PHYSIOL. PLANT, 55: 377-382. Copenhagen 1982

Potassium transport in wheat seedlings grown with differentpotassium supplies.II. The effects of metabolic and transport inhihitors on K"*uptake and translocation

Zoltin Olali, Alajos Berczi and Laszlo Erdei

Olah,, Z., Berczi, A. and Erdei, L. Potassinm transport in wheat seedlings grown withdifferent potassiun^ supplies. IL The effects of metabolic and transport inhibitors onK+ uptake and translocation. - PhysioL Plant. 55: 377-382.

The K^C'Rb) uptake into the roots and the transloeation to tbe shoots of 11-day-oldintact wheat seedlings {Triticum aestivunj L. cv. Martonvasari 8) were investigatedusing plants grown with different K* supplies. The effects of environmental condi-tions (darkness, humidity) and of metabolic and transport inhibitors (oligomyein,disalicylidene-propanediamine, 2,4-dinitriphenol, diethylstilbestrol, colchicine) werealso studied. Plants with K content of about 0.2 mmoi/g dry weight in the root and0.5 mmol/g dry weight in the shoot (low K status) showed high K* uptake into theroots and high translocation rates to the shoots. Both transport processes were ver>'low in plants, with K content of more than 1.5 and 2.2 mmol/g dty weight in the rootand shoot, respectively (high K status).Darkness and a relative humidity of the air of 100% did not influence K* uptake byroots, but did inhibit upward translocation and water transport. Inhibition of photo-synthesis and treatments with diethylstilbestroi (10"* mol/dm^), as well as with col-chicine resulted in inhibition of translocation in plants of low K status, but theseinhibitors had little effect on K* uptake by the roots. Oligomyein, 2,4-dinitrophenoland diethylstilbestrol (10^ mol/dm^), however, inhibited K* uptake by the roots. Ingeneral, K* transport processes were almost unchanged in plants of high K status. Itis concluded that only plants of low K status operating with active K* transportmechanisms are responsive to environmental factors. In high K* plants the transportprocesses are passive and are uncoupled from the metabolic energy flow.

Additional key word - Translocation.

Z. Olah, A. Berczi and L. Erdei (reprint requ£sts), Inst. of Biophysics, BiologicalResearch Center, Hungarian Academy of Sciences, Szeged, Hungary.

J , plasmaletntna of the root cells and did not take inton TO uc ion account the long-distance translocation to the shoot.

Regulation of the K* uptake into roots has recently Regulation of the translocation process in the intactbecotne a relatively well-understood process. Ion up- plant is thus much less vi el] understood, and the ques-take studies with roots of different K status have re- tion arises as to whether the ion status in the shootvealed that the influx is governed by cytoplastnic K* determined the upward translocation (Cram 1976). Theconcentrations via an allosteric negative feedbaek interrelation between salt status, translocation and wa-tneehanisH) (Glass 1976, ,Jensen and Pettersson 1978, ter flow (transpiration) is still basically unresolved andPettersson and Jensen 1978, 1979). These short-term is regarded as "complex" (Sutcliffe 1976, Lauchliexperitnents involved the ion flux rates through the 1979).

Received 14 December, 1981; revised 9 March, 1982

Physiol. Plani. 5.S 0031-9317/82/070377-06 $03.00/0 © 1982 Physiologia Plantarum 377

In this paper the uptake of K* and its translocation tothe shoot of young winter wheat plants of different Kcontents are studied in relation to water flow andmetabolism. The connections between uptake, translo-cation and tnetabolism are investigated using severalinhibitors of metabolic and transport processes, such asoligottiycin, 2,4-DNP, DSPD, DES and colchicine.

Abbreviations - DES, diethylstilbestrol; 2,4-DNP, 2,4-dinit-rophenol; DSPD, disalicyhdene-propanediamine; RH, relativehumidity.

Materials and methods

Intact seedlings of winter wheat (Triticum aestivumL. cv. Martonvasari 8) grown with different K* supplieswere used in the experiments. Det,ails concerning theplant cultivation ,and the ionic compositions of thegrowth solutions and of the plants are given by Berczi etal. (1982). Plants used in the present experimentsoriginated from the same series as in that paper.

K"''(*^Rb) uptake and translocation were carried outin 100 ml of 1 mmol/dm' KCl solution with *" RbCl astracer (37 MBq/dm^, i.e. 1 mCi/1). Usually 3 seedhngswere taken from each type of plant in the K* supplyseries, and were placed in a common beaker with aplastic foam holder having 8 to 12 cuts in it. After anuptake period of 5 h the roots were rinsed in distilledwater three times for 1 min each. Afterwards the rootsand shoots were separated and the amount of "Rbtaken up was measured using a y-spectrometer. The 3seedlings were measured together. In the experimentsshown in Figs 1 and 2 the average values ± SD of 4independent plant series are given. In the experimentswith the inhibitors, 3 independent measurements wereperformed;, one of these was selected for the presenta-tion.

The experiments were carried out in a laminar flowbox, providing air movement at a constant rate and 10W m" illumination (Tungsram 40 W F-7 daylightfluorescent tubes). The temperature was 25°C, and therelative humidity of the air was 45 ± 5%. To eliminatetranspiration the beakers with the plants were em-bedded in wet cotton and were covered by glass vessels(100% RH). During the experiments the temperatureand relative humidity were controlled continuously.

The inhibitors were applied in the uptake solution asethanolic stock solutions (with the exception of2,4-DNP, which was used in ,aqueous solution), withfinal concentrations as follows (mol/dm'): oligomyein10-*; 2,4-DNP lO"*; DSPD lO""; DES 10^ and lO"^;colchicine 10~*. The ethanol concentration of the up-take solution did not exceed 0.4%, and alcoholic con-trols were also made.

Results

The uptake of K^C'Rb) by roots and the translocationto shoots in intact plants of three different K status asthe function of time are given in Fig. 1. The influx ratesin roots decreased after 1 h, especially in the case ofplants grown in a nutrient solution containing 10^mol/dm^ K"*". Translocation to the shoot, however, re-quired a longer uptake time because of the lag period.In further experiments, therefore, the 5 h period waschosen and the ion uptake by roots was regarded as ionretention in the roots rather than as a theoretical influxterm. The connection between influx rates and ioncontents has been discussed earlier (Berczi et al. 1982).Qualitatively the uptake pattern obtained during 5 h inroots of intact plants corresponds to that obtained for1 h experiments using either intact plants or excisedroots.

6 0 2Time, bour

Fig. 1. The K*(""Rb) uptake and translocation in intact wheatseedlings of different K status as the function of time. Plantswere grown in complete nutrient solution with 10^ (•) , 10"(A) or 10" (•) mol/dm^ KCI. Uptake solution contained 10"'mol/dm^ KCL One experiment with three plants in each sam-ple.

I0.1 !

K concentration, mmol/dm^Fig. 2. The K+(*''Rb) uptake into the roots of the intact wheatseedlings on the llth day after germination as the function ofthe K* supply in the complete nutrient solution. Experimentswere carried out in the light (open symbols) and in darkness(filled symbols) for 5 h under the condition of 45% (A) and100% (B) RH. Results presented are average values ± SD offour independent series of experiments (except • which wasmeasured only once).

378 Physioi. Pianl. 55

0.1K concentration,

Fig. 3. The K+C'Rb) transloeation from the roots to the shootsof the intact wheat seedlings on the llth day after germinationas the function of the K"*" supply in the complete nutrient sol-ution. Symbols are the same as in Fig. 2.

The K^C^Rb) uptake into the roots of intact plants ofdifferent K status is shown in Fig. 2. The K" absorptiondecreased with increasing K status of plants under aliconditions used. No influence of low or high humidity,and little if any effect of light or darkness on the K"uptake could be detected.

The rate of K" translocation to the shoot dependedon the K status of the plant and also on the environ-mental conditions (Fig. 3). In general, plants of high Kstatus (plants grown in a medium with K* concentration> 1 mmol/dm^) displayed a low translocation rate, andthose of low K status a high translocation rate. Thislatter process varied under the different conditions. Themaximum translocation rate was observed in the light at

AOO

200

co5Q.'Ul

cto

fr.

Ol

OCM

XIJl

E

hourFig. 4. The transpiration of the iotact wheat seedlings in light(O) and in darkness (•) as the function of time. Plants weregrown in complete nutrient solution with 0.4 mmol/dm' KCl.The transpiration was measured under the same conditions asthe K^ translocation experiments. Transpiration rate is ex-pressed in mg HjO/g fresh weight of shoots.

low humidity, and it decreased by about 30 to 50% inthe dark (Fig. 3A). The translocation in plants grown ina nutrient solution containing 5 x 10^ mol/dm-* KClwas practically unaffected by darkness.

At 100% relative humidity the K* translocation ratefell by 50 to 75% (Fig. 3B). Plants grown in nutrientsolutions containing 5 x 10" and 5 x 10^ mol/dm^KCl showed a lower rate decrease and two relativemaxima were observed in the ctirve. Darkness hardlyaffected the translocation rate under these conditions.

The transpiration influences the ion translocation tothe shoot (Russell and Barber 1960). As an example, inFig. 4 the transpiration in one type of plant from theseries ([K^j = 4 x 10^ moi/dm' in the growth solution)is shown in the light and in the dark, with a relativehumidity of the air 50%. Darkness decreased the trans-piration by 47% in this case, but generally by 35 to 50%in the series. At a relative humidity of 100%, water lossover the experimental error could not be measured, butguttation was observed.

In order to clarify the metabolic relations of thetransport processes the effects of several types of in-hibitors were studied. The actions of inhibitors weremeasured in the light at a relative humidity of the air of50%. Results relating to the uptake into the roots areshown in Fig. 5.

The well-known uncoupler, 2,4-DNP, inhibited theK* uptake by the roots in low-salt plants resulting innearly the same rates as those in high-salt plants.Oligomyein, an inhibitor of respiration, brought about asomewhat lower reduction in the uptake. DSPD, a po-tent inhibitor of NADPH formation by acting on fer-

01 1K concentration, mmol/dm''

10

Fig. 5. The effects of metabolic (A) and transport (B) in-hibitors on the K*(*'Rb) uptake by roots of intact wheatseedlings as a function of K* supply in the complete nutrientsolution. DES was applied in two concentrations, 10"mol/dm^ (A) and 10^ mol/dm' (A), while the other agentswere applied in 10"' mol/dm^ concentration. The experimentat 0°C was carried out in a cold room. Dotted lines stand forcontrol and are from Fig. 2A.

Physiol Pianl. 55 379

T ' 2 n oligomyein.o^ \ A 2.4-DNP

0.1 1 10K concentralion, mmol/dm''

Fig. 6. The effects of metabolic (A) and transport (B) in-hibitors on K^C'Rb) translocalion from roots to shoots of in-tact wheat seedlings as a function of K"*" supply in the completenutrient solution. Symbols and concentrations of agentsapplied are the same as in the Fig. 4. Dotted lines stand forcontrol and are taken from Fig. 3A.

redoxin (Huber and Edwards 1976, Droppa et al.1981), gave only little inhibition and only in roots of lowK status. Colchicine, which destroys plasmodesmata(Hart and Sabnis 1973, Sabnis and Hart 1973), showedno effect on the K* uptake into the roots. DES in con-centrations of 10"^ and 10^ mol/dm^ caused a mild andtotal inhibition, respectively (Balke and Hodges1979a, b, c). The uptake also decreased at low temp-erature.

The effects of the same series of agents on the K+translocation to the shoot are illustrated in Fig. 6. It isseen that 2,4-DNP and oligomyein decreased thetranslocation to the passive level. DSPD influencedplants of low K status in the same manner as did dark-ness. DES, even in the lower concentration used,greatly reduced the translocation while low temperaturestopped it completely. Interestingly, colchicine which

did not influence the uptake into the roots, inhibited theK"'' translocation to the shoot.

Discussion

According to Luttge and Higinbotham (1979), thefeedback mechanism regulating ion transport consists ofthree components; the transport process itself, theenergy status of the plant and a signalling system. Ofthese components the transport process in connectionwith the energy status was studied in wheet seedlings ofdifferent K status.

The plants used in the present work are characterizedby increasing K levels of 0.1 to 1.5, and 0.2 to 2.5 mmolK per g dry weight for the roots and the shoots, respec-tively (Berczi et al. 1982). Both the K+ uptake into theroots and the translocation to the shoots depended onthe K content of the plants. Generally, the initially hightransport rates in plants of low K status decreased to aconstant low value in plants of high K status. In theroots there are two transfer steps; uptake into the corti-cal cells and secretion by the xylem parenchyma cellsinto the xylem. The former is composed of active andpassive processes (Jensen 1981); the secretion is prob-ably active (Lauchli 1976). The subsequent upwardtranslocation depends greatly on the rate of transpira-tion and can be regarded as passive. Finally, the uptakeof ions by the leaf cells can also be a controlled step.

Let us now consider how the K" uptake and translo-cation rates are influenced by the environmental factorsand by the different inhibitors of metabolism. Whendarkness and/or high relative humidity of the air in thelight decrease the rate of transpiration by about 50 and100%, respectively, the uptake into the roots is practi-cally unaffected (Fig. 2). It must be emphasized, how-ever, that the translocated 1 to 2 jimol K* (Fig. 3) isonly a small proportion of the total K"*" taken up (40' to50 (imol), and falls within the error of the uptake ex-periments. The upward translocation rate in Ught and in45% RH showed an asymmetric optimum curve as thefunction of the K status (Fig. 3A). The increasing side ofthe curve may be due to the promoting effect of the

dark, DSPD

•energy supplynutrientsolution

influx radial secretion• transport •

oligo tnyciin,2,4-DNP,

-ADESdO M)

1colchicine

oligomyein,

DES(IO^M)

(sugar)shoot

(leaves)water

transportf

humidity,dark

Fig. 7. Seheme of theinteractions among the K*transport steps, themetabolic and transportinhibitors, the energy suppl)and environmental factors.

380 Piiysioi. Plani. 55

water stream on ion translocation and also to the avail-ability of photosynthetic energy sources. The decline inthe rate can be regarded as the negative feedback regu-lation of the transport process, possibly by an allosterictype of inhibition in plants of high K status (Glass 1976,Jensen and Pettersson 1978, Jensen 1981). In darknesswater transport decreases by about 50% (Fig. 4 andRussell and Barber 1960), and the photosyntheticenergy sources will also be exhausted, resulting in de-creased translocation as well (Crapo and Ketellapper1981). The further inhibition in plants of high K statusis again because of the negative feedback inhibitionback to probably a passive level.

A similar consideration may hold for the resultsobtained for translocation under 100% RH (Fig. 3B).Translocation was reduced both in light and darknessbut showed two maxima as the function of the K statusof plants. It is necessary to note that at this high RHguttation was observed, thus a certain water stream wasstill available for ion translocation. The first peak maybe attributed to the effects of the increasing ion con-centration on transport sites which are then opposed bythe decreased water flux, while the second peak may bedue to the effect of concentration compensation for lowwater flux followed by the allosteric type of inhibition athigh concentrations. These considerations are sup-ported by the earlier works of Pettersson (1966a, b),who found that the rate of water stream can influencesulfate uptake by a change in the turnover of bindingsites. For K"'' transport system a similar signallingtnechanism between shoot and root has been suggestedby Jensen and Kylin (1980) when studying the effect ofhumidity on the efflux of K* from wheat, oat andcucumber roots.

Inhibitors of energy metabolism, oligomyein and theuncoupler 2,4-DNP, reduced both the influx into theroots and the translocation to the shoots (Figs 5 and 6).With these agents, metabolic and non-metabolic trans-port processes can be differentiated (Jensen 1981).Since 2,4-DNP is a H^-earrier, its effect is rapid andinvolves all membranes energized by a pH gradient. Inthe presence of oligomyein the phosphorylation in thetnitochondria is blocked and, because of the exhaustionof the cytoplasmic ATP pool, the transport processgradually ceases.

The action of DSPD is different. Both uptake andtranslocation patterns become very similar to thoseobtained in darkness. DSPD acts on ferredoxin in thephotosynthetic electron transport chain and thus blocksthe formation of NADPH. In this way the limited sugarsupply for phloem transport to the roots will reduce theion uptake. This view is in agreement with the work ofCrapo and Ketellapper (1981), who reported on therelationship between photosynthetic activity, energysupply to the roots, respiration and potassium uptake.They concluded that with the restriction of photosyn-thesis the K"'' uptake is also strongly decreased becauseof the lack of carbohydrates supplied by the shoot. In

addition, sugars delivered from the phloem are moreeffective than sugars from the storage pool (Vakhmis-trov and Ali-Zade 1973). Hence, DSPD influences the"energy status" of the plant which, in the nomenclatureof Luttge and Higinbotham (1979), is a part of thefeedback mechanism in the root-shoot cooperation.

Finally, the actions of the two kinds of transport in-hibitors should be discussed. According to Balke andHodges (1979a, b, c), 10-= mol/dm^ DES inhibits K+adsorption and ATPase activity in oat roots, while 10"mol/dm-* DES causes irreversible inhibition of K" influxand of oxidative phosphorylation. In our case 10~mol/dm^ DES brought about a partial decrease in theK* influx into the roots and a total inhibition of thetranslocation in plants of low K status. In high K*plants, however, neither the influx nor the translocationwere influenced, enabling us to distinguish between ac-tive and passive transport processes.

DES (10^ mol/dm^) blocked both uptake and trans-location in plants of low K status. Besides the inhibitionof the ATP supply, however, the structure of the mem-branes could also be damaged by this DES concentra-tion (Balke and Hodges 1979a).

The other type of transport inhibitor used was col-chicine, which attaches to tubulin and destroys tubularand filemantous structures (Hart and Sabnis 1973,Sabnis and Hart 1973). Colchicine showed no effect onthe K* uptake into the roots, but inhibited the K*translocation to the shoots in plants of low K status. Thetarget of the action of eolchicine is suggested to be theplasmodesmata, resulting in blocking of the two-direc-tional radial transport of K* and sugar across the Cas-parian strip. The interactions between metabolic in-hibitors and transport processes are summarized in Fig.7.

it can be concluded that only plants of low K statusare capable of direct regulation of the K^ transport pro-cesses. The primary uptake mechanism and the secret-ory system (ATPases?) are sensitive to the changes inionic and energy flow; in other words, the K and energytransports are coupled. The translocation is the out-come of the cooperation between the primary uptakeand the radial (medium-distance) transport. Finally, theK" translocation is tightly coupled to the water trans-port.

The opposite statements hold for plants of high Kstatus. Here the primary uptake mechanism is allosteri-cally inhibited and does not respond to small changes inion and energy flows. Only passive transport proceeds.Ion and energy flows are uncoupled. Should the watertransport be high, only low ion influx and upwardtranslocation occur in these plants.

Acknowledgements — This work was supported by the Depart-ment of Plant Protection and Agrochemistry, Ministry of Ag-riculture and Food ( M £ M NAF) under contract No. MUFAVIII/A-Sl -4-1. The assistance of Mrs. Eva Izso and Ilona Birois gratefully acknowledged. Crop Nutrition Research Report,publ. No. 2.

25 Physiot, Piani. 55, i9,S2 381

ReferencesBalke, N. E. & Hodges, T. K. 1979a. Effect of diethylstibestrol

on ion fluxes in oat roots. - Plant Physiol. 63: 42^7.- & Hodges, T. K. 1979b. Inhibition of adenosine triphos-

phatase activity of the plasma membrane fraction of oatroots by diethylstilbestrol. - Plant Physiol. 63: 48-52.

- & Hodges, T. K. 1979c. Comparison of reductions inadenosine triphosphate content, plasma membrane-as-sociated adenosine triphosphatase aetivity, and potassiumabsorption in oat roots by diethylstilbestrol. — PlantPhysiol. 63: 53-56.

Berczi, A., Olah, Z., Fekete, A. & Erdei, L. 19«2. Potassiumtransport in wheat seedlings grown with different potas-sium supplies. I. Ion contents and potassium influx. -Physiol. Plant. 55: 371-376.

Cram, W. J. 1976. The regulation of nutrient uptake by cellsand roots. —In Transport and Transfer Processes in Plants(Wardlaw, I. F. and Passioura, J. B., eds), pp. 113-124.Academic Press, Inc., New York. ISBN 0-12-734850-6.

Crapo, N. L. & Ketellapper, H. J. 1981. Metabolic prioritieswith respect to growth and mineral uptake in roots ofHordeum, Triticum and Lycopersicon. — Am. J. Bot. 68:10-16.

Droppa, M., Demeter, S., Rozsa, Zs. & Horwath, G. 1981.Reinvestigation of the effects of disalicylidenepropane-diamine (DSPD) and 2-heptyl-4-hydroxyquinoline-N-oxide (HONO) on photosynthetic electron transport. - Z.Naturforsch. 36c: 109-114.

Glass, A. D. M. 1976. Re,gulation of potassium absorption inbarley roots. An allosteric model. - Plant Physiol. 58:33-37.

Hart, J. W. & Sabnis, D. D. 1973. Colchicine- binding proteinfrom phloem and xylem of a higher plant. - Planta 109:147-152.

Huber, S. C. & Edwards, G. E. 1976. Studies on the pathwayof cyclic electron flow in mesophyll chloroplasts of a C4plant. - Biochim. Biophys. Acta 449: 420-433.

Jensen, P. 1981. Separation of metabolic and non-metabolicsteps of Rb"*" uptake in spring wheat roots with different K^status. -Physiol. Plant. 52: 437-441.

- & Kylin, A. 1980. Effects of ionic strength and relativehumidity on the efflux of K+^Rb) and Ca^+C=Ca) fromroots of intact seedlings of cucumber, oat and wheat. -.Physiol. Plant. 50: 199-207.

- & Pettersson, S. 1978. Ailosteric regulation of potassiumuptake in plant roots. - Physiol. Plant. 42: 207-213.

Lauchli, A. 1976. Symplasmic transport and ion release to thexylem. — In Transport and TraF3s,fer Processes in Plants(Wardlaw, I. F. and Passioura, J. B., eds), pp. 101-112.Academic Press, Inc., New York. ISBN 0-12-734850-6.

- 1979. Absorption and translocation of mineral ions inhigher plants. - Prog. Bot. 41: 44-54.

Liittge, U. & Higinbotham, N. 1979. Transport in Plants. -Springer Verlag, New York, Heidelberg, Berlin. ISBN0-387-90383-6.

Pettersson, S. 1966a. Active and passive components of sulfateuptake in sunflower plants. — Physiol. Plant. 19: 459-492.

- 1966b. Artificially induced water and snifate transportthrough sunflower roots. - Physiol. Plant. 19: 581-601.

- & Jensen, P. 1978. Allosteric and non-allosteric regulationof rubidium influx in barley roots. — Physiol. Plant. 44:110-114.

-. & Jensen, P. 1979. Regulation of rubidium uptake insunflower roots. - Physiol. Plant. 45: 83-87.

Russell, R. S. & Barber, D. A. 1960. The relationship betweensalt uptake and the absorption of water by intact plants. -Annu. Rev. Plant Physiol. 11: 127-140.

Sabnis, D. D. & Hart, J. W. 1973. P-protein in sieve elements1. Ultrastructure after treatment with vinblastine and col-chicine. -Planta, 109: 127-133.

Sutcliffe, J. F. 1976. Regulation in the whole plant. -In Trans-port in Plants, Encyclopedia of Plant Physiology, NewSeries, Vol. II. B: 393-417. Springer Verlag, Berlin. ISBN3-540-07453-8.

Vakhmistrov, D. B. & Ali-Zade, V. M. 1973. Transition pro-cesses during liberation of bleeding sap by root system ofsunflower. Physiological analysis. - Fiziol. Rast. 20:773-774.

Edited by A.K.

382 Physioi. Piani. 55