TOXIC INFLUENCE OF ALUMINUM ON ROOT MICROSTRUCTURE OF RED PEPPER (CAPSICUM ANNUUM L.)

Agata Konarska
Departament of Botany, University of Life Sciences in Lublin, Poland

ABSTRACT

Toxic effect of aluminum on roots  was studied using seedlings of Capsicum annuum L. Subjected to water culture with different concentrations of AlCl₃·6 H₂O (10, 20, 30 and 40 mg·dm^-3) for a period of 14 days. Observations of red pepper root tissues were carried out by light microscopy and scanning electron microscopy. The roots Al-treated were characterised by an increased diameter, the shortening or absence of the cap and cracks on the surface. The length of the elongation and meristematic region was reduced. Disturbances in the arrangement, size and shape of the cells of the apical section of the root were observed. The cells of the growing point were characterised by disturbed division planes. The cells of precortex and primary cortex underwent necrosis or showed symptoms of destruction. Many cells were marked by hypertrophy and strong vacuolisation. In the vacuoles of the cells of the investigated root section, numerous dark bodies were observed.

Key words: Capsicum annum, Al toxicity, root morphology and anatomy.

INTRODUCTION

The acidification of the soil solution is effected of use of mineral fertilisers and the emission of acid and acid-forming substances released into the atmosphere by industrial facilities and mechanical vehicles, is currently a problem of many countries of the world, also including Poland. The decline in soil pH causes the release of aluminum ions from the sorption complex. These ions are extremely toxic for most species of cultivated plants. The root system of young plants, which easily and quickly absorbs and accumulates aluminum ions in its tissues, is the most exposed to damage [8,10,22,30,35]. As a consequence, the elongation growth of roots is inhibited, and the greatest changes are observed in the apical meristems of both primary and lateral roots [2,19,21,24,36,37]. Species and cultivars differ significantly in their tolerance to excessive aluminum in the substrate, what has been described by many researchers [12,25]. Tolerance to aluminum is determined genetically or can be results of the activity of extra- or intercellular resistance mechanisms, mainly involving the immobilisation of Al ions by fixing them with different compounds [14,29,33].

Over the recent years, the acreage of red pepper cultivated in Poland has increased significantly, the direct consumption and use of this plant’s fruit in food processing have also been growing, therefore, it is important to determine the resistance of this species to aluminum toxicity in order to ensure the most optimal possible conditions for red pepper growth and yielding. An earlier study of Konarska [20] relating to the rate of growth roots of three cultivated plant species under the influence of aluminum shows that red pepper cv. 'Trapez' was characterised by the highest sensitivity to the presence of aluminum in the substrate. In this paper, which is a continuation of the earlier studies, the symptoms of aluminum toxicity are presented with respect to the root morphology and anatomy of this cultivar. In literature, no reports have been found relating to the response of red pepper organs to the increased aluminum content in the environment.

EXPERIMENTAL PROCEDURES

Red pepper (Capsicum annuum L.) seedlings cv. 'Trapez' were cultivated in plastic containers of 4 dm³ volume which were filled with modified Knop's nutrient solution [6]. Aluminum was added as AlCl₃·6 H₂O at four concentrations: 0 (no Al⁺³ was added, control), 10, 20 and 40 mg dm^-3 of the medium, what corresponds to 0; 1.1; 2.2 and 4.5 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. Sterile seeds of plant germinated on wet filter paper of Petri dishes. After four days from germination, the seedlings were transferred to water-hydroponics 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 with a PAR of 300 µmol·m^-2·s^-1, with day and night temperatures of 24°C ±1°C and 20°C ±1°C respectively, and a 70% relative humidity. The experiment was run in three 14^th day replications. And in the end of each experiment, ten top fragments of main roots (about 10 mm lenght) were cut to prepare microscopic slides.

Scanning electron microscopy (SEM). Samples of the roots after 14 days of growth of the control plants and treated with 40 mg AlCl₃·dm^-3 were fixed with a 4% solution of glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 at a temperature of 4°C for 12 hours. Subsequently, the fragments were rinsed four times with the buffer and dehydrated in an ethanol series of ethanol concentrations: 30%, 40%, 50%, 60% and 70%. After being dehydrated, the root fragments were transferred to acetone, dried at the critical point in liquid CO₂ and dusted with gold using the CS 100 Sputter Coater. The preparations were observed using the Tesla BS-340 microscopy.

Light microscopy (LM). For the anatomic observations, sections from the apical parts of main roots were sampled and permanent semi-thin slides with 0.5-0.7 µm thick there cut with glass knifes. They were stained with 1% methylene blue with 1% azure II in a 1% aqueous solution of sodium tetraborate. The images were observed and recorded using the Jenaval Contrast microscopy.

RESULTS AND DISCUSSION

Two higher aluminum concentration (20 and 40 mg·dm^-3 AlCl₃) induced numerous disturbances in the structure of the roots of 'Trapez' red pepper, in proportion to the level of concentration. These changes prove the high sensitivity of the studied taxon to aluminum toxicity, what corresponds with the earlier results obtained by Konarska [20].

Structure of the root surface. The primary roots of the control red pepper seedlings in the investigated section (0-1 cm from the apex) had a slightly striated surface, formed by the prosenchymatic, densely packed epiblema cells. Few exfoliating cells of this tissue were observed at places. A clearly-developed cap formed the apical portion (Photo 1).

The primary roots of red pepper formed in the presence of 40 mg·dm^-3 AlCl₃ were strongly thickened and they were characterised by the absence of the cap and a partial destruction or dieback of the apical meristem, as a result of which the apical region was widened and the apex was flattened (Photos 2, 3). According to Bennet and Breen [1], plants with lower resistance to Al are characterised by roots devoid of the cap in the presence of aluminum.

On the apical surface of the root region, numerous, deep cracks were visible, running perpendicular to the axis of the organ, through which masses of disorderly arranged cells were released, evidencing the disorganisation of the apical meristem (Photos 2, 3). Similar symptoms in other plant species were also observed by De Lima & Copeland [11], Hirano & Hiji [16] and Konarska [19,21].

Moreover, in a very close distance of the growing zone, before the root hair region, the beginnings of lateral roots were observed (Photo 2). In existing literature, no case is described that under the influence of aluminum the first lateral roots are growing above root hairs zone. These deviations could result from the accelerated differentiation and maturation of the tissues, as well as the quick dying-off of the outer cell layers, including the epiblema, what was confirmed by the authors of this paper in light microscopy investigations.

Changes in root anatomy. The tissues in the roots of the control plants, observed in longitudinal sections by using light microscopy, were properly developed and they formed regular rows, characteristic for the root meristem (Photos 4, 5). The cells of the meristematic tissue, situated above the appropriately developed cap (Photo 6), were regular in shape and the presence of a large, centrally located cell nucleus or the occurrence of different stages of mitosis (Photo 5).

In the presence of aluminum ions of concentrations, an increase in the root diameter, as a result of cell hypertrophy, as well as differences in the structure of the apical region and the higher located regions of the root were observed. The cell hypertrophy of the apical region of the root, observed in this study, can be effect by the isotropic growth in all directions. Microtubules, whose structure and activity are disturbed in the presence of aluminum, are responsible for this process [17].

At 20 mg·dm^-3 AlCl₃, the cell division region was clearly shortened, and its cells were characterised by darker staining of the protoplasts (Photo 7). The root cap was also shortened markedly, and it was characterised by larger cells, often with thickened walls (Photo 8). Several layers of the externally located cells of the cap were affected by destruction or necrosis.

At 40 mg·dm^-3 AlCl₃, the absence of the cap, as well as the dying-off and exfoliation of the outer cell layers of the apical meristem caused the root to be bluntly ended (Photo 9). The meristematic region was limited to a group of irregular-shaped and different-sized cells forming spherically contoured clusters (Photos 9, 10). Such an untypical shape of the meristematic region is a feature which has not been hitherto described in rich literature on aluminum related problems.

In the growing zone of the root, disorders in the cell division planes of the meristem and the varying size of daughter cells formed were also observed (Photo 11). The divisions in different directions were probably the cause of the formation of cells with the shapes of irregular polygons (seen in longitudinal section). The arrangement of these cells significantly differed from the topography of a typical meristem of the taproot of the control plants (Photo 5). In the presence of aluminum, many authors also found a disordered pattern of mitosis as a result of disturbed DNA replication and the blocking of the formation of the mitotic spindle, as well as the sticking together of chromosomes [5,13].

The substantial reduction in the length of particular sections of the red pepper roots, proven in this study, in particular of the meristematic and elongation regions, could be the cause for the formation of lateral roots close to the apical part of the primary root, what is confirmed by the studies of Clune & Copeland [8] relating to other plant species. But existing literature does not describe any case that under the influence of aluminum root hairs form above the place where the first lateral roots are growing. The above deviations could result from the accelerated differentiation and maturation of the tissues, as well as the quick dying-off of the outer cell layers, including the epiblema, what was presented by the authors of this paper in light microscopy investigations.

The outer cell layers forming the primary epidermis, the parenchyma of precortex and of the higher located primary cortex (Photos 12, 13) underwent the greatest changes. An irregular arrangement of particular layers of cells was observed, which were often characterised by an untypical shape or showed hypertrophy (Photos 12, 13). Large-sized intercellular spaces were also visible, formed as a result of wall separation or cell dying (Photos 12, 13). In addition, cracks and the loss of tissue, as well as the detachment of the outer cortex layers were observed. Many cells of this sector of the root were characterised by the undulation or damage of the cell walls. Observations of many researchers show that the cell wall is the main and earliest compartment of aluminum accumulation, and ions of this metal, displacing calcium from pectin chains building middle lamella, can result in a substantial limitation of the elasticity of walls and their damage [4,26]. Other authors sugest that under aluminum stress conditions an intensified synthesis of compounds composing cell walls occurs, and that cell walls also increase their rigidity, what in consequence may lead to the disruption of the walls [30,34,38]. In turn, in opinion of McQuattie & Schier [27] and Oleksyn et al. [31] the wall undulation may be caused by the loss of turgor and the shrinkage of protoplasts.

Many cells of the investigated root fragment showed the destruction of protoplasts or necroses (Photo 12). According to Hecht-Buchholz & Foy [15] and Pan et al. [32], necroses may occur as a result of the dying-off (induced death) or disintegration and self-digestion (autolysis) of cells. In the opinion of Lee & Pritchard [23] and McQuattie & Schier [27] , necroses found in the higher portions of the root may result from the destruction of cells and the disintegration of their cytoplasm caused by the disruption of the plasmatic membranes.

The more ordered rows, than the primary cortex cells, formed the tissues of the central cylinder where, at a height of about 1 mm, the formation of the first xylem elements was observed earlier than in the control object (Photo 14).

In the presence of the increased aluminum concentrations, the cell protoplasts were characterised by strong vacuolisation in all the sectors of the root. The obtained results correspond to observations of other researchers who tested different plant species [18,28]. Such strong vacuolisation may result, among others, from the premature aging of tissues [3], induced by the activation of the resistance mechanism, involving the quick immobilisation of aluminum in the vacuoles.

In the vacuoles of the cells of the investigated root fragments, the occurrence of spherical or merging bodies was found, with dark staining and various shapes, probably containing aluminum deposits (Photos 11, 12). In the higher located parts of the root, the number of these formations decreased and they developed in to smaller forms (Photos 13, 14). Papers of other researchers contain similar data [7,8]. These deposits were present in the vacuoles of different-level cells in all the investigated regions of the red pepper root, starting from the epiblema up to finish with the central cylinder, what proves a very high sensitivity of this cultivar to aluminum ions, what was found earlier by Konarska [20]. Such a deep penetration of Al⁺³ may also result from quite a high concentration of aluminum ions in the medium and the long duration of the effect of the toxic element. The influence of these factors on the amount and place of aluminum accumulation is reported by Crawford et al. [9] and Wheeler [39].

At the highest concentration of the aluminum ions (40 mg·dm^-3 AlCl₃·6H₂O), above the mitotic region of the primary root, few leucoplasts were observed containing aggregates of starch grains, whereas their larger number was observed in the cells of the cortex and the cylinder in the higher located regions of the root (Photos 14, 15). But at this concentration, the occurrence of amyloplasts in the roots of the control plants was not found, also at the lowest aluminum concentration (10 mg·dm^-3 AlCl₃·6H₂O).

CONCLUSIONS

1. The root system of 14 day red pepper seedlings 'Trapez' characterises strong sensitivity on a toxic influence of aluminum iones.

2. The morphology and anatomy changes of a root under the aluminum influences regarded the top part of a root; the cap, the meristematic and the elongation zones, especially.

3. The structure disturbances of red pepper roots treated with aluminum were observed in all root layers: epidermis, primary cortex and central cylinder, too.

REFERENCES

Accepted for print: 23.10.2008

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