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Accumulation of ammonium ion in cadmium tolerant and sensitive cultivars of Oryza sativa

Yi Ting Hsu and Ching Huei Kao

Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China, E-mail: kaoch@ntu.edu.tw

Abstract

Cd-tolerant and Cd-sensitive rice cultivars were used to study the role of NH4+ accumulation in Cd-induced toxicity. NH4+ accumulation seems to be involved in regulating the toxicity of rice seedlings caused by CdCl2. This conclusion was based on the observations that (a) on treatment with CdCl2, NH4+ content increased rapidly in the leaves of the Cd-sensitive cultivar (cv. Taichung Native 1, TN1) but not in the Cd-tolerant cultivar (cv. Tainumg 67, TNG67), (b) pretreatment with abscisic acid (ABA) enhanced Cd tolerance and reduced Cd-induced NH4+ accumulation in TN1 seedlings, (c) exogenous application of the ABA biosynthesis inhibitor, fluridone, decreased Cd tolerance and increased NH4+ content in leaves of TNG67.

Media summary

NH4+ accumulation is associated with the toxicity of rice seedlings caused by CdCl2.

Key words

Ammonium ion, Cadmium, Glutamine synthetase, Oryza sativa L.

Introduction

Cadmium (Cd) is a divalent heavy metal cation and is one of the most toxic heavy metals with no described physiological function. In Taiwan, inappropriate disposal of industrial waste has given rise to widespread Cd contamination of irrigated water (higher than 10 ppm). Thus, there is urgent need to study the mechanism of Cd tolerance of rice plants. The effect of Cd stress on nitrogen metabolism has been little investigated (Boussama et al. 1999). Relatively little work has been done to study the effect of Cd on NH4+ accumulation and we know little about the relationship between NH4+ accumulation and Cd-induced toxicity. Thus it is of great interest to examine the role of NH4+ in regulating Cd-induced toxicity of plants.

Methods

Two rice (Oryza sativa L.) cultivars, an Indica type cultivar, TN1 and a Japonica cultivar, TNG67, were used in this study. The hydroponically cultivated seedlings were grown in a Phytotron with natural light at 30 day/25 night and 90% relative humidity. The concentrations of N, P, K, S, Ca and Mg in the hydroponic solution are 11.5, 2.9, 7.2, 15.0, 7.4 and 8.7 ppm, respectively. Twelve-day-old seedlings with three leaves were used in all experiments. Cd, chlorophyll and protein contents were determined according to Hsu and Kao (2003). NH4+ content and glutamine synthetase activity were extracted and determined by the methods described in Chien and Kao (2000).

Results

Cd toxicity in the leaves caused by excess Cd was indicated by a decrease in chlorophyll and protein contents. Figure 1 shows that the decrease in chlorophyll and protein contents in TN1 leaves is more pronounced than that in TNG67 leaves, indicating that TNG67 seedlings Cd-tolerant, whereas TN1 seedlings are a Cd-sensitive. Cd content in the second leaves of TNG67 seedlings remained unchanged after Cd treatment. In contrast, a marked increase in Cd content in Cd-treated TN1 leaves was observed. It seems that avoiding the build-up of Cd in the second leaves of TNG67 prevents the toxic effect of Cd.

Figure 1. Changes in chlorophyll, protein and Cd contents in the second leaves of rice seedlings either untreated or treated with CdCl2 (0.5 mM). Vertical bars represent standard errors (n= 4).

Ammonium ion (NH4+) content in the second leaves remained unchanged in TN1 seedlings treated without CdCl2 (Figure 2). It is clear that accumulation of NH4+ in the second leaves of TN1 induced by CdCl2 was evident at 1 day after CdCl2 treatment (Fig. 2). However, no NH4+ accumulated in the second leaves of TNG67 throughout the CdCl2 treatment (Fig. 2).

Glutamine synthase (GS) is the primary enzyme responsible for NH4+ assimilation in plants (Miflin and Lea 1976). We observed that GS activity in the second leaves of TN1 treated with CdCl2 decreased compared with that given no CdCl2 treatment (Fig. 2). In contrast, CdCl2 had no effect on the GS activity of the second leaves of TNG67 (Fig. 2).

Figure 2. Changes in NH4+ and GS activity in the second leaves of rice seedlings either untreated or treated with CdCl2 (0.5 mM). Vertical bars represent standard errors (n = 4).

We have observed that Abscisic acid (ABA) plays an important role in Cd tolerance and pretreatment of TN1 seedlings with ABA counteracted Cd-induced toxicity (Hsu and Kao 2003). If accumulation of NH4+ is important in regulating toxicity in TN1seedlings, then pretreatment with ABA is expected to reduce Cd toxicity and NH4+ content in TN1 leaves. Since some chlorosis was observed in the second leaves of TN1 treated with ABA for 2 days, Cd toxicity was evaluated by using the third leaves and 1.5 mM CdCl2. As expected, ABA pretreatment reduced Cd toxicity and reduced Cd-induced NH4+ accumulation in the third leaves of TN1 seedlings (Figure 3). Figure 3 also shows that ABA pretreatment reduced the decrease in GS activity in the third leaves of TN1 seedlings.

Figure 3. Effect of ABA-pretreatment on the contents of chlorophyll, protein, NH4+ and the activity of GS in the third leaves of TN1 rice seedlings. TN1 rice seedlings were pretreated with ABA for 2 days and then either untreated or treated with CdCl2 (1.5 mM) for 2 days. Vertical bars represent standard errors (n = 4).

The results presented in Table 1 show that treatment with fluridone, an inhibitor of ABA biosynthesis, inhibited the increase in ABA content and, with the additional presence of Cd in the growth medium, enhanced Cd toxicity, increased NH4+ content, and decreased GS activity in the second leaves of TNG67 seedlings.

Table 1. Effect of fluridone on the contents of ABA, chlorophyll, protein and NH4+ and the activity of GS in the second leaves of TNG67 rice seedlings.

Treatment

ABA
(pmol g-1 FW)

Chlorophyll
(mg g-1 FW)

Protein
(mg g-1 FW)

NH4+
(μmol g-1 FW)

GS activity
(units g-1 FW)

CdCl2 Fluridone

- -

538.6 30.6

3.3 0.46

12.4 1.0

4.7 0.41

3.2 0.24

- +

252.0 34.9

3.1 0.10

12.6 0.41

4.4 0.40

3.3 0.17

+ -

991.4 117.8

2.8 0.21

12.5 0.31

4.3 0.39

2.8 0.21

+ +

548.8 111.9

2.2 0.07

8.0 0.79

6.1 0.86

2.1 0.02

CdCl2 (0.5 mM) or fluridone (0.2 mM) was added to the culture solution for 2 days. The data represent mean values standard errors, n = 4.

Conclusion

The conclusion to be drawn from the present work is that NH4+ accumulation is an effect of the toxicity of rice seedlings caused by CdCl2.

References

Boussama N, Ouariti O and Ghorbal MH (1999) Changes in growth and nitrogen assimilation in barley seedlings under cadmium stress. Journal of Plant Nutrition22, 731-752.

Chien H-F and Kao CH (2000) Accumulation of ammonium in rice leaves in response to excess cadmium. Plant Science 156, 111-115.

Hsu YT and Kao CH (2003) Role of abscisic acid in cadmium tolerance of rice (Oryza sativa) seedlings. Plant, Cell and Environment 26, 867-874.

Miflin BJ and Lea PJ (1976) The pathway of nitrogen assimilation in plants. Phytochemistry 15: 873-885.

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