THE RELATIONSHIP BETWEEN PHOSPHATE ABSORPTION AND OXYGEN UPTAKE IN EXCISED BEECH MYCORRHIZAS

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1 New Phytol. (\98\)S8, 67-68\ 67 THE RELATIONSHIP BETWEEN PHOSPHATE ABSORPTION AND OXYGEN UPTAKE IN EXCISED BEECH MYCORRHIZAS BY J. L. HARLEY AND C. C. MCCREADY Deprtments of Forestry nd Botny, South Prks Rod, Oxford OXl RB, U.K. {Accepted 11 November 198) SUMMARY When mycorrhizl rootlets of beech re ged for severl dys in wter, their respirtion rte flls. If phosphte is pplied, the respirtory rte rises nd the rise in oxygen consumption is proportionl to phosphte uptke in replicte smples ged for equivlent times. The rtio of ywg-tom phosphorus to fig-tom oxygen bsorbed is usully round or just below unity, but it vries with the stte of the tissues to vlues s low s 28. High vlues of this rtio in the lter stte of geing my be spurious becuse, in senescent mycorrhizs, phosphte penetrtes non-metboliclly. High vlues in fresh mycorrhizs occur becuse the rte of phosphte-induced oxygen uptke is low but it rises fster thn tht of phosphte uptke in the erly stges of geing. The conclusion is drwn tht the processes of phosphte bsorption nd polyphosphte formtion re often 2 to o efficient with respect to respirtory oxidtive phosphoryltion. The liner reltionship of totl oxygen consumption to phosphte bsorption observed in fresh mycorrhizs is due to the pre-emption of portion of totl phosphoryltion by polyphosphte production. INTRODUCTION The demonstrtion tht the bsorption of phosphte by beech mycorrhizs is ssocited with the formtion of polyphosphte grnules in the fungl sheth (Chilvers nd Hrley, 198) hs number of importnt implictions in the functioning of these orgns. First, it explins the ccumultion of phosphte in the sheth which my double its content within few hours (see Hrley, 1978) without dmge, since no gret osmotic or cidity chnge is to be expected. Secondly, it explins why phosphte bsorption is extremely oxygen-sensitive. Hrley, McCredy nd Brierley (19) nd Hrley et l. (196) showed tht there my be direct liner reltionship between respirtory oxygen uptke nd phosphte bsorption in some circumstnces. In the present pper, we give the rtios of phosphte bsorbed to oxygen consumed for number of experiments. Hrley etl. (196) pointed out tht, when mycorrhizl roots hd ged in wter for severl dys, their oxygen uptke fell to low vlue but when nutrient slts, sugrs or the uncoupling gent, dinitrophenol (DNP), were pplied to them, it rose to higher rte which depended on concentrtion nd nture of the substnce pplied. In one experiment, phosphte incresed respirtion in proportion to the phosphte bsorbed such tht the men rtio of phosphorus bsorption to oxygen consumed ws 61 (Tble 1). Assuming the mximum possible rtio is, the efficiency of the uptke process is bout 2%. This is further exmined below. In other experiments (Hrley, McCredy nd Brierley, 19; Hrley et l., 196), the uptke of phosphte by freshly excised mycorrhizs ws linerly relted X/81/ $2./ 1981 The New Phytologist

2 676 J. L. HARLEY AND C. C. MCCREADY Tble 1. Fffect of different concentrtions of phosphte on oxygen nd phosphorous uptke (/ig-tom h"^ per 1 mg dry zvt) of beech mycorrhizs ged 8 dys in erted wter t room temperture (c. 18 C) [Phosphte] (mm) Bsl.. uptke Induced P uptke * Men -61 * Owinfi to low reding; of induced O.^ uptke, this is \er\ pproximte \-lue. to totl oxygen uptke in conditions where the incresed oxygen uptke occsioned by the presence of phosphte ws miniml. This mtter is lso re-exmined. MATERIALS AND METHODS Mycorrhizs were obtined from the surfce lyers of beech forest. They were wshed nd smpled s previously described (Hrley nd McCredy, 192). Respirtory gs exchnges were estimted by stndrd W^rburg procedures using smples of bout mycorrhizl tips (4 to 6 mg dry wt) in 2* ml of fluid. Phosphorus uptke ws estimted on the sme smples s were used for mesurement of oxygen uptke by introducing ^-P-lbelled phosphte into the side rm of Wrburg flsk (Hrley et l., 196). Mycorrhizl smples were ged in wter through which strem of ir bubbles ws pssed, either t room temperture (18 to 2 C) or t controlled tempertures of 4-, 2 or 2 C.,, RESULTS F/O rtios during geing Tble 2 gi\ es the results of n experiment in which mycorrhizs were ged for 1 dys in three conditions - in erted wter t 2 C, in erted w ter t 4- C nd on dmp filter pper t 4 C. The bsl respirtion rte ws estimted t intervls nd the phosphte-induced oxygen uptke nd phosphte uptke mesured fter the ppliction of 64 mm KH.^POj. The smples kept t 2 C exhibited rtio of little oxer unity until the th dy, fter xxhich it rose. The smples t 4- C hd rtios of unity or below from the 2nd dy but there ws some sign of rise in rtio t the end ofthe geing period. The rises from the th dy onwrds t 2 C nd t the end of the experiment t the lower tempertures my be explined by senescence of the tissues nd the non-metbolic penetrtion of phosphte into them. In seprte experiment on this point, fresh mycorrhizs treted xvith 64 mm phosphte for 4 min relesed fr less phosphte on prolonged wshing with wter thn roots ged for 2 dys; 4//g P per 1 mg dry wt s opposed to 69//g P. The high rtios fter the th dy t 2 C must therefore be suspect. In the experiment shown in Tble, mycorrhizs were ged in wter or in -64 mm phosphte t 2 C. In hoth cses, rtios gin rose toxvrds the end

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4 678 J. L. HARLEY AND C. C. MCCREADY Tble. Chnges in rtes of respirtion nd phosphte bsorption by excised beech mycorrhizs during storge uptke Stored in wter stored in 64 rrim KH2PO4 J uptke Dy Bsl Induced P uptke Bsl Induced P uptke 1 2 ' Smples tken in Mrch, stored t 2 C. Oj uptke nd P uptke given s/^g-tom per 1 mg dry tissue h-'. KH^PO^ (64 mm) pplied. The initil rtio of phosphte uptke to totl oxygen uptke = 14. of the geing period. The high vlues from the 9th dy in wter or the th dy in phosphte must be ignored on grounds of senescence s before. In both cses over the min period ofthe experiment, however, vlues for were bout unity or little less. High vlues of rtio re usully observed in freshly smpled mycorrhizs in which the bsl respirtion is high nd ppliction of phosphte induces reltively low response (see Hrley et l., 196). As the tissues ge, slt-induced oxygen uptke rises more rpidly thn phosphte uptke, hence the rtio flls. It is s if phosphte ppliction pre-empts portion ofthe totl respirtion in fresh tissues so tht the clcultion of rtio bsed on slt induction of oxygen uptke gives unrelible estimtes. Tble 4 gives series of single estimtes of rtio mde from the results of number of experiments not originlly designed to investigte this mtter. Tble 4. Estimtes of rtios of mycorrhizl roots smpled t different times of the yer nd ged for vrious times in vrious conditions Ageing /^g-tom P Dys Conditions /<g-tom O Mrch My My My My June June July 8 Wter 18 C Wter 18 C Wter 2 C Wter 4 C Dmp pper 4 C Wter 18 C Wter 18 C Wter 18 C Wter 18 C Wter 18 C Wter 18 C Except for the first entry when 2 mm ws pplied, 64 mm phosphte pplied.

5 uptke of oxygen nd phosphte by mycorrhizs 679 Excluding the mesurements on freshly smpled mycorrhizs, the vlues rnge from -28 to For the resons given bove concerning senescence, it seems likely tht credible vlues would not exceed little bove unity. rtios of fresh mycorrhizs It hs been indicted bove tht misledingly high vlues hve been obtined with fresh mycorrhizs when rtios re clculted using slt-induced oxygen uptke. On the other hnd, it hs been previously pointed out tht uptke by fresh mycorrhizs my be relted pproximtely linerly to totl oxygen uptke (Hrley, McCredy nd Brierley, 19; Hrley et l., 196). Tbles () nd (b) give the rtios of phosphte uptke to totl oxygen consumption for two such experiments, where totl oxygen uptke ws vried by vrying the concentrtion of oxygen iri the tmosphere of Wrburg fisks. ^^p phosphte ws tipped from the side rms to give concentrtions of KHgPO^ of 1, 2 nd 2 mm. Very low rtios were obtined. There ws some indiction tht the rtios were greter the higher the phosphte concentrtion pplied but, t the lowest phosphte concentrtion, the rtios were fr from constnt nd incresed s the supplied oxygen concentrtion decresed. When the rtios of phosphte uptke to totl oxygen uptke for the fresh smples in Tbles 2 nd re clculted, 64 mm phosphte gives higher rtios 14 nd 1, thn ny in Tble. Tble. Effect of oxygen concentrtion on phosphte bsorption nd oxygen uptke nd on rtios of freshly-smpled mycorrhizs. Phosphte uptke corrected for uptke in N^ () Phosphte concentrtion 1 mm. Temperture 2 C. Oxygen concentrtion in the tmosphere s precentge prtil pressure. [Oxygen] P uptke O^ uptke (%) (jig-ditom) (//g-tom) (b) Phosphte concentrtions 2 nd 2 mm. Temperture 2 C. Oxygen concentrtion in the tmosphere s percentge prtil pressure. -2 mm phosphte 2 mm phosphte [Oxygen] (%) O2 uptke (/fg-tom) P uptke (//g-tom) O2 uptke (jwg-tom) P uptke. (/<g-tom)

6 68o J. L. HARLEY AND C. C. MCCREADY DISCUSSION Replicte smples of mycorrhizs ged for the sme time gve consistent rtios of phosphte bsorbed nd induced oxygen consumption when supplied with different phosphte concentrtions. This indictes close ssocition of the processes of phosphte uptke nd respirtion. If it is ssumed tht the former involves incorportion into nucleotide phosphte in the metbohc pool (see Loughmn, 1966), reltion between uptke of oxygen nd phosphte would be expected. In beech mycorrhizs, portion of the bsorbed phosphte is incorported into polyphosphte in the fungl tissues (Chilvers nd Hrley, 198), process medited by n ATP-requiring kinse (see Dwes nd Senior, 196). A significnt ccumultion of orthophosphte lso occurs in loction, presumbly the vcuoles, seprted from the metbolic pool of phosphte (Hrley, Brierley nd McCredy, 194), but fter hving pssed through this pool (Hrley nd Loughmn, 196). The rtes of both these processes of ccumultion must depend upon respirtory energy. The theoreticl vlue of the rtio would be three ssuming primry incorportion into ATP, nd hence the frequently estimted vlues of bout 6 to 1- suggest n efficiency of 2 to %, with lowest vlue of bout 1%. During geing, both phosphte-induced oxygen uptke nd the rte of phosphte bsorption commonly rise; the oxygen uptke proportiontely more thn the phosphte bsorption, so tht higher P / O rtios were observed with freshly smpled mycorrhizs. The erly rise in rte of phosphte bsorption during geing ws not due to the loss of phosphte into the erted wter for it lso occurred in smples kept in dilute phosphte solution or on dmp filter pper (Tble 2), lbeit in the ltter somewht lter thn in its replicte in wter. It is resonble to ssume tht, in freshly smpled mycorrhizs, the respirtion rte is sufficiently high tht the ppliction of phosphte my pre-empt prt of the existing ATP production for polyphosphte synthesis with no equivlent increse of oxygen uptke. This results in misledingly high rtios for freshly smpled roots, e.g. 2-8 in Tble nd 2 6 nd 1 67 in Tble 4. Quite soon fter smpling, the respirtion rte of the tissues flls shrply nd rtios of the order of unity re observed. After prolonged period of geing, the mesured rtios rise to higher vlues. It seems these re probbly spurious becuse phosphte my penetrte nonmetboliclly into such ged tissues nd not be removed by the stndrd wshing procedures. The possibility tht the ppliction of phosphte to fresh mycorrhizs my pre-empt prt of the current ATP production for uptke nd synthesis of polyphosphte my explin the observtions tht phosphte uptke my be linerly proportionl to totl oxygen uptke. It ws stted by Hrley et l. (19) tht, when sufficiently high phosphte concentrtions were presented, totl oxygen consumption nd phosphte uptke ppered to be directly correlted. However, phosphte uptke from solutions of less thn mm ws not correlted with O2 uptke when mycorrhizs were kept in oxygen concentrtions of more thn 1% by volume. In Tble (), the rtios re not constnt nd phosphte bsorption is less ffected by oxygen supply t this low concentrtion of phosphte (1 mm). When the rtios (t 21 % o J obtined with 1, -2, 2 nd 64 mm phosphte re compred, they re greter t high concentrtions. This indictes gin tht, during bsorption of phosphte by fresh mycorrhizs, portion of respirtory ctivity is pre-empted by phosphte uptke, i.e. portion of vilble ATP is used

7 uptke of oxygen nd phosphte by mycorrhizs 681 in phosphte bsorption. Indeed, if we ssume tht the rtio of phosphte uptke is one nd the theoreticl mximum is three, we cn clculte the percentge of O2 uptke tht is used in phosphte uptke by fresh mycorrhizs. The vlues obtined, 2,, 1- nd % of totl respirtion for uptke from -1, 2, 2 nd 64 mm phosphte, show tht the pre-emption is smll for uptke from physiologiclly probble concentrtions. REFERENCES CHILVERS, G. A. & HARLEY, J. L. (198). Visuliztion of phosphte ccumultion in beech mycorrhizs. New Phytologist, 84, DAWES, E. A. & SENIOR, P. J. (196). The role nd regultion of energy reserve polymers in micro-orgnisms. Advnces in Microbil Physiology, 1, HARLEY, J. L. (1978). Ectomycorrhizs s nutrient bsorbing orgns. Proceedings of the Royl Society London, B, 2, HARLEY, J. L., BRIERLEY, J. K. & MCCREADY, C. C. (194). The uptke of phosphte by excised roots of the beech. V. The exmintion of possible sources of misinterprettion of the quntities of phosphte pssing into the host. New Phytologist,, HARLEY, J. L. & LOUGHMAN, B. C. (196). The uptke of phosphte by excised roots of the beech. IX. The nture of the phosphte compounds pssing into the host. New Phytologist, 62, -9. HARLEY, J. L. & MCCREADY, C. C. (192). The uptke of phosphte by excised mycorrhizl roots of the beech. II. Distribution of phosphorus between host nd fungus. New Phytologist, 1, HARLEY, J. L., MCCREADY, C. C. & BRIERLEY, J. K. (19). The uptke of phosphte by excised mycorrhizl roots of the beech. IV. The effect of oxygen concentrtion upon host nd fungus. New Phvtologist, 2, HARLEY, J. L., MCCREADY, C. C, BRIERLEY, J. K. & JENNINGS, D. H. (196). The slt respirtion of excised beech mycorrhizs. II. The reltionship between oxygen consumption nd phosphte bsorption. New Phytologist,, LOUGHMAN, B. C. (1966). The mechnism of bsorption nd utiliztion of phosphte by brley plnts in reltion to subsequent trnsport to the shoot. New Phytologist, 6,

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