- Email: [email protected]
Increased metal tolerance in Salix by nicotinamide and nicotinic acid
Available online at www.sciencedirect.com
Plant Physiology and Biochemistry 46 (2008) 655e664 www.elsevier.com/locate/plaphy
Research article
Increased metal tolerance in Salix by nicotinamide and nicotinic acid Anna B. Ohlsson a,*, Tommy Landberg b, Torkel Berglund a, Maria Greger b a
Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden b Department of Botany, Stockholm University, SE-106 91 Stockholm, Sweden Received 11 October 2007 Available online 22 April 2008
Abstract We have earlier shown that nicotinamide (NIC) and nicotinic acid (NiA) can induce defence-related metabolism in plant cells; e.g. increase the level of glutathione. Here we investigated if NIC and NiA could increase the metal tolerance in metal sensitive clones of Salix viminalis and whether this would be mediated via increased glutathione level. Salix clones, sensitive or tolerant to zinc (Zn), copper (Cu) and cadmium (Cd) were grown in the presence of heavy metals (Cd, Cu or Zn) or NIC and NiA as well as in combination. In addition, the influence of Nacetyl-cystein (NAC) and L-2-oxothiazolidine 4-carboxylate (OTC), stimulators of reduced glutathione (GSH) biosynthesis, and the glutathione biosynthesis inhibitor buthionine sulfoximine (BSO) was analysed. Tolerance was measured as effects on root and shoot dry weight, and the glutathione and metal concentrations in the tissues were analysed. Results showed that NIC and NiA decreased the toxic effects of Cd, Cu and Zn on growth significantly in sensitive clones, but also to some extent in tolerant clones. However, the glutathione level and metal concentration did not change by NIC or NiA addition. Treatment with NAC, OTC or BSO did not per se influence the sensitivity to Cd, although the glutathione level increased in the presence of NAC and OTC and decreased in response to BSO. The results suggest that NIC and NiA increased the defence against heavy metals but not via glutathione formation per se. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Glutathione; Heavy metal defence; Nicotinamide; Nicotinic acid; Salix viminalis; Toxicity
1. Introduction Heavy metals in the environment may influence the growth of plants and cause toxic effects if the plants are sensitive [10]. Salix viminalis and other Salix spp. have been shown to evolve clones with huge variation in tolerance to and accumulation of heavy metals [15,20]. It has also been shown that these properties may vary in the same clone for different metals [20]. Salix viminalis is therefore a good candidate for investigations on mechanisms behind metal tolerance in plants. One important effect of heavy metals in biological systems is the induction of oxidative stress, which occurs when the
Abbreviations: BSO, L-buthionine (S,R)-sulfoximine; GSH, glutathione; NAC, N-acetyl-cystein; NIC, nicotinamide; NiA, nicotinic acid; OTC, L-2oxothiazolidine 4-carboxylate; PARP, poly(ADP-ribose)polymerase; PC, phytochelatin; ROS, reactive oxygen species. * Corresponding author. Fax þ46 8 5537 8468. E-mail address: [email protected] (A.B. Ohlsson). 0981-9428/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.04.004
formation of reactive oxygen species (ROS) is too high in relation to the protective antioxidants [13,14]. Glutathione (GSH) is one of the most important non-protein molecules protecting the cell from oxidative damage by neutralizing ROS [14]. There are many potential mechanisms behind metal tolerance in plants [32]. The biological variation in types of defence mechanisms is probably an advantage from an adaptive evolutionary point of view. The ability to synthesise GSH is one important factor for protection against heavy metal toxicity [28]. One suggested mechanism is biosynthesis of metal binding peptides, phytochelatins (PC), at the expense of GSH [16]. This requires the activity of PC synthase genes and the presence of GSH in sufficient amounts. Although present in most other plants studied, we showed recently that no phytochelatin (PC2 or PC3) could be found in Salix before or after metal exposure [22]. Accordingly, PC does not appear to be a main reason for metal tolerance in this genus. Metallothionein (MT) is a cystein-rich protein which can be induced by metal exposure both in animals and plants [11,38]. However, neither
656
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
PC nor MT biosynthesis can completely explain the mechanisms of metal tolerance in plants. Nicotinamide (NIC) has been hypothesised as a signalling link in the response to oxidative stress in eukaryotic cells [5]. The antioxidative defence is tightly associated with GSH metabolism. We have shown earlier that NIC [7,8] and its natural metabolite nicotinic acid (NiA) (unpublished) can induce several defence responses in plants and plant cell cultures of different species, for example a pronounced increase in GSH content and protection against oxidative stress-induced damage. Nicotinamide may protect cells via different types of mechanisms: poly(ADP-ribose)polymerase (PARP) inhibition, which slows down NAD decomposition and thus improves energy metabolism [12]; as a precursor for salvage synthesis of NAD [17]; counteracting histone deacetylation [3,19]; activation of defence genes and defence related metabolism [6]. A considerable interest has recently focused on nicotinamide in medical as well as plant science, since it was shown that it is involved in the regulation of cellular longevity [3,17,19]. The aim of the present investigation was to find out whether NIC and NiA could increase the resistance to toxic effects on plant growth caused by metal exposure, and if so, whether this effect could be related to glutathione content or synthesis. 2. Materials and methods 2.1. Plant material and cultivation Two clones of Salix viminalis, one sensitive and one tolerant to Zn, Cu and Cd were cultivated from cuttings in nutrient solution. Cuttings from 1-year-old shoots of the two clones, 80 mm in length, were rooted and cultivated. The cuttings were mounted six by six in styrofoam plates and each plate was placed in a pot with 900 ml 100 mM Ca(NO3)2 solution [35]. Water loss was compensated when 1% of the water had evaporated. The pH decreased from 6.5 to 6.0 during the cultivation and was not compensated. Cuttings were grown in a controlled climate chamber equipped with metal halogen lamps (Osram, Powerstar HQI-R) providing a photon flux density of 200 mmol m2 s1 for 16 h at 25 C. During the 8 h dark period the temperature was 21 C. The relative humidity was 70e80%. 2.2. Experimental design 2.2.1. Effect of NIC and NiA on metal tolerance The effect of nicotinamide (NIC) and nicotinic acid (NiA) on heavy metal tolerance and accumulation was studied. From the first day of cultivation plants were treated with either 3 mM CuCl2, 7 mM CdCl2, 70 mM ZnCl2, 2 mM NIC, 2 mM NiA in the following combinations: Control (no addition), NIC, NiA, Cd, Cu, Zn, Cd þ NIC, Cu þ NIC, Zn þ NIC, Cd þ NiA, Cu þ NiA or Zn þ NiA. 2.2.2. Effect of BSO, OTC and NAC on metal tolerance and GSH levels The effect of L-buthionine (S,R)-sulfoximine (BSO), L-2oxothiazolidine 4-carboxylate (OTC) and N-acetyl-cysteine
(NAC) on heavy metal tolerance and level of glutathione (GSH) in roots and shoots was studied. OTC and NAC stimulate the production of GSH, while BSO decreases the formation of GSH. From the first day of cultivation plants were treated with either 7 mM CdCl2, 3 mM CuCl2, 70 mM ZnCl2, 0.1 mM BSO, 1 mM OTC and 1 mM NAC in the following combinations; Control (no addition), BSO, OTC, NAC, Cd, Cu, Zn, Cd þ BSO, Cd þ OTC, Cd þ NAC, Cu þ BSO, Cu þ OTC, Cu þ NAC, Zn þ BSO, Zn þ OTC, Zn þ NAC. 2.2.3. Time study of BSO and NIC additions This experiment was performed to analyse the effect on cadmium tolerance by adding BSO and NIC at different time points (day 1, 7 and 14) during the cultivation. Plants were treated with 7 mM CdCl2, 0.1 mM BSO and 2 mM NIC in different order and at different points of time during the cultivation according to Table 1. 2.3. Plant harvest and determination of toxic effects on growth After 21 days treatment plants were harvested and divided in root and shoot, which were dried in 105 C for 48 h. In order to measure the toxic effect on growth the fresh and dry weight were measured as well as the length of root and shoot. 2.4. Analysis of reduced glutathione Reduced glutathione was analysed using HPLC (high performance liquid chromatography) and Ellmans reagent. Fresh plant materials (0.1e 0.5 g) were ultramixed (Polytron, PT Table 1 Treatment protocol in the time study of BSO and NIC additions. Plants were treated 21 days without (e) or with 7 mM CdCl2 (Cd), 2 mM nicotinamide (NIC) and/or 0.1 mM buthionine sulfoximine (BSO) added at various time points separate or in combination. The cultivation solution was changed at day 7 and day 14 Day 1
Day 7
Day 14
e Cd NIC BSO e e e e e e Cd Cd NIC BSO e e e e e e
e Cd NIC BSO Cd Cd NIC NIC BSO BSO Cd þ NIC Cd þ BSO NIC þ BSO BSO þ NIC Cd þ NIC Cd þ BSO NIC þ BSO Cd þ NIC Cd þ BSO NIC þ BSO
e Cd NIC BSO Cd þ NIC Cd þ BSO NIC þ Cd NIC þ BSO BSO þ Cd BSO þ NIC Cd þ NIC þ BSO Cd þ BSO þ NIC NIC þ BSO þ Cd BSO þ NIC þ Cd Cd þ NIC Cd þ BSO NIC þ BSO Cd þ NIC þ BSO Cd þ BSO þ NIC NIC þ BSO þ Cd
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
2000) in 1.5 ml HCl (0.01 M). The samples were then centrifuged at 16,000 g for 5 min at 4 C. The supernatant was filtered with a 0.45 mm Millipore filter and immediately injected (manually) into the HPLC. As internal standards 40 nM GSH (reduced glutathione, EC-200-725-4) and 40 nM cysteine (EC200-158-2) were used. The HPLC (Beckman) was equipped with an A C-18 column (ODS Hypersil 5 mm, Hewlett-Packard, Palo Alto, CA, USA) 250 4 mm. The flow rate was 1.0 ml min1. The analyses procedure was performed according to Tukendorf and Rauser [31]. The column was equilibrated with 0.1% trifluoricacetic acid (TFA) in water. The sample was injected (50 mL) and the column was developed with a linear gradient from 0 to 60% (v/v) acetonitrile with 0.1% TFA for 30 min. Post column the effluent was mixed with 1.8 mM 5,50 dithiobis(2-nitrobenzoic acid) (DTNB, i.e. Ellman’s reagent) in a mixing chamber (Micro mixer, Beckman Coulter, Inc., Miami, FL, USA). The DTNB was delivered at a rate of 0.5 ml min1 by a Beckman 110B Solvent Delivery Module. The reaction was then allowed in a 2.4 m 0.5 mm Teflon tube at 37 C. The DTNB was daily prepared with 14 mg DTNB in 200 ml cold (4 C) 300 mM potassium buffer (pH 7.8) and 15 mM K-EDTA. The solution was kept dark and on ice during the analysis. The absorbance was measured at 405 nm (with the Beckman 166 detector, Beckman Coulter, Inc., Miami, FL, USA). 2.5. Analysis of metals The dried material was wet digested (HNO3:HClO4, 7:3) and analysed on the contents of Cd, Cu and Zn by atomic absorption spectrometry with a flame atomiser (AA-1275, Varian, Springvale, Australia) using standard addition. 2.6. Calculations and statistics All experiments were performed in three replicates where each replicate consisted of six pooled plants. The tolerance index was calculated 1according to Eq. (1) Tolerance index ¼
Dry wieghttreated plant Dry wieghtcontrol
ð1Þ
where either shoots or roots are considered. Statistical treatments were performed by using Tukeye Kramers test and ANOVA at p < 0.05. 2.7. Chemicals L-buthionine (S,R)-sulfoximine (BSO), N-acetyl-cystein (NAC), L-2-oxothiazolidine 4-carboxylate (OTC), nicotinic acid (NiA) and nicotinamide (NIC) were purchased from Sigma-Aldrich Chemical Co. USA.
3. Results Effects on metal tolerance in two Salix viminalis clones were investigated; one clone sensitive to Cd, Cu and Zn and
657
another clone tolerant to the same metals. The clones were exposed to the three metals as well as to nicotinamide (NIC) and nicotinic acid (NiA) in the combinations presented in Fig. 1. All additions were made at the first day of cultivation. Significant difference in tolerance when compared to untreated plants (tolerance index 1.00) is marked with a dot in the bar. The growth of the tolerant clone was only in two cases marginally affected by the metal; Zn in shoot and Cu in root. Addition of only NIC showed a slight inhibitory effect on the growth of shoots in the tolerant clone. However, nearly all treatments affected the growth of the sensitive clone; the decrease in tolerance index caused by Cd, Cu or Zn was in most cases reduced by addition of NIC or NiA, the latter being most effective (significance illustrated by *). For example, in some cases a tolerance index of approximately 0.25 was increased to higher than 0.5 by NiA in this clone. Nicotinamide had no protecting effect towards Cu. The metal content was analyzed in the plants treated with Cd, Cu, Zn, NIC and NiA, solely or in combinations (Table 2). The contents of Cd and Cu were higher in roots than in shoots. On the contrary, the Zn content was about the same in roots and shoots of the tolerant clone and lower in roots when compared with shoots in the sensitive clone. The control levels of Cd and Cu were principally about the same in the tolerant and the sensitive clone of shoot and roots, respectively. The Zn content, on the other hand, was lower in the roots of the sensitive clone, when compared to the tolerant clone. Addition of each metal increased the concentration of that metal, on a percentage basis to the same extent in both clones. No influence of NIC or NiA on the metal levels was detected. To investigate if glutathione could play a role in the tolerance caused by NIC, the tolerant and sensitive clones were exposed to L-2-oxothiazolidine 4-carboxylate (OTC), N-acetylcysteine (NAC), or L-buthionine (S,R)-sulfoximine (BSO), alone and in combination with each metal. OTC is by the enzyme 5-oxo-prolinase converted to S-carboxy-L-cysteine, which via L-cysteine is used for GSH synthesis [36]. NAC is also a cysteine precursor that increases the intracellular GSH level [4]. BSO inhibits the activity of g-glutamylcysteine synthetase, leading to reduced glutathione levels [37]. In general, the addition of the glutathione biosynthesis modifying compounds BSO, OTC or NAC had no effect on tolerance, neither when added alone or in combination with metals (Fig. 2). In the same experiment, the level of glutathione (Fig. 3) increased, with varying significance, by the addition of OTC or NAC, which are known to stimulate glutathione biosynthesis by providing cysteine for GSH biosynthesis. The glutathione level decreased significantly by the addition of the glutathione biosynthesis inhibitor BSO, irrespective of the presence (*) or absence (black dots) of metal. In order to further investigate a possible role of glutathione in NIC-induced metal tolerance, Cd, NIC and BSO were added at different points of time, and tolerance index as well as GSH content were analysed (Figs. 4 and 5). In neither of the clones, Cd nor NIC did change the GSH content, while BSO, independent of addition time, decreased the GSH level in all tissues (black dots). The experiment showed that the Cd tolerance
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
658 1.25
Shoot
Shoot
Tolerant clone
1.25
Sensitive clone
†
Tolerance index
1.00
0.75
†
*
0.50
†
0.75
*
† †
*
*
0.50
0.25
0.25 0 Zn
Zn
Zn
N IC N iA
C u C u C u N IC N iA
C d C d C d N IC N iA
-
-
IC N iA
N
Zn
Zn
Zn
N IC N iA
C u C u C u N IC N iA
N IC N iA
N IC N iA
-
C d C d C d
0
0 0.25
0
0.25 †
0.50
†
*
†
0.75
*
†
0.50 †
*
1.00
0.75
Tolerance index
Tolerance index
Tolerance index
* *
1.00
1.00 Root
Tolerant clone
Root
Sensitive clone
1.25
Fig. 1. Effects of nicotinamide (NIC) and nicotinic acid (NiA) on heavy metal tolerance in one sensitive and one tolerant clone of Salix viminalis. Tolerance is given as tolerance index based on dry weight of roots or shoot. Plants were treated during 21 days and from the first day of cultivation with combinations of 3 mM CuCl2, 7 mM CdCl2, 70 mM ZnCl2, 2 mM NIC and/or 2 mM NiA. n ¼ 3 SE. *Indicate significant differences between treatment with metal and metal þ NIC or NIA. y Denotes significant differences between NIC and NIC þ metal or NiA and NiA þ metal. Black or white dot in bars denotes significant difference from tolerance index 1.00. p < 0.05.
Table 2 Metal content in treated plants. Heavy metal accumulation in roots and shoots of one tolerant and one sensitive clone of Salix viminalis untreated or treated with 3 mM CuCl2, 7 mM CdCl2, 70 mM ZnCl2, 2 mM nicotinamide (NIC) and 2 mM nicotinic acid (NiA) solely or in combinations. n ¼ 3 SE. Different letters show significant difference p < 0.05 between clones with same treatment Analysed metal treatment
Tolerant clone Root
Cd, mg gDW1 e e e Cd Cd Cd
e NIC NiA e NIC NiA
0.025 0.003 0.027 0.003 0.023 0.002 11.930 1.440 12.650 1.081 12.380 1.053
Cu, mg gDW1 e e e Cu Cu Cu
e NIC NiA e NIC NiA
0.686 0.026 0.696 0.023 0.687 0.004 a 13.890 0.790 12.780 1.119 13.460 0.675
Zn, mg gDW1 e e e Zn Zn Zn
e NIC NiA e NIC NiA
11.67 0.233 a 12.07 0.939 a 11.97 0.376 a 620.30 23.511 a 621.70 11.865 a 628.70 6.489 a
Sensitive clone Shoot
Root
Shoot
0.013 0.001 0.011 0.001 0.016 0.001 1.095 0.048 0.981 0.088 0.978 0.048
0.024 0.002 0.021 0.003 0.023 0.003 10.620 0.648 9.412 0.157 10.357 0.413
0.013 0.001 0.014 0.001 0.014 0.001 1.148 0.084 1.015 0.036 1.096 0.013
0.259 0.019 0.267 0.009 a 0.255 0.020 0.974 0.003 0.917 0.081 1.011 0.051
0.735 0.030 0.774 0.035 0.812 0.018 b 11.760 0.197 11.960 0.439 11.080 0.449
0.222 0.016 0.183 0.011 b 0.196 0.025 0.935 0.071 0.987 0.064 0.913 0.021
8.73 0.145 b 9.40 0.321 b 8.80 0.493 b 468.70 17.340 b 425.30 12.170 b 427.70 1.450 b
12.0 0.503 13.1 0.602 13.2 0.145 720.0 19.280 725.3 41.010 716.7 29.670
12.83 0.524 12.93 0.913 13.20 0.945 656.30 12.601 652.70 3.480 666.00 20.033
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
659
1.25
1.25 Sensitive clone
Tolerant clone
Shoot
1.00
1.00
0.75
0.75 †
0.50
†
†
†
†
†
0.50 †
†
†
0.25
0.25
0 Zn
Zn N AC
Zn
O O TC
Zn –
BS
C u C u C u C u –
BS O O TC N AC
C d C d N AC
O
TC O
C d C d –
BS
–
– –
BS O O TC N AC
–
Zn N AC
Zn
Zn
O O TC
Zn –
BS
C u C u C u C u –
BS O O TC N AC
C d C d N AC
O
TC O
C d C d –
BS
–
–
BS O O TC N AC
–
0
0
0
0.25
† †
†
† †
0.50
†
0.75
†
†
†
†
0.50
0.75
†
1.00
Tolerance index
Tolerance index
0.25
1.25
Tolerance index
Tolerance index
Shoot
1.00 Root
Tolerant clone
Sensitive clone
Root
1.25
Fig. 2. Effects of L-buthionine (S,R)-sulfoximine (BSO) and GSH stimulating substances N-acetyl-cystein (NAC) and L-2-oxothiazolidine 4-carboxylate (OTC) on heavy metal tolerance in roots and shoots of 21 day old sensitive and tolerant clones of Salix viminalis. From the first day of cultivation plants were not treated or treated with combinations of 3 mM CuCl2, 7 mM CdCl2, 70 mM ZnCl2, 1 mM NAC, 1 mM OTC and 0.1 mM BSO. n ¼ 3 SE. yDenotes significant differences between no metal treatment and metal treatment when comparing bars of the same colour. Black or white dot in bars denotes significant difference from tolerance index 1.00. p < 0.05.
(Fig. 4) in the sensitive clone was increased by NIC, added at any time point, even though the GSH content (Fig. 5) was not influenced by NIC (light grey bars). Another observation was, that when also BSO was included, NIC was not able to protect against Cd toxicity (medium grey bars). This was valid both for shoot and root, and was most obvious in the sensitive clone, but the tendency could be seen in the tolerant clone as well. There was no difference in tolerance whether NIC was added before or after BSO, but the tolerance index was higher when Cd was added after the two other substances. 4. Discussion Oxidative stress is a well documented effect of metal toxicity in plants [28]. Cadmium, Cu and Zn ions cause oxidative stress and activate various antioxidative responses in Salix viminalis [21]. Our previous studies have shown that nicotinamide (NIC) and nicotinic acid (NiA) can protect plant cells from damage caused by oxidative stress (Berglund and Ohlsson, unpublished). Accordingly, one way to protect plants from detrimental oxidative effects of metal ions could be treatment with NIC or its metabolites, for example NiA.
Indeed, we show in the present investigation that both NIC and NiA did increase the metal tolerance in Salix. However, this protection does not seem to be performed simply via increased glutathione biosynthesis; we did not detect any effect on GSH content by NIC or NiA and, interestingly, OTC, NAC and BSO treatments did not influence metal tolerance despite effects on the glutathione level. There was one indication, though, that GSH may influence the protective action by NIC; the results from the combined NIC/BSO treatments point to some kind of connection between NIC, GSH and metal tolerance, which we cannot explain. The glutathione biosynthesis inhibitor BSO decreased the protective action of NIC against growth inhibition by Cd, while BSO alone did not show any considerable influence on metal tolerance. Phytochelatins (PCs), and hence glutathione metabolism, have been shown to be involved in metal tolerance in several plant species. Phytochelatin biosynthesis is dependent on GSH availability and in PC synthesizing plants increased levels of GSH promote PC biosynthesis [16]. For example, in Arabidopsis thaliana the two glutathione synthesis genes gsh1 and gsh2 and the phytochelatin synthase gene pcs1 were induced after Cd exposure [30]. It was noted that the
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
660
Shoot
Tolerant clone
*
* *
Shoot
*
30
40
Sensitive clone
* *
30
*
*
20
*
*
20
* *
*
*
*
10
10
0
–
–
–
BS
–
O
–
TC AC N O
d C
d C
–
d d C C
O TC AC BS O N
u C
u C
u u C C
O C C – BS OT NA
Zn
Zn
Zn O
– BS
Zn
–
TC AC O N
–
–
O
– BS
–
TC AC O N
d d C C
–
d d C C
O TC AC BS O N
u C
–
u u C C
u C
O TC AC BS O N
Zn
Zn
Zn O
– BS
TC AC O N
5
0
0
5
*
*
*
*
15
* Root
*
*
*
10
20
Zn
15
*
Tolerant clone
10
*
GSH, µg gFW-1
GSH, µg gFW-1
0
GSH, µg gFW-1
GSH, µg gFW-1
40
* * Root
Sensitive clone
* 20
Fig. 3. Effects of L-buthionine (S,R)-sulfoximine (BSO) and GSH stimulating substances N-acetyl-cystein (NAC) and L-2-oxothiazolidine 4-carboxylate (OTC) on level of glutathione (GSH) in roots and shoots of 21 day old sensitive and tolerant clones of Salix viminalis. From the first day of cultivation plants were not treated or treated with combinations of 3 mM CuCl2, 7 mM CdCl2, 70 mM ZnCl2, 1 mM NAC, 1 mM OTC and 0.1 mM BSO. n ¼ 3 SE. *Indicates significant differences between the white bar and the coloured bars within each bar group. yDenotes significant differences between no metal treatment and metal treatment when comparing bars of the same colour. Black or white dot in bars denotes significant difference from control (no treatment). p < 0.05.
level of glutathione did not increase. Any glutathione produced had obviously been used for the synthesis of PCs, which accumulated. However, phytochelatin biosynthesis is apparently not necessary for metal tolerance in Salix, as PC was not found in this system [22] either in tolerant or sensitive clones, before or after metal treatment. This is in line with our present results, showing that increased GSH level, caused by treatment with NAC and OTC, did not influence metal tolerance. Although an increase in GSH content above a control level does not influence metal tolerance in some plants, a decrease below this level may weaken the tolerance. For example, in suspension-cultured azuki bean cells, which were hypersensitive to Cd and did not synthesize PC, GSH treatment did not show any positive effect on Cd tolerance. Still, Cd-toxicity was dependent on the GSH-level, because BSO potentiated growth inhibition caused by Cd [18]. This is similar to our result where BSO reduced the protective action of NIC towards Cd (Fig. 4). As discussed by Ortega-Villasante et al. [26] and Pa´l et al. [27], the research concerning the relationship between phytochelatins and metal tolerance is not clearcut. It is assumed that PC, in addition to a metal-detoxification function, also has a more general role in cellular homeostasis. Overexpression
of PC synthase in Arabidopsis plants even led to hypersensitivity to Cd [23]. Even though the purpose of this investigation was not to clarify the mechanisms behind the metal tolerance-induced effect of NIC and NiA, it is tempting to speculate about reasons for this effect. It is known that exposure to metal ions may bring about an increased formation/presence of ROS [13,14], which in turn, due to increased formation of single strand breaks (SSB) in DNA, can activate PARP mediated consumption of NAD, with a concomitant release of NIC from NAD [2]. ROS/PARP-mediated NAD depletion is a well known phenomenon; especially in medical/toxicological science. Due to the plant NAD salvage pathway from NIC via NiA to NAD [17,24], added NIC or NiA can counteract NAD depletion. The importance of nicotinamidase, converting NIC to NiA in plants, for the NAD salvage pathway in Arabidopsis, has been shown by Wang and Pichersky [34]. It has been indicated that nicotinamidase expression can be increased by stress related treatment, e.g. abscisic acid [17]. Some physiological effects of NIC are shared by NiA, e.g. defence induction (Berglund and Ohlsson, unpublished). NIC added to plants appears to be readily metabolized to NiA, which in turn can be metabolized to trigonelline (N-methyl nicotinic acid) or further to NAD [24]. Accordingly, the effect
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
Tolerance index 1.25
1.00
0.75
0.50
Treatment 0.25
0
Day 1
Day 7
Day 14
Tolerance index 0
0.25
0.50
nb nb nb nb nb nb nb
0 –> NIC+BSO –> Cd+NIC+BSO 0 –> Cd+BSO –> Cd+NIC+BSO 0 –> Cd+NIC –> Cd+NIC+BSO BSO –> NIC+BSO –> Cd+NIC+BSO NIC –> NIC+BSO –> Cd+NIC+BSO Cd –> Cd+BSO –> Cd+NIC+BSO Cd –> Cd+NIC –> Cd+NIC+BSO BSO –> BSO –> BSO NIC –> NIC –> NIC Cd –> Cd –> Cd
Tolerant shoot
661
0.75
1.00
1.25
Sensitive shoot c c
nb
0 –> NIC+BSO –> NIC+BSO 0 –> BSO –> NIC+BSO 0 –> NIC –> NIC+BSO BSO –> BSO –> BSO NIC –> NIC –> NIC 0 –> Cd+BSO 0 –> BSO 0 –> Cd BSO –> BSO Cd –> Cd
b
–> Cd+BSO –> Cd+BSO –> Cd+BSO –> BSO –> Cd
b b c b n
0 –> Cd+NIC –> Cd+NIC 0 –> NIC –> Cd+NIC 0 –> Cd –> Cd+NIC NIC –> NIC –> NIC Cd –> Cd –> Cd
0 –> NIC+BSO 0 –> Cd+BSO 0 –> Cd+NIC BSO –> NIC+BSO NIC –> NIC+BSO Cd –> Cd+BSO Cd –> Cd+NIC BSO –> BSO NIC –> NIC Cd –> Cd
n n
cn cn
n nc c n
n cb
–> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> BSO –> NIC –> Cd
nb nb nc b nc b nb nb c
0 –> NIC+BSO –> NIC+BSO 0 –> BSO –> NIC+BSO 0 –> NIC –> NIC+BSO BSO –> BSO –> BSO NIC –> NIC –> NIC
n n
Tolerant root
0 –> Cd+BSO 0 –> BSO 0 –> Cd BSO –> BSO Cd –> Cd
1.00
0.75
0.50
0.25
0
Tolerance index
Sensitive root
b
–> Cd+BSO –> Cd+BSO –> Cd+BSO –> BSO –> Cd
b b
c b
0 –> Cd+NIC –> Cd+NIC 0 –> NIC –> Cd+NIC 0 –> Cd –> Cd+NIC NIC –> NIC –> NIC Cd –> Cd –> Cd 1.25
c
nb
nc nc nc c n 0
0.25
0.50
0.75
1.00
1.25
Tolerance index
Fig. 4. Effects of L-buthionine (S,R)-sulfoximine (BSO) and nicotinamide (NIC) on cadmium tolerance in tolerant and sensitive clones of Salix viminalis, where 7 mM CdCl2, 0.1 mM BSO and 2 mM NIC were added in different combinations and at different time points (day 1, 7 and 14) during the 21 days cultivation. Tolerance is given as tolerance index based on dry weight of roots or shoot. n ¼ 3 SE. n, c and b denotes significant differences compared with solely NIC, Cd and BSO treatment, respectively, within each bar group. Black dot in bars denotes significant difference from tolerance index 1.00. p < 0.05.
of added NIC could as well be an effect exerted by NiA or its metabolite trigonelline. However, trigonelline does not appear to show protection against oxidative stress in plant cells (Berglund and Ohlsson, unpublished). It could be interesting to use
a plant lacking nicotinamidase, like plants homozygous for the null AtNIC1 allele [34], in studies on NIC induced defence and the importance of NIC metabolization to NiA for this process.
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
662
GSH, µg gFW-1 30
25
20
15
10
5
0
cn cn cn cn cn cn bcn cn b b n n bn n b c
Tolerant shoot
c c c
b
Day 1
Day 7
cn cn cn cn cn b cn n
5
10
n n n b c
0 –> NIC+BSO 0 –> BSO 0 –> NIC BSO –> BSO NIC –> NIC
–> NIC+BSO –> NIC+BSO –> NIC+BSO –> BSO –> NIC
nb n
0 –> Cd+BSO 0 –> BSO 0 –> Cd BSO –> BSO Cd –> Cd
–> Cd+BSO –> Cd+BSO –> Cd+BSO –> BSO –> Cd
cb c
0 –> Cd+BSO 0 –> BSO 0 –> Cd BSO –> BSO Cd –> Cd
c c c
b
15
–> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> BSO –> NIC –> Cd
20
25
30
n cb n cb nc n cb nc nc n cb nc
b b
n n b Sensitiv
e
shoot
c
c b
–> Cd+NIC –> Cd+NIC –> Cd+NIC –> NIC –> Cd –> 0
ncb nc nc n cb
nc nc nc nc b b
0 –> NIC+BSO –> NIC+BSO 0 –> BSO –> NIC+BSO 0 –> NIC –> NIC+BSO BSO –> BSO –> BSO NIC –> NIC –> NIC
n
nb n n n b cb c
–> Cd+BSO –> Cd+BSO –> Cd+BSO –> BSO –> Cd
c
Sensitive root
c b
0 –> Cd+NIC –> Cd+NIC 0 –> NIC –> Cd+NIC 0 –> Cd –> Cd+NIC NIC –> NIC –> NIC Cd –> Cd –> Cd 0 –> 0 –> 0
c n
15
0
0 –> NIC+BSO –> Cd+NIC+BSO 0 –> Cd+BSO –> Cd+NIC+BSO 0 –> Cd+NIC –> Cd+NIC+BSO BSO –> NIC+BSO –> Cd+NIC+BSO NIC –> NIC+BSO –> Cd+NIC+BSO Cd –> Cd+BSO –> Cd+NIC+BSO Cd –> Cd+NIC –> Cd+NIC+BSO BSO –> BSO –> BSO NIC –> NIC –> NIC Cd –> Cd –> Cd
bcn
Tolerant root
Day 14
0 –> NIC+BSO 0 –> Cd+BSO 0 –> Cd+NIC BSO –> NIC+BSO NIC –> NIC+BSO Cd –> Cd+BSO Cd –> Cd+NIC BSO –> BSO NIC –> NIC Cd –> Cd
0 –> Cd+NIC 0 –> NIC 0 –> Cd NIC –> NIC Cd –> Cd 0 –> 0
bc bn
GSH, µg gFW-1
Treatment
10
5
GSH, µg gFW-1
0
0
5
10
15
GSH, µg gFW-1
Fig. 5. Effects of L-buthionine (S,R)-sulfoximine (BSO) and nicotinamide (NIC) on level of glutathion (GSH) in 21 days old roots and shoots of tolerant and sensitive clones of Salix viminalis, where 7 mM CdCl2, 0.1 mM BSO and 2 mM NIC were added in different combinations and at different time points (day 1, 7 and 14) during the 21 days cultivation. n ¼ 3 SE. n, c and b denotes significant differences compared with solely NIC, Cd and BSO treatment, respectively, within each bar group. Black dot in bars denotes significant difference from control (no treatment). p < 0.05.
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
NIC is also known to be a strong PARP inhibitor, while NiA is not [29]. Nevertheless, both have a metal tolerancestimulating effect and this discrepancy indicates that PARP inhibition may not be the basis for the metal tolerance enhancing effect of NIC and NiA. DNA methylation is another possible target for NIC and NiA. Various kinds of abiotic stress can cause changes in the level of global DNA methylation and DNA methylation at specific sites in the plant genome; e.g. osmotic stress, water deficiency, low temperature or paraquat treatment [9]. It has also been shown that a decrease in DNA methylation is often associated with an increased expression of stress related genes [33]. Most types of environmental stress are accompanied by oxidative stress. We have shown that oxidative stress in plants can induce a decrease in DNA methylation [25]. This is interesting with respect to possible effects of metal ions in plants. It is well known that metal ions can cause oxidative stress in plants and furthermore, it has been shown that Cd can reduce the level of DNA methylation in sensitive as well as tolerant plant species [1]. Taken together this points at the possibility that at least one effect of Cd (and probably other metal ions) in plants could be a decrease in the level of DNA methylation and maybe this effect of Cd is mediated via an increased level of tissue reactive oxygen species. It is hard to say whether this decrease in DNA methylation is a toxic or defensive effect in response to Cd. If we hypothesise that it is an induced defensive effect, this can constitute a connection to NIC or NiA, because we have shown that these compounds can cause a decrease in DNA methylation as well as an increase in plant defensive metabolism [25, Berglund and Ohlsson, unpublished]. Thus, we speculate that one way for the natural compounds NIC and NiA to protect plants against metal ions could be by a defence that is induced via a decrease in DNA methylation. We have earlier shown that various clones of Salix viminalis accumulate different high levels of metals and that there is no relation between tolerance to and accumulation of a certain metal [20]. It was therefore expected, and also shown here, that NIC or NiA treatment did not in general influence the metal levels in tolerant or sensitive clones (Table 2) even though these treatments counteracted the growth inhibition caused by the respective metal (Fig. 1). Obviously, decreased metal uptake is not an explanation to the protective action of NIC and NiA (Table 2). On the contrary, these metabolites may increase the metal uptake per plant by improving growth in metal exposed sensitive clones. It seems like NIC and NiA influence tolerance against Cd and Cu as well as Zn via a mechanism which is probably not tied to any of the metal ions in particular. 4.1. Summary The goal of the present study was to investigate whether NIC and NiA could influence metal tolerance in Salix and if so, whether this effect was dependent on increased levels of GSH. We conclude that NIC and NiA can improve metal tolerance in sensitive clones of Salix and that this tolerance is not dependent on increased GSH levels per se. The metal uptake
663
is not influenced by NIC or NiA treatment, but the metal content per plant is increased, due to increased growth. We conclude that the increased metal tolerance is mediated by some other mechanism than via increased glutathione content. This is, to our knowledge, the first report of a single treatment, which gives increased metal tolerance in plants. Even though the mechanisms are not clarified yet, the results may hopefully contribute to the understanding of metal tolerance mechanisms. Acknowledgements This work was supported by grants from Carl Tryggers Stiftelse and Magn. Bergvalls Stiftelse. References [1] R. Aina, S. Sgorbati, A. Santagostino, M. Labra, A. Ghiani, S. Citterio, Specific hypomethylation of DNA is induced by heavy metals in white clover and industrial hemp, Physiol. Plant 121 (2004) 472e480. [2] Y. Amor, E. Babiycchuk, D. Inze´, A. Levine, The involvement of poly(ADP-ribose) polymerase in the oxidative stress response in plants, FEBS Lett. 440 (1998) 1e7. [3] R.M. Anderson, K.J. Bitterman, J.G. Wood, O. Medvedik, D.A. Sinclair, Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae, Nature 423 (2003) 181e185. [4] M. Arakawa, N. Ushimaru, N. Osada, T. Oda, K. Ishige, Y. Ito, N-acetylcysteine selectively protects cerebellar granule cells from 4-hydroxynonenal-induced cell death, Neurosci. Res. 55 (2006) 255e263. [5] T. Berglund, Nicotinamide, a missing link in the early stress response in eukaryotic cells: a hypothesis with special reference to oxidative stress in plants, FEBS Lett. 351 (1994) 145e149. [6] T. Berglund, A.B. Ohlsson, Defensive and secondary metabolism in plant tissue cultures, with special reference to nicotinamide, glutathione and oxidative stress, Plant Cell Tissue Organ Cult. 43 (1995) 137e145. [7] T. Berglund, A.B. Ohlsson, J. Rydstro¨m, Nicotinamide increases glutathione and anthocyanin in tissue culture of Catharanthus roseus, J. Plant Physiol. 141 (1993) 596e600. ˚ . Strid, Effect of [8] T. Berglund, A.B. Ohlsson, J. Rydstro¨m, B.R. Jordan, A nicotinamide on gene expression and glutathione levels in tissue cultures of Pisum sativum, J. Plant Physiol. 142 (1993) 676e684. [9] C.-S. Choi, H. Sano, Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants, Mol. Genet. Genomics 277 (2007) 589e600. [10] S. Clemens, Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants, Biochimie 88 (2006) 1707e1719. [11] P. Coyle, J.C. Philcox, L.C. Carey, A.M. Rofe, Metallothionein: the multipurpose protein, Cell. Mol. Life Sci. 59 (2002) 627e647. [12] M. De Block, C. Verduyn, D. De Brouwer, M. Cornelissen, Poly(ADPribose) polymerase in plants affects energy homeostasis, cell death and stress tolerance, Plant J. 41 (2005) 95e106. [13] K. Durcekova´, J. Huttova´, I. Mistrı´k, M. Olle´, L. Tama´s, Cadmium induces premature xylogenesis in barley roots, Plant Soil 290 (2007) 61e68. [14] P.L. Gratao, A. Polle, P.J. Lea, R.A. Azevedo, Making the life of heavy metal-stressed plants a little easier, Functional Plant Biol. 32 (2005) 481e494. [15] M. Greger, T. Landberg, Use of willow in phytoextraction, Int. J. Phytorem. 1 (1999) 115e123. [16] K. Hirata, N. Tsuji, K. Miyamoto, Biosynthetic regulation of phytochelatins, heavy metal-binding peptides, J. Biosci. Bioeng. 100 (2005) 593e599. [17] L. Hunt, F. Lerner, M. Ziegler, NAD e new roles in signalling and gene regulation in plants, New Phytol. 163 (2004) 31e44. [18] M. Inouhe, R. Ito, S. Ito, N. Sasada, H. Tohoyama, M. Joho, Azuki bean cells are hypersensitive to cadmium and do not synthesize phytochelatins, Plant Physiol. 123 (2000) 1029e1036.
664
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
[19] D.W. Lamming, J.G. Wood, D.A. Sinclair, Small molecules that regulate lifespan: evidence for xenohormesis, Mol. Microbiol. 53 (2004) 1003e 1009. [20] T. Landberg, M. Greger, Can heavy metal tolerant clones of Salix be used as vegetation filters on heavy contaminated land?, in: Aronsson P., Perttu K., (Eds.), Willow Vegetation Filters for Municipal Wastewaters and Sludges. A Biological Purification System. Proc. of a Study Tour, Conference and Work Shop in Sweden, 5e10 June 1994. Ultuna, Uppsala, Rapport 50, Avd. f. Skoglig intensivodling, SLU. ISBN 91-576-4916-2, 1994, pp. 133e144. [21] T. Landberg, M. Greger, Differences in oxidative stress in heavy metal resistant and sensitive clones of Salix viminalis, J. Plant Phys. 159 (2002) 69e75. [22] T. Landberg, M. Greger, No phytochelatin (PC2 and PC3) detected in Salix viminalis, Phys. Plant 121 (2004) 481e487. [23] S. Lee, J.S. Moon, T.-S. Ko, D. Petros, P.B. Goldsbrough, S.S. Korban, Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress, Plant Physiol. 131 (2003) 656e663. [24] A. Matsui, Y. Yin, K. Yamanaka, M. Iwasaki, H. Ashihara, Metabolic fate of nicotinamide in higher plants, Physiol. Plant 131 (2007) 191e200. [25] A.B. Ohlsson, T. Kraska, J. Rydstro¨m, T. Berglund, Influence of nicotinamide and its metabolites on plant defense-related metabolism; facts and fiction, XVIII Congress of The Scandinavian Society for Plant Physiology (SPPS), Uppsala, Sweden, 12e17 June (1997) Poster abstract P57. [26] C. Ortega-Villasante, R. Rella´n-Alvarez, F.F. Del Campo, R.O. CarpenaRuiz, L.E. Herna´ndez, Cellular damage induced by cadmium and mercury in Medicago sativa, J. Exp. Bot. 56 (2005) 2239e2251. [27] M. Pa´l, E. Horva´th, T. Janda, E. Pa´ldi, G. Szalai, Physiological changes and defense mechanisms induced by cadmium stress in maize, J. Plant Nutr. Soil Sci. 169 (2006) 239e246. [28] A. Polle, A. Schu¨tzendu¨bel, Heavy metal signalling in plants: linking cellular and organismic responses, in: H. Hirt, K. Shinozaki (Eds.),
[29] [30]
[31]
[32]
[33]
[34] [35] [36]
[37]
[38]
Topics in Current Genetics, Plant Responses to Abiotic Stress, vol. 4, Springer-Verlag, Berlin Heidelberg, 2003, pp. 187e215. M.R. Purnell, W.J.D. Whish, Novel inhibitors of poly(ADP-ribose) synthetase, Biochem. J. 185 (1980) 775e777. B. Semane, A. Cuypers, K. Smeets, F. Van Belleghem, N. Horemans, H. Schat, J. Vangronsveld, Cadmium responses in Arabidopsis thaliana: glutathione metabolism and antioxidative defence system, Physiol. Plant 129 (2007) 519e528. A. Tukendorf, W.E. Rauser, Changes in glutathione and phytochelatins in roots of maize seedlings exposed to cadmium, Plant Sci. 70 (1990) 155e166. J.A.C. Verkleij, F.E.C. Sneller, H. Schat, Metallothioneins and phytochelatins: ecophysiological aspects, in: Y.P. Abrol, A. Ahmad (Eds.), Sulphur in Plants, Kluwer Academic Publishers, The Netherlands, 2003, pp. 163e176. Y. Wada, K. Miyamoto, T. Kusano, H. Sano, Association between up-regulation of stress-responsive genes and hypomethylation of genomic DNA in tobacco plants, Mol. Gen. Genomics 271 (2004) 658e666. G. Wang, E. Pichersky, Nicotinamidase participates in the salvage pathway of NAD biosynthesis in Arabidopsis, Plant J. 49 (2007) 1020e1029. D.A. Wilkins, The measurement of tolerance to edaphic factors by means of root growth, New Phytol. 80 (1978) 623e633. J.M. Williamson, B. Boettcher, A. Meister, Intracellular cysteine delivery system that protects against toxicity by promoting glutathione synthesis, Proc. Natl. Acad. Sci. USA 79 (1982) 6246e6249. B. Zechmann, M. Mu¨ller, G. Zellnig, Intracellular adaptations of glutathione content in Cucurbita pepo L. induced by treatment with reduced glutathione and buthionine sulfoximine, Protoplasma 227 (2006) 197e209. H. Zhang, W. Xu, J. Guo, Z. He, M. Ma, Coordinated responses of phytochelatins and metallothioneins to heavy metals in garlic seedlings, Plant Sci. 169 (2005) 1059e1065.
Plant Physiology and Biochemistry 46 (2008) 655e664 www.elsevier.com/locate/plaphy
Research article
Increased metal tolerance in Salix by nicotinamide and nicotinic acid Anna B. Ohlsson a,*, Tommy Landberg b, Torkel Berglund a, Maria Greger b a
Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, SE-106 91 Stockholm, Sweden b Department of Botany, Stockholm University, SE-106 91 Stockholm, Sweden Received 11 October 2007 Available online 22 April 2008
Abstract We have earlier shown that nicotinamide (NIC) and nicotinic acid (NiA) can induce defence-related metabolism in plant cells; e.g. increase the level of glutathione. Here we investigated if NIC and NiA could increase the metal tolerance in metal sensitive clones of Salix viminalis and whether this would be mediated via increased glutathione level. Salix clones, sensitive or tolerant to zinc (Zn), copper (Cu) and cadmium (Cd) were grown in the presence of heavy metals (Cd, Cu or Zn) or NIC and NiA as well as in combination. In addition, the influence of Nacetyl-cystein (NAC) and L-2-oxothiazolidine 4-carboxylate (OTC), stimulators of reduced glutathione (GSH) biosynthesis, and the glutathione biosynthesis inhibitor buthionine sulfoximine (BSO) was analysed. Tolerance was measured as effects on root and shoot dry weight, and the glutathione and metal concentrations in the tissues were analysed. Results showed that NIC and NiA decreased the toxic effects of Cd, Cu and Zn on growth significantly in sensitive clones, but also to some extent in tolerant clones. However, the glutathione level and metal concentration did not change by NIC or NiA addition. Treatment with NAC, OTC or BSO did not per se influence the sensitivity to Cd, although the glutathione level increased in the presence of NAC and OTC and decreased in response to BSO. The results suggest that NIC and NiA increased the defence against heavy metals but not via glutathione formation per se. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Glutathione; Heavy metal defence; Nicotinamide; Nicotinic acid; Salix viminalis; Toxicity
1. Introduction Heavy metals in the environment may influence the growth of plants and cause toxic effects if the plants are sensitive [10]. Salix viminalis and other Salix spp. have been shown to evolve clones with huge variation in tolerance to and accumulation of heavy metals [15,20]. It has also been shown that these properties may vary in the same clone for different metals [20]. Salix viminalis is therefore a good candidate for investigations on mechanisms behind metal tolerance in plants. One important effect of heavy metals in biological systems is the induction of oxidative stress, which occurs when the
Abbreviations: BSO, L-buthionine (S,R)-sulfoximine; GSH, glutathione; NAC, N-acetyl-cystein; NIC, nicotinamide; NiA, nicotinic acid; OTC, L-2oxothiazolidine 4-carboxylate; PARP, poly(ADP-ribose)polymerase; PC, phytochelatin; ROS, reactive oxygen species. * Corresponding author. Fax þ46 8 5537 8468. E-mail address: [email protected] (A.B. Ohlsson). 0981-9428/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.04.004
formation of reactive oxygen species (ROS) is too high in relation to the protective antioxidants [13,14]. Glutathione (GSH) is one of the most important non-protein molecules protecting the cell from oxidative damage by neutralizing ROS [14]. There are many potential mechanisms behind metal tolerance in plants [32]. The biological variation in types of defence mechanisms is probably an advantage from an adaptive evolutionary point of view. The ability to synthesise GSH is one important factor for protection against heavy metal toxicity [28]. One suggested mechanism is biosynthesis of metal binding peptides, phytochelatins (PC), at the expense of GSH [16]. This requires the activity of PC synthase genes and the presence of GSH in sufficient amounts. Although present in most other plants studied, we showed recently that no phytochelatin (PC2 or PC3) could be found in Salix before or after metal exposure [22]. Accordingly, PC does not appear to be a main reason for metal tolerance in this genus. Metallothionein (MT) is a cystein-rich protein which can be induced by metal exposure both in animals and plants [11,38]. However, neither
656
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
PC nor MT biosynthesis can completely explain the mechanisms of metal tolerance in plants. Nicotinamide (NIC) has been hypothesised as a signalling link in the response to oxidative stress in eukaryotic cells [5]. The antioxidative defence is tightly associated with GSH metabolism. We have shown earlier that NIC [7,8] and its natural metabolite nicotinic acid (NiA) (unpublished) can induce several defence responses in plants and plant cell cultures of different species, for example a pronounced increase in GSH content and protection against oxidative stress-induced damage. Nicotinamide may protect cells via different types of mechanisms: poly(ADP-ribose)polymerase (PARP) inhibition, which slows down NAD decomposition and thus improves energy metabolism [12]; as a precursor for salvage synthesis of NAD [17]; counteracting histone deacetylation [3,19]; activation of defence genes and defence related metabolism [6]. A considerable interest has recently focused on nicotinamide in medical as well as plant science, since it was shown that it is involved in the regulation of cellular longevity [3,17,19]. The aim of the present investigation was to find out whether NIC and NiA could increase the resistance to toxic effects on plant growth caused by metal exposure, and if so, whether this effect could be related to glutathione content or synthesis. 2. Materials and methods 2.1. Plant material and cultivation Two clones of Salix viminalis, one sensitive and one tolerant to Zn, Cu and Cd were cultivated from cuttings in nutrient solution. Cuttings from 1-year-old shoots of the two clones, 80 mm in length, were rooted and cultivated. The cuttings were mounted six by six in styrofoam plates and each plate was placed in a pot with 900 ml 100 mM Ca(NO3)2 solution [35]. Water loss was compensated when 1% of the water had evaporated. The pH decreased from 6.5 to 6.0 during the cultivation and was not compensated. Cuttings were grown in a controlled climate chamber equipped with metal halogen lamps (Osram, Powerstar HQI-R) providing a photon flux density of 200 mmol m2 s1 for 16 h at 25 C. During the 8 h dark period the temperature was 21 C. The relative humidity was 70e80%. 2.2. Experimental design 2.2.1. Effect of NIC and NiA on metal tolerance The effect of nicotinamide (NIC) and nicotinic acid (NiA) on heavy metal tolerance and accumulation was studied. From the first day of cultivation plants were treated with either 3 mM CuCl2, 7 mM CdCl2, 70 mM ZnCl2, 2 mM NIC, 2 mM NiA in the following combinations: Control (no addition), NIC, NiA, Cd, Cu, Zn, Cd þ NIC, Cu þ NIC, Zn þ NIC, Cd þ NiA, Cu þ NiA or Zn þ NiA. 2.2.2. Effect of BSO, OTC and NAC on metal tolerance and GSH levels The effect of L-buthionine (S,R)-sulfoximine (BSO), L-2oxothiazolidine 4-carboxylate (OTC) and N-acetyl-cysteine
(NAC) on heavy metal tolerance and level of glutathione (GSH) in roots and shoots was studied. OTC and NAC stimulate the production of GSH, while BSO decreases the formation of GSH. From the first day of cultivation plants were treated with either 7 mM CdCl2, 3 mM CuCl2, 70 mM ZnCl2, 0.1 mM BSO, 1 mM OTC and 1 mM NAC in the following combinations; Control (no addition), BSO, OTC, NAC, Cd, Cu, Zn, Cd þ BSO, Cd þ OTC, Cd þ NAC, Cu þ BSO, Cu þ OTC, Cu þ NAC, Zn þ BSO, Zn þ OTC, Zn þ NAC. 2.2.3. Time study of BSO and NIC additions This experiment was performed to analyse the effect on cadmium tolerance by adding BSO and NIC at different time points (day 1, 7 and 14) during the cultivation. Plants were treated with 7 mM CdCl2, 0.1 mM BSO and 2 mM NIC in different order and at different points of time during the cultivation according to Table 1. 2.3. Plant harvest and determination of toxic effects on growth After 21 days treatment plants were harvested and divided in root and shoot, which were dried in 105 C for 48 h. In order to measure the toxic effect on growth the fresh and dry weight were measured as well as the length of root and shoot. 2.4. Analysis of reduced glutathione Reduced glutathione was analysed using HPLC (high performance liquid chromatography) and Ellmans reagent. Fresh plant materials (0.1e 0.5 g) were ultramixed (Polytron, PT Table 1 Treatment protocol in the time study of BSO and NIC additions. Plants were treated 21 days without (e) or with 7 mM CdCl2 (Cd), 2 mM nicotinamide (NIC) and/or 0.1 mM buthionine sulfoximine (BSO) added at various time points separate or in combination. The cultivation solution was changed at day 7 and day 14 Day 1
Day 7
Day 14
e Cd NIC BSO e e e e e e Cd Cd NIC BSO e e e e e e
e Cd NIC BSO Cd Cd NIC NIC BSO BSO Cd þ NIC Cd þ BSO NIC þ BSO BSO þ NIC Cd þ NIC Cd þ BSO NIC þ BSO Cd þ NIC Cd þ BSO NIC þ BSO
e Cd NIC BSO Cd þ NIC Cd þ BSO NIC þ Cd NIC þ BSO BSO þ Cd BSO þ NIC Cd þ NIC þ BSO Cd þ BSO þ NIC NIC þ BSO þ Cd BSO þ NIC þ Cd Cd þ NIC Cd þ BSO NIC þ BSO Cd þ NIC þ BSO Cd þ BSO þ NIC NIC þ BSO þ Cd
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
2000) in 1.5 ml HCl (0.01 M). The samples were then centrifuged at 16,000 g for 5 min at 4 C. The supernatant was filtered with a 0.45 mm Millipore filter and immediately injected (manually) into the HPLC. As internal standards 40 nM GSH (reduced glutathione, EC-200-725-4) and 40 nM cysteine (EC200-158-2) were used. The HPLC (Beckman) was equipped with an A C-18 column (ODS Hypersil 5 mm, Hewlett-Packard, Palo Alto, CA, USA) 250 4 mm. The flow rate was 1.0 ml min1. The analyses procedure was performed according to Tukendorf and Rauser [31]. The column was equilibrated with 0.1% trifluoricacetic acid (TFA) in water. The sample was injected (50 mL) and the column was developed with a linear gradient from 0 to 60% (v/v) acetonitrile with 0.1% TFA for 30 min. Post column the effluent was mixed with 1.8 mM 5,50 dithiobis(2-nitrobenzoic acid) (DTNB, i.e. Ellman’s reagent) in a mixing chamber (Micro mixer, Beckman Coulter, Inc., Miami, FL, USA). The DTNB was delivered at a rate of 0.5 ml min1 by a Beckman 110B Solvent Delivery Module. The reaction was then allowed in a 2.4 m 0.5 mm Teflon tube at 37 C. The DTNB was daily prepared with 14 mg DTNB in 200 ml cold (4 C) 300 mM potassium buffer (pH 7.8) and 15 mM K-EDTA. The solution was kept dark and on ice during the analysis. The absorbance was measured at 405 nm (with the Beckman 166 detector, Beckman Coulter, Inc., Miami, FL, USA). 2.5. Analysis of metals The dried material was wet digested (HNO3:HClO4, 7:3) and analysed on the contents of Cd, Cu and Zn by atomic absorption spectrometry with a flame atomiser (AA-1275, Varian, Springvale, Australia) using standard addition. 2.6. Calculations and statistics All experiments were performed in three replicates where each replicate consisted of six pooled plants. The tolerance index was calculated 1according to Eq. (1) Tolerance index ¼
Dry wieghttreated plant Dry wieghtcontrol
ð1Þ
where either shoots or roots are considered. Statistical treatments were performed by using Tukeye Kramers test and ANOVA at p < 0.05. 2.7. Chemicals L-buthionine (S,R)-sulfoximine (BSO), N-acetyl-cystein (NAC), L-2-oxothiazolidine 4-carboxylate (OTC), nicotinic acid (NiA) and nicotinamide (NIC) were purchased from Sigma-Aldrich Chemical Co. USA.
3. Results Effects on metal tolerance in two Salix viminalis clones were investigated; one clone sensitive to Cd, Cu and Zn and
657
another clone tolerant to the same metals. The clones were exposed to the three metals as well as to nicotinamide (NIC) and nicotinic acid (NiA) in the combinations presented in Fig. 1. All additions were made at the first day of cultivation. Significant difference in tolerance when compared to untreated plants (tolerance index 1.00) is marked with a dot in the bar. The growth of the tolerant clone was only in two cases marginally affected by the metal; Zn in shoot and Cu in root. Addition of only NIC showed a slight inhibitory effect on the growth of shoots in the tolerant clone. However, nearly all treatments affected the growth of the sensitive clone; the decrease in tolerance index caused by Cd, Cu or Zn was in most cases reduced by addition of NIC or NiA, the latter being most effective (significance illustrated by *). For example, in some cases a tolerance index of approximately 0.25 was increased to higher than 0.5 by NiA in this clone. Nicotinamide had no protecting effect towards Cu. The metal content was analyzed in the plants treated with Cd, Cu, Zn, NIC and NiA, solely or in combinations (Table 2). The contents of Cd and Cu were higher in roots than in shoots. On the contrary, the Zn content was about the same in roots and shoots of the tolerant clone and lower in roots when compared with shoots in the sensitive clone. The control levels of Cd and Cu were principally about the same in the tolerant and the sensitive clone of shoot and roots, respectively. The Zn content, on the other hand, was lower in the roots of the sensitive clone, when compared to the tolerant clone. Addition of each metal increased the concentration of that metal, on a percentage basis to the same extent in both clones. No influence of NIC or NiA on the metal levels was detected. To investigate if glutathione could play a role in the tolerance caused by NIC, the tolerant and sensitive clones were exposed to L-2-oxothiazolidine 4-carboxylate (OTC), N-acetylcysteine (NAC), or L-buthionine (S,R)-sulfoximine (BSO), alone and in combination with each metal. OTC is by the enzyme 5-oxo-prolinase converted to S-carboxy-L-cysteine, which via L-cysteine is used for GSH synthesis [36]. NAC is also a cysteine precursor that increases the intracellular GSH level [4]. BSO inhibits the activity of g-glutamylcysteine synthetase, leading to reduced glutathione levels [37]. In general, the addition of the glutathione biosynthesis modifying compounds BSO, OTC or NAC had no effect on tolerance, neither when added alone or in combination with metals (Fig. 2). In the same experiment, the level of glutathione (Fig. 3) increased, with varying significance, by the addition of OTC or NAC, which are known to stimulate glutathione biosynthesis by providing cysteine for GSH biosynthesis. The glutathione level decreased significantly by the addition of the glutathione biosynthesis inhibitor BSO, irrespective of the presence (*) or absence (black dots) of metal. In order to further investigate a possible role of glutathione in NIC-induced metal tolerance, Cd, NIC and BSO were added at different points of time, and tolerance index as well as GSH content were analysed (Figs. 4 and 5). In neither of the clones, Cd nor NIC did change the GSH content, while BSO, independent of addition time, decreased the GSH level in all tissues (black dots). The experiment showed that the Cd tolerance
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
658 1.25
Shoot
Shoot
Tolerant clone
1.25
Sensitive clone
†
Tolerance index
1.00
0.75
†
*
0.50
†
0.75
*
† †
*
*
0.50
0.25
0.25 0 Zn
Zn
Zn
N IC N iA
C u C u C u N IC N iA
C d C d C d N IC N iA
-
-
IC N iA
N
Zn
Zn
Zn
N IC N iA
C u C u C u N IC N iA
N IC N iA
N IC N iA
-
C d C d C d
0
0 0.25
0
0.25 †
0.50
†
*
†
0.75
*
†
0.50 †
*
1.00
0.75
Tolerance index
Tolerance index
Tolerance index
* *
1.00
1.00 Root
Tolerant clone
Root
Sensitive clone
1.25
Fig. 1. Effects of nicotinamide (NIC) and nicotinic acid (NiA) on heavy metal tolerance in one sensitive and one tolerant clone of Salix viminalis. Tolerance is given as tolerance index based on dry weight of roots or shoot. Plants were treated during 21 days and from the first day of cultivation with combinations of 3 mM CuCl2, 7 mM CdCl2, 70 mM ZnCl2, 2 mM NIC and/or 2 mM NiA. n ¼ 3 SE. *Indicate significant differences between treatment with metal and metal þ NIC or NIA. y Denotes significant differences between NIC and NIC þ metal or NiA and NiA þ metal. Black or white dot in bars denotes significant difference from tolerance index 1.00. p < 0.05.
Table 2 Metal content in treated plants. Heavy metal accumulation in roots and shoots of one tolerant and one sensitive clone of Salix viminalis untreated or treated with 3 mM CuCl2, 7 mM CdCl2, 70 mM ZnCl2, 2 mM nicotinamide (NIC) and 2 mM nicotinic acid (NiA) solely or in combinations. n ¼ 3 SE. Different letters show significant difference p < 0.05 between clones with same treatment Analysed metal treatment
Tolerant clone Root
Cd, mg gDW1 e e e Cd Cd Cd
e NIC NiA e NIC NiA
0.025 0.003 0.027 0.003 0.023 0.002 11.930 1.440 12.650 1.081 12.380 1.053
Cu, mg gDW1 e e e Cu Cu Cu
e NIC NiA e NIC NiA
0.686 0.026 0.696 0.023 0.687 0.004 a 13.890 0.790 12.780 1.119 13.460 0.675
Zn, mg gDW1 e e e Zn Zn Zn
e NIC NiA e NIC NiA
11.67 0.233 a 12.07 0.939 a 11.97 0.376 a 620.30 23.511 a 621.70 11.865 a 628.70 6.489 a
Sensitive clone Shoot
Root
Shoot
0.013 0.001 0.011 0.001 0.016 0.001 1.095 0.048 0.981 0.088 0.978 0.048
0.024 0.002 0.021 0.003 0.023 0.003 10.620 0.648 9.412 0.157 10.357 0.413
0.013 0.001 0.014 0.001 0.014 0.001 1.148 0.084 1.015 0.036 1.096 0.013
0.259 0.019 0.267 0.009 a 0.255 0.020 0.974 0.003 0.917 0.081 1.011 0.051
0.735 0.030 0.774 0.035 0.812 0.018 b 11.760 0.197 11.960 0.439 11.080 0.449
0.222 0.016 0.183 0.011 b 0.196 0.025 0.935 0.071 0.987 0.064 0.913 0.021
8.73 0.145 b 9.40 0.321 b 8.80 0.493 b 468.70 17.340 b 425.30 12.170 b 427.70 1.450 b
12.0 0.503 13.1 0.602 13.2 0.145 720.0 19.280 725.3 41.010 716.7 29.670
12.83 0.524 12.93 0.913 13.20 0.945 656.30 12.601 652.70 3.480 666.00 20.033
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
659
1.25
1.25 Sensitive clone
Tolerant clone
Shoot
1.00
1.00
0.75
0.75 †
0.50
†
†
†
†
†
0.50 †
†
†
0.25
0.25
0 Zn
Zn N AC
Zn
O O TC
Zn –
BS
C u C u C u C u –
BS O O TC N AC
C d C d N AC
O
TC O
C d C d –
BS
–
– –
BS O O TC N AC
–
Zn N AC
Zn
Zn
O O TC
Zn –
BS
C u C u C u C u –
BS O O TC N AC
C d C d N AC
O
TC O
C d C d –
BS
–
–
BS O O TC N AC
–
0
0
0
0.25
† †
†
† †
0.50
†
0.75
†
†
†
†
0.50
0.75
†
1.00
Tolerance index
Tolerance index
0.25
1.25
Tolerance index
Tolerance index
Shoot
1.00 Root
Tolerant clone
Sensitive clone
Root
1.25
Fig. 2. Effects of L-buthionine (S,R)-sulfoximine (BSO) and GSH stimulating substances N-acetyl-cystein (NAC) and L-2-oxothiazolidine 4-carboxylate (OTC) on heavy metal tolerance in roots and shoots of 21 day old sensitive and tolerant clones of Salix viminalis. From the first day of cultivation plants were not treated or treated with combinations of 3 mM CuCl2, 7 mM CdCl2, 70 mM ZnCl2, 1 mM NAC, 1 mM OTC and 0.1 mM BSO. n ¼ 3 SE. yDenotes significant differences between no metal treatment and metal treatment when comparing bars of the same colour. Black or white dot in bars denotes significant difference from tolerance index 1.00. p < 0.05.
(Fig. 4) in the sensitive clone was increased by NIC, added at any time point, even though the GSH content (Fig. 5) was not influenced by NIC (light grey bars). Another observation was, that when also BSO was included, NIC was not able to protect against Cd toxicity (medium grey bars). This was valid both for shoot and root, and was most obvious in the sensitive clone, but the tendency could be seen in the tolerant clone as well. There was no difference in tolerance whether NIC was added before or after BSO, but the tolerance index was higher when Cd was added after the two other substances. 4. Discussion Oxidative stress is a well documented effect of metal toxicity in plants [28]. Cadmium, Cu and Zn ions cause oxidative stress and activate various antioxidative responses in Salix viminalis [21]. Our previous studies have shown that nicotinamide (NIC) and nicotinic acid (NiA) can protect plant cells from damage caused by oxidative stress (Berglund and Ohlsson, unpublished). Accordingly, one way to protect plants from detrimental oxidative effects of metal ions could be treatment with NIC or its metabolites, for example NiA.
Indeed, we show in the present investigation that both NIC and NiA did increase the metal tolerance in Salix. However, this protection does not seem to be performed simply via increased glutathione biosynthesis; we did not detect any effect on GSH content by NIC or NiA and, interestingly, OTC, NAC and BSO treatments did not influence metal tolerance despite effects on the glutathione level. There was one indication, though, that GSH may influence the protective action by NIC; the results from the combined NIC/BSO treatments point to some kind of connection between NIC, GSH and metal tolerance, which we cannot explain. The glutathione biosynthesis inhibitor BSO decreased the protective action of NIC against growth inhibition by Cd, while BSO alone did not show any considerable influence on metal tolerance. Phytochelatins (PCs), and hence glutathione metabolism, have been shown to be involved in metal tolerance in several plant species. Phytochelatin biosynthesis is dependent on GSH availability and in PC synthesizing plants increased levels of GSH promote PC biosynthesis [16]. For example, in Arabidopsis thaliana the two glutathione synthesis genes gsh1 and gsh2 and the phytochelatin synthase gene pcs1 were induced after Cd exposure [30]. It was noted that the
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
660
Shoot
Tolerant clone
*
* *
Shoot
*
30
40
Sensitive clone
* *
30
*
*
20
*
*
20
* *
*
*
*
10
10
0
–
–
–
BS
–
O
–
TC AC N O
d C
d C
–
d d C C
O TC AC BS O N
u C
u C
u u C C
O C C – BS OT NA
Zn
Zn
Zn O
– BS
Zn
–
TC AC O N
–
–
O
– BS
–
TC AC O N
d d C C
–
d d C C
O TC AC BS O N
u C
–
u u C C
u C
O TC AC BS O N
Zn
Zn
Zn O
– BS
TC AC O N
5
0
0
5
*
*
*
*
15
* Root
*
*
*
10
20
Zn
15
*
Tolerant clone
10
*
GSH, µg gFW-1
GSH, µg gFW-1
0
GSH, µg gFW-1
GSH, µg gFW-1
40
* * Root
Sensitive clone
* 20
Fig. 3. Effects of L-buthionine (S,R)-sulfoximine (BSO) and GSH stimulating substances N-acetyl-cystein (NAC) and L-2-oxothiazolidine 4-carboxylate (OTC) on level of glutathione (GSH) in roots and shoots of 21 day old sensitive and tolerant clones of Salix viminalis. From the first day of cultivation plants were not treated or treated with combinations of 3 mM CuCl2, 7 mM CdCl2, 70 mM ZnCl2, 1 mM NAC, 1 mM OTC and 0.1 mM BSO. n ¼ 3 SE. *Indicates significant differences between the white bar and the coloured bars within each bar group. yDenotes significant differences between no metal treatment and metal treatment when comparing bars of the same colour. Black or white dot in bars denotes significant difference from control (no treatment). p < 0.05.
level of glutathione did not increase. Any glutathione produced had obviously been used for the synthesis of PCs, which accumulated. However, phytochelatin biosynthesis is apparently not necessary for metal tolerance in Salix, as PC was not found in this system [22] either in tolerant or sensitive clones, before or after metal treatment. This is in line with our present results, showing that increased GSH level, caused by treatment with NAC and OTC, did not influence metal tolerance. Although an increase in GSH content above a control level does not influence metal tolerance in some plants, a decrease below this level may weaken the tolerance. For example, in suspension-cultured azuki bean cells, which were hypersensitive to Cd and did not synthesize PC, GSH treatment did not show any positive effect on Cd tolerance. Still, Cd-toxicity was dependent on the GSH-level, because BSO potentiated growth inhibition caused by Cd [18]. This is similar to our result where BSO reduced the protective action of NIC towards Cd (Fig. 4). As discussed by Ortega-Villasante et al. [26] and Pa´l et al. [27], the research concerning the relationship between phytochelatins and metal tolerance is not clearcut. It is assumed that PC, in addition to a metal-detoxification function, also has a more general role in cellular homeostasis. Overexpression
of PC synthase in Arabidopsis plants even led to hypersensitivity to Cd [23]. Even though the purpose of this investigation was not to clarify the mechanisms behind the metal tolerance-induced effect of NIC and NiA, it is tempting to speculate about reasons for this effect. It is known that exposure to metal ions may bring about an increased formation/presence of ROS [13,14], which in turn, due to increased formation of single strand breaks (SSB) in DNA, can activate PARP mediated consumption of NAD, with a concomitant release of NIC from NAD [2]. ROS/PARP-mediated NAD depletion is a well known phenomenon; especially in medical/toxicological science. Due to the plant NAD salvage pathway from NIC via NiA to NAD [17,24], added NIC or NiA can counteract NAD depletion. The importance of nicotinamidase, converting NIC to NiA in plants, for the NAD salvage pathway in Arabidopsis, has been shown by Wang and Pichersky [34]. It has been indicated that nicotinamidase expression can be increased by stress related treatment, e.g. abscisic acid [17]. Some physiological effects of NIC are shared by NiA, e.g. defence induction (Berglund and Ohlsson, unpublished). NIC added to plants appears to be readily metabolized to NiA, which in turn can be metabolized to trigonelline (N-methyl nicotinic acid) or further to NAD [24]. Accordingly, the effect
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
Tolerance index 1.25
1.00
0.75
0.50
Treatment 0.25
0
Day 1
Day 7
Day 14
Tolerance index 0
0.25
0.50
nb nb nb nb nb nb nb
0 –> NIC+BSO –> Cd+NIC+BSO 0 –> Cd+BSO –> Cd+NIC+BSO 0 –> Cd+NIC –> Cd+NIC+BSO BSO –> NIC+BSO –> Cd+NIC+BSO NIC –> NIC+BSO –> Cd+NIC+BSO Cd –> Cd+BSO –> Cd+NIC+BSO Cd –> Cd+NIC –> Cd+NIC+BSO BSO –> BSO –> BSO NIC –> NIC –> NIC Cd –> Cd –> Cd
Tolerant shoot
661
0.75
1.00
1.25
Sensitive shoot c c
nb
0 –> NIC+BSO –> NIC+BSO 0 –> BSO –> NIC+BSO 0 –> NIC –> NIC+BSO BSO –> BSO –> BSO NIC –> NIC –> NIC 0 –> Cd+BSO 0 –> BSO 0 –> Cd BSO –> BSO Cd –> Cd
b
–> Cd+BSO –> Cd+BSO –> Cd+BSO –> BSO –> Cd
b b c b n
0 –> Cd+NIC –> Cd+NIC 0 –> NIC –> Cd+NIC 0 –> Cd –> Cd+NIC NIC –> NIC –> NIC Cd –> Cd –> Cd
0 –> NIC+BSO 0 –> Cd+BSO 0 –> Cd+NIC BSO –> NIC+BSO NIC –> NIC+BSO Cd –> Cd+BSO Cd –> Cd+NIC BSO –> BSO NIC –> NIC Cd –> Cd
n n
cn cn
n nc c n
n cb
–> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> BSO –> NIC –> Cd
nb nb nc b nc b nb nb c
0 –> NIC+BSO –> NIC+BSO 0 –> BSO –> NIC+BSO 0 –> NIC –> NIC+BSO BSO –> BSO –> BSO NIC –> NIC –> NIC
n n
Tolerant root
0 –> Cd+BSO 0 –> BSO 0 –> Cd BSO –> BSO Cd –> Cd
1.00
0.75
0.50
0.25
0
Tolerance index
Sensitive root
b
–> Cd+BSO –> Cd+BSO –> Cd+BSO –> BSO –> Cd
b b
c b
0 –> Cd+NIC –> Cd+NIC 0 –> NIC –> Cd+NIC 0 –> Cd –> Cd+NIC NIC –> NIC –> NIC Cd –> Cd –> Cd 1.25
c
nb
nc nc nc c n 0
0.25
0.50
0.75
1.00
1.25
Tolerance index
Fig. 4. Effects of L-buthionine (S,R)-sulfoximine (BSO) and nicotinamide (NIC) on cadmium tolerance in tolerant and sensitive clones of Salix viminalis, where 7 mM CdCl2, 0.1 mM BSO and 2 mM NIC were added in different combinations and at different time points (day 1, 7 and 14) during the 21 days cultivation. Tolerance is given as tolerance index based on dry weight of roots or shoot. n ¼ 3 SE. n, c and b denotes significant differences compared with solely NIC, Cd and BSO treatment, respectively, within each bar group. Black dot in bars denotes significant difference from tolerance index 1.00. p < 0.05.
of added NIC could as well be an effect exerted by NiA or its metabolite trigonelline. However, trigonelline does not appear to show protection against oxidative stress in plant cells (Berglund and Ohlsson, unpublished). It could be interesting to use
a plant lacking nicotinamidase, like plants homozygous for the null AtNIC1 allele [34], in studies on NIC induced defence and the importance of NIC metabolization to NiA for this process.
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
662
GSH, µg gFW-1 30
25
20
15
10
5
0
cn cn cn cn cn cn bcn cn b b n n bn n b c
Tolerant shoot
c c c
b
Day 1
Day 7
cn cn cn cn cn b cn n
5
10
n n n b c
0 –> NIC+BSO 0 –> BSO 0 –> NIC BSO –> BSO NIC –> NIC
–> NIC+BSO –> NIC+BSO –> NIC+BSO –> BSO –> NIC
nb n
0 –> Cd+BSO 0 –> BSO 0 –> Cd BSO –> BSO Cd –> Cd
–> Cd+BSO –> Cd+BSO –> Cd+BSO –> BSO –> Cd
cb c
0 –> Cd+BSO 0 –> BSO 0 –> Cd BSO –> BSO Cd –> Cd
c c c
b
15
–> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> Cd+NIC+BSO –> BSO –> NIC –> Cd
20
25
30
n cb n cb nc n cb nc nc n cb nc
b b
n n b Sensitiv
e
shoot
c
c b
–> Cd+NIC –> Cd+NIC –> Cd+NIC –> NIC –> Cd –> 0
ncb nc nc n cb
nc nc nc nc b b
0 –> NIC+BSO –> NIC+BSO 0 –> BSO –> NIC+BSO 0 –> NIC –> NIC+BSO BSO –> BSO –> BSO NIC –> NIC –> NIC
n
nb n n n b cb c
–> Cd+BSO –> Cd+BSO –> Cd+BSO –> BSO –> Cd
c
Sensitive root
c b
0 –> Cd+NIC –> Cd+NIC 0 –> NIC –> Cd+NIC 0 –> Cd –> Cd+NIC NIC –> NIC –> NIC Cd –> Cd –> Cd 0 –> 0 –> 0
c n
15
0
0 –> NIC+BSO –> Cd+NIC+BSO 0 –> Cd+BSO –> Cd+NIC+BSO 0 –> Cd+NIC –> Cd+NIC+BSO BSO –> NIC+BSO –> Cd+NIC+BSO NIC –> NIC+BSO –> Cd+NIC+BSO Cd –> Cd+BSO –> Cd+NIC+BSO Cd –> Cd+NIC –> Cd+NIC+BSO BSO –> BSO –> BSO NIC –> NIC –> NIC Cd –> Cd –> Cd
bcn
Tolerant root
Day 14
0 –> NIC+BSO 0 –> Cd+BSO 0 –> Cd+NIC BSO –> NIC+BSO NIC –> NIC+BSO Cd –> Cd+BSO Cd –> Cd+NIC BSO –> BSO NIC –> NIC Cd –> Cd
0 –> Cd+NIC 0 –> NIC 0 –> Cd NIC –> NIC Cd –> Cd 0 –> 0
bc bn
GSH, µg gFW-1
Treatment
10
5
GSH, µg gFW-1
0
0
5
10
15
GSH, µg gFW-1
Fig. 5. Effects of L-buthionine (S,R)-sulfoximine (BSO) and nicotinamide (NIC) on level of glutathion (GSH) in 21 days old roots and shoots of tolerant and sensitive clones of Salix viminalis, where 7 mM CdCl2, 0.1 mM BSO and 2 mM NIC were added in different combinations and at different time points (day 1, 7 and 14) during the 21 days cultivation. n ¼ 3 SE. n, c and b denotes significant differences compared with solely NIC, Cd and BSO treatment, respectively, within each bar group. Black dot in bars denotes significant difference from control (no treatment). p < 0.05.
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
NIC is also known to be a strong PARP inhibitor, while NiA is not [29]. Nevertheless, both have a metal tolerancestimulating effect and this discrepancy indicates that PARP inhibition may not be the basis for the metal tolerance enhancing effect of NIC and NiA. DNA methylation is another possible target for NIC and NiA. Various kinds of abiotic stress can cause changes in the level of global DNA methylation and DNA methylation at specific sites in the plant genome; e.g. osmotic stress, water deficiency, low temperature or paraquat treatment [9]. It has also been shown that a decrease in DNA methylation is often associated with an increased expression of stress related genes [33]. Most types of environmental stress are accompanied by oxidative stress. We have shown that oxidative stress in plants can induce a decrease in DNA methylation [25]. This is interesting with respect to possible effects of metal ions in plants. It is well known that metal ions can cause oxidative stress in plants and furthermore, it has been shown that Cd can reduce the level of DNA methylation in sensitive as well as tolerant plant species [1]. Taken together this points at the possibility that at least one effect of Cd (and probably other metal ions) in plants could be a decrease in the level of DNA methylation and maybe this effect of Cd is mediated via an increased level of tissue reactive oxygen species. It is hard to say whether this decrease in DNA methylation is a toxic or defensive effect in response to Cd. If we hypothesise that it is an induced defensive effect, this can constitute a connection to NIC or NiA, because we have shown that these compounds can cause a decrease in DNA methylation as well as an increase in plant defensive metabolism [25, Berglund and Ohlsson, unpublished]. Thus, we speculate that one way for the natural compounds NIC and NiA to protect plants against metal ions could be by a defence that is induced via a decrease in DNA methylation. We have earlier shown that various clones of Salix viminalis accumulate different high levels of metals and that there is no relation between tolerance to and accumulation of a certain metal [20]. It was therefore expected, and also shown here, that NIC or NiA treatment did not in general influence the metal levels in tolerant or sensitive clones (Table 2) even though these treatments counteracted the growth inhibition caused by the respective metal (Fig. 1). Obviously, decreased metal uptake is not an explanation to the protective action of NIC and NiA (Table 2). On the contrary, these metabolites may increase the metal uptake per plant by improving growth in metal exposed sensitive clones. It seems like NIC and NiA influence tolerance against Cd and Cu as well as Zn via a mechanism which is probably not tied to any of the metal ions in particular. 4.1. Summary The goal of the present study was to investigate whether NIC and NiA could influence metal tolerance in Salix and if so, whether this effect was dependent on increased levels of GSH. We conclude that NIC and NiA can improve metal tolerance in sensitive clones of Salix and that this tolerance is not dependent on increased GSH levels per se. The metal uptake
663
is not influenced by NIC or NiA treatment, but the metal content per plant is increased, due to increased growth. We conclude that the increased metal tolerance is mediated by some other mechanism than via increased glutathione content. This is, to our knowledge, the first report of a single treatment, which gives increased metal tolerance in plants. Even though the mechanisms are not clarified yet, the results may hopefully contribute to the understanding of metal tolerance mechanisms. Acknowledgements This work was supported by grants from Carl Tryggers Stiftelse and Magn. Bergvalls Stiftelse. References [1] R. Aina, S. Sgorbati, A. Santagostino, M. Labra, A. Ghiani, S. Citterio, Specific hypomethylation of DNA is induced by heavy metals in white clover and industrial hemp, Physiol. Plant 121 (2004) 472e480. [2] Y. Amor, E. Babiycchuk, D. Inze´, A. Levine, The involvement of poly(ADP-ribose) polymerase in the oxidative stress response in plants, FEBS Lett. 440 (1998) 1e7. [3] R.M. Anderson, K.J. Bitterman, J.G. Wood, O. Medvedik, D.A. Sinclair, Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae, Nature 423 (2003) 181e185. [4] M. Arakawa, N. Ushimaru, N. Osada, T. Oda, K. Ishige, Y. Ito, N-acetylcysteine selectively protects cerebellar granule cells from 4-hydroxynonenal-induced cell death, Neurosci. Res. 55 (2006) 255e263. [5] T. Berglund, Nicotinamide, a missing link in the early stress response in eukaryotic cells: a hypothesis with special reference to oxidative stress in plants, FEBS Lett. 351 (1994) 145e149. [6] T. Berglund, A.B. Ohlsson, Defensive and secondary metabolism in plant tissue cultures, with special reference to nicotinamide, glutathione and oxidative stress, Plant Cell Tissue Organ Cult. 43 (1995) 137e145. [7] T. Berglund, A.B. Ohlsson, J. Rydstro¨m, Nicotinamide increases glutathione and anthocyanin in tissue culture of Catharanthus roseus, J. Plant Physiol. 141 (1993) 596e600. ˚ . Strid, Effect of [8] T. Berglund, A.B. Ohlsson, J. Rydstro¨m, B.R. Jordan, A nicotinamide on gene expression and glutathione levels in tissue cultures of Pisum sativum, J. Plant Physiol. 142 (1993) 676e684. [9] C.-S. Choi, H. Sano, Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerophosphodiesterase-like protein in tobacco plants, Mol. Genet. Genomics 277 (2007) 589e600. [10] S. Clemens, Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants, Biochimie 88 (2006) 1707e1719. [11] P. Coyle, J.C. Philcox, L.C. Carey, A.M. Rofe, Metallothionein: the multipurpose protein, Cell. Mol. Life Sci. 59 (2002) 627e647. [12] M. De Block, C. Verduyn, D. De Brouwer, M. Cornelissen, Poly(ADPribose) polymerase in plants affects energy homeostasis, cell death and stress tolerance, Plant J. 41 (2005) 95e106. [13] K. Durcekova´, J. Huttova´, I. Mistrı´k, M. Olle´, L. Tama´s, Cadmium induces premature xylogenesis in barley roots, Plant Soil 290 (2007) 61e68. [14] P.L. Gratao, A. Polle, P.J. Lea, R.A. Azevedo, Making the life of heavy metal-stressed plants a little easier, Functional Plant Biol. 32 (2005) 481e494. [15] M. Greger, T. Landberg, Use of willow in phytoextraction, Int. J. Phytorem. 1 (1999) 115e123. [16] K. Hirata, N. Tsuji, K. Miyamoto, Biosynthetic regulation of phytochelatins, heavy metal-binding peptides, J. Biosci. Bioeng. 100 (2005) 593e599. [17] L. Hunt, F. Lerner, M. Ziegler, NAD e new roles in signalling and gene regulation in plants, New Phytol. 163 (2004) 31e44. [18] M. Inouhe, R. Ito, S. Ito, N. Sasada, H. Tohoyama, M. Joho, Azuki bean cells are hypersensitive to cadmium and do not synthesize phytochelatins, Plant Physiol. 123 (2000) 1029e1036.
664
A.B. Ohlsson et al. / Plant Physiology and Biochemistry 46 (2008) 655e664
[19] D.W. Lamming, J.G. Wood, D.A. Sinclair, Small molecules that regulate lifespan: evidence for xenohormesis, Mol. Microbiol. 53 (2004) 1003e 1009. [20] T. Landberg, M. Greger, Can heavy metal tolerant clones of Salix be used as vegetation filters on heavy contaminated land?, in: Aronsson P., Perttu K., (Eds.), Willow Vegetation Filters for Municipal Wastewaters and Sludges. A Biological Purification System. Proc. of a Study Tour, Conference and Work Shop in Sweden, 5e10 June 1994. Ultuna, Uppsala, Rapport 50, Avd. f. Skoglig intensivodling, SLU. ISBN 91-576-4916-2, 1994, pp. 133e144. [21] T. Landberg, M. Greger, Differences in oxidative stress in heavy metal resistant and sensitive clones of Salix viminalis, J. Plant Phys. 159 (2002) 69e75. [22] T. Landberg, M. Greger, No phytochelatin (PC2 and PC3) detected in Salix viminalis, Phys. Plant 121 (2004) 481e487. [23] S. Lee, J.S. Moon, T.-S. Ko, D. Petros, P.B. Goldsbrough, S.S. Korban, Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress, Plant Physiol. 131 (2003) 656e663. [24] A. Matsui, Y. Yin, K. Yamanaka, M. Iwasaki, H. Ashihara, Metabolic fate of nicotinamide in higher plants, Physiol. Plant 131 (2007) 191e200. [25] A.B. Ohlsson, T. Kraska, J. Rydstro¨m, T. Berglund, Influence of nicotinamide and its metabolites on plant defense-related metabolism; facts and fiction, XVIII Congress of The Scandinavian Society for Plant Physiology (SPPS), Uppsala, Sweden, 12e17 June (1997) Poster abstract P57. [26] C. Ortega-Villasante, R. Rella´n-Alvarez, F.F. Del Campo, R.O. CarpenaRuiz, L.E. Herna´ndez, Cellular damage induced by cadmium and mercury in Medicago sativa, J. Exp. Bot. 56 (2005) 2239e2251. [27] M. Pa´l, E. Horva´th, T. Janda, E. Pa´ldi, G. Szalai, Physiological changes and defense mechanisms induced by cadmium stress in maize, J. Plant Nutr. Soil Sci. 169 (2006) 239e246. [28] A. Polle, A. Schu¨tzendu¨bel, Heavy metal signalling in plants: linking cellular and organismic responses, in: H. Hirt, K. Shinozaki (Eds.),
[29] [30]
[31]
[32]
[33]
[34] [35] [36]
[37]
[38]
Topics in Current Genetics, Plant Responses to Abiotic Stress, vol. 4, Springer-Verlag, Berlin Heidelberg, 2003, pp. 187e215. M.R. Purnell, W.J.D. Whish, Novel inhibitors of poly(ADP-ribose) synthetase, Biochem. J. 185 (1980) 775e777. B. Semane, A. Cuypers, K. Smeets, F. Van Belleghem, N. Horemans, H. Schat, J. Vangronsveld, Cadmium responses in Arabidopsis thaliana: glutathione metabolism and antioxidative defence system, Physiol. Plant 129 (2007) 519e528. A. Tukendorf, W.E. Rauser, Changes in glutathione and phytochelatins in roots of maize seedlings exposed to cadmium, Plant Sci. 70 (1990) 155e166. J.A.C. Verkleij, F.E.C. Sneller, H. Schat, Metallothioneins and phytochelatins: ecophysiological aspects, in: Y.P. Abrol, A. Ahmad (Eds.), Sulphur in Plants, Kluwer Academic Publishers, The Netherlands, 2003, pp. 163e176. Y. Wada, K. Miyamoto, T. Kusano, H. Sano, Association between up-regulation of stress-responsive genes and hypomethylation of genomic DNA in tobacco plants, Mol. Gen. Genomics 271 (2004) 658e666. G. Wang, E. Pichersky, Nicotinamidase participates in the salvage pathway of NAD biosynthesis in Arabidopsis, Plant J. 49 (2007) 1020e1029. D.A. Wilkins, The measurement of tolerance to edaphic factors by means of root growth, New Phytol. 80 (1978) 623e633. J.M. Williamson, B. Boettcher, A. Meister, Intracellular cysteine delivery system that protects against toxicity by promoting glutathione synthesis, Proc. Natl. Acad. Sci. USA 79 (1982) 6246e6249. B. Zechmann, M. Mu¨ller, G. Zellnig, Intracellular adaptations of glutathione content in Cucurbita pepo L. induced by treatment with reduced glutathione and buthionine sulfoximine, Protoplasma 227 (2006) 197e209. H. Zhang, W. Xu, J. Guo, Z. He, M. Ma, Coordinated responses of phytochelatins and metallothioneins to heavy metals in garlic seedlings, Plant Sci. 169 (2005) 1059e1065.