Photopolarization of the Fucus sp. Zygote by Blue Light lnvolves a Plasma Membrane Redox Chain

Plant Physiol. (1994) 105: 519-527

Photopolarization of the Fucus sp. Zygote by Blue Light lnvolves a Plasma Membrane Redox Chain'

Frederic Berger* and Colin Brownlee Marine Biological Association, The Laboratory, Citadel Hill, Plymouth, PL1 2PB, United Kingdom (F.B., C.B.);

and Laboratoire de Biologie Moleculaire et du Developpement, Ecole Normale Superieure de Lyon, 46 allee d'ltalie, F-69364 Lyon Cedex 07, France (F.B.)

Zygotes of fucoid algae are photopolarized by unidirectional blue light (BL). Polar axes are formed, fixed, and expressed by germination of a rhizoid. Hexacyanoferrate(ll1) ions (HCF) specifically inhibit transduction of the BL signal. HCF reduction by Fucus sp. zygotes occurs on the outer surface of the plasma membrane at higher rates in BL than in dark. These observations suggest that BL signal transduction involves a redox chain in the plasma membrane. Low doses of HCF (e50 pmol cell-') inhibit photopolarization but not germination, hence uncoupling both processes. Expo- sure during the photosensitive period to higher doses of HCF together with BL significantly inhibits germination. Further results suggest that BL transduction is dependent on photosynthetic products that could also interact with redox processes.

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Fucoid zygotes are apolar following fertilization (Jaffe, 1958; Brawley et al., 1976). Polarity is acquired within a few hours according to environmental cues (reviewed by Kropf, 1992). A reversible polar axis is initially formed, then fixed irreversibly and expressed as the gennination of a rhizoid that anchors to the substratum. The first asymmetric cell division subsequently occurs perpendicular to the polar a i s and establishes the apical-basal polarity of the multicellular plant. A major environmental factor involved in polarization is light (Dring, 1987). The rhizoid of the Fucus zygote ger- minates on the side opposite to the prevailing light source (Hurd, 1920). Photopolarization in fucoid algae is specifically induced by BL and near-UV light (Hurd, 1920; Jaffe, 1958; Bentrup, 1963). BL also controls many aspects of plant development including growth, tropism, and sexual maturation (Dring, 1987; Kaufman, 1993). Although mutants of Arabi- dopsis defective in their response to BL have been characterized (Chory, 1993) and BL has been shown to affect the phosphorylation state of specific proteins (Short and Briggs, 1990; Reymond et al., 1992a, 1992b; Palmer et al., 1993a, 1993b), the mechanism of BL action is still largely unknown (Chory, 1993) and the nature of BL receptors is still under investigation (Song, 1987; Vani and Raghavendra, 1989; Ahmad and Cashmore, 1993; Quinones et al., 1993).

HCF (femcyanide) has been used as an extemal electron acceptor in severa1 studies of plasma membrane redox proc-

This research was supported by the Marine Biological Assoaation and the Ecole Normale Superieure de Lyon.

* Corresponding author; fax 44-752-226-865.

esses (Mdller and Crane, 1990). BL-induced opening of stomatal guard cells (Zeiger and Hepler, 1977) is associated with proton extrusion and changes in membrane conductance (Assmann et al., 1985; Shimazaki et al., 1986; Gautier et al., 1992). These responses to BL are inhibited by extemal appli- cation of HCF (Gautier et al., 1992), which is reduced to HCF(I1) on the outer side of the plasma membrane by guard cells (Lascève et al., 1993) and guard cell protoplasts (Gautier et al., 1992). These observations have been interpreted in terms of the presence of a redox chain in the plasma membrane as a putative candidate for BL signal transduction (Raghavendra, 1990; Gautier et al., 1992; Rubinstein and Luster, 1993).

Photopolarization of the Fucus zygote involves the establishment of localized gradients of free cytoplasmic calcium ions (Ca2+vt) (Speksnijder et al., 1989; Berger and Brownlee, 1993), which may originate from spatial regulation of ion channels (Kropf, 1992). Therefore, we have investigated the effect of HCF on photopolarization of the Fucus zygote. We report here that low doses of HCF specifically inhibit photopolarization and uncouple this process from gennination, which is affected only by higher doses, probably via altered metabolic status.

MATERIALS AND METHODS

Handling of Cametes and Zygotes

Male and female plants of Fucus serratus L. and Fucus spiralis L. were washed and stored in D at 4 O C . The fronds were induced to shed gametes in FSW (0.2-pm filter; Milli- pore, Watford, Hertsfordshire, UK) by a brief light treatment (Jaffe, 1954). Eggs of F. serratus L. were filtered through a 90-pm nylon mesh and washed three times with FSW. Sperm were collected from droplets of spermatophores from the surface of dry male receptacles and activated in FSW. Addi- tion of sperm suspension to batches of eggs was taken as the time of fertilization ( t = O). F. spiralis L. is monoecious and fertilization occurs readily during gamete release (t = O), after which zygotes were treated as for F. serratus L. eggs.

In most experiments, 2- to 3-h-old zygotes were settled in small Petri dishes (3.5 cm in diameter) and incubated in D

Abbreviations: BL, blue light; D, dark; FSW, filtered sea water; HCF, hexacyanoferrate(II1); HCF(II), hexacyanoferrate(I1); RL, red light; WL, white light.

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until the beginning of the experiment. Unless otherwise stated, zygotes were incubated at 17OC in 3 cm3 of extemal medium. Mutual polarization was avoided by keeping zygote density below 50 zygotes cm-' (Whitaker and Lowrance, 1936).

HCF Treatments

A 1 0 0 - m HCF stock solution was freshly made before each experiment. Light and HCF treatments were given 6 h after fertilization unless otherwise stated. HCF doses are expressed as mo1 HCF cell-' together with the duration of the treatment. Total dose per cell was varied by varying the volume of HCF solution (between 2 and 20 cm')). HCF is reduced by zygotes so that [HCF] cannot be used in dose- response experiments unless the total number of mo1 of HCF exceeds the reduction capacity of the cells, which was not the case in some experiments (see below). WL (50 pmol m-' s-') was provided in the incubator by cool-white fluorescent tubes. RL (610 nm; 250 pmol m-' s-') and BL (450 nm; 30 pmol m-' s-') were provided by a cold light source through 10-nm band-pass interference filters (Andover, Salem, NH).

Percentage zygote germination (n > 200) was scored after 24 h and percentage photopolarization was calculated as [(no. of rhizoids germinated from the hemisphere away from the light in the batch)/(total no. of rhizoids germinated)]. Zygotes with their rhizoid in the hemisphere opposite the light source were counted as 1 and others as O. Zygotes with their rhizoid perpendicular to the light vector were counted as one-half. Thus, a total inhibition of photopolarization occurs at 50% polarization. Experiments were repeated at least three times. In a11 experiments SE values were < 5%.

To study the polarization of zygotes toward their nearest neighbor (group effect [Hurd, 1920]), high zygote densities (>200 zygotes cm-') were used to form small groups of zygotes. HCF was added as zygotes settled in the Petri dish. The efficiency of the group effect was assessed after 26 h by counting the number of zygotes that had germinated in the half plane oriented toward the group.

under various light regimes by the change in A420 (E420 = 1 m-' cm-'). Optical density of the blank (FSW solution) was automatically subtracted from the measurements.

Localization of HCF Reduction

Three-hour-old F. spiralis L. zygotes were settled on cover- slips and incubated for 3 h at 17OC under unilateral WL (50 pmol m-" s-'). A 1.2% (m/v) agar gel(2 mm thick) containing 100 p~ IHCF was placed carefully over a coverjlip coated with zyE;otes so that they were sandwiched and slightly embedded in the gel. After 12 h of incubation in D or under unilateral WL, gels were exposed for 3 h to 10 m FeCL solution. Excess Fe3+ forms a blue precipitate with HCF(I1) (Prussian blue) that is stable at acidic pH. At higher pH values (:>4), Prussian blue forms colloidal suspensions and diffuses slowly in agar gels (Bell et al., 1988). Fe3+ forms a brown precipitate with HCF.

RESULTS

HCF Effect on Photopolarization

The period when zygotes respond to unidirectionai WL by polarizing according to the light vector (photosensitive period) (Whitaker and Lowrance, 1936) was detennined to be 5 to 1 2 h after fertilization (Fig. 1). Six-hour-old F. serratus zygotes photopolarized according to the light vector when submitted to 2 h or to continuous WL (Fig. 2). When HCF (3 x 103 pmol cell-') was added during the light treatment, half of the zygotes germinated on the side facing the light source (i.e. no photopolarization), which was identicai to results with D controls. This treatment reduced perceniage germination by 21%. However, when HCF was applied transiently for 2 h in D, germination and photopolarization ieached the leve1 of the continuously irradiated control.

A dose-response curve was obtained by subm tting Fucus zygotes i(6- to 8-h-old) to a 3-h light pulse with increasing doses of HCF (up to 6 X 103 pmol cell-') (Fig. 3). Both F. serratus ,and F. spiralis gave similar results. Mean half inhibition of photopolarization occurred at 47.8 pmol HCF cell-'

Determination of the Photosensitive Period

Zygotes were incubated in FSW at 17OC in D for various times followed by exposure to a 2-h unidirectional WL pulse and retumed to D. Photopolarization was scored as described above.

Spectrophotometric Measurement of HCF Reduction

Three hours after release, approximately 5 X 103 F. spiralis L. zygotes were introduced into a 1-cm3 cuvette placed hori- zontally so that they attached to the side parallel to the light beam of a spectrophotometer (Cary lE, Varian, Victoria, Australia). After 2 h the cuvette was inverted so that remaining zygotes attached to the opposite side, producing a cuvette coated with a layer of zygotes on two sides parallel to the light path. This avoided the need to agitate the solution during the experiment after the initial addition of HCF, eliminating the need to monitor the turbidity of the suspension (Gautier et al., 1992). HCF reduction was monitored

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Figure 1. Determination of the photosensitive period for Fucus zygote photopolarization. Fucus zygotes were kept in 13. At various times after fertilization (x) a population was treated with 2 h of unidirectiional WL and returned to D. Percentage photopolarization was scored at 28 h postfertilization, independent of when the WL treatment was given. Mean percentage germination W , ~ S 95%.

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Redox Processes in Fucus Zygote Polarization by Blue Light

%Germination H %Photopolarization

1 2 3 4 5 Treatment

B

D Vo Photopolarization D Yo Germination

100 ,

26 t (h) after fertilization

6 8

1 1 I s2- 5 3 1 ; 4 [ H C F

c

E

5- I

Figure 2. Effect of HCF on Fucus zygote photopolarization and germination. Treatments are described in 8. Polarization and germination were scored at 26 h postfertilization. White boxes (in B) indicate illumination with unilateral WL unless otherwise stated. Black boxes correspond to incubation in D. Zygotes were kept in D prior to the onset of the HCF and/or light treatment. HCF dose was 3 x 103 pmol cell-'.

(SE = 8.5, n = 6). Higher doses of HCF (up to 3 X 104 pmol cell-') reversibly inhibited germination and rhizoid growth without any visible deterioration of the zygote. Half inhibition of germination required higher doses (at least 10 times higher than was required for half inhibition of polarization). Very high doses (>3 X 104 pmol cell-') of HCF applied for long illumination periods (>5 h) were eventually lethal, producing visible deterioration leading to zygote death. When the length of the HCF treatment was reduced to 2 h, germination was not inhibited by more than 60% even with high doses (>3 X 103 pmol cell-I). For any given HCF concentration, higher doses (obtained by varying the volume of incubation solution) were more efficient at inhibiting photopolarization (not shown).

Reversibility of HCF-mediated inhibition of photopolarization and germination was tested by submitting photosensitive 6-h-old F. spiralk zygotes to HCF (103 pmol cell-I) + unilateral WL for 1 h. HCF was subsequently washed out and zygotes were maintained under unilateral WL for increasing durations followed by retum to D. Germination was scored at 28 h postfertilization (Fig. 4). The second WL treatment relieved the initial inhibition of both photopolarization and germination induced by HCF. However, full re-

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Log(pmo1 HCF ce~t-')

Figure 3. Dose response of the effect of HCF on photopolarization and germination (mean k SE, n = 6) . Dashed lines represent HCF doses for half inhibition of photopolarization and germination. At 8 h zygotes were submitted to 3 h of unidirectional WL and variable HCF doses. Responses were measured at 28 h postfertilization.

Z XGermlnatIon ---e-- %Photopolarisatlon

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Figure 4. Recovery from HCF treatment by WL illumination. Six- hour-old zygotes were submitted for 1 h to 103 pmol cell-' HCF + WL, followed by washing with FSW and illumination with unidirectional WL for various times (x), then returned to D. Photopolariza- tion and germination were scored 28 h postfertilization.

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522 Berger and Brownlee Plant Physiol. Vol. 105, 1994

covery required unilateral WL for at least 5 h (cf. 2 h incontrols, Fig. 2A) to obtain near-maximal photopolarization.

HCF Reduction by Fucus Zygotes

In the presence of Fe3+ and HCF, blue staining character-istic of HCF(II) was observed in gels containing zygotesexposed to WL (Fig. 5A). Gels containing zygotes and incu-bated in D (Fig. 5B) and gels without zygotes (Fig. 5C) giventhe same light treatment showed an overall brown stainingindicative of absence of significant HCF photoreduction.Zygotes that remained viable following staining were sur-rounded by blue precipitate, which was more prominent onthe side facing away from the incident light (Fig. 5E). Thisappeared to be the side receiving the highest light intensity(Fig. 5D). Germinated rhizoids showed an apparently higherdegree of HCF reduction as indicated by intense blue staining(Fig. 5F). Blue staining was not observed inside the cell,which is consistent with the impermeability of membranesto HCF (Izawa, 1980).

Reduction of HCF by Fucus zygotes was confirmed byspectrophotometric measurements (Fig. 6). Five experiments

conducted on different batches of zygotes gave qualitativelyconsistent, although quantitatively variable, results. Four- to8-h-old F. spiralis zygotes were kept in D for at least 5 hbefore addition of 500 JJM HCF. After an initial enhancedrate of HCF reduction, zygotes reduced HCF in D at a steadyrate of 21.3 nmol h"1 cm"2 plasma membrane (SE = 5.4, n =4). Under continuous RL or BL the steady rate of HCFreduction following the initial enhanced rate was increased(RL: 34.2 nmol h"1 cm"2 plasma membrane [SE = 8.8,n = 4]; BL: 41.8 nmol h"1 cm"2 plasma membrane [SE = 12.85,n = 4]). The steady-state reduction rate in BL was not alteredby subsequent RL (Fig. 6A). However, after an initial illumi-nation with RL (100 min), BL triggered a large initial increasein HCF reduction rate followed by a steady-state reductionrate higher than under initial RL conditions (1.4-2.8 times)(Fig. 6B).

HCF Inhibition of Rhizoid Germination IsDependent on BL

Figure 7 shows the light dependence of HCF inhibition ofrhizoid germination. HCF + BL during the photosensitive

Figure 5. Localization of HCF reduction by Fucus zygotes. A-C, Photographs of agar gels containing 100 JIM HCFobserved after reaction with 10 mM FeCI3 at 28 h postfertilization. A, Twelve hours of unidirectional WL + zyogtes. B, D+ zygotes. C, Twelve hours of unidirectional light (without zygotes). D, Light path in a zygote illuminated laterally withunidirectional WL (arrow). E and F, Microscopical observations of zygotes embedded in gel A. Arrows indicate the lightdirection. Scale bars represent 5 mm in A-C, and 10 pm in D-F.

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Redox Processes in Fucus Zygote Polarization by Blue Light 523

500- 4.

y 400- LL

300 1 I

during the light treatments indicated above t h e traces. HCF at 500 PM was introduced at t = O h. A and B show the results from two different populations of zygotes from the same batch. g f q LL

Y 400

300

period severely inhibited germination (60.2% inhibition of BL control) and reduced rhizoid growth rate (not shown). In contrast, HCF + D and HCF + RL gave only 16.0% and 7.9% inhibition of germination, respectively, and had little effect on subsequent zygote development. HCF inhibition of germination thus appears to be specific to BL. Inhibition of germination by HCF (500 pmol cell-') increased with duration of BL treatment (Fig. 8, treatments 4, 5, 6), being hardly detectable after 1 h and increasing strongly for light treatments longer than 2 h. Inhibition mediated by HCF + BL was also markedly dependent on the photosensitive status of zygotes (Fig. 8, treatments 1, 2, 3, 6), being effective only if applied during and not prior to the photosensitive period.

Effect of HCF on Mechanisms lnvolved in Polarization

HCF(II), which is produced during treatment with HCF, could act as an electron donor that may interfere with a redox chain and inhibit polarization. However, zygotes illuminated for 3 h with HCF(I1) (300 pmol cell-') developed and photopolarized correctly. Zygotes kept in D germinated toward their neighbors (group effect) when separated by less than 300 pm (97%, n = 250) (Fig. 9A). HCF treatment (100 pmol cell-') that fully inhibited photopolarization (see Fig. 11, column 6) did not inhibit the group effect (96.2% conformed

- . <L . BL +'HCF . RL :HCF . D + 'HCF .

Treatment

Figure 7. BL dependence of HCF-mediated inhibition of germination. Six-hour-old zygotes were treated as indicated. HCF dose was 500 pmol cell-'. Light treatments were for 3 h, followed by 15 h of D, after which percentage germination was scored.

to the group effect, n = 186) (Fig. 9B). HCF thus appears to inhibit photopolarization specifically.

We examined the effects of HCF on axis formation and axis fixation (Fig. 10). During axis formation (6-9 h), a 3-h pulse of unidirectional light oriented 92.6% of rhizoids. How- ever, a subsequent treatment with HCF (with the same light vector) substantially reduced the number of zygotes germinating according to the first light pulse, whereas a subsequent treatment with HCF in D had little effect. HCF thus inhibits axis formation and can partially disrupt a preformed axis. Moreover, these effects are light dependent. During axis fixation HCF is as inefficient at resetting axes previously fixed

A

1 2 3 4 5 6 Treatmsnt

B o 2 5 6 7 8 9 12 26 t (h) afier

IPhotosensitive petiod] I fertilization

1 I I I - 2 -- iii e 4 -!%i 5 -- 6 - HCr - - c

E 3 I- I-

Figure 8. Dependence of HCF effect on the photosensitive period. Percentage germination was scored at 26 h postfertilization (A). Zygotes were treated with BL + HCF (500 pmol cell-') before or during the photosensitive period as shown (B).

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Figure 9. HCF action on polarization due tothe "group effect." A, Mutual zygote polariza-tion in a dense culture incubated in FSW in D(26 h postfertilization). B, As in A but with 20 hof incubation in FSW + HCF (100 pmol cell"').Scale bars represent 20 fim.

as a second light treatment in the opposite direction. Weconclude that HCF inhibits axis formation but not axisfixation.

The end of the photosensitive period coincides with thebeginning of axis fixation. This was determined using 3-htreatments with unilateral WL at various times after fertiliz-

ation (Fig. 11). At 10 h, a 3-h treatment combining WL andHCF (100 pmol cell"1) inhibited photopolarization. Similarly,a 7-h treatment with HCF fully inhibited photopolarization.At the end of such a treatment (17 h after fertilization) axesare fixed in control untreated zygotes. However, a subsequent3-h light pulse photopolarized previously HCF-treated zy-gotes to the same extent as during the physiological photo-

B

3 4 5Treatment

: Light direction

c Axisformation n Axis

fixation

9 10 12 13 16 28 t (h) afterfertilization

Figure 10. Effect of HCF on axis formation and fixation (A). Zygoteswere kept in D prior to treatment. After an initial treatment withunilateral WL, zygotes were given a second treatment of equalduration (B). HCF dose was 300 pmol cell"'. Control treatmentsconsisted of single WL irradiation. Percentage photopolarizationaccording to the first light vector was scored at 28 h postfertilization.

B

2 3 4 5Treatment

: Light direction

formationAxisfixation

10 13 16 17 20 28 t (h) afterfertilization

Figure 11. Uncoupling of axis formation and fixation by HCF. A,Zygotes were submitted to HCF (100 pmol cell"') at various timesand for variable periods. Timing of the photosensitive period wasdetermined (1, 3, 4) and treatment with HCF begun during thephotosensitive period and ended after the beginning of axis fixation(6). A subsequent treatment with FSW + WL in the oppositedirection tested percentage axis fixation (5). Percentage photopo-larization according to the first light vector was scored at 28 hpostfertilization. B, Treatment schemes were as shown.

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sensitive period, suggesting that HCF treatment prolonged or delayed the photosensitive period.

DlSCUSSlON

HCF Specifically lnhibits Axis Formation during fucus Zygote Photopolarization

Low HCF doses (<50 pmol cell-I) inhibit photopolarization of Fucus zygotes without significantly affecting germination of the apically growing rhizoid. Thus, HCF uncouples photopolarization from germination. Moreover, HCF does not inhibit mutual zygote polarization (the group effect) and thus appears to inhibit a process specific to photopolarization. Photopolarization is achieved in two steps, axis formation followed by axis fixation (Quatrano, 1973; Kropf, 1992). We have shown that low doses of HCF that inhibit photopolarization can also reset an already formed photopolar axis but are inactive on a fixed axis. Resetting the photopolar axis requires the presence of light during HCF treatment. HCF thus appears to specifically prevent axis formation in response to BL. Under the conditions employed here, axis formation occurs over severa1 hours and it is not clear to what extent transduction of BL is distinct from axis formation. However, there is evidence for coincidence between the two phenomena. It has been shown that a polar axis can be formed with very short pulses of high-intensity BL and that the duration required to form an axis is inversely dependent on BL intensity (Bentrup, 1963). This suggests that BL signal is transduced continuously until an axis is fonned. We also report here that photopolarization is insensitive to HCF before the onset of the photosensitive period, i.e. before mechanisms for BL transduction are active. Moreover, HCF treatment delays the end of the photosensitive period during which axis formation takes place. In other words, HCF inhibits axis formation continuously and since the inhibition is reversible, axis formation occurs when the inhibition is relieved, however long its duration. The data suggest that axis formation occurs downstream from BL perception.

HCF Interferes with Redox Processes

HCF is reduced extemally by Fucus zygotes. Spectropho- tometric measurements show that reduction takes place both in D and under BL or RL. However, steady-state HCF reduction rates in D are lower than under continuous RL or BL, and a pulse of BL after RL illumination triggers an increase in reduction rate. Reduction rates were of the order of 5 to 50 nmol h-' cm-* plasma membrane, comparable with those reported in other systems (Neufeld and Bown, 1987; Gautier et al., 1992). Thus, at very low HCF doses (<5 pmol cell-') that did not inhibit photopolarization (Fig. 3), virtually a11 HCF would be reduced to HCF(I1) within 2 h. Since the inhibition is reversible (see Figs. 4 and ll) , zygotes would be expected to photopolarize during the remaining hour of a 3- h treatment. Confirming this assumption, we also have observed that for the same HCF concentration, higher doses inhibit photopolarization more efficiently. This also shows that HCF reduction by the zygotes (and not HCF itself) mediates the inhibition of photopolarization.

Redox chains are present in the plasma membranes of

plant cells (Rubinstein and Luster, 1993). Although secretion of peroxidase into the extracellular medium by stomatal guard cell protoplasts can account for some reduction (Pantoja and Willmer, 1991), there is convincing evidence that the plasma membrane is itself the site of exogenous HCF reduction in numerous cell types (Rubinstein and Luster, 1993). HCF reduction has been localized by staining to the outer face of the guard cell plasma membrane (Vani and Raghavendra, 1989; Lascke et al., 1993). Photopolarizing Fucus zygotes stained for HCF(I1) showed HCF reduction sites on the outer side of the plasma membrane. We propose that Fucus zygotes reduce HCF by a redox chain present in the plasma membrane as has been proposed for stomatal guard cells (Vani and Raghavendra, 1989; Gautier et al., 1992) and other plant cell types (Rubinstein and Luster, 1993). Furthermore, the degree of reduction was higher at the side of the zygote receiving higher irradiance, i.e. the side facing away from the light source. Although some scattering artifacts could be present in Figure 5D (T. C. Vogelmann, personal communi- cation), further observations (F. Berger and C. Brownlee, unpublished data) indicate strongly that this is not the case and that a mechanism of light focusing exists in Fucus zygo tes .

61 Transduction 1s Dependent on the Redox State of the Cell

HCF inhibits BL-induced swelling of guard cell protoplasts (Gautier et al., 1992). HCF thus appears to specifically inhibit BL signal transduction both in Fucus zygotes and in guard cells. In guard cells, BL on a RL background transiently increases acidification of the extemal medium (Shimazaki et al., 1986; Gautier et al., 1992) and oxygen consumption (Gautier et al., 1992). HCF increases the acidification rate and abolishes the responses to BL. Acidification of the extemal medium has been interpreted in terms of proton efflux induced by a BL-stimulated ATP-dependent outward current (Assmann et al., 1985). A proton pump is responsible for a major part of the BL-induced current (Assmann et al., 1985; Amodeo et al., 1992), although it has also been hypothesized that a redox chain could account for proton extrusion (Rha- gavendra, 1990; Gautier et al., 1992; Kaufman, 1993; Rubin- stein and Luster, 1993). If a unidirectional proton extrusion were involved, BL and HCF would trigger a cytoplasmic alkalinization. This does not occur in Elodea densa (Marré et al., 1988) or in the zygote of another fucoid alga, Pelvetia (Gibbon and Kropf, 1993). Unlike a proton pump, a redox chain can acidify both externa1 and interna1 media simulta- neously (Marré et al., 1988).

In guard cell protoplasts RL influences the intensity of the response to BL (Gautier et al., 1992). Reduced equivalents could be used by a redox chain providing electrons on the cytoplasmic side of the plasma membrane (Raghavendra, 1990; Gautier et al., 1992). Such a process would tend to lower the leve1 of cytoplasmic reduced equivalents. There is indirect evidence for the involvement of a similar mechanism in Fucus zygote photopolarization. High doses of HCF (3 x 103 pmol cell-') inhibit rhizoid germination. Thk inhibition is specifically dependent on co-treatment with BL and is not effective before the photosensitive period. Inhibition of ger-

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mination by HCF thus appears to be a side effect of its action on photopolarization. Preliminary observations indicated that this inhibition could be a consequence of the alteration of a factor or factors produced by light. HCF-mediated inhibition of germination is higher in D than in RL, although the HCF reduction rate is higher in RL. HCF inhibition is reversible but subsequent illuminations for more than 5 h (instead of 2 h in controls) are necessary to obtain maximal photopolarization. HCF treatment thus delays subsequent recovery of photopolarization, presumably by altering some light- dependent intracellular factors. Such factors must be essential for cell viability, since prolonged HCF inhibition of germination eventually leads to cell death. Reduced equivalents such as NADH + H+ would be likely candidates. Increases in HCF reduction triggered by low-intensity BL after a high- intensity RL period indicates that BL increases the activity of a putative redox chain in the plasma membrane. This would also lead to increased consumption of reduced equivalents and could account for the toxic effect of the combination BL + HCF.

BL + HCF could inhibit photopolarization according to the following mechanism. BL focused to the rear side of the zygote increases plasma membrane redox chain activity in this region, thereby providing spatial information. HCF could activate the redox chain over the whole surface of the zygote and consequently erase the spatial information provided by BL. Altematively, HCF could uncouple BL-induced electron transport directly from signal transduction. The nature of this spatial information remains unknown. However, it is likely that BL creates a labile memory of a map of BL intensities on the plasma membrane.

A transient memorization of BL signal has been demon- strated in stomatal guard cells (Vavasseur et al., 1990). This phenomenon could be explained by BL-induced phosphorylation of specific proteins (Chory, 1993). Various proteins with molecular masses around 110 kD whose phosphorylation state is affected by BL have been characterized (Short and Briggs, 1990; Reymond et al., 1992a, 1992b; Palmer et al., 1993a; Short et al., 1993), but their functions remain to be established. Tissue-specific control of phosphorylation of a 114-kD protein is involved in BL-induced phototropism of maize coleoptiles (Palmer et al., 1993b). Moreover, reducing agents enhance BL-induced phosphorylation in maize coleoptiles (Hager et al., 1993). It remains to be seen whether spatially regulated phosphorylation is involved in Fucus zygote photopolarization.

HCF as a To01 for Further lnvestigations

HCF uncouples photopolarization from germination. Moreover, HCF treatments prolong the photosensitive period, i.e. HCF prevents axis formation and consequently delays axis fixation. This demonstrates that axis fixation is not pro- grammed at a fixed time but depends on the completion of axis formation. Therefore, HCF can be used to manipulate axis formation and more generally photopolarization of fucoid zygotes. This could be useful in molecular studies to characterize gene products, the early transcription of which has been shown to be crucial for photopolarization (Kropf et al., 1989).

ACKNOWLEDCMENTS

We are grateful to Alain Vavasseur, Gerard Lasche, Darryl Kropf, Alison Ta ylor, and Sarah Assmann for their helpful suggestions during this work and their comments during the preparation of the manuscript. We also thank Anne Matthews for her help with spectrophotometric measurements.

Received November 22,1993; accepted February 16,1994. Copyright Clearance Center: 0032-0889/94/105/0519/D9.

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Photopolarization of the Fucus sp. Zygote by Blue Light lnvolves a Plasma Membrane Redox Chain

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