Potassium Fluxes in Chlamydomonas reinhardfii - NCBI

Plant Physiol. (1 995) 108: 1537-1 545

Potassium Fluxes in Chlamydomonas reinhardfii' II. Compartmental Analysis Bhupinder Malhotra and Anthony D. M. Class* Department

of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 124 large chloroplast as an additional compartment. [K+ls in chloroplasts have been reported to be quite high (Larkum, 1968; Robinson and Downton, 1984), but the functions of this element within this compartment are not well understood. Furthermore, the extent to which intact chloroplasts are able to maintain a constant [K+] independent of changes in the externa1 medium is unknown. If the [K+l of the chloroplast is maintained at a constant level, it would Seem essential that Some form Of transport regu1ation OCCurS at the inner boundary of the chloroplast. K+ is believed to play an important role in photosynthesis within the chloroplast at a concentration of approximately 50 to 100 mM (Kaiser et al., 1980; Demmig and ~ i ~1983;~rier land ~Berkowitz, ~ , 1987).co, fixation was strongly inhibited in intact ch~oroplastsin a medium containing only low [K+] (Kaiser et al., 1980). Within this organelle, K+ may be involved in enzyme activation and protein synthesis, since it is within the cytoplasm (Leigh and Wyn Jones, 1984). Kt efflux analysis has been undertaken using alga1 cells (Barber, 1968; Wagner, 1974) and higher plants (Pfruner and Bentrup, 1978; Memon et al., 1985a, 198513) to determine the number of subcellular compartments and the subcellular distribution of K+. Compartmental analysis of Cklorella cells showed the presence of two cellular compartments (cell wall and cytoplasm), whereas that of Mougeotia showed the presence of three compartments (cell wall, cytoplasm, and vacuole). In Mougeotia most of the cytoplascompartment is occupied by the large c h ~ o r o p ~ a s t ~ Nevertheless, compartmental analysis showed only three compartments. Values for [K+]s and membrane potentials across the chloroplast envelope have been determined in isolated chloroplasts (Bulychev et al., 1972; Robinson and Downton, 1984). K+ has been suggested to move acroSSthe chloro-

42K+ and "'Rb+ were used to determine the subcellular distribution of potassium in Chlamydomonas reinhardtii by compartmental analysis. In both wild type and a mutant strain, three distinct compartments (referred to as 1, 11, m d 111) were apparent. Using 4zK+, we found that these had half-lives for K+ exchange of 1.07 min, 12.8 min, and 2.9 h, respectively, in wild-type cells and 0.93 min, 14.7 min, and 9.8 h, respectively, for the mutants. Half-lives were not different when 8 6 ~ b +was used to trace K+. Compartments I and I1 probably correspond to the cell wall and cytoplasm, respectively. Based on the lack of a large central vacuole in Chlamydomonas, the effect of a dark pretreatment on the kinetic properties of compartment 111 and the similarity between the [K+l of compartment III and that of isolated chloroplasts, this slowly exchanging compartment was identified as the chloroplast. Growth of wild-type cells at 1O 0 PM (instead of 10 mM K+) caused no change of cytoplasmic [K+] but reduced chloroplast [K+l very substantially. l h e mutants failed to grow at 100 p~ K+.

K t is present in a11 compartments of plant cells but its distribution and roles have not been well characterized. Within the cytoplasm and vacuole its concentrations have been determined by the use of severa1 different techniques, including comPartmental analYsist N M R ~and electrondisPersive x-raY microanalYsis (Pitmany 1963; HarveY et al.f 1981; Pitman et al., 1981; Rona et al., 1982).The cytoplasmic functions Of K+ aPPear not to be reP1aceable by Other and plants appear to maintain rK+]c at a constant level. This is achieved by increasing their capacity for K+ influx at the and by mobilizing K+ from vacuolar reserves (Glass and Fernando, 1992). Within the cytoplasm the primary function of K+ is thought t o be in Protein 'Ynthesis (Leigh and WYn TOnes, 1984). Leigh and Wyn Jones in short proposed that' when K+ s'PPIY~ as the tK+] in the tissue as a whole the concentration in the cytoplasm is initially maintained, until that of the vacuole decreases to a lower limit of approximately 20 mM. During this decrease of vacuolar [K+], turgor within the vacuole is maintained by accumulating alternative solutes. However, certain microalgae, such as Ch'amydomonas' lack a prominent vacuole but have a single'

Abbreviations: ApH+, electrochemical potential difference for H f ; ApK+, electrochemical potential difference for Kt;A$: membrane electrical potential difference; HATS, high-affinity transport system; J, net flux; 4, unidirectional flux; subscript c, cytoplasm; subscript cchl, flux from cytoplasm to chloroplast; subscript chl, chloroplast; subscript chlc, flux from chloroplast to cytoplasm; subscript co, flux from cytoplasm to outside; subscript o, outside; subscript oc, flux from outside to cytoplasm; subscript Q, quantity of Kf in that compartment; subscript T, total quantity of K C in the cell; TAPM, Tris acetate phosphate modified medium; t r k l , mutants defective in K+ transport.

' This research was supported by the Potash and Phosphate Institute of Canada and the Natural Sciences and Research Council of Canada. * Corresponding author; e-mail ag1assQunixg.ubc.ca; fax 1-604-822-6089.

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Malhotra and Glass

plast envelope by passive H'/K+ exchange (Maury et al., 1981) or through K' channels (Wu and Berkowitz, 1991). Yet, no single hypothesis is adequate to explain the mechanism(s) and regulation of K' movement across this barrier. Clearly, more work needs to be done to adequately describe the transport of K t to and from the chloroplast. In the experiments described in this communication, [K+]s were determined in the major subcompartments of Chlamydomonas cells by use of compartmental analysis. Both wild-type strains of Chlamydomonas and a putative K+ transport mutant, f r k l (Polley and Doctor, 1985), were used to probe the basis of defects in the mutant and to explore the mechanism(s) of K+ uptake in normal cells. The trkl strains have a specific requirement for high [K+],. They fail to grow at low ambient [K+] and they fail to respond to K+ deprivation by the normal enhancement (derepression) of K t influx. A model is proposed that attempts to explain the mechanisms of K' transport in wild-type cells and in mutant strains under conditions of adequate and insufficient K+ supply. MATERIALS A N D METHODS Strains and Culture Conditions

Wild-type Chlamydomonas reinhardtii Dang. (CC125') and strains of trkl were used for a11 experiments. Cells were grown in 6-L culture flasks in TAPM at pH 7.0 (Polley and Doctor, 1985) and synchronized by using a 16-h light/ 8-h dark regimen. Irradiance was maintained at 200 pmol m-z s-l at 25°C within a controlled environment room. Cultures were grown mixotropically and aerated with filtered air throughout the study period. Cells were used for efflux experiments between the mid-log and late-log phases of growth at a cell density of 1.5 to 2.5 X 106 cells mL-'. Cell growth was determined by measuring cell number (Coulter Counter model TA 11; Coulter, Hialeah, FL), Chl content (Harris, 1988), or fluorescence (Fluorometer, model 10-000R; Turner Designs Inc., Sunnyvale, CA) during the growth period. K+(Rb+) lnflux and Efflux in CC125+ and frkl Cells of Chlamydomonas

K'(Rbf) influx was obtained by direct determinations as described in detail by Malhotra and Glass (1995) and by calculation from efflux analysis as described by Rygiewicz et al. (1984). Because trkl cells are unable to grow at low ambient [K+], fluxes associated with the HATS were obtained in both strains by growing cells in 10 mM [K+l, and resuspending them in 0.1 mM K' for influx measurements. Efflux analysis was performed under two conditions: (a) both types of cells were grown and maintained in 10 mM K+ throughout the experiment, i.e. grown, loaded, and eluted at 10 mM K+, and (b) wild-type cells were grown to log phase at an externa1 [Kf] of 0.1 mM and resuspended in fresh 0.1 mM K' medium before the tracer was added for loading the cells. The frkl mutants failed to grow at 0.1 mM [K'],; hence, only wild-type cells were used at low [K+],. In most of the experiments, 86Rb+was used for the efflux analysis. To check these data in light of the reported dis-

Plant Physiol. Vol. 108, 1995

crimination against Rbt (Polley and Doctor, 1985), one set of efflux experiments was undertaken using 42K+ with both wild-type and f r k l mutants, grown at 10 mM [Kt],. The cells were allowed to accumulate 86Rbt or 42K' from TAPM containing KCI(O.1 or 10 mM, pH 7.0) and "Rb' or 42K+ (2 pCi mL-' final volume; 0.2 pCi pmol-') for about 24 h. After the cells were loaded for 24 h, a small volume of approximately 10 mL of cell suspension, containing approximately 30 to 40 X 106 cells, was layered onto a Millipore filter (pore size 1.2 pm) in the efflux apparatus (Nalgene [Rochester, NY] filtering funnel). Millipore filters were used for a11 experiments because they bound the least amount of radioactivity compared to other filters tested, such as glass fiber and Whatman No. 1 filter paper. Radiolabeled cells were eluted with TAPM, supplied from a reservoir through a fine silicone tube having a screw valve to control the flow rate of medium into the Nalgene funnel. The flow rate through the Millipore filter was maintained at 2 mL min-' throughout the experiment, except toward the end of the experiment when blocking of the Millipore filter sometimes reduced the flow rate. Vials used to collect the filtrate (eluate) were placed in a bell jar, which was connected to a vacuum pump with a two-way valve to maintain a reproducible suction for replicate experiments. Low suction was applied to the Nalgene funnel to wash the cells briefly during the first 30 s of elution with unlabeled TAPM (pH 7.0) to remove the radiolabeled medium in which cells were suspended. The cells were then continuously eluted with wash solution supplied from the reservoir. This contained 0.1 mM K' (in the case of cells loaded at 0.1 mM K+) or 10 mM K' (in the case of cells loaded at 10 mM K+). Throughout the remainder of the experiment, the filtrate was removed by gravity filtration. Experiments typically lasted for 6 h, during which the rate of delivery of the TAPM was adjusted so that a constant leve1 of 1 mm of medium covered the cells at a11 times. The delay in the arrival of radioactivity in vials because of the volume of solution in the funnel was calculated as follows: surface area of funnel = 1.13cm2; depth of liquid at a11 times = 0.1 cm; volume of liquid = 0.1 mL; flow rate = 2 mL min-'; hence, residence time in the funnel of the 0.1 mL of eluate = 3 s. The filtrate from this elution was collected in glass vials during the earlier intervals. At later times, when samples were taken after 30- to 60-min intervals, eluates were collected in flasks and subsampled for radioactivity determinations. Efflux in the Dark using 86Rb+ and KCI in Wild-Type Cells

Cells grown in 10 mM K+ were transferred to a 250-mL flask and placed in darkness. The presumption of this experiment was that the characteristics of ion fluxes to and from the chloroplast would be altered in dark-grown cells. Cells grown in the dark swam as rapidly as cells grown in the light for up to 5 d and appeared to have the same shape; by 10 d cells had become immobile. Thus, a dark period of 4 d was selected as preconditioning prior to conducting the efflux experiment. These cells were incubated in 10 mM K' in the dark for 4 d and then 86Rbt (0.2 pCi) was added to the flask. The cells were allowed to

K+ Fluxes in Chlamydomonas

grow in the dark for another 24 h to label the cells before elution for 7 h with a washing solution containing 10 mM K+. The [Kt] of 10 mM was chosen for purposes of comparison with the data of other experiments that had been performed at this [Kt]. The efflux experiments were conducted in complete darkness using the procedure described above, except that a green safelight was used while changing vials during collection of the eluates. Calculations of Fluxes and Subcellular Distribution of K+ in Wild-Type and Mutant Cells

Unidirectional fluxes, net fluxes, and subcellular distributions of K+ in wild-type and mutant cells were calculated using standard methods (Walker and Pitman, 1976). An automated computer methodology for calculations and graphic analysis (Rygiewicz et al., 1984) was modified to provide estimates of fluxes between subcompartments. [K+]s were estimated by assuming the size of the cytoplasm to be 40% of the total cell volume (Harris, 1988).Cell dimensions were measured by light microscopy and Coulter Counter (model TA 11); cell numbers were determined by hemocytometry and Coulter Counter. Cell volume was approximated by treating the cell as a sphere. Precise determinations for use in subsequent calculations were based on incubation of cells in 3H,0 and ['4C]inulin (Malhotra and Glass, 1995). These gave a value of 200 t 13 pm3 cell-'. The volume of isolated chloroplasts was taken as 40% of cell volume (Harris, 1988).

[K+]s of lntact Chloroplasts Intact chloroplasts were isolated from wall-less mutants of Cklamydomonas, strain CC-400 (cw 15) according to the methods of Mason et al. (1991) and Price and Reardon (1982).Cells, grown under the same conditions of light and temperature as described above, were harvested in the late-log phase by centrifugation at 2000 rpm for 2 min. The pellets were washed in 20 mM Hepes-KOH (pH 7.5) buffer and resuspended in ice-cold breaking buffer (300 mM sorbitol, 50 mM Hepes-KOH, pH 7.5, 2 mM Na-EDTA, 1 mM MgCl,, 1%BSA) at a cell density of 20 X 107 cells mL-'. These cells (10 mL) were broken immediately by one passage through a 27-gauge stainless steel needle at a flow rate of 0.5 mL s-l (Mason et al., 1991). The broken cells were then centrifuged in a Sorvall (New Town, CT) HB-4 rotor for 2 min at 2000 rpm to pellet whole cells and intact chloroplasts. The pellet was resuspended in 2 mL of breaking buffer and layered on top of discontinuous Perco11 gradients in 30-mL Corex (Corning, NY) tubes (Price and Reardon, 1982).The gradients were centrifuged in a Sorvall HB-4 rotor at 5000 rpm for 15 min. The 45 to 65% interface, containing intact chloroplasts, was collected and diluted 4-fold with breaking buffer. Intact chloroplasts were concentrated by centrifugation at 2000 rpm for 1 min and resuspended in 0.5 mL 50 mM Hepes-KOH buffer (0.3 M sorbitol, pH 8.0). O, evolution from these intact chloroplasts was measured in a temperature-controlled chamber using an O, electrode and an O, monitor (Hansatech, Norfolk, UK). Intactness of the isolated chloroplasts was determined by

1539

using the ferricyanide exclusion test (Lilley et al., 1975). Rates of O, evolution were monitored in the intact chloroplasts (control) and in the chloroplasts subjected to osmotic shock (buffer with no sorbitol) so that their envelopes would rupture to release the thylakoids in the presence of 1 mM ferricyanide (Mason et al., 1991). Comparisons of the rates of O, evolution observed before and after osmotic shock were used as an intactness assay. Photon flux density was 400 pmol mp2 spl. This irradiance was used to compare our results with those of previous workers (Mason et al., 1991). [K+]s were determined in these intact chloroplasts for comparison with the values obtained from compartmental analysis. Isolated chloroplasts were disrupted in 1 M HC10, and the Kt thus released was measured by flame photometry (model 443; Instrumentation Laboratory, Lexington, MA). Values of [K+] estimated by this method were significantly lower than the estimates of compartment I11 (considered to be the chloroplast) derived from compartmental analysis. However, it has been documented that [K+l of the chloroplasts declines during isolation because of its mobility across the chloroplast envelope (Robinson and Downton, 1984). To minimize this loss, K+ was precipitated in intact cells by treatment with sodium cobaltinitrite before isolation. Sodium cobaltinitrite stock solution was filtered and added to cell culture (to generate a final concentration of 2.5 g L-'), and the culture was incubated in a 3-L flask on a shaker for 5 min. This concentration of sodium cobaltinitrite caused a11 of the cells to stop moving because of precipitation of K+. The cells were centrifuged at 2000 rpm for 2 min, washed in 20 mM Hepes-KOH (pH 7.5) buffer to remove extra salt, and resuspended in the breaking buffer. Intact chloroplasts were then isolated from these cells as described above. Chl contents of the whole cells and the isolated chloroplasts were estimated according to the method of Harris (1988). RESULTS Compartmental Analysis of Wild-Type and trkl Mutant Cells

Compartmental analysis performed on wild-type cells grown at 0.1 or 10 mM Kt and labeled with 86Rb+under the same conditions of K+ supply (steady-state conditions) revealed the presence of three compartments (I, 11, and 111) with half-lives for exchange of 0.7 +- 0.06 min, 15 2 1.12 min, and 3.3 t 0.5 h, respectively, for 100 p~ grown cells and 0.56 ? 0.03 min, 12.8 ? 1.61 min, and 3.5 t 0.83 h, respectively, for 10 mM K+ grown cells (Fig. 1; Table I). These compartments probably correspond to the cell wall, cytoplasm, and chloroplast, respectively, and will be referred to as such in subsequent discussions. Compartmental analysis undertaken with trkl cells under steady-state conditions of K+ supply (10 mM [K+l,) also showed three compartments, with half-lives of 1.1 t 0.5 min, 16.7 2 1.73 min, and 8.6 h ? 1.59, respectively (Fig. 2; Table I). According to Students' t test analysis, the halflives of compartment I11 in wild-type and trkl cells were significantly different, but those of compartment I and

Malhotra and Class

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Plant Physiol.

Vol. 108, 1995

3. O f -I

2.

w

o

7

2.

O

+-ta

H

2.

4

H

I:

f .

f'

,

2. 5 --

o c3

J

I

2. 6

-4-

'+.

-

2. 4 -

O

'3. 2.

TIME (h) Figure 1. Linear regression of semi-log plot of 86Rh+elution data from wild-type Chlamydomonas cells grown at 1 O m M K t . The main plot shows linear regression of the final straight line portion of semi-log data to determine the parameters of compartment 111; inset shows the linear regression of log cpm min-' remaining in cells after suhtraction of compartment 111.

compartment I1 were not significantly different at P = 0.05. Efflux analyses undertaken using K'(86Rb') were repeated four times, and the data given for half-lives, fluxes, and compartmental parameters are the means of these determinations. Data derived from the single experiment in which K'(*'K') was used for both wild-type and trkl mutants gave excellent correspondence with values obtained using Kf(86Rb'). Comparisons of half-lives using 42Kf and 86Rbt are shown in Table I. However, because of the extensive replication of 86Rb' data and consequent greater confidence in the derived values, these are shown throughout the remainder of the paper.

tant trkl strains grown at 10 mM [K+],; their values are recorded in Table 11. It is evident that the [K'] of compartment 111 ([K+],,,) of wild-type cells was almost double (223 2 31.4 mM) that of the corresponding compartment of trkl cells (127 +- 14.2 mM), even when both strains were grown at 10 mM K+. By contrast, the [K'ls of compartment I1 ([K'],) were similar for the two strains (wild type, 77.6 -t 13.8; trkl, 71.2 5 4.9). The total K' contents of compartments I1 and I11 ([Kl, plus [K],,,) were calculated to be 24.1 % 1.4 X 10-6 nmol cell-' in wild-type cells and 16.03 X 10-6 nmol cell-' in trkZ cells. These differences were confirmed by independent K' analyses using flame photometry, which indicated that the K' contents of wild-type and mutant cells were 24 X 1OP6 and 15.4 X 1Op6nmol cell -I, respectively . K+(86Rb') influxes, derived from the same compartmental analysis, revealed only slight differences between wild-

Fluxes and Subcellular Distribution of K+ in Wild-Type and frkl Cells

The subcellular distributions and fluxes of K+ were estimated by compartmental analysis in wild-type and mu-

Table I. Compartmental analysis of K+a6Rb+ or 42Kf) efflux at 10 mM [KcI, in wild-type IWT) and mutant (trkl) strains of C. reinhardtii Results are means 2 SE of four separate experiments. Half-Life Compartment I I

Compartment I

Wr

trkl

86Rh*

0.56 2 0.03 1.07 t 0.07

1.1 2 0.5 0.93 2 0.2

1 2 . 8 ? 1.6 12.8 2 0.90

rrkl

WT

h

min

min 4 2 ~ +

Compartment 111 trkl

WT

16.7 2 1.7 14.7 ? 1.5

3.5 2 0.8 2.9 2 0.62

8.6 ? 1.6 9.8 2 1.9

"I 4.

Kt Fluxes in Chlamydomonas

1541

o

3.9

k

+ 'i

+.

,

c.

3.8

2.50

"

'

1

1

20

'

I

1

40

1

1

t

eo

I

J

T I M E (min)

3. 7

3.

3.6

TIME (h) Figure 2. Linear regression of semi-log plot of 86Rb+ elution data from trkl Chlamydomonas cells grown at 10 mM K+. The main plot shows linear regression of the final straight line portion of semi-log data to determine the parameters of compartment 111; inset shows the linear regression of log cpm min-' remaining in cells after subtraction of compartment 111.

type and trkl cells that were not significantly different at P = 0.05. Likewise, the values of K+f6Rbt) efflux were not significantly different. Net uptake of K+(86Rb+)by trkl cells was 31% lower than by wild-type cells. When K-c(86Rb+)influx was measured directly in 0.1 miv [K+J, in the two strains after growth at i 0 mM [K+],, influx values were again lower in trkl cells but not significantly different (P = 0.05), with values of 2.78 2 0.07 and 2.12 ? 0.51 X 10-6 nmol h-'cell -',respectively.

Table II. Unidirectional Kf fluxes (TO-' nmol h- cell- ' I [K+I (in m ~ i and , Q, and Qch, (in nmol 106 cell-') calculated from compartmental analysis at 10 mM lKfl, in wild-type and trkl cells of Chlamydomonas Wild Type

trkl

11.24 i 3.96 10.38 ? 3.85 0.865 5 0.1 1 7.77 i 2.8 7.01 -C 2.68 0.81 2 0.15 6.25 i 1.10 17.9 ? 2.53 24.15 i 1.42 77.6 i 13.8 223.5 i 31.4

9.1 ? 2.76 8.55 i 2.7 0.6 i 0.05 0.6 i 0.05 1.O6 2 0.21 0.42 -C 0.04 5.72 i 0.42 10.44 i 1.34 16.03 i 1.64 71.2 i 4.96 127 2 14.2

Fluxes to compartment 111, by contrast, were very different in the two strains. for wild-type and trkl cells were 7.77 2 0.28 and 1.51 ? 0.22 X 10-6 nmol h-' cell-', respectively. The J,, of K+("Rb+) to this compartment in wild-type cells was almost double that of the equivalent flux in trkl cells. When wild-type cells were grown, loaded, and eluted in solutions containing 0.1 miv [K+l,, estimates of the [K+l,,, were found to be significantly lower (64 mM) than in cells maintained at 10 miv [K+], (223 mM). The lowered [K+],, however, produced virtually no change in the estimated [K*l, (77.6 and 65.1 mM, respectively, for 10 and 0.1 mM grown cells). Data for compartment I1 ([K],) and compartment I11 ([Klchl) for trkl cells maintained in 0.1 mM K+ were not obtained because the trkl cells failed to grow at this concentration of [K+],.

&,,

Efflux in the Dark in Wild-Type Cells

When the efflux experiments were conducted in the dark, the same three compartments were evident as in light-grown cells. The half-lives of compartment I (1.15 min) and compartment I1 (13.8 min) were rather similar to those determined in the light (Table 111). Moreover, the fluxes between these two compartments also remained unchanged (Table IV). The half-life of exchange of compartment 111, however, was 2.4 ? 0.3 h in darkness com-

Malhotra and Glass

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Table 111. Compartmental analysis of K'(86Rb') and in dark Results are means

If; SE


efflux at 10 mM [K+lo in C. reinhardtii in light

of three separate experiments. Half-Life

Compartment I Light

Compartment li Dark

Light

min

0.6 2 0.10

Light

min

1.15 t 0.6

14 t 0.05

pared to 4.15 ? 0.6 h in the light. K+("Rb+) fluxes to compartment I11 also diminished in the dark and resulted in a lower J to this compartment in the dark (0.41 nmol h-' 106 cell-') than in the light (0.81 X 10-6 nmol h-' cell-'). Total K+ content in the dark-grown cells was lower (15.93 X 1OP6 nmol h-' cell-') than in light-grown cells (24.15 X 1OP6 nmol h-' cell-'1. In dark-grown cells, IKfls in compartment I1 remained unchanged compared to the lightgrown cells, but the [K'] of compartment 111 ([KI,,,) decreased to a value of 124 2 21.2 mM (compared to 223 ? 31.4 mM in the light-grown cells; Table IV). [K+l of lsolated Chloroplasts

That isolated chloroplasts retained their ability to photosynthesize was shown by their capacity for O, liberation. The rate of O, evolution was 22 pmol O, mg-' Chl h-' in intact chloroplasts. According to the ferricyanide exclusion test, 93% of the chloroplasts obtained were intact. The isolated chloroplasts were also examined by light microscopy (X600 magnification) and were found to retain their cup shape. The yield of chloroplasts was 12%. This is in agreement with earlier published results of Mason et al. (1991), who also used the syringe method to isolate chloroplasts. In an attempt to prevent K+ loss from chloroplasts during isolation, Kt was precipitated in whole cells by use of sodium cobaltinitrite prior to disruption of the cells and chloroplast fractionation. The yield of intact chloroplasts by this method was lower than for the standard isolation method, but estimates of the [Kf] were much higher (165 -C Table IV. Unidirectional K'(''R6') fluxes (4 10-' nmol cell-'), [K'I (in mM), and Q, and Qch,(10-6 nmol cell-') calculated from compartmental analysis in wild-type cells of Chlamydomonas in the light and the dark K+ Fluxes

Compartment 111

Dark

Light

Dark

11.24 2 3.96 10.38 t 3.85 0.865 ? 0.1 1 7.77 ? 2.8 7.01 rt 2.68 0.81 t 0.15 6.25 2 1.10 17.9 -C 2.53 24.1 5 ? 1.42 77.6 t 13.8 223.5 rt 31.4

13.5 2 3.4 12.9 -C 3.53 0.58 t 0.06 2.99 t 0.31 2.57 t 0.24 0.41 t 0.06 6.03 -C 0.19 9.95 ? 1.67 15.93 2 1.44 75.38 2 2.46 124 2 21.22

Dark

h

13.8

?

0.7

4.15 2 0.65

2.4 rt 0.3

21 compared to 53 ? 3.09 mM). This value compared favorably with the estimates obtained by compartmental analysis (223 2 31.4 mM).

A p K + across the Plasma Membrane and Chloroplast Envelope To evaluate the electrochemical potential gradients for

K+ between the major compartments of wild-type and trkl cells, A$ values were taken from our previous paper (Malhotra and Glass, 1995) together with values for [K+] from the present paper. For wild-type cells two values of [K+],, 0.1 and 10 mM, were used. For trkl cells, it was possible only to use 10 mM because this strain failed to grow at 0.1 mM. Under these conditions, [K+I, was estimated to be 65.1 and 77.6 mM, respectively, for wild-type cells grown at 0.1 and 10 mM [K+], whereas corresponding A$ values were -136 and -129.5 mV, respectively. A p K + had a positive value at low [Ktl, in the range of O to 200 p ~ whereas , from 0.2 to 200 mM, the A p K + assumed a negative value (Malhotra and Glass, 1995). At 0.1 mM [K+],, A p K + across the plasma membrane was 2.7 kJ mol-', whereas at 10 mM [K'],, A p K + was -7.5 kJ mol-' in wild-type cells. Assuming that A$ in trkl cells was the same as in wild-type cells, we estimated A p K + for trkl cells (Table V). Since [K+],s were similar in the two strains, this assumption appears to be valid. The A p K + c c h , were ~ also calculated based on the assumption that there was no electrical potential difference between chloroplast and cytoplasm. Across the chloroplast envelope, A p K + was calculated to be 2.6 kJ mol-' in wildtype cells and 1.4 kJ mol-' in trkl cells when [K'], was 10 mM (Table V). Ap,+ across the chloroplast membrane, estimated on the basis of a ApH value of 1.5 units and zero A$ was calculated to be -8.4 kJ mol-'. DlSCUSSlON

Three compartments (I, 11, and 111) detected by compartmental analysis in C. reinhardtii probably correspond to the cell wall, cytoplasm, and chloroplast, respectively. Halflives of the first and the second compartments were very close to values that have been reported for the cell wall and the cytoplasm in other organisms (Pfriiner and Bentrup, 1978).It is also evident that the half-lives of the first and the second compartments had similar values in wild type and trkl. By contrast, the half-life of exchange for the third compartment of trkl cells was significantly longer than that in wild-type cells. The half-life of the third compartment

K+ Fluxes in Chlamydomonas

Table V. (kJ mo/-') for K+ between cytoplasm and external media ( A ~ ~ + oand c ) cytoplasm and chloroplast (ApK+oc)at 1 O and O. 1 mM [K+], were calculated for both wild-type (WT)and trkl cells of Chalmvdomonas 10 mM [K+],

lK+l

AkK+oc ANLLy+cchl

0.1 mM lK+],

WT

trkl

-7.5

-7.7 1.4

2.6

WT

2.7 -0.05

trkl

N Da ND

"ND, Not determined.

was severa1 hours, which is similar to the vacuolar half-life of exchange in vacuolated cells (Pfriiner and Bentrup, 1978). C. reinkardtii does not contain a large vacuole. However, it does contain a huge chloroplast, which occupies about 40 to 50% of the cell volume (Harris, 1988).Thus, the most likely candidate for compartment 111 was the chloroplast. This hypothesis was tested in two ways: (a) by comparing the [K+] of isolated chloroplasts to the [K+] of compartment 111 estimated by compartmental analysis and (b) by estimating the [K+] of compartment I11 in darkness (also by compartmental analysis). The rationale for this approach was that this treatment was anticipated to affect chloroplast development and hence the characteristics of K' distribution in this organelle. The [Kfl of isolated chloroplasts, immobilized by sodium cobaltinitrite, was found to be 165 5 21 mM, which is reasonably close to the [Kf] obtained for compartment I11 from compartmental analysis (223 2 31.4 mM) and is consistent with the hypothesis that the third compartment is the chloroplast. These values are in the range of those reported in the literature for the [KC] of isolated chloroplasts. For example, Robinson and Downton (1984) reported values that were up to 200 mM for chloroplasts from spinach, sugar beet, and pea leaves. Half-lives of K+ exchange for compartment I11 and fluxes of Kt to and from this compartment were significantly lower in dark-grown than in light-grown cells. It was observed that when Chlamydomonas cells were grown in the dark, chloroplast structure was severely affected. Thus, our observation, that growth of Cklamydomonas cells in the dark altered the characteristics of compartment I11 but not of the other compartments is consistent with the hypothesis that the third compartment is, indeed, the chloroplast. In the following discussion, therefore, compartments I, 11, and I11 will be referred to as the cell wall, cytoplasm, and the chloroplast, respectively . Compartmental analysis revealed that the most dramatic differences between the wild-type and mutant strains were associated with the chloroplast. Half-lives, and J to this compartment as well as [K+] were a11 very significantly lower in mutant than wild-type cells. By contrast, fluxes across the plasma membrane were only slightly reduced in the mutant and the small difference in J may have resulted from the reduced flux to this large chloroplast compartment. When cells were grown at 10 mM [K+],, the [K+Ich,, although reduced by comparison to wild-type cells appeared to be sufficient to sustain normal growth. However, transfer of these cells to lower levels of [K+l, caused ces-

+,

1543

sation of growth. In an earlier report by Polley and Doctor (1985), influx isotherms for K+ influx by wild-type and mutant lines grown in 10 mM [K+], showed virtually no differences. Only after transfer to TAPM medium with no K+ added were growth rates and Kf fluxes diminished. The failure of the mutants to grow at low external Kf may therefore be due to their inability to load and unload Kf at an appreciable rate to and from the chloroplast. It is evident that the chloroplast was, indeed, serving as a reservoir for Kt when wild-type cells were grown at low [K+],. When cells were grown at 0.1 mM Kt, [K+l, was maintained at an almost constant level, whereas [K+lch, decreased from 223 to 64 mM. In the case of the mutant, the initial K+ reserve within the chloroplast was much lower than for wild-type cells, and hence the capacity to "offload" K+ to the cytoplasm may have been severely limited. Because the chloroplasts used for direct analysis of [K+l were isolated from wall-less mutants (cw 151, it was necessary to check the subcellular compartmentation of K+ and half-lives of K+ exchange in this strain by efflux analysis. The half-lives of these compartments in cw 15 cells were identical with those of the corresponding compartments of wild-type cells (data not shown). It was surprising that even the half-life of the cell wall did not differ significantly in these cells (0.5 2 0.12 min) from that of the wild-type cells (0.56 ? 0.03), although it has been shown that these wall-less mutants produce greatly reduced quantities of cell wall material. It may be that the cell wall layers that are absent in these wall-less mutants (W2-W6 layers) fail to bind significant amounts of Kf . We were concerned that the two types of cells (wild type and t r k l ) might discriminate to different extents against 86Rb+ and that this difference might account for the marked difference in half-lives of the chloroplast compartment observed in wild-type and the trkl cells. Thus, the efflux experiments were repeated using 42Kf as the tracer in the two types of cells. It is clear (Table I) that the half-lives of the three compartments were very similar, whether estimated by the use of 86Rb+or 42Kf and that the differences in the half-lives observed between the wildtype and trkl cells are real. ApK+sacross the major cell membranes (shown in Table V) were calculated using the estimated values of [K'], and A$ obtained from tetraphenylphosphonium distribution (Malhotra and Glass, 1995). In wild-type cells, ApK+ across the plasma membrane at 0.1 mM [Kt], was calculated to have a positive value (2.7 kJ mol-I), which means that Kf is at a higher electrochemical potential inside the cell. Thus, it must move actively into such a cell, against the electrochemical potential gradient, and a channel-mediated transport at or below this concentration is unlikely (cf. Hedrich and Schroeder, 1989). At higher [K+], (20.3 mM), Kf is at a lower electrochemical potential inside the cell and could move into such a cell down its electrochemical potential gradient via K'-specific channels. For trkl cells, ApK+ across the plasma membrane was calculated for cells grown at 10 mM only. At this concentration, ApK+ was -7.7 kJ mol-'. Thus, in the mutant, as in wild-type cells, K+ entry is considered to be passive.

Malhotra and Glass

1544

A p K + across the chloroplast envelope was calculated to be -0.04 and 2.5 kJ mol-', respectively, in wild-type cells grown in 0.1 and 10 mM [K'],. In arriving at this calculation, we assumed that the electrical potential difference across the chloroplast envelope was close to zero (Bulychev et al., 1972). In trkl cells A p K + was calculated only for cells grown at 10 mM [K+],. Under these conditions ApK+ was 1.4 kJ mol-' (Table V). Thus, at 10 mM [K+l,, K+ is at a higher electrochemical potential in the chloroplast and has to move against the electrochemical potential gradient. Considering A p K + values across the plasma membrane and the chloroplast, we propose a model to explain the movement of K+ in wild-type and tukl cells (Fig. 3). At 10 mM [K+], (Fig. 3, a and b), wild-type and trkl cells grow normally and K t enters cells of both strains through Ktspecific channels, down the electrochemical potential gradient. Under these conditions [K+], reaches 70 to 80 mM in both strains, and the transfer of K+ to the chloroplast brings [K+Ichlto 223 and 127 mM, respectively, in wild-type and trkl cells. The maintenance of these high concentrations requires that the flux to the chloroplast be active, mediated perhaps by a K + / H + symporter. Based on a ApH,,,, of 3.5 units and AI,!J~,~,of O mV, ApH+ was calcu-

WT

frkl


lated to be -8.4 kJ mol-' for both strains. Thus, in energetic terms there is probably adequate proton motive force to sustain this flux. When wild-type cells were transferred to 0.1 mM [K+],, they grew normally, whereas trkl cells failed to grow at all. Entry of K+ from this reduced [K+], requires active transport, possibly via a K+/H' symporter (Fig. 3c). Continued growth of wild-type cells at 0.1 mM K+ led to increased activity of the HATS, possibly as a result of derepression of the gene coding for this transport system. Interestingly, the increase of HATS activity in Chlamydomonus was relatively small by comparison to the pattern of putative derepression of HATS activity observed in barley. However, in barley increased HATS activity is accompanied by increased transport of K t from vacuole to cytoplasm (Glass and Fernando, 1992). In Chlamydomonus, increased HATS activity appeared to be associated with transport of K+ from the chloroplast to the cytoplasm, probably moving "downhill" via K+ channels. Wild-type cells can withdraw K+ from the chloroplast at an appreciably higher rate than trkl cells (hence the shorter half-life of exchange; Table I) so that [Kf], was maintained at an almost constant level. Hence, at steady state, [K+l, was only slightly reduced (from 77 to 65 mM), whereas [K+Ich, was substantially lowered (from 223 to 64 mM). By contrast, trkl cells appeared to withdraw K+ from the chloroplast at a rate (as indicated by the longer half-life of exchange) that is perhaps inadequate to maintain [K+], at a level required for protein synthesis (Leigh and Wyn Jones, 1984; Memon et al., 1985a, 1985b).Alternatively, at low [K+], the flux to the chloroplast may be insufficient to sustain chloroplast function with concomitant effects on a11 aspects of metabolism. Even at 10 mM [K+],, the Kt flux to the chloroplast was much lower in trkl cells than in wild-type cells (Table II), and [K+Ichlwas approximately 50% of the wild-type value. However, our inability to grow trkl cells at 0.1 mM [K+], prevented determination of [K+lc and [KtIchl for these mutants; therefore, their values in cells grown at this [K+], remain uncertain. The proposed model would therefore localize the genetic lesion responsible for the trkl phenotype at the chloroplast inner membrane (Fig. 3d). The simplest explanation of these data is that the lesion takes the form of a defective K + / H + symporter.

ACKNOWLEDCMENTS

It is a pleasure to thank Dr. Yaeesh Siddiqi and Jarnail Mehroke for their assistance during this work. Received December 1, 1994; accepted May 1, 1995. Copyright Clearance Center: 0032-0889/95/l08/1537/09. (d)

(C)

H+

K+

e

: K+/H+ symporter

K+m : K+channel

Figure 3. A model to explain the mechanisms of K f transport across the plasma membrane (PM) and chloroplast envelope (CHL ENV) at 10 mM (a and b) and 0.1 mM (c and d) [K+I, in wild-type (WT) and trkl cells. The genetic lesion responsible for trkl phenotype is shown as a closed symbol in the chloroplast envelope. CYTO, Cytoplasm.

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Barber J (1968) Measurement of the membrane potential and evidente for active transport of ions in Chlorella pyrenoidosa. Biochim Biophys Acta 150: 618-625 Bulychev AA, Andrianov VK, Kurella GA, Litvin FF (1972) Micro-electrode measurements of the transmembrane potential of chloroplasts and its photoinduced changes. Nature 236: 175-176

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utilization by barley varieties: activation of pyruvate kinase. J EXPBot 3 6 79-90 Pfriiner H, Bentrup FW (1978) Fluxes and compartmentation of K+, Na+ and C1-, and action of auxins in suspension-cultured Petroselinum cells. Planta 143: 213-223 Pier PA, Berkowitz GA (1987) Modulation of water stress effects on photosynthesis by altered leaf K+. Plant Physiol 85: 655-661 Pitman MG (1963) The determination of the salt relations of the cytoplasmic phase in cells of beetroot tissue. Aust J Biol Sci 16: 647-668 Pitman MG, Lauchli A, Stelzer R (3981) Ion distribution in roots of barley seedling measured by electron probe x-ray microanalysis. Plant Physiol 68: 673-679 Polley LD, Doctor DD (1985) Potassium transport in Cklamydomonas reinkardtii: isolation and characterization of transport-deficient mutant strains. Planta 163: 208-213 Price CA, Reardon EM (1982) Isolation of chloroplasts for protein synthesis from spinach and Euglena gracilis by centrifugation in silica sols. In M Edelman, RB Hallick, eds, Methods in Chloroplast Molecular Biology. Elsevier, Amsterdam, The Netherlands, p p 189-209 Robinson SP, Downton JS (1984) Potassium, sodium and chloride content of isolated intact chloroplasts in relation to ionic compartmentation in leaves. Arch Biochem Biophys 228 197-206 Rona JP, Come1 D, Grignon C, Heller R (1982) The electrical potential difference across the tonoplast of Acer pseudoplatanus cells. Physiol Veg 20: 459-463 Rygiewicz PT, Caroline SB, Glass ADM (1984) A comparison of methods for determining compartmental analysis parameters. Plant Physiol 76 913-917 Wagner G (1974) Fluxes and compartmentation of potassium and chloride in the green alga Mougeotia. Planta 118 145-157 Walker NA, Pitman MG (1976) Measurement of fluxes across membranes. In U Liittge, MG Pitman, eds, Encyclopedia of Plant Physiology. Springer-Verlag, Berlin, pp 93-126 Wu W, Berkowitz GA (1991) Stromal pH and photosynthesis are affected by electroneutral K+ and H+ exchange through chloroplast envelope ion channels. Plant Physiol 98: 666-672