Planta (2000) 211: 474±483

Calci®cation and measurements of net proton and oxygen ¯ux reveal subcellular domains in Acetabularia acetabulum Kyle A. Serikawa1*, D. Marshall Porter®eld2, Peter J. S. Smith2, Dina F. Mandoli1,3 1

Box 355325, Department of Botany, University of Washington, Seattle, WA 98195-5325, USA BioCurrents Research Center, The Marine Biological Laboratory, Woods Hole, MA, 02543, USA Center for Developmental Biology, University of Washington, Seattle, WA 98195, USA

2 3

Received: 5 January 2000 / Accepted: 28 January 2000

Abstract. Vegetative adults of Acetabularia acetabulum (L.) Silva were studied as a model system for subcellular patterning in plants, and a description of several phenotypic and physiological characteristics that reveal patterns of subcellular di€erentiation in this unicellular macroalga was undertaken. Initially, calci®cation patterns were studied. Under favorable conditions, the rhizoid and most of the stalk calci®ed. Only the apical 10±20% of the stalk and a small region adjacent to the rhizoid remained uncalci®ed. Calci®cation in algae has been reported to result from a biologically mediated local increase in alkalinity. To test this model extracellular pH and extracellular hydrogen ion gradients were examined with ion-selective, self-referencing, electrodes. In the light, A. acetabulum displayed a general pattern of extracellular alkalinity around the entire alga, although in some individuals the region near the rhizoid and the rhizoid itself displayed extracellular acidity. Acetabularia acetabulum also displayed net hydrogen ion in¯ux at the rhizoid and the apical half of the stalk, variable ¯ux in the lower part of the stalk, and net hydrogen ion e‚ux at the base of the stalk next to the rhizoid. The lack of complete correlation between external pH patterns and calci®cation suggests that other factors contribute to the control of calci®cation in this alga. To examine whether net hydrogen ion ¯ux patterns correlated with photosynthetic or respiration patterns, oxygen ¯ux was measured along the stalk using self-referencing O2 electrodes. Photosynthetic oxygen evolution occurred at comparable levels throughout the stalk, with less evolution in the rhizoid. Respiration mainly occurred near and in the rhizoid, with less O2 consumption occurring more apically along the stalk. Our studies of calci®cation patterns, net hydrogen ion ¯ux and O2 ¯ux *Present address: Box 357350, Department of Biochemistry, University of Washington, Seattle, WA 98195, USA Abbreviations: DCMU = 3-(30 ,40 -dichlorophenyl)-1,1-dimethylurea; SHAM = Salicylhydroxamic acid Correspondence to: K. A. Serikawa; E-mail: [email protected]; Fax: +1-206-543 4822

revealed several overlapping patterns of subcellular di€erentiation in A. acetabulum. Key words: Acetabularia (subcellular di€erentiation) ± Calci®cation ± Oxygen ¯ux ± Proton ¯ux ± Subcellular di€erentiation

Introduction Di€erentiation within single cells is a highly important process in development. Through subcellular di€erentiation, organisms can accomplish tasks including pattern formation, polar transport of important molecules, and morphogenesis. In plants, the mechanisms by which subcellular di€erentiation can occur are still generally unknown. Algae, some of which contain large cells and display patterns of subcellular di€erentiation and morphogenesis, provide useful model systems in which to approach the question of how plant cells create distinct subcellular regions (Fisahn and Lucas 1995; Bouget et al. 1996). The green unicellular macroalga Acetabularia acetabulum may be one such system. Early cell biological experiments in which adult algae were dissected into subcellular portions demonstrated that this alga possesses an asymmetrically distributed developmental potential for regeneration (HaÈmmerling 1936). Given recent advances in the culture and experimental amenability of this alga (Hunt and Mandoli 1992, 1996; Mandoli and Larsen 1993; Mandoli 1998b), we decided to examine its utility for studies of how cells create subcellular domains. As one avenue of approach to this problem, we began by examining calci®cation patterns in A. acetabulum. Calci®cation is a process in which calcium salts are deposited onto or within biological structures. In algae, calci®cation has been linked to inorganic carbon uptake, competition for sunlight, sequestration of nutrients, structural support and protection from predation (Boro-

K. A. Serikawa et al.: Subcellular di€erentiation in A. acetabulum

witzka 1987; Marin and Ros 1992; Pennings and Paul 1992; Buddemeier 1994; Hay et al. 1994). In the wild, A. acetabulum calci®es in a speci®c pattern (Marin and Ros 1992) which we hypothesized might re¯ect underlying subcellular di€erentiation. In algae, calci®cation often occurs through biologically mediated creation of local regions of increased alkalinity and/or supersaturation of calcium and carbonate ions (Borowitzka 1987). Under alkaline conditions, the concentration of carbonate ions (CO23 ) in water increases, allowing carbonate to precipitate with calcium ions in the form of calcium carbonate (CaCO3). Thus, we began our studies with the hypothesis that the calci®ed and uncalci®ed regions of A. acetabulum would correspond to underlying subcellular patterns of extracellular alkalinity and acidity, respectively. Materials and methods Culture of Acetabularia All experiments used heterogeneous wild-type strains of A. acetabulum (L.) Silva, either Aa0006 (Ladenburg #5) or Aa0005 (Ladenburg #17). Axenic cultures were obtained by decontaminating mature caps (Hunt and Mandoli 1992) and ``mass-mating'' the resulting axenic gametangia (Mandoli and Larsen 1993; Mandoli 1998b). The progeny of the matings were grown in an arti®cial seawater, Ace-27, which was the same as Ace-25 (Hunt and Mandoli 1996; Mandoli 1998b) except that the KCl pre-stock was puri®ed over a Chelex 100 column and urea hydrogen peroxide was added to the medium to a ®nal concentration of 10)15 M. Except where noted, algae were grown under cool-white ¯uorescent lights at a photon ¯ux density of approx. 170 mmol m)2 s)1 on a 14:10 h light:dark photoperiod at 21 °C. Zygotes resulting from ``mass matings'' were initially thinned 1:5 in 5-mL snap-cap tubes until they became siphonous juveniles. Juveniles were then thinned once to approx. 300 per mL (1:10 into 50-mL Falcon tubes) until most had developed one to two whorls, and a second time to a ®nal population density of 1 adult/2.25 mL Ace-27 [either into square, polycarbonate boxes (Sigma Chemical Company) or square polystyrene Petri dishes (100 mm ´ 100 mm ´ 15 mm, Nunc Inc.)]. Individuals in boxes and dishes were used for experiments when they had reached the desired phase of growth (Mandoli 1998a). Light micrographs Microphotographs were taken using Kodak Ektar 160 T slide ®lm and a dissecting scope coupled to a camera. Slides were scanned into Adobe Photoshop using a Nikon LS-1000 35-mm Film Scanner. In some instances, contrast and intensity values were adjusted to increase clarity but otherwise images were unaltered. Low-phosphate growth experiments Ordinarily, Ace-27 contains phosphate at a concentration of 50 lM (Hunt and Mandoli 1996). To test the e€ect of decreased phosphate levels on calci®cation, populations of adult A. acetabulum (strain Aa0005, each with 9±12 whorls) were placed into either Ace-27, Ace-27 with 10 lM phosphate, or Ace-27 with 0 lM phosphate (20 individuals per treatment). Every 3 d algae were examined for signs of calci®cation on and around the rhizoid. Observations continued for 2 weeks, until all algae in the 10 lM phosphate medium had begun calcifying. The experiment was repeated three times.

475 Measurements of ion ¯uxes Operation of the ion-selective self-referencing electrode has been detailed elsewhere (Smith et al. 1994, 1999; Smith 1995). Brie¯y, a silanized glass microelectrode was front-®lled with an ion-selective ionophore (in this case a 30 lm column of the H+-selective ionophore FLUKA Hydrogen ionophore cocktail B) and back®lled with 100 mM KCl. The ®nal tip diameter was between 2 and 4 lm and the resistance was between 1 and 3 GW. Electrode potentials were measured with a high-impedance preampli®er with a unity gain (model AD515; Analog Devices). A 3% agar bridge containing 3 M KCl was used to complete the circuit. Signals were ampli®ed 1000-fold and digitized using an analog-to-digital board (DT 2800 series; Data Translations). Equipment and software are products of the BioCurrents Research Center (Marine Biological Laboratory, Woods Hole, Mass., USA). The extracellular H+ gradient of adult A. acetabulum (strain Aa0006) was measured using an H+-selective, self-referencing, electrode. Algae were of approximately the same age as those used in low-phosphate growth experiments and had never been calci®ed. All measurements were taken during the daylight phase of the light:dark cycle, between the hours of 9 a.m. and 8 p.m. No correlation between gradient patterns and time of day was observed (data not shown). All measurements were made in Ace-27. The light source for these experiments was a combination of the microscope condenser lamp and a cool-white ¯uorescent bulb with a photon ¯ux density of approx. 80 mmol m)2 s)1 (approximately equal contributions of each source). For experiments, the electrode was brought to within 1 lm of the cell wall and oscillated over a distance of approx. 10 lm along an axis perpendicular to the surface of the wall as a square wave at a frequency of 0.3 Hz. Voltages, related to the H+ activity, were measured near to and away from the wall. At least 25 measurements were made at each position. The di€erence between these voltages was converted into an H+ ¯ux value and direction using the Fick equation (Smith et al. 1999). This H+-¯ux value should be viewed as the sum of all transmembrane ion ¯uxes that a€ect extracellular concentrations of H+ and should not be taken as a direct measurement of H+ movement alone. This ¯ux value re¯ects the activity of the hydrogen ions and does not take into account the bu€ering capacity of Ace-27 (Ferrier 1980). Before and after experimental measurements the Nernst slope of the electrode was measured with standards at speci®c pH values to allow correction for electrode drift. Voltage measurements can be converted to pH values through comparison with the Nernst slope. Measurements were taken at 5 locations along the stalk for each of 15 adults: at the stalk apex, one-third of stalk length from the stalk apex, two-thirds of stalk length from the stalk apex, immediately adjacent to the rhizoid and at the rhizoid (Fig. 3). To ensure that algae would remain stationary during measurements, each individual was gently fastened to the bottom of a culture dish with a small amount of vacuum grease. For 10 of the adults, measurements of H+ gradients began at the apex and proceeded basipetally to the rhizoid while for the other 5 the order of measuring was reversed. The general pattern of H+ gradients appeared independent of the order of measuring. For two samples the microelectrode broke before the Nernst slope could be calculated after measurements, and data for those two samples were not included in the calculation of ¯ux values (Fig. 4). The electrode measurements were subsequently converted to pH and net ¯ux values using the software supplied by the BioCurrents Research Center (BRC, Woods Hole, Mass., USA). Background measurements were made 1±2 cm away from the cell wall in the bulk medium. To examine the e€ect of light on net H+ ¯uxes, continuous measurements were taken of adult individuals subjected to a 10- to 12-min dark period. Only the individual being measured was subjected to dark periods; the remaining population remained in normal light conditions. Measurements were taken at three points along the stalk: the stalk apex, immediately adjacent to the rhizoid, and at the rhizoid. Each individual was only measured for one of

476 the three positions. At least ®ve individuals were measured for each position. To examine the e€ect of 3-(30 ,40 -dichlorophenyl)-1,1dimethylurea (DCMU) treatment on the net H+ ¯uxes, measurements were taken of adults before and after addition of DCMU to the medium. The ®nal concentration of DCMU was 10 lM. The same points and approximately the same number of individuals for each point were measured as for the light experiments. Measurements of oxygen ¯uxes were performed as described by Land et al. (1999). Membrane-tipped, Whalen-style polarographic oxygen microelectrodes (Model 723) with tip diameters of 2±3 lm were purchased from Diamond General Development Corp. (Ann Arbor, Mich., USA). Oxygen electrodes were employed in a similar fashion to H+-selective microelectrodes ± oscillated as a square wave near plants while measurements of O2 concentrations were taken, with measurements near and away being used to derive the oxygen ¯ux. The probe was moved over 10 lm. Measurements at least 200 lm away from the cell wall gave background values. For oxygen experiments, measurements were initially taken at the ®ve positions along the stalk; the lights turned o€; and the measurements taken again to provide baseline light and dark O2 ¯uxes. Adults were then subjected to treatment by either the photosynthetic inhibitor DCMU in the light or the respiratory inhibitors KCN and salicylhydroxamic acid (SHAM) in the dark. To examine the e€ect of DCMU on O2 ¯ux, DCMU was added to the medium to a ®nal concentration of 10 lm and the oxygen ¯ux measured again at the ®ve positions. To examine the e€ect of respiratory inhibitors, KCN was added to a ®nal concentration of 1 mM and the oxygen ¯ux at the ®ve positions measured. Next, SHAM was added to a ®nal concentration of 15 mM and the oxygen ¯ux measured a ®nal time at the ®ve positions. Ten plants were measured for each treatment (DCMU or KCN/SHAM).

Results Patterns of calci®cation Characterizing the calci®cation patterns of A. acetabulum required the development of culture conditions that allow reliable calci®cation in laboratory-grown populations. Although calci®cation occurred sporadically in the culture conditions normally used, it was neither reproducible nor predictable (data not shown). One possible explanation why calci®cation did not appear reproducibly was that some component(s) of the arti®cial medium inhibited the nucleation and spread of calci®cation. Phosphate, which can act as a ``crystal poison'' (M. Borowitzka, personal communication), was a good candidate for an inhibitory factor. To test this hypothesis, we transferred adults into medium with reduced phosphate concentrations and studied the initiation and propagation of calci®cation. Calci®cation rapidly appeared in all adults grown in medium lacking phosphate (Fig. 1). Medium with 10 lM phosphate also allowed reproducible, rapid calci®cation of adults, although not as quickly as in the absence of phosphate. Approximately 25% of plants grown in 50 lM phosphate (Ace-27) showed signs of calci®cation by the end of the experiment, but the proportion of individuals within a given population, the rate of appearance of calci®cation, and the degree of calci®cation were all much less than that in either 10 lM or phosphate-lacking medium (Fig. 1 and data not shown). Low-phosphate media allowed the characterization of calci®cation patterns in adults of A. acetabulum. When algae were transferred to 10 lM or phosphate-lacking

K. A. Serikawa et al.: Subcellular di€erentiation in A. acetabulum

Fig. 1. E€ect of phosphate concentrations on the progression of calci®cation. Populations of 20 adults were grown in medium with 50, 10 or 0 lM phosphate and monitored every 3 d for the percentage of the population that had initiated calci®cation. Values are the means ‹ SE of three separate trials. When standard error bars are not visible, they are obscured behind the symbol

media, calci®cation initially appeared on the rhizoid, often in the form of crystals resting on or enclosed within the digits of the rhizoid (Fig. 2A,B). Subsequently, the majority of the stalk calci®ed, progressing acropetally over time (from the rhizoid towards the apex, Fig. 2C). Calci®cation along the stalk could ®rst appear in streaks oriented along the stalk axis, and sometimes hairs also calci®ed (data not shown). After several days the only uncalci®ed regions were the apical 10±20% of the stalk (Fig. 2D), and often a small region of the stalk immediately adjacent to the rhizoid (Fig. 2E). The patterns of calci®cation seen in the laboratory are similar to those seen in the wild (Marin and Ros 1992, personal observation), except that in the wild the clear area near the base of the rhizoid does not occur. Hydrogen ion gradients and extracellular pH in A. acetabulum Given the correlation between alkalinity and calci®cation in species such as Chara and Halimeda (Lucas and Smith 1973; Borowitzka and Larkum 1976), the calci®cation patterns we observed suggested that there might be a corresponding pattern of extracellular pH or net H+-¯ux patterns. To address this possibility ion-selective, self-referencing electrodes were used to measure extracellular H+ gradients and pH around A. acetabulum. These electrodes measure gradients of H+ activity near the surface of the cell. These gradient measurements are then converted into an H+-¯ux value, hereafter denoted as ``net H+ ¯ux'' that is the sum of all transmembrane ion ¯uxes (H+, OH), HCO3 , etc.) which contribute to extracellular H+ gradients. The H+ values we report represent the ``free H+ ¯ux,'' and do not take into account the bu€ering capacity of Ace-27 (Ferrier 1980). We measured H+ gradients and extracellular pH at ®ve positions along the long axis of the alga. Typical traces are shown in Fig. 3. Non-calci®ed plants were used for all measurements. For all plants measured, the

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477

Fig. 3. Hydrogen ion gradients and pH along the axis of A. acetabulum. Positions of measurements are indicated with representative tracings for both net H+ ¯ux and pH. As an internal control for probe drift, measurements at each position alternated with measurements of the surrounding medium away from the cell wall (indicated by arrows along the top of the ®gure). Measurements at each position lasted 1±2 min. Each position exhibited invariant direction of ¯ux for the duration of the measurement. The mean net H+ ¯ux (in pmol/ cm2 s) and pH values are indicated at each position. The direction of net hydrogen ion ¯ux is indicated by the sign of the ¯ux value. Positive values represent e‚ux; negative values in¯ux

Fig. 2A±E. Calci®cation patterns of A. acetabulum. A An uncalci®ed rhizoid grown in Ace-27. B A rhizoid of the same age as that seen in A 10 d after transfer to 0 lM phosphate medium. Calcium carbonate crystals have accumulated over the surface of the rhizoid. C An adult showing typical patterns of calci®cation after transfer to 0 lM phosphate medium for 1±2 weeks. Note the absence of calci®cation of the stalk at the apex and near the rhizoid. D Closer (approx. 2´ higher magni®cation) view of the apex of a calcifying plant, showing the junction between calci®ed and non-calci®ed regions. In some cases calci®cation does not end abruptly but forms a gradient near the apex (not shown). E Closer (approx. 2´ higher magni®cation) view of the non-calci®ed region near the rhizoid. Although both stalk and rhizoid are well calci®ed, this region often remains free of calcium deposits. Bars = 400 lm (A, B), 2 mm (C), 1 mm (D, E)

net H+ ¯ux at each position was invariant in terms of direction of ¯ux for the duration of the measurement (Fig. 3). Surprisingly, in this and other individuals measured, the relative magnitude of net H+ ¯ux along the cell did not always correlate with the magnitude of

extracellular pH. The source of this discrepancy was unclear (but see Discussion). The patterns and relative magnitudes of extracellular pH and net H+ ¯uxes in the light for the entire population are summarized in Fig. 4. A picture of an adult, calci®ed individual is shown to indicate the general locations of measurements. The pH measurements made at each position for 13 plants are summarized in histograms with the mean pH at each position for the population listed alongside (left half of Fig. 4A). In the histogram, alkalinity and acidity are relative to Ace-27, which in this set of experiments had a measured pH of 8.1. The measured net H+ ¯uxes have been summarized in a similar fashion (right half of Fig. 4A). The various speci®c combinations of pH and ¯ux patterns we observed have been represented digramatically in Fig. 4B. In general, individuals of A. acetabulum exhibited extracellular alkalinity along the entirety of the stalk and rhizoid, although in some individuals the rhizoid or the region near the rhizoid exhibited extracellular acidity. Also, in general, the majority of the stalk and the rhizoid demonstrated net H+ in¯ux while the region near the rhizoid exhibited e‚ux. These are overall patterns; on an individual level, however, there was not a strict correlation between a plant's external pH pattern

478

K. A. Serikawa et al.: Subcellular di€erentiation in A. acetabulum

Fig. 4A,B. pH and net H+ ¯uxes along the stalk of A. acetabulum. A The pH and net H+ ¯ux values measured at each position along the stalk for each of 13 plants are plotted in histograms next to the mean value for that position. Alkalinity and acidity for the pH values is designated relative to Ace-27 (pH 8.1 in these experiments). Values for net H+ ¯ux represent the ``free H+ ¯ux'' and do not take into account the bu€ering capacity of the media. A picture of a calci®ed A. acetabulum individual illustrates how calci®cation patterns

compare to pH and net H+ ¯uxes. Mean values ‹SE are presented. B The various overall patterns of pH and net H+ in¯ux/e‚ux are diagrammed with the number of algae that showed that particular pattern indicated underneath. Regions showing alkalinity or H+ in¯ux are shaded grey; regions showing acidity or e‚ux are clear. Although overall patterns are similar between extracellular pH and net H+ ¯uxes, on a plant-by-plant basis ¯ux direction and extracellular pH did not always correlate

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and its net H+ ¯ux patterns. That is, the patterns in the left and right halves of Fig. 4B do not correspond in a one-to-one fashion. In addition, the relative magnitudes of external pH along the alga and the mean magnitudes of ¯ux do not clearly correspond (compare mean pHs and ¯uxes for positions 1, 2 and 3). Interestingly, the patterns of extracellular alkalinity do not correlate completely with calci®cation. Although, as expected, the rhizoid and the calcifying portions of the stalk exhibited mean extracellular alkalinity, so did the apex and region near the rhizoid. Thus, while demonstrating that calci®cation does not result from a simple model of extracellular alkalinity/acidity, our data also revealed the existence of a second pattern, one of net H+ ¯ux, which presumably re¯ects another subcellular pattern of di€erentiation.

to dark breaks are shown in Fig. 5. In general, darkness caused a rapid change in the direction of net H+ ¯ux at positions 1 and 5 and a transitory increase in e‚ux at position 4. When lights were turned on, positions 1 and 5 again behaved similarly to each other, with the ¯ux direction reverting to in¯ux. The magnitude of e‚ux at position 4 decreased temporarily. These measurements demonstrate that net H+ ¯ux at each of these positions is responsive to light. We used the photosynthetic uncoupler DCMU to examine how the response of net H+ ¯uxes to darkness was coupled to photosynthesis. Individuals were measured while DCMU was added to the medium. Addition of DCMU led to a rapid change in ¯ux direction at the apex, but had marginal e€ects at and near the rhizoid (Fig. 5). We were surprised to ®nd, given that our previous measurements suggested an incomplete correlation between net H+ ¯uxes and pH (Fig. 4), that pH often responded rapidly and in synchrony to changes in net H+ ¯ux direction.

Light and net H+ ¯uxes To understand the e€ect of light on net H+ ¯uxes, we measured individuals while subjecting them to a dark break. Three points along the stalk axis were measured (Fig. 5). For each measurement, the ion-selective electrode was placed near one of the three positions and net H+ ¯ux measured before, during and after a 12- to 15-min dark break. Typical patterns of ¯ux response

Fig. 5. E€ect of light and DCMU on H+ ¯uxes at positions 1, 4 and 5 along the stalk of A. acetabulum. Representative traces of net H+ ¯ux and extracellular pH at three positions along the stalk are shown for both light/dark and DCMU experiments. The number of adults showing the representative trace pattern is given. In the light experiments, the one adult that did not display the predominant response pattern at position 4 showed a decrease in e‚ux during the dark break. At position 5, the adult that did not show

Oxygen ¯ux in A. acetabulum Oxygen ¯ux measured in the light re¯ects photosynthetic oxygen evolution less respiratory oxygen consumption.

the predominant response pattern showed no change in ¯ux during the dark break. In the DCMU experiments, the one adult that did not show the predominant pattern at position 1 showed no change in ¯ux after DCMU addition. At position 4, the one adult that did not show the predominant pattern exhibited a change in ¯ux direction from e‚ux to in¯ux upon DCMU addition. At position 5, the two adults that did not show the predominant pattern exhibited a slow change from net H+ in¯ux to H+ e‚ux

480

K. A. Serikawa et al.: Subcellular di€erentiation in A. acetabulum Table 1. O2 ¯ux measurements and DCMUa Position

1 2 3 4 5

Oxygen ¯ux (pmol/cm2s ‹ SD) in White light

Darkness

White light + DCMU

184 244 310 212 23

)72 )51 )28 )144 )161

)64 )56 )22 )122 )148

‹ ‹ ‹ ‹ ‹

15 10 14 12 4

‹ ‹ ‹ ‹ ‹

10 3 11 9 11

‹ ‹ ‹ ‹ ‹

9 5 6 12 9

a

Positive numbers denote O2 e‚ux, negative numbers in¯ux

Fig. 6. Direction and approximate magnitudes of photosynthetic oxygen evolution and respiratory oxygen consumption measured along the stalk of A. acetabulum. Lengths and directions of arrows are approximately proportional to mean ¯ux values at each position. Note that the respiratory oxygen consumption we measured (left side of Fig. 6) is equivalent to the oxygen in¯ux measured in the dark (Table 1, right column). The rhizoid and stalk immediately adjacent to the rhizoid consume the most oxygen, 2-fold or more than consumed elsewhere. Gross photosynthetic oxygen evolution is represented by the arrows on the right of the ®gure. Gross oxygen evolution was calculated as the sum of the oxygen e‚ux measured in the light added to the absolute magnitude of the oxygen in¯ux measured in the dark or in the light in the presence of 10 lM DCMU. For this ®gure we used the oxygen in¯ux measurements made in the dark (i.e. left and middle columns of Table 1). The rhizoid produces only a little more than half as much oxygen as other regions

Oxygen ¯ux in the dark re¯ects respiratory oxygen consumption. Thus, the total amount of photosynthetic oxygen evolution is the sum of the oxygen e‚ux Table 2. Oxygen ¯ux measurements and inhibitors of respirationa

Position

Oxygen ¯ux (pmol/cm2s ‹ SD) in White light

1 2 3 4 5 a

measured in the light and the absolute magnitude of the oxygen in¯ux measured in the dark. To examine how photosynthesis and respiration may be tied to net H+ ¯uxes, oxygen ¯ux was measured along the stalk (Table 1, Fig. 6). First, to characterize overall photosynthesis and respiration patterns, oxygen ¯ux was measured in the light and in the dark, and also in the light after DCMU addition (Table 1). Net photosynthetic oxygen evolution occurs almost evenly along the stalk, with a slightly increasing gradient of oxygen production from apex to base (Fig. 6). The rhizoid, however, produced only a little more than half as much as other regions (Fig. 6). Addition of DCMU mimicked the e€ect of darkness at each position (Table 1, cf. Fig. 6). Both darkness and addition of DCMU demonstrated that the rhizoid and the region of the stalk immediately adjacent to the rhizoid consume the most oxygen, up to twice as much as that consumed by other regions of the stalk (Fig. 6). A shallow, decreasing gradient of oxygen consumption was measured from the apex towards position 3 (Fig. 6), suggesting that di€erent regions of the body plan do not have identical respiratory needs. Plants, some fungi and protists possess two pathways for respiration ± through the phosphorylating cytochrome pathway and the non-phosphorylating alternative pathway. To test whether H+ ¯uxes correlate with either respiratory pathway, oxygen ¯uxes in algae were measured in darkness after the addition of KCN, which inhibits the cytochrome pathway, and again after the addition of SHAM, which inhibits the alternative pathway (Table 2). Addition of KCN reduced but did not abolish oxygen in¯ux. Further addition of SHAM reduced oxygen in¯ux to nearly undetectable levels. The

190 248 300 203 25

‹ ‹ ‹ ‹ ‹

9 11 9 11 9

Darkness

+KCN

)69 )55 )32 )145 )162

)31 )19 )5 )39 )49

‹ ‹ ‹ ‹ ‹

9 9 12 10 9

‹ ‹ ‹ ‹ ‹

9 11 10 11 11

+KCN and SHAM )4 3 1 )3 )6

‹ ‹ ‹ ‹ ‹

2 3 2 1 2

Alternative respiration (%)b

45 35 16 27 30

Positive numbers denote O2 e‚ux, negative numbers in¯ux Approximate alternative respiration was calculated as (¯ux in darkness + KCN)/(¯ux in darkness) ´ 100 b

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481

asymmetric distribution of di€erent ion transporters, organelles, photoreceptors and/or other macromolecules. For example, we have recently discovered that some mRNAs are asymmetrically distributed within adults of A. acetabulum (Serikawa and Mandoli 1999). Untangling how subcellular patterns are maintained and how they interact will be an exciting challenge for future studies. Calci®cation in A. acetabulum

Fig. 7. Subcellular patterns in the A. acetabulum cell as revealed through observations of calci®cation and measurements of H+ and oxygen ¯ux. The calci®cation, net H+ ¯ux, respiration and photosynthesis patterns we measured are diagrammed with arrows showing direction and relative magnitude (for a given ¯ux) of ¯uxes. For net H+ ¯uxes, shaded areas represent predominant in¯ux, hatched areas represent equal probability of an alga showing in¯ux or e‚ux, and clear areas represent predominant e‚ux

rhizoid and stalk near the rhizoid were a€ected more, proportionally, by KCN addition than the apical and upper region of the stalk (positions 1 and 2) as re¯ected in the percentage of oxygen consumption that we calculated as attributable to alternative respiration (Table 2, right column). Discussion Spatial separation in a giant unicell Our data show that several subcellular patterns are organized and maintained within the A. acetabulum cell, including calci®cation, the distribution of ion transporters in the plasma membrane, photosynthesis and respiration (Fig. 7). Surprisingly, the pattern we began with ± calci®cation ± appears to be controlled in a more complex manner than originally hypothesized. The identi®cation and de®nition of these patterns provides us with a starting point from which to analyze the molecular and cellular mechanisms underlying subcellular di€erentiation in this alga. Like Chara, which also has been investigated as a system for understanding acid and alkaline domains, photosynthesis and subcellular patterning (Lucas and Smith 1973; Lucas 1983; Chau et al. 1994; Plieth et al. 1994; Fisahn and Lucas 1995), A. acetabulum is a giant cell with highly active transmembrane ionic behaviors (Bowles and Allen 1986; Gradmann et al. 1982; Gradmann 1989), and is capable of withstanding substantial dissection and manipulation (HaÈmmerling 1936; Mandoli and Hunt 1996; Mandoli 1998b), features which will facilitate our investigations of the behavior of its subcellular regions. Although virtually nothing is known about the mechanisms of subcellular di€erentiation in A. acetabulum, these regional di€erences could arise through the

Calci®cation occurs in a stereotypical pattern in adults of A. acetabulum (Fig. 2). The uncalci®ed region near the base of the stalk is the one feature that di€ers from the pattern seen in the wild. However, some individuals in our experiments did calcify in that region. Further, we only examined the establishment and propagation of calci®cation over a 2-week period in late adults. Algae in the wild calcify over a period of months, starting during early adulthood and under highly variable conditions, and this may account for the di€erence. In Chara and other species, calci®cation arises where the alga creates extracellular regions of alkalinity (Borowitzka 1977, 1987). Based on the calci®cation patterns we observed, we expected that the extracellular pH of the stalk apical region and the region immediately adjacent to the rhizoid would be acidic relative to the surrounding medium while that of the rest of the stalk and the rhizoid would be alkaline. Our pH measurements suggest that this simple model is insucient to explain all of the calci®cation patterns in A. acetabulum (Fig. 4). The entire alga shows extracellular alkalinity. While this correlates with a simple model of extracellular alkalinity driving calci®cation at the rhizoid and the majority of the stalk, it does not ®t in with the lack of calci®cation at the apex and near the rhizoid. However, the region near the rhizoid displays the lowest mean extracellular pH and several (5 of 13) individuals actually exhibited extracellular acidity, which may contribute to maintaining this region free of calci®cation. In another contradiction of a simple alkalinity model for calci®cation, the magnitude of extracellular alkalinity does not correspond to the order in which calci®cation normally appears ± although the rhizoid is the ®rst part of the alga to calcify, its average external pH is not as high as that along the apical two-thirds of the stalk. One possible explanation for why calci®cation patterns do not always agree with external pH could be that the cell wall plays an important role in controlling calci®cation. One of the most energetically unfavorable steps in calci®cation is the initial nucleation of crystals (Borowitzka 1982, 1987). The cell wall in the apical region is thinner than that of the rest of the stalk (M. von Dassow, personal communication) and cell wall compounds favorable for calci®cation may not be present in these thinner walls. Alternatively, the apical region may exude compounds that actively inhibit crystal formation, thus keeping it uncalci®ed. A similar

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explanation may underlie why the rhizoid calci®es ®rst ± it may contain favorable, biologically assembled nucleation sites. Another possible factor a€ecting calci®cation may be the behavior of net hydrogen ion ¯uxes in the dark. Net ¯uxes at positions 1, 4 and 5 all respond to shifts from light to dark and back again (Fig. 5), and these changing behaviors may also a€ect calci®cation. Thus, rather than simply re¯ecting subcellular patterns of extracellular pH as we initially assumed, calci®cation patterns may re¯ect a complex relationship among factors including pH, light and subcellular di€erences in cell wall composition. The magnitude of transmembrane calcium ¯uxes and their potential role in promoting calci®cation were not examined in this study. However, a previous study using the vibrating voltage electrode examined the distribution of net currents along adult algae before and after addition of calcium blockers (O'shea et al. 1990). This allows a crude estimation of the magnitude and direction of calcium ¯ux. Along the stalk, putative calcium ¯ux was fairly constant (not varying more than 2-fold) and directed outward. At the rhizoid, calcium ¯ux of the same approximate magnitude was directed inward. Thus, direction and magnitude of calcium ¯uxes do not correlate with calci®cation patterns and calcium ¯ux does not appear to be a likely candidate to explain the discrepancy between pH and calci®cation.

species undergo circadian rhythms in aspects of metabolism such as photosynthesis (Terborgh and McLeod 1967). As all of these measurements were taken during the day, the behavior of ¯uxes in the middle of the night was not characterized and might provide other insights into the roles and control of net H+ ¯uxes in the alga.

Extracellular pH and net hydrogen ion ¯ux Surprisingly, our data did not show an exact correlation between net H+ ¯ux and extracellular pH. Overall patterns were consistent: positions 1, 2, 3 and 5 exhibited mean extracellular alkalinity and mean net H+ in¯ux; and position 4 showed the lowest mean extracellular pH and mean net H+ e‚ux. Some speci®c aspects of the relationship seem contradictory however. For example, even though the apex had the strongest and most invariant ¯ux pattern, it also displayed the lowest mean extracellular pH of the three apical stalk positions. In addition, at position 3, the opposite occurred: although the mean ¯ux was essentially zero, position three exhibited high extracellular alkalinity. In contrast to these contradictions between net hydrogen ion ¯ux and extracellular pH in our steadystate measurements, removal of light and addition of DCMU induced rapid changes in net H+ ¯ux direction which were often accompanied by a corresponding change in extracellular pH (Fig. 5). A useful next step in resolving the relationship between net H+ ¯ux would be to examine proton ¯ux on a ®ner scale along the A. acetabulum stalk. Although measuring at ®ve positions provides a general overview of net H+ ¯ux and extracellular pH, it may not reveal subtleties in the distribution of ¯ux patterns that might explain the discrepancies between pH and net H+ ¯ux values we saw during steady-state measurements. A variable that we did not assess, and which would be another fruitful area for future study is the e€ect of circadian rhythms on net H+ ¯uxes. Acetabularia

Oxygen ¯uxes in A. acetabulum In other organisms, both extracellular pH and calci®cation patterns correlate with photosynthetic activity. For example, in Chara, acidic and alkaline bands disappear in the dark (Lucas and Smith 1973) and H+ e‚ux is linked to increased photosynthesis (Plieth et al. 1994). In A. acetabulum, net hydrogen ion ¯uxes were a€ected by light, but not all were a€ected equally by photosynthesis (Fig. 5). Photosynthetic oxygen evolution occurred all along the stalk in A. acetabulum with lower but substantial levels in the rhizoid (Fig. 6 and Table 1). This ®nding is in agreement with some (Vanden Driessche 1974), but not all previous studies of photosynthesis in A. acetabulum (Issinger et al. 1971). Prior studies did not measure respiration, and this may explain the di€erences between previous work and the data presented here. The rhizoid and region of the stalk immediately adjacent to the rhizoid respired more than the rest of the stalk (Fig. 6). Although oxygen ¯ux measurements revealed subcellular di€erences within A. acetabulum, net H+ ¯ux patterns are not easily linked to either photosynthesis, respiration, or the utilization of the alternative oxidase (Table 2, Fig. 6). For example, although the apical region had the greatest magnitude of net H+ in¯ux and the direction of that ¯ux clearly depended upon photosynthesis, oxygen evolution occurred at a greater rate in the lower regions of the stalk, with the highest levels near the rhizoid at position 4, where control of net H+ ¯ux appears to be independent of photosynthesis. However, net H+ ¯ux could still be important for photosynthesis. For example, the net H+ e‚ux at the base of the rhizoid may lower the extracellular pH, resulting in a higher concentration of extracellular CO2 available for photosynthesis. Concluding remarks Ionic relations in Acetabularia acetabulum have been examined before, and the value of this organism for studies of ion ¯uxes has been described (Bowles and Allen 1986; Dazy et al. 1986; Gradmann et al. 1982; Gradmann 1989; O'shea et al. 1990). Previous studies often used indirect means to measure speci®c ion contributions to overall transmembrane current distribution. Our studies measured net H+ and oxygen ¯uxes directly and highlight the utility of A. acetabulum for studies of ion movement across the plasma membrane. Clearly, an important next step is to identify the molecular mechanisms which control H+ ¯uxes in the various regions of the body plan of this giant unicell.

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We thank the BioCurrents Research Center for allowing us to use the self-referencing electrode apparatus. We especially thank Kasia Hammar and Roger Lew for help and discussions while at the BRC, and Robert Cleland, Richard Ivey, Rene Kratz, Katie Mitchell and Mickey von Dassow for critical comments. We also thank Dr. Michael Borowitzka for helpful comments and suggestions at the beginning of this project. This work was supported by an NIH grant (P41-RR01395) to P.J.S., an NSF grant (IBN9630618) to D.F.M. and an NSF postdoctoral fellowship to K.A.S.

Hunt BE, Mandoli DF (1996) A new arti®cial seawater that facilitates growth of large numbers of cells of Acetabularia acetabulum (Chlorophyta) and reduces the labor inherent in cell culture. J Phycol 32: 483±495 Issinger O, Maass I, Clauss H (1971) PhotosyntheseintensitaÈt der Stielregionene von Acetabularia. Planta 101: 360±364 Land SC, Porter®eld DM, Sanger RH, Smith PJS (1999) The selfreferencing oxygen-selective microelectrode: detection of transmembrane oxygen ¯ux from single cells. J Exp Biol 202: 211± 218 Lucas WJ (1983) Photosynthetic assimilation of exogenous HCO3 by aquatic plants. Annu Rev Plant Physiol 34: 71±105 Lucas WJ, Smith FA (1973) The formation of alkaline and acid regions on the surface of Chara corallina cells. J Exp Bot 24: 1±14 Mandoli DF (1998a) Elaboration of body plan and phase change during development of Acetabularia: How is the complex architecture of a giant unicell built? Annu Rev Plant Physiol Plant Mol Biol 49: 173±198 Mandoli DF (1998b) What ever happened to Acetabularia? Bringing a once-classic model system into the age of molecular genetics. Int Rev Cytol 182: 1±67 Mandoli DF, Hunt BE (1996) kurkku, a phenotype of Acetabularia acetabulum which is stuck in vegetative growth, can be rescued with wildtype cytoplasm. Plant Cell 8: 323±332 Mandoli DF, Larsen T (1993) Improved mating eciency in Acetabularia acetabulum: recovery of 48±100% of the expected zygotes. Protoplasma 176: 53±63 Marin A, Ros JD (1992) Dynamics of a peculiar plant-herbivore relationship: the photosynthetic ascoglossan Elysia timida and the chlorophycean Acetabularia acetabulum. Mar Biol 112: 677± 682 O'shea P, Goodwin B, Ridge B (1990) A vibrating electrode analysis of extracellular ion currents in Acetabularia acetabulum. J Cell Sci 97: 503±508 Pennings SC, Paul VJ (1992) E€ect of plant toughness, calci®cation and chemistry on herbivory by Dolabella auricularia. Ecology 73: 1606±1619 Plieth C, Tabrizi H, Hansen U-P (1994) Relationship between banding and photosynthetic activty in Chara corallina as studied by the spatially di€erent induction curves of chlorophyll ¯uorescence observed by an image analysis system. Physiol Plant 91: 205±211 Serikawa K, Mandoli DF (1999) Aaknox1, a kn1-like homeobox gene in Acetabularia acetabulum undergoes developmentally regulated subcellular localization. Plant Mol Biol 41: 785± 793 Smith PJS (1995) Non-invasive ion probes ± tools for measuring transmembrane ion ¯ux. Nature 378: 645±646 Smith PJS, Sanger RH, Ja€e LF (1994) The vibrating Ca2+ electrode: a new technique for detecting plasma membrane regions of Ca2+ in¯ux and e‚ux. Methods Cell Biol 40: 115± 133 Smith PJS, Hammar K, Porter®eld DM, Sanger RH, Trimarchi JR (1999) Self-referencing, non-invasive, ion-selective electrode for single cell detection of trans-plasma membrane calcium ¯ux. Micros Res Tech 46: 398±417 Terborgh J, McLeod GC (1967) The photosynthetic rhythm of Acetabularia crenulata. I. Continuous measurements of oxygen exchange in alternating light-dark regimes and in constant light of di€erent intensities. Biol Bull 133: 659±669 Vanden Driessche T (1974) Circadian rhythm in the Hill reaction of Acetabularia. In: Avron M (eds) Proceedings of the 3rd international congress on photosynthesis. Elsevier, Amsterdam, pp 745±751

References Borowitzka M (1977) Algal calci®cation. In: Barnes H (ed) Oceanography and marine biology, an annual review. Aberdeen University Press, Aberdeen, pp 189±224 Borowitzka M (1982) Morphological and cytological aspects of algal calci®cation. Int Rev Cytol 74: 127±162 Borowitzka M (1987) Calci®cation in algae: mechanisms and the role of metabolism. In: Conger BV (ed) CRC critical reviews in plant sciences. CRC Press, Boca Raton, pp 1±46 Borowitzka MA, Larkum AWD (1976) Calci®cation in the green alga Halimeda: III. The sources of inorganic carbon for photosynthesis and calci®cation and a model of the mechanism of calci®cation. J Exp Bot 27: 879±893 Bouget F-Y, Gerttula S, Shaw SL, Quatrano RS (1996) Localization of actin mRNA during the establishment of cell polarity and early cell division in Fucus embryos. Plant Cell 8: 189±201 Bowles EA, Allen NS (1986) A vibrating probe analysis of lightdependent trancellular currents in Acetabularia. In: Nuccitelli R (ed) Ionic currents in development: proceedings of a satellite meeting to the Tenth International Congress of the International Society of Developmental Biologists. Liss, New York, pp 113±121 Buddemeier RW (1994) Symbiosis, calci®cation and environmental interactions. In: Doumenge F (ed) Past and present biomineralization processes. Considerations about the carbon cycle. Musee Oceanographique, Monaco, pp 119±136 Chau R, Bisson MA, Siegel A, Elkin G, Klim P, Straubinger RM (1994) Distribution of charasomes in Chara: re-establishment and loss in darkness and correlation with banding and inorganic carbon uptake. Aust J Plant Physiol. 21: 113±123 Dazy A-C, Borghi H, Garcia E, Puiseux-Dao S (1986) Transcellular current and morphogenesis in Acetabularia mediterranea grown in white, blue and red light. In: Nucitelli R (ed) Ionic currents in development: proceedings of a satellite meeting to the tenth international congress of the international society of developmental biologists. Liss, New York, pp 123±130 Ferrier JM (1980) Extracellular ion transport. J Theor Biol 85: 739± 743 Fisahn J, Lucas WJ (1995) Spatial organization of transport domains and subdomain formation in the plasma membrane of Chara corallina. J Memb Biol 147: 275±281 Gradmann D (1989) ATP-driven chloride pump in giant alga Acetabularia. Methods Enzymol 174: 490±504 Gradmann D, Tittor J, Goldfarb V (1982) Electrogenic Cl) pump in Acetabularia. Philos Trans R Soc London Ser B 299: 447±457 HaÈmmerling J (1936) Studien zum Polaritatsproblem I-III. Zool Jahrb Abt Allg Zool Physiol Tiere 56: 439±486 Hay ME, Kappel QE, Fenical W (1994) Synergisms in plant defenses against herbivores: Interactions of chemistry, calci®cation and plant quality. Ecology 75: 1714±1726 Hunt BE, Mandoli DF (1992) Axenic cultures of Acetabularia (Chlorophyta): a decontamination protocol with potential application to other algae. J Phycol 28: 407±414