Abscisic acid accumulation and cadmium tolerance in rice
seedlings
Yi Ting Hsu and Ching Huei Kao*
Department of Agronomy, National Taiwan University, Taipei, Taiwan
Correspondence *Corresponding author, e-mail: [email protected]
Received 6 September 2004; revised 8 January 2005
doi: 10.1111/j.1399-3054.2005.00490.x
Rice (Oryza sativa L.) seeds were soaked for 18 h in distilled water in the absence (–PBZ) or presence (þPBZ, a triazole) of 100 mg l1paclobutrazol and then air dried. These air-dried seeds were germinated in the dark and then cultivated in a Phytotron. Twelve-day-old –PBZ and þPBZ seedlings were treated or not with CdCl2. Cd toxicity was judged by the decrease in
biomass production, decrease in chlorophyll and protein content, increase in NH4þ content and induction of oxidative stress. The results indicated that
PBZ applied to seeds was able to protect rice seedlings from Cd toxicity. On treatment with CdCl2, the abscisic acid (ABA) content increased in
þPBZ leaves, but not in –PBZ leaves. The decrease in the transpiration rate of –PBZ seedlings by CdCl2was less than that of þPBZ seedlings. Exogenous
application of the ABA biosynthesis inhibitor, fluridone (Flu), reduced ABA accumulation, increased the transpiration rate and Cd content, and decreased the Cd tolerance of þPBZ seedlings. The effects of Flu on the Cd toxicity, transpiration rate and Cd content were reversed by the application of ABA. It seems that the PBZ-induced Cd tolerance of rice seedlings is mediated through an accumulation of ABA.
Introduction
Paclobutrazol [PBZ, (2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazolyl)-pentan-3-ol], a member of the triazole plant growth regulator group, is an inhibitor of gibberellin (GA) biosynthesis (Graebe 1987, Rademacher et al. 1987, Davis and Curry 1991) and a retardant of shoot growth (Cox and Keever 1988, Davis et al. 1988, Davis and Curry 1991). The primary mode of action of PBZ is inhibition of ent-kaurene oxidase to ent-kaurenoic acid in the early pathway of GA biosynth-esis (Graebe 1987, Rademacher et al. 1987). PBZ has been used to provide plant protection against environ-mental stresses, such as chilling (Whitaker and Wang 1987, Lurie et al. 1994, Pinhero and Fletcher 1994,
Pinhero et al. 1997), heat (Kraus and Fletcher 1994, Pinhero and Fletcher 1994, Kraus et al. 1995, Gilley and Fletcher 1998, Vettakkorumakankav et al. 1999), flooding (Webb and Fletcher 1996), salt (Abou El-Khashab et al. 1997) and gaseous sulphur dioxide (Lee et al. 1985).
Cadmium (Cd) is a heavy metal that is toxic to humans, animals and plants, and is a widespread pollu-tant with a long biological half-life (Wagner 1993). This metal enters the environment mainly from industrial processes and phosphate fertilizers and is transferred to animals and humans through the food chain (Wagner 1993). Taken up in excess by plants, Cd directly or indirectly inhibits physiological processes, such as
Abbreviations – ABA, abscisic acid; AOS, active oxygen species; APX, ascorbate peroxidase; ASC, ascorbate; CAT, catalase; d. wt., dry weight; ELISA, enzyme-linked immunosorbent assay; Flu, fluridone; f. wt., initial fresh weight; GA, gibberellin; GR, glutathione reductase; GS, glutamine synthetase; GSH, reduced glutathione; MDA, malondialdehyde; PAL, phenylalanine ammonia-lyase; PBZ, paclobutrazol; POX, peroxidase; SOD, superoxide dismutase; TN1, Taichung Native 1.
respiration, photosynthesis, cell elongation, plant–water relationships, nitrogen metabolism and mineral nutri-tion, resulting in poor growth and low biomass (Sanita´ di Toppi and Gabbrielli 1999).
In Taiwan, inappropriate disposal of industrial waste has given rise to widespread Cd contamination of irrigated water (higher than 10 mg l1). Thus, there is an urgent need to study the mechanism of Cd tolerance of rice plants.
It has been demonstrated that uniconazole, another potent member of the triazole family, induces Cd tolerance in wheat (Singh 1993). However, this is the only report describing the protective effect of triazole against Cd toxicity. Here, we show that PBZ protects rice seedlings from Cd toxicity, confirming the ability of PBZ to induce stress tolerance in plants.
In higher plants, abscisic acid (ABA) is a well-known stress hormone leading to the induction of various pro-tective reactions, which are adaptations for coping with abiotic environmental stresses, such as ozone (Lin et al. 2001), freezing (Guy 1990), chilling (Lee et al. 1993), drought (Zeevaart and Creelman 1988) and salt (La Rosa et al. 1987). Recently, a role of ABA in the Cd tolerance of rice seedlings has been demonstrated (Hsu and Kao 2003a, 2003b, Kuo and Kao 2004). We found that rice seedlings of cultivar Tainung 67 were more tolerant to Cd than were those of cultivar Taichung Native 1 (TN1), and the increase in endogenous ABA content was closely related to the Cd tolerance of rice seedlings (Hsu and Kao 2003a). It is not known whether PBZ-induced Cd tolerance of rice seedlings is mediated through ABA accumulation. Thus, we also investigated the role of ABA in the PBZ-induced Cd tolerance of TN1 rice seedlings.
Materials and methods Plant material and treatment
Rice (Oryza sativa L., cv. TN1) seeds were sterilized with 2.5% sodium hypochlorite for 15 min and washed extensively with distilled water. These seeds were soaked for 18 h at room temperature in 100 mg l1 PBZ or in distilled water (–PBZ), as described by Fletcher and Hosfsta (1990), and then were air dried for 5 days. These air-dried seeds were then germinated in Petri dishes with wet filter papers at 37C in the dark.
After 48 h of incubation, uniformly germinated seeds were selected and cultivated in a 250 ml beaker con-taining half-strength Kimura B solution including the following macro- and microelements: 182.3 mM (NH4)2SO4, 91.6 mM KNO3, 273.9 mM MgSO47H2O,
91.1 mM KH2PO4, 182.5 mM Ca(NO3)2, 30.6 mM
Fe-citrate, 0.25 mM H3BO3, 0.2 mM MnSO4H2O,
0.2 mM ZnSO4.7H2O, 0.05 mM CuSO45H2O and
0.07 mM H2MoO4(Chu and Lee 1989). The
hydroponi-cally cultivated seedlings were grown in a Phytotron (Agricultural Experimental Station, National Taiwan University, Taipei, Taiwan) with natural sunlight at 30C (day)/25C (night) and 90% relative humidity.
Twelve-day-old seedlings with three leaves were used in all experiments.
For Cd, ABA or fluridone (Flu) treatment, 12-day-old seedlings were grown in basic nutrient solution plus 1.5 mM CdCl2, 5 mM ABA or 0.2 mM Flu.
Growth analysis
At the end of treatment, the seedlings were divided into their separate parts (shoot, adventitious roots and primary roots). The length of the shoot and primary roots and the fresh weight (f. wt.) of the shoot and roots (adventitious roots plus primary roots) were then measured. For dry weight (d. wt.) estimation, the shoot and roots were dried at 65C for 48 h.
Cd determination
For the determination of Cd, leaves were dried at 65C
for 48 h. Dried material was ashed at 550C for 20 h.
The ash residue was incubated with 31% HNO3 and
17.5% H2O2at 72C for 2 h, and dissolved in distilled
water. Cd was then quantified using an atomic absorp-tion spectrophotomer (Model AA-6800, Shimadzu, Kyoto, Japan). The amount of Cd was expressed on a d. wt. basis.
Determination of chlorophyll, protein, NH4+,
malondialdehyde (MDA), ascorbate (ASC) and reduced glutathione (GSH)
The chlorophyll content was determined according to Wintermans and De Mots (1965) after extraction in 96% (v/v) ethanol. For protein determination, leaves were homogenized in a 50 mM sodium phosphate buffer (pH 6.8). The extracts were centrifuged at 17 600 g for 20 min, and the supernatants were used for determina-tion by the method of Bradford (1976). NH4þwas
mea-sured in the crude extract by the Berthelot reaction, modified according to Weatherburn (1967). The detailed procedure has been described previously (Lin and Kao 1996). MDA, routinely used as an indicator of lipid peroxidation, was extracted with 5% (w/v) trichlor-oacetic acid and determined according to Heath and Packer (1968). ASC in 5% (w/v) trichloroacetic acid
and GSH in 3% sulphosalicylic acid extract were determined as described by Laws et al. (1983) and Smith (1985), respectively.
Enzyme extraction and assays
For the extraction of enzymes, leaf tissues were homogenized with 0.1 M sodium phosphate buffer (pH 6.8) in a chilled pestle and mortar. The homogenate was centrifuged at 12 000 g for 20 min and the resulting supernatant was used for the determination of the enzyme activity. The whole extraction procedure was carried out at 4C. Superoxide dismutase (SOD) was determined according to Paoletti et al. (1986). One unit of SOD was defined as the amount of enzyme that inhibited by 50% the rate of NADH oxidation observed in a blank sample. Peroxidase (POX) activity was measured using a modification of the procedure of MacAdam et al. (1992). The activity was calculated using the extinction coefficient (26.2 mM1cm1 at 470 nm) for tetraguaiacol. One unit of POX was definedas the amount of enzyme that caused the form-ation of 1 mmol tetraguaiacol per minute. Glutamine synthetase (GS) was assayed by the method of Oaks et al. (1980). One unit of GS was defined as the amount of enzyme that caused the formation of 1 mmol L-glutamate g-monohydroxamate per minute.
Phenylalanine ammonia-lyase (PAL) was extracted and determined according to Hyodo and Fujinami (1989). The activity was calculated using the extinction coefficient (9500 M1cm1at 290 nm) for trans-cinna-mate. One unit of PAL was defined as the amount of enzyme that caused the formation of 1 mmol trans-cinnamate per hour.
ABA determination
For the extraction of ABA, leaves were homogenized with a pestle and mortar in extraction solution (80% methanol containing 2% glacial acetic acid). To remove plant pigments and other non-polar compounds which could interfere in the immunoassay, extracts were first passed through a polyvinylpyrrolidone column and C18 cartridges. The eluates were concentrated to dryness by vacuum evaporation and resuspended in Tris-buffered saline before enzyme-linked immunosorbent assay (ELISA). ABA was quantified by ELISA (Walker-Simmons 1987). The ABA immunoassay detection kit (PGR-1) was purchased from Sigma Chemical Co. (St. Louis, MO) and was specific for (þ)-ABA. By evaluating
3H-ABA recovery, ABA loss was less than 3% by the
method described here.
Transpiration rate
The transpiration rate was measured according to Greger and Johansson (1992). The transpiration rate was calculated from the water loss during each interval and converted to a per day per seedling basis.
Expression of data and statistical analysis
In the present study, the third leaves of rice seedlings were used to determine chlorophyll, protein, NH4þ,
MDA, ASC, GSH and ABA. As the f. wt. of –PBZ leaves was no different from that of þPBZ leaves, data were expressed on the basis of initial fresh weight (f. wt.). Statistical differences between measurements (n 5 4) for different treatments or for different times were analysed following theLSDtest.
Results
Growth analysis
PBZ-treated rice seedlings exhibited typical characteristics of triazole treatment, such as reduced shoot length and f. wt. and enhanced primary root length (Table 1; Pinhero and Fletcher 1994). Shoot d. wt., root d. wt. and root f. wt. of PBZ-treated rice seedlings were not significantly different from those of the controls (–PBZ) (Table 1). Less adventi-tious roots were visually observed in þPBZ than in –PBZ seedlings. Both adventitious roots and primary roots were used to determine the f. wt. and d. wt. of roots. This may explain why the d. wt. and f. wt. of þPBZ roots were not significantly different from those of –PBZ roots.
Evaluation of Cd toxicity Biomass production
Cd is readily taken up by rice seedlings, leading to growth reduction (Chen and Kao 1995). Thus, in the Table 1. Growth analysis of rice seedlings 12 days after planting. Seeds were soaked for 18 h in water (–PBZ) or PBZ (þPBZ) and dried. The seedlings were cultivated for 12 days in a Phytotron with natural sun-light at 30C (day)/25C (night) and 90% relative humidity. Significant
difference ataP < 0.01 andbP < 0.05, respectively. PBZ, paclobutrazol.
Parameter –PBZ þPBZ
Shoot length (cm) 13.7 0.3 9.4 0.1a
Root length (cm) 7.9 0.2 13.2 0.3a
Shoot fresh weight (mg seedling1) 53.6 3.1 44.0 1.7b
Shoot dry weight (mg seedling1) 12.7 0.2 12.9 0.1 Root fresh weight (mg seedling1) 47.1 3.9 40.5 3.2
present study, Cd toxicity was first evaluated by biomass production (shoot and root d. wt.). The effect of CdCl2
concentration on shoot and root d. wt. of rice seedlings is presented in Fig. 1. In –PBZ seedlings, the shoot d. wt. was decreased by 0.5 mM CdCl2 and no further
decrease was observed at 1 and 1.5 mM (Fig. 1A). However, CdCl2 (0.5 – 1.5 mM) had no effect on the
shoot d. wt. of þPBZ seedlings (Fig. 1B). Increasing concentrations of CdCl2 from 0.5 to 1.5 mM
progres-sively decreased the root d. wt. of –PBZ seedlings, but had no effect on the root d. wt. of þPBZ rice seedlings. Fig. 2 shows the time courses of biomass production of –PBZ and þPBZ seedlings in the presence or absence of 1.5 mM CdCl2. The d. wt. of the shoot and roots of –
PBZ seedlings treated with CdCl2 was significantly
lower than that of the –PBZ seedlings not treated with CdCl2. However, the d. wt. of the shoot and roots of
þPBZ seedlings was only slightly affected by CdCl2. All
of these results suggest that þPBZ seedlings are Cd tolerant.
Chlorophyll and protein loss
In plants, the most general symptom of Cd toxicity is chlorosis (Das et al. 1997). In previous work, we have shown that rice seedlings treated with CdCl2at high (0.5
– 1.5 mM) and low (10–50 mM) concentrations show chlorosis and protein loss (Hsu and Kao 2003a). However, a longer period (more than 6 days) was
required to show chlorosis when rice seedlings were treated with low concentrations of CdCl2 (Hsu and
Kao 2003a). When seedlings were treated with 0.5 mM CdCl2, chlorosis was first shown in the second
leaves, but not the third leaves, of rice seedlings in a short-term experiment (3 days) (Hsu and Kao 2004). In order to show chlorosis in the third leaves in a short-term experiment, 1.5 mM CdCl2was required. Thus, in
the present study, Cd toxicity in the third leaves exposed to 1.5 mM CdCl2was assessed by the decrease in
ophyll and protein content. A marked decrease in chlor-ophyll and protein was observed in –PBZ leaves after Cd treatment (Fig. 3A,C). In contrast, only a slight decrease in chlorophyll and protein content caused by CdCl2was
observed in þPBZ leaves (Fig. 3B,D). NH4+accumulation
NH4þis a central intermediate of nitrogen metabolism
in plants (Miflin and Lea 1976), but a high content of NH4þ is known to have toxic effects on plant cells
(Givan 1979). Recent studies have demonstrated that NH4þ accumulation in the leaves of rice seedlings is
linked to Cd toxicity (Hsu and Kao 2003b). In this study, we showed that, on treatment with CdCl2, the NH4þ
content increased markedly in –PBZ leaves (Fig. 4A), but only slightly in þPBZ leaves (Fig. 4B). These results suggest that PBZ protects rice seedlings from NH4þ
accumulation caused by Cd toxicity.
GS is the key enzyme in NH4þ assimilation and
catalyses the ATP-dependent condensation of NH4þ
Shoot d. wt. (mg seedling –1 ) 0 2 4 6 8 10 12 14 16 0 0.5 1 1.5 0 2 4 6 8 10 0 2 4 6 8 10 12 14 16 –PBZ +PBZ a b b b a a a a a a a a A B D CdCl2 (mM) 0 0.5 1 1.5 Root d. wt. (mg seedling –1) 0 2 4 6 8 10 a b bc c C
Fig. 1. Effect of CdCl2concentration on the dry weight (d. wt.) of
shoot (A, B) and roots (C, D) of –PBZ and þPBZ rice seedlings (PBZ, paclobutrazol). The d. wt. of shoot and roots were measured 2 days after treatment. Bars indicate the standard error (n 5 4). Values with the same letter are not significantly different at P < 0.05.
0 1 2 3 0 1 2 3 Root d. wt. (mg seedling –1) 0 2 4 6 8 10 12 14 0 5 10 15 20 +CdCl2 –CdCl2 Shoot d. wt. (mg seedling –1) Time (h) 0 2 4 6 8 10 12 14 0 5 10 15 20 –PBZ +PBZ b b A B C D b a c d b b b a c cd d c a a b bc bc c d a b ab cd d c bc
Fig. 2. Changes in the dry weight (d. wt.) of shoot (A, B) and roots (C, D) of –PBZ and þPBZ rice seedlings (PBZ, paclobutrazol) in the presence or absence of CdCl2(1.5 mM). Bars indicate the standard error (n 5 4).
with glutamate to produce glutamine (Miflin and Lea 1976). PAL catalyses the elimination of NH4þ
from phenylalanine and produces trans-cinnamate (Hahlbrock and Grisebach 1979). NH4þ released from
PAL reaction has been shown to be trapped in gluta-mine molecules by the action of GS (Razel et al. 1996, Van Heerden et al. 1996). Sakurai et al. (2001) have provided evidence to show that GS is partly coupled to the reaction of PAL in developing rice leaves. Previous
work has indicated that PAL and GS are the enzymes responsible for NH4þ accumulation in rice leaves
caused by Cd toxicity (Hsu and Kao 2003b). In this study, we demonstrated that the increase in PAL specific activity and the decrease in GS activity caused by CdCl2
were more pronounced in –PBZ than in þPBZ leaves (Fig. 5A,B). These results further support the conclusion that PBZ-treated seedlings are Cd tolerant.
NH4þcan also be produced during nitrate reduction
and photorespiration (Miflin and Lea 1976). It is not known whether NH4þ accumulation in rice leaves
caused by Cd toxicity is mediated via the promotion of nitrate reduction and photorespiration. Further research in this area is likely to be highly rewarding.
Oxidative stress
Unlike Cu and Fe, Cd is not a redox metal, and therefore cannot catalyse Fenton-type reactions yielding active oxygen species (AOS). However, Cd can induce oxida-tive stress indirectly by producing a disturbance in chloroplasts. Thus, Cd produces the degradation of chlorophyll and carotenoids, as well as an inhibition of their biosynthesis (Bazzaz et al. 1974), which can produce disturbances in the electron transport rates of PSI and PSII, leading to the generation of AOS. In a
0 1 2 3 1 2 3 Protein (mg g –1 f. wt.) 0 10 20 30 40 50 60 0 1 2 3 4 5 +CdCl2 –CdCl2 Chlorophyll (mg g –1 f. wt.) Time (h) 0 0 10 20 30 40 50 60 0 1 2 3 4 5 –PBZ +PBZ A B C D a a a a b b c c ab ab a bc c c d a a b b c d c bc a ab bc c d
Fig. 3. Changes in the contents of chlorophyll (A, B) and protein (C, D) in the third leaves of –PBZ and þPBZ rice seedlings (PBZ, paclobutrazol) in the presence or absence of CdCl2(1.5 mM). Bars indicate the
stan-dard error (n 5 4). Values with the same letter are not significantly different at P < 0.05. f. wt., fresh weight.
0 1 2 3 MDA (nmol g –1 f. wt.) 0 20 40 60 80 100 120 140 Time (h) 0 1 2 3 0 20 40 60 80 100 120 140 NH 4 + (nmol g –1 f. wt.) 0 3 6 9 12 15 18 0 3 6 9 12 15 18 +CdCl2 –CdCl2 A B D C –PBZ +PBZ b b b a c d e ab b ab a b c d b b b a a c d b b a a ab b c
Fig. 4. Changes in the contents of NH4þ(A, B) and malondialdehyde
(MDA) (C, D) of –PBZ and þPBZ rice seedlings (PBZ, paclobutrazol) in the presence or absence of CdCl2(1.5 mM). Bars indicate the standard
error (n 5 4). Values with the same letter are not significantly different at P < 0.05. f. wt., fresh weight. GS activity (units g –1 f. wt.) 0 1 2 3 4 5 PAL specific activity (units
mg –1 protein) 0 1 2 3 c b a a b a a a CdCl2 PBZ – + – + – – + + A B
Fig. 5. Effect of CdCl2on phenylalanine ammonia-lyase (PAL) specific
activity (A) and glutamine synthetase (GS) activity (B) in the third leaves of –PBZ and þPBZ rice seedlings (PBZ, paclobutrazol). Rice seedlings were either untreated or treated with CdCl2(1.5 mM) for 2 days. Bars
indicate the standard error (n 5 4). Values with the same letter are not significantly different at P < 0.05. f. wt., fresh weight.
recent review, Schu¨tzendu¨bel and Polle (2002) sug-gested that the depletion of GSH was a critical step in Cd-induced AOS generation.
In previous work, it has been demonstrated that Cd can induce oxidative stress in rice leaves, characterized by an increase in the content of MDA (an indicator of lipid peroxidation) (Hsu and Kao 2004, Kuo and Kao 2004). In the present study, we observed that the increase in the content of MDA (Fig. 4) caused by CdCl2was more pronounced in –PBZ leaves (Fig. 4C)
than in þPBZ leaves (Fig. 4D).
The striking increase in lipid peroxidation seen in –PBZ leaves treated with CdCl2 (Fig. 4C) may reflect
changes in the activities of antioxidant enzymes and contents of antioxidants. Previously, it has been observed that increases in SOD and POX specific activ-ities in rice seedlings take place prior to the occurrence of Cd toxicity (decrease in protein content) (Kuo and Kao 2004). In this study, we showed that the increase in the specific activities of SOD and PO, caused by CdCl2, was more pronounced in –PBZ leaves than in
þPBZ leaves (Fig. 6A,B). ASC is a major antioxidant in photosynthetic and non-photosynthetic tissues, which reacts directly with AOS and is utilized as a substrate for APX-catalysed H2O2 detoxification (Noctor and
Foyer 1998). GSH is involved in ASC regeneration and also functions as a direct scavenger of AOS (Noctor and Foyer 1998). It is clear from Figs 6C and 6D that the decrease in ASC and GSH contents caused by CdCl2
was greater in –PBZ leaves than in þPBZ leaves. However, the depletion of ASC and GSH caused by CdCl2 could not be prevented completely in þPBZ
leaves, suggesting that there was still some oxidative stress in þPBZ seedlings when exposed to CdCl2. All
of the results presented here indicate that PBZ can be used to protect rice seedlings from oxidative stress caused by Cd toxicity.
ABA accumulation in +PBZ leaves
Previously, we have shown that an increase in endo-genous ABA content is closely related to the Cd
tolerance of rice seedlings (Hsu and Kao 2003a). In this study, we showed that CdCl2treatment resulted in
an increase in endogenous ABA in þPBZ leaves, but not in –PBZ leaves (Table 2), suggesting that ABA may play a role in Cd tolerance.
Flu treatment
The role of ABA in PBZ-induced Cd tolerance was tested by using an inhibitor of ABA biosynthesis, Flu, which blocks the conversion of phytoene to phytofluene in the carotenoid biosynthesis pathway (Kowalczyk-Schro¨der and Sandmann 1992). Flu was observed to inhibit the increase in ABA content (Fig. 7E) and to enhance Cd toxicity (as judged by biomass production and the contents of chlorophyll and protein) in þPBZ seedlings (Fig. 7A – D). The effect of Flu on Cd toxicity in þPBZ seedlings was reversed by the application of
CdCl2 PBZ – + – + – – + + – + – + – – + +
SOD specific activity (units
mg –1 protein) 0 1 2 a a a b ASC (nmol g –1 f. wt.) 0 5 10 15 20 25 a a b c GSH (nmol g –1 f. wt.) 0.0 0.2 0.4 0.6 a b c d A
POX specific activity (units
mg –1 protein) 0 1 2 a a b c C B D
Fig. 6. Effect of CdCl2on the specific activities of superoxide dismutase
(SOD) (A) and peroxidase (POX) (B) and the contents of ascorbate (ASC) (C) and reduced glutathione (GSH) (D) in the third leaves of –PBZ and þPBZ rice seedlings (PBZ, paclobutrazol). Rice seedlings were either untreated or treated with CdCl2(1.5 mM) for 2 days. Bars indicate
the standard error (n 5 4). Values with the same letter are not signifi-cantly different at P < 0.05. f. wt., fresh weight.
Table 2. Effect of CdCl2on abscisic acid (ABA) content, transpiration rate and Cd content in the third leaves of –PBZ and þPBZ rice seedlings. Rice
seedlings were either untreated or treated with CdCl2(1.5 mM) for 2 days. Values with the same letter are not significantly different at P < 0.05. d.
wt., dry weight; f. wt., fresh weight; PBZ, paclobutrazol.
Treatment ABA content (pmol g1f. wt.) Transpiration rate (mg H
2O seedling1day1) Cd content (mg g1d. wt.)
–PBZ – CdCl2 334.8 21.0 (b) 566 39.0 (d) 4.15 0.34 (a)
þ CdCl2 337.6 20.7 (b) 145 9.1 (b) 33.68 1.24 (c)
þPBZ – CdCl2 255.1 27.6 (a) 456 20.5 (c) 5.33 0.84 (a)
ABA (Fig. 7A – D). It should be noted that CdCl2þABA
treatment resulted in a similar biomass production as CdCl2 treatment (Fig. 7A,B). These results suggest that
the ABA content in the leaves of þPBZ seedlings treated with CdCl2 is sufficient to exert an effect on biomass
production.
Cd content
Table 2 shows the effect of CdCl2on the Cd content in –
PBZ and þPBZ leaves. The Cd content in þPBZ leaves increased about four-fold after Cd treatment (Table 2). However, an eight-fold increase in Cd content in Cd-treated –PBZ leaves was observed (Table 2). Flu treat-ment caused an increase in Cd content in the leaves of þPBZ seedlings exposed to CdCl2(Fig. 8A). The effect
of Flu on the Cd content in Cd-treated leaves of þPBZ seedlings was reversed by the application of ABA (Fig. 8A).
Transpiration rate
In the absence of CdCl2, the transpiration rate of –PBZ
seedlings was observed to be higher than that of þPBZ seedlings (Table 2). Cd treatment decreased the tran-spiration rate in both –PBZ and þPBZ seedlings (Table 2). However, the decrease in the transpiration rate in response to CdCl2 was less pronounced in
–PBZ seedlings than in þPBZ seedlings (Table 2). Flu treatment resulted in an increase in the transpiration rate in þPBZ seedlings treated with CdCl2(Fig. 8B). The
effect of Flu on the transpiration rate in þPBZ seedlings treated with CdCl2 was reversed by ABA application
(Fig. 8B). Discussion
Cd causes biomass reduction (Chen and Kao 1995), chlorophyll and protein loss (Hsu and Kao 2003a), NH4þaccumulation (Hsu and Kao 2003b) and oxidative
stress (Kuo and Kao 2004) in rice seedlings. In the present study, we evaluated Cd toxicity by the decrease in biomass production, decrease in chlorophyll and protein content, increase in NH4þ content and
Root d.wt. (mg seedling –1) 0 2 4 6 8 10 Shoot d.wt. (mg seedling –1) 0 5 10 15 20 a a CdCl2 Flu ABA – + + + + – – + – + – – – + + a a a b a ab b c A B Protein (mg g –1 f. wt.) 0 10 20 30 40 50 Chlorophyll (mg g –1 f. wt.) 0 1 2 3 4 5 6 CdCl2 Flu ABA – + + + – – + + – – – + a b c d a a a b ABA (pmol g –1 f. wt.) 0 200 400 600 C D E c b a c
Fig. 7. Effect of fluridone (Flu, 0.2 mM) and abscisic acid (ABA) (5 mM) on the dry weight (d. wt.) of shoot (A) and root (B) and the contents of chlorophyll (C), protein (D) and ABA (E) in the third leaves of þPBZ rice seedlings (PBZ, paclobutrazol) treated or not with CdCl2(1.5 mM). All
measurements were made 2 days after treatment. Bars indicate the standard error (n 5 4). Values with the same letter are not significantly different at P < 0.05. f. wt., fresh weight.
Transpiration rate (mg H2 O seedling –1 day –1) 0 100 200 300 400 500 600 CdCl2 Flu ABA – + + + – + + – – – – + c b a c Cd ( µ g g –1 d.wt.) 0 10 20 30 40 50 60 b c a b A B
Fig. 8. Effect of fluridone (Flu, 0.2 mM) and abscisic acid (ABA) (5 mM) on the Cd content (A) in the third leaves and the transpiration rate (B) of þPBZ rice seedlings (PBZ, paclobutrazol) treated or not with CdCl2
(1.5 mM). The Cd content was measured 2 days after treatment, and the transpiration rate was measured 1 day after treatment. Bars indicate the standard error (n 5 4). Values with the same letter are not signifi-cantly different at P < 0.05. d. wt., dry weight.
induction of oxidative stress. On the basis of these criteria, we demonstrated that PBZ applied to seeds was able to protect rice seedlings from Cd toxicity. The protective effect of uniconazole, a member of the triazole family, against Cd stress has also been described previously (Singh 1993).
The present study indicated that ABA was involved in the Cd tolerance of þPBZ seedlings. This conclusion was based on the following observations: (1) the increase in the endogenous ABA content in response to Cd in þPBZ leaves was more pronounced than that in –PBZ leaves (Table 2); (2) Flu treatment led to a decrease in the ABA content, as well as Cd tolerance, of þPBZ seedlings (Fig. 7); and (3) the effect of Flu on the Cd toxicity of þPBZ seedlings was reversed by the application of ABA (Fig. 7). These results suggest that the regulation of endogenous ABA biosynthesis under Cd stress is correlated with the tolerance of þPBZ seed-lings. As Flu is an inhibitor of ABA biosynthesis through the carotenoid pathway (Kowalczyk-Schro¨der and Sandmann 1992), the effects of this inhibitor on þPBZ leaves may imply that the ABA biosynthesis pathway in response to Cd appears to be the same as that estab-lished in other stress conditions (Zeevaart and Creelman 1988, Seo and Koshiba 2002). In addition, the defect in ABA accumulation in –PBZ leaves may account for the Cd intolerance of –PBZ seedlings.
Plants have a range of potential mechanisms at the cellular level that may be involved in the detoxification of, and thus tolerance to, heavy metals. These all appear to be involved primarily in avoiding the build-up of toxic concentrations at sensitive sites within the cell, and thus preventing damaging effects (Hall 2002). In this context, reduced translocation of Cd to the shoot appears to be a possible mechanism of Cd tolerance in the shoot. Cd translocation to the shoot has been sug-gested to be driven by transpiration (Salt et al. 1995). Cd has been shown to decrease the transpiration rate in several plants (Bazzaz et al. 1974, Kirkham 1978, Lamoreaux and Chaney 1978, Hagemeyer et al. 1986, Schlegel et al. 1987, Hsu and Kao 2003a). Here, we also observed that Cd treatment decreased the transpiration rate of –PBZ and þPBZ seedlings (Table 2). Cd treat-ment decreased the transpiration rate in –PBZ and þPBZ seedlings to about 74% and 88% of the control value, respectively (Table 2). Thus, the decrease in the transpiration rate of –PBZ seedlings (which were unable to accumulate ABA) caused by Cd was less than that in þPBZ seedlings (which accumulated ABA), and conse-quently resulted in a higher Cd content in –PBZ than in þPBZ seedlings (Table 2). The effect of Flu on þPBZ seedlings indicated that not only was ABA biosynthesis blocked, but the transpiration rate and Cd content were
increased (Figs 7E and 8). Furthermore, the effect of Flu on the transpiration rate and Cd content of þPBZ seed-lings was reversed by the application of ABA (Fig. 8). It appears that the increase in endogenous ABA content is closely related to the Cd tolerance of þPBZ seedlings. ABA may exert its regulatory effect on the transpiration rate, decreasing the translocation of Cd to the shoot.
Stress-tolerant plants often grown more slowly than stress-intolerant plants. It has been hypothe-sized that þPBZ plants show a better quality of growth than –PBZ plants under stress conditions as a result of the slower growth rate or metabolism of the former (Abou El-Khashab et al. 1997). Thus, the possibility that ABA may also exert its effect on the metabolism of þPBZ seedlings cannot be excluded.
PBZ inhibits GA biosynthesis (Graebe 1987, Rademacher et al. 1987). It has been shown that the application of GA counters both the growth inhibitory and stress-protective effects of triazoles (Guoping 1997, Vettakkorumakankav et al. 1999, Sarkar et al. 2004). An interesting question then arises: is a decrease in both endogenous GA content and shoot length essential to enhance Cd tolerance in rice seedlings? Future work will focus on this question by examining the Cd tolerance in GA-responsive and GA non-responsive dwarf mutants of rice.
Acknowledgements – This work was supported financially by the National Science Council of the Republic of China.
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