studies phosphorus nitrogen compounds marine … that the titration curve ... absence andpresence of...

Plant Physiol. (1989) 89, 1380-13870032-0889/89/89/1 380/08/$01 .00/0

Received for publication July 12, 1988and in revised form December 10, 1988

Phosphorus-31 and Nitrogen-14 NMR Studies of the Uptakeof Phosphorus and Nitrogen Compounds in the Marine

Macroalgae Ulva lactuca1

Peter Lundberg, Raer G. Weich, Paul Jensen, and Hans J. Vogel*Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4 (P.L., H.J.V.); and the

Department of Plant Physiology, University of Lund, S-221 00 Lund, Sweden (R.G.W., P.J.)

ABSTRACT

Cytoplasmic phosphomonoesters and inorganic phosphate, aswell as vacuolar inorganic phosphate and polyphosphates, gaverise to the major peaks in 31P nuclear magnetic resonance (NMR)spectra of the marine macroalgae Enteromorpha sp., Ceramiumsp., and UJva lactuca which were collected from the sea. Incontrast, NMR-visible polyphosphates were lacking in Pylaieliasp. and intracellular vacuolar phosphate seemed to act as themain phosphorus store in this organism. In laboratory experi-ments, polyphosphates decreased in growing U. lactuca whichwas cultivated in continuous light under phosphate-deficient con-ditions. In contrast, the same organism cultivated in seawaterwith added phosphate and ammonium, accumulated phosphatemainly in the form of polyphosphates. When nitrate was providedas the only nitrogen source, accumulation of polyphosphates inthe algae decreased with increasing extemal nitrate concentra-tion. From the chemical shift of the cytoplasmic Pi peak, thecytoplasmic pH of superfused preparations of Ulva was estimatedat 7.2. The vacuolar pH, determined from the chemical shifts ofthe vacuolar Pi and the terminal polyphosphate peaks, was be-tween 5.5 and 6.0. The intracellular nitrate and ammonium levelsin U. lactuca were determined by 14N NMR. Both nitrogen sourceswere taken up and stored intracellularly; however, the uptake ofammonium was much faster than that of nitrate.

High-resolution NMR has rapidly evolved to become a

useful method for the study of metabolism in plants (10, 18,20). In particular, phosphorus-3 1 NMR spectroscopy has beenutilized for the study of metabolism in different plants andplant tissues (for reviews, see 20, 24). Although, to our knowl-edge, no 31P NMR studies have been reported for macroalgaeto date, microalgae have received some attention. For exam-

ple, Elgavish and Elgavish (4) have investigated intracellularphosphate pools in intact Cosmarium sp. cells. Their 31P

NMR measurements revealed the presence of polyphosphates(linear polymers of phosphate); subsequent electron micros-copy studies indicated that these were primarily localized incytoplasmic granules (4). However, in 31P NMR studies of

These projects were supported by research grants provided by theNatural Science Research Councils of Sweden and Canada (to P. J.and H. J. V.). P. L. and H. J. V. are presently supported by a

studentship and a scholarship from the Alberta Heritage Foundationfor Medical Research.

Chlorellafusca (21), evidence was presented for the existenceof polyphosphates on the outside of the cytoplasmic mem-brane which were complexed with metal-ions. In addition tothese studies, other NMR work has focused on the effects ofphotosynthesis on the 3'P NMR spectra of a variety of mi-croalgae (5, 11, 12, 22) and blue-green algae (7, 16, 23). Ingeneral, two intracellular phosphate signals were observed.They originate from the cytoplasm and other intracellularorganelles such as the vacuoles and/or the chloroplasts. Theyhad light-dependent chemical shift values, indicating changesin the intracellular pH during photosynthesis. The aim of thepresent investigation was to compare phosphorus and nitro-gen uptake in different species of marine macroalgae and tostudy the dynamics ofthe various pools in Ulva lactuca grownin seawater with different phosphorus concentrations andnitrogen sources. Both phosphorus-3 1 and nitrogen- 14 NMRexperiments were performed.

MATERIALS AND METHODS

Plant Material and Cultivation Procedure

Four species of marine macroalgae were collected duringthe summer of 1985. Ulva lactuca and Enteromorpha sp. werefound in the mouth of the river Saxan south of Landskrona(Sweden) and Pylaiella sp. and Ceramium sp. were obtainedfrom the sea at Borstahusen, Landskrona. U. lactuca andEnteromorpha sp. grew free floating or were partly coveredby sediment. River water concentrations of ammonium, ni-trate, and phosphate were 7 to 10 ylM, 280 to 700 AM, and 1to 10AM, respectively (annual average, 1984). After collection,the plant material was directly used for 3IP NMR measure-ments. U. lactuca was kept in the laboratory in 120 L glass-tanks with aerated seawater which was collected over sandybottom at Borstahusen. The salinity of the seawater variedbetween 9 and 13 ppt.2 One-third of the seawater in the tankswas changed once a week. One week before the seawater wasused in the phosphorus and nitrogen uptake experiments itwas placed in aerated glass-tanks in the dark at 13C. Thephosphate concentration in the seawater varied between 1and 5 AM. U. lactuca grown in the tanks, was illuminated bytwo 40 W Gro-lux tubes. The photoperiod was 16 h and the

'Abbreviations: ppt, parts per thousand; Pi(c), cytoplasmic inor-ganic phosphate; Pi(v), vacuolar inorganic phosphate; PME, phos-phomonoesters; PP, polyphosphates.

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31P AND 14N NMR OF MARINE MACROALGAE

temperature was about 1 3°C. The algae were normally usedfor uptake experiments within 2 weeks of collection.For the uptake experiments discs (25 mm diameter) were

punched out of the algae and kept in natural seawater in thedark. After 12 to 15 h, they were transferred into glass-tanks(25 discs in 5 L) with aerated seawater supplemented withNaH2PO4 and NH4NO3, NaNO3 or NH4Cl at varying concen-trations. The 5 L tanks were maintained in growth chambersat 20 ± 1°C with a continuous illumination of about 20 Wm-2 (87 uE cm-2 s-') between 400 and 700 nm given by 400W HQI lamps. The growth of the algae was measured as theincrease of the surface area in percentage of the value at thestart of the experiment.

NMR Measurements

For each 31P NMR experiment with U. lactuca, 25 algae-discs with diameters between 25 and 40 mm-the disc sizebeing dependent on the amount of growth during the uptakeexperiment-were inserted into a plastic sample holder hav-ing a length of 35 mm and a 20 mm diameter. The cells werewashed free of medium prior to NMR analysis. An NMRprobe, equipped with a horizontal solenoidal coil with adiameter of 20 mm (27), was used in all experiments. Maxi-mum time efficiency was achieved using 600 pulses (55 ,spulse length) and a 1 s interval was used. Using these condi-tions, all resonances were recorded at better than 90% of theirfull intensity (29). The spectra were obtained using a home-built spectrometer equipped with a 6 tesla Oxford magnet(widebore). The 31P NMR resonance frequency was 103.2MHz. The spectral width was +10,000 Hz and 8K memorywas used. Each experiment consisted of 3,600 pulses, unlessotherwise indicated, resulting in a total measuring time persample of 1 h. Normally, 30 Hz was introduced throughdigital filtering. Chemical shifts were measured with respectto a phosphocreatine standard which was included in somesamples. All chemical shifts are reported with respect to 85%H3PO4 (0 ppm), giving upfield shifts a negative sign. Thehomebuilt solenoidal probe gave about 30 Hz linewidth foran aqueous phosphate solution, which was used for shimmingthe magnetic field. With algae in the probe, the linewidth ofthe extracellular phosphate increased to 100 Hz. The majorityof the NMR measurements were performed in the dark atroom temperature, without superfusion ofthe cells. However,the sample holder was furnished with an inlet and an outletallowing the tube to be perfused with oxygenated seawater inthe course of the experiment (27). In some experiments, thesample was illuminated in the NMR probe during the meas-urements, using a 100 W lamp and a mirror and a lens tofocus the light on the sample.A Nicolet NT 360 WB spectrometer with an 8.55 T super-

conducting Oxford magnet was used to acquire nitrogen-14spectra at 26.14 MHz. A 12 mm vertical, standard, broadbandprobe from Nicolet Instruments was used without perfusion.Pulses of 55 jAs (corresponding to 900) were used, and the timebetween pulses was 0.8 s to allow for complete relaxation.Sixteen K datapoints were used, and a 20 Hz linebroadeningwas introduced before transformation. The preacquisitiondelay was 70 ius, and the number of scans was 1000 for mostspectra unless otherwise indicated. Data were acquired at

room temperature (26°C). Ammonium was used as an inter-nal chemical shift reference assigned to 0 ppm, upfield shiftsare given a negative sign.

Analysis of the 31P NMR SpectraAssignments of resonances were made by comparing with

standards (27) and with spectra reported for microalgae andblue-green algae. For the purpose of determining the averagechain length of NMR-visible polyphosphates, the integratedareas for the PPI, PP2, and PP, resonances were measured.The average polyphosphate chain length was calculated foreach spectrum using the formula 2(PPI + PP2 + PPn)/PPI.The accuracy of this determination varies between ±1 and±3 and is mainly dependent on the signal to noise ratio ofthe PP1 resonance.The cytoplasmic and vacuolar pH were determined from

the chemical shifts measured for their respective Pi peaks,using a standard pH calibration curve (26, 27). The cyto-plasmic pH was further confirmed by measuring the shift ofthe PME resonance. Assuming that the titration curve forthese compounds is similar to that of glucose-6-phosphate(27), the pH estimated from this resonance agreed to within0.2 pH units with that measured from Pi(c). The vacuolar pHwas further determined from the titration behaviour of theterminal phosphate of the polyphosphates. The polyphos-phate PPn and PP2 resonances are very sensitive to the bindingof Mg2e (9). Hence, similar to the way in which the ATPshifts are often used to provide an estimate ofthe cytoplasmicMg2e level (27), the PP shifts can be used to determine theextent of Mg2e binding to this polymer. In addition, pHtitration curves were determined for a polyphosphate sample(200 mM KCI) with an average chain length of 18 in theabsence and presence of 1 equivalent of Mg2" (P:Mg = 1:1).The pKa determined for these two conditions were 6.6 and4.8 by fitting the data using nonlinear procedures (Simplex)to a simple titration curve (1 pKa). The magnesium-dependentshift in the pKa is normal and is also observed for ATP andits analogs (25). Both the PP, and PP2 resonances were foundto be sensitive to changes in pH (Appm 4.4 and 2.2, respec-tively, for Mg2+/polyphosphate mixture), whereas the PP,resonance was relatively insensitive (Appm = 0.1).3 Conse-quently, plots of the chemical shift difference between PP,nand PP1 could be used to determine the vacuolar pH. Tomake use of a plot which gives the difference in shift is anadvantage, as this difference is not dependent on any errorsin the calibration of the chemical shift scales. A comparisonbetween the vacuolar pH values determined from Pi(v) andpolyphosphate showed that they generally agreed to within0.3 pH units. Such differences may be caused by different saltconcentrations which are known to affect the shifts and pKavalues (20, 27).

Determination of Total PhosphorusAfter some of the 31P NMR experiments, the algae were

dried (48 h at 65C) and wet-combusted in a mixture of3The PP3 resonance can be resolved in standard solutions contain-

ing Mg2+-polyphosphates, but it could not be resolved in the in vivospectra-except for Ceramium (see Fig. 1C)- and it is thereforeincluded in PPn throughout this paper.

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Plant Physiol. Vol. 89, 1989

concentrated HNO3 and HC104 (2:1 v/v). After dilution ofthe samples, the total P concentrations in the algae-digestswere determined at 680 nm with a spectrophotometer (VarianTechtron, series 634) using the molybdenum blue method(13).

RESULTS

Phosphate Uptake and Storage

Figure 1 show a series of 31P NMR spectra recorded for themarine macroalgae directly after they were collected from thesea. In order to allow for a direct comparison of the spectra,approximately 3 g fresh weight (0.3 g dry weight) of thedifferent species was used for each measurement. The 31PNMR resonances for Enteromorpha sp. were assigned tophosphomonoesters (PME, presumably mainly sugar-phos-phates), cytoplasmic [Pi(c)] and vacuolar inorganic phosphate[Pi(v)] (these three resonances are unresolved), NAD(H) andpolyphosphates (PPI, PP2, and PPn) (Fig. IA). In U. lactuca(Fig. 1B), the resonances for the phosphomonoesters and thetwo intracellular Pi signals from two compartments were

APPn

P1(c)NPM /AATpP ATrPaATPP

B

D

0 -20 ppmFigure 1. 31P NMR spectra for Enteromorpha sp. (A), U. lactuca (B),Ceramium sp. (C), and Pylaiella sp. (D) sampled directly from theirnatural habitats.

resolved [Pi(c) and Pi(v)]. In addition, some resonances forATP were observed. The chemical shifts determined for ATPare consistent with the notion that it is more than 90%saturated with Mg2+.4 Although polyphosphates are detectedin both these algae and in Ceramium (Fig. IC), the intensityofthe PP, resonance compared to the PP2 and PP, resonanceswas much higher in the latter, thus indicating that the averagechain length was shorter than in Enteromorpha and Ulva (6,12, and 13, respectively). The chemical shift of the PP,resonance and the separation between the PP2 and PPn peaksindicate that the polyphosphates are saturated with Mg2+.Other 3'P NMR detected resonances for Ceramium (Fig. IC)were phosphomonoesters and some intracellular phosphate(Pi), which appeared to be mainly cytoplasmic. The poly-phosphates which dominated the spectra of the green and redalgae were absent- from the spectrum of the brown algaePylaiella (Fig. ID). Only resonances for phosphomonoestersand an intracellular phosphate signal which is mainly vacuolarwere detected. The total phosphorus concentrations in Enter-omorpha, Ulva, Ceramium, and Pylaiella were 89.5 ± 2.0,119 ± 1.5, 116.5 ± 2.3, and 120.0 ± 5.8 ,mol/g dry weight,respectively (values ±SE for three replicates), showing that thetotal phosphorus pools were comparable. Hence, the spectrain Figure 1 directly reflect differences in intracellular Pi stor-age. To study if other compounds appeared in the spectra ifthe algae were incubated in high Pi containing media, asample of U. lactuca was cultivated for 48 h in seawater with10 mm phosphate (Fig. 2). The same resonances as beforewere observed [PME, Pi(c), Pi(v), ATP, and polyphosphateswith an average chain length of7 phosphate groups]. Althoughthese algae had been grown for 48 h at a high phosphateconcentration, the spectrum was qualitatively similar to the31P NMR spectrum shown in Figure lB for U. lactuca col-lected from the natural habitat, except that the vacuolar Piresonance had increased somewhat, and that the average chainlength of the polyphosphate resonances was slightly shorter.

In 3'P NMR spectra recorded for U. lactuca after a 48 h

Pm(v)

I I I I

0 -20 ppm

Figure 2. 31P NMR spectra recorded for U. lactuca (10 h, 36,000scans) which had been grown for 48 h in aerated seawater suppliedwith 10 mm phosphate and 0.25 mm ammonium nitrate (final pH 6.0).The algae were grown in continuous light.

4Although it is stated here that Mg2" is the main cation that bindsto the polyphosphates, the possibility cannot be excluded that bindingof other cations may cause a similar shift. However, high Mg2"concentrations are often found in vacuoles (8), making this cationthe most likely candidate.

1 382 LUNDBERG ET AL.

PPn

Pi(,




incubation in seawater supplied with 10 to 100 ,uM phosphate,the same resonances for phosphomonoesters, cytoplasmic andvacuolar Pi, and polyphosphates were detected (data notshown). With external phosphate concentrations of 10, 20,50, 80, and 100 ,uM, the PP1 signal (which gives the numberof polyphosphate chains) gradually increased in intensity,whereas in addition at the higher levels, the average chainlength of the polyphosphates was somewhat longer ( 1 versus20, see Table 1). Also, the cytoplasmic Pi peak was somewhathigher at the higher Pi concentration. The total phosphorusconcentrations in the algae were also about the same (TableI). The surface area had increased by about 110% for the algaetreated at phosphate concentrations in the range 10 to 80 ,AMcompared with the surface area at the start of the experiment(data not shown). However, for U. lactuca grown at 0.1 mMphosphate, the surface area increased only by about 70%.The influence of various concentrations (0.1, 0.3, 0.5, 1.0,

3.0, and 5.0 mM) ofammonium and nitrate on the phosphoruspools in U. lactuca were studied during a 24 h incubation.Some of the results are shown in Figure 3. The 31P NMRresonances for intracellular phosphate and polyphosphates inthe algae at the start of the experiment (Fig. 3A) indicate lowconcentrations of the phosphorus compounds. After 24 hincubation in seawater supplied with 100 uM NaH2PO4 and100 ,uM NaNO3, a small accumulation of cytoplasmic phos-phate and polyphosphates was detected. However, a gradualdecrease of the polyphosphate resonances was readily appar-ent at increasing external nitrate concentrations, leading tothe total disappearance of the polyphosphate pool at 5.0 mmN03- (Fig. 3C). When ammonium was used as the solenitrogen source, intracellular Pi and, in particular, the poly-phosphate concentrations increased. Although the totalamount of phosphate incorporated in the polyphosphatesappeared quite similar at all NH4' concentrations tested, theaverage chain length of the polyphosphates varied graduallybetween 17 and 10 in going from 0.1 to 5.0 M NH4' (Fig. 3,D and E). If both NH4' and N03- were supplied at a concen-tration of 0.25 mM, polyphosphates did build up.Under phosphate deficient conditions (natural seawater

without any additions of phosphate and nitrogen), the poly-phosphate pool was reduced by 75% after 72 h, while the

Table I. Total Phosphate Concentrations, Relative Amount ofPolyphosphate and Average Polyphosphate Chain Length in U.lactucaThe experimental conditions were as follows: cells were grown at

varying phosphate concentrations for 24 h in continuous light. Noextra nitrogen sources were added. The values for the total phos-phorus represent the average and SE for 3 replicates

Extemal Total RelativeSizeof Average ChainPhosphate Phosphorus RelativeSie of Length of

Concentration Concentration PolyphosphatesMM mol/g drywt %

Control 65.5 ± 2.5 51 1110 72.4 ± 3.5 49 1020 64.8±3.4 63 1450 70.3±0.2 72 2080 66.8 ± 0.2 95 17100 107.9 ± 3.8 100 19

A

B

C

D

E

IPPn

P1(c)

II I II0 -20 ppm

Figure 3. 31P NMR spectra for U. lactuca at the start of the experi-ment (A) and after 24 h treatment in seawater supplied with 0.1 mMphosphate and 0.1 (B) and 5 mm (C) NaNO3 or 0.1 (D) and 5 mm (E)NH4CI. The algae were grown in continuous light.

average chain length was virtually unaltered (17 and 20,respectively). Furthermore, the intracellular phosphate poolremained at about the same level. The surface area increaseof U. lactuca in the course of this experiment was about140%. When U. lactuca was incubated for 6 d in the dark,the intracellular polyphosphate pool was unchanged and nogrowth occurred (data not shown).

Determination of pH

A series of experiments were performed to investigate theinfluence of cultivation in light and dark on the intensity andthe chemical shift of the intracellular Pi resonances. Spectrafor U. lactuca cultivated for 24 h in light or dark under

1 383




phosphate limiting and high phosphate (100 Mm) conditionsare shown in Figure 4. It is noteworthy that the cytoplasmicPi and the phosphomonoester resonances, as well as thepolyphosphate PP1 resonance, were higher in the samples thathad been incubated for 24 h in the light. This is paralleled byour observation that the cells growing in the light at both Piconcentrations had increased in surface area by 40% in 24 h,while no growth occurred in the cells that had been kept inthe dark (not shown). Apparently, cell growth and phosphateuptake only occur in cells that are actively photosynthesizing.The cytoplasmic and vacuolar pH in these samples is, how-ever, remarkably similar in these spectra as indicated by thevirtually indistinguishable chemical shift for the Pi(c) andPi(v) resonances, respectively. The chemical shifts measuredin these spectra are consistent with a cytoplasmic pH of 6.6± 0.2 and a vacuolar pH of 5.7 ± 0.2. These values weredetermined using the Pi(c) and the PME peak for the cyto-plasmic pH and the Pi(v) and terminal polyphosphate peakfor the vacuolar pH (see "Materials and Methods"). Similarvalues for the cytoplasmic and vacuolar pH were obtainedfrom the spectra for the various algae shown in Figures 1, 2,and 3 except that the vacuolar pH appeared to vary between5.5 and 6.0.The rather low cytoplasmic pH determined from the spectra

in Figures 1 and 4 suggested that the conditions under whichthe spectra were collected were not optimal to determine a

physiologically relevant pH value. Therefore, we decided toacquire spectra ofsome Ulva samples that were perfused withaerated seawater in the presence and absence of light. Figure5 shows the 31P NMR spectra recorded for U. lactuca whichwas perfused with aerated seawater in the NMR probe duringthe measurement. One spectrum was recorded while shininglight on the sample; the other one was obtained without

A

i!iiiiii

P,(v)

Pi(c) PPIPME

B

I I -5 0 5ppm

DI I I I

5 p

Figure 4. 31P NMR spectra for U. lactuca cultivated for 24 h inaerated seawater supplied with 0.25 mm ammonium nitrate withoutphosphate addition in light (A) and in darkness (B) or with 0.1 mMphosphate in light (C) and in darkness (D).

PPI

Pi (c)

PME

A

B

5 0 -5 -10 ppm

Figure 5. 3'P NMR spectra for U. lactuca during 1 h (3600 scans) ofperfusion with oxygenated seawater and light (A) or darkness (B).The sample was pre-grown in light at 200C in natural seawatersupplied with 100 Mm phosphate and 250 Mm NH4NO3.

illumination. Compared to Figure 4, where spectra are shownfor nonperfused samples, there is only a major change of thechemical shift of the Pi(c) resonance, which now indicates anintracellular pH of 7.2 ± 0.1. The resonances for the vacuolarPi and the terminal polyphosphate under these conditionsdoes not experience any shift, indicating that the vacuolar pHremains at 5.7 ± 0.1. Irradiation of the algae in the probe didnot give rise to any detectable changes in the cytoplasmic pH;however, a decrease of cytoplasmic Pi was noted (Fig. 5).Because ofthe high cell density which had to be used in theseexperiments to obtain a sufficient signal to noise ratio, it isunlikely that all cells were evenly irradiated, and thus, we didnot pursue this further. Results which were completely con-sistent with the patterns shown in Figures 4 and 5 wereobtained when cells grown at 500 ,M Pi were used (data notshown).

Uptake of Nitrogen Compounds

Only resonances for ammonium and nitrate were observedin the 14N NMR spectra recorded for U. lactuca (Fig. 6). Forthese experiments, the algae were cultivated for 36 h inseawater supplied with 0.5 mm phosphate and ammonium ornitrate at the following concentrations: 0.1, 0.3, 0.5, 1.0, and5.0 mm. The results obtained are listed in Table II. Followinggrowth in 0.1 mm external ammonium concentration, only asmall amount of ammonium was detected in the algae. Atammonium concentrations higher than 0.1 mm, a large peakfor intracellular ammonium was detected. Surprisingly, theintensity of this peak was the same over the whole range of





A

NO3

BNH+

--

. . I., . I I I I I I I I a . a I I,

300 200 100 0 ppm

Figure 6. 14N NMR spectra (1000 scans) for U. lactuca cultivated for36 h in aerated seawater supplied with 0.5 mm phosphate and 0.1mM (A) or 5.0 mm (B) KNO3. No ammonium was added. No otherresonances were detected if up to 5000 scans were collected.

Table II. Nitrate and Ammonium Levels as Determined by Nitrogen-14 NMR in U. lactucaSamples were incubated for 36 h in media containing different

nitrogen sources. The samples were kept under light and the uptakemedium (200C) was bubbled with air. The peak heights of the majorpeaks in the spectra (nitrate at 355 ppm and ammonium at 0 ppm)are expressed in relative units.

Conditions Concentration Nitrate AmmoniummM

KNO3 0.1 0.03 <0.010.3 0.07 <0.010.5 0.27 <0.011.0 0.44 <0.015.0 0.71 0.16

NH4CI 0.1 <0.01 0.030.3 0.02 0.860.5 0.03 0.891.0 0.05 0.925.0 0.05 1.00

ammonium concentrations that were tested (0.3-5.0 mM).Only a small amount of nitrate was seen in the spectra of thealgae grown on ammonium which barely exceeded the levelthat was observed in control samples. When nitrate wasprovided as the only nitrogen source, increasing intracellularnitrate resonances were detected in U. lactuca, which variedwith the external nitrate concentration (Table II). A smallamount of ammonium was detected in the sample incubatedat the highest nitrate concentration (Fig. 6), suggesting thatreduction of nitrate to NH4' is occurring in the cells and thatsome of the produced NH4 was stored and not directlyincorporated. When the time course of uptake was followedin an experiment where nitrate and ammonium ions werepresent simultaneously at 0.25 mm, the ammonium resonanceexceeded that of nitrate and it reached its maximum in 5 hand remained at the same level during a 30 h incubation. Theintracellular nitrate increased only very slightly in the courseof this experiment, which is consistent with its behavior inthe previous experiment. In separate experiments where cellsthat had stored N03- and/or NH4' were transferred into freshseawater, a decrease of the internal stores was noted duringgrowth (data not shown).

DISCUSSION

Our results concerning the phosphate uptake and storageclearly established that polyphosphates form the main intra-cellular store for Pi in the marine macroalga U. lactuca. ThePi which can only be taken up during active photosynthesisis accumulated and stored intracellularly mainly as polyphos-phate. The NMR-visible polyphosphate pool appears to belocalized in the vacuole which, as judged from the polyphos-phate chemical shifts, is also rich in Mg2e. Our data conclu-sively indicate that polyphosphates can be synthesized whilethe organisms are growing in seawater which is supplementedwith Pi (see Table I) and that it is utilized to support growthif the organism is transferred to phosphate-deficient media(Fig. 4). In yeast, microalgae and certain bacteria, polyphos-phates play an identical role as a phosphate store (2, 8, 14). Ithas been suggested that they can sometimes also act as anenergy store (8, 14), but our present data do not allow us todraw any conclusions on this. The average chain length ofthepolyphosphates that are NMR visible in U. lactuca, Cera-mium sp. and Enteromorpha sp. is relatively short (6-20).This is considerably less than the polyphosphates found inyeast (14) or certain microalgae (21), leaving the question iflong chain polyphosphates are perhaps present, but not de-tected. These compounds could become NMR invisible ifthey exist in an immobilized form (21, 24). Attempts to detectlonger polyphosphates in cell extracts have failed, becausehydrolysis of polyphosphates to Pi took place during theextraction (data not shown). However, we consider it unlikelythat such compounds are present since a close correlationbetweenNMR intensities and direct phosphate measurementswas observed (see also ref. 29).The brown algae Pylalliela appears to store Pi not as

polyphosphates, but as vacuolar Pi. This strategy would besimilar to that employed by some higher plants such asCatharanthus roseus (26) and Nicotiana tabacum (30). Never-theless, it is generally considered to be an exception to findhigh Pi levels in the vacuolar sap of freshwater, brackish, andmarine species (19).

In U. lactuca, a decrease in the level of polyphosphates isobserved at high external nitrate concentrations (Fig. 3). Thissuggests that under these conditions no Pi is taken up andintracellular polyphosphate stores are being utilized to main-tain growth. The concentrations of nitrate and ammoniumused in our experiments exceeded in general the naturalconcentrations, although U. lactuca occasionally can be ex-posed to high nitrate concentrations in river water. The sim-plest explanation of our observations is that nitrate inhibitsthe phosphate uptake directly, for example, by binding to andblocking the phosphate transporter. However, it is also pos-sible that when nitrate was present as the only nitrogen source,the energy required for the uptake and reduction of nitratein the chloroplast might have limited the polyphosphatesynthesis, which is an energy-requiring process (8). It is wellknown that nitrate can act as a hydrogen acceptor in light,thus offering an alternative to CO2 (15). Detailed kineticexperiments will be needed to determine the exact mode ofinhibition.

In U. lactuca, two Pi resonances could readily be distin-

1 385




guished in all spectra. In these experiments, we have assignedthese Pi resonances to cytoplasmic and vacuolar Pi. It is likelythat the resonance for chloroplast Pi-which could not beresolved here-overlaps with the cytoplasmic Pi peak. Theassignment of the two Pi resonances to cytoplasmic andvacuolar Pi was based on the pH determined from these twopeaks (7.2 and 5.5-6.0), which was similar to the pH valuesdetermined from the phosphomonoester and the terminalpolyphosphate peal (see "Materials and Methods"). The for-mer compound has been used in other studies as a pH-indicator (20, 29). However, the terminal polyphosphate peakhas, to our knowledge, not been used before as a pH indicator,although its parallel behavior with Pi(v) was noted previously(14). Because of the low pKa of the Mg2`_ polyphosphatecomplex and the large chemical shift difference of the PP,peak (Appm > 4.0), it provides a good pH probe for thevacuole because it allows one to accurately measure pH valuesbelow pH 5.8 all the way down to pH 4.0, provided that theterminal polyphosphate is clearly identifiable as it was in thespectra shown here. The Pi resonance is most sensitive to pHchanges between 7.8 and 5.8 and is therefore less suitable tomeasure in a similarly low pH range. It should be kept inmind though, while using the terminal polyphosphate reso-nance as a pH probe, that its saturation with Mg2" needs tobe determined in order to obtain a correct pH calibrationcurve, since its chemical shift and pKa are strongly dependenton its presence (see "Materials and Methods," also 9).Although the chemical shift of both Pi resonances was not

dependent on the growth in, or the presence of light, theposition of the cytoplasmic Pi peak was clearly dependent onperfusion with aerated seawater (Figs. 4 and 5),. Similarbehavior has been noted before for higher plant tissues, whereperfusion with oxygenated media also produced a 0.6 pH unitrise in the cytoplasmic pH (26, 30). Presumably, the circulat-ing medium removes products that otherwise would result inacidification of the cytoplasm. Apparently, the vacuolar pHwas not altered by these different treatments as both the Pi(v),and the terminal polyphosphate resonance were not affected.

Nitrogen-14 NMR has been used here for the detection ofintracellular nitrate and ammonium. Because of efficientquadrupolar relaxation, '4N NMR peaks are generally verybroad, but molecules with a high symmetry, such as nitrateand ammonium, are exceptions (1, 10). Nevertheless, ourspectra were somewhat disappointing because only N03- andNH4' resonances were detected: in '4N NMR studies of otherplant material resonances for amino acids have also beendetected (1; P Lundberg and HJ Vogel, unpublished obser-vations). Possibly Ulva has a higher intracellular viscositythan higher plants which could broaden these signals beyondthe detection limit. Be that as it may, we feel that 14N NMRis more useful than the more commonly used '5N NMR todetermine NH4' and N03--but not amino acids-accumu-lation in plants, as it can be readily quantitated in a shortmeasuring time because ofthe relatively short T,. Conversely,problems with hydrogen exchange, nuclear Overhauser ef-fects, and long relaxation times render difficult the quantita-tive detection ofNH4' and N03- in high resolution '5N NMRstudies of plant tissues. However, free amino acid pools can

be studied readily in plant material by '5N NMR (17, PLundberg, HJ Vogel, unpublished observations).When nitrate and ammonium were present in equal con-

centrations in the seawater, higher levels ofammonium thannitrate were detected in U. lactuca. This has also been ob-served for other algae. For example, D'Elia and De Boer (3)have found similar effects for nitrate and ammonium uptakein the red algae Neoagardhiella bailleyi and Gracilaria foli-ifera. Similarly, in Baltic macroalgae, the ammonium uptakeexceeded the nitrate uptake when the algae were treated atecologically representative concentrations (28). Our observa-tion that the total amount of NHE' which can be taken up inU. lactuca is limited is somewhat surprising in view of otherreports on Enteromorpha sp. where the ammonium uptakewas found to be a linear function of the concentration (6).The latter can be readily explained if one assumes that am-monium in the uncharged NH3 form can readily pass themembrane, thus giving rise to an equilibrium between intra-and extracellular NH4A. Given the fast uptake of NH4V, thiscould also play a role in U. lactuca. However, as it reaches anupper limit (Table II), it appears to be a more complicatedprocess than that in Enteromorpha. Nevertheless, the fact thatconsiderable amounts ofNO3- and NH4 accumulate, meansthat more of these nutrients are taken up (and stored) thanare immediately needed for incorporation into amino acidpools.

In conclusion, the data presented in here clearly demon-strate that U. lactuca and other marine macroalgae are capableof accumulating and storing nutrients (Pi, NH4+, NO3-),which may be growth-limiting under a variety of conditions.The organisms can sustain their growth by utilizing theseintracellular stores when they are faced with a situation ofdeficiency. It is therefore anticipated that the increased nu-trient load in the sea and ocean could eventually lead to awidespread increase in the growth of these seaweeds. In ad-dition to the above, our data establish that '4N NMR is auseful tool to follow the levels of NH4+ and NO3- in algae.Furthermore, we have shown that the chemical shift of theterminal polyphosphate resonance can provide a useful probefor determining the vacuolar pH.

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