THE ABSORPTION OF IONS BY MICRO- ORGANISMS AND EXCISED ROOTS

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1 New Phytol. (1974) 73, THE ABSORPTION OF IONS BY MICRO- ORGANISMS AND EXCISED ROOTS BY D. A. BARBER Agricultural Research Council, Letcombe Laboratory, Wantage, Berks. {Received i^ June 1973) SUMMARY Micro-organisms are present in sufficient numbers on barley roots to account for the differences previously attributed to them in experiments comparing the uptake of rubidium by sterile and non-sterile excised roots. Experiments with thallium, which is highly phytotoxic at concentrations above 0.2 mm, provide further grounds for rejecting the postulate, based on 'dual absorption isotherms', that under physiological conditions different mechanisms of absorption operate depending on the external concentration. INTRODUCTION The effects of micro-organisms in the root environment on nutrient absorption by intact plants has been demonstrated in several investigations (Rovira and Bowen, 1966; Bowen and Rovira, 1966; Barber, 1966, 1969; Barber and Loughman, 1967). This encouraged Barber and Frankenburg (1971) to examine detached barley roots grown under comparable conditions to those used in investigations into the 'dual isotherm' of absorption by plants (Epstein, 1966; Laties, 1969). They showed that the uptake of both phosphate and rubidium was appreciably greater in roots infected with micro-organisms at the ambient laboratory level than in those grown under rigidly sterile conditions and they attributed the difference largely to absorption by the micro-organisms. The effects were particularly pronounced at low external concentrations and led Barber and Frankenburg (1971) to suggest that experiments designed to investigate the nature of uptake process by plants should be carried out under sterile conditions. Epstein (1972) considered that the conclusions of Barber and Frankenburg 'must be rejected'. His principal grounds were that micro-organisms were incapable of accumulating the quantities of rubidium which Barber and Frankenburg had suggested and that 'roots with a complement of associated bacteria should be considered normal, sterile roots being in the nature of experimental artifacts'. Epstein's views on these matters are here examined taking account both of information which was available at the time of his publication and more recent results. EXPERIMENTAL METHODS Barley seedlings were grown for 7 days either infected with bacteria at the ambient laboratory level or under rigidly sterile conditions by the methods described previously (Barber and Frankenburg, 1971). In some experiments, the magnitude of the bacterial flora from the non-sterile roots was then examined by making plate counts on ^ strength 91

2 92 D. A. BARBER tryptone soya agar, a medium widely used because of its low selectivity (A. D. Rovira, personal communication). In other experiments, measurements of the respiration rates of excised roots, both sterile and non-sterile, and of micro-organisms isolated from roots were made using a Gilson respirometer or ion absorption was measured over a period of I h. The methods for plant culture under non-sterile conditions and for measuring ion uptake are closely similar to those used by Epstein and others in the studies which led to the opinion that two mechanisms of uptake are operative respectively at high and low concentration (Epstein, 1966; Welch and Epstein, 1968, 1969; Osmund and Laties, 1968; Edwards, 1968, 1970; Kannan, 1971). RESULTS AND DISCUSSION The extent and significance of the bacterial contamination of roots Epstein's (1972) rejection of Barber and Frankenburg's conclusion on the extent of the absorption of rubidium by micro-organisms rested primarily on his assumption, which was unsupported by direct evidence, that the volume of bacteria is o.oi % of that of the roots; this he regarded as 'surely a large estimate'. Barber and Frankenburg (1971) had shown that 1.5 /imol more rubidium per gram fresh weight of root were absorbed during i h by non-sterile than sterile roots when exposed to o.i mm solutions. Thus, on the basis of this value and his assumption of the volume of bacteria present, Epstein calculated that the concentration of rubidium in them was 15 M. This he not unnaturally rejected as an unreasonable value. The acceptability of Epstein's stricture depends on the validity of his assumption as to the magnitude of the microbiological population on the roots; quantitative data being hitherto unavailable this matter has been investigated. Plate counts showed that the population of bacteria was about 5x10^ per gram fresh weight on washed roots of barley seedlings 7 days old (i.e. when roots are normally excised for measurements of ion uptake). Since the average fresh weight of a bacterial cell may be taken as about 1.5 x io~^^ g (Alexander, 1961) this implies that bacteria represent about 0.8% of the fresh weight of the roots, a factor of 80 greater than that assumed by Epstein. Adjusting his calculation for this discrepancy Barber and Frankenburg's results in fact suggest that the concentration of rubidium in the micro-organisms was in the order of 200 mm which is well within the range reported by other workers. The work of Rorem (1955) provides an alternative basis for estimating the ability of bacteria to absorb rubidium. He showed that different species of bacteria could absorb from 15 to 400 m-equiv. rubidium per 3 x 10"* cells in i h. On this basis the 5x10^ cells associated with i g fresh weight of roots could absorb /imol; the quantity of rubidium bacteria appeared to absorb in Barber and Frankenburg's work, 1.5 ;<mol, falls well within this range. Furthermore, although no precise biological meaning can be ascribed to measurements of cation exchange capacity, it is of interest that values quoted for common soil bacteria vary between 95 and 340 m-equiv. per 100 g dry weight (Zwarun, Bloomfield and Thomas, 1971; Zwarun and Thomas, 1973) which greatly exceed the 12.3 m-equiv. determined for barley roots (Drake, Vengris and Colby, 1951). The bacterial counts here reported are probably minimal estimates of the mean population present during the one hour for which absorption by the excised roots was measured since, not only is the plating medium likely to be selective but the numbers reported relate to organisms present at beginning of the uptake period. The loss of organic material from the roots following excision is likely to stimulate their rate of

3 Absorption of ions 93 multiplication. Evidence for this is provided by the fact that a mixed population of micro-organisms isolated from roots and inoculated into ioo ml o.i % glucose in freshly prepared 0.2 mm calcium sulphate, increased in 24 h from 2 x 10* to i x 10^ compared with 3x10^ for a similar inoculum in 100 ml o.i % glucose in calcium sulphate in which sterile roots had previously been grown (final fresh weight of roots 5.3 g in 800 ml solution). Measurements of respiration point to the same conclusion. Respiration due to micro-organisms associated with roots (i.e. the difference between the respiration rates of sterile and non-sterile roots) was considerably greater than that of a population of O 300. j; Fig. I. Uptake of oxygen by micro-organisms present on excised barley roots (l,) [derived by difference from the values obtained for sterile ( ) and non-sterile ( ) roots] and by an equivalent quantity of micro-organisms isolated from non-sterile roots ( ). micro-organisms, similar in number to that originally present on the non-sterile roots, when they were cultured in the absence of roots but with 3 mm glucose as substrate (Fig. 1). That the microbial flora is so large is not surprising. Lundegardh (1924) demonstrated that the carbon dioxide evolved by the roots of wheat plants grown under non-sterile conditions was 80 o greater than that evolved by equivalent quantities of sterile roots; Barker and Broyer (1942) made similar observations with squash roots. Zwarun (1972) showed that when glucose labelled with '*C was added to the solutions in which excised sterile and non-sterile soya bean roots were suspended the evolution of ^"^COj was six times greater with the non-sterile roots and that in the absence of roots there was little multiplication of the bacteria. The nature and physiological significance of 'dual absorption isotherms' Epstein's (1972) suggestion, that sterile roots are 'in the nature of experimental artifacts', must be considered in the context of Barber and Frankenburg's (1971) comments to which he referred. These workers alluded to the physiological inferences which had been drawn from the dual absorption isotherm by Epstein and others, namely that two 60

4 94 D. A. BARBER mechanisms of absorption operate in plant cells, one at low concentrations (below c. i.o nim) and the other at higher concentrations; Barber and Frankenburg did not suggest that the dual isotherm was caused by bacteria. They did draw, however, the obvious inferences from the facts that (i) micro-organisms can be responsible for an appreciable fraction of the apparent absorption by plants under non-sterile conditions, and (ii) the proportionate contribution of micro-organisms can be much enhanced when the ionic concentration is low (Barber, 1969). Accordingly they thought it prudent not to ignore the variable effects of micro-organisms in attempts to interpret the dual absorption isotherm. Sterile culture was therefore suggested. Epstein's main reason for rejecting this view appears to be that he and his colleagues had observed great similarities in the absorption of ions by roots and excised leaf tissues 0 05 Externoi concenfrction (mw) Fig. 2. Absorption during i h of thallium ( ) and rubidium ( ) by excised barley roots grown under sterile conditions. (Smith and Epstein, 1964). This would be relevant to the discussion only if microorganisms were absent from leaves. However, there is ample evidence of an active and varied phyllosphere microflora on leaves (Preece and Dickinson, 1971) and it is well known that metabolites, which can form a substrate for micro-organisms, are readily leached from intact leaves (Tukey, Wittwer and Tukey, 1957) especially when they are illuminated; slicing of leaf tissues such as Epstein and his colleagues practised might be expected to increase this substantially. Thus, Epstein's comments have no direct bearing on the central point that Barber and Erankenburg discussed. A further paper by Barber (1972), which was in press when Epstein's (1972) paper was prepared clarifies the issue. Under rigidly sterile conditions the dual absorption isotherm was observed (see Eig. 2), though with some quantitative difference from that found in plants contaminated with micro-organisms. The elimination of microbiological effects simplified interpretation and Barber showed that the mechanism of uptake postulated to be operative at the higher concentration range, i.e. above mm, was due to passive

5 Absorption of ions 95 diffusion into the tissue down a concentration gradient. In contrast the absorption at lower concentrations reflected the movement of ions against a concentration gradient and was dependent on metabolism; thus it alone appeared to deserve consideration in physiological studies. Further evidence for this view has been provided by experiments on the absorption of thallium by excised barley roots. A dual isotherm of absorption comparable to that shown for rubidium is evident (Fig. 2). However, as is well known, thallium is toxic to plant tissues at moderate to high concentration (Bange and van Iren, 1971). Table i shows effects on respiration of concentrations of i mm or higher after 30 min; lesser Table i. Effect of thallous sulphate on the uptake of oxygen by excised barley roots grown under sterile conditions (0.2 mm calcium sulphate was present in all treatments) Concentration of TI2SO4 (mm) O IO.O Oxygen uptake (//I 02/g dry vrt/h) Per cent inl o 74-5 effects which appeared more slowly were observed with concentrations down to o. i mm as reported by Bange and van Iren (1971). In short, the upper part of the dual isotherm for thallium described in Epstein's terminology as mechanism 2 occurs under conditions of considerable toxicity. This observation supports the view that absorption at high concentrations is not relevant to the interpretation of mechanisms whereby ions enter plants under physiological conditions. There thus appears to be no grounds for assuming that different metabolically mediated processes operate in different concentration ranges. In physiological terms, the postulate that this occurs in growing plants might be regarded as resting on an 'experimental artifact'. ACKNOWLEDGMENTS I thank Dr R. Scott Russell, Dr J. M. Lynch and Dr M. G. T. Shone for much helpful discussion during the preparation of this manuscript. REFERENCES ALEXANDER, M. (1961). Introduction to Soil Microbiology. Wiley, New York. BANGE, G. G. J. & IREN, F. VAN (1971). The absorption of thallium ions by excised barley roots. Acta hot. neerl., 19, 646. B.'VRBER, D. A. (1966). Effect of micro-organisms on nutrient absorption by plants. Nature, Lond., 212, 638. BARBER, D. A. (1969). The influence of the microflora on the accumulation of ions by plants. In: Ecological Aspects of the Mineral Nutrition of Plants (Ed. by I. H. Rorison), p Blackwell Scientific Publications, Oxford. B.\RBER, D. A. (1972). 'Dual isotherms' for the absorption of ions by plant tissues. New Phytol., 71, 255. BARBER, D. A. & LOUGHMAN, B. C. (1967). The effect of micro-organisms on the absorption of inorganic nutrients by intact plants. II. Uptake and utilization of phosphate by barley plants grown under sterile and non-sterile conditions. X exp. Bot., 18, 170. BARBER, D. A. & FRANKENBURG, U. C. (1971). The contribution of micro-organisms to the apparent absorption of ions by roots grown under non-sterile conditions. New Phytol., 70, BARKER, H. A. & BROYER, T. C. (1942). Notes on the influence of micro-organisms on growth of squash plants in water culture with particular reference to manganese nutrition. Soil Sci., 53,.467. BowEN, G. D. & RoviRA, A. D. (1966). Microbial factor in short-term phosphate uptake studies with plant roots. Nature, Lond., 211, 665. DRAKE, M., VENGRIS, J. & COLBY, W. G. (1951). Cation exchange capacity of plant roots. Soil Sci., 72,139.

6 96 D. A. BARBER EDWARDS, D. G. (1968). The mechanism of phosphate absorption by plant roots. Trans, gth int. Cot^r. Soil ScL, 2, 183. EDWARDS, D. G. (1970). Phosphate absorption and long-distance transport in wheat seedlings. Attst.J. biol. Sci., 23, 255. EPSTEIN, E. (1966). Dual patterns of ion absorption by plant cells and by plants. Nature, Lond., 212, EPSTEIN, E. (1972). Ion absorption by roots: the role of micro-organisms. New Phytol., 71, 873. KANNAN, S. (1971). Kinetics of iron absorption by excised rice roots. Planta, 96, 262. LATIES, G. G. (1969). Dual mechanisms of salt uptake in relation to compartmentation and long-distance transport. A. Rev. PL PhysioL, 20, 89. LUNDEGARDH, H. (1924). Der Kreislauf der Kohlensaure in der Nature. Gustav Fischer, Jena. OSMUND, C. B. & LATIES, G. G. (1968). Interpretation of dual isotherm for ion absorption in beet tissue. PL PhysioL, Lancaster, 43, 747. PREECE, T. F. & DICKINSON, C. H. (1971). Ecology of Leaf Surface Micro-Organisms. Academic Press, New York and London. RoREM, E. S. (1955). LTptake of rubidium and phosphate ions by polysaccharide producing bacteria. J. Bacteriol., 70, 691. RoviRA, A. D. & BowEN, G. D. (1966). Phosphate incorporation by sterile and non-sterile plant roots. Aust. J. biol. Sci., 19, SMITH, R. C. & EPSTEIN, E. (1964). Ion absorption by shoot tissue: kinetics of potassium and rubidium absorption by com leaf tissue. Plant PhysioL, Lancaster, 39, 992. TUKEY, H. B. Jr., WITTWER, S. H. & TUKEY, H. B. (1957). Leaching of carbohydrates from plant foliage as related to light intensity. Science, N.Y., 126, 120. WELCH, R. M. & EPSTEIN, E. (1968). The dual mechanism of alkali cation absorption by plant cells: their parallel operation across the plasmalemma. Proc. natn. Acad. Sci. U.S.A., 61, 447. WELCH, R. M. & EPSTEIN, E. {1969). The plasmalemma: site of the type 2 mechanisms of ion absorption. PL PhysioL, Lancaster, 44, 301. ZwARUN, A. A. (1972). Microbial competition for glucose in excised root experiments. Proc. Soil Sci. Soc. Am., 36, 968. ZwARUN, A. A., BLOOMFIELD, B. J. & THOMAS, G. W. (1971). Effect of soluble and exchangeable aluminium on a soil Bacillus. Proc. Soil Sci. Soc. Am., 35, 460. ZwARUN, A. A. & THOMAS, G. W. (1973). Effect of soluble and exchangeable aluminium on Pseudomonas stutzeri. Proc. Soil Sci. Soc. Am., 37, 386.

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