Triacontanol can protect Erythrina variegata from cadmium toxicity

J. Plant Physiol. 158. 1487 – 1490 (2001)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

Short Communication Triacontanol can protect Erythrina variegata from cadmium toxicity Krishnasamy Muthuchelian1, Massimo Bertamini2, Namachevayam Nedunchezhian2, 3 * 1

School of Energy, Environment and Natural Resources, Madurai Kamaraj University, Madurai – 625 021, India

2

Istituto Agrario di San Michele all’ Adige, 38010 San Michele all’ Adige, Italy

3

Current address: Government Higher Secondary School, Vellimedupettai – 604 207, Tindivanam, India

Received May 22, 2001 · Accepted July 16, 2001

Summary The simultaneous effect of 0, 10, 100 and 1000 µmol/L Cd2 + [Cd(NO3)2 × 4H2O] and 1 mg kg –1 (H2O) triacontanol [TRIA] spray on certain parameters of growth, pigments, starch, 14CO2 fixation, ribulose1,5-bisphosphate carboxylase (Rubisco), nitrate reductase (NR) and photosynthesis in Erythrina variegata seedlings was studied. With increasing Cd2 + concentration in the nutrient solution the monitored activities decreased. When the seedlings were subsequently sprayed with triacontanol the cadmium effect was partially or completely reversed indicating that TRIA can protect from cadmium toxicity. Key words: carotenoids – chlorophyll – electron transport – Rubisco – triacontanol Abbreviations: BQ p-benzoquinone. – DCPIP 2,6-dichlorophenol indophenol. – DPC diphenyl cabazide. – DTT dithiothreitol. – MV methyl viologen. – PS photosystem. – Rubisco ribulose-1,5bisphosphate carboxylase

Introduction Over the last few years, heavy metal toxicity in plants has received considerable attention as a consquence of increased environmental pollution (Adriano 1986). In the soil, they function as stress factors causing physiological disorders after having been absorbed by the root system (Moustakas et al. 1994). Cadmium is a major environmental contaminant (Friberg et al. 1974). In high concentrations, this * E-mail corresponding author: [email protected]

metal manifests considerably more phytotoxicity than other heavy metals (Baszynski 1986). Physiological responses of plants to toxic Cd concentrations include growth inhibition, but also changes in various biochemical characteristics, such as higher activity of dark respiration (Lee et al. 1976) and hydrolytic enzymes (Van Assche et al. 1988) or decreased content of soluble proteins (Sheoran et al. 1990). At a low concentration, Cd is not toxic to plants, but with increasing concentration (Usha-Keshan and Mukher 1997) leaf chlorosis accompanied by a retardation of plastid devel0176-1617/01/158/11–1487 $ 15.00/0

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Krishnasamy Muthuchelian, Massimo Bertamini, Namachevayam Nedunchezhian

opment (Ghoshroy and Nadakavukaren 1990) and degradation of their ultrastructure (Stoyanova and Tchakalova 1997) has been observed. Substantial inhibition of PS II activity by Cd has been reported and is accompanied or followed by the disapperance of granal stacks, degradation of thylakoid acyl lipids, release of some polypeptides associated with the oxygen evolving complex and disorganization of LHC II antenna system (Baszynski et al. 1980, Krupa and Baszynski 1985, Krupa et al. 1993, Horvath et al. 1996, Ghorbanli et al. 1999, Purohit and Singh 1999). Triacontanol [CH3(CH2)28CH2OH] (TRIA) and its second messenger L( + )-adenosine possess a growth stimulating activity in plants (Ries 1991). Most profound effect of TRIA is increase in growth, biomass, free amino acids, reducing sugars, increase in photosynthetic activities and soluble proteins (Muthuchelian et al. 1995). The objective of this study was to investigate if TRIA can protect from Cd toxicity by assessing the concerted effect of TRIA and cadmium on Erythrina variegata seedlings with particular emphasis on growth, pigments, Rubisco, and photosynthetic activity.

Solutions were renewed after 10 days. Plants were grown in a growth chamber under irradiance of 350 µmol m – 2 s –1, 16 h photoperiod, temperature of 25 ± 2 ˚C, and relative humidity of 80 %. Twenty days old seedlings were analyzed. Dry mass was determined after drying the plant material at 105 ˚C for 10 h. Concentrations of chlorophylls and carotenoids were determined spectrophotometrically by the method of Lichtenthaler (1987). Soluble starch and soluble proteins were extracted and concentrations determined following the method of McCready et al. (1950) and Bradford (1976). Thylakoid membranes were isolated from leaves as described by Berthhold et al. (1981). Whole chain electron transport (H2O → MV) and partial reactions of photosynthetic electron transport mediated by PS II (H2O → BQ), PS I (DCPIPH2 → MV) and the rate of DCPIP photoreduction were measured as described by Nedunchezhian et al. (1997). Fully expanded leaves were cut into small pieces and homogenized in a grinding medium of 50 mmol/L Tris-HCl, pH 7.8, 10 mmol/L MgCl2, 5 mol/L DTT and 0.25 mmol/L EDTA. The extract was clarified by centrifugation at 10,000 ×g for 10 min. The clear supernatant was decanted slowly and used for Rubisco analysis. The assay for Rubisco activity was carried out as described by Nedunchezhian and Kulandaivelu (1991). The rate of 14CO2 fixation and nitrate reductase activity were measured according to the method of Muthuchelian et al. (1995).

Materials and Methods Seeds of Erythrina variegata Lam. were germinated for 7 days in a Petri dishes with distilled water at 27 ˚C. Young seedlings were transferred to 1000 cm3 plastic containers with nutrient solution containing 5 mmol/L KNO3, 5 mmol/L Ca(NO3) × 4H2O, 2 mmol/L MgSO4 × 4H2O, 1 mmol/L KH2PO4, 0.09 mmol/L NH4Fe(SO4), and micronutrients. The plants (five per container) were divided into eight equal groups. Plants in the 1st group (control) were treated with a nutrient solution without cadmium. Plants in the 2nd, 3rd and 4th groups were grown at three different concentrations of Cd2 + (Cd(NO3)2 × 4H2O : 10, 100 and 1000 µmol/L, respectively). Seedlings of the 5th group were sprayed with growth stimulator TRIA [1 mg kg –1 (H2O)] (NOCIL, India) and Tween 20 (1 g dm – 3) added as a surfactant by hand pump sprayer. Care was taken to wet both sides of the leaf. The 6th, 7th and 8th groups of seedlings were treated with Cd2 + (10, 100 and 1000 µmol/L, respectively) and simultaneously sprayed with TRIA. All solutions were kept at pH 5.8, aerated and adjusted daily with distilled water.

Results and Discussion Plants treated with different concentrations of Cd2 + had reduced fresh and dry masses, chlorophyll and carotenoid contents (Table 1). The reduction increased with increasing concentration of Cd2 + . The marked reduction of total chlorophyll in Cd2 + treated seedlings is mainly due to decrease of both chlorophyll a and chlorophyll b. Also, Cd2 + probably enhanced the chlorophyllase activity in leaves. These observations are in good agreement with reports on Abelmoschus esculentus (Purohit and Singh 1999) and Glycine max (Ghorbanli et al. 1999). TRIA treatment resulted in significantly higher fresh and dry masses, chlorophyll and carotenoid contents in control plants and in all Cd2 + treatments. Even at

Table 1. The effect of different concentrations of cadmium on growth [mg plant –1], total chlorophyll, carotenoids and starch [mg g –1 (f. m.)] contents in non-treated and TRIA treated 20 days old Erythrina variegata seedlings. Values in parentheses show percent reduction relative to 0 Cd or 0 Cd + TRIA treatments. Each value represents the average ± SD of at least 4 replicates. Cd concentration (µmol/L)

fresh mass

dry mass

chlorophyll

0 10 100 1000

653.6 ± 20.1 588.2 ± 15.2 418.3 ± 16.4 235.2 ± 9.8

(0) (10) (36) (64)

81.7 ± 4.1 71.9 ± 3.5 49.8 ± 2.8 26.1 ± 1.2

(0) (12) (39) (68)

1.82 ± 0.98 1.55 ± 0.06 1.02 ± 0.04 0.49 ± 0.01

0 + TRIA 10 + TRIA 100 + TRIA 1000 + TRIA

791.3 ± 24.3 759.6 ± 19.8 593.5 ± 17.5 395.6 ± 12.3

(0) (4) (25) (50)

96.5 ± 4.1 91.7 ± 4.4 71.4 ± 3.9 46.3 ± 2.1

(0) (5) (26) (52)

2.43 ± 0.11 2.24 ± 0.10 1.70 ± 0.08 1.02 ± 0.05

carotenoids

starch

(0) (15) (44) (73)

0.56 ± 0.01 (0) 0.49 ± 0.02 (12) 0.30 ± 0.01 (46) 0.16 ± 0.003 (71)

16.82 ± 0.60 14.46 ± 0.42 9.58 ± 0.31 4.37 ± 0.18

(0) (14) (43) (74)

(0) (8) (30) (58)

0.70 ± 0.02 0.66 ± 0.03 0.48 ± 0.02 0.31 ± 0.01

23.56 ± 1.04 21.60 ± 1.02 16.49 ± 0.76 9.65 ± 0.31

(0) (8) (30) (59)

(0) (6) (32) (56)

Triacontanol reduces cadmium toxicity

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Table 2. The effect of different concentrations of cadmium on whole chain (H2O → MV), PS II (H2O → BQ; H2O → DCPIP; DPC → DCPIP) and PS I (DCPIPH2 → MV) electron activities [µmol(O2) mg Chl –1 h –1] in non-treated and TRIA treated 20 days old Erythrina variegata seedlings. Values in parentheses show percent reduction relative to 0 Cd or 0 Cd + TRIA treatments. Each value represents the average ± SD of at least 4 replicates. Cd concentration (µmol/L)

whole chain (H2O → MV)

PS II (H2O → BQ)

PS II (H2O → DCPIP)

PS II (DPC -- > DCPIP)

PS I (DCPIPH2 -- > MV)

0 10 100 1000

92.1 ± 3.2 (0) 73.7 ± 2.9 (20) 47.9 ± 1.6 (48) 23.0 ± 1.0 (75)

124.6 ± 4.6 102.2 ± 3.9 76.0 ± 3.1 39.8 ± 1.5

(0) (18) (39) (68)

102.2 ± 4.2 85.8 ± 2.6 59.2 ± 2.2 30.6 ± 1.4

(0) (16) (42) (70)

110.8 ± 3.5 97.5 ± 2.2 87.5 ± 2.4 70.9 ± 1.9

(0) (12) (21) (36)

260.4 ± 6.5 247.3 ± 5.6 231.7 ± 6.2 213.5 ± 4.5

(0) (5) (11) (18)

0 + TRIA 10 + TRIA 100 + TRIA 1000 + TRIA

122.7 ± 3.5 107.9 ± 3.8 83.4 ± 3.1 53.9 ± 2.1

155.7 ± 4.2 141.6 ± 4.6 115.2 ± 3.3 79.4 ± 2.1

(0) (9) (26) (49)

125.7 ± 3.8 113.1 ± 2.9 87.9 ± 2.5 60.3 ± 2.1

(0) (10) (30) (52)

129.2 ± 3.9 118.8 ± 3.5 98.1 ± 2.5 71.0 ± 2.6

(0) (8) (24) (45)

314.8 ± 7.1 308.5 ± 6.3 295.9 ± 5.5 280.2 ± 7.2

(0) (2) (6) (11)

(0) (12) (32) (56)

high Cd2 + (1000 µmol/L), fresh and dry masses, chlorophylls and carotenoids were protected. The higher values of pigments in TRIA treated leaves, even in the presence of Cd2 + , suggest that TRIA can prevent symptoms of Cd phytotoxicity. TRIA increases the synthesis of both chlorophyll a and chlorophyll b in flooded Erythrina variegata seedlings (Muthuchelian et al. 1995). The content of starch decreases in Cd2 + treated seedlings (Table 1). TRIA reverses the effect. TRIA application also increases the amount of starch, sugar and soluble proteins in salt stressed and flooded Erythrina variegata seedlings (Muthuchelian et al. 1995, 1996). The whole chain electron transport was markedly inhibited in Cd2 + treated seedlings. However, the PS I activity was much less diminished (Krupa and Baszynski 1985). In contrast to PS I, the PS II activity measured by both benzoquinone (BQ) and DCPIP was significantly inhibited (Table 2). DCPIP collects electrons after plastoquinone (PQ) but BQ at the reducing side of PQ (Lien and Bannister 1971). As the PS II activity loss due to Cd2 + was similar in the systems H2O → BQ and H2O → DCPIP, the site of Cd2 + action must be prior to PQ in the electron transport. DPC, an artificial electron donor for PS II, donates electrons close to the PS II reaction center (Packham et al. 1982). Thus, the inhibition of PS II could be due to an alteration of the water-splitting system, since the addition of DPC restored significantly its activity. So Cd2 + acts on the donor side of PS II. These observations are in good agreement with reports by Tukendorf and Baszynski (1991), Siedlecka and Baszynski (1993) and Purohit and Singh (1999). TRIA application reduced the inhibition by Cd2 + in both whole chain and PS II activities. The effects on PS I activity were much smaller in size but of a similar character (Table 2). Total soluble protein content and Rubisco activity were reduced markedly in Cd2 + treated leaves, whereas TRIA treatment increased it (Table 3). The relatively low level of soluble proteins in Cd2 + treated seedlings may have been due to decrease in the synthesis of Rubisco, the major soluble protein

Table 3. The effect of different concentrations of cadmium on 14CO2 fixation, Rubisco activity [µmol(CO2) mg protein –1 h –1] and nitrate reductase activity [µmol(NO2) mg Chl –1] in non-treated and TRIA treated 20 days old Erythrina variegata seedlings. Values in parentheses show percent reduction relative to 0 Cd or 0 Cd + TRIA treatments. Each value represents the average ± SD of at least 4 replicates. Cd concentration (µmol/L)

14

0 10 100 1000

28.1 ± 0.9 24.3 ± 1.0 16.0 ± 0.4 8.9 ± 0.1

(0) (14) (43) (68)

33.3 ± 1.2 29.3 ± 0.9 19.6 ± 0.5 9.3 ± 0.2

(0) (12) (41) (72)

112.4 ± 2.6 96.6 ± 2.3 68.5 ± 1.9 39.3 ± 1.1

(0) (14) (39) (65)

0 + TRIA 10 + TRIA 100 + TRIA 1000 + TRIA

34.5 ± 1.1 31.3 ± 1.0 23.8 ± 0.9 15.8 ± 0.5

(0) (9) (33) (54)

42.2 ± 1.5 34.8 ± 1.1 30.4 ± 0.9 17.7 ± 0.4

(0) (8) (28) (58)

140.5 ± 3.5 130.6 ± 2.5 105.3 ± 3.1 68.8 ± 1.9

(0) (7) (25) (51)

CO2 fixation

Rubisco activity

nitrate reductase activity

of leaf. A loss of protein in Cd2 + treated leaves would partially account for damaged chloroplasts or could be the result of inhibition of protein synthesis. The reduction in the overall photosynthetic rates correlates well with the decreased Rubisco activity in treated leaves. A marked reduction of Rubisco activity was observed in 1000 µmol/L Cd2 + treated leaves (Table 3). Such reduction was due to inhibition of protein synthesis induced by Cd2 + . The reduction in Rubisco activity in Cd2 + treated plants and improvement by TRIA treatment correlated with the 14CO2 fixation (Table 3). The reduction in 14CO2 fixation of Cd treated seedlings was probably an indirect effect due to the destruction of photosynthetic pigments (as evidenced by the present results). Seedlings grown in the presence of Cd2 + had a relatively low nitrate reductase activity (Table 3) that was ameliorated by TRIA application. The reduction in nitrate reductase activity may reflect a balance between synthesis or inactivation on one hand, and degradation or inactivation on the other. The

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changes in intercellular pH values due to Cd2 + stress might decrease the transfer of nitrate (substrate) from a storage pool to an active cytoplasmic pool accessible to the enzyme. The inhibition of nitrate reductase activity might be also due to the inhibition of protein synthesis or it might have stemmed out from decreased rate of photosynthetic supply in the Cd2 + treated leaves. The enhancement of growth by TRIA might result from an increase in effective leaf area, stimulation of photosynthesis, Rubisco and nitrate reductase activities, modification in partitioning of photosynthates, or from their cooperative effects (Muthuchelian et al. 1994, 1995, 1996). In the present work, the application of TRIA to cadmium treated seedlings reduced the Cd2 + effects by partially restoring/maintaining the photosynthetic machinery. In these plants increases in growth, pigments, Rubisco, nitrate reductase and photosynthetic activities were demonstrated. In conclusion, TRIA application can effectively counterbalance the toxicity of Cd2 + containing nutrient solutions in E. variegata. Acknowledgements. This work was in part supported by a grant from Provincia Autonoma di Trento and National Council of Research (CNR).

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