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Role of rice shoot vacuoles in copper toxicity regulation
Environmental and Experimental Botany 39 (1998) 197 – 202
Role of rice shoot vacuoles in copper toxicity regulation Fernando C. Lidon *, Fernando S. Henriques Plant Biology Unit, Faculdade de Cieˆncias e Tecnologia, Uni6ersidade No6a de Lisboa, 2825 Monte da Caparica, Lisboa, Portugal Accepted 4 September 1997
Abstract Rice (Oryza sati6a L. cv. Safari) plants were grown over a 30-day period in nutrient solutions containing 0.002–1.25 mg l − 1 Cu concentrations. It was found that increasing Cu concentrations led to increasing Cu contents in the plant’s shoot and to an inhibition of its growth. Transmission electron microscopy showed no obvious ultrastructural changes in the shoot tissues, except for electron-dense deposits adherent to the tonoplast, appearing from the 0.25 mg l − 1 Cu treatment onwards. On a protein basis, Cu and SH groups concentrations in isolated shoot vacuoles increased by 41% and 120%, respectively, from the 0.01 to the 1.25 mg l − 1 Cu treatments. In spite of increased vacuolar Cu contents, the activities of NADH-cytochrome c reductase and acid phosphatase were not inhibited, apparently because of metal binding to sulphydryl groups contained in the vacuole. © 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Copper toxicity; Oryza sati6a; Shoot; Vacuole; Acid phosphatase; NADH-cytochrome c reductase
1. Introduction Heavy metal pollution is one of the current most troublesome environmental problems due to the widespread use of metals for industrial and agricultural purposes (Nriagu and Pacyna, 1988; Fernandes and Henriques, 1991). Cu is an essential micronutrient for plants, but most species are very sensitive to high concentrations of this metal, which cause metabolic disturbances and growth inhibition (Fernandes and Henriques, 1991; Lidon and Henriques, 1991; Lidon et al., 1993; Ouzounidou, 1994). Plants growing in Cu-enriched sub* Corresponding author. Tel.: + 351 1 4416855 or +351 1 2954464 ext. 1101; fax: +351 1 4416011 or + 351 1 2954461.
strates developed a variety of defense mechanisms against its toxicity, the most common being the metal sequestration in cell compartments where it least interferes with vital metabolism and/or its inactivation by reaction with various cellular components (Verkleij and Schat, 1990; Fernandes and Henriques, 1991). Increased production of the metal-binding compounds metallothioneins and phytochelatins has been reported to occur in response to high cellular levels of Cu, being particularly important for metal detoxification in non-tolerant plants (Schultz and Hutchinson, 1988; Verkleij et al., 1989; Schat and Kalff, 1992; Murphy and Taiz, 1995). Our previous work with rice grown in Cu-enriched nutrient solutions has shown that the
S0098-8472/98/$19.00 © 1998 Published by Elsevier Science B.V. All rights reserved. PII S0098-8472(97)00025-7
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threshold toxic concentration for this metal was 35 mg Cu g − 1 tissue dw and also that this threshold value was reached when the plants were grown in 0.01 mg l − 1 Cu solutions (Lidon and Henriques, 1992). The roots of plants subjected to higher Cu concentrations accumulated most of the excess metal inside their vacuoles, precipitated in the form of granules randomly scattered within them (Lidon and Henriques, 1994). The present work was undertaken to test the hypothesis that excess Cu translocated to the shoot was also preferentially localized inside the vacuoles and, if so, how it affected enzyme activities in this cell compartment.
2. Materials and methods Rice (Oryza sati6a L. cv. Safari) seeds were washed, sterilized and germinated as described previously (Lidon and Henriques, 1992). The seedlings (50 per pot) were hydroponically grown for 30 days in cylindric 2 l pots at 35 – 37oC/25– 27oC day/night temperatures, under 250 mm m − 2 s − 1 PAR irradiance over a 12 h-day period. The nutrient solution was that of Yoshida et al. (1976) containing (mg l − 1) 40 N, 10 P, 40 K, 40 Ca, 40 Mg, 0.5 Mn, 0.2 B, 0.05 Mo, 0.01 Zn. Iron was added as a hexahydrated FeCl3 at 2 mg l − 1, chelated by 50 mM monohydrated citric acid. Cu concentrations were (mg l − 1) 0.002, 0.01, 0.05, 0.25 and 1.25. The solutions were adjusted daily to pH 5.5, the volume kept constant at the original level and the solutions were renewed every 5 days. For elemental analysis, shoot samples were dried for 24 h at 80oC followed by 3 days at 100oC and 1 g of dry material was successively digested in a nitric:perchloric (5:2, v/v) and nitric:sulfuric:perchloric (10:1:10 v/v/v) acid mixtures (Jackson, 1958). Cu concentrations were determined by atomic absorption spectrophotometry using a Perkin-Elmer model 3030. For electron microscopy, pieces of leaf tissue were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.0) for 2 h, buffer-washed and post-fixed in cacodylate-buffered 1% osmium tetroxide for 1 h. After buffer-washing and dehy-
dration in a graded series of methanol, the samples were treated with propylene oxide and embedded in Epon (Luft, 1961). Ultrathin sections were cut with a LKB ultramicrotome, double stained with aqueous uranyl acetate and lead citrate (Reynolds 1963) and examined in a Philips TEM 300 electron microscope at 80 kV. Isolation of leaf protoplasts followed the methods of Rubinstein (1978), as modified by Kelly and Weskich (1988). Vacuoles were obtained following the procedure of Kringstad et al. (1980). The concentration of SH groups was determined according to Habbeb (1972), using the Ellman reagent. The assay for acid phosphatase (E.C. 3.1.3.1) followed the method of Parida and Mishra (1980), with some minor modifications. The enzyme activity was calculated on the basis of net inorganic phosphorus released during the incubation period, following Jaffe and Galston (1966). The assay for NADH cytochrome c reductase (E.C. 1.6.99.3) followed King and Khanna (1980), after enzyme extraction according to Kamada and Harada (1984). Enzyme activity (1 U) was defined as the change of 1.0 in the absorbance at 550 nm per mg protein. Protein concentration was determined according to the method of Bradford (1976), using a BSA standard curve.
3. Results Table 1 shows that rice shoot lengths decreased when Cu concentrations in the nutrient solutions surpassed 0.05 mg l − 1 and that these decreases were particularly prominent for the two highest Cu treatments. Such decreases in shoot lengths were parallel to increases in tissue Cu concentrations (Table 1), again particularly marked for the last two treatments, reflecting the metal interference with normal growth processes. Examination by transmission electron microscopy of shoot tissues revealed electron dense deposits adherent to the tonoplast of a large number of cells (Fig. 1B) in plants grown in solutions containing 0.25 mg l − 1 or higher Cu concentrations. Leaves from lower Cu treatments exhibited no apparent ultra-
F.C. Lidon, F.S. Henriques / En6ironmental and Experimental Botany 39 (1998) 197–202
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Table 1 Lengths and Cu concentrations in shoot tissues of Cu-treated rice plants Cu treatments (mg Cu/l)
0.002 0.01 0.05 0.25 1.25
Shoot lengths (cm 9S.E.)
43 9 3.0 439 3.0 399 2.5 329 2.5 23 9 1.5
(a) (a) (a, b) (b, c) (c)
Cu concentration in the shoot mg Cu g−1 9S.E.
ng Cu mg−1 Protein9 S.E.
17.5 91.2 21.5 91.6 27.0 92.1 46.5 93.3 95.0 9 6.9
367 925 (c) 376 928 (c) 524 941 (c) 978 969 /(b) 2672 9194 (a)
(c) (c) (c) (b) (a)
Each value is the mean+ 6 S.E. based on three replicates of three independent series. In a one way ANOVA, the F-ratio test indicated the existence of differences among treatments for a PB0.001; different letters indicate significant differences among the treatments in a multiple range analysis for a 95% confidence level.
structural changes (Fig. 1A). Vacuoles isolated from leaf tissues showed that their Cu levels increased from the 0.05 mg l − 1 Cu treatment on-
wards, to reach a maximum value in the 1.25 mg l − 1 Cu treatment (Table 2). The concentrations of SH groups in isolated shoot vacuoles followed a pattern similar to the Cu concentrations, but with a more pronounced increase in the 1.25 mg l − 1 Cu treatment (Table 2). Thus, the SH/Cu ratio increased continuously with increasing Cu concentrations, as it is shown in the last column of Table 2. Activities of acid phosphatase and NADH-cytochrome c reductase from leaves grown at increasing Cu supply are shown in Table 3. When expressed per unit protein, the activity of the acid phosphatase displayed a continuous increase with increasing Cu exposure. The NADHcytochrome c reductase activity reached a maximum at 0.05 mg l − 1 Cu and decreased thereafter; it is worth noting, however, that the enzyme activity remained much higher at 0.25 and 1.25 mg l − 1 Cu than in the 0.002 and 0.01 mg l − 1 Cu treatments.
4. Discussion
Fig. 1. Cell ultrastructure of leaves from rice plants grown in (A) 0.01 and (B) 1.25 mg l − 1 Cu treatments. Note the electron-dense deposits lining the vacuoles of high Cu-treated plants.
The data presented here show that between the lowest and the highest Cu treatments tested, the shoot region of rice plants registered a 7-fold copper increase, whereas Cu content in vacuoles increased only 1.4 times, on a protein basis. The physiological meaning of this comparison may be debatable, as Cu in the shoot and particularly that accumulated in the vacuole, is most certainly also bound to compounds other than proteins,
200
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Table 2 Cu and SH group concentrations in vacuoles isolated from leaves of Cu-treated rice plants Cu treatments
Cu (ng Cu mg−1 protein)
SH groups (mg SH mg−1 protein 9S.E.)
SH/Cu (mol ratio×10−5)
0.002 0.01 0.05 0.25 1.25
0.17 9 0.015 0.17 9 0.025 0.19 9 0.015 0.21 9 0.035 0.24 9 0.035
26.9 9 3.94 28.7 9 3.74 33.1 9 3.55 36.7 9 3.06 59.2 9 7.07
3.04 3.25 3.35 3.36 4.74
(b) (b) (a, b) (a) (a)
(b) (b) (b) (b) (a)
Each value is the mean+ 6 S.E. based on three replicates of three independent series. In a one way ANOVA test different letters indicate significant differences among the treatments in a multiple range analysis for 90 and 95% confidence intervals for Cu and SH concentrations, respectively.
but we interpret the data to indicate that shoot vacuoles do not appear to play a major role in the sequestration of excess Cu, in contrast to the situation we previously reported for the roots (Lidon and Henriques 1994). Indeed, in this part of the plant, Cu was mostly found inside the vacuoles, where it could be seen by electron microscopy as small, individual granules randomly scattered throughout the vacuolar sap (Lidon and Henriques 1994). It is concluded, therefore, that shoots and roots of rice resort to different strategies for dealing with excess Cu, probably reflecting differences in their contribution to the structure and function of the whole plant. In general, plants retain excess copper in their roots with only small amounts being translocated to the shoot (Fernandes and Henriques, 1991), very few exceptions to this pattern having been reported (Hogan and Rauser, 1981; MacNair, 1981; Lolkema et al., 1984). Recently, Neumann et al. (1995) studied the intracellular distribution of copper in Armeria maritima grown in soils derived
from a Cu-mine dump and found that a great part of the absorbed Cu was retained inside vacuoles, both in roots and shoots. This observation of Cu accumulation in shoot vacuoles disagrees with our data, but it should be noted that Armeria maritima ssp. halleri is a heavy-metal tolerant subspecies, capable of accumulating extraordinarily high amounts of Cu in comparison to non-tolerant species, such as most cultivated plants are. It should also be pointed out that a significant part of Cu taken up by Armeria maritima was localized inside the chloroplasts and nuclei (Neumann et al., 1995), which imposes adjustments in the plant’s metabolic functioning in order for the metal not to affect its growth and development. We believe that strategies adopted by metal tolerant species to survive high metal contents in the growth substrate cannot be directly extrapolated to non-tolerant plants of a different species and so, it should constitute no surprise that rice differs from Armeria maritima in its pattern of metal intracellular compartmentation.
Table 3 Activities of acid phosphatase and NADH-cytochrome c reductase from leaves of Cu-treated rice plants Cu treatments (mg Cu l−1)
Acid phosphatase (mmol Pi mg−1 protein min 9S.E.)
NADH-cytocrome c reductase (U mg protein min−1 9S.E.)
0.002 0.01 0.05 0.25 1.25
0.36290.035 0.3829 0.036 0.4619 0.046 0.75390.076 1.8219 0.217
0.214 9 0.026 0.305 9 0.026 0.711 9 0.076 0.605 9 0.045 0.514 9 0.025
(c) (c) (b, c) (b) (a)
(c) (c) (a) (a, b) (b)
Each value is the mean+ 6 S.E. based on three replicates of three independent series. In a one way ANOVA, the F-ratio test indicated the existence of differences among treatments for a PB0.001; different letters indicate significant differences among the treatments in a multiple range analysis for a 90% confidence level.
F.C. Lidon, F.S. Henriques / En6ironmental and Experimental Botany 39 (1998) 197–202
Neumann et al. (1995) have also shown the presence of osmiophilic precipitates in leaf vacuoles with high Cu contents, which resembled in general appearance and fine structure the electron dense material adherent to the tonoplast shown in our micrograph of the shoot tissue. They dismiss a possible high Cu-related origin for these precipitates, which they concluded to be artefacts resulting from the fixation procedure; however, our observation that these precipitates were only visible in the highest Cu treatments casts doubts on their conclusion. It has been previously reported (Long, 1961; Hasegawa et al., 1976; Juma and Tabatabai, 1988) that the activities of NADH-cytochrome c reductase and acid phosphatase are strongly inhibited by Cu(II) but can recover in the presence of thiol groups. In this work we observed no inhibition of these enzymes activities in spite of increasing Cu concentrations in the vacuoles, which suggested that the metal sequestered in the vacuole is in a non-reactive form. Our additional observation that the content of SH groups in the vacuole increased with its Cu content, further suggested that the Cu fraction contained in the vacuole, is inactivated by binding to thiol groups which render it harmless to enzyme activity.
References Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72, 248 – 253. Fernandes, J.C., Henriques, F.S., 1991. Biochemical, physiological, and structural effects of excess copper in plants. Bot. Rev. 57, 246–273. Habbeb, A.F.S.A., 1972. Reaction of protein sulphydryl groups with Ellman’s reagent. In: Hirs, C.W., Timasheff, S.N. (Eds.), Methods in Enzymology XXV. Academic Press, New York, pp. 457–464. Hasegawa, Y., Lynn, K.R., Brockbank, W.J., 1976. Isolation and partial characterization of cytoplasmic and wall-bound acid phosphatase from wheat roots. Can. J. Bot. 54, 1163 – 1169. Hogan, G.D., Rauser, W.E., 1981. Role of copper binding, absorption and translocation in copper tolerance of Agrostis gigantea Roth. J. Exp. Bot. 32, 27–36. Jackson, M.L., 1958. Soil Chemical Analysis. Prentice-Hall, Englewood Cliffs, NJ.
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Jaffey, M.J., Galston, A.W., 1966. Physiological studies on pea tendrils. II. The role of light and ATP in contact coiling. Plant Physiol. 41, 1152 – 1158. Juma, N.G., Tabatabai, M.A., 1988. Phosphatase activity in corn and soybean roots: conditions for assay and effects of metals. Plant Soil 107, 39 – 47. Kamada, H., Harada, H., 1984. Studies on nitrogen metabolism during somatic embryogenesis in carrot. II. Changes in nitrate reductase activity. Biochem. Physiol. Pflanzen 179, 403 – 410. Kelly, B.M., Weskich, J.T., 1988. Respiration of mitochondria isolated from leaves and protoplasts of A6ena sati6a. Plant Physiol. 87, 705 – 710. King, J., Khanna, V., 1980. A nitrate reductase-less variant isolated from suspension cultures of Datura innoxia (Mill.). Plant Physiol. 66, 632 – 636. Kringstad, R., Kenyon, W.H., Black, C.C., 1980. The rapid isolation of vacuoles from leaves of Crassulacean acid metabolism plants. Plant Physiol. 66, 379 – 382. Lidon, F.C., Henriques, F.S., 1991. Limiting step on photosynthesis of rice plants treated with varying copper levels, J. Plant Physiol. 138, 115 – 118. Lidon, F.C., Henriques, F.S., 1992. Copper toxicity in rice: a diagnostic criterium and its effect on Mn and Fe contents. Soil Sci. 154, 130 – 135. Lidon, F.C., Ramalho, J., Henriques, F. S., 1993. Copper inhibition of rice photosynthesis, J. Plant Physiol. 142, 12 – 17. Lidon, F.C., Henriques, F.S., 1994. Subcellular localization of copper and partial isolation of copper proteins in roots from rice plants exposed to excess copper, Aust. J. Plant Physiol. 21, 427 – 436. Lolkema, P.C., Donker, M.H., Schouten, A.J., Ernst, W.H.O., 1984. The possible role of metallothioneins in copper tolerance of Silene cucubalus. Planta 162, 174 – 179. Long, C., 1961. Biochemist Handbook. Van Nostrand, Princeton, New Jersey. Luft, J., 1961. Improvement in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409 – 414. MacNair, M.R., 1981. The uptake of copper by plants of Mimulus guttatus differing primarily at a single major copper tolerance locus. New Phytol. 88, 723 – 730. Murphy, A., Taiz, L., 1995. Comparison of metallothionein gene expression and non-protein thiols in ten Arabidopsis ecotypes. Plant Physiol. 109, 945 – 954. Neumann, D., Nieden, U.Z., Lichtenberger, O., Leopold, I., 1995. How does Armeria maritima tolerate high heavy metal concentrations? J. Plant Physiol. 146, 704 – 717. Nriagu, J.O., Pacyna, J.M., 1988. Quantitative assessment of worldwide contamination of air water and soils by trace metals. Nature 333, 134 – 139. Ouzounidou, G., 1994. Copper-induced changes on growth, metal content and photosynthetic functions of Alyssum montanum L. plants. Env. Exp. Bot. 34, 165 – 172. Parida, R.K., Mishra, D., 1980. Acid phosphatase and adenosine triphosphatase activities during rice leaf development and senescence. Photosynthesis 14, 431 – 436.
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Reynolds, E., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208 – 212. Rubinstein, B., 1978. Use of lipophilic cations to measure the membrane potential of oat leaf protoplasts. Plant Physiol. 62, 927 – 929. Schat, H., Kalff, M.A., 1992. Are phytochelatins involved in differential metal tolerance or do they merely reflect metalimposed strain? Plant Physiol. 99, 1475–1480. Schultz, C.L., Hutchinson, T.C., 1988. Evidence against a key-role for metallothionein-like protein in the copper tolerance mechanism of Deschampsia cespitosa (L.) Beauv. New Phytol. 110, 163–171. Verkleij, J.A.C., Koevoets, P., Van’t Riet, J., Van Rossenberg,
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M.C., Bank, R., Ernst, W.H.O., 1989. The role of metalbinding compounds in the copper tolerance mechanism of Silene cucubalus. In: Winge, D., Hamer, D. (Eds.), Metal Ion Homeostasis: Molecular Biology and Chemistry. Alan R. Liss, New York, pp. 225 – 260. Verkleij, J.A.C., Schat, H., 1990. Mechanisms of metal tolerance in higher plants. In: Shaw, A.J. (Ed.), Heavy Metal Tolerance in Plants: Evolutionary Aspects. CRC Press, Boca Raton, FL. CRC Press, Boca Raton, FL, pp. 179 – 193. Yoshida, S., Forno, D.A., Cook, J.H., Gomes, K.A., 1976. Routine procedure for growing rice plants in culture solution. In: Laboratory Manual for Physiological Studies of Rice, 3rd ed. The International Rice Research Institute, Los Ban˜os, Laguna, Philippines, pp. 61 – 65.
Role of rice shoot vacuoles in copper toxicity regulation Fernando C. Lidon *, Fernando S. Henriques Plant Biology Unit, Faculdade de Cieˆncias e Tecnologia, Uni6ersidade No6a de Lisboa, 2825 Monte da Caparica, Lisboa, Portugal Accepted 4 September 1997
Abstract Rice (Oryza sati6a L. cv. Safari) plants were grown over a 30-day period in nutrient solutions containing 0.002–1.25 mg l − 1 Cu concentrations. It was found that increasing Cu concentrations led to increasing Cu contents in the plant’s shoot and to an inhibition of its growth. Transmission electron microscopy showed no obvious ultrastructural changes in the shoot tissues, except for electron-dense deposits adherent to the tonoplast, appearing from the 0.25 mg l − 1 Cu treatment onwards. On a protein basis, Cu and SH groups concentrations in isolated shoot vacuoles increased by 41% and 120%, respectively, from the 0.01 to the 1.25 mg l − 1 Cu treatments. In spite of increased vacuolar Cu contents, the activities of NADH-cytochrome c reductase and acid phosphatase were not inhibited, apparently because of metal binding to sulphydryl groups contained in the vacuole. © 1998 Published by Elsevier Science B.V. All rights reserved. Keywords: Copper toxicity; Oryza sati6a; Shoot; Vacuole; Acid phosphatase; NADH-cytochrome c reductase
1. Introduction Heavy metal pollution is one of the current most troublesome environmental problems due to the widespread use of metals for industrial and agricultural purposes (Nriagu and Pacyna, 1988; Fernandes and Henriques, 1991). Cu is an essential micronutrient for plants, but most species are very sensitive to high concentrations of this metal, which cause metabolic disturbances and growth inhibition (Fernandes and Henriques, 1991; Lidon and Henriques, 1991; Lidon et al., 1993; Ouzounidou, 1994). Plants growing in Cu-enriched sub* Corresponding author. Tel.: + 351 1 4416855 or +351 1 2954464 ext. 1101; fax: +351 1 4416011 or + 351 1 2954461.
strates developed a variety of defense mechanisms against its toxicity, the most common being the metal sequestration in cell compartments where it least interferes with vital metabolism and/or its inactivation by reaction with various cellular components (Verkleij and Schat, 1990; Fernandes and Henriques, 1991). Increased production of the metal-binding compounds metallothioneins and phytochelatins has been reported to occur in response to high cellular levels of Cu, being particularly important for metal detoxification in non-tolerant plants (Schultz and Hutchinson, 1988; Verkleij et al., 1989; Schat and Kalff, 1992; Murphy and Taiz, 1995). Our previous work with rice grown in Cu-enriched nutrient solutions has shown that the
S0098-8472/98/$19.00 © 1998 Published by Elsevier Science B.V. All rights reserved. PII S0098-8472(97)00025-7
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threshold toxic concentration for this metal was 35 mg Cu g − 1 tissue dw and also that this threshold value was reached when the plants were grown in 0.01 mg l − 1 Cu solutions (Lidon and Henriques, 1992). The roots of plants subjected to higher Cu concentrations accumulated most of the excess metal inside their vacuoles, precipitated in the form of granules randomly scattered within them (Lidon and Henriques, 1994). The present work was undertaken to test the hypothesis that excess Cu translocated to the shoot was also preferentially localized inside the vacuoles and, if so, how it affected enzyme activities in this cell compartment.
2. Materials and methods Rice (Oryza sati6a L. cv. Safari) seeds were washed, sterilized and germinated as described previously (Lidon and Henriques, 1992). The seedlings (50 per pot) were hydroponically grown for 30 days in cylindric 2 l pots at 35 – 37oC/25– 27oC day/night temperatures, under 250 mm m − 2 s − 1 PAR irradiance over a 12 h-day period. The nutrient solution was that of Yoshida et al. (1976) containing (mg l − 1) 40 N, 10 P, 40 K, 40 Ca, 40 Mg, 0.5 Mn, 0.2 B, 0.05 Mo, 0.01 Zn. Iron was added as a hexahydrated FeCl3 at 2 mg l − 1, chelated by 50 mM monohydrated citric acid. Cu concentrations were (mg l − 1) 0.002, 0.01, 0.05, 0.25 and 1.25. The solutions were adjusted daily to pH 5.5, the volume kept constant at the original level and the solutions were renewed every 5 days. For elemental analysis, shoot samples were dried for 24 h at 80oC followed by 3 days at 100oC and 1 g of dry material was successively digested in a nitric:perchloric (5:2, v/v) and nitric:sulfuric:perchloric (10:1:10 v/v/v) acid mixtures (Jackson, 1958). Cu concentrations were determined by atomic absorption spectrophotometry using a Perkin-Elmer model 3030. For electron microscopy, pieces of leaf tissue were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.0) for 2 h, buffer-washed and post-fixed in cacodylate-buffered 1% osmium tetroxide for 1 h. After buffer-washing and dehy-
dration in a graded series of methanol, the samples were treated with propylene oxide and embedded in Epon (Luft, 1961). Ultrathin sections were cut with a LKB ultramicrotome, double stained with aqueous uranyl acetate and lead citrate (Reynolds 1963) and examined in a Philips TEM 300 electron microscope at 80 kV. Isolation of leaf protoplasts followed the methods of Rubinstein (1978), as modified by Kelly and Weskich (1988). Vacuoles were obtained following the procedure of Kringstad et al. (1980). The concentration of SH groups was determined according to Habbeb (1972), using the Ellman reagent. The assay for acid phosphatase (E.C. 3.1.3.1) followed the method of Parida and Mishra (1980), with some minor modifications. The enzyme activity was calculated on the basis of net inorganic phosphorus released during the incubation period, following Jaffe and Galston (1966). The assay for NADH cytochrome c reductase (E.C. 1.6.99.3) followed King and Khanna (1980), after enzyme extraction according to Kamada and Harada (1984). Enzyme activity (1 U) was defined as the change of 1.0 in the absorbance at 550 nm per mg protein. Protein concentration was determined according to the method of Bradford (1976), using a BSA standard curve.
3. Results Table 1 shows that rice shoot lengths decreased when Cu concentrations in the nutrient solutions surpassed 0.05 mg l − 1 and that these decreases were particularly prominent for the two highest Cu treatments. Such decreases in shoot lengths were parallel to increases in tissue Cu concentrations (Table 1), again particularly marked for the last two treatments, reflecting the metal interference with normal growth processes. Examination by transmission electron microscopy of shoot tissues revealed electron dense deposits adherent to the tonoplast of a large number of cells (Fig. 1B) in plants grown in solutions containing 0.25 mg l − 1 or higher Cu concentrations. Leaves from lower Cu treatments exhibited no apparent ultra-
F.C. Lidon, F.S. Henriques / En6ironmental and Experimental Botany 39 (1998) 197–202
199
Table 1 Lengths and Cu concentrations in shoot tissues of Cu-treated rice plants Cu treatments (mg Cu/l)
0.002 0.01 0.05 0.25 1.25
Shoot lengths (cm 9S.E.)
43 9 3.0 439 3.0 399 2.5 329 2.5 23 9 1.5
(a) (a) (a, b) (b, c) (c)
Cu concentration in the shoot mg Cu g−1 9S.E.
ng Cu mg−1 Protein9 S.E.
17.5 91.2 21.5 91.6 27.0 92.1 46.5 93.3 95.0 9 6.9
367 925 (c) 376 928 (c) 524 941 (c) 978 969 /(b) 2672 9194 (a)
(c) (c) (c) (b) (a)
Each value is the mean+ 6 S.E. based on three replicates of three independent series. In a one way ANOVA, the F-ratio test indicated the existence of differences among treatments for a PB0.001; different letters indicate significant differences among the treatments in a multiple range analysis for a 95% confidence level.
structural changes (Fig. 1A). Vacuoles isolated from leaf tissues showed that their Cu levels increased from the 0.05 mg l − 1 Cu treatment on-
wards, to reach a maximum value in the 1.25 mg l − 1 Cu treatment (Table 2). The concentrations of SH groups in isolated shoot vacuoles followed a pattern similar to the Cu concentrations, but with a more pronounced increase in the 1.25 mg l − 1 Cu treatment (Table 2). Thus, the SH/Cu ratio increased continuously with increasing Cu concentrations, as it is shown in the last column of Table 2. Activities of acid phosphatase and NADH-cytochrome c reductase from leaves grown at increasing Cu supply are shown in Table 3. When expressed per unit protein, the activity of the acid phosphatase displayed a continuous increase with increasing Cu exposure. The NADHcytochrome c reductase activity reached a maximum at 0.05 mg l − 1 Cu and decreased thereafter; it is worth noting, however, that the enzyme activity remained much higher at 0.25 and 1.25 mg l − 1 Cu than in the 0.002 and 0.01 mg l − 1 Cu treatments.
4. Discussion
Fig. 1. Cell ultrastructure of leaves from rice plants grown in (A) 0.01 and (B) 1.25 mg l − 1 Cu treatments. Note the electron-dense deposits lining the vacuoles of high Cu-treated plants.
The data presented here show that between the lowest and the highest Cu treatments tested, the shoot region of rice plants registered a 7-fold copper increase, whereas Cu content in vacuoles increased only 1.4 times, on a protein basis. The physiological meaning of this comparison may be debatable, as Cu in the shoot and particularly that accumulated in the vacuole, is most certainly also bound to compounds other than proteins,
200
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Table 2 Cu and SH group concentrations in vacuoles isolated from leaves of Cu-treated rice plants Cu treatments
Cu (ng Cu mg−1 protein)
SH groups (mg SH mg−1 protein 9S.E.)
SH/Cu (mol ratio×10−5)
0.002 0.01 0.05 0.25 1.25
0.17 9 0.015 0.17 9 0.025 0.19 9 0.015 0.21 9 0.035 0.24 9 0.035
26.9 9 3.94 28.7 9 3.74 33.1 9 3.55 36.7 9 3.06 59.2 9 7.07
3.04 3.25 3.35 3.36 4.74
(b) (b) (a, b) (a) (a)
(b) (b) (b) (b) (a)
Each value is the mean+ 6 S.E. based on three replicates of three independent series. In a one way ANOVA test different letters indicate significant differences among the treatments in a multiple range analysis for 90 and 95% confidence intervals for Cu and SH concentrations, respectively.
but we interpret the data to indicate that shoot vacuoles do not appear to play a major role in the sequestration of excess Cu, in contrast to the situation we previously reported for the roots (Lidon and Henriques 1994). Indeed, in this part of the plant, Cu was mostly found inside the vacuoles, where it could be seen by electron microscopy as small, individual granules randomly scattered throughout the vacuolar sap (Lidon and Henriques 1994). It is concluded, therefore, that shoots and roots of rice resort to different strategies for dealing with excess Cu, probably reflecting differences in their contribution to the structure and function of the whole plant. In general, plants retain excess copper in their roots with only small amounts being translocated to the shoot (Fernandes and Henriques, 1991), very few exceptions to this pattern having been reported (Hogan and Rauser, 1981; MacNair, 1981; Lolkema et al., 1984). Recently, Neumann et al. (1995) studied the intracellular distribution of copper in Armeria maritima grown in soils derived
from a Cu-mine dump and found that a great part of the absorbed Cu was retained inside vacuoles, both in roots and shoots. This observation of Cu accumulation in shoot vacuoles disagrees with our data, but it should be noted that Armeria maritima ssp. halleri is a heavy-metal tolerant subspecies, capable of accumulating extraordinarily high amounts of Cu in comparison to non-tolerant species, such as most cultivated plants are. It should also be pointed out that a significant part of Cu taken up by Armeria maritima was localized inside the chloroplasts and nuclei (Neumann et al., 1995), which imposes adjustments in the plant’s metabolic functioning in order for the metal not to affect its growth and development. We believe that strategies adopted by metal tolerant species to survive high metal contents in the growth substrate cannot be directly extrapolated to non-tolerant plants of a different species and so, it should constitute no surprise that rice differs from Armeria maritima in its pattern of metal intracellular compartmentation.
Table 3 Activities of acid phosphatase and NADH-cytochrome c reductase from leaves of Cu-treated rice plants Cu treatments (mg Cu l−1)
Acid phosphatase (mmol Pi mg−1 protein min 9S.E.)
NADH-cytocrome c reductase (U mg protein min−1 9S.E.)
0.002 0.01 0.05 0.25 1.25
0.36290.035 0.3829 0.036 0.4619 0.046 0.75390.076 1.8219 0.217
0.214 9 0.026 0.305 9 0.026 0.711 9 0.076 0.605 9 0.045 0.514 9 0.025
(c) (c) (b, c) (b) (a)
(c) (c) (a) (a, b) (b)
Each value is the mean+ 6 S.E. based on three replicates of three independent series. In a one way ANOVA, the F-ratio test indicated the existence of differences among treatments for a PB0.001; different letters indicate significant differences among the treatments in a multiple range analysis for a 90% confidence level.
F.C. Lidon, F.S. Henriques / En6ironmental and Experimental Botany 39 (1998) 197–202
Neumann et al. (1995) have also shown the presence of osmiophilic precipitates in leaf vacuoles with high Cu contents, which resembled in general appearance and fine structure the electron dense material adherent to the tonoplast shown in our micrograph of the shoot tissue. They dismiss a possible high Cu-related origin for these precipitates, which they concluded to be artefacts resulting from the fixation procedure; however, our observation that these precipitates were only visible in the highest Cu treatments casts doubts on their conclusion. It has been previously reported (Long, 1961; Hasegawa et al., 1976; Juma and Tabatabai, 1988) that the activities of NADH-cytochrome c reductase and acid phosphatase are strongly inhibited by Cu(II) but can recover in the presence of thiol groups. In this work we observed no inhibition of these enzymes activities in spite of increasing Cu concentrations in the vacuoles, which suggested that the metal sequestered in the vacuole is in a non-reactive form. Our additional observation that the content of SH groups in the vacuole increased with its Cu content, further suggested that the Cu fraction contained in the vacuole, is inactivated by binding to thiol groups which render it harmless to enzyme activity.
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