Aluminium localization in conifers growing on highly acidic soils in Ontario, Canada
M.J. Hodson1 and A.G. Sangster2
1School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane, Headington, Oxford, OX3 0BP, UK; 2Division of Natural Sciences, Glendon College, York University, Toronto, M4N 3M6, Canada

Two year old needles were sampled from healthy trees of Eastern white pine (Pinus strobus L.) growing in a previously heavily polluted environment with acidic soils at Sudbury, Ontario, Canada. Mineral distribution in the needles was investigated in a cryo-SEM with x-ray microanalysis. The epidermal and transfusion cells and the xylem walls were major sites of aluminium accumulation, and Al increased towards the tip of the needle. Silicon was concentrated in the tip with the epidermal and mesophyll walls and the transfusion cells being the major silicon localization sites. The heaviest concentrations of calcium occurred in the epidermis, mesophyll and endodermis. The results are compared with previous work on trees from uncontaminated sites, and the possible role of silicon in alleviating the toxicity of aluminium in the conifers is discussed.

Key words: Pinus strobus L.; Eastern white pine; aluminium; silicon; x-ray microanalysis.

     It is widely accepted that acidic precipitation has been responsible for the die-back of trees in Western Europe and North America, but the mechanisms are not well understood. Free Al is mobilized in affected soils, and Al toxicity is almost certainly a major contributory factor in the die-back. Our own work has shown that silicon can alleviate Al toxicity in cereals (Hodson and Sangster, 1993; Hodson and Evans, 1995; Cocker et al., 1998a, 1998b), but the conifers have received little attention. Over the last six years we have been carrying out some research on biomineralization in the needles of the conifers (Sangster et al., 1997; Hodson and Sangster, 1998). Microanalytical work, on needles of several conifer species, indicated that Al is always codeposited with Si (Hodson and Sangster, 1999), in the outer tangential wall of the epidermis and in the transfusion cells. Silica distribution is fairly even along the length of the needle in spruce (Hodson and Sangster, 1998), whilst the heaviest mineralisation is at the needle tip in pine (Hodson and Sangster, 1999).
     Thus far all of our investigations have concerned trees growing in relatively unpolluted environments. Sudbury in N. Ontario, Canada, is well known as a site that has been heavily polluted in the past, and was at one time a major world source of sulphur dioxide and acidic pollution. We decided to extend our previous work on the white pine needle (Hodson and Sangster, 1999) to include plants growing at the Sudbury site. The aims of this investigation were: to assess aluminium and silicon distribution in needles from Sudbury trees; to compare the results with those from previous work on trees from uncontaminated sites; and to consider the possible role of silicon in ameliorating the toxicity of aluminium in the conifers.

Species and Site Description
     Pinus strobus L. (Eastern white pine) is amongst the conifers (division Coniferophyta) exhibiting needles in clusters. It has needles of 5-10 cm in length, in groups of five, the only Canadian pine in this category. An important commercial species, it ranges across southern Canada from Newfoundland to Manitoba, preferring sandy or loamy soils, but it may also grow on rocky substrates and sphagnum bogs. It occurs as pure stands or in association with other conifers and hardwoods (Petrides, 1972; Hosie, 1979).
     The Sudbury collection site is in the Hemlock-White Pine- Northern Hardwood Forest Region, the biotic transition between northern boreal forest and the more southern deciduous forest of N. America (Delcourt and Delcourt, 1991). The site is on the southern portion of the Precambrian Laurentian Shield, an ice-scoured plain resulting from Pleistocene glaciation. With subsequent ablation of the ice, the land mass was overlain by till and glaciofluvial deposits. The Sudbury collection site was located in the Lake Laurentian Conservation Area (46o 26' 52" N; 80o 58' 12" W) which is covered by a thin layer of glacial till broken by frequent rock outcrops. A major smelter complex at Sudbury, engaged in the production of nickel and copper from sulphide ores, is located 8 km NNW of the collection site. The combined historical impacts of logging, brush fires and smelting processes removed the original pine forests and other vegetation which covered the region to produce barren areas. Subsequently, soil erosion washed away the humus rich soil horizons. Acidification and elevated copper, zinc and nickel levels resulting from smelter emissions contaminated the remaining soil. Soil pH in the region is in the 3.5-4.0 range. The ground under the trees at the collection site was covered by an 8-10 cm thick duff layer composed of pine needles and mosses. Soil and duff combined to form a thin overburden on the granitic rock. Over the past 30 years advances in smelting technology, such as flue gas desulphurization, have reduced emissions considerably. Reclamation projects have employed liming as a technique to elevate the soil pH, decrease the uptake of metals and increase the activity of the soil microflora (Lautenbach, 1985). Reclamation, along with natural regeneration, has contributed greatly to the recolonization of the Sudbury barrens. Environmental modification had previously prevented regeneration because of frost heaving of seedlings, slow growth and die-back. Eastern white pine, such as those at the collection site, were uncommon so close to the smelter operations (except for plantings in the reclamation plots). The relatively few present appeared to be healthy except for occasional browning of some needle tips, which may have been drought related. These specimens grew in a thin sandy podzol underlain by granitic rock.
Collection of plant material in the field and sample storage
     Healthy trees were sampled in May 1999. Branches growing 3 m above ground level were sawn from trees which were approximately 10 m in height. Severed branch bases were submerged in water for transport to the laboratory. Mean needle length (n=10) was 5.8 cm. Two year old needles were removed from the branches, cleaned in a detergent solution, and wiped dry with absorbent tissue. They were stored in a cryo-biological storage unit, maintained at the temperature of liquid nitrogen, until examined.
Cryo-SEM and microanalysis
     Preparation of the frozen hydrated needles for examination in the cryo-SEM followed the procedures used previously for gymnosperm needles by Hodson and Sangster (1998). The P. strobus needles were divided into basal, middle and tip sections. Elements detected in the needle specimens were: magnesium, aluminium, silicon, sulphur, chlorine, potassium, calcium and manganese. Here we will only briefly consider results for calcium and we will concentrate on Al and Si. Quantitative data were obtained for Al and Si in six tissue locations: the epidermis outer tangential wall (OTW); the hypodermis OTW; the mesophyll radial wall; the endodermis OTW; the transfusion walls and cytoplasm; and the xylem cell walls. Analyses were conducted under the following conditions: accelerating voltage 15 kV; beam current, 1 nA; beam area, 2 x 2 µm at x 3000 magnification; working distance 35 mm. Other specifications were as described previously (Sangster et al., 1997; Hodson and Sangster, 1998). Contaminant checks on the embedding medium surrounding the specimens and the column indicated that no spurious heavy metal peaks were present. Five replicate analyses per tissue were transformed into concentrations (mmol kg-1) using a ZAF-PB program.

 Figure 1 shows mineral localization in the tip of a white pine needle. The secondary electron image (Fig. 1A) illustrates the thickened epidermal and hypodermal layers surrounding the mesophyll chlorenchyma. Internally the endodermis borders the vascular tissue including the large transfusion tissue cells. The heaviest concentrations of calcium occur in the epidermis, mesophyll and endodermis (Fig. 1B). Figure 1C indicates that the major sites of silicon localization were the epidermal and mesophyll walls and the transfusion cells. Aluminium accumulates mostly in the epidermal and transfusion cells (Fig. 1D).
     The results of a quantitative microanalytical survey of Al and Si distribution in the needles of white pine are shown in Figures 2 and 3. Aluminium was found in all of the tissues, but was particularly high in the transfusion cells and the xylem walls (Fig. 2 ). The data also suggest that Al increases towards the tip of the needle. Silicon distribution in white pine needles is very much concentrated in the tip (Fig. 3). It is worth noting that the tissues with the highest Si in the tip region, the xylem walls and transfusion, were also the tissues with the highest Al (Fig. 2).

     In our previous investigation (Hodson and Sangster, 1999) we reported that Al was found in all of the tissues in white pine needles from Muskoka, Ontario, but was particularly high in transfusion cells. The data suggested that Al increased towards the tip of the needle. This has also been found by Giertych et al. (1997), using chemical analysis of Scots pine needles. The patterns of Al distribution observed in the Sudbury pine needles were very similar (Figs 1D, 2). Silicon in Sudbury white pine needles is very much concentrated in the tip (Fig. 3). The tissues with the highest Si in the tip region, the xylem walls and transfusion, were also the tissues with the highest Al (Fig. 2). Muskoka needles showed a similar pattern of Si distribution (Hodson and Sangster, 1999).
 Table 1 shows a comparison between Al and Si analyses of the transfusion layer (the area of heaviest Al accumulation) in white pine needles collected from three sites in Ontario, Canada. The Sudbury site is described above, and the soil has a pH in the range 3.5-4.0. The Muskoka and Glendon sites have been previously described (Hodson and Sangster, 1998), and the soil pHs at 40 cm were determined to be 4.2 and 6.7, respectively. The results suggest that Al concentrations in the transfusion cells are higher in tips of the needles from plants growing in the two sites with acidic soils. It may also be significant that Al concentrations were highest in the tips of needles from the previously polluted Sudbury site. The Glendon plants show no accumulation of Al in the needle tips. Plants from all three sites showed increased Si in the needle tips, but the Glendon plants showed this to the least extent.
     As yet it is not known whether the association of Al with Si observed in these and other conifer needles (see Hodson and Sangster, 1999) has any physiological significance, or is just a coincidence. In the cereals very little Al is transported to the shoot, and we believe that the observed amelioration of Al toxicity by Si in these plants is probably the result of interactions in the root apoplast (Cocker et al. 1998b). In the conifers, however, much more Al is transported to the shoot tissues (Hodson and Sangster, 1999) and codeposition with Si may serve as a mechanism for isolating toxic Al from the other tissues. Unfortunately, there are, as yet, no experimental data on Al/Si interactions in the conifers. We hope to obtain such data in the near future.

The authors thank: Profs. M.E. McCully, M.J. Canny of the Biology Dept. and Dr. Cheng Huang, Lewis Ling and P. Jones of the Research Facility for Electron Microscopy, Carleton University, Ottawa, Ontario.

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