Article

ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, No. 10, pp. 1455-1466 © Pleiades Publishing, Ltd., 2023.

Published in Russian in Biokhimiya, 2023, Vol. 88, No. 10, pp. 1761-1774.

1455

Electrical Signals at the Plasma Membrane

and Their Influence on Chlorophyll Fluorescence

of Chara Chloroplasts in vivo

Alexander A. Bulychev

1,a

*, Stepan Yu. Shapiguzov

, and Anna V. Alova

Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

e-mail: bulychev@biophys.msu.ru

Received May 31, 2023

Revised June 30, 2023

Accepted July 2, 2023

Abstract— Action potentials of plant cells are engaged in the regulation of many cell processes, including photosynthesis

and cytoplasmic streaming. Excitable cells of characean algae submerged in a medium with an elevated K

content are

capable of generating hyperpolarizing electrical responses. These active responses of plasma membrane originate upon the

passage of inward electric current comparable in strength to natural currents circulating in illuminated Chara internodes.

So far, it remained unknown whether the hyperpolarizing electrical signals in Chara affect the photosynthetic activity.

Here, we showed that the negative shift of cell membrane potential, which drives K

influx into the cytoplasm, is accom-

panied by a delayed decrease in the actual yield of chlorophyll fluorescence F′ and the maximal fluorescence yield F

′

under low background light (12.5 μmol m

–2

–1

). The transient changes in F′ and F

′ were evident only under illumination,

which suggests their close relation to the photosynthetic energy conversion in chloroplasts. Passing the inward current

caused an increase in pH at the cell surface(pH

), which reflected high H

/OH

–

conductance of the plasmalemma and

indicated a decrease in cytoplasmic pH due to the H

entry into the cell. The shifts in pH

arising in response to the first

hyperpolarizing pulse disappeared upon repeated stimulation, thus indicating the long-term inactivation of plasmalem-

mal H

/OH

–

conductance. Suppression of plasmalemmal H

fluxes did not abolish the hyperpolarizing responses and

the analyzed changes in chlorophyll fluorescence. These results suggest that K

fluxes between the extracellular medium,

cytoplasm, and stroma are involved in the functional changes of chloroplasts reflected by transients of F′ and F

′.

DOI: 10.1134/S0006297923100048

Keywords: Chara, electrical signaling, hyperpolarization, plasmalemmal K

conductance, chlorophyll fluorescence changes,

/OH

–

transport, plasmalemma–chloroplast interactions

Abbreviations: AP,action potential; APW,artificial pond water; Chl,chlorophyll; HR,hyperpolarizing response; NPQ,non-pho-

tochemical quenching; pH

,pH in water layers adjacent to the cell surface; PET,photosynthetic electron transport; PS,photosys-

tem; PSA,photosynthetic apparatus; YII,quantum yield of electron transfer.

* To whom correspondence should be addressed.

INTRODUCTION

Many processes in plant cells, such as photosyn-

thesis, signal transduction, and intracellular regulation,

are associated with changes in the electrical potentials

across the plasmalemma and organellar membranes.

Excitable membranes generate action potentials (APs)

in response to mechanical, chemical, and electrical

stimuli. Thylakoid membranes perform photosynthetic

electron transport(PET) coupled with the H

transfer

and electric potential generation. Drachev etal. [1] have

proposed an effective method for studying electrogen-

esis of phototrophic bacteria using polymer films and

adsorbed pigment–protein complexes and chromato-

phores. The use of short flashes allowed to overcome

some kinetic limitations, such as dark inactivation of

the photosystem I (PSI) acceptor side and slowing

of PET by the increasing thylakoid ΔpH. At the same

time, such limiting factors have a pronounced effect on

PET in isolated plastids and chloroplasts of whole cells,

BULYCHEV et al.1456

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

thus affecting the induction curves of membrane poten-

tial and chlorophyll (Chl) fluorescence [2, 3]. This ex-

plains a significant interest in the invivo studies of PET

regulation mechanisms, which are much more diverse

than in the preparations of thylakoids and isolated intact

plastids. One of these mechanisms involves regulation of

photosynthesis by electrical signals of the plasma mem-

brane.

Pulse-amplitude-modulated microfluorometry pro-

vides the means to monitor the effective quantum yield

of PSII photoreaction (YII) and relative rates of linear

PET on microscopic cell areas, as well as to follow rapid

transients in non-photochemical quenching(NPQ) that

reflect ΔpH in the thylakoids. The studies on the excit-

able cells of the alga Chara have revealed the involve-

ment of plasma membrane electrogenesis in the regula-

tion of photosynthetic electron flow. The generation of

AP in illuminated cells caused a rapid (~30 s) decrease

in YII by nearly 50% and increased NPQ for a period

of up to 15min [4,5]. Apparently, the composition of

the cytoplasmic constituents (ions and metabolites) is

labile and depends on the state of plasma membrane ion

channels. Experiments with alternating local and total

illumination of the internodal cell demonstrated that the

spatial heterogeneity of the cytoplasm, which is attribut-

ed to the presence of photosynthetically active and inac-

tive zones, remains hidden under stationary conditions,

when fluorescence is recorded in a given cell area, but

becomes apparent after excitation-induced reversible ar-

rest of cyclosis [6].

Changes in the cytoplasm composition are only one

factor in the transformation of plasmalemmal signals to

the functional manifestations in the chloroplasts. Anim-

portant stage in this process is the cytoplasm-to-stroma

transfer of ions, first of all, H

and Ca

[7, 8]. The in-

fluence of Ca

on Chl fluorescence is mediated by its

effect on the activity of Calvin cycle enzymes and by

accompanying pH shifts in the stroma and thylakoid

lumen. During AP generation, the cytoplasmic content

ofCa

in plant cells increases from 0.1 to 10-40 μM [9],

which promotes Ca

entry into illuminated plastids

due to the negative electric potential of the stroma [10].

Theincrease in stromal [Ca

] suppresses the Calvin cy-

cle and decreases consumption of ATP and NADPH in

its reactions [11]. Consequently, ΔpH in the thylakoids

increases, which promotes NPQ and slows down linear

electron flow [12].

The inner membrane of the chloroplast envelope

contains ion channels permeable to mono- and divalent

cations, as well as K

antiporters (KEA1/2) [13-15].

An important role of ion exchange mediated by KEA1/2

becomes evident as photosynthesis disorders, e.g., in

mutant plants grown on media with different salt com-

positions. In experiments with isolated chloroplasts,

removal from the external medium lowered stromal

pH and inhibited photosynthetic O

evolution due to

theK

exchange [16]. At the same time, it remains

unclear whether the treatments that elevate the cytoplas-

mic and stromal K

levels affect the functional param-

eters of chloroplasts in short-term experiments invivo.

The electrical activity of characean cells is not lim-

ited to the AP generation. Stimulation of internodal cells

by inward electric current in the presence of elevated K

concentrations in the medium induces active hyperpo-

larizing response (HR) manifested as nonmonotonic

changes in the membrane potential [17, 18]. It should be

noted that electric currents inducing HRs are compara-

ble in their strength to natural electric currents circulat-

ing between different parts of illuminated cell. Thein-

creasing shift in the membrane potential during HR

results from partial suppression of K

conductance and

activation of other yet poorly studied electrogenic mech-

anisms. So far, there are no data on a possible influence

of HR on Chl fluorescence in charophytes. However,

this information is important because ion fluxes associ-

ated with the plasmalemmal HR differ principally from

the AP-triggered fluxes. During HR measurements, the

studied part of the cell is bathed with a K

-rich medi-

um (K

concentration, ~100 mM) and is completely

depolarized. The negative shift of the cytoplasm electric

potential during the passage of inward current drives K

into the cell in amounts that exceed the fluxes of other

ions due to the increased membrane permeability for

and high K

concentration in the medium. Because

of the presence of envelope transporters, accumulation

of K

in the cytoplasm should induce K

entry into the

chloroplast stroma in exchange for H

leaving the stroma.

In this case, the HR can modulate chloroplast fluorescence

via the K

exchange across the plastid membrane.

In this work, we studied the influence of plasma

membrane electrical signals in Chara and Nitella algal

cells on Chl fluorescence that served as an indicator of

photosynthetic apparatus(PSA) performance. We found

that hyperpolarization affected the actual fluorescence

yield F′ and maximum fluorescence yield F

′ in the

cells exposed to continuous background illumination.

The results revealed the relationship between the plas-

malemmal HR, H

influx into the cytoplasm, and Chl

fluorescence parameters. These findings expand current

knowledge on the regulation of photosynthesis by the

plasma membrane electrical activity.

MATERIALS AND METHODS

Experiments were performed on the internodal cells

of Chara australis R. Br. and Nitella translucens (Pers.)C.

Agardh obtained from the laboratory of Professor Ilse

Foissner (University of Salzburg). The algae were

grown in glass vessels under room lighting. The inter-

nodal cells 40-50 mm in length and 0.9 mm (Chara)

and 0.3 mm (Nitella) in diameter were excised from the

FLUORESCENCE OF Chara CELLS UPON HYPERPOLARIZATION 1457

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

thalli and placed in artificial pond water (APW) con-

taining 0.1 mM KCl, 1 mM NaCl, and 0.1 mM CaCl

NaHCO

was added to adjust the solution pH to 7.0.

Isolated cells were kept in APW for at least one day after

their excision from adjacent internodes.

Chara internodes that have completed their growth

are structurally heterogeneous and contain cytoplasmic

regions with a high and low content of charasomes that

alternate along the internode length. Charasomes are

special organelles enriched with H

-ATPase that facili-

tate the entry of photosynthetic substrate CO

into the

cells [19]. The spatial heterogeneity of internodes is also

manifested as an alternation along the cell axis of calci-

fied and crystal-free areas on the cell surface. Thecal-

cified areas are typically located in the regions with low

photosynthetic activity and high pH in the perimem-

brane layer of the outer medium. Chl fluorescence and

plasma membrane potential were measured in noncalci-

fied or weakly calcified cell areas.

An internodal cell was mounted in a three-section

transparent chamber (Fig. 1) and placed on a stage of an

Axiovert 25-CFL inverted microscope (Zeiss, Germany)

equipped with a Microscopy-PAM fluorometer (Walz,

Germany). The sections of the experimental chamber

were separated with air gaps 4 mm in diameter to ensure

their electrical insulation. The surface area of a Chara

cell segment occupying the central section was approx-

imately 0.1 cm

. The solution in the central section was

in contact with an Ag/AgCl reference electrode (sur-

face area, ~15 cm

) via a flexible salt bridge filled with

1 M KCl solution and 2% agar. The electric potential dif-

ference across the plasma membrane was measured using

Pyrex glass capillary micropipettes in the cell segment

occupying the central compartment of the chamber and

recorded with a U5-9 electrometric amplifier (Russia).

The voltage from the amplifier was fed to aPCI-6024E

analog-to-digital converter (National Instruments,

USA) and a computer monitor.

The side compartments of the chamber contained

connected Ag/AgCl electrodes to which a command

voltage from the ADC/DAC (PCI-6024E) was applied

through a load resistance of 1.2 MΩ; these electrodes

served for passing stepwise pulses of electric current.

The current strength varied from 3 to 8 μA, which cor-

responded to the current density from 30 to 80 μA cm

–2

in the central part of Chara cell.

Chl fluorescence was measured on cell regions with

a diameter of ~100 μm. The actual yield of Chl fluores-

cence F′ was recorded under background illumination

of the entire cell with modulated blue light emitted by a

LED of a Microscopy-PAM instrument (Walz, Germa-

ny). Measuring light flashes with a duration of 5 μs were

emitted at a frequency of 24 Hz. The duration of the sat-

urating flashes produced by the same LED was800 ms.

A 2mm-thick red light filter RG645 (Schott, Germany)

was placed in front of the photomultiplier input window

(see description of a Microscopy-PAM fluorometer on

the Walz website https://www.walz.com/downloads.html).

Chl fluorescence of microscopic cell regions was mea-

sured as described by Goh et al. [20]. Background illu-

mination of the whole cell was provided by the micro-

scope upper light source through a blue-green (SZS-22,

λ < 580 nm) and neutral density (NS-3) glass filters at

the intensity of 12.5 μmol quanta m

–2

–1

. The effective

quantum yield of the electron transfer in PSII was es-

timated using saturating light pulses and WinControl-3

program (Walz, Germany). The PSII quantum yield

(YII) at background irradiance under control conditions

ranged from 0.7 to 0.745. The output signal from the

photomultiplier was processed with the WinControl-3

and WinWCP programs (Strathclyde Electrophysiology,

United Kingdom) and displayed on a computer monitor.

The influence of HRs on the maximum fluores-

cence yield F

′ was studied using saturating light pulses

separated by 10-s intervals. Initiation of a series of sat-

urating pulses was usually accompanied by a noticeable

decrease in F

′. For this reason, the cells were stimu-

lated with a stepwise current pulse after the reduction in

′ caused by saturating light pulses had slowed down.

pH at the cell surface (pH

) was measured with

glass-insulated H

-specific antimony microelectrodes

and a U5-9 electrometric amplifier [4]. The raw read-

ings of the pH microsensor were processed by subtract-

ing the background voltage drop created by the electric

current on a series resistance of the medium, salt bridge,

and reference electrode.

Cytochalasin D (Sigma-Aldrich, USA) was dis-

solved in dimethyl sulfoxide (DMSO) to obtain a stock

solution that was then diluted to the final concentra-

tion of 10-20 μM. The final concentration of DMSO

did not exceed 0.2%. At this concentration, DMSO did

not affect the measured parameters of Chl fluorescence,

membrane potential, and cytoplasmic streaming.

Fig. 1. Scheme of the three-section experimental chamber with the

internodal cell and electrodes: 1) central compartment filled with

KCl-enriched medium; 2)side compartments filled with APW; 3)in-

ternodal cell of Chara (Nitella) alga; 4)Ag/AgCl electrodes used for

passing electric current; 5) Ag/AgCl reference electrode common

forthe measuring circuit and current pathway; 6)flexible salt bridge;

7)electrode microsensor (intracellular capillary electrode for measur-

ing plasma membrane potential or pH-sensitive extracellular micro-

electrode for measuring local pH shifts on the cell surface); 8)air gaps

providing electrical insulation of the central and side compartments.

BULYCHEV et al.1458

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 2. HRs of Chara cells caused by passing an inward electric current in the presence of 0.1M KCl in the medium. a)HR kinetic curves at various

current densities: 1)33μAcm

–2

, 2)45μAcm

–2

, and 3)50μAcm

–2

. b)HR kinetic curves in the presence of 20μM cytochalasin D after transferring

the cell to darkness: 1)in the background light (photon flux density, 12.5μmol m

–2

s

–1

); 2-5) 0.5, 5, 10, and 15min, respectively, after switch-

ing off the background light. c)HR kinetic curves for the same cell in the presence of cytochalasin D after transition from the dark to the light:

1-3)0.5, 5, and 10min after transferring the cell to the light. Current density during electric pulses in(b) and(c) was 33μAcm

–2

. d)Effect of

light conditions on the time of attaining the HR peak (counted from the onset of inward current) in the absence of inhibitors and after arrest of

cyclosis with 20μM cytochalasinD(CD). The moment of CD addition is marked with an arrow. Vertical light and shaded areas mark the light

and dark periods, respectively; current density during the pulse was 58μAcm

–2

The results presented in the figures were obtained

from at least four cells. The averaged kinetic curves for

F′ and standard errors are presented with indication

of the total number of measurements (n) on different

cells (N). For averaging the F′ values obtained on dif-

ferent cells, the curves were normalized to the average

fluorescence level within the time interval of 0-25s re-

corded before the pulse application. The average F

′ ki-

netics for different cells was obtained after normalizing

′ values recorded in response to the first saturating

pulse in aseries of 30 flashes.

RESULTS

Under standard experimental conditions (cells

bathed in APW), the membrane potential of illuminated

Chara cells ranged from –170 to –210 mV. Depolariz-

ing current pulse (2μA, 150ms) elicited an AP with the

amplitude of up to 200 mV, which was accompanied by

a short-term stoppage of cytoplasmic streaming followed

by its recovery within ~5min at 25°C.

Effects of light and cytoplasmic streaming on the HR.

At the beginning of each experiment, we verified that

FLUORESCENCE OF Chara CELLS UPON HYPERPOLARIZATION 1459

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

the membrane potential at rest and during excitation

fell into the characteristic ranges and that the effective

quantum yield of the PSII-driven electron transfer was

0.7-0.745 to ensure an adequate physiological state of

the cell. After APW in the central section of the chamber

was replaced with APW containing 0.1 M KCl, the pas-

sage of a standard supra-threshold current pulse caused

a stepwise cell depolarization to ~0 mV. This state is

characterized by a high K

conductance of the plasma-

lemma [21] and the membrane potential corresponding

to zero level of equilibrium Nernst potential.

Subsequent passage of a rectangular inward current

pulse (30-80 μA cm

–2

; pulse duration, 50-75 s) was ac-

companied by the generation of an active HR (Fig. 2).

During the HR development, the negative membrane

potential increases at an accelerating rate towards a sharp

spike that is followed by partial depolarization [22].

At equal concentrations of K

in the medium and in the

cytoplasm (~100 mM), the negative shift in the mem-

brane potential causes K

ions to enter the cell due to the

high K

conductivity of the membrane.

The time required to achieve the peak hyperpolar-

ization reduced upon the increase in the density of in-

ward current (Fig.2a). At the same time, the stationary

potential established after the spike was nearly indepen-

dent of the current density. Both peak hyperpolariza-

tion and subsequent rapid depolarization were markedly

smoothed over after cell treatment with cytochalasinD,

an inhibitor of actin polymerization (Fig. 2, b and c).

Similar suppression was previously observed for the ac-

tion of cytochalasinB on the surface membrane ofiso-

lated chloroplast-free cytoplasmic droplets ([22] and ref-

erences therein). In our experiments, addition of 20 μM

cytochalasin D to the medium caused cessation of

cytoplasmic streaming. In the presence of this inhibi-

tor, the HR curves at a constant current density strong-

ly depended on the illumination conditions (Fig. 2,

b andc).

In the dark, the time toward the peak hyperpolar-

ization increased within approximately 20min (Fig. 2b),

but then restored back to shorter times after the cells

were illuminated again (Fig. 2c). Interestingly, the effect

of illumination on the HR was observed only in the pres-

ence of cytochalasin D but not under the control con-

ditions. As can be seen from Fig. 2d, the time required

to achieve the HR peak in the light and in the dark was

almost the same if cytoplasmic streaming remained un-

disturbed, but differed significantly when cyclosis was

blocked by cytochalasinD.

Thus, chloroplasts were able to affect plasmalem-

mal ion channels when the actin cytoskeleton was dis-

turbed. It is possible that the light–dark transitions al-

tered the composition of the cytoplasm, which modified

plasma membrane channels involved in the HR gener-

ation. However, this effect was prevented by active cy-

toplasmic streaming that smoothed over the differences

in the cytoplasmic composition between cell parts with

different photosynthetic activity.

Influence of hyperpolarizing response on the Chl

fluorescence yield F′. We have focused our attention

mainly on a possible impact of HR on Chl fluorescence

parameters indicative of the redox state of the PET

chain and energy-dependent dissipation of Chl excited

states(NPQ). Emergence of the driving force for theK

influx from the medium to the cytoplasm upon the neg-

ative shift in the plasma membrane potential should

be accompanied by K

accumulation in the cytoplasm,

which, in turn, can induce the K

exchange across

the chloroplast envelope membranes and affect Chl flu-

orescence.

Figure 3a and b shows changes in the fluores-

cence yield F′ induced by the pulse of an inward current

in C. australis and N. translucens internodes exposed

to low-intensity background light (12.5 μmol m

–2

–1

In both characean species, the F′ changes comprised

adelayed stage of fluorescence yield decrease that start-

ed approximately 25s after the onset of the current pulse

and continued for ~50s after the end of HR.

The decrease in F′ in N. translucens cells was pre-

ceded by the increase in fluorescence that started at

the first seconds after the current pulse application.

InC. australis cells, the increase in F′ preceding F′ re-

duction was observed occasionally, but not in the aver-

aged recordings. The opposite changes in F′ associated

with the HR indicate the occurrence of several processes

of different origin. Among them, only slow changes in

F′ can result from the K

exchange across the chlo-

roplast envelope membranes, since high cytoplasmic

concentration ([K

]

cyt

, ~100 mM) excludes its rapid

change. The reasons for the rapid increase in fluores-

cence in N. translucens cells remain unclear. It is pos-

sible that the inward electric current is associated with

the electroosmotic water inflow and visually inconspic-

uous swelling of the plastids. This is indirectly indicated

by the fact that the increase in F′ was clearly evident in

small-diameter cells (N. translucens) featuring a com-

paratively large surface to volume ratio.

The stage of delayed fluorescence decrease during

the HR generation was observed only in illuminated

C. australis and N. translucens cells, but not in the cells

placed in the dark (Fig. 3, c and d). In contrast, the

stage of F′ increase clearly visible in N. translucens cells

was independent of illumination conditions. This con-

firms the assumption that the described opposite chang-

es in fluorescence have different nature.

Influence of HR on the fluorescence yield F

′.

In Chara internodes placed into standard APW with

0.1 mM K

, F

′ in low light approached the highest val-

ues, which indicated the absence of NPQ. Replacement

of APW with the medium containing 0.1 M KCl led to a

noticeable decrease in F

′, i.e., to the NPQ development.

In this connection, we examined the effect of plasma

BULYCHEV et al.1460

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 3. Changes in Chl fluorescence yield F′ caused by the inward current in C.australis(a,c) and N.translucens(b,d) cells. a)Changes in (1)F′

and (2)membrane potential caused by the inward current. Curve1 (black line) represent an averaged recording of F′ (n=45, N=9) on the back-

ground of standard error of the mean (SEM). Curve2 shows changes in the membrane potential caused by a pulse of inward current with a density

of 35 μAcm

–2

. b)Changes in Chl fluorescence in N.translucens cells (curve1) caused by a pulse of inward current with a density of 100μAcm

–2

(curve2). The averaged recording of F′ is presented on the background of SEM (n=11, N=4). c,d)Light dependency of hyperpolarization-

induced decrease in F′ for C.australis and N.translucens cells (n=9-11) in the (1)light and (2)dark. Curves3 show voltage changes during HR

in panel(c) and current density in the central cell region in panel(d).

membrane hyperpolarization on F

′ as an NPQ indicator.

Figure4 shows the results of a representative experiment

in which saturating light pulses were applied either with

or without electrical stimulation. The cell was alternately

subjected to several cycles of such treatment and the re-

cordings with the HR induction were alternated with the

recordings during stationary depolarization. The onset of

saturating pulses released at 10-s intervals was often ac-

companied by the initial decrease in F

′ (Fig.4, curves3

and4, segments at t ≤ 75 s). After the F

′ decline slowed

down (within 75s after the start of recording), a step-like

current pulse was applied to induce the HR in the cell

area under study (curve 1). Under the same conditions

but in the absence of current, the plasmalemma remained

in the depolarized state (curve 2).

As can be seen in Fig. 4, the curves3 and 4 almost

coincided in their initial segments (t ≤ 75 s), i.e., before

the HR induction, but diverged significantly at t > 100 s

depending on the occurrence or absence of HR. When

the plasmalemma remained steadily depolarized, the F

′

values reached a constant level after a smoothed maxi-

mum (curve 4), while after plasma membrane hyper-

polarization, a slight increase (or shoulder) in F

′ was

transiently observed that was replaced by a strong de-

crease in F

′ (curve3). Subsequent recovery of F

′ took

~10 min. The kinetic curves of F

′ showed some sim-

ilarity with the F′ changes during the HR. Under a

low-intensity background illumination, the quantum ef-

ficiency of the PSII-driven electron transfer (YII) slight-

ly decreased (≤ 0.03). At the same time, at the light

intensity of 25 μmol m

–2

–1

, the HR-induced decrease

in the PSII quantum yield reached substantial values

(~0.15 units). In cells placed in the dark, the HR had

noeffect on the PSII quantum yield.

FLUORESCENCE OF Chara CELLS UPON HYPERPOLARIZATION 1461

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 4. HR of the plasma membrane in the K

-conductive state and

its effect on F

′. Hyperpolarization of the plasma membrane (curve1)

was induced by an inward current pulse (42μAcm

–2

, duration 75s) un-

der weak background irradiance (12.5μmol m

–2

s

–1

). In the absence

of electrical stimulation, the membrane remained steadily depolar-

ized (curve2). Curves 3 and 4 represent F

′ changes observed with or

without HR generation (curves3 and 4, respectively). The monotonic

decrease in F

′ over t ≤ 75-100 s time interval was caused by a series

of saturating light pulses. Curves 3 and 4 were obtained in a repre-

sentative experiment by averaging the results of five measurements

per treatment.

As shown in Fig. 5, the influence of HR on F

′

was only evident in the light but disappeared after the

light was switched off. In the illuminated cell, the ki-

netics of F

′ changes differed clearly in the presence

and absence of the hyperpolarizing stimulus (Fig. 5a).

However, after the cell was placed in darkness, chang-

es in F

′ observed upon the current passage and in the

absence of electrical stimulation were almost the same

(Fig. 5b). Thus, PET coupled with the transmembrane

flows is a necessary condition for the signal trans-

mission from the plasma membrane to chloroplasts.

Transmembrane H

fluxes during hyperpolarization

and their inactivation. When the plasmalemma is in a

-conductive state, the transmembrane K

fluxes are

predominant. At the same time, the negative shift of cy-

toplasmic electric potential during HR may in principle

cause the redistribution of H

between the cytoplasm

and outer medium, as the plasma membrane of illumi-

nated Chara cells is permeable to H

and/or OH

–

ions

[4,5]. We tested this possibility by measuring pH values

near the cell surface (pH

) during the current-induced

hyperpolarization of Chara plasmalemma.

Curve 1 in Fig.6a shows the pH

shift induced by

passing a stepwise inward current through the plasma-

lemma of Chara cell. The hyperpolarizing pulse caused

an increase in pH

by approximately 0.5 units, which in

some cases, but not always, continued for a while after

the pulse termination. The rate of pH

shift increased

during the stimulus, which was presumably due to pro-

gressive hyperpolarization and the increasing driving

force for H

entry into the cytoplasm. After pulse termi-

nation, the pH

values usually fell below the initial level

(Fig.6a, curve1); however, the pH

depression was re-

leased with time (Fig.6a, curve2).

Unlike the impact of the first electrical stimulus on

, the second or third current pulses did not cause

any discernible pH

changes (Fig.6a, curve 2), which

indicated that the first hyperpolarizing stimulus caused

an almost complete inactivation of the plasmalem-

ma passive conductance for H

. In some cells, the H

conductance was restored after a resting period of~1 h.

However, in many cases, no restoration of the H

Fig. 5. Light dependency of hyperpolarization-induced changes in F

′. a)Plasma membrane hyperpolarization in illuminated cells causes a de-

layed decrease in F

′. b)Effect of hyperpolarization on F

′ disappears after transferring the cell to darkness. 1)Changes in the membrane po-

tential caused by a rectangular current pulse (83μAcm

–2

); 2)F

′ changes caused by saturating light pulses and HR generation: (a)in the light

and (b)in the dark; 3)F

′ changes caused by saturating light pulses in the resting cell in the absence of electrical stimulation and HR induction.

Traces2 and3 represent average values obtained from two measurements per treatment.

BULYCHEV et al.1462

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 6. Changes in the cell surface pH during induced hyperpolarization. a)Changes in pH at the Chara cell surface (pH

) caused by passing a

hyperpolarizing current pulse through the plasmalemma: 1)changes in pH

in response to the first pulse with a density of 58μAcm

–2

; 2)dis-

appearance of pH

shift upon subsequent application of current pulses of the same strength. b)Suppression of pH

changes in response to the

repeated electrical stimulation of plasmalemma (curve1, pH

) did not eliminate hyperpolarizing shifts of the membrane potential (curve2,V

Results presented in(a) and (b) were obtained on different cells. Dashed vertical lines mark the moments when the inward electric current

was switched on and off.

Fig. 7. Changes in NPQ(a), effective quantum yield of PSII reaction(YII)(b), and pH on the Chara cell surface(pH

) caused by a hyperpolar-

izing pulse with a current density of 33μAcm

–2

. The medium (APW) in the central compartment of the chamber was supplemented with 30mM

KCl; background light intensity was 25μmol m

–2

s

–1

. Vertical dashed lines mark the moments when the electric current was switched on and off.

conductance was observed in continuous experiments.

Nevertheless, pH

changes in response to the current

pulse restored on the next day provided the cells were

kept overnight in standard APW. The data presented in

Fig. 6b show that suppression of plasmalemmal H

con-

ductance (curve 1) had no inhibitory effect on the HR

development (curve2).

Figure 7 shows that the pH

shifts associated with

the hyperpolarizing pulses could be as high as 1.5 units

and were accompanied by a significant increase in NPQ

(Fig.7a) and a transient decrease in the effective quan-

tum yield of PSII reaction (Fig. 7b). The emergence

of HR under these particular conditions was verified

in parallel experiments. The decrease in YII indicated

transient deceleration of the photosynthetic linear elec-

tron flow.

DISCUSSION

Cells of many plants respond to physiological and

damaging stimuli by generating electrical signals (action

FLUORESCENCE OF Chara CELLS UPON HYPERPOLARIZATION 1463

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

potentials, variable potentials) that influence photosyn-

thesis and Chl fluorescence [4, 5, 23]. The mechanisms

of signal transduction from the plasma membrane to

chloroplast membranes have been only partly clarified.

The effects of the electrical activity on the PSA perfor-

mance can be studied on characean internodes since

they combine excitability, cytoplasmic streaming, and

spatially nonuniform distribution of H

fluxes and pho-

tosynthesis.

The ability of plasmalemma to generate active HRs

has been long known [17,24], but the relation of HRs to

other cellular events and their effect on PSA remained

unexplored. We found that the time from the onset of

electric pulse to the HR peak was altered during the

light–dark transitions, provided cyclosis was arrested

by cytochalasin D (inhibitor of actin cytoskeleton po-

lymerization). The effect of dark–light transitions on

the rate of HR development (Fig. 2) was probably due

to changes in the cytoplasm composition during the on-

set and cessation of the light-dependent transport across

the chloroplast envelope membranes. The distribution

along the cell length of cytoplasmic components trans-

ported through the plastid membrane seems to become

more heterogeneous in the absence of cyclosis, because

intense streaming smooths down the concentration pro-

files of these components. This explains the lack of a

visible effect of illumination on the HR development

during active cytoplasmic streaming, as well as a mark-

ed HR dependence on light conditions after cessation

ofcyclosis.

According to the earlier study of isolated cytoplas-

mic droplets, the disturbance of actin cytoskeleton by

cytochalasin B modified the HR shape by eliminating

the sharp peak tentatively ascribed to the activity of the

electrogenic pump ([22] and references therein). Theef-

fect of cytochalasin D on whole cells also smoothed

down the hyperpolarization peak and the depolarization

stage of HR (Fig. 2). These observations suggest that

the state of actin cytoskeleton controls the conductance

of channels involved in the HR formation. The mech-

anisms of interaction between ion channels and actin

cytoskeleton have been discussed in [25]. These mech-

anisms can be applied to explain the effect of cortical

actin on ion channels in the plasma membrane of Chara.

HR modulation by light conditions might be based on

changes in the cytoplasmic [Ca

] and [H

] that arise

from the photoinduced fluxes of these ions across the

plastid membrane and exert direct or indirect action on

the activity of ion channels. The mechanisms of Ca

and H

influence on the cytoskeleton of plant cell have

been described in the review by Hepler [26].

We investigated the effects of HR on the PSA by

assessing changes in F′ and F

′. Here possible relations

between HR and cell physiological events are worth

noting. In illuminated Chara and Nitella cells, nat-

ural electric currents circulate between the cell parts

that produce external acidic and alkaline zones [27].

The density of light-dependent inward currents in the

alkaline zones of Chara cells is 20-60 μA cm

–2

[28].

In our study, the HR of the plasma membrane was in-

duced by electric current with a density of 30-80 μA cm

–2

i.e., in the physiological range. Hence, the observed

interactions between the plasma membrane and chlo-

roplasts can, in principle, arise upon the increase

in the external K

level. When an illuminated cell is

challenged by a solution with a high K

concentration

(10-100mM), the inward current might lead to a pro-

tective decrease in the K

conductance due to the HR

generation and also block the H

conductance (Fig. 6)

to prevent operation of the H

pump in a short-circuited

mode.

The HRs of charophytes show some similarities

with the hyperpolarization signals in higher plants,

which arise when a leaf is heat-treated or damaged

with an open flame. In particular, moderate heating of

wheat leaves resulted in gradual cell hyperpolarization

by 10-20 mV for 20 min, which was accompanied by

the increase in NPQ [29]. The high-amplitude HRs in

Chara cells (~250mV) developing within 10-75s caused

a marked increase in the regulated energy dissipation

in PSII (Fig. 7a), as well as a decline in the quantum

yield of PSII reaction and slowing of the linear electron

flow (Fig.7b).

The effects of HRs on cellular processes attract a

particular interest because the negative voltage arising

during HR creates a driving force for the K

entry into

the cytoplasm through the plasma membrane pre-con-

verted to the K

-conductive state. Therefore, HR can

be used as a tool to elevate the cytoplasmic K

level

and to examine the outcomes of the increased [K

]

cyt

on the PSA condition.

We used relatively long intervals between the cur-

rent pulses (600 s) to facilitate the recovery of [K

]

cyt

during transitions from the HR to the resting state.

The cells subjected to recurrent HR generation re-

mained viable on the next day after the experiment and

all measured parameters (cytoplasmic streaming velocity,

effective quantum yield of PSII, and resting potential)

remained within their normal ranges. The redistribu-

tion of ions due to the HR generation did not usually

lead to the irreversible cell damage. Nevertheless, the

HR generation was followed by a long-term inactiva-

tion of H

/OH

–

fluxes. Under physiological conditions,

the H

/OH

–

conductance depends on illumination.

The H

/OH

–

fluxes caused by the HR generation oc-

curred both in the light and, occasionally, in the dark.

Presently, it remains unclear whether the potential-driv-

en H

/OH

–

fluxes are equally pronounced in photosyn-

thetically active and inactive cell areas.

An elevation in [K

]

cyt

by itself is insufficient to affect

PSA. However, it can influence the ionic composition

of the stroma via carriers located in the plastid envelope.

BULYCHEV et al.1464

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Thus, the inner envelope membrane contains K

antiporters [13, 14, 30]. When [K

]

cyt

increases, these

transporters should transfer K

to the stroma in ex-

change for H

from the plastid stroma. Importantly, the

activity of KEA1/2 is retained in the dark [31]. An in-

crease in the stromal pH is known to stimulate the re-

actions of carbon dioxide fixation, which can shift the

redox states of plastoquinone and Q

acceptor towards

oxidation, thus decreasing the actual yield of Chl fluo-

rescence F′ (Fig.3). According to the proposed scheme,

delayed changes in F′ and F

′ in response to the plasma

membrane hyperpolarization are due to the induction of

exchange across the chloroplast envelope.

At the same time, hyperpolarizing electric pulses

can potentially affect the water balance of the cell and

chloroplasts. If the pore of the plasma membrane K

channel contains fixed negative charges, electroosmotic

entry of water into the cell and corresponding swelling of

chloroplasts cannot be ruled out. The redistribution of

granal system in a larger volume might underlie rapid F′

increase in N. translucens cells that feature an increased

surface to volume ratio. In addition, K

entry into the

stroma of plastids in exchange for H

efflux might be

accompanied by the osmotic regulation of chloroplast

volume. Taking into account various factors occurring

simultaneously during the current flow will help to clar-

ify the mechanistic relations between the electrical pro-

cesses at the plasmalemma and chloroplast activity.

Hyperpolarization creates a driving force not only

for the K

entry but also for the H

influx into the cy-

toplasm. The dependence of cytosolic pH on the shift

in the membrane potential seems likely, because the

plasmalemma of Chara cells contains passive conduct-

ing pathways for H

or OH

–

[5, 27]. Previously, the ex-

istence of plasmalemmal H

/OH

–

conductance was

deduced from the formation of narrow alkaline zones

on the surface of illuminated internodal cells bathed in

a low-K

medium (i.e., under conditions ensuring sus-

tained circulation of natural electric currents at low

conductance of the plasma membrane). Here, we

demonstrated the existence of H

fluxes caused by hyper-

polarization at a high K

conductance of the plasmalemma

(Figs.6 and7).

The results presented in Figs. 6 and 7 reveal two

principally different types of pH

changes taking place

after switching off the inward current. The instant re-

versal of the sign of pH changes at the peak in Fig.7

and the ongoing pH rise after termination of the current

pulse in Fig. 6 indicate that the hyperpolarization-in-

duced pH changes comprise the stages of different or-

igin. The changes in pH reverted after the shift of the

membrane potential to zero apparently reflect the volt-

age-dependent transfer of H

(or OH

–

) along the plas-

malemmal conducting pathways. In contrast, pH changes

proceeding at almost the same rate after removal of the

driving force (Fig. 6) indicate a different nature of the

post-hyperpolarization pH shift. Such increase in pH

similar to the formation of a local alkaline zone with-

in approximately 30 s after microperforation of Chara

cell [32]. Parallel measurements of local shifts in pH

and O

concentration in the area of cell microinjury in

the absence and presence of inhibitors indicated that the

pH increase concurrent with the sharp drop in O

level

was due to the enzymatic transfer of electrons through

the plasmalemma from NAD(P)H to O

and subsequent

consumption of H

in the course of O

•

reduction to

[33]. Based on these data, it is reasonable to con-

clude that the pH

rise during plasma membrane hy-

perpolarization can involve different mechanisms, i.e.,

transmembrane transport of H

under the influence of

electrochemical proton gradient, as well as a combined

mechanism that includes biochemical processes insensi-

tive to the transmembrane H

gradient.

Interestingly, pH

changes occurred only in re-

sponse to the first and, rarely, to the second current

pulse, while subsequent stimulation had almost no ef-

fect on pH

. Since the effect of HR on F′ and F

′ re-

mained after application of multiple electrical stimuli, it

appears that H

fluxes caused by hyperpolarization did

not play a critical role in the signal transmission from

the plasmalemma to chloroplasts upon repeated stim-

ulation. However, considering possible changes in the

cytoplasmic pH during HR development, it seems im-

portant to examine the role of cytoplasmic pH in PSA

rearrangements in more detail.

The HR-induced F

′ transients are somewhat sim-

ilar to the AP-induced F

′ changes that take place at a

moderate light intensity. It is known that hyperpolariza-

tion can activate Ca

-permeable channels [34], thus al-

lowing Ca

entry to the cytoplasm. According to one of

the hypotheses, the depolarization stage occurring after

the HR peak in cytoplasmic droplets of charophytes is

similar to the front of the AP in intact cells and is as-

sociated with an increase in the cytoplasmic Ca

[22].

However, this hypothesis is not entirely convincing with

respect to intact cells, as cytoplasmic streaming does not

stop at the peak of HR, even though cyclosis is extreme-

ly sensitive to the increase in cytoplasmic Ca

[35].

The role of thylakoid ΔpH in the regulation of F′ and

′ is evidenced by the fact that the impact of HR on

these parameters was manifested only in illuminated

cells and disappeared in the dark along with the PET

(Figs.3 and5).

The long lag period preceding the HR-induced

changes in F′ and F

′ reflects the multistep transfor-

mation of plasmalemmal electrical signal into fluores-

cence response in the thylakoid membranes. The pro-

posed intermediate steps include changes in [K

]

cyt

exchange across the chloroplast envelope, mod-

ulation of activity of stromal enzymes, and pH effect

on the electron transport and fluorescence quenching

in the antenna.

FLUORESCENCE OF Chara CELLS UPON HYPERPOLARIZATION 1465

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

In conclusion, this study revealed new interactions

between the plasma membrane and chloroplasts, which

are mediated by ion fluxes mobilized due to the elec-

trical cell signaling. Hyperpolarization of the plasma-

lemma in the K

-conductive state leads to noticeable

changes in F′ and F

′ of Chl fluorescence pointing to

the existence of regulatory mechanisms that differ from

previously discovered pathways triggered by the AP gen-

eration. Our experimental observations are consistent

with the notion that the K

exchange on the inner

envelope membrane has a regulatory impact not only in

the long-term experiments (e.g., in plants grown at el-

evated salinity), but also in the case of relatively short-

term treatments. Further studies are needed for deeper

understanding of the intracellular interactions underly-

ing the effect of electrical events at the plasmalemma on

the photosynthetic performance of chloroplasts.

Contributions. A.A.B. designed the study, conduct-

ed experiments, and wrote the draft manuscript; S.YuSh.

carried out experiments and processed raw data; A.V.A.

discussed the results and supervised the study.

Funding. This work was supported by the Rus-

sian Foundation for Basic Research (RFBR) (project

no.20-54-12015NNIO_a) and carried out as part of the

Scientific Project of the State Order of the Government

of the Russian Federation to the Lomonosov Moscow

State University (no.121032500058-7).

Ethics declarations. The authors declare no con-

flict of interest. This article does not contain descrip-

tion of studies with human participants or animals per-

formed by any of the authors.

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