- Top of page
- Materials and Methods
Aluminium (Al) is the most abundant metal in the world and it is the third most abundant element in the earth's crust. Al generally becomes phytotoxic in acid soils (pH ≤ 4.5) and is one of the major factors limiting crop production worldwide. It is therefore of great importance to understand the mechanisms of Al resistance in plants.
The most important and primary manifestation of Al phytotoxicity is an inhibition of growth of the root apex and root hairs; mature root regions appear to be relatively unaffected by exposure to Al (Kochian, 1995). Al probably causes the primary injury in the apoplast of the peripheral root cells and acts on several cellular processes, such as cell wall assembly, ion fluxes, plasma membrane stability and function (Horst, 1995; Kochian, 1995; Rengel, 1996; Wenzl et al., 2001), lipid peroxidation (Yamamoto et al., 2001), cytoskeletal dynamics (Blancaflor et al., 1998) and callose synthesis (Zhang et al., 1994).
Callose (β-1,3-glucan) is a plant polysaccharide that is a natural constituent of sieve plates, pit fields and developing cell plates (Verma & Hong, 2001). Increased synthesis of callose is a well known early indicator of Al injury in several plant species (Horst, 1995). In maize (Zea mays), both aluminium and callose accumulate in the zone 1–2 mm behind the root tip (Sivaguru & Horst, 1998). The Al induced growth inhibition might in part be due to a translocation of sugars from cellulose to callose production (Zhang et al., 1994; Kaneko et al., 1999). It is thought that Al increases intracellular calcium levels, which in turn lead to induction of callose synthesis and increased callose deposits in plasmodesmatal pores, causing impaired intercellular transport and cell-to-cell communication (Jones et al., 1998; Sivaguru et al., 2000).
The molecular mechanisms of Al toxicity remain poorly understood (Delhaize & Ryan, 1995; Kochian, 1995; Richards et al., 1998). Aluminium stress has been shown to up-regulate pathogenesis related (PR) genes (Hamel et al., 1998) and genes that are part of a general stress response in plants. In particular, peroxidases have been shown to be induced by Al, and are involved in oxidative stress, which in turn leads to browning reactions as well as being part of the signal transduction cascade involved in callose production (Cakmak & Horst, 1991; Richards et al., 1998; Jan et al., 2001; Milla et al., 2002).
Most of what we know about Al tolerance in plants comes from work on Al sensitive species such as Arabidopsis, maize, rice and wheat. In general, tree species are more tolerant to Al than herbs, grasses and cereals. For example, it was found there was no reduction in root growth in seedlings of Norway spruce (Picea abies), silver birch (Betula pendula) and Scots pine (Pinus sylvestris) at Al concentrations below 0.3, 3, and 6 mm, respectively (Göransson & Eldhuset, 1987, 1991). In the field, 3 yr of artificial Al addition, producing potentially toxic Al concentrations in the soil solution, had no impact on fine root growth in Norway spruce trees (De Wit et al., 2001). Norway spruce thrives on naturally acid soil where its growth leads to a further acidification of the soil. It is therefore likely that Norway spruce has developed adaptive mechanisms to tolerate high Al conditions; however, there have been few attempts to elucidate Al tolerance mechanisms in tree species, and such studies could yield valuable new information about Al tolerance mechanisms in plants in general.
The main goal of this study was to investigate fundamental characteristics possibly involved in Al tolerance mechanisms in Norway spruce seedlings. Young seedlings exposed to Al solutions for up to 1 wk were used to study the effects of Al on callose production, root growth, and root tip browning and swelling. Various microscopy staining techniques were used to identify cellular changes and callose deposits, and the expression of chitinase and peroxidase isoforms was examined by polyacrylamide gel electrophoresis – isoelectric focusing.
- Top of page
- Materials and Methods
The initial and most dramatic symptom of Al toxicity is inhibition of root growth. Our study shows that root growth in Norway spruce was strongly reduced after exposure to 5 mm Al, while in the 0.5 mm Al treated plants reduced root growth only occurred after prolonged exposure. Compared with what many other plant species can tolerate, an Al concentration of 0.5 mm is considered as a high dosage and is greater than that typically found in solutions of acid soils (5–200 µm). However, our observations are consistent with previous studies on Al stress in Norway spruce. For example, in a study on Norway spruce nursery seedlings, Godbold & Kettner (1991) found no impact on root growth after 5 d at 0.4 mm Al, while 60% of the seedlings had ceased growing already after 2 d exposure to 0.8 mm Al. The reduced root growth may be related to the fact that Al alters cytoskeletal dynamics and signal transduction pathways which may eventually lead to inhibition of cell division and elongation (Jones & Kochian, 1995; Blancaflor et al., 1998; Frantzios et al., 2001). Additionally, when Al binds to and modulates the cell walls, the wall expansion and cell extension can be affected (Le Van et al., 1994; Zhu et al., 2003).
The browning reaction observed in the seedling root tip, right behind the apical meristematic zone, is a characteristic of Al injury caused by oxidative stress and the accumulation of phenolic substances (Richards et al., 1998). Al-induced root tip browning, the concomitant collapse in cell structure, accumulation of polyphenols, and the development of intercellular air spaces, are similar to the symptoms observed during the hypersensitive response (localized cell death) at the site of initial contact with a pathogen after infection (Nicholson & Hammerschmidt, 1992). We observed localized cell death in the area where the phloem starts to differentiate. This phenomenon may lead to decreased transport of carbohydrates from the shoot to the root apical meristem, and could partially explain the observed decline in starch content in the roots of Al treated seedlings as a lack of soluble carbohydrates may induce starch degradation.
It was evident from observing stained roots that Al was located primarily in the walls of the root cap cells, border cells and the outer cortical cells of Norway spruce roots, even at 5 mm concentrations. Al may be confined to the cell wall region, most likely due to an occupation of carboxyl groups in pectin (Hamel et al., 1998). An inhibition of Al diffusion through the cell walls prevents it from reaching the symplast and the vascular system at high concentrations. A greater cell wall Al binding capacity could help explain the higher resistance observed in Norway spruce compared with other, more Al sensitive species. This ability to retain Al may be due to a high cationic exchange capacity of the cell wall of Al tolerant species (Godbold & Jentschke, 1998). Another prominent feature of spruce roots is the large root cap cells, which have a high mucilage production. A high mucilage production has been hypothesized to increase the capacity of the roots to bind and exclude Al from sensitive growing areas (Zhu et al., 2003). In Al sensitive species, for example the Graminae (grasses), the root cap cells are small and produce less mucilage, something that could explain their reduced capacity to scavenge and detoxify Al (Crawford & Wilkens, 1997; Zhu et al., 2003).
Callose concentration in root tips increased only at 5 mm Al, and even then the increase in callose production was much lower than what is commonly observed in other species, for example maize, treated with much lower Al concentrations (DL Jones, unpublished). At exposure to lower Al concentration no significant change in the callose levels was detected. In species like soybean, Al concentrations as low as 10 µm induce callose production in root tips within minutes (Wissemeier et al., 1987). In 5-wk-old Norway spruce seedlings, callose in root tips has been observed after 3 h treatment with 170 µm Al (Jorns et al., 1991). Callose has also been measured in root tips from field grown spruce trees where the soil solution Al concentration ranged from less than 10 to 200 µm (Wissemeier et al., 1998). Corresponding to the biochemical measurements, we also detected Al induced callose production in the form of callose deposits in the cell walls and interstitial spaces of root cap and cortical cells at 5 mm Al. These callose deposits may lead to an occlusion of pores and plasmodesmata, which effectively block symplastic transport (Sivaguru et al., 2000). Callose occlusion would perhaps act as a barrier to Al influx into the cortex of the root, but is also a very probable factor in the root growth inhibition of Al (Sivaguru et al., 2000). We found a correlation in callose increase and root growth cessation at 5 mm Al. It should be noted that root growth in 0.5 mm Al decreased without any production of callose above that of the control. This contradicts the findings of others (Wissemeier et al., 1998) that callose increases in concentration before root growth decreases. It is uncertain whether stress protein and callose production in response to increased Al is symptomatic of susceptible or of tolerant species (Jan et al., 2001). Ezaki et al. (2001) found a lower callose content in more Al resistant Arabidopsis lines compared with the wild type. Our study strongly indicates that in 10-d-old Norway spruce seedlings, callose is not an important inducible factor in Al tolerance; increased callose production is triggered only at extreme Al stress.
Genes corresponding to peroxidases and other pathogenesis and stress related proteins were previously reported to be up-regulated upon Al exposure in wheat (Triticum aestivum L. cv.) and Arabidopsis (Ezaki et al., 1996, Hamel et al., 1998). Our results show that in 5-wk-old Norway spruce only very strong and prolonged Al stress would lead to an up-regulation resulting in increased activity of several peroxidase and chitinase isoforms. The initial fall in enzyme activity during the first 1–12 h may be due to reduced enzyme activity as a direct inhibitory effect of Al (as many peroxidases are known to be extra cellular), increased degradation or reduced expression of the corresponding gene(s). In barley (Hordeum vulgare), Tamás et al. (2003) report an increased activity of at least five anionic (acidic) and four cationic (basic) peroxidase isozymes in response to Al exposure. In the present work, one highly basic and one highly acidic peroxidase isoform, and four basic and three acidic chitinase isoforms increased in response to 5 mm Al treatment after 7 d. In other stresses, such as drought and pathogen infection, we have found a strong increase in similar chitinase and peroxidase isoforms within 4 d (Fossdal et al., 2001; Nagy et al., 2004). It is possible that Norway spruce may not need to produce peroxidases and chitinases above the already high constitutive levels as a response to Al concentrations at or below 0.5 mm. These proteins are involved not only in stress responses, but also in normal development and morphogenesis. For example, peroxidase activities occur in the cell wall, were these enzymes have been suggested to modulate cell wall rigidity and extensibility, thus reducing Al diffusion through cell wall (Hamel et al., 1998). Even though the main role of chitinases is in defence against pathogenic fungi, evidence is suggesting that some chitinolytic enzymes play a role in normal growth and development acting on arabinogalactan proteins (Passarinho et al., 2001), and affect cell growth and cell-to-cell communication (Wiweger et al., 2003). The high ability of Norway spruce to grow on acidic soils could be explained by a higher capacity to tolerate Al, maybe in part by a generally high constitutive level of these and other stress related proteins. This is, to our knowledge, the first report indicating that isoforms of peroxidases and chitinases are affected only by extremely strong Al exposure in Norway spruce.
Based on a total evaluation of our results, we postulate that with external Al concentrations lower than 0.5 mm, an increased production of callose, peroxidase or chitinase (above constitutive levels) is not necessary for the Al tolerance in young Norway spruce seedlings. Callose seems to be important only at very high Al concentrations. It is possible that the high constitutive levels of peroxidase and chitinase confer to this species cell wall properties that help to exclude Al, and that increased production of these proteins are necessary only when external Al increases to unusual levels. In addition, physical barriers in the rhizosphere, for example mucilage, root cap (border) cells, ectomycorrhiza, and cation exchange capacity may be so effective that the more sophisticated inducible mechanisms is not effectuated. Only if the external Al concentration exceeds a limit so that the external defence barriers break down, and Al gets access further into the roots, increased callose production and enzyme activity will be apparent. The ability to endure within cell Al may also be associated with the high content of phenolics in the vacuoles of epidermal cells in Norway spruce roots (Ofei-Manu et al., 2001), a possible accumulation of Al in complexes with phenolics deserves further studies.