uptake of 24mg by excised pine roots: a preliminary study

Plant and Soil 172: 323-326, 1995. ~) 1995 Kluwer Academic Publishers. Printed in the Netherlands.

Short Communication

Uptake of 24Mg by excised pine roots: A preliminary study

Peter H6gberg l, Paul Jens6n 2, Torgny N~isholm 3 and Helene Ohlsson 1 l Deptartment of Forest Ecology, Swedish University of Agricultural Sciences (SUAS), S-901 83 Umed, Sweden, 2Department of Horticulture, SUAS, P O. Box 55, S-230 53 Alnarp, Sweden and 3Department of Forest Genetics and Plant Physiology, SUAS, S-901 83 Umed, Sweden

Received 5 July 1994. Accepted in revised form 2 December 1994

Key words: ICP-MS, Mg uptake, Pinus sylvestris, stable isotopes, roots

Abstract

Uptake of 24Mg by excised roots of Pinus sylvestris L. during up to 4 h long incubations in 99.9 atom % 24Mg (50/IM) was measured by ICP-MS. A rapid initial uptake phase (30 min) was followed by a slower uptake. This was interpreted as a shift from a phase dominated by saturable ion exchange (free space uptake), to a non-saturable phase, during which the rate of uptake was 0.077=t=0.0.012/~mol Mg g-~ (d.wt.) h -1. The metabolic uncoupler DNP (2,4-dinitrophenol) at 50/~M decreased the Mg uptake rate by 35% only, but the effect of DNP was significant (p<0.01). Several problems related to a high variability in the experimental material were encountered, and further refinement of this approach in studies of plant Mg uptake is suggested.

Introduction

Magnesium deficiency has sometimes been identified as a cause of forest decline in Middle Europe (e.g. Schulze, 1989; Z6ttl and Htittl, 1986). Problems with Mg nutrition are associated with an excessive supply of N through deposition and low availability of Mg in acid soils. High concentrations of H + and A13+ could potentially interfere with uptake of Mg 2+ by occupying exchange sites in the root apoplast (e.g. Marschner, 1991) and by blocking sites of active uptake into the symplast (Rengel, 1990; Rengel and Robinson, 1989). So far, the most detailed studies of Mg uptake by roots have been those of depletion in nutrient solutions (e.g. Rengel and Robinson, 1989). The radioactive isotope 28Mg with a half-life of 21.3 h would be potentially useful, but has according to our knowledge not been used for this purpose.

Recent developments of inductively coupled plasma-mass spectrometers (ICP-MS) suggested the possibility of using stable Mg isotopes in studies of this kind. We tested (i) if one of the stable Mg isotopes, 24Mg, could be used for short-term studies, and (ii) if an active uptake component could be identified.

We also attempted to see (iii) if uptake of Mg was influenced by the Mg status of the plants. 24Mg is the most common of the stable Mg isotopes (77-78 atom % at natural abundance), and hence theoretically less useful in a tracer experiment, but is much cheaper than 25Mg and 26Mg.

Materials and methods

Plant material and growth conditions

Surface sterilized (30% H202, 7 min) seeds of Pinus sylvestris L. were sown onto acid-washed siliceous sand in 1.5 L plastic pots. These were watered with deionized water for 6 days, and only one seedling was left in each pot after germination. Thereafter the pots were watered thrice a week with 250 m L pot -I of a complete nutrient solution (pH=6.3-7.8) with 1.424 mM NH4NO3, and the other elements in what was considered as optimal weight proportions in relation to N (Ingestad and Lurid, 1986). In the experiments the ratio between Mg and N was varied from 8.5:100 down to 0:100. Six different Mg:N ratios, here denoted 100

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(Mg:N = 8.5:100), 50, 10, 5, 2.5 and 0 (Mg:N = 0:100), respectively, were used in our study. There were nine replicate plants per treatment.

The plants were kept in a growth chamber under the following conditions: 18 h day, photon flux den- sity 300-400 /lmol m -2 s - l , temperature 20°/15 ° (day/night) and 70% relative humidity. The positions of the pots in the chamber were changed once a week. Two uptake experiments were performed after 120 days.

Experiment 1: Time course of uptake

One large plant from each Mg:N cultivation treatment was harvested. Individual root systems were divided into five samples of 5-150 mg (d.wt.) each in the case of Mg:N cultivation treatments 5-100, four samples in the case of Mg:N = 2.5, and three samples in case of Mg:N = 0. The root samples were preincubated for 1 h in 0.5 mM CaCI2 (pH=8.3) to remove ions from the apoplast and to maintain integrity of membranes. Thereafter they were incubated in 50/~M MgCI2 (99.92 atom % 24Mg, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA) and 0.5 mM CaC12 (pH=8.3), at 25°C in stirred solution for 0, 0.5, 1,2 and 4 h. After the uptake period the roots were washed for 0.25 h in 0.5 mM CaC12 (pH=8.3) to remove 24Mg2+ from root surfaces. In an additional experiment root systems of intact plants were used, in which case shoots were carefully prevented contact with solutions, to see if 24Mg tracer was translocated to shoots within 2 h.

Experiment 2: Inhibition of uptake by 2, 4-dinitrophenol ( DNP )

Five plants from each Mg:N treatment were used. Each root system was divided into two samples. One sam- ple per plant was preincubated, incubated for 2 h, and washed as described above. Preincubation and incuba- tion solutions used for the other set of samples contained 50 #M DNP, an uncoupler of oxidative phos- phorylation, and hence of active uptake.

Analyses

Concentrations of Mg and atom % 24Mg were deter- mined on roots from the two experiments, and shoots from the study using intact plants, plus as a baseline, roots and shoots from each Mg:N treatment and seeds never exposed to 24Mg tracer. The samples were anal- ysed on an ICP-MS (Perkin-Elmer Elan 5000, Nor-

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Fig. 1. Biomass (a), biomass allocation (b), and Mg content (c) of seedlings of Pinus sylvestris in relation to Mg:N supply ratio during cultivation (n=9, mean 4- I S.E.). The ratio 100 corresponds to a weight ratio Mg:N of 8.5:100, which was the optimum recommended by lngestad and Lund (1986).

walk, Connecticut, USA) after digestion in a mixture (10:1) of nitric and perchloric acids. The substrate, sand,was dissolved in hydrofluoric acid before analy- sis, while nutrient solutions were admitted directly to the ICP-MS. Weight corrections for mineral particles adhering to root surfaces were made after weighing minerals left in the acid digests, and uptake rates of Mg were calculated as pmol Mg g- l root (d.wt.) h- t based on changes in atom % 24Mg. The precision of determi- nations of atom % 24Mg was ~0.05% (S.D.).

Results

Plant growth and Mg content

There were large differences in growth as a result of differences in Mg supply, but only the Mg:N ratios 0 and 2.5 differed from the others (ANOVA: p<0.001; Fig. la), and were also the only treatments with a pro-

nounced yellowing of needles. The other treatments will, therefore, sometimes be averaged to represent plants not deficient in Mg. There was a tendency that plants given a low supply of Mg had a higher shoot:root ratio than plants with a high supply of Mg (Fig. lb, ANOVA: p=0.067). The Mg content of plants increased rapidly with increasing supply at very low rates of supply, and thereafter more slowly (Fig. lc). Seeds contained only about 0.77#mol Mg seed -1. The Mg content of plants not given any Mg, i.e. the Mg:N = 0:100 cultivation treatment was, however, 9.23 gmol seedling- 1 at harvest. This additional Mg must have come from the acid-washed sand (0.003 % Mg), because seedlings in a solution culture experiment using the same chemicals only increased their Mg content 0.15/~mol during a period of 73 days.

Atom 24Mg in different compartments

Seeds contained 76.96-/-0.06 atom % 24Mg, while the nutrient solution and the sand contained 77.16 and 77.024-0.07 atom % 24Mg, respectively. Passage through the sand did not cause any shift in the atom % 24Mg of the solution. Root systems never exposed to 24 Mg tracer contained 76.894-0.06 atom % 24 Mg, and there was no trend in relation to the Mg:N treatments. A clear trend in this regard was, on the other hand, observed in the shoots, in which atom % 24Mg increased from 77.2 to 77.5 with increasing supply of Mg (rZ=0.67, p<0.001). Moreover, shoots had on an average 0.44:t:0.04 % higher atom % 24Mg than roots of the same plant. The average atom % 24Mg in roots, 76.89, was therefore used as the baseline for calcula- tions of uptake of 24Mg tracer by roots.

Experiment I

There was a rapid initial increase in atom % 24Mg in roots in the 24MgClz solution (Fig. 2). After 0.5 h the uptake rate of label was 4-5 times lower than the initial rate. This lower rate corresponded to 0.077+0.012 /~mol Mg g-1 root (d.wt.) h -l. The total changes in atom % 24Mg were on an average around 0.5 atom %, i.e. 10 times the analytical precision. In the study using intact plants, no increase in atom % 24Mg was observed in the shoots after 2 h.

Experiment 2

Labelling by 24Mg during this experiment as expressed in atom % was highest in plants given a low supply of

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Fig. 2. Uptake of 24Mg by roots of Pinus sylvestris expressed as change in atom % 24Mg, (a) cumulative uptake (b) and rate of uptake (e). Closed circles show seedlings (n--4, mean 4- 1 S.E.) cultivated at high Mg:N supply ratios (Mg:N = 5-100, where 100 corresponds to the optimum weight ratio 8.5:100 according to Ingestad and Lund, 1986). Open circles, seedlings cultivated at Mg:N = 2.5; triangles, seedlings cultivated at Mg:N = 0.

Mg before the experiment (Fig. 3a). This larger isotope effect was associated with the lower initial Mg content of these plants. Hence, when uptake was calculated per unit dry weight of root, there were no differences in uptake rates. There was an effect of DNP, although there was a large variability. The calculated average effect of DNP on uptake (Fig. 3b), 0.027-1-0.006 ~mol Mg g- l root (d.wt.) h-1 was nevertheless significant (t-test, p=0.006).

Discussion

Above all, these results demonstrated the possibility of using Z4Mg for short-term Mg uptake studies. The changes in atom % 24Mg were, however, only ten times the analytical errors in Experiment 1 (Fig. 2), which means that great homogeneity of the experimental material is a demand in studies of this kind. Hence, larger root samples than those used by us should be preferred. This would also enable a more precise base-

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Fig. 3. Effect of DNP on uptake of 24 Mg by roots of Pinus sylvestris. Open circles, control samples; closed circles, roots treated with DNP. a) symbols denote mean 4- 1 S.E. of samples from five seedlings; b) symbols denote differences between control and DNP-treated roots from the same seedling (n=5, means4- 1 S.E.). The Mg:N supply ratio 100 corresponds to a weight ratio Mg:N = 8.5:100 during cultivation, which was the optimum recommended by Ingestad and Lund (1986).

line determination of natural levels of atom % 24Mg in duplicates of roots exposed to tracer 24Mg. Shoots did not provide a proper baseline, and we believe there was also some important variation within root systems. Such variation could result from older parts of the root system being dominated by seed Mg, while younger parts could get an isotopic signature more related to Mg supplied with the nutrient solution.

There was probably an active component of uptake, as shown by the effect of DNP (Fig. 3). However, DNP at this concentration only decreased the uptake rate during the non-saturable phase by one third (Figs. 2 and 3).

Our data did not show an increased capacity for uptake of Mg in Mg-deficient seedlings. If this is true, the results is in contrast to the response of plants deficient to P, K and N, which have significantly increased capacity for uptake of these elements (Harrison and Helliwell, 1979; Jones et al., 1987, 1991). The larger increase in atom % 24Mg in Mg-deficient plants during incubations in tracer solutions was reflecting the dilution of tracer in a smaller initial Mg pool, as pre- viously discussed for 15N (H~3gberg et al., 1994), and was borne out when uptake was calculated per unit root dry weight.

We conclude that 24Mg and the other stable isotopes of Mg may have potentials in studies of interac- tions between Mg 2+ and AI 3+, Ca 2+, K + and NH + (Marschner, 1986).

Acknowledgements

This work was financially supported by the Swedish Environmental Protection Agency. We would like to thank O Emteryd and L Olsson for help with deter- minations of % Mg and 24Mg, Prof P B Tinker for constructive comments, Dr T Ericsson for valuable information and Dr T Hedlund for help with convert- ing MgO to MgCI2.

References

Harrison A F and Helliwell D R 1979 J. Appl. Ecol. 16, 491-505. H6gberg Pe t al. 1994 New Phytol. 127, 515-519. Ingestad T and Land A-B 1986 Scand. J. For. Res. 1, 439453. Jones H E et al. 1987 New Phytol. 107, 695-708. Jones H E et al. 1991 For. Ecol. Manage. 42, 267-282. Marschner H 1986 Mineral Nutrition of Higher Plants. Academic

Press, London, 674 p, Marschner H 1991 Plant and Soil 134, 1-20. Rengel Z 1990 Plant Physiol. 93, 1261-1267. Rengel Z and Robinson D L 1989 Plant. Physiol. 91, 1407-1413. Schulze E-D 1989 Science 244, 776--783. Z6ttl H W and Htittl F 1986 Water Air Soil Pollut. 31,449--462,

Section editor: R F Huettl

uptake of 24mg by excised pine roots: a preliminary study

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