Some Observations on the Leaf Ultrastructure of Halimione Portulacoides (L.) Aellen Grown in a Medium Containing Copper

J. Plant Physiol. Vol. 137. pp. 717-722 (1991)

Some Observations on the Leaf Ultrastructure of Halimione Portulacoides (L.) Aellen Grown in a Medium Containing Copper F.

REBOREDO

and F.

HENRIQUES

Universidade Nova de Lisboa, F.C.T.,

Sec~ao

de Biologia Vegetal, 2825 Monte da Caparica, Portugal

Received July 24, 1990 . Accepted November 12, 1990

Summary The effects of copper (5 ppm) on the leaf ultrastructure as well as on chlorophyll and protein content of Halimione plants grown in hydroponic cultures were studied in vitro. No morphological changes were detected at the 15th day of treatment with Cu. Chlorophyll a and b and total protein contents were not affected by the treatment, compared with control values. Ultrathin sections of control and Cu-treated plant leaves exhibit regular fine structure of mitochondria, chloroplasts, nucleus and rough endoplasmic reticulum, indicating the absence of cellular damage. However, the presence of starch grains in the chloroplasts of some phloem cells of the Cu-treated plants was noted, generally, contrasting with the absence in chloroplasts of control cells.

Key words: Halophytes, Copper effects, Ultrastructural studies, Protein and chlorophyll contents. Introduction Copper, although an essential nutrient for plants, when absorbed in excess amounts can be responsible for various damage at the morphological, biochemical and ultrastructural levels. For example, excess Cu was found to induce a severe leaf drop, interveinal chlorosis and alter leaf pigmentation (Heale and Ormrod, 1982), to reduce both Fe translocation to shoots and leaf catalase activity (Agarwala et al., 1977), to reduce the activity of ribulose-1,5-biphosphate carboxylase (Stiborova et al., 1986), to interact with ferredoxin causing inhibition of ferredoxin-dependent reactions (Shioi et al., 1978) and to affect chloroplast ultrastructure (Brinkhuis and Chung, 1986). Heavy metal toxicity on aquatic plants has been studied (Mortimer and Czuba, 1982; Sela et aI., 1988) with particular emphasis on unicellular algae (De Filippis et al., 1981; Les and Walker, 1984), due to their key-role in ocean primary production and support of numerous aquatic food chains. Little information about Cu toxicity on halophytic species is available in spite of the well documented importance of © 1991 by Gustav Fischer Verlag, Stuttgart

several halophytes in the accumulation of heavy metals (Newell et aI., 1982; Reboredo, 1985, 1988). In the present study we investigated the effects on leaf ultrastructure, as well as on chlorophyll a and b and total protein contents of Halimione plants grown in hydroponic culture supplied with 5 ppm Cu for 15 days.

Materials and Methods Plant material and growth conditions Plants used in hydroponic cultures, previously obtained from cuttings, were maintained in a controlled chamber illuminated for a 14 h-day period in a continuously aerated nutrient solution (Baumeister and Schmidt, 1962) with 5ppm Cu (as CuCh) or without Cu (control). Solutions were renewed every 2 days and pH adjusted at that time. In previous studies (Reboredo, 1988), it was observed that the maximum Cu level in the soil marshes of the Sado river estuary, extracted with double distilled water, was similar to the above-mentioned concentration, leading to the choice of this level.

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REBOREDO

and F.

HENRIQUES

Table 1: Chlorophyll, total protein and copper contents of the leaves of Halimione plants grown in hydroponic culture after 5, 10 and 15 days of exposure to 5 ppm Cu.

5 days 10 days 15 days

Chl.a l

Control plants Total protein 2 ChI. b l

Cu leveP

ChI. a l

Copper-treated plants ChI. b l Total protein2

Cu leveP

296.3*±62.1 263.4*±49.1 283.1*±56.4

121.6*±37.6 109.8""±29.3 132.2" ±32.0

10.2±0.51 11.5± 1.36 13.8±2.45

312.0*±54.3 270.1*±51.3 284.4" ±69.4

126.9* ±28.9 134.1""±41.3 133.6* ±47.0

16.7±0.88 26.3±5.48 43.2±5.54

10.5± 1.7 10.2± 1.0 10.0±0.6

8.8±0.5 9.3±1.0 10.0± 1.0

Mean value ± S. deviation (n - 30); expressed as JLg/g fresh weight. Mean value ± S. deviation (n = 3); expressed as %. 3 Mean value ± S. deviation (n = 3); expressed as JLg/g dry weight. "" Mean values not significant at the 0.05 significance level (P > 0.05) . ..,. Mean values significantly different at the 0.05 significance level (P < 0.05). I

2

Fig. 1: A - Transverse section of the control plant leaf showing the epidermis (E), chlorenchyma (Ch) and vascular bundle (VB). x 170; B - Aspect of some Ch. cells showing regular organization of the chloroplast (C), one of them with a starch grain (SG). Nucleus (N), nucleolus (Nu) and mitochondria (M) are also visible. x21,500. C - Aspect of some phloem cells with regular organization of the thylakoidal system of the chloroplasts (C) without starch grains x 10,000. D - Detail of a phloem cell showing dense cytoplasm with ribosomes, rough endoplasmic reticulum (RER), nucleus (N), vacuole (V) the tonoplaste (T) and cell wall (CW), which is common to a parenchyma cell. x 40,000.

Leaf ultrastructure in copper medium

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Fig.2: A - Copper-treated plant showing some cells of the Ch. with abundance of chloroplasts (C) and with starch grains (SG). x 19,500; B - Detail of a Ch. cell showing the nucleus (N), nucleolus (Nu), mitochondria (M), vacuole (V) and chloroplasts (C) x 29,250; C - Section of a Ch. cell with regular organization of the thylakoidal system of the chloroplast (C), grana stacks (G) and plastoglobuli. x 32,250.

Protein and chlorophyll determination For chlorophyll and protein analysis, leaves were collected (yellow-green leaves and young leaflets were not considered for that purpose) after 5, 10 and 15 days of treatment with Cu. These analysis were carried out in parallel with the collection of leaves for Cu determination by atomic absorption spectrophotome· try. Chlorophyll was extracted in 80% (v/v) aqueous acetone and the absorption measured in a Shimadzu UV-160 spectrophotometer at 645 and 663 nm, according to Arnon (1949). Total protein was measured according to the method described by Lowry et al. (1951), using bovine serum albumin as standard.

Each determination was carried out in triplicate. The results from several halophytes in the accumulation of heavy metals (Newell et aI., 1982; Reboredo, 1985, 1988).

Copper determination by atomic absorption spectrophotometry Each gram of dry leaf was placed in a 100 mL borosilicate beaker and digested with NH0 3-HCI0 4 (4 : 1) until dryness. The residue was dissolved in a 2 % aqueous solution of HCI, filtered and diluted to a final volume of 25 mL (Agemian and Chau, 1976). The metal was determined by A.A.S. using a Perkin-Elmer model 5000 fitted with a deuterium background corrector. The operating conditions

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REBOREDO

and F. HENRIQUES

.

Fig. 3: A - Aspect of some phloem cells of a copper-treated plant exhibiting a large nucleus (N), nucleolus (Nu) and dense cytoplasm. Some cells contained chloroplasts with a few grana (G) while others did not present them. x 10,500. B - Detail of a phloem cell and a phloem parenchyma cell (Ppc), the latter showing three chloroplasts, two of them with starch grains (SG), and a tiny bending of the grana stacks (-) x 16,800. were those recommended by the manufacturer; p.a. reagents were used in every case and standards were prepared by serial dilution of commercially available stock solutions. Each analysis was carried out in triplicate.

lead citrate (Reynolds, 1963) and examined in a Jeol100C transmission electron microscope at 80KV.

Results and Discussion Electron microscopy Small segments (ca. 1 mm) of the young leaves of Halimione plants were collected after 15 days exposure of 5 ppm Cu and fixed in 4.0 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 14 h, buffer-washed and post-fixed in phosphate-buffered 2.0 % OS04 for 1 h. After buffer-washing and dehydration with ethanol, the leaves were treated with propylene oxide and embedded in Epon-Araldite (Mollenhauer, 1964). Ultrathin sections were cut with a LKB ultramicrotome, stained with aqueous uranyl acetate (Watson, 1958) and

A decrease of ChI. a content was observed for plants treated with 5 ppm Cu, while ChI. b content remained approximately constant (Table 1). Similar observations were made with control plants. Comparing the mean values (n = 30) for ChI. a and b of treated and control plants no statistically significant differences at 0.05 significance level were detected, which means that the species belong to the same population.

Leaf ultrastructure in copper medium

In spite of the differences between the total protein content of Cu-treated and control plants at the 5th day of treatment, at the end of the experiment these levels were equal (Table 1). It can be concluded that the synthesis of chlorophyll and protein was not affected by the tested level and the different exposures. Leaves of Cu-treated plants presented approximately 3 times more Cu at the end of the experiment than leaves of control plants (Table 1); however, these levels were not sufficient enough to induce morphological and ultrastructural changes. Furthermore, Howeler (1983) stated that the toxicity limit for the plant leaves ranges between 15 and 50 ppm Cu, our mean value (43.2 ppm) being lower than the upper limit. These results are in agreement with observations at the ultrastructural level. Ultrathin sections of control and Cu-treated plants leaves are shown in Figs. 1,2 and 3. Chloroplasts of chlorenchyma of control and treated plants exhibit a typical arrangement of higher plants with grana stacks, plastoglobuli and starch grains, generally one grain per plastid. Other organelles like mitochondria and nucleus are both recognized with well preserved membranes. The great majority of the phloem cells presented a dense cytoplasm with the nucleus occupying a large volume of the cell (Figs. 1 C and 3 A). Some of these cells do not exhibit chloroplasts although they may occur occasionaly. Phloem parenchyma cells are also seen in Figs. 1 D, 3 A and 3 B. These cells may contain starch (Fig. 3 B), and if completely differentiated are almost occupied by the vacuole, being much larger than adjacent phloem cells. The cells of the phloem and associated parenchyma of control and Cu-treated plants exhibited similar profiles. However, it can be emphasised that starch grains are absent in chloroplasts of the phloem cells of control plants (Fig. 1 C). Chloroplasts of parenchyma cells presented a regular organization of the thylakoidal system (Fig. 1 D), while in Cutreated phloem cells one starch grain per chloroplast with few internal membranes is generally observed (Fig. 3 A), and in a particular case a tiny bending of the grana stacks of a chloroplast parenchyma cell was detected (Fig. 3 B). Earlier reports on the inhibitory effects of heavy metals on chlorophyll levels were commonly found in the literature (De Filippis et al., 1981; Stiborova et aI., 1986; Prasad and Prasad, 1987), contrasting with our results in which the tested Cu level did not interfere with chlorophyll synthesis. This fact can probably be explained by the short-term exposure, since previous results in vitro obtained from longterm exposures (8 weeks) indicated that 5 ppm Cu (as CuClz) induced a secondary deficiency of Fe in plant leaves with a concomitant decrease in ChI. a content (Reboredo, 1988). Cellular responses to pollutants are one of the most interesting features in toxicological studies. Chloroplast ultrastructural changes in young sporophytes of Laminaria saccharina L. treated with 100 and 500 J.lg Cu per liter of media, after 7 days exposure, were observed (Brinkhuis and Chung, 1986). These changes included slight swelling and detachment of thylakoid membranes and the rupture and diffusion of the thylakoid membrane, and were concomitant with the decrease of chlorophyll content approximately 12 and 86 % lower than the control value

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for sporophytes treated with 100 and 500 J.lg. L -\, respectively. Although we detected no disturbances at the 15th day of Cu-exposure, it does not exclude that other metabolic pathways are affected. For example, Agarwala et al. (1977) observed that an excess of Zn does not induce marked visible symptoms on Hordeum vulgare L. but was quite effective in depressing growth and dry matter yield. The same authors also observed that chlorophyll and soluble protein contents were not reduced by excess Zn, but catalase activity of young leaves decreased approximately 25 % compared with control plants. Although this data is for zinc, it might be applied for this study. Under natural conditions it is practically impossible to predict the effects of contaminants on biota. Stress situations arise without previous indications of changes, e.g. on the relative abundance of a given community, diversity and biomass production. In all cases, a turning-point may exist, i.e. a moment in which lethal or sub-lethal effects are expressed. More than to carry out studies on the bioaccumulation of a given toxic agent by plants or animals living in stressed areas, the future of field research must be focused on the search for a ratio, index or some equivalent that allows us to detect the appearance of that turning-point. This work was done on that basis, taking into account that the tested level induced a secondary deficiency of Fe as stated previously (Reboredo, 1988). We also observed a significant difference at the 0.05 significance level between ChI. a content of control and Cu-treated plants after 4 weeks exposure, while after 2 weeks these differences were not significant at the 0.05 significance level. Since those results were obtained from plants cultivated in pots with a soil composition somewhat similar to that observed in soil salt-marshes of the Sado river estuary, we developed an in vitro experiment with hydroponic plant cultures, during 2 weeks, in a media containing the same Cu concentration. From the results presented here it seems that studies on size and number of starch grains per chloroplast, complemented by some biochemically related work, must be carefully developed.

Acknowledgements The authors are grateful to the director of the Laboratorio Nacional de Investiga~ao Veterinaria (LNIV) and the head and staff of the Electron Microscopy Laboratory for providing the con· ditions for the effectiveness of this work. A special acknowledgement is offered to Mrs. Maria Lapao for her support during the stay of one of us (F.R.) in the LNIV, to Dr. Azinhais Mendes (UNL) for correcting the English version and helpful indications on computer management and Dr. J. P. Silva (UNL) for chlorophyll analysis. This work was financed by the Instituto Nacional de Investiga~ao Ciendfica (INIC), Portugal.

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