CHANGES IN THE ULTRASTRUCTURE OF CAPSICUM ANNUUM L . SEEDLINGS ROOTS UNDER ALUMINUM STRESS CONDITIONS

The effect of aluminum was investigated on the cell ultrastructure of roots of fourteen-day-old `Trapez` red pepper seedlings grown in water culture. Disorders in the cell structure were observed using transmission electron microscopy (TEM). An analysis of longitudinal sections of the apical region of the control plants’ roots showed that cells of the cap and cells of the meristematic region had an arrangement and shape typical for these root regions, and cell organelles were properly developed. Changes in the cell structure under the infl uence of aluminum involved a reduction in the number of starch grains in the leucoplasts of the cap, the formation of lobate nuclei and a reduction in the number of cisternae in the dictyosomes as well as the damage of the cell membranes. Moreover, the swelling of mitochondria was observed with a simultaneous reduction in the number of mitochondrial cristae or the bursting of the membrane of these structures. In the vacuoles of the investigated root section, the presence of numerous electron-opaque large-sized formations was found, being most probably aluminum deposits. The cell wall, often thickened, was wavy or the formation of two walls in close distance was observed. The obtained results prove the high sensitivity of the studied red pepper cultivar to aluminum stress.


INTRODUCTION
Most plants respond to the presence of mobile aluminum in the substrate by a signifi cant limitation of growth and yielding induced by development anomalies and metabolic disturbances as well as by insuffi cient supply of the plant with water and nutrients.Studies of many authors have demonstrated that the greatest disorders occur in the place of toxicant application, i.e. in the root, in particular in its apical portion (C l u n e and C o p e l a n d , 1999; M i c h a ł e k , 2002; K o n a rs k a , 2004a).The most characteristic morphological symptoms of aluminum toxicity are as follows: the shortening of the length of roots, their thickening and brown- The purpose of this study was to determine the effect of aluminum on the cell ultrastructure of the apical portion of roots of red pepper cv.`Trapez` using transmission electron microscopy.

MATERIALS AND METHODS
Red pepper (Capsicum annuum L.) seedlings cv.`Trapez` were cultivated in plastic containers 4 dm 3 in capacity which were fi lled with modifi ed Knop's nutrient solution (B r a u n e r and B u n k a t s h 1987).Aluminum was added as AlCl 3 6H 2 O at two concentrations: 0 (control) and 20 mg dm -3 of the medium, what corre- Figs 5, 6.Cells of the meristematic region of the root of 14-day-old red pepper seedlings in the control; visible a cell nucleus (N) with a nucleolus (Nu), numerous mitochondria (M), proplastids (P), small vacuoles (V), endoplasmic reticulum (ER) and a Golgi body (G).Bar -2 μm (Fig. 5), bar -1 μm (Fig. 6).sponds to 0 and 1.1 mg dm -3 of pure aluminum.The pH of the solutions was set to 4.3 by using 0.1 M HCl or 0.1 M NaOH.Disinfected seeds of tested plant germinated on wet fi lterpaper of Petri dishes.After four days from germination, the seedlings were transferred to water culture.Each treatment comprised 30 plants cultured for fourteen days.During the experiment, the nutrient solution was aerated and its depletion was supplemented, and after a week it was replaced.The experiment was carried out in a phytotron under a 14-hour photoperiod, at a temperature of 24°C ±1°C and a 70% relative humidity.
Transmission electron microscopy (TEM).Top fragments of red pepper main roots after 14 d of growth of the control plants and treated with 20 mg AlCl 3 dm -3 were fi xed in a fi xative containing 2% paraformaldehyde and 2.5% glutaraldehyde buffered at pH 7.4 in 0.1 M cacodylate buffer.Fixation was performed at room temperature for two hours, followed by 12 hr at 4 o C. When fi xed, the samples were rinsed with 0.1 M cacodylate buffer at 4 o C for 24 hr and then treated with 1% OsO 4 .Subsequently, the samples were transerred to re-distilled water and stained with a 0.5 aqueous solution of urany acetate.After passage through increasing concentrations of propylene oxide in ethanol and fi nally through pure propylene oxide, the samples were embedded for 12 hr in Spurr Low Viscosity resin at 70 o C. Ultrathin sections (60nm thick) were treated with a 8% solution of uranyl acetate in acetic acid and with lead citrate.The images were observed and recorded using the Tesla BS-500 electron microscopy.

RESULTS
An analysis of electronograms from the longitudinal sections of the apical region of the red pepper root showed that the cap cells of the control plants were characterised by the presence of a centrally located nucleus with one or two nucleoli and a varied number of small vacuoles.Mitochondria, Golgi bodies, smooth and rough endoplasmic reticulum (ER), many ribosomes and amyloplasts, fi lled with small starch grains, also occurred in the cells (Figs 1, 2).
In the presence of 20 mg dm -3 AlCl 3 , the cytoplasm of the cap cells seen in TEM was characterised by the occurrence of organelles which in many cases had an altered structure (Figs 3, 4).The amyloplasts contained a smaller number of starch grains, but with much larger sizes compared to the control (Fig. 3).In the small vacuoles of the cap cells, the occurrence of dark, electron-dense, spherical-like formations was found (Fig. 3), which were also observed by using light microscopy.Numerous mitochondria were characterised by the electron brighter matrix, as well as a reduced number of cristae and often degenerating contents (Fig. 4).
In the control object, the dividing cells in the meristem region were surrounded by a thin cell wall, and their protoplast, in addition to a nucleus, contained proplastids, mitochondria, Golgi bodies, ER, numerous ribosomes and small vacuoles (Figs 5, 6).
On the other hand, the cells of the meristematic tissue of the plants treated with aluminum (20 mg dm -3 AlCl 3 ) were distinguished by the presence of small, but more numerous, vacuoles containing electron-opaque formations (Figs 7,8), similar to those described in the vacuoles of the cap.The lobate cell nuclei had irregular, folded contours with deep invaginations (Fig. 8), and at some place a partial degradation of the nucleus membrane occurred (Fig. 9).
The occurrence of swelling mitochondria showing destructive changes, similar to those in the root cap, as well as the bursting of the membrane of these organelles, was also found (Figs 9,10).
Moreover, the cell walls were marked by varied thickness or undulation.The formation of two walls in close vicinity was also observed (Figs 11,12).

DISCUSSION
The increased aluminum concentration induced numerous disorders in the cell ultrastructure of roots of red pepper cv.`Trapez`.These changes prove the high sensitivity of the studied cultivar to aluminum toxicity, which is confi rmed by earlier results obtained by K on a r s k a (2004a) relating to the index of tolerance (IT) for the root system of this cultivar.Less numerous and enlarged grains of statolith starch occurring in the cap of the plants treated with aluminum could be related to the disturbed positive geotropism of the roots and their hook-like curvature, which was observed in earlier studies by K o n a r s k a (2004b, 2005).
The destruction of plasmatic membranes of many organelles, observed in the study, could also result from the formation of permanent bonds of ions of the toxicant with proteins and phospholipids composing these membranes, which was described by other researchers (C h a and L e e , 1996; L i u et al. 1996).Anomalies in the structure of mitochondria, noted in the study, could be related to the disorders in phosphorus uptake and its defi ciency, many times observed in experiments with aluminum (H o l o p a i n e n et al. 1992; M a lk a n t h i et al. 1999).
Electron-opaque bodies, the presence of which was found in the vacuoles of cells of the root at its different levels, were probably aluminum deposits.Such form of their deposition is reported by other researchers (C l u n e and C o p e l a n d , 1999; M a n g a b e i r a et al. 1999; V á z q u e z et al. 1999).In the vacuoles, aluminum ions may be bound by organic acids or phosphorus (M a and H
ing as well as the bending of the terminal section of the root (W e r y s z k o -C h m i e l e w s k a et al. 1999; M a l a t h i et al. 2001; T a b u c h i and M a t s um o t o , 2001; K o n a r s k a , 2004b; 2005).Many researchers also observed peeling off or falling off of the cap as well as a reduction in the length of lateral roots (B u d i k o v á , 1999; W e n z l et al. 2001).The effect of aluminum was frequently accompanied by the shortening of root regions and of the length of root hairs as well as their deformation (S z a t a n i k -K l o c , 1999; M i c h a ł e k , 2002).An increased thickness of cell walls in the outer layers of the root was also observed, as well as the formation of cracks and losses in these tissues (W e r y s z k o -C h m i e l e w s k a and C h w i l , 1998; B u d i k o v á , 1999; V á z q u e z et al. 1999; K o n a r s k a , 2005).At the cell level, strong vacuolisation of cells, a decrease in the number of endoplasmic reticulum tubules and a reduced number of vesicles produced by Golgi bodies were a frequent reaction to excess aluminium (De Lima and C o p el a n d , 1994; B e n n e t , 1998).In the presence of aluminum ions, the programmed death of PCD cells was also observed (P a n et al. 2001), as well as the disintegration of microtubules and a change in polymerisation of the cytoskeleton (H o r s t et al. 1999; S i v a g u r u et al. 1999).
i r a d a t e , 2000; Ya n g et al. 2000), silicon (V á z q u e z et al. 2002) or tannins (S t o u t j e s d i j k et al. 2001).But in literature we fi nd reports on the deposition of aluminum deposits also in cell nuclei (S i l v a et al. 2000) and cell walls (C r a w f o r d and W i l k e n s , 1997; H o r s t et al. 1999; S c h m o h l and H o r s t , 2001), which is confi rmed by research of the present paper's author.Also, the conducted study does not unequivocally confi rm changes in the number of dictyosomes and vesicles of Golgi bodies or disorders in the structure and number of endoplasmic reticulum tubules, which were described by other authors (C r a w f o r d and W i l k e n s , 1997; C l u n e and C o p e l a n d , 1999).