THE UPTAKE OF UREA BY CHLORELLA

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$SYRETT Department f Btany and Micrbilgy, University Cllege f Swanseay Wales, {Accepted 2 June 1978) SUMMARY The uptake f ^^C by urea-grwn cells f Chlrella (strain 211/8p) supplied with [^"^ was$

1 Nezu Phytl. (1979) 82, THE UPTAKE OF UREA BY CHLORELLA BY LA. BEKHEET* AND P. J. SYRETT Department f Btany and Micrbilgy, University Cllege f Swanseay Wales, {Accepted 2 June 1978) SUMMARY The uptake f ^^C by urea-grwn cells f Chlrella (strain 211/8p) supplied with [^"^ was studied. Uptake was rapid in light with an apparent half-saturatin value (ic^,) fr urea f 16 /im. Uptake was inhibited cmpetitively by thiurea. Uptake f ^'^C was inhibited by 6x ' DGMU in light and was very much slwer in darkness; nevertheless, under these cnditins, rate f lss f ^^G frm the medium was much the same as in illuminated nninhibited cultures. [^**G]urea was metablized rapidly by the cells and, in illuminated cells after a min expsure t [^*G]urea, less than 0 % f the sluble ^*G within the cells was in [^ *G]urea. It is cncluded that much f the measured ^^G uptake may have been due t cnversin f [^ "Clurea t ^"^GOa fllwed by phtsynthetic ^'*GG2 fixatin. Nevertheless, when [^'^Gjurea in the cells was extracted and separated by thin-layer chrmatgraphy, there was clear evidence f accumulatin at a cncentratin abve that in the external medium and f light stimulatin f uptake. It is suggested that, in view f the rapid metablism f [^'*C]urea by Chlrella, it may be preferable t study the uptake mechanism f this rganism by using the urea analgue, thiurea. INTRODUCTION The study f the mechanisms f uptake f substances by cells is greatly aided when the substance is radiactive and is nt metablized rapidly. In these circumstances, estimatin f the appearance f radiactivity within the cells is a rapid and cnvenient measure f uptake f the substance. This is nt s when the substance absrbed is metablized quickly because then the rate f appearance f radiactivity within the cells may reflect the subsequent metablism f the substance rather than uptake. This bjectin can smetimes be vercme by studying the uptake f a nn-metablizable analgue. Thus, fr example, 6-dexyglucse and 3-O-methylglucse have been used t study the system that transprts glucse int Chlrella (Haass and Tanner, 1974). Pateman et al. (1973) used thiurea as a nn-metablizable analgue f urea t measure the activity f the urea uptake system ^ Aspergillus. Syrett and Bekheet (1977) studied the uptake f thiurea by Chlrella; thiurea was absrbed actively, with Michaelis-Menten kinetics, and uptake was inhibited cmpetitively by urea. Syrett and Bekheet cncluded, therefre, that thiurea was taken up by Chlrella by a mechanism which was respnsible fr urea uptake. Hwever, Williams and Hdsn (1977) using Chlamydmnas and Rees and Syrett (1979) using Phaedactylum have shwn that, with these algae, it is pssible t use radiactive urea t study the uptake f urea directly. This is because rate f uptake exceeds greatly the rate f metablism and mre than 80% f the radiactivity taken up as urea remains in the cells as unchanged urea, at least, during experiments f up t min duratin. Measurement * Present address: Department f Btany, University f Alexandria, Alexandria, Eg>'pt X/79/ JO2.OO/O 1979 The New Phytlgist

2 i8 I. A. BEKHEET AND P. J. SYRETT f appearance f radiactivity in the cells thus gives an adequate measure f the activity f the uptake mechanism. In this paper we describe experiments in which we attempted t measure the uptake f radiactive urea by Chlrella. MATERIALS AND METHODS Chlrella fusca var. vaculata (strain 211/8p) was grwn with urea as nitrgen surce and cell suspensins fr experiments were prepared as described by Syrett and Bekheet (1977). Measurements f ^*C-uptake were made in the same way as described in that paper except that [^*C]urea was used instead f [^^CJthiurea. Generally, at each sampling time, 1 ml samples were remved, in duplicate, and sucked thrugh 2 mm Whatman GF/C glass-fibre filters. Smetimes the filtrate was als cllected fr assay. Cells n the filter were washed with x ml nitrgen-free medium and radiactivity in them estimated by scintillatin cunting. Calibratin was by additin f 12- nci p^cjurea t the vials. Er separatin f the radiactive cmpunds in the cells, cells after separatin frm medium were extracted in ethanl. The cncentrated extracts were chrmatgraphed n thin-layer cellulse plates using butanl:acetic acid:water (12:3:) as slvent. Radiactive cmpunds were lcated by leaving the develped plates in cntact with X-ray film fr 7 days. RESULTS Time curse f ^*C uptake int cells Figure 1 shws the time curse f appearance f ^*C within the cells. Uptake was almst linear with time ver 60 min. Althugh rate f uptake was rapid at 2 C in light, in darkness at 2 C it was slw and was nt very much faster in dark aerbic 300 S : J X r ; r Time (min) Fig. 1. Uptake f radiactivity frm [^^CJurea. A 1 ml cell suspensin ( x 'cells was incubated with 1-/tml (/tci) ["C]urea. O, (18 Wm'^), aerbic, 2 X dark, aerbic, 2 C; A, dark, anaerbic, 2 C; T, dark, aerbic, 2 C.

3 uptake f urea by Chlrella i8i cultures than in dark anaerbic nes r in dark cultures held at 2 ""C. With Chlamydmnas (Williams and Hdsn, 1977) and Phaedactylum (Rees and Syrett, 1979), incubatin f the cultures fr sme hurs in nitrgen-free medium markedly increases the rate at which they subsequently take up urea. Such nitrgen deprivatin, fr up t 24 h, did nt have a similar effect in ur experiments with Chlrella. Disappearance f ^^C frm medium In anther experiment, incrpratin f ^'*C int cells in light r in darkness was fllwed alng with disappearance f ^*C frm the medium (Table 1). Incrpratin f ^*C int cells was much the same as in the experiment f Figure 1 althugh nt s Table 1. Lss f ^^C frm medium and uptake int cells Incubatin time (min) Cells A Medium Cells Darkness Medium ml samples f urea-gr\vn cells (-3 x 'cells ml"^) were incubated with 1-/iml (000 nci) ["CJurea at 2 C in either light (18 W m~^) r darkness. Results are given as ttal nci recvered in cells and medium; the sums f these values at any sampling time are thus cmparable with the 000 nci added at zer time. linear with time. Althugh incrpratin in light was much greater than in darkness, disappearance f ^"^C frm the medium was much the same in light as in darkness. After 60 min, practically all f the ^**C added (000 nci) had disappeared frm the medium. In the light, abut ne-third f this radiactivity was recvered in the cells but in darkness less than % was recvered in cells and medium tgether. It must be cncluded that [^^CJurea was metablized rapidly in darkness and ^"^C lst, presumably as The effect f DCMU n uptake f i*c Figure 2 shws the effect f 6 x "^ M DCMU n uptake f ^^C in light under either aerbic r anaerbic cnditins. Inhibitin f uptake by DCMU was marked, particularly in air. Measurements f the disappearance f ^*C frm the medium shwed that the rate was much the same in light under anaerbisis r with DCMU as it was in aerbic cultures withut DCMU. Incrpratin f ^^C int ther cmpunds An experiment was carried ut in which cells were incubated with P'^Cjurea in light, aerbically r anaerbically and in the presence r absence f 6 x ^ M DCMU. Samples were taken at intervals and the cells remved, washed and extracted with ethanl. Incrpratin f ^^C int ethanl-sluble and ethanl-insluble cmpunds was measured. The ethanl extracts were then cncentrated and chrmatgraphed t separate [^^CJurea frm ther cmpunds. After detectin f the radiactive spts n the chrmatgrams by autradigraphy, the radiactivity in each spt was determined

4 182 LA. BEKHEET AND P. J. SYRETT and the quantity present in P'CJurea expressed as a percentage f the ttal (Tables 2 and 3). Table 2 shws that, under all cnditins, ^*C was incrprated int insluble cmpunds but the percentage s incrprated was less under anaerbisis. In the 300- JH 200- % - 0- Time (min) Fig. 2. Effect f DCMU (6 x "^ M) n uptake f radiactivity frm P^C]urea. A 1 ml cell suspensin ( x ' cells ml~^) was ineubated with 1- fiml ( /tci) [^^Cjurea in light (18 W m~^) at 2 C under either air r nitrgen. O> Cntrl in air; A, cntrl under nitrgen; #, plus DCMU in air; A, plus DCMU under nitrgen. presence f DCMU, less ^^C was taken int the cells (cf. Fig. 2) and, under dark aerbic cnditins, very little. Table 3 shws that, in light with DCMU and in darkness, few sluble ^^C cmpunds were in the cells. By cntrast, in light in the absence f DCMU, after incubatin with p^c]urea, there were 11 cmpunds with appreciable radiactivity n the chrmatgrams and P^CJurea itself accunted fr nly % f the radiactivity present in the cells as sluble cmpunds. In the presence f DCMU, abut 90% f the sluble ^^C was present as P'^CJurea after min incubatin and, even after, 0 t 60% f the sluble ^'*C cnsisted f P'*C]urea; the darkened cells shwed similar behaviur. Frm these results, and knwing the vlume f the cells, it is pssible t calculate the rati f internal: external urea cncentratin under varius cnditins (Table 4). Accumulatin ratis in light under air are lw, i.e. abut 4. They are higher (abut ) in light under nitrgen. The ratis in the presence f DCMU are f the same rder despite the greater prprtin f internal ^^C that remains as P^C]urea. This is because, in the presence f DCMU, ttal uptake f ^^C is markedly lwer than in its absence (Figure 2 and Table 2). The kinetics f ^^C incrpratin Figure 3 shws the results f tw experiments. Fr the first [Fig. 3(a)], the cell density was x ' cells nil~^ and urea was supplied t give initial cncentratins ranging frm 2 t 0 /^M. Uptake f ^^C was fllwed ver 1 min. The initial rate f uptake was much the same with 2, 0 r 0 /^i urea. At lwer cncentratins, rates were slwer but, as the [^'^CJurea was fully utilized within min, the initial rates

uptake f urea by Chlrella 183 Table 2. Fate f ^^C taken int cells time (min) Insl. Ligbt (aerbic) 1. Sl. i^c / sl. Insl. i^c Ligbt (anaerbic) Sl. "C / sl. (aerbic + DCMU) Insl. ^'C Sl. lie / sl.

5 uptake f urea by Chlrella 183 Table 2. Fate f ^^C taken int cells time (min) Insl. Ligbt (aerbic) 1. Sl. i^c / sl. Insl. i^c Ligbt (anaerbic) Sl. "C / sl. (aerbic + DCMU) Insl. ^'C Sl. lie / sl t time (min) 1 30 Insl. i.c (anaerbic + DCMU) Sl. I'C / sl Insl Dark (aerbic) A, Sl.»c / sl A 30 ml cell suspensin (4-6 x ' cells ml~^) was incubated with 3 fivax (30 /ici) [^^C]urea under the cnditins shwn. ( intensity, 18 W m"^; DCMU cne. 6 x ~* M). Five ml sampls were remved at inter\'als and centrifuged rapidly. Supernatants were retained fr cunting. Cells were washed twice and then extracted with ethanl. Radiactivity in the extract (sluble - Sl.) and in the residual pellet (insluble - Insl.) was estimated. Results aie given as nci per cells and % sl. Incubatin time (min) 1 30 (aerbic), 4(4) 36 () 32(6) (11) Table 3. Fate f^^c taken ifit cells (anaerbic), 7(3) 61(4) 43(6) (11) (aerbic + DCMU), 89(2) 94(2) 67(3) 60(3) (anaerbic + DCMU), 9(2) 88 (2) 84(2) 0 (3) Dark (aerbic) (2) 66(2) 74(2) 7(2) Cncentrated ethanl extracts (see Table 2) were chrmatgraphed tgether with marker spts f [^^CJurea n thin-layer cellulse plates. After lcatin f radiactive spts by autradigraphy, cellulse in these areas was scraped frm the plates and cunted. The percentage f the radiactivity recvered which was present as [^''CJurea is given. The numbers in parentheses shw the number f radiactive spts seen and cunted. derived frm the curves fr 2, r //M urea are unreliable. Cnsequently, the experiment was repeated using a lwer cell density and mre rapid sampling [Fig. 3(b)]. Since bth experiments were dne with 2 /^M urea, their results can be cmbined t give the curve shwn in Figure 4, T btain this nly the rates btained between zer time and the first sampling time have been used. The hyperbla drawn in Figure 4 was fitted t the data by the iterative cmputer f Bliss and James (1966). It gives an apparent half-saturatin value (K^^,) f //-M and a maximum rate f uptake f 16-3 ± 1-6 nci/^ cells/min. If this uptake was due nly t uptake f [^^C]urea, this rate crrespnds t the uptake f 4-9 ±0- nml urea/^ cells/min. Table shws the effect f the presence f nn-radiactive thiurea n the rate f

184 I. A. BEKHEET AND P. J. SYRETT Table 4. Accumulatin f urea in cells nml urea per ml cell vlume nml urea per ml external medium Rati internal cne external cne.

6 184 I. A. BEKHEET AND P. J. SYRETT Table 4. Accumulatin f urea in cells nml urea per ml cell vlume nml urea per ml external medium Rati internal cne external cne. aerbic min anaerbic min aerbic + DCMU min anaerbic + DCMU min Darkness, aerbic min By cmbining the results in Tables 2 and 3, the quantity f [^^C]urea in the cells was fund. The vlume f ^ urea-grvi/n cells was ml. Using this value and assuming that the urea is unifrmly distributed thrughut cell vlume, the cncentratin f urea in the cells was calculated. Thesefiguresare given abcve. Radiactivity in the mediunn at each sampling time was determined. Assuming that this is due t residual urea, the external urea cncentratin was calculated. Hence the rati f the {internal cncentratin): (external cncentratin) was estimated fr the and samples. J c Time (min) Fig. 3. Uptake f radiactivity frm [^^Cjurea supplied at different cncentratins. Incubatin was in light (18 W m"^) in air at 2 "C. The urea slutin supplied cntained 3 /^mles and lo/ici per ml. The quantities supplied gave the initial urea cncentratins (in /tm) shwn against each curve. Experiment {a) was carried ut with a cell suspensin cntaining x* cells ml"^; fr Experiment (6) cell density was 2 x * cells ml^^

7 uptake f urea by Chlrella 18 uptake f ^'*C frm [^'^CJurea at urea cncentratins ranging frm 1 t /fm. With //M [^^C]urea present, even 120//M thiurea did nt inhibit uptake f ^'^C; indeed there was apparently sme stimulatin. Cnversely, with 1 //M [^^C]urea, additin f 60 r 120 /^M thiurea inhibited ^"^C uptake cnsiderably. These are the characteristics f cmpetitive inhibitin and wuld be expected if urea and thiurea are transprted by the same mechanism fr which they cmpete. It is evident frm the results in Table that, if this is s, the affinity f the mechanism fr urea must be cnsiderably greater than its affinity fr thiurea but the data are nt sufficiently gd fr an accurate estimate f a K: value t be made. CD O c urea cncentralin 7 Fig. 4. Effectf [^*C]urea cncentratin n rate f uptake f "C. Data are frm the experiments shwn in Eigure 3 with values fr 0 and 0/iM urea frm Figure 3 (a) and the thers frm Figure 3(b). Only the rates between zer time and the first sampling time were used. Table. The effect f thiurea n rate f incrpratin f LTrea cne. (//M) 0 Thiurea cncentratin (// A 60 M) LV ml cell suspensin (2 x ' cells ml"*^) was incubated in light (18 W m ^) at 2 C with zer, 60 r 120/(M thiurea. At zer time, ['^CJurea (2/iml and //Ci per ml) was added t give the cncentratins shwn. Tw minutes later, samples were remved fr determinatin f ^^C uptake. Results are expressed as a percentage f the rate with /im urea and zer thiurea; this rate was -0 nci per ^ cells per min. DISCUSSION The results presented here shw that ^*C cntained in p^c]urea was taken up rapidly by urea-grwn cells f Chlrella. The rate f uptake was cnsiderably greater than that allwed by passive diffusin (cmpare the result in darkness at 2 C in Figure i) and was much mre rapid in light than in darkness. The rate f uptake was related t urea cncentratin in a hyperblic manner and the apparent /v^,, fr uptake was

i86 LA. BEKHEET AND P. J. SYRETT 16- +4*8/iM.

8 i86 LA. BEKHEET AND P. J. SYRETT *8/iM. This value is smewhat higher than the value f -1/lu fr the urea uptake mechanism f Chlamydmnas (Williams and Hdsn, 1977) and the value f /fm deduced fr Chlrella by Syrett and Bekheet (1977) by study f the cmpetitive inhibitin f thiurea uptake. The value is lwer than the apparent A'^, fr thiurea uptake which is 8/^M. The V^^^ value fr urea uptake f 4-9 nml/^ cells/min agrees with the V^^ fr thiurea uptake fund by Syrett and Bekheet (1977); this was 4-4 nml/^ cells/min. Hwever, further examinatin f ur results casts dubt n what was being measured when ^^C was taken up frm P^CJurea. Williams and Hdsn (1977) fund that, after min, all the ^^C taken up frm p^cjurea by ChlamydmJtas culd be recvered frm the cells as [^-^CJurea. Cnsequently, their measurements f ^^C uptake were a reliable measure f urea uptake. This was nt s in ur experiments. Table 3 shws that, after min uptake in light, there were fur significantly labelled radiactive cmpunds within the cells and nly 4 % f the -^^C taken up was recvered in urea. Mrever, althugh the rate f ^^C uptake was very much lwer in darkness than in light, the rate at which ^*C disappeared frm the medium was nt very much different (Table 1). In additin, DCMU, an inhibitr f phtsynthetic COg fixatin, inhibited markedly ^*C uptake in light (Fig. 2). These findings suggest strngly that Chlrella metablizes P^CJurea rapidly in either Hght r in darkness with the prductin f ^^COg which, in light, is cnverted t rganic cmpunds by phtsynthesis. It is ntewrthy that with Phaedactyluni in which ver 80% f the ^"^C taken up frm [^^C]urea can be recvered as p*c]urea within the cells, DCMU had very little effect n ^*C uptake althugh uptake was markedly light dependent (Rees and Syrett, 1979). Nevertheless, despite these reservatins in interpretatin f the results, it is clear that light stimulatin f urea uptake did ccur. Table 4 shws the quantities f P^C]- urea fund in the cells after separatin f radiactive cmpunds by thin-layer chrmatgraphy. It is nt nly clear that accumulatin f urea ccurred under all cnditins but that cnsiderably mre free p^c]urea was present in the cells in light than in darkness. The uptake f ^^C lw cncentratins (c. 2 fm) p^cjurea was inhibited cmpetitively by thiurea (Table ), This finding is cnsistent with the cnclusin f Syrett and Bekheet (1977) that urea and thiurea are transprted int Chlrella by a mechanism which has a higher affinity fr urea than fr thiurea. With Chlrella^ use f P'^CJurea t study this uptake mechanism directly is cmplicated by the rapid metablism f urea and the uncertainties this intrduces int interpretatin f the results. It may well be that it is preferable, in this case, t use the analgue, thiurea, t study the uptake system because this is metablized t nly a limited extent if at all. Against this, hwever, is the undubted lwer affinity f the uptake mechanism fr the analgue as cmpared t the, presumably, natural substrate, urea. REFERENCES BLISS, G. I. & JAMES, A. T. (1966). Fitting the rectangular hyperbla. Bimetrics, 22, 73. HAASS, D. & TANNER, W. (1974). Regulatin f hexse transprt in Chlrella vulgaris. Characteristics f inductin and turnver. Plajit Physilgy^ 3, 14. PATEMAN, J. A., KINGHORN, J. R., DUNN, E. & FORBES, E. (1973). Ammnium regulatin in Aspergilhn nidiilans. Jurnal f Bacterilgy J 114, 943. REES, T. A. V. & SYRETT, P. J. (1979). The uptake f urea by the diatm, Phaedactvhim. Nei Phythgist, 82, 169. SYRETT, P. J. & BEKHEET, I. A. (1977). The uptake f thiurea by Chlrella. Netv Phythgist, 79, 291. WILLIAMS, S. K. & HODSON, R. C. (1977). Transprt f urea at lw cncentratins in Chlamydmnas reinhardi. Jurnal f Bacterilgy, 130, 266.

* Manuscript received August 25, t Horticultural Research Section, CSIRO, Glen Osmond, South Australia.

* Manuscript received August 25, t Horticultural Research Section, CSIRO, Glen Osmond, South Australia. SUCROSE BREAKDOWN AND SYNTHESIS IN THE RIPENING GRAPE BERRY* By P. J. HARDyt Sucrse is the majr translcated sugar in the grapevine (Swansn and EIShishiny 1958), but in the berry glucse and fructse tgether