-3 (forward) and 5 -GCCAGCTCATCCTCATATAAG-3 (reverse) for TIP41-like protein. The following conditions of amplifications were applied: 30 s at 95 ◦ C; 45 cycles of 10 s at 95 ◦ C, 10 s at 55 ◦ C and 15 s at 72 ◦ C, with final melting for 15 s at 65 ◦ C. 2.5. Statistics For each of at least two independent protein and RNA extractions, the measurements of enzyme activity and gene expression were obtained in triplicate and the means ± SD of these values are presented in the figures. The quantitative PCR data were analyzed by the CT – method using the LightCycler® Software 4.1 (Roche). 3. Results The roots of cucumber seedlings were excised from plants and incubated for 60 min in a buffer with or without the addition of 200 mM NaCl. The extracts obtained from roots were then used in
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Fig. 1. Effect of 200 mM NaCl treatment on total (A) and actual (B) NR activity in cucumber roots. Initial NR activity measured in roots at the beginning of experiment (time 0) was used as 100% (1.39 (A) and 0.71 (B) mol NO2 − g−1 FW h−1 ). Presented data are means of 12 replications of 4 independent experiments. Error bars represent SD.
the assays determining total (+EDTA) and actual (+MgCl2 ) NR activities. The total activity of NR in both, control and salt-treated roots was similar (Fig. 1A), whereas the actual enzyme activity was significantly affected by salt (Fig. 1B). Namely, marked stimulation of actual NR activity was observed within 15 min of pre-incubation of roots with sodium chloride. After 30 min of incubation of roots with salt, almost 170% NR activity was detectable comparing to enzyme activity measured at the beginning of the experiment (time 0). On the contrary, the actual nitrate reductase activity in roots incubated without NaCl did not change (Fig. 1B). Moreover, the activation status of NR (activity of dephospho-NR measured in the presence of Mg2+ ions and shown as the percentage of total activity) increased during incubation of roots in the presence of NaCl (Table 1). In order to determine, whether NaCl-induced modifications of NR activity resulted from the ionic and/or osmotic stress, cucumber roots were incubated in buffer containing iso-osmotic concentration of polyethyleneglycol (PEG). No significant changes of both, total and actual nitrate reductase activities were observed during 60 min of tissues treatment with buffer containing 20% PEG (Fig. 2A and B). Stimulating effect of sodium chloride on the actual NR activity was very rapid and reversible after elimination of the stressogenic factor from the environment (Fig. 3). The observed drop in the enzyme activity was very strong. However, the decrease in the
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Fig. 2. Total (A) and actual (B) NR activity in cucumber roots treated with or without 20% PEG. The NR activity measured in cucumber roots at time 0 was used as 100% (1.03 (A) and 0.56 (B) mol NO2 − g−1 FW h−1 ). Every data point is the average of 12 replications of 4 independent experiments. Error bars represent SD.
Fig. 3. Actual NR activity measured in cucumber roots incubated up to 60 min in buffer with 200 mM NaCl () or without addition of salt (). After 15 min of NaCl treatment two sets of roots were transferred to the incubation buffer without salt (䊉) or to the buffer containing 20% PEG () for the next 45 min. Enzyme activity determined in time 0 was used as 100% (0.47 mol NO2 − g−1 FW h−1 ). The data are means of 9 replications of 3 independent experiments. Error bars represent SD.
enzyme activity was not so large, when salt-treated roots were transferred to the environment containing iso-osmotic concentration of PEG (Fig. 3). Rapid changes in the actual NR activity in cucumber roots indicate post-translational modification of NR protein. It is well known that post-translational phosphorylation/dephosphorylation is one of the most common protein
Table 1 Nitrate reductase activation state in cucumber roots during NaCl treatment. NR activation state [%] Control
−NaCl 15
−NaCl 30
−NaCl 60
+NaCl 15
+NaCl 30
+NaCl 60
52
56
54
53
76
86
83
Total and actual nitrate reductase activities were measured in cucumber roots incubated in buffer with or without NaCl. Activation state shows NR actual activity as a % of total enzyme activity.
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Fig. 5. The level of ATP in cucumber roots treated with or without 200 mM NaCl. Presented data are means of 9 replications of 3 independent experiments. Error bars represent SD.
Fig. 4. Effect of microcystin-LR (MC) (A) and cantharidin (canth.) (B) on actual NR activity in cucumber roots. The enzyme activity in extracts obtained from roots incubated without addition of salt and with or without inhibitor was used as 100% (0.46 and 0.26 mol NO2 − g−1 FW h−1 , respectively). The data are means of 6 replications of 2 independent experiments. Error bars represent SD.
modifications that occurs in living cells. It has been also reported that NR activity increase due to the dephosphorylation of enzyme protein [2]. Hence, in the further assay two potent inhibitors of type 1 and 2A protein phosphatases, microcystin-LR and cantharidin [26,27] were used to elucidate, whether the protein phosphatases contribute in the NaCl-induced stimulation of actual NR activity in cucumber roots. The addition of 1 M microcystin-LR into the incubation media completely abolished the stimulatory effect of NaCl on NR activity in cucumber roots (Fig. 4A). Similar effect was observed when 50 M cantharidin was included into the assay (Fig. 4B). In addition to NR activity measurements, the level of ATP in cucumber root tissues subjected to NaCl was also studied. As it is demonstrated in Fig. 5, the level of nucleotide significantly decreased after short treatment of cucumber roots with 200 mM NaCl. Changes in NR activity in roots subjected to salt treatment were very rapid. It suggests that they were not caused by the changes in the transcript level of the NR-coding genes. To confirm that, the expression of genes encoding nitrate reductase in cucumber roots was studied. The cucumber genome has been recently sequenced and submitted to GenBank by Huang et al. [28]. Three cucumber genes: CsNR1, CsNR2 and CsNR3 encoding for putative nitrate reductase have been identified through the screening of cucumber whole genome shotgun reads (NCBI database) using Nucleotide Blast, Protein Blast and two gene prediction programs: FGENESH
and GeneMarkTM (Reda and Migocka, data unpublished). The PCR amplicons were cloned and sequenced and are now available in GenBank database. The GenBank accession numbers are shown in Table 2. To assess the expression level of three putative nitrate reductase genes in cucumber roots treated or untreated with NaCl, the real-time PCR assay was performed. The relative expression of NR genes in cucumber roots was differentially affected due to NaCl treatment. The level of CsNR1 transcript slightly increased during the first 30 min incubation of roots in buffer with or without NaCl. Further incubation of roots with buffer resulted in significant, almost 6 times higher, increase of CsNR1 transcript in control roots, comparing to the expression of the gene measured at the beginning of the experiment. On the contrary, a slight decrease in CsNR1 transcript level was observed in roots treated with NaCl for the same time (Fig. 6A). On the other hand, the expression of CsNR2 in cucumber roots was not affected significantly during the experiment. After 30 min of incubation, a very slight increase in CsNR2 transcript level was observed in both, salt-treated and control roots and it sustained during the next 30 min of the assay (Fig. 6B). Contrary to this, CsNR3 transcript level in roots was enhanced during the first 30 min incubation with and without salt and decreased significantly throughout further incubation time (Fig. 6C). 4. Discussion Salinity is one of the most important environmental factors that limits growth and development of higher plants. It is well known that initial steps of nitrogen metabolism in plants (NO3 − ions uptake, reduction of nitrates and assimilation of ammonium) are sensitive to salt stress [13,14]. In this work, we studied the effect of 200 mM NaCl on NR activity in cucumber roots during the first hour of exposure to salt. The 60 min-long incubation of roots in solution containing 200 mM NaCl did not affect the total NR activity. On the contrary, a 50% stimulation of the actual NR activity was observed in roots after 15 min-long salt treatment. Modifications of nitrate reductase activity under salinity were related with the increase in the activation status of the enzyme in roots (Table 1) and a decrease in the phosphorylated enzyme pool in the cytosol.
Table 2 Nitrate reductase genes family in Cucumis sativus. NR gene
Accession no.
Cucumber genome contig accession no.
Nucleotides
Strand
CsNR1 CsNR2 CsNR3
HM755943 HM755944 HO116134
ACHR01005902.1 ACHR01003989.1 ACHR01003989.1
2925–6128 2816–5791 140–4975
Plus/Plus Plus/Plus Plus/Minus
Bioinformatic analysis was done using Nucleotide Blast and Protein Blast and two gene prediction programs SoftBerry and GeneMarkTM .
M. Reda et al. / Plant Science 180 (2011) 783–788
Fig. 6. Relative expression of nitrate reductase genes in cucumber roots. To determine the expression of CsNR genes, Real-Time PCR was performed. Values are means of 3 replications. Errors bars represent SD.
The stimulation of the actual NR activity (measured in the presence of Mg2+ ) can result from the dephosphorylation of the enzyme protein. Type 1 or 2A protein phosphatases can be directly involved in the stimulation of actual NR activity under salinity, since both of the protein phosphatase inhibitors, microcystin-LR and cantharidin, completely abolished stimulatory effect of salt on NR activity (Fig. 4A and B). Until now, there has been no evidence for a direct, salt-induced modification of protein phosphatases activity. However, Huber et al. [29] observed a strong increase in NR activity in spinach leaves following the addition of different organic and inorganic salts into the enzyme-containing extracts. The authors postulated that the NR stimulation resulted from the effect of high ionic strength caused by the presence of salts. The stimulatory effect of salts was abolished by the addition of protein phosphatases inhibitor [29], suggesting the contribution of dephosphorylation in NR activation. Some recent evidence indicates that NaCl causes a short-term decrease in the cytoplasmic pH and alkalization of vacuole during the first hours of salt stress [30]. In addition, a significant decrease in the activity of protein kinases and phosphatases specific to NR was observed during cytoplasm acidification [31]. These data were supported by further studies revealing that acidification effect was directly mediated via pH-dependent change in
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the relative activities of NR kinases or NR phosphatases [2]. Moreover, it was previously suggested [6] that NR activity in lower pH increases because the protein kinases are more affected by the acidification than protein phosphatases. The salt-induced decrease in ATP levels in cells of cucumber roots (Fig. 5) could also limit the protein kinase activity [29]. Hence, the transient decrease in cytosol pH during salinity could probably induce the increase in actual activity of NR in roots through the dephosphorylation of NR protein. The alterations of NR activity in cucumber roots treated with salt might be also the result of the modifications related with 143-3 proteins. It is well known that 14-3-3 proteins are involved in post-translational down regulation of nitrate reductase [3,4]. They also play an important role in regulation of the activities of a wide array of targets by protein–protein interactions [32]. Some recent studies show that 14-3-3 proteins are encoded by multigene families containing 13 isoforms in Arabidopsis [33] and 12 isoforms in tomato [34]. Recently, Xu and Shi revealed that the expression of genes encoding for 14-3-3 in tomato was altered during salt stress conditions [34]. Namely, NaCl stimulated the expression of four of 14-3-3 genes from 12 members of tomato multigene family. Expression of the remaining 8 genes was not affected by salt [34]. So the above-mentioned assumption could not explain the observed up-regulation of NR activity. In Arabidopsis thaliana, various 14-33 proteins show significantly different affinity to the phosphoNR form that is probably related with their organ and developmental expression pattern [33]. It was also suggested that nitrate-bound NR underlay much stronger inhibition by 14-3-3 proteins [33]. Treatment of plants with NaCl reduces the level of nitrate ions in the cytoplasm since it also inhibits nitrate uptake by plants [35]. At low nitrate concentrations, 14-3-3 dissociation from pNR might occur leading to the dephosphorylation of nitrate reductase and subsequent release of fully active enzyme [33]. Beside the posttranslational level of nitrate reductase regulation, the expression of NR genes may also be affected by external factors. However, the observed modification of NR activity in our assay is probably the result of very rapid, salt-induced protein modification occurring within the first minutes of salt stress. Moreover, the expression of NR encoding genes observed in cucumber roots during short-term salinity did not correlate with changes in enzyme activity measured in the tissue. Namely, the presence of salt in the incubation media did not enhance NR genes expression in comparison to the control conditions. Moreover, NaCl abolished the observed induction of CsNR1 expression in control roots. This marked induction of CsNR1 expression in roots incubated without salt might be the effect of NO3 − present in the incubation buffer. Such response of CsNR1 gene to nitrate may suggest that CsNR1 demonstrate higher sensitivity towards nitrate present in the environment than the two other CsNR genes. It is well known that NR genes are nitrate inducible [36]. Microarray analysis of nitrate-inducible genes in Arabidopsis thaliana indicated that NIA1 shows higher induction than NIA2 after 20 min of nitrate treatment [37]. On the contrary to CsNR1, the expression of CsNR3 after 60 min of incubation decreased in both, NaCl-treated and untreated roots, whereas CsNR2 expression remained nearly unaffected. The decrease in CsNR3 expression observed in cucumber roots might be the effect of diurnal fluctuations of NR mRNA levels. It has been previously shown that the highest expression of NR genes occurs at the beginning of the light period [2]. During the light phase, the amount of NR mRNA drops and NR activity increases and rises to the highest level during a few hours. The roots used in the experiment were harvested after 6 h of illumination period, when the amount of NR mRNA transcript in tissue should be relatively low. Thus the continuous lowering of CsNR3 transcript level could be the result of diurnal cycle effect. It might be also suggested, that CsNR3 shows different regulation when compared to the other two genes. However, these assumptions need further investigation.
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Rising stress during salinity results from two overlapping phenomena, namely osmotic and ionic stresses [38]. Therefore, the alterations of NR activity in roots during the first hour of NaCl action could be a result of osmotic or ionic component of salt stress. In further assays, cucumber roots were incubated in the presence of 20% PEG, which reflected the solution osmosity corresponding to 200 mM NaCl solution. Osmotic stress did not affect neither the actual or total NR activity. Hence we suggest that the salt-induced increase in the actual NR activity resulted rather from ionic than from the osmotic component of salt stress. Similar results were obtained during studies on NR activity in tomato seedlings [39]. Activity changes relying on phosphorylation or dephosphorylation of proteins are reversible at post-translational level. The increase in actual NR activity in roots treated with salt appeared to be also reversible, since it was considerably decreased after the transfer of roots to salt-free solution reaching only 60% of control activity determined in roots untreated with salt (Fig. 3). The reasons for such a striking decrease in the NR activity could be multiple. Strong cell hydratation may occur after the transfer of roots from 200 mM NaCl solution to the buffer without salt. This may in turn lead to the dilution of the cell nitrate content which provides substrates for NR. Due to the inhibition of the NO3 − uptake process, the level of nitrates in cytoplasm is rapidly decreased during salinity. The decrease in NO3 − uptake rate is detectable very quickly after introducing salt [35]. It is also possible that this two-fold, very rapid change in osmotic conditions in the cell was unfavorable for cell’s integrity. The decrease in enzyme activity was not so drastic, when salt-treated roots were transferred to the iso-osmoticum solution of 20% PEG (Fig. 3). Therefore, a huge decrease in the activity of nitrate reductase could also be a result of this osmotic change. Reassuming, from the results presented in this work it can be concluded that short-term salinity significantly stimulates the nitrate reductase activity in cucumber roots through posttranslational modification of NR protein involving reversible dephosphorylation. References [1] W.H. Campbell, Nitrate reductase structure, function and regulation: brinding the gap between biochemistry and physiology, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 277–303. [2] W.M. Kaiser, H. Weiner, A. Kandlbinder, C.-B. Tsai, P. Rockel, M. Sonoda, E. Planchet, Modulation of nitrate reductase: some new insights, an unusual case and a potentially important side reaction, J. Exp. Bot. 53 (2002) 875–882. [3] W. Shen, S.C. Huber, Polycations globally enhance binding of 14-3-3omega to target proteins in spinach leaves, Plant Cell Physiol. 47 (2006) 764–771. [4] G.S. Athwal, S.C. Huber, Divalent cations and polyamines bind to loop8 of 14-3-3 proteins, modulating their interaction with phosphorylated nitrate reductase, Plant J. 29 (2002) 119–129. [5] W.M. Kaiser, S.C. Huber, Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers, J. Exp. Bot. 52 (2001) 1981–1989. [6] W.M. Kaiser, H. Weiner, S.C. Huber, Nitrate reductase in higher plants: a case study for transduction of environmental stimuli into control of catalytic activity, Physiol. Plant 105 (1999) 385–390. [7] M. Reda, G. Kłobus, Modifications of the activity of nitrate reductase from cucumber roots, Biol. Plant 50 (2006) 42–47. [8] J. Glaab, W.M. Kaiser, Rapid modulation of nitrate reductase in pea roots, Planta 191 (1993) 173–179. [9] A. Bortel, W.M. Kaiser, Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status, Planta 201 (1997) 496–501. [10] P. De la Haba, E. Agüera, L. Benítez, J.M. Maldonado, Modulation of nitrate reductase activity in cucumber (Cucumis sativus) roots, Plant Sci. 161 (2001) 231–237. [11] M. Reda, G. Kłobus, Effect of different oxygen availability on the nitrate reductase activity in Cucumis sativus L. roots, Biol. Plant 52 (2008) 674–680. [12] B. Ehlting, P. Dłuzniewska, H. Dietrich, A. Selle, M. Teuber, R. Hänsch, U. Nehls, A. Polle, J.P. Schnitzler, H. Rennenberg, A. Gessler, Interaction of nitrogen nutrition and salinity in Grey poplar (Populus tremula x alba), Plant Cell Environ. 30 (2007) 796–811. [13] P. Flores, M.Á. Botella, A. Cerdá, V. Martinez, Influence of nitrate level on nitrate assimilation in tomato (Lycopersicon esculentum) plants under saline stress, Can. J. Bot. 82 (2004) 207–213.
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Kilian, E.A.G. van der Vossen, Y. Wu, J. Guo, J. He, Z. Jia, Y. Ren, G. Tian, Y. Lu, J. Ruan, W. Qian, M. Wang, Q. Huang, B. Li, Z. Xuan, J. Cao, Asan, Z. Wu, J. Zhang, Q. Cai, Y. Bai, B. Zhao, Y. Han, Y. Li, X. Li, S. Wang, Q. Shi, S. Liu, W.K. Cho, J.-Y.Y. Kim, Y. Xu, K. Heller-Uszynska, H. Miao, Z. Cheng, S. Zhang, J. Wu, Y. Yang, H. Kang, M. Li, H. Liang, X. Ren, Z. Shi, M. Wen, M. Jian, H. Yang, G. Zhang, Z. Yang, R. Chen, S. Liu, J. Li, L. Ma, H. Liu, Y. Zhou, J. Zhao, X. Fang, G. Li, L. Fang, Y. Li, D. Liu, H. Zheng, Y. Zhang, N. Qin, Z. Li, G. Yang, S. Yang, L. Bolund, K. Kristiansen, H. Zheng, S. Li, X. Zhang, H. Yang, J. Wang, R. Sun, B. Zhang, S. Jiang, J. Wang, Y. Du, S. Li, The genome of the cucumber, Cucumis sativus L., Nat. Genet. 41 (2009) 1275–1281. [29] S.C. Huber, J.L. Huber, W.M. Kaiser, Differential response of nitrate reductase and sucrose-phosphate synthase-activation to inorganic and organic salts, in vitro and in situ, Physiol. Plant 92 (1994) 302–310. [30] M. 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Effect of short-term salinity on the nitrate reductase activity in cucumber roots ˙ Małgorzata Reda ∗ , Magdalena Migocka, Grazyna Kłobus Department of Plant Physiology, Institute of Plant Biology, University of Wrocław, Kanonia 6/8, 50-328 Wrocław, Poland
a r t i c l e
i n f o
Article history: Received 4 October 2010 Received in revised form 10 February 2011 Accepted 14 February 2011 Available online 21 February 2011 Keywords: Cucumber Nitrate reductase Posttranslational regulation Salt stress
a b s t r a c t In short-term experiments, the effect of high salinity on cucumber (Cucumis sativus) nitrate reductase activity was studied. The 60-min exposure of cucumber roots to 200 mM NaCl resulted in significant increase of the actual NR activity (measured in the presence of Mg2+ ), whereas the total enzyme activity (measured with EDTA) was not affected. NaCl-induced stimulation of the actual NR activity was rapidly reversed upon transfer of roots to salt-free solution. The increase in actual activity was completely prevented by microcystin-LR and cantharidin, protein phosphatases inhibitors. In addition, a significant decrease in ATP level was also observed in roots incubated with NaCl. These data suggest that the reversible protein phosphorylation is involved in the induction of NR activity during the first hour of salt stress. The effect of short-term salinity on the expression of genes encoding for nitrate reductase in cucumber roots was also studied. 200 mM NaCl diminished the increase in CsNR1 expression observed in control roots. During the same time period, the expression of CsNR2 was not affected, whereas the expression of CsNR3 decreased significantly after 1 h incubation of the excised roots in both, control and salt-containing nutrient solutions. Incubation of roots in the presence of iso-osmotic concentration of PEG had no effect on both, NR activity and expression. This indicates that only the ionic component of salt stress was involved in the salt-induced modifications of nitrate reductase activity. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Nitrate reductase (NR – EC 1.6.6.1.) is the first and key enzyme of the nitrate assimilation pathway in higher plants. Its catalytic activity in tissue is subjected to complex regulation in response to different environmental stimuli. The modulation of NR activity by a number of external factors is very rapid and occurs due to the posttranslational modifications of nitrate reductase protein [reviewed by [1,2]]. The best known is reversible phosphorylation catalyzed by specific protein kinase followed by association with 14-3-3 protein in the presence of divalent cations (Mg2+ ) and/or polyamines [3], which leads to an inactive complex formation [4]. Reactivation of NR occurs after dephosphorylation of the enzyme protein mediated by protein phosphatase type 1 and 2A and subsequent dissociation of the inhibitor protein [5]. Such post-translational modifications of NR in response to changes in light conditions, carbon dioxide availability or low temperature have been well described for green tissue [6]. Also in roots, NR activity has been shown to be modified through reversible phosphorylation of the protein due to differ-
Abbreviations: NR, nitrate reductase; PEG, polyethyleneglycol; MC, microcystinLR. ∗ Corresponding author. Tel.: +48 71 3754113; fax: +48 71 3754118. E-mail addresses: [email protected] (M. Reda), [email protected] (M. Migocka), [email protected] (G. Kłobus). 0168-9452/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2011.02.006
ent external and internal stimuli. It has been previously reported that NR activity in root extracts changed rapidly in the presence of adenine nucleotides and Mg2+ [7]. Different aeration and uncoupler treatment of pea [8], barley [9] and cucumber roots [10,11] resulted in post-translational modifications of NR in root tissue. In recent years, excessive salinity became a common environmental factor limiting plant growth and development [12]. Since sodium chloride constitutes the majority of the salts in soil solution, it is considered as the main reason for the increased salinization of the environment. It is well known that excessive salinity affects a number of metabolic processes in plants, including nitrogen assimilation. It has been also reported that the negative effect of NaCl on plant nitrogen assimilation is strictly related to salt-induced modifications of the enzymes involved in NO3 − assimilation pathway [13–15]. Namely, the activity of nitrate reductase has been shown to be modified by salinity. The salt-induced modification of NR activity depends on many factors, such as plant species, nitrogen source availability, salt concentration and the time of plant exposure to stress conditions. A significant decrease of NR activity was observed in roots and leaves of bean [16], leaves of maize [17], sugar beet [18] and tomato [15] after 5–10 days of the exposure of plants to high concentrations of NaCl in the environment. On the contrary, a stimulatory effect of salt stress on the NR activity was reported in sprouting bean seeds [19], soybean roots [20] and tomato roots [15]. Conversely, the enzyme activity remained unaffected in poplar plants treated with NaCl for 1 or 2 weeks [12]. Nevertheless, the
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available reports do not describe the molecular mechanism underlying modifications of NR activity under salt stress conditions. It has been suggested that a decrease in nitrate reductase activity observed during long-term (above 24 h) salinity could be the result of the reduced nitrate uptake [13], lower nitrate content in tissues [15] and a decreased expression of Nia genes under salt stress [16]. Indeed, a high concentration of NaCl in the environment led to the significant decrease of NO3 − uptake by wheat [21], tobacco [22], bean [16] and tomato [13]. However, the information explaining molecular alterations in the process of nitrogen assimilation during the first hours of salt stress is still lacking. Furthermore, the available data concerning the salinity-induced regulation of the nitrate assimilation pathway predominantly focus on leaves. Although nitrate assimilation occurs mainly in green tissue, the activities of nitrate reductase and other nitrate assimilating enzymes have been also detected in roots. Moreover, root is the part of plant, which is directly exposed to the soil solution and thus first affected by high concentrations of salt. Therefore, the initial plant response to salt stress is expected to occur in root cells. In this work we have investigated nitrate reductase activity in cucumber roots subjected to short-term sodium chloride treatment. We present evidence that the rapid modifications of NR activity in response to salinity occur at the post-translational (phosphorylation/dephosphorylation) level. 2. Materials and methods 2.1. Plant material Cucumber plants were grown hydroponically in growth chamber under a 16-h photoperiod (180 mmol m−2 s−1 ) at 25 ◦ C during the day and 22 ◦ C during the night. For the first 6 days plants were grown in N-free solution, pH 6.0, containing 1 mM K2 SO4 , 0.17 mM Ca(H2 PO4 ), 1.5 mM CaSO4 , 0.33 mM MgSO4 and the following micronutrients: 25 M Fe-citrate, 3.3 M MnSO4 , 1.7 M H3 BO4 , 0.3 M CuSO4 , 0.003 M ZnSO4 , 0.017 M Na2 MoO4 . After that, plants were transferred for 1 day into the fresh nutrient solution, pH 6.0, containing 1.7 mM Ca(NO3 )2 , 1.7 mM KNO3 , 0.33 mM KH2 PO4 , 0.33 mM MgSO4 and micronutrients [7]. The roots of 7day-old seedlings were excised from plants after 6 h of illumination period and incubated up to 60 min in 10 mM Mes-KOH, pH 5.5, containing 5 mM KNO3 (incubation buffer) with or without addition of 200 mM NaCl or 20% PEG. In the experiments where the inhibitors of protein phosphatases were used, 1 M microcystin-LR or 50 M cantharidin were added to the incubation solutions and the equal amount of their solvent, DMSO was applied to control assays. After the incubation for the times indicated in the figures, the roots were blotted with paper, frozen in liquid nitrogen and used for the enzymatic assays. 2.2. Nitrate reductase assay For the extraction of NR, 1 g of frozen roots was ground in a chilled mortar with 1.5 ml of 50 mM Hepes-KOH (pH 7.5) containing 1 mM DTT, 1 mM PMSF, 1% BSA and 1 mM PVPP. The homogenate was filtered through 4 layers of cheesecloth and centrifuged at 15,000 × g for 15 min at 4 ◦ C. The obtained supernatant was desalted on Sephadex G-25 Fine. Nitrate reductase activity was measured in the desalted supernatant in the absence of MgCl2 (−Mg2+ , total activity) and/or in the presence of MgCl2 (+Mg2+ , actual activity), according to Kaiser and Huber [23] with some modifications. The reaction medium contained 50 mM Hepes-KOH, pH 7.5, 5 mM EDTA (−Mg2+ ) or 5 mM MgCl2 (+Mg2+ ), 10 mM KNO3 and crude extract. Following the addition of 0.2 mM NADH, the reaction was carried out for 5 min at 27 ◦ C and stopped with 0.066 ml of 1 mM zinc acetate. The mixture was centrifuged
and the amount of nitrite was determined colorimetrically at 540 nm [24]. 2.3. Extraction and determination of ATP Adenosine triphosphate (ATP) was determined according to Glaab and Kaiser [25] with some modifications. Following incubation of roots in 10 mM Mes-KOH, pH 5.5, with or without NaCl, 1 g of the fresh tissue was immediately frozen and ground in liquid nitrogen. Afterward, 5 ml of 4.5% HClO4 was added to the frozen powder and mixed until complete thawing. The mixture was then supplemented with 0.125 ml of 2 mM tris(hydroxymethyl)aminomethane and centrifuged for 5 min at 5000 × g at 4 ◦ C. The pH of the supernatant was adjusted to 7.4 with 5 mM K2 CO3 and samples were centrifuged once again (5 min, 5000 × g, 4 ◦ C). The ATP level in the supernatant was determined luminometrically in a TD-20/20 Luminometer (Turner Designs, USA) using Firefly luciferin-luciferase (Sigma). 2.4. Expression of CsNR genes To evaluate the expression of the genes encoding for nitrate reductase CsNR1 (HM755943), CsNR2 (HM755944) and CsNR3 (HO116134) the Real Time PCR was performed using LightCycler (2.0) system from Roche Diagnostics. For the normalization of each CsNR gene expression, a gene encoding TIP41-like protein (GW881871) was used as the internal standard. Total RNA was isolated from 50 mg of frozen roots tissue with Tri Reagent (Sigma) according to manufacturer’s instruction. Total RNA yield was determined using NanoDrop Spectrophotometer ND-1000 (Thermo Scientific) and the 260/280 nm ratio showed expected values between 1.8 and 2.0. To avoid any DNA contaminations, the RNA samples were treated with RNAse-free DNAseI (Fermentas) and then reversed transcribed into first-strand cDNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following manufacturer’s instruction. The cDNA was then used as the template for PCR amplification with RealTime 2× PCRMaster Mix SYBR® (A&A Biotechnology) kit. Gene specific primers used for PCR were carefully designed using Lightcycler Probe Design program. The CsNRs expression was analyzed with the following primer pairs: 5 -GGACGGTAGAGTAAAGAAGGC-3 (forward) and 5 -TATCCCTTTTACTCCATTCA-3 (reverse) for CsNR1, 5 CGACTCCTCCTCCAACTCC-3 (forward) and 5 -CCACTTCCATGTTGTCCAA-3 (reverse) for CsNR2, 5 -TCCAATGGCGACTGCTG-3 (forward) and 5 -CATCATCATCAATAAGGAGCGG-3 (reverse) for CsNR3 and 5 -CAACAGGTGATATTGGATTATGATTATAC-3 (forward) and 5 -GCCAGCTCATCCTCATATAAG-3 (reverse) for TIP41-like protein. The following conditions of amplifications were applied: 30 s at 95 ◦ C; 45 cycles of 10 s at 95 ◦ C, 10 s at 55 ◦ C and 15 s at 72 ◦ C, with final melting for 15 s at 65 ◦ C. 2.5. Statistics For each of at least two independent protein and RNA extractions, the measurements of enzyme activity and gene expression were obtained in triplicate and the means ± SD of these values are presented in the figures. The quantitative PCR data were analyzed by the CT – method using the LightCycler® Software 4.1 (Roche). 3. Results The roots of cucumber seedlings were excised from plants and incubated for 60 min in a buffer with or without the addition of 200 mM NaCl. The extracts obtained from roots were then used in
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Fig. 1. Effect of 200 mM NaCl treatment on total (A) and actual (B) NR activity in cucumber roots. Initial NR activity measured in roots at the beginning of experiment (time 0) was used as 100% (1.39 (A) and 0.71 (B) mol NO2 − g−1 FW h−1 ). Presented data are means of 12 replications of 4 independent experiments. Error bars represent SD.
the assays determining total (+EDTA) and actual (+MgCl2 ) NR activities. The total activity of NR in both, control and salt-treated roots was similar (Fig. 1A), whereas the actual enzyme activity was significantly affected by salt (Fig. 1B). Namely, marked stimulation of actual NR activity was observed within 15 min of pre-incubation of roots with sodium chloride. After 30 min of incubation of roots with salt, almost 170% NR activity was detectable comparing to enzyme activity measured at the beginning of the experiment (time 0). On the contrary, the actual nitrate reductase activity in roots incubated without NaCl did not change (Fig. 1B). Moreover, the activation status of NR (activity of dephospho-NR measured in the presence of Mg2+ ions and shown as the percentage of total activity) increased during incubation of roots in the presence of NaCl (Table 1). In order to determine, whether NaCl-induced modifications of NR activity resulted from the ionic and/or osmotic stress, cucumber roots were incubated in buffer containing iso-osmotic concentration of polyethyleneglycol (PEG). No significant changes of both, total and actual nitrate reductase activities were observed during 60 min of tissues treatment with buffer containing 20% PEG (Fig. 2A and B). Stimulating effect of sodium chloride on the actual NR activity was very rapid and reversible after elimination of the stressogenic factor from the environment (Fig. 3). The observed drop in the enzyme activity was very strong. However, the decrease in the
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Fig. 2. Total (A) and actual (B) NR activity in cucumber roots treated with or without 20% PEG. The NR activity measured in cucumber roots at time 0 was used as 100% (1.03 (A) and 0.56 (B) mol NO2 − g−1 FW h−1 ). Every data point is the average of 12 replications of 4 independent experiments. Error bars represent SD.
Fig. 3. Actual NR activity measured in cucumber roots incubated up to 60 min in buffer with 200 mM NaCl () or without addition of salt (). After 15 min of NaCl treatment two sets of roots were transferred to the incubation buffer without salt (䊉) or to the buffer containing 20% PEG () for the next 45 min. Enzyme activity determined in time 0 was used as 100% (0.47 mol NO2 − g−1 FW h−1 ). The data are means of 9 replications of 3 independent experiments. Error bars represent SD.
enzyme activity was not so large, when salt-treated roots were transferred to the environment containing iso-osmotic concentration of PEG (Fig. 3). Rapid changes in the actual NR activity in cucumber roots indicate post-translational modification of NR protein. It is well known that post-translational phosphorylation/dephosphorylation is one of the most common protein
Table 1 Nitrate reductase activation state in cucumber roots during NaCl treatment. NR activation state [%] Control
−NaCl 15
−NaCl 30
−NaCl 60
+NaCl 15
+NaCl 30
+NaCl 60
52
56
54
53
76
86
83
Total and actual nitrate reductase activities were measured in cucumber roots incubated in buffer with or without NaCl. Activation state shows NR actual activity as a % of total enzyme activity.
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Fig. 5. The level of ATP in cucumber roots treated with or without 200 mM NaCl. Presented data are means of 9 replications of 3 independent experiments. Error bars represent SD.
Fig. 4. Effect of microcystin-LR (MC) (A) and cantharidin (canth.) (B) on actual NR activity in cucumber roots. The enzyme activity in extracts obtained from roots incubated without addition of salt and with or without inhibitor was used as 100% (0.46 and 0.26 mol NO2 − g−1 FW h−1 , respectively). The data are means of 6 replications of 2 independent experiments. Error bars represent SD.
modifications that occurs in living cells. It has been also reported that NR activity increase due to the dephosphorylation of enzyme protein [2]. Hence, in the further assay two potent inhibitors of type 1 and 2A protein phosphatases, microcystin-LR and cantharidin [26,27] were used to elucidate, whether the protein phosphatases contribute in the NaCl-induced stimulation of actual NR activity in cucumber roots. The addition of 1 M microcystin-LR into the incubation media completely abolished the stimulatory effect of NaCl on NR activity in cucumber roots (Fig. 4A). Similar effect was observed when 50 M cantharidin was included into the assay (Fig. 4B). In addition to NR activity measurements, the level of ATP in cucumber root tissues subjected to NaCl was also studied. As it is demonstrated in Fig. 5, the level of nucleotide significantly decreased after short treatment of cucumber roots with 200 mM NaCl. Changes in NR activity in roots subjected to salt treatment were very rapid. It suggests that they were not caused by the changes in the transcript level of the NR-coding genes. To confirm that, the expression of genes encoding nitrate reductase in cucumber roots was studied. The cucumber genome has been recently sequenced and submitted to GenBank by Huang et al. [28]. Three cucumber genes: CsNR1, CsNR2 and CsNR3 encoding for putative nitrate reductase have been identified through the screening of cucumber whole genome shotgun reads (NCBI database) using Nucleotide Blast, Protein Blast and two gene prediction programs: FGENESH
and GeneMarkTM (Reda and Migocka, data unpublished). The PCR amplicons were cloned and sequenced and are now available in GenBank database. The GenBank accession numbers are shown in Table 2. To assess the expression level of three putative nitrate reductase genes in cucumber roots treated or untreated with NaCl, the real-time PCR assay was performed. The relative expression of NR genes in cucumber roots was differentially affected due to NaCl treatment. The level of CsNR1 transcript slightly increased during the first 30 min incubation of roots in buffer with or without NaCl. Further incubation of roots with buffer resulted in significant, almost 6 times higher, increase of CsNR1 transcript in control roots, comparing to the expression of the gene measured at the beginning of the experiment. On the contrary, a slight decrease in CsNR1 transcript level was observed in roots treated with NaCl for the same time (Fig. 6A). On the other hand, the expression of CsNR2 in cucumber roots was not affected significantly during the experiment. After 30 min of incubation, a very slight increase in CsNR2 transcript level was observed in both, salt-treated and control roots and it sustained during the next 30 min of the assay (Fig. 6B). Contrary to this, CsNR3 transcript level in roots was enhanced during the first 30 min incubation with and without salt and decreased significantly throughout further incubation time (Fig. 6C). 4. Discussion Salinity is one of the most important environmental factors that limits growth and development of higher plants. It is well known that initial steps of nitrogen metabolism in plants (NO3 − ions uptake, reduction of nitrates and assimilation of ammonium) are sensitive to salt stress [13,14]. In this work, we studied the effect of 200 mM NaCl on NR activity in cucumber roots during the first hour of exposure to salt. The 60 min-long incubation of roots in solution containing 200 mM NaCl did not affect the total NR activity. On the contrary, a 50% stimulation of the actual NR activity was observed in roots after 15 min-long salt treatment. Modifications of nitrate reductase activity under salinity were related with the increase in the activation status of the enzyme in roots (Table 1) and a decrease in the phosphorylated enzyme pool in the cytosol.
Table 2 Nitrate reductase genes family in Cucumis sativus. NR gene
Accession no.
Cucumber genome contig accession no.
Nucleotides
Strand
CsNR1 CsNR2 CsNR3
HM755943 HM755944 HO116134
ACHR01005902.1 ACHR01003989.1 ACHR01003989.1
2925–6128 2816–5791 140–4975
Plus/Plus Plus/Plus Plus/Minus
Bioinformatic analysis was done using Nucleotide Blast and Protein Blast and two gene prediction programs SoftBerry and GeneMarkTM .
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Fig. 6. Relative expression of nitrate reductase genes in cucumber roots. To determine the expression of CsNR genes, Real-Time PCR was performed. Values are means of 3 replications. Errors bars represent SD.
The stimulation of the actual NR activity (measured in the presence of Mg2+ ) can result from the dephosphorylation of the enzyme protein. Type 1 or 2A protein phosphatases can be directly involved in the stimulation of actual NR activity under salinity, since both of the protein phosphatase inhibitors, microcystin-LR and cantharidin, completely abolished stimulatory effect of salt on NR activity (Fig. 4A and B). Until now, there has been no evidence for a direct, salt-induced modification of protein phosphatases activity. However, Huber et al. [29] observed a strong increase in NR activity in spinach leaves following the addition of different organic and inorganic salts into the enzyme-containing extracts. The authors postulated that the NR stimulation resulted from the effect of high ionic strength caused by the presence of salts. The stimulatory effect of salts was abolished by the addition of protein phosphatases inhibitor [29], suggesting the contribution of dephosphorylation in NR activation. Some recent evidence indicates that NaCl causes a short-term decrease in the cytoplasmic pH and alkalization of vacuole during the first hours of salt stress [30]. In addition, a significant decrease in the activity of protein kinases and phosphatases specific to NR was observed during cytoplasm acidification [31]. These data were supported by further studies revealing that acidification effect was directly mediated via pH-dependent change in
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the relative activities of NR kinases or NR phosphatases [2]. Moreover, it was previously suggested [6] that NR activity in lower pH increases because the protein kinases are more affected by the acidification than protein phosphatases. The salt-induced decrease in ATP levels in cells of cucumber roots (Fig. 5) could also limit the protein kinase activity [29]. Hence, the transient decrease in cytosol pH during salinity could probably induce the increase in actual activity of NR in roots through the dephosphorylation of NR protein. The alterations of NR activity in cucumber roots treated with salt might be also the result of the modifications related with 143-3 proteins. It is well known that 14-3-3 proteins are involved in post-translational down regulation of nitrate reductase [3,4]. They also play an important role in regulation of the activities of a wide array of targets by protein–protein interactions [32]. Some recent studies show that 14-3-3 proteins are encoded by multigene families containing 13 isoforms in Arabidopsis [33] and 12 isoforms in tomato [34]. Recently, Xu and Shi revealed that the expression of genes encoding for 14-3-3 in tomato was altered during salt stress conditions [34]. Namely, NaCl stimulated the expression of four of 14-3-3 genes from 12 members of tomato multigene family. Expression of the remaining 8 genes was not affected by salt [34]. So the above-mentioned assumption could not explain the observed up-regulation of NR activity. In Arabidopsis thaliana, various 14-33 proteins show significantly different affinity to the phosphoNR form that is probably related with their organ and developmental expression pattern [33]. It was also suggested that nitrate-bound NR underlay much stronger inhibition by 14-3-3 proteins [33]. Treatment of plants with NaCl reduces the level of nitrate ions in the cytoplasm since it also inhibits nitrate uptake by plants [35]. At low nitrate concentrations, 14-3-3 dissociation from pNR might occur leading to the dephosphorylation of nitrate reductase and subsequent release of fully active enzyme [33]. Beside the posttranslational level of nitrate reductase regulation, the expression of NR genes may also be affected by external factors. However, the observed modification of NR activity in our assay is probably the result of very rapid, salt-induced protein modification occurring within the first minutes of salt stress. Moreover, the expression of NR encoding genes observed in cucumber roots during short-term salinity did not correlate with changes in enzyme activity measured in the tissue. Namely, the presence of salt in the incubation media did not enhance NR genes expression in comparison to the control conditions. Moreover, NaCl abolished the observed induction of CsNR1 expression in control roots. This marked induction of CsNR1 expression in roots incubated without salt might be the effect of NO3 − present in the incubation buffer. Such response of CsNR1 gene to nitrate may suggest that CsNR1 demonstrate higher sensitivity towards nitrate present in the environment than the two other CsNR genes. It is well known that NR genes are nitrate inducible [36]. Microarray analysis of nitrate-inducible genes in Arabidopsis thaliana indicated that NIA1 shows higher induction than NIA2 after 20 min of nitrate treatment [37]. On the contrary to CsNR1, the expression of CsNR3 after 60 min of incubation decreased in both, NaCl-treated and untreated roots, whereas CsNR2 expression remained nearly unaffected. The decrease in CsNR3 expression observed in cucumber roots might be the effect of diurnal fluctuations of NR mRNA levels. It has been previously shown that the highest expression of NR genes occurs at the beginning of the light period [2]. During the light phase, the amount of NR mRNA drops and NR activity increases and rises to the highest level during a few hours. The roots used in the experiment were harvested after 6 h of illumination period, when the amount of NR mRNA transcript in tissue should be relatively low. Thus the continuous lowering of CsNR3 transcript level could be the result of diurnal cycle effect. It might be also suggested, that CsNR3 shows different regulation when compared to the other two genes. However, these assumptions need further investigation.
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Rising stress during salinity results from two overlapping phenomena, namely osmotic and ionic stresses [38]. Therefore, the alterations of NR activity in roots during the first hour of NaCl action could be a result of osmotic or ionic component of salt stress. In further assays, cucumber roots were incubated in the presence of 20% PEG, which reflected the solution osmosity corresponding to 200 mM NaCl solution. Osmotic stress did not affect neither the actual or total NR activity. Hence we suggest that the salt-induced increase in the actual NR activity resulted rather from ionic than from the osmotic component of salt stress. Similar results were obtained during studies on NR activity in tomato seedlings [39]. Activity changes relying on phosphorylation or dephosphorylation of proteins are reversible at post-translational level. The increase in actual NR activity in roots treated with salt appeared to be also reversible, since it was considerably decreased after the transfer of roots to salt-free solution reaching only 60% of control activity determined in roots untreated with salt (Fig. 3). The reasons for such a striking decrease in the NR activity could be multiple. Strong cell hydratation may occur after the transfer of roots from 200 mM NaCl solution to the buffer without salt. This may in turn lead to the dilution of the cell nitrate content which provides substrates for NR. Due to the inhibition of the NO3 − uptake process, the level of nitrates in cytoplasm is rapidly decreased during salinity. The decrease in NO3 − uptake rate is detectable very quickly after introducing salt [35]. It is also possible that this two-fold, very rapid change in osmotic conditions in the cell was unfavorable for cell’s integrity. The decrease in enzyme activity was not so drastic, when salt-treated roots were transferred to the iso-osmoticum solution of 20% PEG (Fig. 3). Therefore, a huge decrease in the activity of nitrate reductase could also be a result of this osmotic change. Reassuming, from the results presented in this work it can be concluded that short-term salinity significantly stimulates the nitrate reductase activity in cucumber roots through posttranslational modification of NR protein involving reversible dephosphorylation. References [1] W.H. Campbell, Nitrate reductase structure, function and regulation: brinding the gap between biochemistry and physiology, Annu. Rev. Plant Physiol. Plant Mol. Biol. 50 (1999) 277–303. [2] W.M. Kaiser, H. Weiner, A. Kandlbinder, C.-B. Tsai, P. Rockel, M. Sonoda, E. Planchet, Modulation of nitrate reductase: some new insights, an unusual case and a potentially important side reaction, J. Exp. Bot. 53 (2002) 875–882. [3] W. Shen, S.C. Huber, Polycations globally enhance binding of 14-3-3omega to target proteins in spinach leaves, Plant Cell Physiol. 47 (2006) 764–771. [4] G.S. Athwal, S.C. Huber, Divalent cations and polyamines bind to loop8 of 14-3-3 proteins, modulating their interaction with phosphorylated nitrate reductase, Plant J. 29 (2002) 119–129. [5] W.M. Kaiser, S.C. Huber, Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers, J. Exp. Bot. 52 (2001) 1981–1989. [6] W.M. Kaiser, H. Weiner, S.C. Huber, Nitrate reductase in higher plants: a case study for transduction of environmental stimuli into control of catalytic activity, Physiol. Plant 105 (1999) 385–390. [7] M. Reda, G. Kłobus, Modifications of the activity of nitrate reductase from cucumber roots, Biol. Plant 50 (2006) 42–47. [8] J. Glaab, W.M. Kaiser, Rapid modulation of nitrate reductase in pea roots, Planta 191 (1993) 173–179. [9] A. Bortel, W.M. Kaiser, Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status, Planta 201 (1997) 496–501. [10] P. De la Haba, E. Agüera, L. Benítez, J.M. Maldonado, Modulation of nitrate reductase activity in cucumber (Cucumis sativus) roots, Plant Sci. 161 (2001) 231–237. [11] M. Reda, G. Kłobus, Effect of different oxygen availability on the nitrate reductase activity in Cucumis sativus L. roots, Biol. Plant 52 (2008) 674–680. [12] B. Ehlting, P. Dłuzniewska, H. Dietrich, A. Selle, M. Teuber, R. Hänsch, U. Nehls, A. Polle, J.P. Schnitzler, H. Rennenberg, A. Gessler, Interaction of nitrogen nutrition and salinity in Grey poplar (Populus tremula x alba), Plant Cell Environ. 30 (2007) 796–811. [13] P. Flores, M.Á. Botella, A. Cerdá, V. Martinez, Influence of nitrate level on nitrate assimilation in tomato (Lycopersicon esculentum) plants under saline stress, Can. J. Bot. 82 (2004) 207–213.
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