Effects of aluminum on the cell morphology in the root apices of two pineapples with different Al-resistance characteristics

ABSTRACT

Pineapple (Ananas comosus [L.] Merrill) is a commercial crop commonly planted in strongly acid soils in Taiwan. However, aluminum (Al) in strongly acid soils may be toxic and inhibits the root growth of various pineapples. This study evaluated the cell morphology, 1,3-β-glucan, and malondialdehyde (MDA) of the root apex cells of Al-resistant Cayenne and nonresistant Tainung No. 17 pineapples under two Al concentrations (0 and 300 μM AlCl₃). An observation using transmission electron microscopy indicated no particular variation in the cells of the root apices of the Cayenne pineapple after a treatment with 300 μM AlCl₃. However, apparent changes in the cells of the root apices of the Tainung No. 17 pineapple were observed, for example, separation of the cell wall and cell membrane, enlargement of vacuoles, shrinking and gathering of mitochondria beside the inner cell membrane). The 1,3-β-glucan (callose) and malondialdehyde (MDA) contents in the Cayenne pineapple were not affected by the Al treatment. However, in the Tainung No. 17 pineapple, those contents showed substantial increases under the treatment with 300 μM AlCl₃, indicating definite damage on the root apices of Tainung No. 17. The results further revealed that the non-Al-resistance pineapple cultivars should be cultivated on the soil that contains low toxic aluminum.

KEY WORDS:

• Aluminum
• pineapple
• root apices
• root morphology
• Al resistance

1. Introduction

In strongly acid soils (pH < 5.5), Al exists mainly in the Al³⁺, Al(OH)²⁺, and Al(OH)₂⁺ biotoxic forms (Marion et al. 1976). Thus, Al toxicity is commonly recognized as one of the main causes of damage to plant growth in strongly acidic soils. In general, damage to the root apex cells of plants can be observed using an electron microscope. Goodwin and Sutter (2009) employed scanning electron microscopy to observe the root apices of Arabidopsis species treated with 50 μM AlCl₃, and discovered that the cell wall of the root apices became thicker to defend against Al toxicity. Le Van et al. (1994) studied Al toxicity in the root apices of pumpkins, and Tabuchi and Matsumoto (2001) studied that in wheat. These studies yielded similar results. Because root apex cells are subject to Al toxicity, the hemicellulose content increases gradually, making the cell wall thicker and more tenacious. This phenomenon plays a critical role in the mechanism of resistance to Al toxicity. Minochaa et al. (2001) used transmission electron microscopy (TEM) to observe the internal cells of Picea rubens root apices after a treatment with 0.5 mM Al concentration for 24 h. They observed that the vacuoles in the cells were enlarged, the cell walls were thickened and separated from the cell membrane, and the number of mitochondrian had increased. These signs manifested cell damage when compared with those treated with low Al concentration. When plant roots are subject to Al toxicity, Al blocks the formation of fiber on the cell wall and induces callose synthase on the cell membrane to form callose (Kaneko et al. 1999; Teraoka et al. 2002). As Al concentration increases, the amount of callose on the root also increases (Bhuja et al. 2004). In the case of Al toxicity, the root cellulose synthase stops within 5 min and induces the formation of large quantities of callose (Nakashima et al. 2003). Thus, the formation of callose is a protective mechanism for damaged plants (Jaffe and Leopold 1984). The same phenomenon was found in the studies of other Al-resistant crops, such as oats (Schreiner et al. 1994; Bhuja et al. 2004), corn (Horst et al. 1997; Eticha et al. 2005), tobacco (Chang et al. 1999), barley (Kaneko et al. 1999), and bean (Massot et al. 1999). Al causes lipid peroxidation on the cell membrane of root apex cells (Basu et al. 2001; Yamamoto et al. 2001). This phenomenon leads to the inhibition of root growth in soybeans (Cackmak and Horst 1991), peas (Yamamoto et al. 2001; Kobayashi et al. 2004), rape (Basu et al. 2001), rice (Meriga et al. 2004; Achary et al. 2008), wheat (Darko et al. 2004; Hossain et al. 2005), onions (Achary et al. 2008), and corn (Boscolo et al. 2003). Under normal conditions, the production and removal of free radicals in plants are in a dynamic equilibrium. However, after being intoxicated by Al, this equilibrium is destroyed and the free radicals begin to accumulate, leading to phospholipid peroxidation on the cell membrane and the production of phosphatidylcholine hydroxide. These effects are followed by the conversion to a secondary metabolic product, MDA (Yamamoto et al. 2002). MDA is the final product of the reaction between unsaturated fatty acid and reactive oxygen species, and it is a highly active compound containing aldehyde. The higher the concentration of MDA in a plant, the worse is the damage to its cells or tissue. Therefore, the concentration of MDA can reflect the degree of damage in plants under environmental stress.

Some researches about the examining experiments of Al-resistance for different pineapple cultivars were published. For example, the root elongation, the dry weight and the concentration of calcium and magnesium in Cayenne were increased with increasing Al concentration after treating with AlCl₃ for 4 weeks, however, that of Tainung No.17 were decreased (Lin 2010; Lin and Chen 2011). On the other hand, the Al concentration in Tainung No.17 in whole plant was higher than Cayenne in the treatments of different Al concentrations. Based on these researches, we know that Cayenne was Al-resistance and Tainung No.17 was non-Al-resistance in high Al concentration (300 μM AlCl₃). This study was conducted to evaluate the cell morphology of traditional (Cayenne) and new (e.g., Tainung No.17) pineapple cultivars by non-Al and high-Al environment. The results will provide the knowledge for the farmers to appropriately choose soil properties when cultivating the new pineapple cultivars.

2. Material and methods

2.1. Cultivation of pineapples

Approximately 81 ± 8 g in fresh weight of Cayenne and Tainung No. 17 pineapple was cleaned with deionized water before being planted in a circular plastic container (inner diameter: 25 cm, height: 30 cm) with a hydroponic solution in triplicate. The composition of the hydroponic solution was that of Hoagland and Arnon (1938) with some modifications. The pH was adjusted to 4.5 by using 0.1 N HCl and 0.1 N NaOH. The container with the pineapple cultivar was aerated uniformly by using a pump; this was then moved into a growth chamber (Hipoint, FH-302, Taiwan) regulated at 27°C during daytime (14 h, RH 65%) and 23°C during night-time (10 h, RH 85%). The hydroponic solution was replaced once weekly. The cultivars with different Al concentrations (0 and 300 μM AlCl₃) were cultivated for 4 weeks.

2.2. Effects of Al concentration on the cell morphology of pineapple root apices under TEM observation

The root apices (1 cm) of Cayenne and Tainung No. 17 grown in 0 and 300 μM AlCl₃ solution for 4 weeks were excised using alcohol-cleaned stainless scissors and observed through TEM. The root apices of the pineapples were fixed with 2.5% glutaraldehyde-Na phosphate at a pH of 7 for 2 h and were rinsed with 0.1 M Na phosphate buffer (pH 7) and 5% sucrose solution for 10 min each. The root apices were then fixed with 1% osmium tetroxide and 0.1M Na phosphate at a pH of 7 for 1 h. After rinsing with water three times, alcohol at concentrations of 50%, 70%, 80%, 90%, 95%, and 100% and 100% acetone were used to dehydrate the sample. The dehydrated sample was then enclosed in Spurr’s resin, after which it was cut with an Ultramicrotome (Reichert–Jung Ultracut E), and finally dyed with uranyl acetate and lead citrate. Then the samples were prepared for examination of the root apex cells through TEM (Hitachi, H7100).

2.3. Effects of Al concentration on callose and MDA in the root apices of pineapples with different al resistance

2.3.1. Determination of callose

After treatment with 0 and 300 μM AlCl₃, the root apices (1 cm) of Cayenne and Tainung No. 17 pineapples were cut using alcohol-cleaned stainless scissors to measure the callose. The spectrophotometric method described by Zhang et al. (1994) was used. Root apices were fixed with alcohol (boiling at 70°C for 10 min). After removing the alcohol, the sample was stored at −40°C until the analysis. The samples were immersed in 1 mL NaOH, and then sonicated for 2 min. After being placed in a water bath at 80°C for 30 min, each sample was centrifuged for 15 min at 10,000g. After filtration, the callose was measured using a fluorescence spectrophotometer (Shimadzu, RF-1501, Japan). The excitation wavelength was set at 350 nm and the emission wavelength at 500 nm. Laminarin was employed as a standard, and the callose content was expressed in mg laminarin.

2.3.2. Determination of MDA

The MDA content in the root apices (1 cm) of the Cayenne and Tainung No. 17 pineapples was determined following the method of Heath and Packer (1968). Sample of root apex (0.1 g) were rinsed with 4 mL of trichloroacetic acid (TCA; 5% w/v) and ground. The mixture was centrifuged at 10,000g for 5 min. The filtrate (1 mL) was added to 4 mL of thiobarbituric acid (0.5% w/v, in 20% w/v TCA) and immersed in a water bath at 95°C for 30 min. The sample was quickly frozen and sonicated to remove bubbles before centrifugation at 5,000g for 10 min. The absorption at a wavelength of 532 nm was compared with the standard curve to obtain the MDA content.

3. Results and discussion

3.1. Effects of Al concentration on the morphology of pineapples root apices

Through TEM observation, we found no notable variation in cell nucleus, mitochondria or vacuoles for Cayenne pineapples treated either with 0 or 300 μM AlCl₃ (Fig. 1). However, the vacuole in the cell was enlarged, the cell nucleus became smaller, and mitochondria were sprayed around the cell membrane in Tainung No. 17 pineapples (Fig. 2). Goodwin and Sutter (2009) investigated the change in Arabidopsis species cells due to Al toxicity and found that the cell walls of root apices became thicker and that Al accumulated in the vacuoles when the root apices were treated with 50 μM AlCl₃. This did not occur without Al treatment. This study demonstrates that the cell membrane and cell wall in Tainung No. 17 root apices were separated when treated with 300 μM AlCl₃. Miyasaka obtained similar results regarding Al toxicity with the root apices of Picea rubens after a treatment with 0.5 μM Al for 24 h. They discovered that vacuoles in the cells played a role in the storing of toxic substances and detoxification. Because Tainung No. 17 is not Al-resistant, its vacuoles were spread widely in the cell to mitigate Al toxification when treated with Al solution. To dispel Al from the cell, mitochondria were widely distributed around the cell membrane. These changes in the root apex cells were quite similar to those of rapeseed subject to Al toxicity, as observed by Clune and Copeland (2001).

Figure 1. Observation of root apex cells in Cayenne pineapples by transmission electron microscopy. (a) 0 μM AlCl3, (b) 300 μM AlCl3. (N: cell nucleus, V: vacuole, M: mitochondria, CM: cell membrane, CW: cell wall)

Figure 2. Observation of root apex cells in Tainung No. 17 pineapples by transmission electron microscopy. (a) 0 μM AlCl3, (b) 300 μM AlCl3. (N: cell nucleus, V: vacuole, M: mitochondria, CM: cell membrane, CW: cell wall)

3.2. Effects of Al concentration on callose and MDA in the root apices of pineapples

Under normal conditions, the fiber synthase on the cell membrane of most plants continues to enhance the cell to form fiber. However, the function of fiber synthase is inhibited in response to environmental stress. By contrast, the function of callose synthase is initiated to defend against environmental stress (Delrner, 1987). Bouazizi et al. (2009) treated soybean root apices with 75 μM CuSO₄ and discovered that callose was produced in abundance. They deduced that the increased thickness of the cell wall in root apices was related to the production of callose. Volgger et al. (2009) reported that the root of wheat produces a large quantity of callose to defend against environmental stress due to high permeability. Vicedo et al. (2009) suggested that hexanoic acid can induce the production of callose to stave off powdery mildew. Therefore, the production of callose plays a major role in the defense against environmental stress for plants. Table 1 indicates that no callose was measured in the root apices of Cayenne and Tainung No. 17 pineapples when they were treated with hydroponic solutions without Al. However, when they were treated with 300 μM AlCl₃, the callose content in the root apices of Cayenne and Tainung No. 17 pineapples was 1.1 and 4.9 mg µmole/g, respectively. The MDA content in the root apices of Cayenne and Tainung No. 17 pineapples was approximately equivalent (3.2 and 3.4 μmole/g FW, respectively) when they were not treated with Al. However, when they were treated with Al, the MDA content in the root apices of Cayenne pineapples increased to 8.5 μmol/g FW, whereas that of Tainung No. 17 pineapples increased to 28 μmol/g FW. These results reveal that the damage on the root apices is more severe in Tainung No. 17 pineapples than in Cayenne pineapples. When plants are subject to damage, callose is produced to protect the cell. Therefore, its content can serve as an index for plant damage (Jaffe and Leopold 1984). This study demonstrates that the root apices of Tainung No. 17 pineapples are more severely damaged than are those of Cayenne pineapples when they are treated with 300 μM AlCl₃. This phenomenon is due to the formation of fiber being inhibited on the cell wall of the root apices and to callose synthase being induced on the cell membrane to form callose (Kaneko et al. 1999; Teraoka et al. 2002). MDA can serve as an index for oxidization damage to plant cell or tissue. The higher the MDA content in plants, the more the cell or tissue is damaged (Yamamoto et al. 2001). This study also reveals that the MDA content in the root apices of Tainung No. 17 pineapples is considerably higher under a treatment of 300 μM AlCl₃ than without Al treatment. However, the MDA content in the root apices of Cayenne pineapples is only slightly increased by the treatment of 300 μM AlCl₃ compared with no Al treatment. Therefore, when treated with 300 μM AlCl₃, the root apices of Tainung No. 17 pineapples appear to be more oxidized compared with those of the Cayenne pineapples.

Table 1. Callose and malondialdehyde content in the root apices of Cayenne and Tainung No. 17 pineapples after treatment with 0 and 300 µM AlCl_3.

	Cayenne		Tainung No.17
	0 μM AlCl3	300 μM AlCl3	0 μM AlCl3	300 μM AlCl3
Callose (µmole/g FW)	N.D.	1.1 ± 0.4	N.D.	4.9 ± 1.2
MDA (µmole/g FW)	3.2 ± 1.0	8.5 ± 2.3	3.4 ± 0.7	28 ± 3.2

Callose: 1,3-β-glucan MDA: malondialdehyde

*In this experiment, 3 replicates were used.

4. Conclusion

After observation through TEM, there were no significant changes on the cell morphology of the Cayenne pineapple under a no Al or high Al treatment. However, the cell morphology of the Tainung No. 17 pineapple displayed great changes. Based on the analysis of callose and MDA, the cell in the root apices of Tainung No. 17 was actually damaged under high Al concentration. This study can serve as a reference for Al resistance in pineapples.

Acknowledgments

The authors would like to thank Professor Yu-Chia Chung at the National Sun Yat-sen University, in Kaohsiung, Taiwan, for the critical reading of the manuscript. The English of this article was critically corrected by Wallace Academic Editing.