HEIM Alexander, LUSTER Jörg, BRUNNER Ivano, FROSSARD Emmanuel *

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1 Scientific registration n : 2168 Symposium n : 43 Presentation : poster Root exudation of Norway spruce treated with aluminum, calcium or sodium in solution culture Exsudation racinaire en culture hydroponique d épicéas traités avec l aluminium, le calcium ou le sodium HEIM Alexander, LUSTER Jörg, BRUNNER Ivano, FROSSARD Emmanuel * Swiss Federal Institute for Forest, Snow and Landscape Research, CH-8903 Birmensdorf, Switzerland * Institute of Plant Sciences, Swiss Federal Institute of Technology Zürich, Field Station, CH-8315 Lindau-Eschikon, Switzerland Introduction While root exudation of crops has been studied intensively (reviewed by Marschner, 1995 and Uren and Reisenauer, 1989) there still is little information on the amount and composition of tree root exudates. It has been demonstrated that Al tolerant maize and wheat cultivars react to free Al 3+ in soil solution by excretion of organic acids which can complex Al and thus prevent its uptake (Delhaize et al., 1993; Pellet et al., 1995). For trees, however, it is not known, if they are able to modify their exudation pattern when chemical conditions in the soil solution change. In acid forest soils, increasing acidification can lead to high concentrations of phytotoxic Al species in soil solution. Conifers naturally adapted to these soils therefore must possess mechanisms to tolerate these conditions. Based on the results with crops, our aim was to investigate if root exudation of organic acids is involved in Al tolerance in Norway spruce. Material and Methods Plant material Three-year-old spruce trees from our institute s tree nursery were carefully removed from the soil and their root systems were washed free from soil with tap water. The roots were largely mycorrhizal and typically showed few fresh root tips at that time. Fresh weight of individual plants ranged from 20 to 60 grams. Experimental design Two plants were transferred to one 300 ml Erlenmeyer flask filled with 250 ml of treatment solution. The roots were bathed in the solution while the shoots remained outside. The flasks were sealed with Parafilm. Three groups of five Erlenmeyer flasks 1

2 each were formed. Within one group plants received the same treatment. Care was taken that total biomass of the groups was approximately equal. Plants were treated with 500 µm solutions of AlCl 3, CaCl 2 or NaCl, which were adjusted to ph 4,3 and autoclaved before use. We used these two different control treatments in order to check for differences between the effect of nutrient cations and non-essential cations. Treatments were carried out in a climate chamber (20 C, 16 h light period). Samples of 30 ml were taken four times during the treatment using a sterile syringe and were filtered immediately. At the end of the experiment, plants were separated into shoot and root and dried to constant weight at 60 C. Analysis Twenty ml of the solutions were freeze-dried and redissolved in 0.5 ml of doublydeionized water. To remove chloride, the concentrated solutions were mixed with 50 mg of Ag-saturated strongly acidic cation-exchange resin and shaken for one hour. After centrifugation the supernatants were used for determination of organic acids by capillary electrophoresis (CE). We used a 45 cm x 50 µm capillary. The buffer was composed of 10 mm phthalate and 2,5% Anion-BT (Waters), adjusted to ph 5,6 with 0.5 M LiOH. Indirect UV detection was performed at 215 nm. All other analyses were performed on non-concentrated samples. Samples were analyzed for ph using an ISFET sensor. Total organic carbon was determined by a TOC-Analyzer (TOC-500, Shimadzu). Cations were analyzed by capillary electrophoresis, using the method of Göttlein and Blasek (1996) with a 50 µm ID capillary. Phenolics were determined colorimetrically using the method of Swain and Hillis (1959). In order to compare phenolic C to total C, we assumed an average composition of six carbon atoms per molecule of phenolic substance. Amino acids were determined colorimetrically using the method by Allen (1981). Total sugars were determined by the phenol method according to Chaplin (1994). Results and Discussion Solution conditions Analysis of cations by CE revealed that in Al treatments free Al 3+ had almost completely disappeared from solution after two days. Instead, calcium and magnesium ions were present in this solution (Fig.1). Samples taken three hours after starting the experiment already showed the same results. This could be due to a rapid exchange reaction at the root surface, where Al 3+ displaces exchangeably bound nutrient cations. X-ray microanalysis of root samples indeed showed clear evidence of Al present in fairly high amounts on the surface of Al treated roots whereas Al content of control roots was very low. Calcium was present in high amounts at the surface of control roots and usually much lower in Al treated roots. Adsorption to the glass wall of the Erlenmeyer flasks was tested in a control experiment without plants and found not to be relevant. 2

3 We found that ph values of the treatment solutions changed during autoclaving. While there was a small rise in ph in the CaCl 2 and NaCl solutions, the ph of the AlCl 3 solution fell below 3. When the solutions came into contact with the root system the ph of all solutions rose by an average of about 1 unit within a few hours (Fig.2). In summary, ionic composition of Al and Ca treatment solutions was very similar after the first rapid reactions, whereas ph of Al treatment solutions was about 1 unit below the control treatments. Ca after 2 days of contact to spruce roots Absorption units Mg at the start of the experiment Al Migration time (minutes) Fig.1: Electropherograms of cations in Al treatment solutions days Na Ca Al Fig.2: Course of ph values with time in the three treatments (value at day 0 equals ph of treatment solutions before contact to the roots) 3

4 Exudation There was a good correlation between root dry weight and total organic carbon concentration in the treatment solutions after two days (Fig. 3). However, treatment itself did not show a significant effect on total C exudation. By contrary, differences in exudate composition were observed between the Na treatment and the Ca and Al treatments. The organic carbon released by Na treated plants showed a significantly lower percentage of phenolics than exudates of Ca or Al treated plants. No difference in percentage of phenolics could be observed between the Ca and the Al treatment. However, this might be different if Al concentrations can be kept at a constantly high level in the Al treatment Al Na Ca R 2 = g dry weight Fig.3: Total organic carbon concentration in treatment solutions after two days in relation to root biomass. Different treatments are indicated by different symbols. We detected acetic, lactic, oxalic and formic acids in the samples. As formic acid varied unsystematically and could even be found in water blanks, the occurrence of this acid cannot be attributed to the plant, but must be regarded as a contamination. Acetic acid was present in extremely varying concentrations, ranging from 1 to approx. 500 µm. Despite large variations, average concentrations in Na treated plants were higher than in Ca and Al treatments. Oxalic acid was present at low concentrations (0.5 to 3 µm) in solutions of the Al treatment. As oxalate forms stable complexes with Al (Hue et al.), oxalate exudation could be a mechanism to detoxify Al 3+. However, as Al 3+ disappeared rapidly from solution, it seems unlikely that oxalate exudation was stimulated by high Al 3+ concentrations in solution but rather by low ph in the Al treatments. 4

5 Table 1: Organic acids identified in the treatment solutions after 2 days (µmol /l) n.d. not detected Treatment Acetic Oxalic Al 22 ± ± 1.0 Na 186 ± 170 n.d. Ca 31 ± 27 n.d. All organic compounds identified up to now only amount to 5 to 46% of total C released. As sugars and amino acids are at or below detection limits, they do not seem to contribute substantially to C concentration in our treatment solutions. Therefore, these unidentified C species either consist of high-molecular weight substances or of nonphenolic aromatic organic acids which would neither be detected by our CE method nor by the colorimetric method for phenolics. Conclusions In short-term treatments of 3-year-old spruce trees solution conditions are strongly influenced by rapid proton-consuming reactions in the system at the start of the experiment and by the ability of this system to strongly adsorb highly charged cations. In future treatments therefore ph should be kept constant. In addition, preloading of the root system with the cation under investigation might be useful. Our results suggest that total root exudation of 3-year-old spruce depends mainly on root biomass. Differences in exudate composition observed between the different treatments are to be considered as preliminary results. Their interpretation is made difficult by the unstable solution conditions. Acknowledgments We would like to thank Ursula Beutler for assistance in the lab and Anton Burkart for providing the trees. X-ray microanalysis was performed by Dr. Beat Frey whose cooperation we gratefully acknowledge. The project is funded by the Swiss National Science Foundation (Grant Nr ). References Allen, G. (1981): Laboratory techniques in biochemistry and molecular biology. Sequencing of proteins and peptides. North Holland Publishing Company, Amsterdam Chaplin, M.F. (1994): Monosaccharides. In: Chaplin, M.F. and Kennedy, J.F. (eds.), Carbohydrate Analysis; a Practical Approach, 2nd ed., Oxford University Press, Oxford, pp

6 Delhaize, E., P.R. Ryan, and P.J. Randall (1993): Aluminum tolerance in wheat (Triticum aestivum L.). II. Aluminum-stimulated excretion of malic acid from root apices. Plant physiology 103 (3): Göttlein, A. and R. Blasek (1996): Analysis of small volumes of soil solution by capillary electrophoresis. Soil Science 161 (10): Hue, N.V., G.R. Craddock, and F. Adams (1986): Effect of organic acids on aluminum toxicity in subsoils. Soil Sci. Soc. Am. J. 50: Marschner, H. (1995): Mineral Nutrition of Higher Plants, 2nd ed., Academic Press, London Pellet, D.M., L.D. Grunes, and L.V. Kochian (1995): Organic acid exudation as an aluminum-tolerance mechanism in maize (Zea mays L.). Planta 196(4): Swain, T. and W.E. Hillis (1959): The phenolic constituents of prunus domestica. I. The quantitative analysis of phenolic constituents. J. Sci. Food Agric. 10, Uren, N.C. and H. M. Reisenauer (1989): The role of root exudation in nutrient acquisition. In: Tinker, B. and Läuchli, A. (eds.), Advances in Plant Nutrition, Vol.3, Praeger, New York, pp Keywords : root exudates, Norway spruce, aluminum toxicity, solution culture, organic acids, phenolics, capillary electrophoresis Mots clés:exsudats racinaires, épicéa, toxicité de l aluminium, culture hydroponique, acides organiques, composés phenoliques, électrophorèse capillaire 6