adaptation of citrus cells to salt stress. Abel Piqueras, Jos6 ... the selected cell line also showed an increase in choline and glycine betaine, but to lesser extent.
Plant Cell, Tissueand Organ Culture 45: 53-60, 1996. © 1996Kluwer Academic Publishers. Printed in the Netherlands.
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Changes in antioxidant enzymes and organic solutes associated with adaptation of citrus cells to salt stress A b e l P i q u e r a s , Jos6 A. H e r m i n d e z , E n r i q u e O l m o s , E l a d i o Hellin* & F r a n c i s c a S e v i l l a Departamento de Nutrici6n y Fisiologfa Vegetal, CEBAS-CSIC, Apdo. 4195, E-30080 Murcia, Spain (* requests for offprints) Received4 August 1995;acceptedin revisedform23 January 1996 Key words: Catalase, cell culture, peroxidase, salinity, superoxide dismutase, Citrus limon
Abstract Embryogenic callus cultures of lemon (Citrus limon L. Burm f. cv Verna), were selected for resistance to salt stress (170 mM NaCI). Inorganic analysis showed that selected callus accumulated more Na + and C1- ions than the non-selected one. Moreover, the salt-tolerant C. limon callus exhibited an increase in the activity of antioxidant enzymes involved in oxygen metabolism, with the induction of a new superoxide dismutase isozyme and an increase of the peroxidase activity while the catalase activity was unchanged. Proline and total sugar, mainly sucrose, concentrations increases significantly in salt-tolerant cells as compared to control cells. On the other hand, the selected cell line also showed an increase in choline and glycine betaine, but to lesser extent. Abbreviations: BSA - bovine serum albumin; P5CR - pyrroline-5-carboxylated reductase; QAC - quaternary ammonium compounds; SOD - superoxide dismutase Introduction Salinity affects many aspects of plant metabolism and often results in reduction in plant growth. In vitro selection for cells exhibiting increased tolerance to salt stress is considered a suitable system to investigate plant metabolism and the selective accumulation of inorganic and organic compounds that contribute to turgor maintenance (Heyser & Nabors, 1981; Binzel et al., 1987). Evidence is accumulating which suggests that oxidative stress, which include the superoxide ( 0 2 ) and hydroxyl ('OH) free radicals, as well as hydrogen peroxide (H202), is a major damaging factor in plants exposed to different environmental stresses, such as temperature extremes, herbicides and xenobiotic treatments, drought, nutrient deficiency and toxicity (Monk etal., 1989; Scandalios, 1993; Hern~ndezetal., 1993a, 1993b, 1995). It is also widely known that plants resist the stress-induced production of active oxygen species by increasing components of their defensive system (Rabinowitch & Fridovich, 1983; del Rio et al., 1991;
S alin, 1991; Foyer et al., 1994). Plant survival against these potential cytotoxic effects depends on the presence of reduced molecules and antioxidant enzymes. Superoxide dismutases (SOD; EC 1.15.1.1) are a group of metalloenzymes that catalyze the disproportionation of O~- to H202 and 02 (Fridovich, 1986). Hydrogen peroxide produced by SOD and in the course of other enzymatic and nonenzymatic reactions, is removed by catalase and peroxidase (Fridovich, 1986; Salin, 1991). However, little is known of the effect of salt stress on activated oxygen metabolism and there is little information in particular on activated oxygen metabolism in cultured plant cells. This knowledge can provide information on the possible involvement of activated oxygen species in the mechanism of NaCIinduced damage and could also allow a more complete understanding of the molecular mechanisms of cell adaptation to salt-induced oxidative stress. Studies with P sativum cell lines have also demonstrated an induction of two Cu, Zn-SODs in salt tolerant callus as compared to control (sensitive) cells (Olmos
54
et al., 1994). These results suggest a salt tolerance mechanism operating at the cellular level. Adaptation of cells to high salinity is reported to be associated with reduced cell expansion even though turgor is maintained. In these cells extensive osmotic adjustment occurs principally involving Na + and CI(Binzel et al., 1987), and has also been reported the participation of organic solutes such as glycine betaine and proline (Delauney & Verma, 1993; Rhodes & Hanson, 1993). In this work, we are interested in discovering if there are differences in salt tolerance at the cellular level in embryogenic callus of Citrus limon and if so, were these differences correlated with activatedoxygen related enzymes as well as with the accumulation of organic solutes?
MateriaLs and methods
7~ssue culture Embryogenic callus (control) obtained from nucellar tissue of lemon (Citrus limon (L). Burm f. cv. Verna) was exposed to 170 mM NaC1 for six weeks in a complete medium (Murashige & Tucker, 1969). This concentration proved lethal for most of the cells with only 4.26% surviving. These surviving cells were subcultured and grown for four more weeks until uniform growth was observed. After three subcultures, one cell line showing maximal growth was selected and subcultured at regular intervals of one month. The whole selection process took 18 months. The cell line which showed tolerance to 170 nM NaC1 was named the tolerant callus line, whereas callus unable to grow at the inhibitory 170 mM NaCI concentration was referred to as the sensitive (control) callus line (Piqueras & Hellfn, 1992). For all studies, the sensitive (control) callus line was grown on media without NaCI, whereas tolerant callus line was grown on media containing 170 mM NaC1. Samples were taken at day 28 of each subculture.
Preparation of extracts C. limon nucellar embryogenic callus (1 g), both sensitive and salt-tolerant, were blended for 2-3 min at top speed in 50 mM K-phosphate buffer (pH 7.8), containing 0.1 mM EDTA, 5 mM cysteine, 1% (w/v) polyvinyl pyrrolidone (40T) and 0.2% (v/v) Triton X100 (tissue/medium ratio 1:2; w/v) using a Polytron tis-
sue homogenizer. Homogenates were filtered and centrifuged at 8,000 x g for 15 min. The supernatants were dialyzed for 24 h against 1 1 of 10 mM K-phosphate buffer (pH 7.8), containing 3 mM B-mercaptoethanol, with two changes of volume to remove low-molecularweight compounds interfering with the determination of SOD activity. The dialyzed samples were clarified by centrifugation at 2,500 x g for 10 min, and were immediately used for the enzymatic determinations. All operations were performed at 0--4°C.
Enzyme assays Total SOD (EC 1.15.1.1) activity of the samples was determined spectrophotometrically at 550 nm by the ferricytochrome c method, using xanthine/xanthine oxidase as the source of superoxide radicals (McCord & Fridovich, 1969). Catalase (EC 1.11.1.6) and peroxidase activity (EC 1.11.1.7) was determined by spectrophotometry according to the Aebi (1984) and BarAkiba (1968) methods respectively. All enzyme assays were performed at 25°C and not later than 24 h after dialyzing the samples.
SOD-Isozymes SOD isozymes were separated by isoelectric focusing on cylindrical 8.5% (w/v) polyacrylamide gels containing 2.5% (v/v) ampholytes of a 1:0.73 mixture of Pharmalytes (Pharmacia) pH 4.5--4.9 and pH 4.5-5.4, respectively, according to Almansa etal. (1989). SODs were localized on the gels using the photochemical method of Weissiger & Fridovich (1973). The three types of SOD were identified by separately performing the activity stains in gels previously incubated at 25°C for 45 min, in 50 mM K-phosphate buffer (pH 7.8) containing 2 mM C N - and 5 mM H202. Cu, Zn-SOD are inhibited by C N - and H202, Fe-SOD is inhibited by H202 and Mn-SOD is resistant to both inhibitors (Bridges & Salin, 1981). The stained gels from total activity (withouth inhibitors) were recorded in a Shimadzu CS-9000 densitometer which integrated the activity areas of each band under the transmittance peaks. The isozyme activities (Cu, Zn-SOD, Mn-SOD and Fe-SOD) were calculated by the sum of the relative percentages from the identified bands of total SOD activity.
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Sugar content Sucrose, glucose and fructose were extracted from both types of callus in deionized hot water (60°C) and filtered through a 0.22 #m millipore membrane. Analysis was carried out by HPLC on an interaction CHO-682 carbohydrate column, thermostabilized at 90 °C using a Merck L-6200A HPLC pump. The flow rate was 0.4 ml m i n - l ; the mobile phase was H20; and detection was measure with a Light Scattering Detector SEDEX 4X (S.E.D.E.R.E., France).
Quaternary ammonium compounds and choline contents QAC were quantified according to the method of Grieve & Grattan (1983). Oven dried ( 70°C for 24h and 100-105°C for 4 h) finely-ground callus (either sensitive or tolerant) was shaken with deionized water for 24 h at 25°C and then filtered. The extracts were diluted 1:1 with 2N H2SO4. Aliquots of 0.5 ml were cooled in ice-cold H20 for 1 h and then 0.2 ml of cold KI-I2 was added. The tubes were stored at 4°C overnight and then centrifuged at 10000 x g for 15 min at 0°C. The precipitate was dissolved in 9 ml of 1,2-dichloroethane and the absorbance was measured at 365 nm after about 2.5 h. Glycine betaine was used as a standard. Callus extracts were diluted 1:1 with 0.2 M K- phosphate (pH 6.8) buffer and the choline periodides were precipitated and analyzed as previously described for total QAC. As glycine betaine does not precipitate at this pH, it was quantified by subtracting choline from the QAC concentration.
1,5
-1-
Lu 1,0 "lI.u rig.
0,5
0 0
I0
20
30
40
TIME (day) Fig. 1. Effect of NaCI on growth based on fresh weight of Citrus limon embryogenic callus. Control callus on MT medium (A), control callus on MT medium + NaCI (170 mM) (&) and NaCl-tolerant cells on MT medium + NaCI (170 mM) (1"1). Vertical bars indicate -4- SE
analysis, oven-dried callus (65°C for 24 h and 100°C for 4 h) was extracted in distilled water at room temperature for 1 h and analysed by ionic chromatography in a Dionex chromatograph (Piqueras & Hellln, 1990).
Results Growth parameters
Proline and protein analysis Proline was extracted from fresh callus tissues and assayed according to the method of Bates et al. (1973) using proline as standard. Protein determinations were carried out according to the method of Bradford (1976) using crystalline BSA as standard.
In fig. 1, the results showed that tolerant cell lines grew in 170 mM NaC1, however growth was reduced 5-fold as compared to the control cells. Incorporation of NaC1 in the control medium caused a complete inhibition of the growth of sensitive embryogenic callus.
Antioxidant enzymes Ion analysis Callus samples were oven-dried (65°C for 24 h and 100°C for 4 h) and ashed at 490°C in a muffle furnace for further analytical determinations. Sodium and potassium concentrations of both types of callus were assessed with a flame photometer, while magnesium and calcium concentrations were determinated by atomic absorption spectrophotometry. For chloride
In both control and NaCl-tolerant cells catalase activity was similar. Superoxide dismutase and peroxidase activities showed statistically significant increases in the salt-tolerant cells compared to control cells (Table. 1). It was previously shown, that in C. limon leaves, nine distinct SODs can be characterized by isoelectric focusing in polyacrylamide gels. In lemon leaves, four
56 Table 1. Antioxidantactivities from extract of salt-sensitive(control) and salt tolerant (170 mM NaCI) nucellarembryogeniccallus of Citrus limon. Each valuerepresents mean 4- SE of three differentsamples. Differencesfrom controlvalueswere significantat: p< 0.01 (A); p< 0.05(B); NS, not significant,accordingto Duncan's MultipleRangeTest.
lomm
Cell type
SOD U (mg protein)-1
Catalase nmol(min mg protein)-t
Peroxidase U (mg protein)-1
Control NaCl-tolerant
8.00 4- 0.70 10.454- 0.36B
2.11 4- 0.08 2.68 4- 0.27NS
0.065 4- 0.002 0.099 4- 0.002A
10~'n
1
1
4s 6
( I
l
I
4.31
• I
I
4.63
) I
I
4.95
I
I
5.27
I
I
~,)
J.59
lm
pit Fig. 2. Superoxidedismutases isozymespresent in extracts of saltsensitive(control) Citrus limon nucellarembryogeniccallus. Curve
Fig. 3. Superoxidedismutases isozymespresent in extracts of salt tolerant (170 mM NaCI) Citrus limon nucellarembryogeniccallus.
a, total activity (no inhibitors); curve b, preincubatedwith 2 mM CN- ; curve c, preincubatedwith 5 mM H202. Cu,Zn-SOD(Band 1; pH 4.37), Mn-SOD(Bands 3, 4 and 5; pH 4.94, 5.00 and 5.08), Fe-SOD (Bans 2, 6 and 7; pH 4.85, 5.14 and 5.30)
Curve a, total activity (no inhibitors);curve b, preincubatedwith 2 mM CN-; curve c, preincubatedwith 5 mM H202. Cu,Zn-SOD (Band 1; pH 4.38), Mn-SOD(Bands 3, 4, 5 and 6; pH 4.88, 4.96, 5.03 and 5.13), Fe-SOD (Bans2, 7 and 8; pH 4.83, 5.17 and 5.31)
Cu, Zn-SODs, three Fe-SODs and two Mn-SODs were present in gels (Almansa et al., 1989). One of the FeSOD isozymes has been purified and fully characterized (Almansa e t al., 1994). A different SOD isozyme pattern was found in C. l i m o n callus. Densitometric scans of isoelectric focused gels from control (sensitive) callus extracts, (Fig. 2 curve a), showed the presence of 7 SOD isozymes. Based on sensivity to C N - and H202, as one Cu,Zn-SOD (band 1) inhibited by C N - (curve b), three Mn-SODs (bands 3, 4 and 5) resistant to C N - and H202 (curves b an c) and three Fe-SODs isozymes (bands 2, 6 and 7) resistant to C N but inhibited by H202 (Fig. 2, curve c) were identified.
When crude extracts from NaCl-tolerant callus were subjected to isoelectric focusing without inhibitors, 8 SOD isozymes were observed (Fig. 3, curve a). The new isozyme (band 3) was cyanide and H202 resistant (Fig. 3, curves b and c) and was therefore considered be a Mn-SOD. Thus, the selected line of lemon appeared to show an induction of a manganese-containing isozyme. Table 2 shows the individual SOD-isozymes activities. The Mn-SOD isozymes were increased significantly in salt-tolerant cells. On the other hand, the Cu, Zn-SOD and FeSOD activities showed an increase, although nonsignificant, as compared to control cells.
57 Table 2. Superoxide dismutaseisozyme activities from extracts of saltsensitive(control)and salt-tolerant(170 mM NaCI)nucellarembryogenic callusof Otrus limon. Each valuerepresentsmean4- SE of three different samples. Differencesfrom controlvalueswere significantat: p< 0.01 (A); NS, not significant,accordingto Duncan's MultipleRangeTest. Cell type
Cu, Zn-SOD
Mn-SOD Fe-SOD U (mg protein)-l
Control 4,70 4- 0.44 2.21 4- 0.20 NaCI-tolerant 5.624- 0.19NS 3.604- 0.12A
i .09 4- 0.10 1.23 4- 0.04NS
Table 3. Ion contentsof salt-sensitive(control)and salt-tolerant(170 mM NaCI) nucellarembryogenic callus of Citrus limon. Each value represents mean 4- SE of three differentsamples. Differences from control valueswere significantat: p< 0.001 (A);p
