Plant and Soil 237: 165–171, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands. 165
Relative importance of Na
+, Cl
−, and abscisic acid in NaCl induced
inhibition of root growth of rice seedlings
Chuan Chi Lin & Ching Huei Kao
1Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China.1Corresponding author∗ Received 9 March 2001. Accepted in revised form 12 September 2001
Key words: abscisic acid, NaCl, Oryza sativa, peroxidase, root growth
Abstract
The relative importance of endogenous abscisic acid (ABA), as well as Na+and Cl−in NaCl-induced responses related to growth in roots of rice seedlings were investigated. The increase in ammonium, proline and H2O2
levels, and cell wall peroxidase (POD) activity has been shown to be related to NaCl-inhibited root growth of rice seedlings. Increasing concentrations of NaCl from 50 to 150 mM progressively decreased root growth and increased both Na+ and Cl−. Treatment with NaCl in the presence of 4,4-diisothiocyano-2,2-disulfonic acid (DIDS, a nonpermeating amino-reactive disulfonic acid known to inhibit the uptake of Cl−) had less Cl−level in roots than that in the absence of DIDS, but did not affect the levels of Na+, and responses related to growth in roots. Treatment with 50 mM Na-gluconate (the anion of which is not permeable to membrane) had similar Na+level in roots as that with 100 mM NaCl. It was found that treatment with 50 mM Na-gluconate effected growth reduction and growth-related responses in roots in the same way as 100 mM NaCl. All these results suggest that Cl−is not required for NaCl-induced responses in root of rice seedlings. Endogenous ABA level showed no increase in roots of rice seedlings exposed to 150 mM NaCl. It is unlikely that ABA is associated with NaCl-inhibited root growth of rice seedlings.
Abbreviations: ABA – abscisic acid; DIDS – 4,4-diisothiocyano-2,2-dissulfonic acid; DW – dry weight; ELISA – enzyme-linked immunosorbent assay; FW – fresh weight; POD – peroxidase
Introduction
Soil salinity, particularly due to NaCl stress, can be considered as the single most widespread soil toxicity problem that global rice production faces at present. In rice, it was shown that during vegetative growth the young seedling stage is the most sensitive to NaCl, in comparison to germination and tillering stages (Lutts et al., 1995). Roots play a number of important roles during plant growth and development and typically are the first and critical part of the plant to encounter soil salinity. The inhibition of root growth by salinity is a widespread problem in agricultural practice. How-ever, the mechanisms underlying this inhibition are
∗ FAX No: +886-2-2362-0879. E-mail: Kaoch@ccms.ntu.edu.tw
not yet clear (Greenway and Munns, 1980; Munns and Termaat, 1986; Rengel, 1992).
When growing in saline soils, roots have to cope with two types of stresses. The first of these is an osmotic stress resulting from salt concentration in the soil that results in lowered water potential and a con-sequent loss of cell turgor in roots. The second is ionic stress induced by changes in the concentrations of Na+, Cl−or both in the root-growing medium and within root tissues. These stresses in turn caused a reduction in the water uptake and inhibition of root growth (Kafkafi and Bernstein, 1995).
Recent studies in our laboratory show that NaCl-inhibited root growth of rice seedlings is associated with increase in H2O2level and cell wall peroxidase
(POD) activity (Lin and Kao, 1999, 2001a). This suggests that NaCl-induced cell wall stiffening is a
Figure 1. Photograph of rice seedlings treated with NaCl. Photograph was taken after 5 days of treatment. NaCl concentrations from left were
0, 50, 100 and 150 mM, respectively.
possible mechanism of NaCl-inhibited root growth of rice seedlings. We also reported that increase in cell wall POD activity in NaCl-treated roots is associated with ionic rather than osmotic component of NaCl stress (Lin and Kao, 1999). However, we do not know whether this ionic effect is caused by Na+,Cl−or both. High tissue Cl−content found in salt-treated beans has been considered as the principal causes for the salt-induced growth reduction (Marschner, 1995). Cramer et al. (1994) reported early variety differences in leaf growth rates of maize plants. They showed that maize variety which was more sensitive to early growth inhibtion by salinity accumulated less Na+ than the salt-resistant variety. Thus, greater inhibi-tion of leaf growth was not related to proporinhibi-tionately higher rates of salt accumulation. Recent investiga-tions by Montero et al. (1998) and Sibole et al. (1998) strongly suggest that bean is also extremely sensitive to Na+. Munns et al. (1988) reported that leaf growth of barley was reduced by the higher NaCl treatments, but is not directly controlled by local concentration of Na+ or Cl−, suggesting that some influence ori-ginating elsewhere in the plants. It has been observed that ABA increases in plants under salt stress and in-hibits growth when applied to plants under control conditions (Munns and Sharp, 1993). Thus, growth reduction under stress is possibly related to higher
rates of ABA accumulation. In fact, data reported by Montero et al. (1997) suggest that salt-inhibited leaf expansion growth in bush beans is mediated by ABA rather than Na+or Cl− toxicity. In this study, we in-vestigated the relative importance of Na+, Cl− and ABA in the inhibition of root growth of rice seedlings caused by NaCl.
Materials and methods
Rice (Oryza sativa L., cv. Taichung Native 1) seeds were sterilized with 2.5% sodium hypochlorite for 15 min and washed extensively with distilled water. In order to get more uniformly germinated seeds, rice seeds in Petri dish (20 cm) containing distilled water were pretreated at 37◦C for 1-day under dark condi-tion. Uniformly germinated seeds were then selected and transferred to a Petri dish (9.0 cm) containing two sheets of Whatman No. 1 filter paper moistened with 10 ml of distilled water or NaCl at the desired con-centration as specified in the individual experiments. Root growth of seedlings grown in distilled water is similar to that grown in medium containing inorganic salts, thus seedlings grown in distilled were used as the controls. For the experiments to examine the rel-ative importance of Cl− in regulating NaCl-inhibited
167
Figure 2. Effects of NaCl (150 mM) on root growth and Na+and Cl−levels in roots of seedlings in the presence or absence of DIDS (0.05 mM). Root growth and Na+and Cl−levels were measured after 5 days of treatment. Bars represent standard errors (n=4).
root growth, Na-gluconate and DIDS were used. Each Petri dish contained 20 germinated seeds. Each treat-ment was replicated four times. The germinated seeds were allowed to grow at 27◦C in darkness and 3 ml of distilled water or test solutions was added to each Petri dish on day 3 of the growth.
Cell walls were prepared by homogenizing roots in ice-cold phosphate buffer (50 mM, pH 5.8) using a pestle and mortar. The homogenate was centrifuged at 1000 g, and washed at least four times with 50 mM phosphate buffer (Lee and Lin, 1995). The pellet was collected and used as a cell wall fraction.
POD ionically bound to the walls was extracted with 1 M NaCl. Cell walls were prepared as described above and incubated in 1 M NaCl for 2 h with shaking at 30◦C and centrifuged at 1000 g. The supernatant was considered as the cell wall fraction.
POD activities were measured using a modification of the procedure described by Curtis (1971). The assay
Figure 3. Effects of NaCl and Na-gluconate on root growth and
Na+and Cl− levels in roots of rice seedlings. Root growth and Na+and Cl−levels were measured after 5 days of treatment. Bars represent standard errors (n=4).
medium contained 0.05 M phosphate buffer (pH 5.8), 7.2 mM guaiacol, 11.8 mM H2O2and 0.1 ml enzyme
extract in a final assay volume of 3.0 ml. The reaction was initiated by the addition on H2O2and the change
in absorbance at 470 nm was measured. Activity was calculated using the extinction coefficient (26.6 mM−1 cm−1 at 470 nm) for tetraguaiacol. One unit of POD was defined as the amount of enzyme that causes the formation of 1 µmol tetraguaiacol per min. The activ-ity of cell wall POD was expressed on the basis of DW.
The H2O2level was colorimetrically measured as
described by Jana and Choudhuri (1981). H2O2was
extracted by homogenizing 10 roots with 3 ml of phosphate buffer (50 mM, pH 6.8). The homogenate was centrifuged at 6000 g for 25 min. To determine H2O2 levels, 3 ml of extracted solution was mixed
with 1 ml of 0.1% titanium chloride (Aldrich) in 20% (v/v) H2SO4 and the mixture was then centrifuged
at 6000 g for 15 min. The intensity of yellow color of supernatant was measured at 410 nm. H2O2 level
Figure 4. Effects of NaCl (150 mM) on ammonium, proline and
H2O2levels, and cell wall POD activity in roots of rice seedlings in the presence or absence of DIDS (0.05 mM). All measurements were made after 5 days of treatment. Bars represent standard errors (n=4).
Figure 5. Effects of NaCl (100 mM) and Na-gluconate (50 mM) on
ammonium, proline and H2O2levels, and cell wall POD activity in roots of rice seedlings. All measurements were made after 5 days of treatment. Bars represent standard errors (n=4).
was calculated using the extinction coefficient 0.28
µmol−1cm−1.
Ammonium and proline were extracted and de-termined according to the methods described
previ-Figure 6. Changes in root growth and ABA levels in roots of
rice seedlings treated with distilled water or NaCl. Two-day-old rice seedlings were treated with distilled or 150 mM NaCl. Bars represent standard errors (n=4).
ously (Lin and Kao, 1996a, b). For Na+determination, harvested roots were washed three times (with each 1 min) with distilled water, dried at 65◦C for 2 days, extracted in 1 N HCl at room temperature (Hunt, 1982) and analyzed with a flame photometer (Evans Elec-troselenium Ltd., UK). Chloride was estimated in a separate extract made according to the method de-scribed by Hodson et al. (1985) and estimated using
169 an ion meter (Mittler Delta 350, UK) equipped with
chloride ion electrode. The levels of ammonium, pro-line, H2O2, Na+and Cl−were expressed on the basis
of DW.
For extraction of ABA, root samples were homo-genized with a mortar and pestle in extraction solution (80% methanol containing 2% glacial acetic acid). To remove plant pigments and other non-polar com-pounds which could interfere in the immunoassay, ex-tracts were first passed through polyvinylpyrrolidone column and C18 cartridges. The eluates were concen-trated to dryness by vaccum-evaporation and resus-pended in Tris-buffered saline before enzyme-linked immunosorbent assay (ELISA). ABA was quantitated by ELISA (Walker–Simmons, 1987). ABA immun-oassay detection kit (PGR-1) was purchased from Sigma Chemical Co. (St Louis, MO, USA), is specific for (+)–ABA.
For biochemical analysis, four independent extrac-tions were performed for each treatment, and for each extraction 20 seedlings were used. All experiments de-scribed here were repeated at least three times. The data reported here are from a single experiment.
Results and discussion
Figure 1 shows the effect of NaCl on the growth of rice seedlings. Increasing concentrations of NaCl from 50 to 150 mM progressively decreased root length. Since the pattern of root growth of rice seedlings in response to NaCl is similar to that of FW or DW of roots, FW and DW of roots were used as an indicator of root growth for the subsequent experiments. Figure 2 shows the effect of NaCl on root growth and Na+and Cl−levels in roots of rice seedlings in the presence or absence of DIDS, a nonpermeating amino-reactive di-sulfonic acid known to inhibit the uptake of Cl−(Lin, 1981). NaCl at 150 mM decreased root growth and increased both Na+and Cl−levels in roots. Treatment with NaCl in the presence of DIDS had less Cl−level in roots than that in the absence of DIDS, but did not affect Na+level and root growth when compared with NaCl treatment. This suggests that Cl−is not involved in NaCl-inhibited root growth of rice seedlings. DIDS treatment resulted in about 40% reduction of Cl− (Fig-ure 2). Thus, the lack effect of DIDS on root growth of rice seedlings treated with NaCl can be explained by the possibility that the level of Cl−is still high enough to exert its effect. In order to test further the involve-ment of Cl−, experiments were performed to compare
the effect of NaCl with that of Na-gluconate, the an-ion of which is not able to permeate the membrane (Blumwald and Poole, 1985). The results are shown in Figure 3. Increasing concentrations of NaCl from 50 to 150 mM progressively decreased FW and DW of roots and increased both Na+and Cl−levels in roots. The reduction of root growth is closely correlated with the increase of Na+and Cl−levels in roots. Treatment with 50 mM Na-gluconate had similar Na+level but had much less Cl−level in roots as that with 100 mM NaCl. The reduction of root growth by 50 mM Na-gluconate is similar to that by 100 mM NaCl. These results strongly suggest that Cl− is not required for NaCl-inhibited root growth of rice seedlings.
In previous studies, we have shown that proline ac-cumulation is correlated with root growth inhibition of rice seedlings induced by NaCl (Lin and Kao, 1996b). In the present study, we demonstrated that proline con-tent in roots of rice seedlings treated with NaCl in the presence of DIDS was similar to that in the absence of DIDS (Figure 4). We also demonstrated that roots of rice seedlings treated with 50 mM Na-gluconate accu-mulated similar level of proline as those with 100 mM NaCl (Figure 5).
It is known that ammonium inhibits the growth of many plants (Haynes and Goh, 1978). It has been shown that NaCl was effective in stimulating the accu-mulation of ammonium in roots of rice seedlings, and that accumulation of ammonium in roots preceded in-hibition of root growth caused by NaCl (Lin and Kao, 1996a). If Na+rather than Cl− is important in regu-lating growth reduction of roots caused by NaCl, then ammonium accumulation in roots treated with NaCl is expected to be induced by Na+ instead of Cl−. The results reported in Figures 4 and 5 demonstrate that this is, infact, correct.
Recent studies in our laboratory show that NaCl-inhibited root growth of rice seedlings is associated with increase in H2O2level and cell wall POD activity
(Lin and Kao, 1999, 2001a), suggesting that NaCl-induced cell wall stiffening is a possible mechanism of NaCl-inhibited root growth of rice seedlings. If the proposed mechanism of NaCl-inhibited roots growth of rice seedlings is correct, then the increase in H2O2
level and cell wall POD activity is expected to be caused by Na+ rather than Cl−. It was found that treatment with NaCl in the presence of DIDS or with Na-gluconate increased H2O2level and cell wall POD
activity in roots, in the same way as that NaCl did (Figures 4 and 5). The data reported in the present study clearly indicate that growth-related responses in
rice seedlings caused by ABA, a mechanism similar to that of NaCl-inhibited root growth (Lin and Kao, 1999, 2001a, b). Thus, it is of great interest to know whether NaCl-inhibited root growth of rice seedlings is mediated through the increase of endogenous ABA level. However, there is no increase in ABA level in the roots of rice seedlings treated with NaCl when compared with the control (Figure 6). Moons et al. (1995) observed that ABA level increased slightly in the roots of salt-stressed Taichung Native 1 rice seedlings (the investigated variety of this study). It ap-pears that NaCl-inhibited root growth of rice seedlings is unlikely to be mediated through ABA. However, Montero et al. (1997) reported that endogenous ABA levels are linked to decreased leaf growth of bean plants treated with NaCl.
The fact that ABA did not increase in NaCl-treated roots does not mean that it is not overproduced in roots in response to NaCl if the translocation of ABA from roots to shoot is also stimulated by NaCl. It is not known whether NaCl stimulates the translocation of ABA from roots to shoots. Further work is needed to clarify this issue.
On the basis of the results reported in the present investigation, it can be concluded that Cl− is not in-volved in NaCl-induced growth reduction and growth-related responses of roots of rice seedlings.
Acknowledgements
This work was supported by the National Science Council of the Republic of China (NSC 89-2313-B-166).
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