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Michigan Sim:
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This is to certify that the

dissertation entitled

Light-induced short-term ion flux responses
and associated transport mechanisms in leaf tissues
of pea (Pisum sativum L. cv. Argenteum)

presented by

Bin Yan

has been accepted towards fulﬁllment
of the requirements for

Ph.D. Horticulture

degree in

 

 

Jib; s: Z/JJID

Major professor

 

Date August 16, 2001

 

MS U i: an Afﬁrmative Action/Equal Opportunity Institution 042771

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 eJCIRC/DatoDuepGS-QIS

 

Light-induced short-term ion flux responses and associated
transport mechanisms in leaf tissues of pea (Pisum sativum
L. cv. Argenteum)

By

Bin Yan

AN ABSTRACT OF A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

Doctor of Philosophy

Department of Horticulture

2001

Professor Irvin E. Widders

ABSTRACT

LIGHT -INDUCED SHORT-TERM ION FLUX RESPONSES AND ASSOCIATED
TRANSPORT MECHANISMS IN LEAF TISSUES OF PEA (PISUM SA TIVUM L. CV.
ARGENT E UM)

By Bin Yan
The study was conducted to investigate light-induced rapid ﬂux responses of Ca2+, H+, K“
and Cl' and associated transport mechanisms in leaf tissues of pea (Pisum sativum L. cv.
Argenteum). Light stimulation (450 nmol rn'2 3") resulted in a transient increase in net

1 in mesophyll tissues. The light-response lag time of

Ca2+ inﬂux of about 50 nmol In2 3'
Ca2+ is the most fast (13 s), whereas net inﬂuxes of H+, Cl' and K+ were also rapidly
induced to increase within approximately 29, 45, and 48 s by light exposure, respectively.
After this initial response to light, a rapid Post-Initial Flux Response (PIFR) was
measured for Ca2+ and Cl' within 2-3 min, due most likely to an increase in efﬂux. In
contrast, PIFR of H”, K+ was slower. The light-induced alkalinization of mesophyll
apoplast (0.4-0.5 pH increase) which occurred within initial 5-6 min could be explained
by either C02 uptake into photosynthesizing tissues or increased K+ and Cl' uptake
mediated by HJ'/K’r and H+/Cl' symports, respectively.

LaCl3 (0.1 mM), a Ca2+ channel blocker, and or EGTA (l.0 mM), a carion/Ca2+
chelator inhibited the light-induced initial net Ca2+ inﬂux, and lead to reduction of the
light-induced PIFRs of Cl' and K+ around the mesophyll tissues over time. In contrast,

the addition of Ca2+ ionophore A23187 (5 uM) or high Ca2+ concentration (2.0 mM) in

the bathing solution produced an opposite effect. Therefore, Ca2+ inﬂux resulting from

increased Ca2+ channel activity may be an initial light response which could contribute to
membrane depolarization. The plasma membrane H+-ATPase inhibitors, Na3VO3 (1.0
mM) and DCCD (0.1 mM), inhibited the light-induced initial response in net H+ inﬂux by
50-60 % and eliminated the PIFR of H+, whereas fusicoccin (0.01 mM) signiﬁcantly
increased the light-induced PIFR of H+ and the initial response in net C1" inﬂux. This
suggests that H+-ATPase in the plasma membrane may not be directly involved in the
initial light response in mesophyll tissues. In addition, the K+ outwardly-directed
channels and the R-type anion channels are thought to play a role in the light-induced
PIFR of K+ and Cl', respectively, whereas Cl' PIFR is Cay—dependent.

Light-induced changes in net Ca2+, H+, K+ and Cl' ﬂux from mesophyll tissues
increased in a saturable manner with increasing ﬂuence. However, high ﬂuence (1800
and 2800 umol m’2 8") enhanced the temperature of the bathing solution and produced a
possible heat effect on ion ﬂux. White light (WL) of 450 umol m'2 s'1 stimulated
signiﬁcant changes in net ﬂux of Ca2+, W, K" and Cl' from mesophyll tissues. Blue-light
(BL) and red-light (RL) of similar ﬂuence induced similar time course changes in net
Ca2+, H+, K+ and Cl’ ﬂuxes as WL. There were no signiﬁcant differences in the
magnitude of change in net Ca2+, H+, K+ and Cl’ ﬂuxes between BL and RL. The result

suggests that BL and RL play a similar role in rapid ionic responses to light.

To my parents and my aunt who believe in me

and whom I love.

iv

ACKNOWLEDGEMENTS

I express sincere gratitude to my committee members, Dr. Irv Widders, Dr. Ken Poff, Dr.
Eunice Foster and Dr. Eric Hanson, for the guidance and encouragement they have given
me during my Ph.D. study. Their great helps made me ﬁnish the graduate program. First
of all, I would like to thank my major professor, Dr. Irv Widders, for his didactic advise
and tremendous contribution of his knowledge. His patience and supports are
indispensable to my research. I extend my appreciation to Dr. Poff who enhanced my
understanding of light signal transduction and gave me conﬁdence. Drs. Foster and
Hanson paid a lot attention and contribution to data analysis and experimental suggestion.
Without their support my dissertation might be still an elementary step.

I wish to give a special thanks to Drs. Ian Newman, Sergy Shabala and Olga
Babourina from University of Tasmania, Australia. They provided great help with MIFE
technique. A very warm thanks to Dr. Frank Dennis and Dr. Jose Saunders who edited
my writing. I would like to thank Richard Wolthuis for his assistance in building
wonderful chambers. Bob Forbes made a nice Faraday cage for the MIFE system.

Also I express my appreciation to Dr. Erik Runkle for his friendly help of light
measurements and English editing. Dr. Jose Masanbni gave me a lot of helps during my
entire period in Horticulture. I would like to thank individuals in this Department —
Pricilla Hockin, Joyce Jackson, Lowrie Kristi, Khanh Nguyan, Steve Brooks and HOGS
people. Finally, I want to thank my parents, aunt and ﬁ‘iends for their emotional support,

friendship during my stay at MSU.

TABLE OF CONTENTS

 

Page

ACKNOWLEDGEMENTS ...................... v
LIST OF TABLES .......................... viii
LIST OF FIGURES .......................... x
LIST OF ABBREVIATIONS ..................... xiv
INTRODUCTION .......................... 1
GENERAL INTRODUCTION ..................... 1
LITERATURE REVIEW ....................... 4

1. Generality of Signal Transduction ................ 4

2. Membrane Potential and Electrical Signalling ............ 6

3. Ion Fluxes and Signal Transduction ................ 9

3.1 Ca2+ ﬂuxes and Ca2+ signalling ............... 10

3.2 H+ ﬂuxes ........................ 15

3.3 K ﬂuxes ........................ 19

3.4 CI‘ ﬂuxes ........................ 22

4. Light Signal Transduction .................... 24

4.1 Photoreceptors ...................... 24

4.2 Light effects on membrane potentials and ion ﬂuxes ..... . 26

Chapter 1. Characterization of Light-Induced Ca“, H”, K* and CI' Fluxes from

Mesophyll and Epidermal Tissues of Pea Leaf Tissues ........... 30
ABSTRACT ........................... 31
INTRODUCTION ......................... 32
MATERIAL AND METHODS .................... 34
Plant material ......................... 34
Ion-selective microelectrodes ................... 35
MIF E system .......................... 36
Flux measurements ........................ 39
Experimental protocol ...................... 43
Data statistical analysis ...................... 46
RESULTS ............................ 51
DISCUSSION ........................... 69
LITERATURE CITED ....................... 81

vi

Chapter 11. Possible Mechanism of Light-Induced Ca2+, HZ K+ and Cl‘

Fluxes from Pea Mesophyll Tissues ................... 85
ABSTRACT ........................... 86
INTRODUCTION ......................... 87
MATERIAL AND METHODS .................... 90
Plant materials ......................... 90
Flux measurements ....................... 90
Experimental protocol ...................... 92
Chemicals ........................... 93
Data statistical analysis ...................... 94
RESULTS ............................ 94
DISCUSSION .......................... 109
LITERATURE CITED ....................... 122
Chapter III. Effect of Light Intensity and Quality on Ion Fluxes
from Pea Mesophyll Tissues ..................... 128
ABSTRACT ........................... 129
INTRODUCTION ......................... 129
MATERIAL AND METHODS ................... 132
Plant materials ......................... 132
Flux measurements ....................... 132
Light sources ......................... 133
Experimental protocol ...................... 136
Solution temperature ...................... 136
Data statistical analysis ..................... 137
RESULTS ............................ 137
DISCUSSION .......................... 150
LITERATURE CITED ....................... 155
SUMMARY ............................ 158
BIBLIOGRAPHY .......................... 164

vii

LIST OF TABLES

 

Page

Chapter I.
Table 1. Quantitative characteristics of the light-induced net ﬂuxes and

extracellular concentrations of HI, Ca2+, K+ and C1‘ in pea mesophyll tissues

bathed in 0.5 mM CaSO4 and 1.0 mM KCl at pH 5.2 ........... 56
Table 2. Quantitative characteristics of the light-induced time responses of H“,

Ca2+, K and C1' in pea mesophyll tissues bathed in 0.5 mM CaSO4 and 1.0

mM KCl at pH 5.2 ........................ 58

Table 3. Qualitative characteristics of light-induced ionic responses of pea
mesophyll tissues bathed in 0.5 mM CaSO4 and 1.0 mM KCl at pH 5.2 . . . 59

Table 4. Quantitative characteristics of the light-induced induced net ion ﬂuxes

and extracellular ion concentrations in pea epidermal tissues bathed in 0.5
mM CaSO4 and 1.0 mM KCl at pH 5.2 ................ 63

Table 5. Effect of external pH in bathing solution on the light induced net ﬂuxes
of H”, Ca2+, K+ and Cl’ from pea mesophyll tissues ........... 64

Table 6. Effect of external pH in bathing solution on the light-induced
extracellular concentrations of H‘”, Ca2+, K+ and Cl' and time responses in
pea mesophyll tissues ....................... 65

Table 7. Effect of DCMU on the time responses of light-induced ion ﬂux from
pea mesophyll tissue ........................ 67

Chapter II.

Table 1. Effects of H+-ATPase inhibitors and F C on light-induced net ﬂuxes
of H“, Ca2+, K+ and Cl‘ from pea mesophyll tissues ........ . . . . 97

Table 2. Effects of Ca2+ channel blocker and chelator on light-induced net
ﬂuxes of HI, Ca2+, K” and Cl' and time response in pea mesophyll tissues . . . 100

Table 3. Effects of K+ channel blocker on light-induced net ﬂuxes of H‘”, Ca2+,

viii

K“ and Cl'from pea mesophyll tissues ................ 103

Table 4. Effects of Cl' channel blocker on light-induced net ﬂuxes of H“, Ca2+,
K+ and Cl“ and time response in pea mesophyll tissues .......... 105

Table 5. Effects of external K+ and Cl' concentrations on light-induced net
ﬂuxes of W, Ca”, K+ and Cl‘ from pea mesophyll tissues ......... 108
Chapter 111.

Table 1. Effects of white-, blue- and red-light on net ﬂuxes of H, Ca2+, K+
and Cl' from pea mesophyll tissues . . . . ............. 146

Table 2. Effects of white-, blue- and red- on net ﬂuxes of H“, Ca2+, K and
Cl' from pea mesophyll tissues at similar light ﬂuences ...... . . . 149

ix

LIST OF FIGURES

Page

 

Chapter I.

Figure 1. Schematic illustration of the experimental set-up, the MIFE

system and the position of the light source . . ............

Figure 2. The MIFE system and the position of the microelectrode and light

source ............................

Figure 3. A segment of H+ ﬂux data record from mesophyll to illustrate the

voltage change from which the ﬂuxes were estimated using the MIF E system .

Figure 4. Time course of net ion ﬂuxes and extracellular ion concentrations
of W, Ca2+, Cl' and K+ at 1500-2000 pm from pea leaf surface using the
MIF E system ..........................

Figure 5. Calculation of light-induced change in net HI ﬂuxes and
extracelluler pH around pea mesophyll tissues. F0 or C0 is 5 min average of
net ﬂuxes or extracellular ion concentrations, respectively, before light-on.
Fr is amplitude of light-induced Post-Initial Flux Response (PIFR). Fi is
initial net ion ﬂux or extracellular ion concentrations before PIFR. Ff or Cf

is initial ﬂux or concentration response, respectively, aﬁer light-off .....

Figure 6. Time course of net ﬂuxes and extracellular concentrations of H2
Ca2+, Cl' and K” around pea mesophyll tissues of one leaf sample under

dark .............................

Figure 7. Li ght-induced time course in net ﬂuxes and extracellular
concentrations of H, Ca2+, Cl‘ and K+ around pea mesophyll tissues of one

leaf sample ..........................

Figure 8. Light-induced time course of net ﬂuxes and extracellular
concentrations of H, Ca2+, C1” and K around pea epidermal tissues of

one leaf sample .........................

.44

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Fi

Figure 9. Effect of a photosynthetic inhibitor, DCMU, on light-induced
initial response in net ion inﬂux from pea mesophyll tissues. Data
represents an average of 6 - 9 replicates. Vertical bars represent LSD
values at p<0.05 .......... . . . . . .......... 67

Figure 10. Light-induced time course of net ion ﬂux from pea mesophyll
tissues. Data points represent a leaf sample. Net charge ﬂux is a sum I-F +
Ca” + K+ - Cl' ﬂuxes ........................ 68

Figure 11. Light-induced time course of net ion ﬂux from pea mesophyll and
epidermal tissues. Data points represent an average of 7-12 leaf samples.
Net charge ﬂux is a sum of H” + Ca2+ + K+ - Cl’ ﬂuxes ........... 68

Chapter 11.

Figure 1. Effect of Hi-ATPase inhibitors, vanadate ('Na3VO4) and N, N-
dicyclohexicarbo-diamide (DCCD), and the ATPase stimulator F usicocin
(FC) on net H+ ﬂux under low ﬂuence (50 nmol m'2 5"). Data points
represent an average of 6-8 replicates. Vertical bars represent LSD values

at p<0.05 ............................ 95

Figure 2. Effect of HI-ATPase inhibitors, vanadate (Na3V04) and N, N-
dicycloexicarbodiamide (DCCD), and the ATPase stimulator Fusicocin
(F C) on the time course of light (450 nmol m’2 5“) induced change in net H+
ﬂux from pea mesophyll tissues . . . . ............... 95

Figure 3. Effects of Ca2+ channel blocker (LaCl3), chelator (EGTA) and
ionophore A23178 on net Ca2+ ﬂux from pea mesophyll tissues under low
ﬂuence (50 nmol m‘2 3"). Data points represent an average of 6-8
replicates. Vertical bars represent LSD values at p<0.05 .......... 99

Figure 4. Effect of the Ca2+ chelator (EGTA) and channel blocker (LaCl3) on
the time course of light (450 umol rn'2 s")-induced change in net Ca2+ ﬂux
from pea mesophyll tissues. Data points represent an average of 6-10
replicates. Vertical bars represent LSD values at p<0.05 .......... 99

Figure 5. Effect of K+ channel blockers tetraethylammonium (TEA) and
Cesium chloride (CsCl) on the time course of light (450 umol rn‘2 s")-
induced change in net Ki ﬂux ﬁom pea mesophyll tissues. Data points
represent an average of 6-10 replicates. Vertical bars represent LSD values
at p<0.05 ............................ 102

Figure 6. Effect of Cl‘ channel blockers diisothiocyanatoslibene (DIDS) and
anthracene—9-carboxylic (A-9—C) on the time course of light (450 umol m‘2 s'

xi

Ho

Ch
Fig

Fig

l)-induced change in net Cl' ﬂux from pea mesophyll tissues. Data points
represent an average of 6-10 replicates. Vertical bars represent LSD values

at p<0.05 ...........................

Figure 7. Effect of Ca2+ ionophore A23187 on the light-induced ion ﬂux
response of pea mesophyll tissues. Changes in net K+ and Cl' ﬂux indicate
the amplitude of light (450 nmol rn‘2 s")-induced Post-Initial Flux
Response (PIFR), negative values. Data represents an average of 6-9

replicates. Vertical bars represent LSD values at p<0.05 .........

Figure 8. Effect of external Ca2+ concentration on the light-induced ion ﬂux
response of pea mesophyll tissues. Changes in net K+ and Cl‘ ﬂux
indicate the amplitude of light (450 umol m'2 s")-induced Post-Initial
Flux Response (PIFR), negative values. Data represents an average of 6-9

replicates. Vertical bars represent LSD values at p<0.05 .........

Figure 9. A hypothetical model for light-induced ion ﬂux response in pea

mesophyll cells .........................

Chapter 111.

Figure 1. Fluence of white, blue and red lights over a range of wavelengths . . .

Figure 2. Effect of white-light intensity on ﬂux responses HI, Ca2+, K” and
Cl' around pea mesophyll tissues. Data points represent an average with
3-5 replicates. Vertical bars represent the standard error of the mean.
Change in net ion inﬂux indicates the light-induced initial response (a
positive value). Amplitude of light-induced Post-Initial Flux Response
(PIFR) indicates a decrease in inﬂux or an increase in efﬂux or both (a

negative value) .........................

Figure 3. Effect of increasing solution temperature on net ﬂuxes H+, Ca2+,
K+ and Cl‘ from pea mesophyll tissues. Data points represent an average
of 2-5 replicates. Vertical bars represent standard error of the mean.
Treatment temperature increases: T1 = 1-1.5 °C; T2 = 1.5-2.5 °C; T3 =

3-4 °C; T4 = 4-5°C. The base temperature was 23i1°C .........

Figure 4. Effect of white and red lights on temperature of the bathing solution.

Data points represent an average of 3 replicates ............

Figure 5. Effects of light quality on time course of net ﬂux of H*, Ca2+, K
and Cl‘ ﬁom pea mesophyll tissues. Data points represent an average of 8-
10 replicates. Vertical bars represent LSD test result at p<0.05. Light
intensity of white-light (WL), blue-light (BL) and red-light (RL) are 450,

32 and 225 nmol m‘2 s", respectively .................

xii

. 102

. 107

. 107

. 122

135

. 138

. 140

143

 

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Figure 6. Effect of blue-light intensity on ﬂux responses H+, Ca2+, K” and Cl'
around pea mesophyll tissues. Data points represent an average with 3-5
replicates. Vertical bars represent the standard error of the mean. Change
in net ion inﬂux indicates the light-induced initial response (a positive
value). Amplitude of light-induced Post-Initial Flux Response (PIF R)
indicates a decrease in inﬂux or an increase in efﬂux or both (a negative
value) .............................. 144

Figure 7. Effect of red-light intensity on ﬂux responses H”, Ca2+, K and Cl'
around pea mesophyll tissues. Data points represent an average with 3-5
replicates. Vertical bars represent the standard error of the mean. Change
in net ion inﬂux indicates the light-induced initial response (a positive
value). Amplitude of light-induced Post-Initial Flux Response (PIFR)
indicates a decrease in inﬂux or an increase in efﬂux or both (a negative
value) .............................. 148

xiii

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LIST OF ABBREVIATIONS

A-9-C: Anthracene-9-carboxylic acid
ANOVA: analysis of variance

BL: blue light

Ca2+: calcium ion

Cl': chloride ion

CsCl: Cesium chloride

d: day

DCCD: N-N’-dicyclohexil carbodiumide
DCMU: 3-(3 ,4-dichlorophenyl)- l , 1 -dimethylurea
Di: diffusion coefﬁcient

DIDS: Diisothiocyanatostilbene

DSMO: dimethylsulfoxide

En: Nemst potential

EGTA: Ethyleneglycol-bis(B-aminoethylether)N, N, N’,N’-tetraacetic acid
F: Faraday constant

F C: F usicoccin

H’: hydrogen ion

HEPES: N-(2-Hydroxyrthyl)piperazine-N’-(2-thanesulfonic acid)
K+z potassimn ion

LaCl,: Lanthanum chloride

LSD: Least Signiﬁcant Difference

MIFE: Microelectrode Ion Flux Estimation
min: minus

Na3VO4: sodium vanadate

PIFR: Post-Initial Flux Response

R: gas constant

RL: red light

5: second

SE: standard error

T: temperature

TEA: tetraethylammonium

AV: electrochemical potential gradient
VDI: valid data interval

WL: white light

2: valency

xiv

INTRODUCTION

GENERAL INTRODUCTION

Plants are constantly exposed to ﬂuctuations in various abiotic and biotic factors such as
light, temperature, water, micro-organisms, and to mechanical and chemical stresses.
Most of these signals evoke morphological, biochemical and physiological responses,
indicating that there must be mechanisms in plants to perceive these external stimuli, and
to elicit and transduce signals.

In recent years, a great deal of progress has been made toward understanding signal
transduction in plants (Felle, 1993; Leigh, 1993; Verhey and Lomax, 1993; Gilroy and
Trewavas, 1994; Yang, 1996; Blumwald et al., 1998; Ebel and Mithofer, 1998; Grabov
and Blatt, 1998; Genoud and Métraux, 1999). Most studies indicate that plants have
developed multiple pathways to perceive and transduce signals. The inter-related
molecular, cellular and tissue level events involved in triggering ﬁnal responses have
become important areas of research in plant science. However, the biochemical and
biophysical mechanisms are not yet fully understood.

External stimulation leads to changes in membrane potential and induce rapid
electrical signal propagation either locally or systematically (Davies, 1987a; Wildon et
al., 1992; Thain and Wayne, 1993; Rhodes et al., 1996). Most researchers agree that the
electrical signalling involve ion ﬂuxes across membrane systems (Beilby, 1984; Davies,

1987a; Felle, 1993; Wayne, 1993; Ward and Schroeder, 1997; Grabov and Blatt, 1998).

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Therefore, environmental stimulation most certainly induces changes of ion ﬂux across
cellular membranes.

Calcium, a powerful second messenger, presumably plays a central role in signal
transduction in plants. Recent investigations on stimuli-induced changes in cytosolic
Ca2+ concentration have enhanced the understanding of the mode of Ca2+ action in plants
(Bush, 1995; Webb et al., 1996; Blatt, 1999; Sanders et al, 1999; Trewavas, 1999). The
identiﬁcation of Ca2+- or Ca2+/calmodulin-dependent protein kinases in plant cells
suggests that Ca2+ functions is a critical signalling ion. Further investigation is needed,
however, to examine the role of Ca2+ in plants.

Of the many environmental stimuli affecting plant growth and development, light
may be the most important. Light has two principal functions in plants, energizing
photosynthesis and photomorphogenesis. Plants have developed complex and
sophisticated photosensory machinery that enable them to sense, transduce and respond to
the direction, duration, quality, and quantity of light. Light-induced signalling systems in
higher plants have been demonstrated by several authors (Chamovitz and Deng, 1996;
Batschauer, 1998; Kurana et a1, 1998). The light responses of leaves involve at least two
photoreceptors, a phytochrome and a blue/UV-A photoreceptor.

An early response by leaf cells to light stimulation is a rapid change in the electrical
potential across the plasma membrane (Bentrup, 1974; Cheeseman et al., 1982; Elzenga
et al., 1995; Errnolayeva et al., 1996; Trebacz and Sievers, 1998). Changes in membrane
potential indicate the involvement of ion ﬂuxes in light signal transduction. Studies have

attempted to determine if ion transporters, ion pumps, carriers and ion channels, in the

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plasma membrane are involved in light-induced electrical signals (Mummert and
Gradmann, 1991; Errnolayeva et al., 1996; Elzenga and van Volkenburgh, 1997; Szarek
and Trebacz, 1999). Four inorganic ions were most highly correlated with light-induced
bioelectrogensis, Ca2+, HI, KI and Cl'. Despite extensive research, it is unclear what role
ion transport mechanisms play in the electrical response to light, which ions are important
in electrical signalling, or whether Ca2+ is a key ionic messenger, and which
photoreceptors may be linked to ion transporters and thus regulate their activity in
cellular membrane. The interesting questions in need of answer include, how do leaves
transduce light-elicited signals, what is the role of ion ﬂuxes in the transduction of signals
in photo-responses.

In previous reports, pea leaves displayed rapid membrane depolarization, possibly
involving H+, Cl' and Ca2+ (Elzenga and Van Volkenburgh, 1993; Elzenga et al., 1995;
Stahlberg and Van Volkenburgh, 1999). However, there were no experimental evidence
of ion ﬂuxes for this conclusion. The Microelectrode Ion Flux Estimation (MIFE)
technique has been introduced to investigate ion ﬂux changes in root and leaf tissues
(Lucas and Kochian, 1986; Newman et al., 1987; Shabala et al., 1997; Shabala and
Newman, 1999). This noninvasive approach provides a direct tool to monitor ion
dynamics around the plasma membrane of pea leaf cells, and to reveal possible ionic
transport mechanisms. Ion-selective microelectrodes are utilized broadly in plant cell
biology (F elle, 1992).

This study proposes three hypotheses at the outset: (1) rapid light-induced electrical

signalling results from ions ﬂuxes driven by ion transporters localized in the plasma

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membranes; (2) Ca2+ plays an initial role in affecting activity of other ion transporters
within the plasma membrane; and (3) ion ﬂuxes across the plasma membrane are
inﬂuenced by light quantity and quality. Thus, the ﬁrst goal of this research was to
characterize light-elicited rapid changes in ion (Ca2+, K", H+ and Cl’) ﬂuxes in leaf
tissues, and investigated their relationships to early light electrical signals. Subsequently,
we sought to identify the transport mechanisms involved in regulatory ion ﬂux across the
plasma membranes as well as the role of Ca2+ in light-induced ion ﬂux responses.
Finally, we measured the effects of light quantity and quality on the ﬂuxes of Ca2+, K‘“,
H+ and Cl'. This research should provide experimental evidence to elucidate

photosensory mechanisms in leaves.

LITERATURE REVIEW

1. Generality of Signal Transduction
Like other biological organisms, plants can sense, transduce and respond to external
signals via the built-in signalling systems. When stimulated, speciﬁc receptors induce
one or several intracellular or intercellular sequences of biochemical and /or biophysical
event leading to the modiﬁcation of metabolic activities or the regulation of speciﬁc
genes, which in turn produce a ﬁnal response. Thus, sensory systems of plant cells
typically involve three steps: perception, signal elicitation and transduction, and a
localized or systemic plant response.

Juniper (1976) thought that “perception” is an altered physical state, which is

assumed to pass the ﬁrst physiological phase. The term represents the initial step of

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signalling and involves interaction between the stimulus and the receptors in a plant cell.
However, stimulus-perception may involved a complex of physical, biochemical and
molecular processes. Genoud and Métraux (1999) imaged that perception is analogous to
a group of interacting algorithms, including an input layer, an output layer, and an
intermediate layer linking the output and the input. Thus, they suggested a Boolean
network model. “Signal transduction” infers sequence from stimulus perception to ﬁnal
response, and may involve conversion of energy, alteration of metabolic pathways, and
changes of pH and water potential. Ray (1999) thought that “signal transduction” is the
common term used to deﬁne a diverse topic that encompasses a large body of knowledge
about the biochemical mechanisms that regulate cellular physiology. “Response”
involves downstream steps in signal transduction chains, including morphological,
biochemical, or physical changes.

Research on signal transduction in plants follows animal models. Some important
progress in plants has been made, including the identiﬁcation of the ethylene receptor
(Bleecker and Schaller, 1996), phytochromes (Bowler, 1997), and detection of certain
intermediate proteins, such as receptor-like protein kinases, GTP-binding proteins (G
proteins), phospholipase C and Cazi/cahnodulin-dependent kinases (Gilory and
Trewavas, 1994; Yang, 1996). To study plant sensory systems, molecular genetic,
biochemical and biophysical approaches are used widely in plant research. Signal
transduction mechanisms have been proposed in almost all environmental and hormonal
signals, including gravity (Chen, 1999), water (Bray, 1997), pathogens (Blumwald et al.,
1998, Ebel and Mithofer, 1998), light (Chamovitz and Deng, 1996; Khurana et al., 1998),

mechanical stimuli (Verhey and Lomax, 1993), and abscisic acid (ABA) (Leung and

1‘4

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Giraudat, 1998). However, the mechanistic base of signal transduction in higher plants
remains a “black box”.

With the improvement of research technology, progress has been made toward
understanding plant signal transduction processes. Some proposed models have been
established to explain the biochemical pathways of plant response to external and internal
signals (Gilory and Trewavas, 1994; Yang, 1996; Chang and Stewart, 1998). After
perception by a speciﬁc receptor, the primary signal (ﬁrst messenger) interacts and is
transformed into transduction chains. G proteins serve a key regulatory role in initiating
signal transduction. Next, the secondary messengers amplify the ﬁrst messenger.
Finally, the ampliﬁed signals drive and link cellular signalling pathways that result in
ﬁnal responses. Many secondary messengers/signalling elements are suggested in plants,
including Ca2+, HI, lipids and cAMP (for reviews see Felle, 1993; Verhey and Lomax,
1993; Leckie et al., 1998). Among these second messengers, Ca2+ is best studied and
accepted as a main signalling component. The Ca2+ messenger is thought to connect
many signalling networks in plant cells. However, plant-signalling systems are being
found to be highly complex, as new details are discovered. Interaction between signal
transduction pathways may also occur when plants are stimulated concurrently by

multiple factors.

2. Member potential and electrical signalling
A voltage potential is deﬁned as a voltage difference across a membrane, which is created
by the net separation of positive charges ﬁom negative charges (Wayne, 1993). The

voltage potential represents a quantitative expression of membrane potential. In plant

 

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cells, positive charges are carried by cations, e.g. KI, Ca2+, Na+ and HI, and negative
charges are carried by anions, e.g. Cl', and organic acids. When a plant cell is not
stimulated, the measured membrane potential is considered a resting potential with range
from -100 to -200 mV.

Membrane potential changes whenever the given membrane is perturbed or
stimulated. When plasma membrane depolarization occurs in plant cells, there is a shift
ﬁ'om the normally negative resting potential to less negative potentials. Either a net
positive charge movement into a cell or a net negative charge movement out of a cell is
necessary to effect membrane depolarization. Conversely, membrane hyperpolarization
is a process of either a net positive charge movement out of a cell or a net negative charge
movement into a cell, which shifts membrane voltage potential to more negative. Many
environmental stimuli can alter membrane potential resulting in depolarization or
hyperpolarization (for reviews see Davies, 1987a; Thain and Wildon, 1993).

Electrical signals involving membrane potential changes are thought to “control”
many responses, for instant, plant cell growth (Pickard, 1971; Hush et al., 1991),
metabolic changes associated defense to biotic factors (Wildon et al., 1989), and
activation of ion channels, such as Ca2+, K+, and anion channels. Two classes of electrical
signalling, action potentials and slow-wave potentials, potentially perform local or long-
distance communication (Stankovic and Davies, 1998; Stankovic et al., 1998). Local
electrical signalling occurs when electrical potential changes occur across membranes
within a cell, whereas long-distance signalling involves the systemic transmission and

Propagation of voltage potential changes along membrane systems between cells or

organs.
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organs. Many stimuli induce local membrane potential changes. However, the evidence
of systemic transduction of voltage potential changes seems to be limited to wound-
induced plants (W ildon et al., 1992; Rhodes et al., 1996).

Action potentials in plants are characterized by a large, transient change in the
membrane potential. Slow-wave potentials or variation potentials differ from action
potentials by their amplitude and slower rate of propagation (Frachisse and Desbiez,
1989; Thain and Wildon, 1993; Stahlberg and Cosgrove, 1997). Action potentials are
observed in a wide range of plant cells and thought to play a major role in intercellular
and intracellular communication and in the regulation of ion transport and turgor at the
cellular and organell levels (Retivin and Opritov, 1987; From, 1991). A remarkable
example is Chara algae cell, which exhibits a large and transient action potential (Hope
and Findlay, 1964). Many higher plant species generate action potentials in response to
light (Trebacz and Zawadzki, 1985; Trebacz and Sievers, 1998), cold (Shiina and
Tazawa, 1986; Pyatygin et al., 1992) and wounding (Wildon etal., 1992; Stankovic et al.,
1998)

Action potentials are considered to be the most rapid electrical phenomena in plants.
The electrical signalling suggests that changes in ion ﬂux across the plasma membrane
occur. Wayne (1993) and Davies (1987a) proposed that efﬂuxes of Cl’ and K+ and inﬂux
of Ca2+ give in part rise to the action potentials. Fromm and Spanswick ( 1993) provided
an indirect evidence in willow (Salix viminalis) using ion transporter inhibitors. In light-
induced liverwort (Conocephalum conicum), Cl' channels are involved in the

depolarization event associated with action potentials (Trebacz et al., 1994).

 

 

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3. Ion ﬂuxes and signal transduction

In biology, ion ﬂux is a quantitative expression of the net movement of ions ﬁ'om one site
in a cell to another over a speciﬁc period of time and area. Ion transport across the
plasma membrane is either by passive or active pathways. Passive transport, through ion
channels, protein carriers and facilitated diffusion pathways, depends on electrochemical
gradients of ions across the membrane. In contrast, active transport is an energy-
dependent process.

Membrane potential changes mediated by ion ﬂuxes may be considered primary
events in signal transduction. Ca2+, H", K+ and C1: are believed to be primary ions
involved in effecting changes in membrane potential. Stimulus-induced changes in
membrane potential could be coupled to perception and transduction of extracellular
signals. Consequently, ion ﬂuxes across the plasma membrane may be linked with
cellular signalling pathways. Guard cells provide a valuable model system for study of
photoreception, ion transport, and osmoregulation in plant cells (Serrano and Zeiger,
1989). Stomatal movement is operated through coordination of signalling elements in
guard cell ion channels, and is regulated by ABA and auxin (Grabov and Blatt, 1998).
Membrane depolarization in guard cells is evoked by ABA and auxin and is accompanied
by Ca2+ inﬂux and cytosolic alkalinization (McAinsh et al., 1997). In addition, outward-
rectifying K+ channels are activated by membrane depolarization and a rise in
Cytoplasmic Ca2+ concentration and pH (Plieth et al., 1998; Roelfsema and Prins, 1998;

Blatt, 1999). Opening of anion channels leads to ion efﬂux and further depolarization

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(Ward and Schroeder, 1997). The massive ion ﬂuxes across the guard cells control turgor
and thus stomatal aperture. This indicates that ion ﬂuxes are involved in both signalling
and achievement of the physiological response in guard cells. However, the similar
model of stirnuli-response systems in other leaf tissues are still lacking.

The ionic mechanism is based on the observation of ion inﬂuxes or efﬂuxes across
membranes. However, ion ﬂuxes may be controlled by various transport mechanisms.
Davies (1987b) has already underlined that “ions (...) are likely to play a major role,
especially as local (intracellular) signals”, and that “electrical signals (...) need to attain
respect from a greater number of plant researchers, as candidates for major intercellular
communication devices”. Ion-distribution and ion-channel blocking experiments suggest
that the ﬂuxes of Ca2+, K+, H+, and Cl' across membrane systems may be involved in

speciﬁc electrical signalling events in a transduction pathway.

3.1. Ca2+ﬂwc and Ca“ signalling

Calcium, as a second messenger, plays a central role in signal transduction in plants.
When the primary stimuli perceived by receptors are transduced to intracellular
messengers (e.g., Ca2+), the second messenger ampliﬁes primary signals associated with
modiﬁcation of many enzymes and brings about changes in biochemical and
physiological responses. Numerous reviewers have implicated Ca2+ ﬁinction in plant
signal transduction (Bush, 1995; Blatt, 1999; Sanders et al., 1999; Trewavas, 1999).
Cytoplasmic Ca2+ and Cay-dependent protein kinases have been studied to elucidate

signalling mechanisms.

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A huge Ca2+ concentration gradient exists across the plasma membrane. In resting
cells, cytosolic Ca” concentrations are maintained within the range of 0.1-1.0 uM,
whereas the level of apoplastic Ca2+ for land plants is approximately 1000 uM (Bjorkrnan
and Cleland, 1991; Harker and Venis, 1991). This produces a typical concentration
difference of about 10‘4 to 10'3 fold. Estimation of cytosolic Ca2+ concentration is based
on some reliable techniques, including Ca2+-selective electrodes, ﬂuorescent dyes and
luminescent protein aequorin (Read et al., 1993; Roos , 2000). In general, the electrical
potentials across the plasma membrane are -100 to -200 mV, and the resultant
electrochemical difference in Ca2+, calculated from the Nemst equation, is approximately
50 kJ mol'1 which is comparable to the free energy for ATP hydrolysis, i.e. approximately
-44 kJ mol", in living cells (Miller et al., 1990). The electrochemical gradients for Ca2+
across other cellular membranes, indicating those found the endoplasmic reticulum (ER)
and tonoplast, are also large and rarely reversed (Bush, 1993).

Calcium movement across membranes is facilitated by Cay-permeable channels and
Ca2+ pumps. Passive inﬂux of Ca2+ occurs mainly through Ca2+-permeable channels
found in the plasma membrane. Considering ion selectivity, White (1998) suggested that
there are at least twomajor classes of Ca2+ channels in the plasma membrane. The ﬁrst
class is relatively non-selective with respect to cations, and the other has a high single-
channel conductance and is called a maxi-cation channel (White, 1993; White, 1994).
The second class is referred to as voltage-dependent channels with greater selectivity for
cation (White, 1994; Pineros and Tester, 1995; Pineros and Tester, 1997). In addition,
the third Cay-channel has been reported, a stretch-activated channel, which can be

activated by membrane tension (Cosgrove and Hedrich, 1991; Ding and Pickard, 1993).

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Among the CaZI-permeable channels characterized in the plasma membrane of plant
cells, voltage-activated Ca2+ channels may contribute to calcium signalling. Membrane
depolarization causes activation of Cay-channels. Direct measurement using the patch
clamp technique indicated that depolarization of the plasma membrane in carrot
protoplasts involves activated Ca2+-permeable channels (Thuleau et al., 1994). Similar
observation has been made in maize shoots (White, 1997). Such voltage gating which
activates the channel may be critical during cellular signal transduction. Numerous
stimuli, including blue light (Spalding and Cosgrove, 1992b), red light (Errnolayeva et
al., 1996) and fungal elicitors (Kuchitsu et al., 1993), are known to induce rapid
membrane depolarization. The resultant increase in Ca2+ channel activity and inﬂux
would be expected to enhance cytosolic Ca2+ concentration by inﬂux. Concurrently, the
increase in Ca2+ inﬂux results in membrane depolarization. Therefore, voltage-gated Ca2+
channels provide a connection between stimuli and Ca2+ inﬂux across the plasma
membrane. However, compared with other ion channels (e.g. K“ channels), there is
limited direct evidence that Ca2+ channels operate in the plasma membrane of leaves.
Most conclusions are based on pharmacological and cell biological studies. These results
only provide indirect evidence that voltage-dependent Ca2+ channels in the plasma
membrane are important for the initiation of plant responses to external signals (Hepler
and Wayne, 1985; Leonard and Helper, 1990; Thion et a1, 1996).

Two energy-dependent Ca2+ transport systems have been shown to function. Ca2+-
ATPase provides high afﬁnity Ca2+ transport whereas proton-coupled antiporters provide
low afﬁnity Ca2+-transport (Evans and Williams, 1998). Calcium efﬂux from the plasma

membrane is mediated by Cay-ATPase or ATP-hydrolyzing pumps and calcium

12

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exchangers, which catalyze an exchange of Ca2+ with H+ (Evans et al., 1991; De Michelis
et al, 1992). The nH+/Ca2+ carriers have been reported in the plasma membrane (Rasi-
Caldogno et al., 1987), tonoplast (Schumaker and Sze, 1986), and chloroplast thylakoid
membrane (Ettinger et al., 1999). The plasma membrane CaZI-ATPase itself was
considered to function as a ATP-dependent nH+/Ca2+ antiporter (Rasi-Caldogno et al.,
1987). However, there is no further evidence supporting this. The Ca2+-ATPase is a
primary efﬂux transporter functioning against a Ca2+ concentration gradient. Like the
plasma membrane H+-ATPase, Ca2+-ATPase is also inhibited by vanadate (Williams et
al., 1990; Camelli et al., 1992).

The regulation of cytoplasmic Ca2+ involves a coordination of Ca2+ channels and the
plasma membrane Ca2+-ATPase activity in plant cells. Low cytosolic Ca” concentrations
are required for the stability and activity of many enzymes, and appear to play a critical
role in protein synthesis in the secretory pathways (Rudolph et al., 1989; Gill et al.,
1996). Evans et a1. (1991) suggested that the plasma membrane Ca2+-ATPase plays a
central role in the regulation of Ca2+ homeostasis of the cell, and, in particular, the
restoration of low cytosolic Ca2+ concentrations after a stimulus-induced increase in
concentration. Some intracellular organelles, including vacuoles, endoplasmic reticulum,
mitochondria, and even chloroplasts, are able to store and sequester Ca2+ effectively. The
vacuole occupies 90% of the mature plant cell volume. It contains a high Ca2+
concentration (DuPont et al., 1990) and represents a major intracellular Ca2+ pool. The
voltage-dependent or voltage—gated Ca2+ channels in tonoplasts contribute to the
regulation of cytoplasmic Ca2+ concentrations (Sanders et al., 1999). These channels are

sensitive to inositol-l,4,5-triphosphate (IP3) concentrations, which induce a release of

13

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Ca2+ from the vacuole (Schumaker and Sze, 1987; Alexandre et al., 1990; Gilroy et al.,
1990). Afterward, cyclic ADP-ribose (cADPR) was found to activate tonoplast Ca2+
charmels (Allen et al., 1995), thereby indicating that tonoplast Ca2+ channels are ligand-
regulated. IP3 is also proposed to be an important signalling messenger in cellular signal
transduction (Gilory and Trewavas, 1994; Leckie et al., 1998). Calmodulin (CaM), upon
binding to‘Ca”, is another unique protein which interacts with a number of key enzymes,
and other structural proteins called CaM-binding proteins (Roberts et al., 1986). The
plasma membrane Ca2+-ATPase, a CaM-binding protein, can be activated by CaM
(Colbran and Soderling, 1990; Hsieh et al., 1991). The CaZI-binding-CaM enhances the
capability of the plasma membrane Cay-ATPase to respond to an increase of cytoplasmic
free Ca2+ concentrations.

The spatial and temporal changes in cytosolic Ca2+ indicate that Ca2+ is involved in a
wide range of cellular signalling networks, and Ca2+ oscillations are crucial to the
speciﬁcity of Ca2+ signalling mechanisms (McAinsh and Hetherington, 1998). Increases
in cytosolic free Ca2+ concentrations have been widely observed in response to a number
of stimuli, including that of ABA (McAinsh et al., 1990), mechanical stress and cold
(Knight et al., 1991; Knight et al., 1992), auxin (F elle, 1988a), red light (Shacklock et al.,
1992), and during phototropism and gravitropism (Gehring et al., 1990). The role of Ca2+
in action potentials has been demonstrated (Beilby, 1984; Wayne, 1993). Calcium
signalling is involved in cell grth and development (Brownlee et al., 1999). Ca2+-
/CaM-dependent proteins and protein kinase C, driven by increased cytosolic Ca2+,

regulate protein phosphrylation/dephosphorylation in plant cells (Poovaiah and Reddy,

14

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1993; Trewavas and Malho, 1998), a primary mechanisms of signal transduction.
Changes of in intra- and intercellular Ca2+ concentrations are considered to be a possible
key component of cellular signalling pathways.

Coordination of Ca2+ transporters in membranes is necessary to achieve the dramatic
changes of cytosolic Ca” concentration, and time course in concentration oscillations.
Trewavas (1999) suggested that the function of cytoplasmic Ca2+ oscillation may lie in
constructing a cellular “intelligence”. Determination of changes in Ca2+ ﬂuxes may be an
important approach to the study of Ca2+ signalling mechanisms. Some common Ca2+-
related signalling components, such as Cazidependent protein kinase (CDPK), and
CaZI/CaM-dependent protein kinases, support the role of Ca2+ in cellular signal
transduction. However, CaZI-independent pathways or other signalling systems are

accepted as existing in plants (MacRobbie, 1992; Allan et al., 1994).

3. 2. H + ﬂuxes

Plant cells maintain a pH gradient across the plasma membrane of about 1.5 - 2.0 pH
units, i.e., cytoplasm with pH 7.0-7.5 and apoplast with pH 5.0-5.5. The proton gradient
is believed to energize many important transport systems through H+-coup1ed co-
transporters, including (1) solute uptake into the cells against concentration gradients
mediated by syrnporters, and (2) loading of organic compounds into the phloem for
transport to sink tissues (Palrngren and Harper, 1998). Thus, this electrochemical
gradient, known as the proton driving motive force (dmj), represents the sum of the

membrane potential and the pH difference across a given membrane. The dmf provides

15

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energy for secondary active proton-driven solute transport across the plasma membrane,
e.g. K+ (Marré et al., 1989) and sugars (Serrano, 1985). Mitchell’s chemiosmotic theory
hypothesized that H+ gradients across biological membranes ﬁmction as a source of
energy for cellular work (Mitchell, 1961;, 1976;, 1985).

Proton ﬂuxes across the plasma membrane are chieﬂy catalyzed by the plasma
membrane H+-ATPase (proton pump) and H+-coupled transporters. The plasma
membrane H+-ATPases generate a chemiosmotic H+-gradient and are the main pathway
for H+ efﬂux across the plasma membrane. Studies have demonstrated that the plasma
membrane H+-ATPase is a proton pump and is electrogenic (i.e., it establishes a negative
membrane potential that is positive on the outside, and negative on the inside) (Sze,
1985). The enzyme pumps outwardly H+ across the plasma membrane into the apoplast.
The resulting extracellular acidiﬁcation, i.e. efﬂux of positively charged H+, causes an
initial repolarization of membrane potential. On the contrary, the inward movement of
H” produces a membrane depolarization and cytoplasmic acidiﬁcation. Biochemical and
molecular research indicates that the enzyme is involved in many physiological roles
(Palrngren and Harper, 1998; Sze et al., 1999).

A major function of the plasma membrane H+-ATPase is the establishment of a pH
gradient across the plasma membrane. Although the enzyme plays an indirect role for
nutrient uptake into the cell, it actually serves as a main energy source in plant cells. For
example, from the Nemst equation, En = 58.2 log [Ion]ou,/[Ion]m. A Nemst membrane
potential (E,) of -175 mV (3x 58.2), [Ion].m,/[Ion]in would create a 1000-fold

concentration gradient in a monovalent cation such K+; i.e., the cell can maintain 100

16

m“. K' :r

me 5011 so

 

Protor

imports

abut H‘

mM K+ inside by virtue of a channel protein even though K+ is present at only 100 uM in
the soil solution (Stein, 1990).

Proton inﬂux into the cytoplasm is mediated by symporters, such as H+/K+
(Rodriguez-Navarro et al., 1986; Netting , 2000) and H+/Cl' (Sanders et al., 1985) in the
plasma membrane. Both carriers also facilitate K+ and Cl' uptake by cells. However, the
rate of transport through symporters is much slower than via K+ and anion channels
(Palmgren and Harper, 1999). This is because solute ﬂuxes through symporters are
energetically coupled with H+ transport. The symport enzyme requires energy
transformation for conformational changes. Clearly, both H+-ATPase and H“ coupled
symports regulate proton ﬂuxes across the plasma membrane. Experimental evidence
about H+/K+, H+/Cl' or other symporters remain insufﬁcient.

A high cytoplasm pH is of utmost importance for maintaining cellular metabolic
activities. A number of transporters capable of maintaining a stable cytoplasmic pH are
well characterized (Sze et al., 1999). Except the plasma membrane H+-ATPase, the H‘-
ATPase and H+-pyrophosphase in the tonoplast are also important pathways contributing
to cytoplasmic ‘pH stat’ (Sze et al., 1992; Rea and Poole, 1993; Zhen et al., 1997).
Moreover, the regulation of cytoplasmic pH regulation is thought to be linked to K+
channels (Blatt, 1992; Ilan et al., 1994), and anion channel activity (Johannes et al.,
1998). In corn root tissue, Felle (1998) found that cation channels may contribute to
apoplastic pH regulation. Several other mechanisms are involved in pH regulation,

including the production and consumption of malate via the biochemical pH stat (Leigh,

1993).

17

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pH regulation in plant cells may reﬂect a dynamic equilibrium in ion ﬂuxes across

membranes. Protons are considered to be another second messenger in plants because of
the above pH-dependent processes (Felle, 1989b; Felle, 1993; Grabov and Blatt, 1998;
Leckie et al., 1998). The most remarkable observation is that ABA functions in the
regulation of stomatal movement. The ABA action in stomata is strictly associated with
cytoplasmic alkalinization by 0.2-0.4 pH (Iring et al., 1992; Blatt and Thiel, 1993). Blatt
and Armstrong (1993) conﬁrmed the key role of H+ as a messenger in regulating stomata
aperture when they found that ABA-induced stomatal closure was blocked by low
cytosolic pH. Another hormone, auxin, may also play a role in cytoplasmic pH
signalling. Auxin at low concentrations can elicit stomata opening (Marten et al., 1991)
and reduce cytosolic pH (Irving et al., 1992). This is because auxin stimulates proton
extrusion by enhancing the activity of plasma membrane H+-ATPases (Felle, 1993). The
resulting cytoplasm alkalinization activates a K+-inward rectiﬁer channel, which leads to
osmotic changes in guard cells and ultimate stomatal opening (Thiel and Wolf, 1997). In
contrast, the key role of the H+ messenger for stomatal closure is achieved by activation
of a K+-outward rectiﬁer channel (Lemtiri-Chlieh and Macrobbie, 1994; Grabov and
Blatt, 1997) and anion channels (Blatt and Grabov, 1997).

The experimental evidence for the role of H+ as second messenger is limited to guard
Cells so far. The cytoplasm pH regulation is known to link many ionic transport
meChanisms. It thus is reasonable to assume that H+ messenger plays a similar role in
Other tissues as guard cells. The regulation of plasma membrane ATPase may provide an

connection for H“ ﬂuxes and stimuli. Auxin has been demonstrated to activate gene

18

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expression of H+-ATPase (Theolois, 1986). This enzyme was also phosphorylated in
vitro (Xing et al., 1996). A high-afﬁnity fusicoccin binding protein has also been
identiﬁed in different plant tissues (Weiler et al., 1990). In addition, white light
(Linnemeyer et al., 1990) and blue light (Shimazki et al., 1993) stimulate proton efﬂux,

suggesting that H+-ATPase may be regulated by external factors.

3.3 K ﬂux

Potassium ion (K+) has been demonstrated to serve both biophysical and biochemical
functions in plant cells. In the cytoplasm, K+ within the 50-500 mM range, is an
indispensable cytosolic component in plant cells, a major contributor to osmotic potential
of plant cells (Haschke and Liittge, 1975). Because of the high concentration gradient
across the plasma membrane, K+ also exerts a dominant inﬂuence on the diffusion
potential component of transmembrane electrical potential. Additionally, high
cytoplasmic K” serves as a catalyst for a large number of enzymes (Evans and Wildes,
1971) and is a requirement for protein synthesis (Leigh and Wyn Jones, 1984; Memon et
al., 1985). K+ ﬂuxes across the plasma membrane are essential for many physiological
ﬁmctions, e.g., stomatal opening (I-Iumbel and Hsiao, 1970) and leaf movements (Lee,
1990). The regulation of K+ transport and cytosolic K+ homeostasis is accomplished by
K+ channels and ion-coupled KI symport or antiport. High afﬁnity uptake via a symport
meChansim has been postulated in Neurospora (Blatt, 1987). The symport of HI/K+
results in K+ accumulation in the cytoplasm against its concentration gradient. The

inﬂuxes of KI and W depolarize the membrane and render the apoplast alkaline (Netting ,

l9

den
5110'.

hp:

Char

2000). In contrast, a K+/H+ antiport facilitates H+ efﬂux coupled to K” inﬂux. This
mechanism has been referred to as a capable pump-independent pathway of short-tenn
pH regulation (Marré et al., 1987; Felle, 1988b; F elle, 1989a).

Two classes of K+ channels have been characterized; an outwardly-directed K+
channel facilitating K” efﬂux and an inwardly-directed K+ channel facilitating or K+
inﬂux. The activity of both channels depend strongly upon their membrane potentials,
various effectors, channel. blockers, as well as their respective gating mechanisms
(Schroeder, 1992; Blatt and Thiel, 1993). Known K+ channels are highly permeable to
K”, which is larger than for other monovalent cations, such as Pb+, NH+, and Na+
(Bentrup, 1990). Compared as to other cation channels, important progress has been
made in understanding the molecular mechanisms and regulation of K+ channels
(Czempinski et al., 1999). Two types of inwardly rectifying K+ channels (KTAl , AKTl)
from Arabidopsis thaliana have been cloned (Anderson et al., 1992; Sentenac et al.,
1992)

Potassium inward rectiﬁers, or inwardly-directed K+ channels were originally
demonstrated in guard cells (Schroeder et al., 1987; Blatt, 1992). These channels were
shown to be activated in a voltage- and time-dependent manner when membranes are
hyperpolarized ﬁom -50 mV to -200 mV (Blatt, 1992). Thus the inwardly-directed K"
Channels are considered to represent a pathway for K+ uptake by plant cells. Another
pathway for K+ inﬂux is K“-H+ symport. However, it is not easy to distinguish
SYn'Iporters from K" inward rectiﬁers (Hedrich and Schroeder, 1989).

Another class of K+ channels, outward rectiﬁers, or outwardly-directed K+ channels,

20

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represent a main mechanism for K+ efﬂux across the plasma membrane. This type of K”

channel is activated by depolarization of the membrane potential (Iijima and Hagiwara,

1987; Moran et al., 1988; Schroeder, 1989). In guard cells, outwardly-directed K+

channels play a major role in stomatal closure (Thiel and Wolf, 1997). The same K+
outward currents were also observed in Arabidopsis thaliana mesophyll cells (Miedema
et al. , 2000). The efﬂux of K+ mediated by outwardly rectifying K”r conductance may
result in repolarization of action potentials in both algae and higher plant cells (Sikaoka,
1 966; Beilby and Coster, 1979; Simons, 1981) and takes part in motor cell movement of
Samanea saman (Satter et al., 1988).

In general, K+ ﬂuxes mediated by K+ channels are thought to possibly be involved in
signalling in guard cells, or participate in electrical signalling in excitable plants. Both
classes of K+ channel are sensitive to changes in apoplastic pH and Ca2+ concentration
(Herich et al., 1990; Blatt and Armstrong, 1993; Grabov and Blatt, 1997; Roelfsema and
Prins, 1998). Cytoplasmic acidiﬁcation reduces the conductance of the outward rectiﬁer
but increases that of the inward rectiﬁer. Increased cytoplasmic Ca2+ is known to
inactivate the K“ inward rectiﬁer (Kelly et al., 1995) but to activate another one, i.e., the
K+ outward rectiﬁer (Schroeder and Hegiwara, 1989). The involvement of K” channels in
Sensing numerous stimuli suggests that K+ ﬂuxes or channels play a potential role in
certain signalling pathways similar to that found in guard cells. Assmann (1996)
hYpothesized that G-protein regulation of K+ channels may be a main ABA—signal
transduction pathway. Further regulation of K+ channel gating may be accomplished by

G proteins and protein phosphorylation (F airley and Assmann, 1991; Kelley et al., 1995).

21

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Therefore, K+ ﬂuxes or channels may be involved in many processes, including

regulation of pH and Ca”, and hormone signalling.

3. 4. C1” ﬂuxes

Chloride ion (Cl') is a predominant inorganic anion in plant cells. A large
electrochemical gradient for C1' exists across the plasma membrane. For example, a
positive potential difference of 300-340 mV is typically present across the plasma
membrane in Chara (Smith and Walker, 1976). The concentration gradient drives
passive Cl’ efﬂux from the cytoplasm. Chloride efﬂux also play an important role in
maintaining a negative membrane potential. It thus can be anticipated that anion channels
are a primary route of Cl' efﬂux from the cytoplasm, while the inﬂux or uptake for C1’ is
mediated by a carrier mechanism (Assmann and Zeiger, 1987). Zeiger et a1. (1978)
hypothesized that Cl‘ enters via a Cl'/H+ symport or a Cl'/OH‘ antiport.

Chloride inﬂux requires the input of a considerable amount of metabolic energy. The
pH gradient across the plasma membrane, or proton motive driving force, is thought to
provide this energy. Chloride import through a 2H*/Cl‘ symport against an opposing Cl'
concentration gradient has been proposed and has been demonstrated experimentally in
Chara (Smith and Walker, 1976; Sanders, 1980; Beilby and Walker, 1981; Sanders et al.,
1985) and root-hair cell of Sinapis alba (F elle, 1994). However, experimental proof in
higher plants has remained somewhat scant so far.

Anion channels have higher selectivity for C1‘ than for malate (Hedrich and Martin,
1993; Schmidt and Schroeder, 1994). Many elements, such as nucleotides (Herdich et al.,

1990), pH (Tyermen et al., 1986), malate (Hedrich and Marten, 1993), and

22

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phosphorylation/dephosphorylation events (Terry et al, 1991; Hedrich and Marten, 1993),
affect the activity of anion channels. Cytoplasmic Ca2+ has been shown to regulate anion
channels (Hedrich et al, 1990; Schroeder and Hagiwara, 1990). A CaZI-activated Cl“
channel has been observed in an inside-out membrane patch of Chara plasma membrane
(Okihara et al., 1991), whereas Cl' efﬂux in perfused Chara cells is sensitive to Ca2+
inﬂux (Mimura and Shimmen, 1994). An anion-conducting chloride channel family
(CIC) has been cloned (Lurin et al., 1996; J entsch and Giinther, 1997).

Two modes of anion channel activity have been identiﬁed in guard cells: slow-type
(S-type) and rapid-type (R-type) (Schroeder and Keller, 1992; Schroeder, 1995). The
slow and sustained activation of S-type anion channels would produce a long-term
depolarization which last a range of several minutes or longer (Schroeder and Hagiwara,
1 989; Linder and Raschke, 1992). ABA and Ca2+ cause long-term depolarization (Bisson
and Gutlmecht, 1980; Findlay, 1982; Blatt, 1987), leading to the suggestion that the S-
type anion channels may be activated by either ABA or Ca2+. On the other hand, the
rapid anion channels (R-type), produce a short, transient depolarization of the plasma
membrane (Hedrich et al., 1990). In light of voltage-dependent, anion channels can also
be divided into at least two major classes: depolarization- (Tazawa et al., 1987; Keller et
al., 1989; Schroeder and Hagiwara, 1989) and hyperpolarization-activated (Tyennan et
31-, 1986; Terry et al., 1991). Another class of anion channels can be activated by
Stretching of membrane (Falke et al., 1988; Schroeder and Hedrich, 1989; Cosgrov and
Hedrich, 1991).

Tyerman (1992) and Schroeder (1995) suggested that anion channels play key roles in

Controlling cellular functions, including turgor- and osmoregulation, stomatal movements

23

and anion transport. Most results in studies of guard cells indicate that anion channels in
the plasma membrane may provide a control mechanism for stomatal closure. The
proposed model suggests that anion channel activation and the resulting anion efﬂux
from guard cells cause further membrane depolarization, thereby activating the non-
selective Ca2+ channel which ultimately driving K+ efﬂux through outwardly-directed K+
channels required for stomatal closure (Schroeder and Hagiwara, 1989; Ward and
Schroeder, 1997; Asamann and Shimazaki, 1999). Therefore, in guard cells, anion
channels may be involved in ABA-induced stomatal movement by linking the Ca2+
messenger, Cl' and K+ efﬂuxes. Furthermore, anion channels-controlling Cl' efﬂux
constitutes one of main ionic components in leaf movement (Satter, 1988; Coté, 1995).
Sensitivity of anion channels to a wide range of stimuli provides a possible role for
involvement of Cl‘ ﬂux in cellular signal transduction. Johannes et a1 (1998) proposed
that anion channels play a central role in cytosolic pH regulation in plants, in addition to

their established roles in turgor/volume regulation and signal transduction.

4 Light Signal Transduction

4. 1 Photoreceptors

Light plays a primary role in regulating the growth and development of plants. Two
important processes in plants, photomorphogenesis and photosynthesis are driven directly
by light. Light also provides plants with many different kinds of information about their
environment, such as quantity, quality, and direction. This information, known as light
Signals, is perceived and integrated by a complex of sensory systems, and used to

Optimize a variety of physiological and morphological responses. Recently, much

24

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attention has focused on photosensory mechanisms in plants, and several light signalling
pathways have been proposed (Chamovitz and Deng, 1996; Batschauer, 1998; Khurana et
al., 1998). The most important progress has been identifying photoreceptors.

Three classes of photoreceptor systems have been suggested for the photoperception
in plants. These include phytochromes, blue/UV-A photoreceptors, and UV-B
photoreceptors. The presence of multiple photoreceptors provides an approach to explore
photosensory machinery.

Among these photoreceptors, phytochromes are the best characterized with speciﬁc
' sensitivity for red/far red light in plants. Five phytochrome genes (phyA ~ phyE) are
present in Arabidopsis (Sharrock and Quail, 1989; Quail et al., 1991; Neuhaus et al.,
1993; Clark et al., 1994). Light absorption is mediated by a chromophore covalently
bound to an apOprotein, and each phytochrome can exist in two photo-interconvertible
forms: a red (R) absorbing Pr form and a far-red (FR) absorbing Rfr form (Bowler, 1997).
Several models of phototransduction mediated by phytochromes have been hypothesized
(Bowler et al., 1994b, Chamovitz and Deng, 1996). Biochemical and molecular research
suggested that GTP binding proteins (G-proteins) are major components of signal
machinery (Clark et al., 1993). Ca2+ and cyclic GMP (cGMP) are utilized as second
messengers in phytochrome signalling (Neuhaus et al., 1993; Bowler et al., 1994a).
However, the primary mechanism of phytochrome action remains to be determined.

Progress in understanding photoregulation of plant processes activated by blue-light
has until recently lagged far behind the phytochrome work (Briggs and Liscum, 1997).
Four blue-light responses in higher plants have receiving concentrated attention

(Kaufman, 1993). Two responses occur in the leaf (blue-light-induced increase in

25

stomatal aperture and blue-light-induced change in gene expression) and two responses
occur in the stem (blue-light-induced suppression of stem elongation, blue-light-induced
curvature of the stem, i.e. phototropism). However, blue-fUV-absorbing photosensory
systems are still elusive. Higher plant blue-light photoreceptor have not been deﬁnitively
isolated nor characterized (Short and Briggs, 1994). Based on their absorption properties,
ﬂavins, proteins or carotenoids have been proposed as blue-light receptors
(cryptochromes) in higher plants (Galland and Senger, 1991; Horwitz and Berrocal,
1997). Phototropism is representative of blue-light-induced responses in plants. Briggs
and co-workers have identiﬁed in maize coleptiles and etiolated pea seedlings a plasma-
membrane—associated protein of about 120 kDa, which is connected to protein
phosphorylation following exposure to blue light (Gallagher et al., 1988; Short and
Briggs, 1990; Short et al., 1991; Palmer et al., 1993). Quarmby (1994) and Chamovitz
and Deng (1996) have proposed that Ca2+ and cAMP have a role in blue-light signalling
pathways. However, to our knowledge, the mechanism of blue-light signal transduction

is still unclear.

4.2 Light effects on membrane potentials and ion ﬂuxes

An early response by leaf cells to light stimulation is a transient change in the membrane
potential. Bentrup (1974) found that light induces changes in transmembrane electrical
potential in green tissues. The transition from light to dark usually reduces the
extracellular membrane potential prior to a subsequent increase, while the reverse
transition has an opposite effect in T radescantia albbiﬂora leaves (Giroldini, 1988). A

similar transient depolarization of membrane potential were found in light-induced

26

mesophyll cells of Dionaea muscipula Ellis (Trebacz and Sievers, 1998), pea (Elzenga et
al., 1995), the intact leaves of Atriplex and Chenopodium (Liittge and Pallaghy, 1969),
T radescantia and Commelina (Cheeseman et a1, 1982), Arabidopsis (Spalding et a1,
1992), as well as in several other aquatic and terrestrial plants (Novak and Ivankina,
1975). Most electrical responses to light involve membrane depolarization. However,
light-induced membrane hyperpolarization has also been observed (Mimura and Tazawa,
1986; Takeshige et al., 1992).

The changes in membrane potential indicate that light induces underlying changes in
ion ﬂux across membranes and infer the involvement of ion transporters. In the
Characean algae, the action-potential-like depolarization results from activation of Ca2+
(Weisenseel and Ruppert, 1977) and Cl‘ channels (Lunevsky et a1, 1983). Chloride
pumps as well as Ki and Cl' channels were involved in action potentials in Acetabularia
(Mummert and Gradmann, 1991). In gametophytes of Asplenium trichomances, light-
induced depolarization was suppressed by C1' channel blockers (Szarek and Trebacz,
1999). Blom-Zandstra et a1. (1997) reported transient light-induced changes in ion
channel and proton pump activities in tobacco mesophyll protoplasts, and suggested that
these ion transporters in the plasma membrane were involved in light-induced transient
changes in the membrane potential. In Arabidopsis mesophyll, white light induced an
increase in transient membrane depolarization and in K" channel activity within the
plasma membrane (Spalding et al., 1992). A light-induced anion channel in the plasma
membrane of pea mesophyll cells has been identiﬁed by Elzenga and Van Volkenburgh

(1997). Shabala and Newman (1999) reported a light-induced ﬂux response involving

27

Hi, Ca2+, Cl' and K” around bean mesophyll tissues. Light increased Ca2+ ﬂux into xylem
exudate from excised tomato petioles within less than 4 s after light-on or light-off
treatments (Ries et al., 1995). Light also stimulated changes in the pH of the apoplasm of
pea leaves (Van Volkenburgh and Cleland, 1980; Staal et al., 1994; Stahlberg and Van
Volkenburgh, 1999). Similar result was reported in the cytoplasm of the unicellular
green alga Ermosphaera viridis (Bethmann et al., 1998).

Both red and blue light are thought to stimulate stomatal opening (Assmann and
Shimazaki, 1999). Red light, matching the absorption spectrum of chlorophyll,
stimulates an outward electrical current that may be carried by the plasma membrane H”-
ATPase in Vicia guard cell protoplasts (Serrano et al., 1988). Blue light presented-as a
single pulse stimulates stomatal opening (Zeiger et al., 1987; Zeiger, 1990) and induces
proton extrusion from guard cell protoplasts (Assmann et al., 1985; Shimazaki et al.,
1993). In addition, since an anion channel in Arabidopsis hypocotyl is activated by blue
light (Cho and Spalding, 1996), blue light would probably affect anion channels in guard
cells. Using calcium-sensitive ﬂuorescent dyes, Shacklock et al. (1992) have shown that
red light induces proplast swelling and causes a transient Ca2+ increase followed by a
rapid reduction in cytoplasmic Ca2+ to below resting levels. The blue-light-induced
membrane depolarization in etiolated cucumber hypocotyls involves inhibition of the
proton pump and inﬂux of calcium (Spalding and Cosgrove, 1992b), whereas K+ channels
are activated by both phytochromes (Lew et al, 1990) and blue-light activated (Suh et al. ,
2000)

Clearly, light elicits transient electrical signals facilitated by ion ﬂuxes across the

28

plasma membrane of plant cells. These changes may be one of rapid pathways of light
signal transduction. Athough biochemical and molecular technology are becoming
dominant approaches for the investigation of signalling intermediates and to elucidate the
proposed signal transduction pathways, the ionic approach is still an important and

powerful tool to understand photosensory mechanisms.

29

Chapter I.

CHARACTRIZATION OF LIGHT-INDUCED CA”, H*, K+ AND CL'
FLUXES FROM MESOPHYLL AND EPIDERMAL TISSUES OF

PEA LEAF TISSUES.

30

ABSTRACT

Peeled mesophyll and epidermal tissues ﬁ'om excised leaﬂets of pea (Pisum sativum L.
cv. Argenteum) bathed in 0.5 mM CaSO4 and 1.0 mM KCl at pH 5.2 were exposed to an
approximately 30 min light (450 nmol m‘2 5") period. The net ﬂux rates of Ca2+, Hi, K+
and Cl’ around mesophyll and epidermal tissues were measured simultaneously by
noninvasive ion-selective microelectrodes. Light stimulation resulted in a transient net
Ca2+ inﬂux an increase in inﬂux of about 50 nmol m'2 5'1 within 1 min in mesophyll
tissue. The light-response lag time of Ca2+ is the most fast (13 5), whereas net inﬂuxes of
H, Cl’ and 1C were also rapidly induced to increase within approximately 29, 45, and 48
s by light exposure, respectively. A light-induced extracellular alkalinization (pH
increase of 0.4 - 0.5) was observed that lasted 5 - 6 min. Following the light-induced
initial ﬂux response, a rapid post-initial ﬂux response occurred for Ca2+ and Cl‘ which led
to a net efﬂux within 3 - 4 min. When light was turned off, net ﬂux of all four ions
decreased and then gradually exhibited a small change in magnitude. In contrast, in the
epidermis, light only induced slight changes in the ﬂux of Ca2+, H”, K+ and Cl'.
Compared to pH 5.2, changing pH in the bathing solution to a more acidic conditions (pH
4.0) increased further the light-induced initial response in net K+ and Cl' inﬂux, whereas
alkalinization of external medium (pH 6.0) produced an opposite effect on K+ inﬂux and
enhanced the light-induced Post-Initial Flux Response of Cl' (i.e. a net efﬂux). Inhibition
of ion ﬂux by DCMU (0.05 mM) indicates that the light-induced change in ion ﬂuxes is
dependent upon photosynthesis. These results suggest that the light-induced increase in

net Ca2+ inﬂux is an initial response that may contribute to membrane depolarization.

31

Light-induced initial apoplastic alkalinization could be explained as either CO2 uptake by

mesophyll tissues or increased uptake of K+ and Cl‘.

INTRODUCTION

Light-induced signalling systems have been found to exist in higher plants. One of the
most remarkable light responses is a rapid change of membrane potential. Almost all
green plant cells investigated react to sudden illumination by light-induced electrical
signals. Because of the complexity of the effect and differences among plant species,
either hyperpolarization (Mimura and Tazawa, 1986; Takeshige et al., 1992) or
depolarization (Elzenga et al., 1995; Szarek and Trebacz, 1999) has been measured. Most
results, however, showed that light induces a transient depolarization.

The mechanism of light-induced electrical signalling is not yet entirely understood.
Membrane potential represents a voltage difference between cations and anions across a
given membrane. Membrane potential changes indicate that changes in ion ﬂux across
membrane systems occur. Therefore, it is possible that light also induces changes in ion
ﬂuxes across the plasma membrane of plant cells. Four ions, Ca2+, H“, K“ and Cl“, were
shown to correlate with the light-induced bioelectrogensis. Some researchers tried to
look for whether ion transporters in the plasma membrane are involved in light-induced
electrical signalling (Mummert and Gradmann, 1991; Ermolayeva et al., 1996; Stahlberg
and Van Volkenburgh, 1997 ; Szarek and Trebacz, 1999).

Opinions in previous reports about the ionic basis involved in the light-induced
electrical response are still controversial, and possibly there are somewhat different

mechanisms in different species and even different tissues of the same species. In

32

ior.

66.:

K01

New

1995

addition, experimental technology is often different in some reports. The characterization
of the light-induced ionic responses, the magnitude of the responses, the speciﬁc ions
involved and time responses depend strongly on the ionic compositions (Elzenga et al.,
1995; Johannes et al., 1997) and pH (Prins et al., 1982; Remis et al., 1994; Shabala et al.,
1997) of the medium environment. Pea leaves have been shown to display a rapid
membrane depolarization followed by a slower membrane repolarization (Elzenga and
Van Volkenburgh, 1993; Elzenga et al., 1995). These reports suggest that H, Ca2+ or Cl'
ﬂuxes across the plasma membrane may be involved in the light-induced electrical
signals of pea mesophyll. However, there is an incomplete understanding about which
ion is acting as the depolarizing agent in the initial phases of plasma membrane
depolarization and how the ions are involved in the event.

To study light-induced ionic responses, some research groups have used ion-selective
microelectrodes to measure membrane potential changes. Ion-selective microelectrodes
have been utilized broadly in plant cell biology. The Microelectrode Ion Flux Estimation
(MIF E) technique was introduced to investigate ion ﬂuxes around root (Lucas and
Kochian, 1986; Newman et al., 1987; Shabala et al., 1997) and leaf tissues (Shabala and
Newman, 1999). This non-invasive approach provides a direct tool to monitor ion
dynamics around the plasma membrane of pea leaf cells, and to reveal rapid photosensory
mechanisms. Shabala and Newman (1999) found that light induced a rapid increase in
Ca2+ inﬂux in bean mesophyll tissues and proposed that Ca2+ inﬂux was involved in light-
induced depolarization. However, Cl' efﬂux has also been proposed to be involved in the
depolarization response to light stimulation (Spalding and Cosgrove, 1992; Elzenga et al.,

1995)

33

Reports about the role of proton ﬂux in plasma membrane electrical signalling are
also contradictory. Plasma membrane H*-ATPase activity was reported to increase
following light illumination (Marré et a1, 1989; Linnemeryer et al., 1990; Remis et al.,
1994). The enzyme can mediate H+ efﬂux from leaf cells which leads to apoplastic
acidiﬁcation. However, it has been observed in many cases that light causes an initial
extracellular alkalinization in leaf tissues (Prins et al., 1982; Shabala and Newman, 1999;
Stahlberg and Van Volkenburgh, 1999). The reason for this contradiction could be that
there has not been direct evidence to explain the experimental relationship between H+
ﬂuxes and extracellular pH under light exposure. One can expect that external pH
inﬂuences ion transports across the plasma membrane. Therefore, measurement of ion
ﬂuxes from pea mesophyll in the changing external pH environment may provide insights
into pH and H” ﬂux responses induced by light.

The ﬁrst investigation presented in this chapter deals with the characterization of
light-induced ion ﬂuxes from mesophyll and epidermal tissues of pea leaves. To explain
the ionic basis of the light-induced transience of membrane depolarization of pea leaf
tissues, the kinetics of Ca2+, K+, H“ and Cl‘ ﬂuxes across the plasma membrane was
measured using the non-invasive MIFE technique. External pH effects on light-induced

ion ﬂux changes are discussed.

MATERIAL AND METHODS
Plant Material

Pea (Pisum sativum L. cv. Argenteum) seeds were soaked in distilled water for 6-8 hours,

34

and then germinated in an artiﬁcial clay shale (Turface manufacture) at room temperature
(23i1°C). After 4-5 days of germination, the young seedlings were transplanted and
transferred to a growth chamber with a 16 hour photoperiod (200 nmol m'2 s'1 at the top
of the canopy), day/night temperatures of 20/25 °C and 75% relative humidity. The
plants were cultured in a modiﬁed Hoagland solution (Johnson et al., 1957) following an

increase in concentration from 0 —) ‘A —> V2 at 4-5 d intervals after transplanting.

Ion-Selective Microelectrode
Glass capillaries (catalog no. MIB 150-6; World Precision Instruments, Inc., Sarasota,
FL, USA) were pulled using a two stage puller (PC-10, Narishige, Tokyo, Japan) to
produce micropipettes with a tip diameter of about 2 pm. After pulling, the micropipettes
were dried in the oven at 200°C for 4-5 h, and silanized with N-
dimethyltrimethyldiylarnine (catalog no. 41716, F luka, Milwaukee, WI). Cooled
microelectrodes were back-ﬁlled with 500 mM CaCl2 for Ca”, 500 mM KCI for K+ and
Cl', and 15 mM NaCl plus 40 mM KHZPO, (adjusted to pH 6.0 with NaOH) for H.
Electrode tips were then front-ﬁlled with a 50-80 rim-long column of the appropriate ion-
selective sensor, including H+ (no. 95291), Ca2+ (no. 21048), K“r (no. 60031) and Cl' (no.
24902) cocktails (all from Fluka, Milwaukee, WI). Silanized micropipettes were used
within one week of preparation, while the ﬁlled microelectrodes only were used within
one-day period.

Microelectrodes were calibrated for each ion using a known set of standard solutions

before and after use. The standard solutions contained a pH 4.0 -7.0 for H, 0.1 — 2.0 mM

35

CaSO, for Ca”, or 0.1-5.0 mM KCl for K and CI. The average slope was 52-55 mV /
pIon for monovalent ions and 26-29 mV / pCa2+ for Ca2+ electrodes.

An Ag/AgCl reference electrode was prepared using a 4-5 cm section of 2 mm
diameter polyethylene tubing ﬁlled with 2 % agar in 1 M KCl. A section of silver wire
(diameter 0.38 mm) was coated in 0.2 M HCl. The coated silver wire was then inserted
into one end of the tubing containing 2 % agar and wrapped with paraﬁlm to ﬁx in
position. The reference electrode was stored in 1 M KCl solution and only used 1 (1.
During measurements, the end of the reference electrode with silver wire was connected
to the preampliﬁer, while the open end of the tubing was bathed into solution being

measured.

MIFE system
Ion concentrations at the surface of the leaf mesophyll cells were measured using a non-
invasive Microelectrode Ion Flux Estimation (MIFE) system (MIFE, Unitas Consulting,
Hobart, Australia) (Shabala and Newman, 1997; Shabala et al., 1997). From time course
data obtained by the MIFE system, the directionality and rates of ion ﬂux could be
estimated. Additional information on the MIFE is available at http://www.phys.utas.edu
.au/physics /biophys.

The set-up of the MIFE system is illustrated in Figure 1 and 2. An inverted
microscope (Leitz Wetzlar, Germany) was placed on an anti-vibration table that was
installed inside a Faraday cage to minimize electrical and magnetic noise. Three ion-

selective electrodes were mounted on a coarse multi-microelectrode holder/manipulator

36

with three-dimensional positioning ability. After a tissue ample/solution chamber (8.0x
2.5 x 1.5 cm) was fastened to its holder, the electrodes were positioned at a 50 um
distance parallel to the tissue surface. The distance between adjacent electrode tips was
about 4-5 pm. For ﬁne positioning of the microelectrodes, the holder supporting the
tissue sample/solution chamber was controlled by a three way hydraulic
micromanipulator (WR 88, Narishige, Tokyo, Japan) driven by a computer-controlled
stepper motor (MO61-CE08, Superior Electric, Bristol, CT). Thus, the ion-selective
electrode could be moved to any programmed position through computer control of the
stepper motor. In this study the electrodes were moved vertically from 50 to 90 um
above the leaf surface in a 20-s square-wave cycle with a frequency of 0.1 Hz, i.e. 40 pm
for each movement (a half cycle) with 10 5 pass at each position.

The three ion-selective electrodes and the reference electrode were connected to a pre-
ampliﬁer inside the Faraday cage. The pre-ampliﬁer was then linked to a main ampliﬁer
outside the Faraday cage. A personal computer with an A/D card was used to monitor
and record all measurements (Figure 1, 2).

A small Plexi-glass curved mount in the shape of arc with a diameter 1.2 cm was
positioned at the bottom of the measuring chamber. This mount caused a gentle bending
of the leaf segment mounted in the chamber. Two braces were used in the chamber to
prevent dislocation of the leaf segment from the mount. This set-up allowed for a clear
view of leaf cells for electrode positioning through the inverted microscope. The
microscope light position, tangential to leaf surface, provided background illumination of

approximately 50 nmol m‘2 s'1 (Figure 1). The microscope lamp was turned off prior to

37

 

 

 

Main Ampliﬁer 7W

 

 

 

 

 

 

 

¢ I Computer
['\ “V... m -_, w_ a-

l,/ I Pre-ampllﬁer
/,.’ A
.// l N .
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’ ___.. Bathing solution

/

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i. r I
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Faraday Cage Hydraulic micromanipulator

Stepper motor

Figure 1. Schematic illustration of the experimental set-up, the MIFE
system and the position of the light source

1: Micromanipulator; 2: Microelectrodes; 3: Ag/AgCl reference electrode;
4: leaf segment; 5: Measuring chamber.

38

iiiiliil.‘,'.

 

 

 

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UN
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initiating any experiments in order to allow the tissue to equilibrate the new environment.

A ﬁber-optic light (450 nmol m2 s") was used as a light source (Figure 2). The 250
W lamp was from GS Edmumd Scientiﬁc Company (Barrington, NJ). A lens (no L-16-

16, World Precision Instruments, Inc., Sarasota, FL) was ﬁxed to the top of a light guide
to focus the beam. Light intensity was measured using a quantum sensor (model LI-189,

LI-COR, Lincoln, NE).

Flux measurements

The MIFE system utilizes slowly vibrating-speciﬁc ion microelectrodes, enabling
concurrent measurement of multiple ions. The MIF E data recording system and ﬂux
calculation protocol have been described by Shabala et al. (1997). In brief, a software
CHART package (Unitas Consulting, Hobart, Australia) provides an automatic record,
control all measurement processes in a real-time display on the computer screen (Figure 1
and 2). After measurements are completed, the recorded data ﬁles can be transferred by
CHART to MS—Excel ﬁles. These programs were then used to calculate average ion
concentrations and mean net ﬂuxes at 10-s intervals (each half-cycle of manipulator
movement).

The concentration of each ion in the bathing solution was calculated for each position
by measurement of voltage potential on the basis of Nemst equation. Ion concentrations
close to the leaf were calculated from the calibration data and from the average mV value
for the two positions. The magnitude and the directionality of the difference in estimated

ion activity between the two positions, perpendicular to the leaf tissue surface, the

39

 

the microelectrode and light source

Figure 2. The MIFE system and the position of

the microelectrode and light source

40

concentration gradient, was the basis for estimating both the rate and directionality of the
ion ﬂuxes.

The estimation methods for ion ﬂux using ion-selective microelectrodes have been
described by Newman et al. (1987), Kochian et al. (1992), and Shabala et al. (1997).
Basically, ion ﬂuxes were calculated using F ick’s ﬁrst law of diffusion assuming a planer
diffusion proﬁle:

D, (C, - C2)

J.= —— (1)

where J, is the net ion ﬂux, D, is the self-diffusion coefﬁcient for the ion in equation (in
cm'2 s"); Cl and C2 are the ion activities at the two positions, A r is the amplitude of
electrode movement. In this study, A r is 40 um. The net ion ﬂux is affected by D,,
temperature, leaf shape, Ar and electrochemical potential gradient. Temperature
inﬂuences the Nemst equation according to E, = RT/zF In (C/Ce), where T is temperature,
2 is valency, F is Faraday constant (96,487 J mol" volt“), R is gas constant, and C/Ce is
concentration difference between the membrane. The temperature was ignored due to a
constant room temperature (23 °C). D, was used for Ca2+ (8.0 x 10'2 cm"2 s"), H+ (9.17 x
10'5 cm'2 s"), CI' (2.032 x 10-5 cm'2 s") and K+ (1.9 x 10’5 cm'2 3"). Leaf shape could be
considered as a planar system. The general equation for estimation of planar diffusion
based upon measurements using the MIF E system is:
J = c u F (SS/Nemst slope) (dv/dx) (2)

where c = ion concentration; u = mobility, and dv/dx is voltage gradient over distance x.

41

tier:
elect}
peter
more
001621

that r

The Nemst slope is obtained from a calibrated curve over a range of ionic concentration.
The mobility u is speciﬁc for each ion and is related to the diffusion coefﬁcient D,.
Because D, equals uRT, one can appreciate that Di depends on temperature T (Noble,
1991). Therefore, the net ion ﬂux is dependent on the voltage potential gradient (dv)
when other conditions are constant. The ion ﬂux values were expressed in terms of nmol
m'2 s“.

The calculation procedure for electrochemical potential gradient is illustrated in
Figure 3. The electrometer’s AV is considered as the electrical voltage equivalent of the
electrochemical potential difference over the distance A r. At 30 s the manipulator moved
electrodes to position 1 (90 pm from leaf surface) within 10 s. The electrochemical
potential measured by the electrometer was about 2.3 mV. At 40 s, the manipulator
moved the electrodes to position 2 (40 pm from leaf surface). The electrochemical
potential decreased to about 1.4 mV within 10 5. Position 3 had a higher mV (about 2.8)
that when electrodes were positioned more distant from the leaf surface. The high-low
potential was cycled each 20 3 period. Higher mV at position 1 more distance ﬁom leaf
surface indicates a higher H+ concentration at this position. Hydrogen ion movement
down its electrochemical potential gradient is toward the leaf cell, i.e. inﬂux. An
Opposite movement is an efﬂux, against the electrochemical gradient.

During ﬂux estimation, the ﬁrst step in the calculation of ion ﬂux was the
determination of voltage potential at the extreme position of each manipulator movement.

Since only ion activities at two positions perpendicular to the sample surface were

measured, only net ﬂux could be estimated. The net ﬂux represents a balance between

42

inﬂux“
scalar

Ilux r

calct
talc:
Perle
estirr
ll’lﬁ‘ cf

Kim I)

EXpe

inﬂux and efﬂux. The ﬁrst 4 s (2 s for electrode movement and 2 s for electrochemical
settling of the electrodes) of each half cycle (10 s) were not used for concentration and
ﬂux estimation. This was considered to be a delay time necessary to obtain a stable
measurement following movement of the microelectrodes. Instability was thought to be
due to solution imbalance and vibration noise following movement. The remaining 6 s at
a position is deﬁned as the valid data interval (VDI). Mean voltage potential during three
consecutive VDIs, i.e. VDI,, VDI2 and VDI3 (Figure 3), at position 1, 2, and 3,
respectively, were determined (2.31, 1.40 and 2.80 mV, respectively). The function for
the line connecting the mean mV values for VDI, and VDI3 was established. The AV was
the difference between the mean mV midtime (T2) value for VDI2 and also at T2 to this
line ﬁrnction as shown in Figure 3.

Ten 5 later, the next VDIs were repeated to create the next triplet, to be used to
calculate AV again in similar manner. The electrochemical potential gradients (AV) were
calculated by this process at 10 5 intervals which thus reﬂected running averages for 30 5
period. Finally, net ﬂux was estimated from the AV, i.e. dv in equation (2) A positive
estimated ﬂux value indicates a net inﬂux, while negative one is a net efﬂux.

The above procedure makes reasonable considerations for the settling of voltage at
the electrode tip after each movement. It also allows for drift of the signal, as seen Figure
3, which is to be expected when the tissue ﬂux causes changes in the concentration of the

ion being measured.

Experimental Protocol

43

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44

Young expanding leaﬂets (leaf age about 2 weeks) were harvested from pea plants with a
razor blade. For studies involving ﬂux from mesophyll tissues, leaf sections of approxi-
mately 15 x 10 mm (LxW) with limited veinal tissue were excised from the leaﬂet
lamina. Using a ﬁne forceps, a small strip of epidermal tissue was then detached from the
abaxial surface of the lamina in such a manner so as to avoid major veins and to minimize
mechanical damage. Isolated leaf segments were ﬂoated on buffer solution (0.5 mM
CaSO4, 1.0 mM KCl, 2.5 mM HEPES-HCl, and 10 mM sucrose at pH 5.2) under 60 W
m'2 illumination (Philips lamp). After 2.0-2.5 h, the peeled lamina segments were
mounted within the sample chamber and bathed in 6-7 mL unbuffered solution
(containing 0.5 mM CaSO,, 1.0 mM KCl, pH 5.2). The bathing solution was replaced 3 -
5 times. The sample tissue was then pretreated for 50-60 min in the dark. The dark
treatment allowed for stabilization of ion ﬂux across the plasma membrane as a result of
equilibration of the tissue with the new environment. After dark pre-treatrnent, the leaf
tissue was ready for measurement.

Preliminary experiments showed that ion ﬂuxes can vary signiﬁcantly from one
position to another over the surface of the lamina tissues. To minimize the time course
variability of ﬂux measurements, regions with stable ﬂux were sought by moving
manually the hydraulic manipulator stepper. After a suitable site was selected, ion ﬂuxes
were measured for a minimum 5-10 min before the treatment light was turned on. Data
recording for ﬂux estimation was continued for an average 30 min after the onset of
illumination. The light source was then turned off, and data recording continued for an

additional 10-1 5 min. The sample chamber was then removed and replaced with another

45

 

bairirt;
dined
until

Iii.

dunn,

C011313

Signirt

l 500‘:

chamber with a ﬂesh leaf sample. All experiments were conducted in unbuttered solution
at room temperature (23i1°C). Arif et al. (1995) explained the reasons for not using
buffers in the bath. This is because some of the HI crossing the tissue boundary diffuse
as protoned buffer whose ﬂux is not including in the ﬂux calculated from the H+
electrochemical gradient (Arif et al., 1995). Heat emission from the light source was
negligible (was discussed in Chapter III).

The pH treatments were achieved by adding 0.1 mM HCl / NaOH to the unbuffered
bathing solution. Photosynthetic inhibitor, DCMU (3-(3,4-dichlorophenyl)-1,1-
dimethylurea, from SIGMA) was prepared in 0.5 % (v/v) DMSO and then dissolved in
unbuffered solution adjusted to pH 5.2. DMSO of 0.5 % had no discernible effect on ion
ﬂux. To test effects of DCMU on ion ﬂux, the control (an unbuffered solution) was
replaced with DCMU solution (0.5 mM CaSO,, 1.0 mM KCl, 0.05 mM DCMU, pH 5.2)

during the course on an experiment using a single leaf sample.

Data statistical analysis

By using ion-speciﬁc microelectrodes, net ion ﬂuxes from leaf cells can be measured
directly. One of the major concerns has been whether any artifacts were associated with
ion ﬂux measurement using the MIFE technique. No signiﬁcant artifacts could be
identiﬁed in this study. In most cases, changes in extracellular ion concentrations were
consistent with changes in net ion ﬂux. Secondly, light illumination did not cause
signiﬁcant changes in ion ﬂuxes from leaf tissues when electrodes were lifted far (at least

1500-2000 pm) from leaf surface (Figure 4). Additionally, heat effects induced by light

46

pH

0|' (PM)

K“ (MM)

Ca2+ (11M)
§

 

513
_ H Lighton . ﬂux Light off
5.6 — -9— concentration I

5.4 —

5.2 —

 

5.0 —

LL

 

640 - 032+

620 >—

F l
600 P—

l— ’-1 , ":- A.) l ‘9' l‘.--.;

 

rlrlrlrlrlrlrlrlrl

 

1400 l— CI-

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1300 — l
1200 -
1100

1000

 

1400

1300

1200

1100

 

1000

 

900 l l l l 1 l l l l l

l

 

 

0 2 4 6 8 10 12 14 16 18
Time (min)

Nlrlrlrl

40

20

Net H+ ﬂux (nmol m'2 5'1)

100

Net Ca2+ flux (nmol m'2 5'1)

-100

-200

Net CI' flux (nmol rn'2 3'1)

-300

N (A)
8 8
-2 s' )

S

O

-100

net K+ tlux (nmol m

-200

~300

Figure 4. Time course of net ion ﬂuxes and extracellular ion concentrations

of H+, Ca2+, CI' and K+ at 1500-2000 pm from pea leaf surface using
the MIFE system.

47

treatment were considered to be negligible (see Chapter HI). Fluctuations in ion ﬂux over
time are thought to reﬂect normal ion transport processes and dynamics across the plasma
membrane of leaf cells. Thus, the extracellular ion concentration changes measured by
the MIF E system were considered to be results of real changes in net ﬂuxes across the
plasma membrane.

Over 30 replicated leaf samples were conducted for the control. For analysis of ion
ﬂux response to light stimulation, there was concern about simply averaging replicate
time-course measurements. The problem of averaging multiple replicates is that
important time course changes in ﬂux elicited by the light/dark treatments would be
masked. For example, if two different replicate leaf samples exhibit a similar response in
ion ﬂux, but with a shift in time of several seconds or minutes, those shifts would result
in a much difference response trend if the data were averaged. This, thus, would cause a
loss of valuable information. For this reason, representative individual replicates were
selected and shown in Figure 6 and 7 to best illustrate a typical response.

In order to smooth the treatment data, and to reduce some of the noise for analysis of
the responses, data (including concentration and ﬂux) over 1 min interval were averaged.
The data could be reproduced for replicates of leaf samples.

One-way Analysis of variance (ANOVA) of the data was conducted using SAS 8
(SAS Institute Inc, Cary, NC). To determine if speciﬁc treatments (i.e. pH, DCMU) or
comparisons among the four ions resulted in statistically signiﬁcant differences in the
light-responses for ﬂux and extracellular ion concentration, LSD test (Fisher) at 0.05 was
used. ANOVA results appear in the text and in the ﬁgures.

Figure 5 illustrated how important features of the light-response for net H+ ﬂux and

48

extracellular pH was calculated. The analysis for other ions (Ca2+, K+ and Cl') could also
be made in a similar manner. The following terms were used in this study:

F a or C0: Five min average net ion ﬂux or extracellular ion concentration before light-
on.

Average change during the entire light period: the averaged net ion ﬂux or
extracellular ion concentration during the entire light period minus F0 or C0, respectively.

Average change after 10 min light-off: the averaged net ion ﬂux or extracellular ion
concentration during the entire light period minus F, or C,, where Ff or Cf is 10 min
average net ﬂuxes or extracellular ion concentrations from light-on to light-off,
respectively.

Ion ﬂux and extracellular ion concentration responses:

Initial ﬂux response (net inﬂux): initial maximum net inﬂux or minimum net efﬂux
after light-on minus F0 (Figure 5). A positive value indicates a net inﬂux. Initial
concentration response (decrease): initial minimum extracellular ion concentration after
light-on minus Co (Figure 5). However, pH response is an increase (positive), i.e. H+
concentration decrease.

Maximum ﬂux increase (net inﬂux): maximum net inﬂux or minimum net efﬂux
during the entire light period minus F0. A positive value indicates increase in net inﬂux.
Maximum extracellular ion concentration decrease: minimum extracellular ion
concentration during entire light period minus C0.

Change during the ﬁrst min: the average of net ion ﬂux or extracellular ion

concentration during the ﬁrst min after light-on minus F0 or C0. Change during the ﬁrst

49

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ﬁve min: the average value of net ion ﬂux or extracellular ion concentration during the
ﬁrst 5 min after light-on minus F0 or C0.

Amplitude of light-induced Post-Initial Flux Response (PIFR): the lowest inﬂux or
the highest efﬂux after Fi minus Fi (Figure 5), where F, is the net ﬂux when the light-
induced initial increase in net inﬂux started to decline. PIF R is a negative value.

Time response:

Response lag time (second): the time between the light-on or light-off and change in
net ion ﬂuxes or extracellular ion concentrations.

Initial light response time (min): the time to reach initial maximmn changes for ion
ﬂuxes or for extracellular ion concentrations after light-on (from dark to light) (Figure 5).

T 50,, for initial change (min): the time to reach 50 % of initial response in net inﬂux
or in extracellular ion concentration after light-on.

Time to maximum change (min): the time to reach maximum increase in net inﬂux
or maximum decrease in extracellular ion concentration during the entire light period.

PIFR time: the time for whole PIFR process or the time from Fi to lowest inﬂux or

highest efﬂux after F i (Figure 5).

RESULTS
Ion flux change of mesophyll tissues in darkness
In the dark, net ﬂuxes (a balance between inﬂux and efﬂux) of H", Ca2+, Cl' and K from

pea mesophyll tissues in a bathing solution containing 0.5 mM CaSO4 plus 1.0 mM KCl,

51

5.8

5.6

5.0

540
520

C82+UM
s g s § §

1400

1300

1200

Cl" uM

1100

1000

1600

1500

1400

K+uM

1300

1200

1100

 

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% ‘ d
P r x,. x ‘ ; ' :_ ~ -l
t " t -.
1 l l r l l r r r r-
0 5 10 15 20 25 30 35 40 45 50
Time (min)

Figure 6. Time course of net ﬂuxes and extracellular concentrations of H+,
C32+, Cl' and K+ around pea mesophyll tissues of one leaf sample under dark.

52

40

O N
O

'8
Net H” ﬂux (nmol m‘2 s")

g g g 5; ° 3 s a e g
Net Ca2+ tlux (nmol m'2 3'1)

° §

§
Net Cl' ﬂux (nmol m'2 371)

°§§§§

lb .3
8 8
Net K+ flux (nmol m'2 s'1)

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at pH 5.2, exhibited small ﬂuctuation over time (Figure 6). The extracellular
concentrations of these ions were stable and ﬂuctuated only slightly. Estimation of mean
rates for the entire dark period for PF, K” and C1“ ﬂuxes were -4, -51, -89 nmol m2 s",
respectively. Thus, a net efﬂux of these ions occurred under dark conditions. In. contrast,
a net Ca2+ inﬂux (+9 nmol 111‘2 s") was observed during the dark period, indicating Ca2+

uptake by the mesophyll.

Ion flux response of mesophyll tissues in light

When pea mesophyll tissues were stimulated by exposure to light in the same bathing
solution (0.5 mM CaSO4, 1.0 mM KCl, at pH 5.2), H”, Ca2+, K“ and cr exhibited
different patterns of change in net ﬂuxes and extracellular ion concentration over 30 min
(Figure 7). In spite of some variability in the light-induced ion inﬂux responses,
especially of K+ and Cl', most replicate leaf samples showed similar light responses for
each ion. In general, light induced a rapid increase in net ﬂux (positive) and a decrease in
extracellular ion concentration (negative). After a rapid large initial ﬂux response, a
reduced or slower net ﬂux change was observed over time. The process, called the Post-
Initial Flux Response (PIFR) in this study, typically involved a gradual return to ﬂux rate
closer to or lower than that measured prior to light stimulation. Since only net ﬂux could
be measured using the MIFE system, the PIFR could theoretically involve either
independent changes or concurrent changes in both inﬂux and efﬂux. Both Ca2+ and Cl‘
exhibited a rapid PIFR, whereas H‘” and K+ maintained a net inﬂux for an extended
period, which was then followed by a slow PIFR. Thus, the PIFR is a negative value.

When the light was turned off, the net ﬂux rate for each ion declined leading ultimately to

53

2+

(HM)

 

l..A‘\

40

A

3 N-

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20
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45

40

35

15

10

Time (min)

Figure 7. Light-induced time course in net ﬂuxes and extracellular concentrations
of H+, Ca2+, Cl' and K+ around pea mesophyll tissues of one leaf sample.

54

a net efﬂux. The ﬂux change was small over time as compared to the response to light.
The corresponding apoplastic ion concentrations increased, where pH decreased (Figure
7).

The quantitative features for each ionic response are summarized in Table 1 and Table
2. A simple averaging of different periods (the entire period from dark to light or from
light to dark) for each ion resulted in a smaller magnitude of change in either ion ﬂux or
extracellular ionic concentration (Table la, 1b). For H+, the average ﬂux change over the
entire light period was +11 nmol m'2 5", whereas the light-induced initial net ﬂux change
was +26 nmol m'2 s'l and maximum increase in net inﬂux was +29 nmol m'2 s" (Table
la). Thus, the average change over the entire light period was not used as a parameter to
evaluate light-induced ionic responses. The light-induced initial responses in net ﬂux and
ion concentration were similar to the light-induced maximum changes for each ion (Table
1a and 1b).

By comparison, Ca” ﬂux exhibited the most rapid response to light. A transient
increase in net Ca2+ inﬂux of 48 nmol m”2 s'l occurred after light exposure (Figure 7 and
Table la). This increase is considered a result of the increase in Ca2+ inﬂux and not a
decrease in Ca” efﬂux, because the initial Ca2+ ﬂux before light exposure (F0) is a net
inﬂux. The net ﬂuxes of H", K” and Cl' changed initially from a net efﬂux to a net inﬂux
with a magnitude of 26, 174, and 119 nmol m'2 s“, respectively, following exposure to
light (Table 1a). Extracellular concentration for these ions surrounding mesophyll tissues
also decreased, consistent with the development of their net inﬂuxes. The extracellular
pH around mesophyll tissues increased by 0.4 - 0.5 during the 30 min (Table 1b).

However, the average change in net Cl' ﬂux during the ﬁrst 5 min aﬁer illumination was

55

 

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I PIFR

 

Table 1. Quantitative characteristics of the light-induced net ﬂuxes and
extracellular concentrations of H", Ca2+, K‘“ and CI' in pea mesophyll tissues bathed
in 0.5 mM Case, and 1.0 mM KCl at pH 5.2.

 

 

 

 

 

 

 

a: ion ﬂuxes
1r Caz: K“ cr
(nmol m:2 s“)
F 0“ ~3 +8 ~5 1 ~61
Average change during +Ill +14 +84 +31
the entire light period
Average change after -8 ~21 ~43 ~46
10 min after light-off
Initial ﬂux response (net inﬂux) +26 +48 +174 +119
Change during the ﬁrst min +6 +25 +20 +31
Change during the ﬁrst ﬁve min +14 +31 +55 ~56
Maximum increase (net inﬂux) +29 +50 +208 +190
Amplitude of PIFRt ~20 ~52 ~176 -l 71
b: extracellular ion concentrations
H+ Ca” K“ Cl'
pH uM uM uM
C0§§ 5.02 562 1172 1126
Average change of +’0.36 ~37 ~45 ~30
the entire light period
Average change after ~0.21 +31 +208 +213
' 10 min after light-off
Initial concentration response +0.43 ~49 ~109 ~108
(decrease)
Change during the ﬁrst min +0.04 -9 +11 ~15
Change during the ﬁrst ﬁve min +0.24 ~27 ~50 +38
Maximum extracellular ion +0.47 -59 -128 ~132

concentration decrease

 

All data are means with 30 replicates.

5 F0: ﬁve min average of net ion ﬂux before light-on, where + or - indicate net ion inﬂux or net ion efﬂux,

respectively.
?

- or + indicates decrease or increase of net ion ﬂux and extracellular ion concentration, respectively.

’ PIFR: Light-induced Post-Initial Flux Response, a result of either a decrease in net inﬂux or an increase
in net efﬂux, or both. This value is negative.

§§ Co: ﬁve min average of extracellular ion concentration before light-on.

56

near:

|
most

time

YES:
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lnstar

also \

 

 

 

is St:

 

negative (~56 nmol m'2 s", Table 1a), and extracellular Cl' concentration increased during
the same period (Table 1b). This result indicates that a net Cl' efﬂux response occurred.

Some ions responded more quickly to light than others. Compared to other ions (H2
1C and Cl'), Ca2+ ﬂux was observed to have the smallest Tm, (1.5 min), the time for 50%
change in initial net ion ﬂux. The time to the maximum increase (inﬂux) was also the
most rapid for Ca” (3.5 min) (Table 2a). Chloride ﬂux had a similar initial light response
time (2.3 min) compared to Ca2+ (2.2 min), and both were signiﬁcantly shorter than H”
and K“. In considering light-response lag time, Ca2+ ﬂux response was the most rapid
within 13 s, then followed H+ (29 s) and by C1' (45 s) and K+ (48 3). Similar results were
also observed in extracellular ion concentration (Table 2b).

The PIF R time for Ca2+ (3.4 min) and Cl' (4.1 min) were signiﬁcantly shorter than for
H+ (8.4 min) and K+ (10.3 min) (Table 2a). When light was turned off, the response lag
time for Ca” ﬂux from light to dark was also signiﬁcantly shorter (19 s) than other ions.

The qualitative characteristics of the measured light-induced ionic responses are
presented in Table 3. Changes in net ﬂux and apoplastic ion concentration occurred
concurrently following light stimulation when compared Table 1a with Table 1b. For
instant, light exposure quickly stimulated Ca2+ uptake by the mesophyll within 13 s, and
also caused a reduction in apoplastic Ca2+ concentration within 13 s. Thus, the evidence

is strong that the Ca2+ ﬂux response was not an artifact of some other factors.

Light-induced ion ﬂuxes from epidermal tissues

57

Table 2. Quantitative characteristics of the light-induced time responses of H“, Ca”,
K+ and CI' in pea mesophyll tissues bathed in 0.5 mM CaSO4 and 1.0 mM KCI at pH

 

 

 

 

 

5.2.
a: ion ﬂuxes
IF Caz; K+ Cl'

Response lag time (s) 29 b 13 c 48 a 45 a
(from dark to light)

Response lag time (s) 26 b 19 c 50 a 45 a
(from light to dark)

Initial light response time 4.6 a 2.2 b 4.3 a 2.3 b
(min)

T50.) * 2.0 b 1.5 c 2.7 a 1.9 c
(min)

Time to maximum change 5.9 b 3.5 c 10.1 a 9.3 a
(min)

PIFR‘ time (min) 8.4 a 3.4 b 10.3 a 4.1 b

b: extracellular ion concentrations

H+ Ca2+ K+ Cl'

Response lag time (s) 32 b 13 c 51 a 45 a
(ﬁ'om dark to light)

Response lag time (s) 28 b 18 c 45 a 43 a
(from light to dark)

Initial light response time 5.3 a 3.3 b 5.9 a 3.4 b
(min)

Tm’ 2.6 a 1.6 b 2.7 a 2.6 a
(min)

Time to maximum change 15.1 a 8.7 b 7.0 b 8.1 b
(min)

 

All data are means with 30 replicates.

Different letters indicate signiﬁcant difference across rows according to LSD test at p < 0.05.

’ Tm: time to reach 50 % initial response of net ion ﬂux or extracellular ion concentration, respectively.

t PIFR: Light-induced Post—Initial Flux Response, a result of either a decrease in net inﬂux or an increase
in net efﬂux, or both. PIFR time: the time for whole PIFR process.

58

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59

An isolated strip of epidermal tissue (about 5 x 15 mm) was mounted in the same
measuring chamber and bathed in the same solution as the mesophyll tissue. The
experimental protocol was similar to that described previously. Unlike mesophyll tissues,
light did not elicit a large ﬂux change for any ion (Figure 8). No signiﬁcant difference in
PIFR were observed during about 25-30 min measurement. The data of Table 4
presented a possible characterization of the light-induced ion ﬂuxes and extracellular ion
concentrations around the epidermal tissues. After light stimulation, net Ca2+ inﬂux
increased slightly, net H“, K+ and Cl' efﬂuxes decreased slowly (11+) or changed toward a
net inﬂux (K+ and Cl') during the ﬁrst min or during the ﬁrst 5 min after illumination
(Table 4). Although net ﬂux of each ion was estimated to have positively increased, the
magnitude of increase in epidermal tissues (Table 4a) was much smaller than that of
mesophyll tissues (Table la). In addition, light only induced a small initial increase in

extracellular pH (Figure 8, Table 4b).

External pH effects on light-responses of mesophyll tissues

The mesophyll tissue was pretreated under dark for 2.0 ~ 2.5 h in a buffered solution with
either pH 4.0 or 6.0. The net ion ﬂuxes from mesophyll tissues were measured under
light illumination in the unbuffered solution (containing 0.5 mM CaSO4 and 1.0 mM
KCI) at the same pH as each buffered solution. Light period (30 min) was the same as at
the control (pH 5 .2). Compared to the control, a higher pH (6.0) of the bathing solution
resulted in a reduced light-induced initial response in net inﬂux of both H+ and K+ by

about 45 %, in particular, in a signiﬁcant decrease in net ﬂux change over 5 min

60

5.8
11* .
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5.6 __ Light on . flux
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I
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5.2 ..
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.\(l|lli _
5.0 _ llllllluluullltulk
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l l J J l l l l
*- .—
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i 1
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3 480 — l .-
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N 460 l . ' l ‘ . 4. '
8 440 .. - I ' i . 1 1
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p
400 ’_ l 4 l 1 I 1 J L _
1400 Lcr _
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2 1200 l— 1 __
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Time (min)

40

O N
0

1'0
0
Net H+ ﬂux (nmol m'2 5'1)

63 g g o a s a s g
Net Ca2+ llux (nmol m'2 s")

I L o d N
g 8 8 8
Net 01' flux (nmol m'2 s")

§°§§§§

Net K+ flux (nmol rn'2 5'1)

‘é'é’

Figure 8. Light-induced time course of net ﬂuxes and extracellular concentrations

of H+, C32+, Cl' and K+around pea epidermal tissues of one leaf sample.

61

 

to {'11

“EYE

c0116.

 

mo

S" l

C01

111;

illumination (Table 5). The alkaline medium also declined the light-induced PIFR of
both ions (50 % for H and 36 % for K+). However, the increase in external pH did not
alter the light-induced initial response in extracellular pH (Table 6), as well as in net
inﬂuxes of Ca2+ and Cl' (Table 5).

Acidiﬁcation of the bathing solution (pH 4.0) produced a different response compared
to the pH 5.2 treatment. Net H+ ﬂux was always positive (net inﬂux) while mesophyll
were being illuminated. It started with a large inﬂux (156 nmol m'2 5“) under dark
conditions (Table 5). Light treatment initially induced a large increase in net inﬂux (+44
nmol m‘2 s") which subsequently declined during the remainder of the light period (Table
5). Thus, the light-induced PIFR of H“ was a decrease in net inﬂux (-108 nmol m'2 s")
which was signiﬁcantly larger than at both pH 5.2 and 6.0 (Table 5). Under pH 4.0, the
light-induced initial increase in extracellular pH was much slower and reached to 0.35
within 19.5 min (Table 6). In contrast, the lower external pH resulted in a larger light-
induced initial response in net inﬂuxes of K+ and Cl' as compared to pH 5.2 (Table 5).
During 5 min after illumination, the net ﬂux of Cl' still increased positively (54 nmol m'2
s") at pH 4.0. However, that of the pH 5.2 decreased negatively (-74 nmol m'2 5")
compared with at pH 4.0 (Table 5). The similar difference was observed in change in
extracellular Cl' concentration (Table 6). The light-induced PIFR of K+ was not
inﬂuenced by lower pH (Table 5). However, the PIFR of Cl' was reduced by a large
magnitudefrom 148 to 29 nmol rn‘2 s'l (Table 5).

The pH 4.0 treatment did not affect the magnitude of light-induced Ca2+ initial

response. However, the PIFR of Ca2+ and its initial light response time in either ﬂux or

62

Table 4. Quantitative characteristics of the light-induced induced net ion ﬂuxes and
extracellular ion concentrations in pea epidermal tissues bathed in 0.5 mM CaSO4

 

 

 

 

 

 

 

 

(concentration decrease)

and 1.0 mM KCI at pH 5.2.
a: ion ﬂuxes
H+ Ca2+ Ki CI’
[11110] m-z s.1
F0§ -10 (9)‘ 2 (8) -9 (9) -1 (10)
Initial ﬂux response +l6 (9)+l 8 (8) +81 (8)
(net inﬂux)
Change during the ﬁrst min -5 (9) +5 (8) +23 (8) +36 (8)
Change during the ﬁrst ﬁve min -3 (9) +3 (8) +3 (8) +2 (8)
Maximum increase (net inﬂux) +9 (9) +23 (8) +121(8) +104 (8)
b: extracellular ion concentrations
H+ Ca2+ Ki" Cl'
pH 11M 11M 11M
C0§§ 5.04 (9) 529(8) 1126 (9) 1093 (8)
Initial concentration response +“0.08 (9) ~28 (8) -75 (7) -36 (8)
(decrease)
Change during the ﬁrst min +0.01 (9) O (8) +45 (7) +4 (8)
Change during the ﬁrst +0.03 (9) -7 (8) -ll (7) -1 l (8)
ﬁve min
Maximum extracellular ion +0.10 (9) -38 (8) -75 (7) -47 (8)

 

5 F0: ﬁve min average of net ion ﬂux before light-on, where + or - indicate net ion inﬂux or net ion efﬂux,

respectively.
t

3 The numbers in parentheses indicate the number of replicates.

§§ Co: ﬁve min average of extracellular ion concentration before light-on.

63

- or + indicates decrease or increase of net ion ﬂux and extracellular ion concentration, respectively.

Table 5. Effect of external pH in bathing solution on the light induced net ﬂuxes

flux of H‘, Ca“, K+ and Cl‘ from pea mesophyll tissues.

 

 

 

pH Hi Caz+ Ki Cl'
inhathingiolutiom nmol m'2 s’1
F0§

4.0 +156(8)§§ a +21(8) ~102 (8) a ~232 (7) a

5.2 ~0.4 (9) b 5 (12) ~81 (8) ab ~72 (8) ab

6.0 ~7 (8) b 0 (9) ns 24 (7) b ~26 (5) b
Initial ﬂux response (net inﬂux)

4.0 +‘44 (8) a +50 (8) +272 (8) a +174 (7) a

5.2 +22 (9) ab +46 (12) +167 (8) b +101 (8) b

6.0 +12 (8) b +41 (9) ns +92 (5) b +131 (5) ab
Change during the ﬁrst min

4.0 ~15 (8) a +28 (8) +69 (8) +115 (7) a

5.2 +2 (9) b +25 (12) +39 (8) +17 (8) b

6.0 +1 (8) b +18 (9) us +28 (5) ns +31 (4) ab
Change during the ﬁrst ﬁve min

4.0 ~3 (8) a +15 (8) +89 (8) a +54 (7) a

5.2 +11(9) b +29 ( 12) +62 (8) a ~74 (8) b

6.0 +2 (8) a +12 (9) ns +1 (5) b ~10 (5) ab
Amplitude of PIFR‘

4.0 ~108 (8) a ~74 (8) a ~208 (8) a ~29 (7) b

5.2 ~22 (9) b ~59(12) ab ~203(8) a ~148 (8) a

6.0 ~11 (8)b ~42 (9) b ~131(5)b ~195 (5)a

 

Different letters indicate signiﬁcant difference between external pHs within columns according to LSD test

at p < 0.05.
“ns” indicates no signiﬁcant difference.

§ F : ﬁve min avera e of net ion flux before 1i t-on, where + or — indicate net ion inﬂux or net ion efﬂux,
0

respectively.

§§ The numbers in parentheses indicate the number of replicates.

*

~ or + indicates decrease or increase of net ion ﬂux and extracellular ion concentration, respectively.

t PIFR: Light-induced Post-Initial Flux Response, a result of either a decrease in net inﬂux or an increase

in net efﬂux, or both. This value is negative.

 

Tab.-
con

4“ .. -
mu. C.

11111;

In.

hill

.0 A
31:

D

(ii

p ..
1. m C t
a _ .u .1.

r1;

 

 

Table 6. Effect of external pH in bathing solution on the light-induced extracellular
concentrations of H“, Ca“, K“ and Cl’ and time responses in pea mesophyll tissues.

 

 

 

pH Hi Ca2+ K+ Cl‘
Mammalian (pH) (AM) (.uM) CHM)

4.0 4.46(8) “c 608 (8) 1159 (8) ab 1168 (8)

5.2 5.00 (9) b 558 (12) 1200 (8) a 1065 (8)

6.0 5.40 (8) a 541 (9)ns 1015 (7) b 1175 (5) ns
Initial concentration response (decrease)

4.0 +1035 (8) ~46 (8) ~192 (8) ~192(6) a

5.2 +0.42 (9) ~46(10) ~128(8) ~11 1(8) ab

6.0 +0.30 (8) ns ~44 (9) ns ~92 (5) ns ~66 (5) b
Change on the ﬁrst ﬁve min

4.0 +0.07 (8) b ~24 (8) ~82 (8) ~135 (8) a

5.2 +0.21 (9) a ~37 (12) ~63 (8) ~29 (8) b

6.0 +0.16 (8) ab ~18 (9) ns ~30 (5) ns ~21 (5) b
Initial light response time (min)

4.0 19.5 (8) a 3.1 (8) b 8.3 (8) a 4.4 (8) a

5.2 5.3 (9) b 2.6(12) b 4.6 (8) b 2.3 (8) b

6.0 6.3 (8) b 5.4 (9) a 4.0 (5) b 3.4 (5) ab

 

Different letters indicate signiﬁcant difference between external pHs within columns according to LSD test
at p < 0.05.
“ns” indicates no signiﬁcant difference.

i Co: ﬁve min average of extracellular ion concentration before light-on.
§§ The numbers in parentheses indicate the number of replicates.
' ~ or + indicates decrease or increase of net ion ﬂux and extracellular ion concentration, respectively.

65

6X17
Tn:

COT“.

E111
Phc

“35

Sum
111.172
net 2‘.
ions

DIE.

 

 

extracellular concentration were signiﬁcantly larger at pH 4.0 than pH 6.0 (Table 5, 6).
The lower pH medium prolonged the initial light response time of extracellular K+ and Cl'

concentration compared with pH 5.2 (Table 6).

Effect of photosynthetic inhibitor on light-induced ion ﬂuxes

Photosynthetic inhibitor, DCMU can block electron transfer. This inhibitor at 0.05 mM
was added to the bathing solution (0.5 mM CaSO4 and 1.0 mM KCI) and the pH adjusted
to 5 .2 with HCl. The addition of DCMU signiﬁcantly inhibited the light-induced initial
response in net inﬂuxes of H+, Ca2+ and 1C around mesophyll tissues (Figure 9), and
elonged the light response lag time of H“ and Ca2+ ﬂuxes (Table 7). However, the light-
induced initial response in net Cl‘ inﬂux were not signiﬁcantly inﬂuenced by DCMU

application (Figure 9).

Sum of light-induced net ion ﬂuxes

When the magnitude and the directionality of net charge ﬂux, based upon the combined
net ﬂuxes of W, Ca2+, K+ and Cl' (Figure 7), the light-induced net charge ﬂux of the four
ions increased transiently, lasting for about 5 min, and then declined (Figure 10 and 11).
The light-induced initial increase in net inﬂux (about 300 nmol m'2 3") indicates that a
positive net charge move to the cytoplasm and would be expected to contribute to a
membrane depolarization. Compared with mesophyll tissues, however, net charge ﬂux

rates in epidermal tissues did not exhibit any response to light (Figure 11).

66

Table 7. Effect of DCMU on the light-induced the time responses of H“, Ca“, K+
and CI' in pea mesophyll tissue.

 

 

 

Hi Ci” K+ Cl‘
(8)
Light response lag time
~DCMU (Control) 28 (5)"b 13 (6) b 46(6) 42(5)
+ 50 11M DCMU 57(6) a 32 (6) a 47 (5) ns 46 (5) ns

 

Different letters indicate signiﬁcance between the control and DCMU treatment according to LSD test at
p< 0.05. “ns” indicates no signiﬁcance.

. Response lag time is the time between the light-on or light-off and change in net ion ﬂuxes.

.. The numbers in parentheses indicate the number of replicates.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

‘5; 160 ~ 1:1 control -
: +0.05 mM DCMU
E '7"
(I)
30.1 120 F -
E
9 5 1
s 2
g ‘x’ w l— l —1
g 3
3 5 LSD(0.05)
E 8
z “ 4° " I j
.C
.9 1 ,
_, .
0 WA , //
H“ (2.2+ K” of

Figure 9. Effect of a photosynthetic inhibitor, DCMU, on light-induced initial
response in net ion inﬂux from pea mesophyll tissues. Data represents an
average of 6-9 replicates. Vertical bars represent LSD test result at p<0.05.

67

400

 

 

 

 

+ Mesophyll
Light off
A 300 1— —
'7.» Lighten
N
IS 200 _ —
‘6
S
v 100 — _
m
a)
x
5.3.
c O — _
.9
‘6
Z '100 F- ﬁ
.200 l l l l l J J l l
O 5 10 15 20 25 3O 35 4O 45 50

Time (min)
Figure 10. Light-induced time course of net ion ﬂux from pea mesophyll

tissues. Data points represent a leaf sample. Net charge ﬂux is a sum
of H” + Ca2+ + K” - cr ﬂuxes

 

 

 

 

 

250
+ Mescphyll

200 _ '6- Epidemis _
~70, Lighton Light off
0" 150 -- —l
E
E 100 — -
r:
m
a 50 - ~
:1
a
.5 o _ _
8
Z

-50 _ ...1

-100 1 1 1 1 1 1 1 1 1 1

0 5 10 15 20 25 30 35 40 45 50
Time (min)

Figure 11. Light-induced time course of net ion ﬂux from pea mesophyll
and epidermal tissues. Data points represent an average of 7~12 leaf
samples. Net charge ﬂux is a sum of H+ + Ca2+ + K+ - Cl' ﬂuxes.

68

 

Exp:
(1pr-

al..

1r 11 '
hon
61C.
1111

mt”?

dtp

st :7

Sir:

 

Pl;-

D11,

 

 

DISCUSSION

Which ion is the initial depolarizing agent?

Exposure of leaf mesophyll cells to light has been shown to result in a transient
depolarization of the membrane potential, followed by a slow repolarization (Elzenga et
al., 1995). Similar observations have been reported in other species including in
Arabidopsis (Lewis et al., 1997), Phaseslus vulgaris (Nishizaki, 1992), Asplenium
trichomanes (Szarek and Trebacz, 1999), etc. The membrane depolarization responses,
however, have varied in their magnitude of membrane potential and the time responses,
etc. In general, changes in membrane potential are considered to be the result of ion
ﬂuxes across the plasma membrane. Thus, the ionic component of light-induced
membrane depolarization seems to be a foundation for understanding electrical
signalling. Many authors suggested that Ca2+, H+, K+ and Cl' play a major role in stimuli-
induced electrical signalling. Cation inﬂux or anion efﬂux from leaf cells results in
membrane depolarization. However, the identify of the ions involved in transient plasma
membrane depolarization is still controversial in the literature.

Elzenga et al. (1995) demonstrated that the light-induced plasma membrane
depolarization (about 25 mV) in pea mesophyll tissues occurred at about 1-2 min after
stimulation, which was followed by a slow repolarization. Assuming that four ions (Ca2+,
Hi, Ki and Cl‘) are primarily involved in light-induced membrane potential changes, the
summed net charge ﬂux rates (Figure 10) measured in the present study exhibited a
similar time course trend as the light-induced membrane potential response reported
previously (Elzenga et al., 1995; Stahlberg and Van Volkenburgh, 1999). Therefore, we

propose that the change in the net charge ﬂux involving Ca2+, H“, K“ and Cl' following

69

 

13‘».

ct.

 

 

0C

TC

 

light exposure could potentially be responsible for the reported light-induced membrane
potential response. A light-induced transient increase in the net charge ﬂux could be
expected to depolarize the plasma membrane. Thus, the ﬁndings in this study provide
indirect experimental evidence for the involvement of Ca2+, Hi, K+ and Cl' ﬂuxes in light-
induced electrical signalling.

Shabala and Newman (1999) suggested that Ca2+ is a depolarizing ion based upon
direct measurement of light-induced ion ﬂuxes in bean mesophyll tissues. Their
conclusion was based on time-course data following illumination, i.e., Ca2+ ﬂux exhibited
a more rapid (5 3) change than H”, K+ and Cl’ ﬂuxes. The result in pea mesophyll (Figure
7 and Table 1, 2) agrees with their results in bean leaf tissues. However, they did not
evaluate the transition from light to dark. Our results indicate that the light-induced ﬂux
changes of Ca2+, Hi“, K+ and Cl' declined and returned gradually to the rate which existed
before light treatment when the light was turned off (Figure 7). Considering the time of
the response to light, net Ca2+ inﬂux increased rapidly to the maximum magnitude within
3.5 min aﬁer light exposure. The maximum increase in net H+ and K+ inﬂux, however,
occurred after a 5.9-min and a 10.1-min delay, respectively (Table 2a). The initial light
response time, T50.) and light response lag time of Ca2+ were also relatively short (Table
2a). Among the four different ions measured, Ca2+ should be considered as a main
candidate for membrane depolarization in pea mesophyll. After about 1 min of
illumination, net Ca2+ inﬂux increased by approximately 25 nmol m'2 5'1 near mesophyll
tissues. This result should be explained as an increase in Ca2+ inﬂux, and not a decrease
in Ca2+ efﬂux.

According to the calculation of Nobel (1991), charge transfer (Q) = capacitance of

70

membrane (C) x voltage generated (AE), i.e. Q = CAE. The C of most biological
membranes are approximately are luF cm'2 (Liittge and Pitman 1976). If membrane
potential change (AB) is 25 mV, the Q required is 25 x 10'9 Coulombs cm‘z. One mole of
monovalent ions has charge of 96500 Coulombs, or a mole of divalent ions has
approximately 2 x 105 Coulombs. Therefore, a net transfer (Q) of 0.125 pmol of Ca2+ can
realize 25 mV membrane change. In this study, an increase in_ Ca2+ inﬂux of 25 nmol m'2
s" or 2.5 pmol cm”2 s'1 in pea mesophyll was observed within 1 min after light exposure.
Actual measured membrane depolarization resulting from light exposure has been
reported to rang between 25 and 35 mV (Elzenga et al., 1995; Stahlberg and Van
Volkenburgh, 1999.). In addition, the initial response time for transient Ca2+ inﬂux (about
2 min) measured in this study, thus, is also consistent with light-induced depolarization
(2-3 min) reported by Elzenga et al. (1995). Therefore, Ca2+ can be considered to be an
initial depolarizing ion.

However, one should not exclude other ions from possible involvement in the light-
induced membrane depolarization. A light-induced Arabidopsis plasma membrane
conductance change for C1' has been suggested by Spalding et al. (1992). In pea
mesophyll, Elzenga et al. (1995) found that a transient depolarization depended upon
external Cl' concentrations and was unaffected by changing external Ca” or K
concentration. On the other hand, light-induced membrane depolarization of pea
epidermal cells was enhanced by increasing Ca2+ concentration in the bathing solution.
Thus, these investigators concluded that the ionic mechanism for light-induced

depolarization differs between epidermis and mesophyll. The membrane potential

71

 

 

op.

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1,
pi 1L

168

10:

 

 

changes in the epidermis were explained as being due to changes in Ca2+ ﬂux and activity
of the plasma membrane H+~ATPase (Elzenga at al., 1995). However, their conclusion
was based on experiments in which the ionic concentrations of Ca2+ and Cl‘ were
modiﬁed. Also ion ﬂux was not measured directly. Because of numerous interactive and
feedback regulatory mechanisms for ion transporters in the plasma membrane, the
measurement of only membrane potential is not sufﬁcient to reveal involvement of the
speciﬁc ion ﬂuxes.

Only a few groups have directly measured light-induced ion ﬂux changes in leaf
tissues. Johannes et al. (1997) observed that Ca2+, K+ and anion-permeable channels were
open at the peak of light-induced membrane depolarization in red-light-induced moss
Phycomitrella patens. While Ca2+ inﬂux and anion efﬂux coincided with the depolarizing
phase (2-15 5), K+ inﬂux occurred only during the ﬁrst 30 s after light stimulation. Their
results indicated that a transient K” efﬂux appeared later and induced the plasma
membrane repolarization. However, their conclusions on ion ﬂux dynamics are
questionable because they measured ion ﬂuxes using 30 s or 1 min intervals which is
longer than the time necessary to attain the peak of membrane depolarization (2-15 5).

Shabala and Newman ( 1999) rejected Cl' efﬂux and 1C inﬂux as depolarizing agents
aﬁer measuring ion ﬂuxes using the MIFE system. They found both large Cl' and K
inﬂuxes after a 2 min delay between the onset of light stimulation and the onset of ﬂux
changes in bean mesophyll. In this study, changes in net Cl' and K+ inﬂux did not occur
until approximately 50 s (response lag time) aﬁer illumination (Table 2a). The
magnitudes of increase in net inﬂux from pea mesophyll were smaller than that from bean

mesophyll (about 1000 nmol m'2 3"). After 2.3 min, the net Cl’ inﬂux declined to a net

72

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ill=

 

efﬂux below the F0 (Table 2a). Thus, this process, i.e. the PIFR, could be considered to
be a result of an increase in C1' efﬂux not a decrease in C1' inﬂux. This is because the
amplitude of PIFR (~171nmol m'2 s") was greater than the magnitude of light-induced
initial increase in net Cl' inﬂux (119 nmol m'2 3") (Table 1a). The anion efﬂux could be
expected to be involved in membrane depolarization. Elzenga and Van Volkenburgh
(1997) tested anion channels by using patch-clamp techniques and found high anion
channel activity in pea mesophyll cells after a 30 3 light treatment. They concluded that
Cl' efﬂux is involved in light-induced membrane depolarization. In this study, however,
light induced a rapid initial response in net Cl‘ inﬂux. This net Cl' inﬂux could be
involved in the repolarization process and might counteract the light-induced
depolarization. However, summation of net charge ﬂuxes (including Ca2+, H", K+ and Cl'
) in Figure 10 and Figure 11 suggests that light still caused a net inﬂux increase that
contributes to a transient depolarization. Thus, Cl' efﬂux is involved in light-induced
membrane depolarization but not in the initial transient depolarization event.

The K“ exerts a dominating inﬂuence on the diffusion potential component of
transmembrane electrical potential differences by virtue of its relatively high permeability
in biological membranes. Even under dark conditions, K+ maintained a variable net ﬂux
rate around £100 nmol m2 s'1 which is larger than H and Ca2+ (Figure 6). Among Ca2+,
K+ and Cl‘, which the electro-chemical potential for K+ is the lowest at the unperturbed
membrane voltage of approximately ~150 mV. This is because the equilibrium voltage
for K+ (Ex) is probably near ~100 mV (Leigh and Wyn Jones, 1984). Spalding and
Cosgrove (1992) put forward that K+ is not a major depolarizing ion in blue-light-induced

cucumber hypocotyls because TEA, an inhibitor of K+ channels, did not affect the onset

73

of 1
P351
mer

efll

C11

ICE

[it

I

in:

 

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of depolarization. In most cases, K+ is considered as a charge balancing ion, moving
passively to compensate for light—induced ion movement of C1' or Hi. However,
membrane depolarization of Desmodium motorium appeared to be associated with K+
efﬂux (Antkowiak et al., 1991). This conclusion was not based on direct ﬂux
measurement. In the present study and in bean mesophyll (Shabala and Newman, 1999),
direct K+ measurement using the MIFE system indicated that K+ does not ﬁmction in
initial light-induced electrical signalling. The data also suggested that H+ is not an initial
depolarizing ion. Net H” inﬂux and apoplast pH exhibited only relatively small increases
within 1 min after light stimulation (Table la). The time-course response of H+ was
slower than that of Ca2+ (Table 2). The delay between light application and the beginning
of the H”, K+ and Cl' ﬂux responses could be explained as the time required to elevate
cytosolic free Ca2+ concentrations to a level that can elicit a cellular signalling cascade
response. This observation is in agreement with the hypothesis that Ca2+ is a major ion

initiating the plasma membrane depolarization in pea mesophyll.

Light-induced alkalinization and photosynthetic effects

Initial extracellular alkalinization in response to light was observed in leaf tissues of
many species including sunﬂower (Hoffmann and Kosegarten, 1995), pea (Stahlberg and
Van Volkenburgh, 1999), and bean (Muhling et al., 1995; Shabala and Newman, 1999).
The same result was observed in Figure 7. A light-induced increase in H+ inﬂux occurred
after about a 30 s lag time (Table 2a), whereas Shabala and Newman (1999) found a lag
time of l~2 min in bean mesophyll. The magnitude of the increase in apoplastic pH, 0.4 ~

0.5, is similar to the ﬁnding of Stahlberg and Van Volkenburgh (1999). In addition, they

74

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re
P 1

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an
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also reported an extracellular acidiﬁcation after 15 min of light exposure with the parallel
occurrence of a gradual hyperpolarization of membrane potential. They concluded that
growth stimulation of pea leaves is due to H+ efﬂux mediated by a plasma membrane Hi-
pump. Unfortunately, however, H+ ﬂuxes were not measured in their study. In the
present experiment, direct H” ﬂux measurement does not support their speculation. Light
treatment caused a rapid increase in net H" inﬂux, not an efﬂux. Light-induced
alkalinization was slowed down and extracellular pH started to decline after
approximately 5-6 min, whereas net H+ inﬂux was decreased or reversed to net efﬂux or
both, i.e. PIFR. These results seem to correspond to the “acidiﬁcation” response
described by Stahlberg and Van Volkenburgh (1999). Their observation, however,
occurred as a result of a 1.0-1.5 h light treatment and the “acidiﬁcation” was due to a
decrease in apoplastic pH from the maximum initial light-induced pH. It should be noted
though that the reported reduction in pH was not lower than the original level measured
before light was turned on. Termination of light exposure resulted in a decrease of
apoplastic pH (Figure 7) in agreement with Stahlberg and Van Volkenburgh (1999).

In bean mesophyll tissues, Shabala and Newman (1999) showed a light-induced
initial net H+ efﬂux but not a net inﬂux, even though extracellular pH increased. They
explained that this apparent inconsistency could be a result of rapid CO2 uptake by
photosynthesizing tissues. Another possible consideration is the effect of other ion
transporters which regulate extracellular pH. Several mechanisms can be involved in
apoplastic pH regulation (Leigh, 1993), including production and consumption of malate

via the biochemical pH ‘stat’, transport of protons to and from the vacuole, and ﬂuxes of

75

 

 

 

811'

[1.11

 

H+ at the plasma membrane. Other ions, such as K“, Ca2+ and anions may be involved in
cytoplasmic acidiﬁcation or extracellular alkalinization which contribute to an initial
depolarization of membrane potential (inﬂux of positively charged H“) followed by a
repolarization as the H+ pump is activated (Franchisse and Desbiez, 1989). Gating of the
K+~inward and Kl~outward rectiﬁers in guard cells is sensitive to external pH (Thiel and
Wolf, 1997). Therefore, it is possible that the activity of other ion transporters in the
plasma membrane also inﬂuence Hi ﬂux and extracellular pH.

The photosynthetic inhibitor DCMU is often used to test the effects of photosynthesis
on light-induced electrical signalling in leaf tissues (Nishizaki, 1992; Elzenga et al., 1995;
Stahlberg and van Volkenburgh, 1999; Szarek and Trebacz, 1999). In pea mesophyll,
0.05mM DCMU inhibited to a large extent membrane depolarization (Stahlberg and van
Volkenburgh, 1999). The presence of DCMU at the same concentration inhibited the
light-induced initial response in not H, Ca2+ and K+ inﬂuxes (Figure 9) and delayed the
initial light response (Table 7). This result experimentally demonstrates the correlation
between photosynthesis and light-induced ion ﬂux change.

Carbon dioxide (C02) uptake was thought to be one reason for light-induced apoplast
alkalinization (Gotow et al., 1985; Shimazaki and Zeiger, 1987; Shabala and Newman,
1999). In lower plants, light-induced pH increase is due to the uptake of HCO‘ ions in
(Merrett et al., 1996). Formation of HCO’ ions and CO2 is expected from the following
equilibrium: CO2 + H20 <:> HZCO3 ¢:> H” + HCO‘3 <=> CO'3 + 2H“. Reduction in either
CO2 or HCO‘3 results in a decline H+ concentration or pH increase in solution. In the

bathing solution, CO2 reacts with H20 to form HCO‘3 with a combined pK of 6.3

76

1N:

by

11111“

1‘1‘
611:

1101

 

 

 

(Neumann and Levine, 1971). In this study, when light was turned on, the uptake of CO2
by mesophyll cells shifted the equilibrium switch toward production of H2C03. This
resulted in a decrease in H+ concentration and thus alkalinization. Therefore, DCMU
inhibition of photosynthesis which lowers CO2 uptake may signiﬁcantly inhibit light-
induced H+ inﬂux as suggested by this study (Figure 9, Table 7).

Additional evidence for the light-induced alkalinization comes from external pH
effects. High extracellular pH (7.0) initiated further alkalinization (0.6-0.7 pH unit, data
not shown) compared with pH 5.2 (0.4 ~ 0.5 pH unit) of Table 1b. Enhanced pH shiﬁs
the equilibrium toward H2C03 formation (Yin et al., 1996) resulting in a further increase
in pH. On the other hand, a shift to a more acidic environment (pH 4.0) would be
expected to reduce HZCO3 formation and inhibit the rapid pH rise illustrated in Table 6.
After 5 min of illumination, only a small pH increase (0.07) was observed in the acidic
bathing solution (Table 6), whereas at pH 7.0, extracellular pHiwas transiently increased
by 0.4 ~ 0.5 during the same period (data not shown). Similar observations were made in
light-induced Cyanidium cadarium (Kura-Hotta and Enami, 1981) and Dunaliella
acidophia (Remis et al., 1994). Therefore, it seems reasonable to suggest that CO2 uptake
might cause apoplastic alkalinization in the light-induced pea mesophyll tissues.
Moreover, the results using different external pH treatment indicate that the regulation of
extracellular pH is not solely dependent on H+ ﬂux across the plasma membrane. The
high net H+ ﬂux under pH 4.0 was similar to the increase in extracellular pH as under pH

6.0 and pH 5.2 (Table 6).

77

13+

 

Furthermore, another possible explanation may be that ion uptake is driven by the
proton motive potential. H" inﬂux into cytoplasm is mediated by symporters within the
plasma membrane, e.g. H+/K+ (Hedrich and Schroeder, 1989; Netting, 2000) and H+/C1'
(Sanders et al., 1985). The uptake of H+ by plant cells is energetically favorable due to
the negative membrane potential and a low cytosolic H+ concentration in the cytoplasm
compared to the apoplast. Thus, H+ ﬂuxes are believed to serve as the energy source for
membrane conformational changes that accompany solute transport involving
symporters. The inﬂux or uptake of Cl' is mediated by active transports (Assmann and
Zeiger, 1987), and hypothesized to occur via a Cl'/H" symport or a Cl'/OH' antiport
(Zeiger et al., 1978). The C1“ import requires the input of a considerable amount of
metabolic energy. To meet this requirement, 2Hl/Cl' co-transport against an opposing Cl'
gradient has been postulated which has indeed been demonstrated and kinetically
modeled for Chara (Sanders et al., 1985) and root-hair cell of Sinapis alba (F elle, 1994).
The experimental evidence in higher plants, however, has remained somewhat scant to
date. In pea mesophyll, a light-induced initial response in net Cl‘ inﬂux was observed
within 2-3 min (Table 1a, 2a). Extracellular Cl' concentration declined following the net
Cl” inﬂux (Table 1b). With decreasing pH or increasing H+ concentration in the bathing
solution (4.0), the light-induced initial response in net Cl' inﬂux was signiﬁcantly
enhanced as compared to that at pH 5.2 and pH 6.0 (Table 5). However, high external pH
(6.0) resulted in a higher light-induced PIFR of Cl' than at pH 4.0 (Table 5). Similar
results were observed for the Kl response to external pHs (Table 5). Therefore, Cl'/H+

and H+/K+ symport might be hypothesized to mediate light-induced apoplastic

78

 

 

 

C1

 

"(J

{/J

 

alkalinization of pea mesophyll. More experimental evidence is required to clarify this
issue though.

No direct attempt has been made to experimentally determine the inﬂuence of
extracellular pH on Ca2+ ﬂux ﬁom leaf cells so far. The Hl/Ca2+ antiporter is a likely
candidate for H+ ~ Ca2+ exchange which in principle mediates transport of Ca2+ against its
concentration gradient and requires a H+/Ca2* stochiometry of at least three (Schumaker
and Sze, 1987). However, activity of Hl/Ca” activity frequently was associated with
membranes of mitochondria and chloroplast and only rarely with the plasma membrane
(Bush, 1995). In this experiment, external pH did not inﬂuence the light-induced initial
Ca2+ ﬂux change (Table 5). Compared with pH 4.0, the alkalinizing environment (pH
6.0) inhibited the light-induced PIFR of Ca2+ (Table 5). Presumably changes in external
pH alter the pH gradient across the plasma membrane or affect feedback regulation by
cytoplasmic pH, thus inﬂuencing cytosolic Ca“ homeostasis.

The light-induced PIFR is evidenced in the current study. Shabala and Newman
(1999) did not discuss this event. The PIFR may reﬂect a combined response of Ca2+, H“,
K” and Cl' ﬂux in leading to membrane repolarization which could be a cellular
mechanism to regulate external pH. Another possible function of the PIFR could be to
regulate a cytoplasmic Ca2+ messenger which is involved in light-induced signal
transduction. Further investigation on the interaction between ion transporters in the

plasma membrane is needed.

Light-induced ion ﬂuxes from epidermal cells.

Shabala and Newman (1999) only measured a relatively small change of net ion ﬂuxes

79

from bean epidermal strip when exposed to light. They suggested that the epidermis is an
effective barrier to ion ﬂux but not for CO2 diffusion into the leaf. Elzenga et al. (1995)
proposed that the ionic mechanisms of light-induced depolarization for mesophyll and
epidermis of pea are different. In the current research, the light-induced initial ion ﬂux
changes in epidermal tissues were also smaller than in mesophyll (Figure 8, Table 4).
Light initially induced a small decrease in net Hl efflux resulting in apoplastic
alkalinization (0.08 pH unit), and a reduction in extracellular Ca2+ concentration (28 11M)
as well. The summation of light-induced net ﬂux Ca2+, Hl, K+ and C1' in epidermal
tissues was smaller than in mesophyll tissues (Figure 11). This result was similar to the
relatively small light-induced membrane potential change reported by Elzenga et al.
(1995). Although light-induced ion ﬂux changes were observed, it is impossible to
speculate on the ionic mechanism for electrical signalling in epidermal tissue.

The results in this study explain in part the ionic basis for light-induced
bioelectrogenesis. However, more evidence is needed on the speciﬁc ion transporters
involved in electrical signal transduction. Further investigations should also focus on the

role of ion channels and proton pumps in light responses.

80

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84

Chapter II.

POSSIBLE MECHANISM OF LIGHT-INDUCED CA”, H‘, K‘ AND

CL' FLUXES FROM MESOPHYLL TISSUES.

85

ABSTRACT

Mechanisms of light-induced rapid changes in net Ca2+, H“, K+ and C1” ﬂux around
mesophyll tissues from excised leaﬂet of pea (Pisum sativum L. cv. Argenteum) were
analyzed. Calcium channel blocker LaCl3 (0.1 mM) or chelator EGTA (1.0 mM)
inhibited 42-44 % of the light-induced initial response of net Ca2+ inﬂux and the light~
induced Post-Initial Flux Response (PIFR) of Cl’ and K“ over time. In contrast, the Ca2+
ionophore A23187 (5 11M) and increased exogenous Ca2+ (2.0 mM) signiﬁcantly
enhanced the light-induced (PIFR) of Cl', whereas low external Ca” (0.1 mM) produced
an opposite effect. The evidence suggests that Ca2+ inﬂux, resulting from increased Ca2+
channel activity, might be an initial step in the rapid response by leaf mesophyll to light.
The plasma membrane H+~ATPase inhibitors, Na3VO4 (1.0 mM) and DCCD (0.1 mM),
inhibited the light-induced initial response in net H+ inﬂux by 50 - 60 % and eliminated
the PIFR of H“. The presence of Na3VO4 in the bathing solution also signiﬁcantly
suppressed the light-induced increase in net K+ inﬂux. However, these inhibitors did not
affect the light-induced response in net Ca2+ inﬂux. Furthermore, fusicoccin (0.01 mM)
signiﬁcantly increased the light-induced PIFR of Hi and the initial response in net Cl'
inﬂux. These results suggest that H+~ATPase in the plasma membrane is not involved
directly in the initial light response, whereas H“-K+ and H+~Cl' symports may contribute
to the light-induced initial extracellular alkalinization and the uptake of K+ and C1' by
mesophyll tissues. In addition, TEA (5.0 mM) signiﬁcantly blocked most of the light-
induced PIFR of K. The R-type anion channel blocker DIDS (0.1 mM) completely
suppressed light-induced net Cl' efﬂux within the ﬁrst 5 min of illumination, but the S-

type blocker A~9~C (0.1 mM) did not. These observations indicate that the outwardly-

86

directed K“ channels and the R-type anion channels may play a role in light-induced PIFR

of K+ and Cl', respectively, whereas that of Cl' is Cay-dependent.

INTRODUCTION

Rapid light-induced changes in membrane potential are ubiquitous in higher green plants.
This bioelectrogenesis can be explained as light-induced changes in ionic ﬂuxes across
the plasma membrane. Light has been shown to elicit rapid changes in ion ﬂux in leaves
of Dunalliella acidophila (Remis et al., 1994), Physcomitrella patens (Johannes et al.,
1997), bean (Vicia fava) (Shabala and Newman, 1999). These reports propose that ion
transport across the plasma membrane is highly regulated, involving speciﬁc transport
mechanisms during periods of light exposure. However, few studies have related ion
transport activity and regulatory mechanisms to responses to light.

In general, plant physiological responses to light depend on cellular signalling systems
that link light perception to terminal responses. Therefore, 1i ght-induced ion ﬂux changes
may link one or several signalling pathways. Ion ﬂuxes across cell membrane systems
are controlled by many ion channels, carrier proteins, and electrogenic ion pumps. These
ion transporters regulate transport of ions across the plasma membranes and maintain ion
homeostasis, and large chemical potential gradients. Light stimulation may activate or
up/down regulate certain mechanisms that mediate changes of ion ﬂuxes across
membranes of plant cells. Light-induced leaf movement has been shown to involve K+,
Cl‘ and H‘“ ﬂuxes mediated by K+ channels and proton pumps in extensor and ﬂexor
tissues (Coté, 1995; Mayer et al., 1997). Other studies have focused on stomatal guard

cell changes in turgidity controlled by ion ﬂuxes across the plasma membrane of guard

87

cells (Grabov and Blatt, 1998; Assmann and Shimazaki, 1999). The dominant ions
involved in signal transduction in guard cells include Hi, Ca2+, K+ and Cl‘. Ion channels
(such as Ca2+~, inward K+ rectiﬁer and outward K+ rectiﬁer and anion-channels) and H1
ATPase are considered to play important roles in light-responses of guard cells.
However, research is lacking on the regulation and responsiveness of ion transport
mechanisms in other leaf tissues to environmental stimuli. Data on the ionic basis of
light-induced membrane potential changes are also controversial and vary with plant
species and experimental conditions.

To date, it is not fully understood which ion transporters are associated with light-
induced ion ﬂux responses in leaf tissues. Many possibilities exist because multiple ions
are involved in the regulation of plasma membrane electrical potential. Shabala and
Newman (1999) measured directly ion ﬂuxes from bean mesophyll tissues and suggested
that light-induced Ca2+ inﬂux is a main depolarizing agent. They did not, however,
explain the relationship between Ca2+ channel activity and the light-induced transient net
Ca2+ inﬂux. The results of Johannes et al. (1997) support a role for Ca“ in the red-light-
induced ﬂux change in Physcomitrella patens. Blom-Zandstra et al. (1997) reported
transient light-induced changes in ion channel and proton pump activities in the plasma
membrane of tobacco mesophyll protoplasts, and suggested that ionic transporters in the
plasma membrane might be involved in the light-induced transient changes in membrane
potential. In Arabidopsis mesophyll, white light induced an increase in K“ channel
activity in the plasma membrane and a transient membrane depolarization (Spalding et
al., 1992). In pea mesophyll cells, the light-controlled anion channels in the plasma

membrane were proposed as a mechanism for membrane depolarization (Elzenga and

88

Van Volkenburgh, 1997). Further investigation indicated that the plasma membrane Hi-
ATPase is not directly involved in rapid light-induced membrane depolarization
(Stahlberg and Van Volkenburgh, 1999). They also suggested that several different
mechanisms participate in light-induced membrane depolarization in the mesophyll and
the epidermis (Elzenga at al., 1995). However, ion ﬂux in leaf cells was not directly
measured in the above studies. In Chapter I, the light-induced rapid changes in the net
ﬂux of H", Ca2+, K+ and Cl' were demonstrated in pea mesophyll tissues. The transient
net Ca2+ inﬂux and the concomitant apoplastic alkalinization were thought to involve Caz’
channels, or Hi-K+ or Hl-Cl' symporter, as well as or H+~ATPase in the plasma
membrane. The light-induced Post-Initial Flux Response (PIFR) of Cl‘ from a net inﬂux
to a net efﬂux suggests that anion channels may also play a role whereas the function of
K“ ﬂuxes in light response has still not been conﬁrmed in pea mesophyll. Differential
changes in ionic ﬂux reveal that there are several ion transporters involved in the cellular
mechanisms regulating light signal transduction. Therefore, ﬁrrther research is necessary
to determine which transport mechanisms are associated with the light-induced ion ﬂux
changes.

Speciﬁc chemicals that inhibit or activate ion channels or ion pumps are used widely
to determine the effect of environmental stimuli on speciﬁc ion transporters in the plasma
membrane. However, most previous studies have only measured light-induced
membrane potential change. The role of ion transporters cannot be elucidated from only
membrane potential data alone. To test our hypothesis, two different approaches were
used. The ﬁrst consisted of treating pea mesophyll tissues with selective inhibitors or

activators of H“, Ca2+, K" and Cl’ transporters in the plasma membrane before light

89

illumination. Light-induced dynamic ion ﬂux changes of H, Ca2+, K+ and Cl' were then
monitored using the Microelectrode Ion Flux Estimation (MIFE) system. The second
approach was to observe ionic ﬂux responses to light in different external ionic solution
environments. In this way, one is able to study possible mechanisms involved in the

ionic response to light.

MATERIALS AND METHODS

Plant Material

Seeds of pea (Pisum sativum L. cv. Argenteum) were germinated 4-5 (1 and then
transferred to a growth chamber with a 16 h photoperiod, day/night temperatures of 25/25
°C, 7 5% relative humidity, and a radiance of 200 pmol m’2 s" at the top of the canopy.
The plants were cultured using modiﬁed Hoagland’s solution (Johnson et al., 1957),

gradually increasing the concentration from 0 to 1A to I/2 strength at 4-5 (1 intervals.

Flux measurements

Ion ﬂuxes and concentrations at the surface of the leaf mesophyll were measured using
the MIFE system. Technical details were generally as described in Chapter I and Shabala
et a1. (1997). Additional information on the MIFE system is available at
http://www.phys. utas.edu.au/physics/biophys. In brief, micropipettes with a tip diameter
of 2-3 um were pulled ﬁom 1.5-mm-diameter borosilicate glass capillaries (catalog no.
MIB 150-6; World Precision Instruments, Inc., Sarasota, FL) with a horizontal electrode
puller (PC-10, Narishige, Tokyo, Japan). After silanization, cooled microelectrodes were

back-ﬁlled with 500 mM CaCl2 for Ca“, 500 mM KCI for K+ and Cl', and 15 mM NaCl

90

plus 40 mM KH,,PO4 (adjusted to pH 6.0 with NaOH) for H”. Electrode tips were then
front-ﬁlled with a liquid-membrane-type of the appropriate ion-selective sensor, Hi (no.
95291), Ca2+ (no. 21048), K+ (no. 60031) and Cl‘ (no. 24902) cocktails (all from F luka,
Milwaukee, WI), until a 50- to 80- um long column of ionophore was produced.
Microelectrodes were calibrated for each ion using a known set of standards before and
after use (see Chapter I). The Ag/AgCl reference electrode was made using a plastic tube
containing 1000 mM KCl in 2% agar.

Calibrated ion-selective electrodes were positioned over the desired cells in a leaf
segment using a micromanipulator with three-dimensional control. The set-up was
mounted on the stage of an inverted microscope (Leitz Wetzlar, Germany) which
provides a clear view for electrode positioning. The sample chamber with mounted leaf
segment was placed on a three-way hydraulic micromanipulator (WR 88, Narishige,
Tokyo, Japan) driven by a computer-controlled stepper motor (MO61-CE08, Superior
Electric, Bristol, CT). The MIFE system allows for measurement of three ions
simultaneously. Three microelectrodes can be slowly vibrated at any angle in a two-
dimensional plane by separately controlling the amplitude of the vibration of the
micromanipulator. I

The MIFE system measured the voltage differences at two extreme positions of a 50
um vibration above the leaf tissue surface. The magnitude and the directionality of the
difference in estimated ion activity between the two positions was the basis for estimating
both the rate and directionality of the ion ﬂuxes. In this research the electrodes were
moved in a 20—s square wave cycle between 50 to 90 um above the leaf surface at a low

frequency (0.1 Hz). The concentrations of ions were calculated from their

91

electrochemical potentials at each position. The ﬂux for each speciﬁc ion was calculated
from the measurements of the difference in the electrochemical potential between these
positions (Shabala et al., 1997, or see Chapter I). During analysis, the ﬁrst 4 s of each
half cycle (10 s duration) were not used for concentration and ﬂux estimation. This 4 3
period accounted for the time for electrode movement to a new position and for 'voltage

potential stability.

Experimental protocol

Young expanding leaﬂets (leaf age about 2 weeks) were harvested and a leaf section of
approximately 15 x 10 mm (LxW) with limited veinal tissue was cut surgically from the
lamina. A small strip of epidermal tissue was peeled from the abaxial surface of the
lamina of each section so as to prevent mechanical damage to the underlying veinal and
mesophyll tissue (Long and Widders, 1990). Isolated leaf segments were ﬂoated on the
buffered solution (0.5 mM CaSO4, 1.0 mM KCI, 2.5 mM HEPES-HCI, and 10 mM
sucrose at pH 5.2) under 60 W m‘2 illumination (Philips lamp). After 2.0-2.5 h, the cut
segment was mounted in a sample chamber. The sample chamber contained 6-7 mL
unbuffered solution or bathing solution (0.5 mM CaSO,, 1.0 mM KCl, pH 5.2), and the
segments then were exposed to 50—60 min of darkness. After 5-10 min of measurement,
the light was turned on. Flux measurement was continued for an average 30 min after the
start of illumination. After light was turned off, the measurement was conducted for
another lO-15 min. The sample chamber was then removed and a new leaf sample was

installed.

92

A ﬁber-optic light source (delivering 450 pmol m'2 s") as measured by a quantum
sensor, model LI-189, LI-COR, Lincoln, NE) was used with a 250 W halogen lamp (GS
Edmumd Scientiﬁc Company, Barrington, NJ). A lens (no L~16~16, World Precision

Instruments, Inc., Sarasota, FL) was ﬁxed on the top of light guide to focus the beam.

Chemicals

Chemical treatments were selected according to their effects on ion transporters. These
treatments included a Ca2+ channel blocker (LaC13), a Ca“ chelator (EGTA), a Ca2+
ionophore A23187, H+~ATPase inhibitor (vanadate and DCCD), a Hl-ATPase activator
fusicoccin (FC), K+ channel blockers (TEA and CsCl), and anion channel blockers (A-9-
C and DIDS). Vanadate (Fisher, Lawn, NJ), CsCl, EGTA, and LaCl3 (SIGMA, St. Louis,
M0) were dissolved directly in an unbuffered solution (0.5 mM CaSO,,, 1.0 mM KCI, pH
5.2). Others including FC, DCCD, A-9-C, DIDS, Ca2+ ionophore A23187 and TEA
(from SIGMA, St. Louis, M0) were prepared in DMSO as stock solutions.

To test the effects of these chemicals (V anadate, DCCD, F C, EGTA, LaC13, Ca2+-
ionophore A23187, A-9-C, DIDS, TEA, CsCl) on ion ﬂuxes under light stimulation
(450umol m'2 s“), all chemicals from their stock were dissolved with the unbuffered
solution adjusted to pH 5.2. The ﬁnal DSMO concentration was below 0.5 % (v/v) in the
bathing medium. The leaf segment was incubated in the unbuffered bathing solution
(containing one of these chemicals) under darkness for 50-60 min. Following the
procedure of experimental protocol described above, ion ﬂux was measured over an
average 30 min period of light exposure.

Vanadate, DCCD, FC, EGTA, LaC13, Ca2+ ionophore A23187, A-9-C, DIDS, TEA,

93

CsCl were added to bathing solution, respectively, to test then individual effects on a
speciﬁc ion ﬂux under low ﬂuence (50umol m'2 5").

Different external concentrations of Ca2+ (0.1 and 2.0 mM), K” (0.2, 5.0 and 10 mM)
and Cl’ (0.2, 2.0, and 10 mM) were achieved by changing the salt concentrations in the

bathing solution.

Data statistical analysis
Data analysis has been described in Chapter 1. Analysis of variance (ANOVA) of the

data was conduced using SAS 8 (SAS Institute Inc, Cary, NC).

RESULTS

Effects of H+-ATPase inhibitor and FC.

To test the possible involvement of a plasma membrane proton pump in the light-induced
ﬂux change, two plasma membrane H+~ATPase inhibitors, DCCD (at 0.1 mM) and
Na3VO, (at 1.0 mM), as well as a stimulator of H“-ATPase, Fusicoccin (FC) at 0.01 mM,
were applied to the bathing solution (0.5 mM CaSO,, 1.0 mM KCI, adjusted to pH 5.2).
The PC is a powerﬁrl effector that stimulates the plasma membrane IP-ATPase and is
used as an experimental tool to investigate the mechanism of physiological modulation of '
this enzyme (Marré, 1979). Under low background light (50 pmol m‘2 s"), the net H“ ﬂux
from mesophyll tissues of the control (Figure 1) exhibited a small change around zero.
However, the addition of DCCD and Na3VO4 inhibited H+ efﬂux and resulted in a high
net H+ inﬂux of 16-20 nmol m'2 s“. In contrast, FC stimulated H+ efﬂux and resulted in

decrease in net H+ ﬂux of 18 nmol m'2 s'l after approximately 30 min (Figure l).

94

 

 

1' -0- control ‘
+ 0.1 mM DCCD

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A 30 _ + 1mMNa v04 -*
...— + 0.01 mM FC
to " .l
N
'E 20 — ‘_._____________. —
‘5 Add N
E I - II
5
x 10 - 1 -~ —
3
t: ' v -
s 0 - r
C
.E ‘ " ‘
a)
3» ~10 — ~- _
‘0 _-
5 - LSD(0,05) \‘ q

~20 — _

1 1 1“ 1 1 1 1 1
0 5 10 15 20 25 30 35 40
Time (min)

Figure 1. Effect of H+-ATPase inhibitors. vanadate (Na3VO )and N, N-dicyclohexi cabodiamide
(DCCD), and the ATPase stimulator Fusicocin (FC) on net H ﬂux under fluence (50 pmol

111‘2 s'1 ). Data points represent an average of 4-6 replicates. Vertical bars represent LSD
values at p<0.05.

 

 

 

 

 

 

 

20
<>-ammx
+ 0.1 mM DCCD .
r, -O— 1 mM Na V04 Light-off ..
,5" + 0.01 mM F0
.m l
‘1‘ 10 l— —
E Light-on
o
e - 1 w
25
O — _.
+¢ A
I
E . .
c
.E
81 ~10 — -
c
cu
.1:
O 1 .
-20 1 1 1 1 1 1 1 1 1 1
0 5 10 15 20 25 30 35 40 45

Time (min)

Figure 2. Effect of H+-ATPase inhibitors, vanadate (N33VO4) and N. N-dicyclohexicabodiamide
(DCCD), and the ATPase stimulator Fusicocin (FC) on the time course of light (450 pmol rn'2 s‘1)
-induced change in net H+ ﬂux from pea mesophyll tissues. Data points represent an average

of 6~1O replicates.

95

After DCCD, NagVO, and FC were added (at the same concentrations as in low light)
to the bathing solution (0.5 mM CaSO,, 1.0 mM KCl, adjusted to pH 5.2), the leaf tissue
was incubated for 50-60 min in the dark. Net H+ ﬂux and extracellular pH around the pea
mesophyll tissues were then measured following light stimulation (450 pmol rn‘2 3").
Light induced a rapid increase in both net H+ inﬂux and extracellular pH. After 5-10 min
this inﬂux (the control) decreased or changed to a net efﬂux, which is known as the light-
induced PIFR (Figure 2, Table l). The PIFR could involve a gradual return to ﬂux rate
closer to or lower than that measured prior to light stimulation. Either decrease in inﬂux
or increase in efﬂux (or both) caused the PIFR. Addition of DCCD and Na,VO4 resulted
in a signiﬁcant inhibition of the light-induced initial response (61% and 54% of the
control respectively) in net H+ inﬂux (Table 1) and. extracellular pH (data not shown). A
50-60 % reduction in H+ PIFR was observed in the presence of DCCD or Na.,VO4 (Table
l). The F C also reduced the light—induced initial response in net H+ inﬂux within 5 min
of exposure to light and suppressed the light-induced extracellular alkalinization. As
compared to the control (~22 nmol m'2 s"), F C caused a higher amplitude of PIFR (~46
nmol 111‘2 s") from net inﬂux to net efﬂux (Table 1), indicating that F C had stimulated H+
efﬂux from the plasma membrane.

As discussed previously in Chapter I, light induced a transient increase in net Ca2+
inﬂux. Both DCCD and Na,VO4 treatments signiﬁcantly reduced the light-induced PIF R
of Ca2+ (38 % of the control), whereas Na,VO4 signiﬁcantly inhibited the light-induced
initial response (28 %) in net K+ inﬂux (Table 1). FC increased light-induced initial
response in net Cl' inﬂux (240 %) but inhibited that of net Ca2+ inﬂux (Table 1). During

the ﬁrst 5 min of light treatment, the light-induced net Cl' ﬂux change was 27 nmol m'2 s'1

96

Table 1. Effects of H+-ATPase inhibitors and FC on light-induced net ﬂuxes of 11“,
Ca“, K" and Cl' from pea mesophyll tissues.

 

11* Ca” K: cr
Qmol m'2 8")
Initial ﬂux response (inﬂux)
control +126(10) §a +48(10) a +171 (8) a +101 (8) b
1.0 mM Na3VO, +12 (10) b +57 (7) ab +132 (8) b +72 (7) b
0.1 mM DCCD +12 (8) b +52 (8) ab +139 (7) ab +67 (7) b

0.01 mM FC +24 (7) a +30 (6) b +200 (6) a +242 (6) a
Flux change during the ﬁrst min
control +4 (10) +26 (10) a +24 (8) a +13 (8) ab
1.0 mM Na:,VO4 +2 (10) +35 (9) a +19 (8) a -9 (7) b
0.1 mM DCCD +4 (8) +7 (10) ab +27 (7) a ~20 (7) b
0.01 mM FC ~1 (8) ns +1 (8) b ~62 (6) b +69 (6) a
Flux change during the ﬁrst ﬁve min
control +13 (10) +29 (10) a +67 (8) ~60 (8)
1.0 mM Na3VO, +4 (10) +41 (8) a +16 (7) ~55 (7)
0.1 mM DCCD +6 (8) +17 (10) ab +17 (7) -36 (7)
0.01 mM FC +4 (8) ns ~13 (6) b +9 (6) ns +27 (6) ns
Amplitude of PIFRi
control ~22 (10)a -63 (10) a ~188 (8) ab -151 (8)
1.0 mM Na3VO4 ~9 (8) c ~40 (8) b ~138 (8) a -213 (5)
0.1 mM DCCD ~10 (8) c -49 (8) b ~200 (7) b ~l73 (7)
0.01 mM FC ~46 (6) b ~53 (6) ab ~181 (6) ab -207 (6) ns

 

Different letters indicate signiﬁcant difference between treatments within columns at p < 0.05 according to
LSD test.
“ns” indicates no signiﬁcant difference.

§ The numbers in parentheses indicate the number of replicates.

1 ~ or + indicates decrease or increase of net ion ﬂux and extracellular ion concentration, respectively.

‘ PIFR: Light-induced Post-Initial Flux Response, a result of either a decrease in net inﬂux or an increase
in net efﬂux, or both. This value is negative.

97

under F C, indicating a net inﬂux. However, in other treatments, net Cl' ﬂux changes
were negative, indicating a net efﬂux. Additionally, the light-induced PIFR of Cl' (-207
nmol rn’2 5") occurred after 5.3 min, whereas the control only needed 2.1 min. FC did not
affect the light-response of K” ﬂux. These results indicated that the inhibition of H1
ATPases caused a decrease in the light induced initial inﬂuxes of H+ and KL, and in the
PIFRs of IF and Ca2+. On the other hand, an increase in IF-ATPase activity enhanced the

light induced initial inﬂuxes of Cl' and PIFR of H”.

Effects of Ca2+ channel blocker and chelator

The Ca“ channel blocker (LaC13) or chelator EGTA were used to evaluate their effects on
Ca2+ channel activity. The Ca2+~ionophore A23187 is thought to mediate Cazl/ZH+
exchange across the membrane (Pressman, 1976). When at the same low light
background (50 pmol In2 8"), net Ca2+ ﬂux from pea mesophyll tissues in the control only
displayed a small change (Figure 3). However, the net Ca2+ inﬂux was inhibited by both
LaCl3 (0.1 mM) and EGTA (1.0 mM) but increased by Cay-ionophore A23187 of 5 11M
(Figure 3).

Light (450 pmol In2 S") elicited a transient increase in net Ca2+ inﬂux followed by a
PIFR (Figure 4, or see Chapter 1). Compared with the control, both LaCl3 and EGTA
signiﬁcantly reduced the light-induced initial response in net Ca2+ inﬂux (by 42-44% of
the control) and corresponding response in extracellular Ca” concentration (64% and
39%, data not shown), as well as the light-induced PIFR of Ca2+ around pea mesophyll

(27 % and 37 %, respectively). Its initial light response time also was delayed by both

98

 

 

 

 

 

 

 

 

 

 

 

60 T -O-1mMEGTA ..
+ 0.1mM
‘_-~ j + SuM Calﬁc‘ignophore -
(:0 40 _ + 5 Ca-ionOphore _
'E . .
‘o
E 20 ~ _
5
x 1 .
.2
+ 0 l" -
N
(U
0 h 1
2 ~20 — -
.E
O F .
g -40 _ _
6 . .
‘60 " 1 1 1 1 1 1 l 1 T
0 5 10 15 20 25 30 35 40
Time (min)

Figure 3. Effects of Ca 2+ channel blocker (LaCl3). chelator (EGTA) and ionophore A23178
on net Ca2+ ﬂux from pea mesophyll tissues under low fluence (50 pmol rn'2 3'1). Data points
represent an average of 6-8 replicates. Vertical bars represent LSD values at p<0.05.

40

 

 

 

 

b)
O

 

N
O

..L
o

 

Change in net C32+ ﬂux (nmol m'2 s'1)
O

 

 

 

 

-10
'20 _ Lsoom. _
-30 _ ..
1 1 1 1 1" 1 1 I 1 1
0 5 10 15 20 25 3O 35 40 45
Time (min)

Figure 4. Effect of the C82+ chelator (EGTA) and channel blocker (LaCl3) on the time course
of light (450 pmol m”2 3'1)-induced change in net 032+ ﬂux from pea mesophyll tissues. Data
points represent an average of 6~10 replicates. Vertical bars represent LSD values at p<0.05.

99

Table 2. Effects of Ca“ channel blocker and chelator on light-induced ﬂuxes of H“,
Ca“, K1 and Cl' and time response in pea mesophyll tissues.

 

 

H1 Ca2+ K+ Cl‘
Initial ﬂux response (inﬂux) (nmol m'2 5")
control +126(10)§ a +45 (8) a +169 (8) a +96 (8)
1.0 mM EGTA +7 (8) b +25 (9) b +121 (8) b +138 (8)
0.1 mM LaCl, +13 (8) b +26 (10)b +75 (8) 0 +85 (9) ns
Flux change during the ﬁrst min (nmol m"2 3")
control +3( 10) +24 (8) a +22 (8) +10 (8)
1.0 mM EGTA +1 (8) +13 (9) b +52 (8) +71 (7)
0.1 mM LaCl, +3 (8) ns +4 (10) b +29 (8) us +12 (9) ns
Flux change during the ﬁrst ﬁve min (nmol m’2 s")
control +13 (10) a +32(8) a +63 (8) ~57 (8) b
1.0 mM EGTA +1 (8) b +9 (9) b +30 (8) +61 (7) a
0.1 mM LaCl, +8 (8) a +100) 0 +13 (8) ns -4 (9) c
Amplitude of PIFR‘ (nmol rn‘2 8“)
control ~26 (10) a ~60 (8) a ~201 (8) a ~143 (8) a
1.0 mM EGTA ~18 (8) ab -38 (9) b -121 (8) b -34 (4) b
0.1 mM LaCl3 -1l (7) b -44(10) b -99 (8) b -50 (9) b
Initial light response time (min)
control 3100) 2.1 (8) b 3.9 (8) 2.3 (8) b
1.0 mM EGTA 4.5 (8) 3.7 (9) a 6.6 (8) 4.8 (8) b
0.1 mM LaCl, 3.4 (8) ns 3.8(10) a 4.4 (8) ns 5.6 (9) a

 

Different letters indicate signiﬁcant difference between treatments within columns at p < 0.05 according to

LSD test.
“ns” indicates no signiﬁcant difference.

§ The numbers in parentheses indicate the number of replicates.
I ~ or + indicates decrease or increase of net ion ﬂux and extracellular ion concentration, respectively.
‘ PIFR: Light-induced Post-Initial Flux Response, 8 result of either a decrease in net inﬂux or an increase

in net efﬂux, or both. This value is negative.

100

(Table 2). This indicates that Ca2+ channel blocker and chelator inhibit the light response
in net Ca2+ inﬂux.

Simultaneously, the light-induced initial responses of net H+ and K“ inﬂux were also
signiﬁcantly suppressed in the presence of LaCl3 and EGTA (Table 2). Neither Ca2+
inﬂux inhibitor affected the light-induced initial response of net Cl' inﬂux. However, the
light-induced PIFR of Cl‘ was greatly inhibited by LaCl3 and EGTA (Table 2). During
the ﬁrst 5 min after illumination, a negative change in net Cl' ﬂux, a net efflux, had been
observed in the presence of EGTA. The change was signiﬁcantly higher than that of
LaCl3 and EGTA. Like Cl', LaCl3 and EGTA also produced a decrease in the light-

induced PIFR of K+ and H+ (only LaC13) (Table 2).

Effects of K“ channel blocker

Tetraethylammonium (TEA) is a powerful blocker for both classes of K+ channels,
inward— and outward-rectiﬁers (Bentrup, 1990). CsCl blocks inward K+ current (Moran et
al., 1990). To identify whether K” channels are involved in the light-induced Kl ﬂux
change, TEA-Cl' (5.0 mM) and CsCl (2.0 mM) were added to the bathing solution (0.5
mM C380,, 1.0 mM KCl, adjusted to pH 5.2). In general, light stimulation produced a
rapid increase in net Kl inﬂux followed by a PIFR which may be due to a decrease in net
K+ inﬂux or an increase in net K or both (Figure 5 or see Chapter I). Compared to the
control, TEA signiﬁcantly blocked the light-induced initial response (35 % of the control)
in net K+ inﬂux and the PIFR of K” (66 %) (Table 3). The effects of CsCl on the light-
induced K+ ﬂux change were similar but smaller in the magnitudes as compared to that of

TBA.

10]

 

120

 

   

 

 

 

 

 

 

 

 

-0- control
+ 2 mM CsCl . -
A 100 + 5mM TEA L'QM‘O" _
f...” l- -l
,. l
E 80 — _.
'5
g ” .
:g 60 - -
a t l
+
x 40 _ _
8
C - .
.E
G
C . .1
2
o 0 — _
r- J- -l
_20 1 1 1 1 1 l 1 1 1 1
0 5 1O 15 20 25 30 35 40 45
Time (min)

Figure 5. Effect of K+ channel blockers tetraethylammonium (TEA) and Cesium chloride
(CsCl) on the time course of light (450 pmol rn'2 s'1Hnduced change in net K+ ﬂux from
pea mesophyll tissues. Data points represent an average of 6-10 replicates. Vertical bars
represent LSD values at p<0.05.

 

200

 

i

1mMA-9-C

-O-
" -O- 0.
+ 0.1mMDlDS _

 

 

150 -
- LSD(0.05) 4

10° ‘- Licht- -
. Light-off .
o

 

 

Change m net 01' flux (nmol m'2 s'1)
C
1
O
c
o
1

-100 r-

 

 

,
-150 i 1 1 1 1 1 1 1 1 1
0 5 10 15 20 25 30 35 4O 45

Time (min)
Figure 6. Effect of Cl' channel blockers diisothiocyanatoslibene (DIDS) and anthracene~9~
carboxylic (A-9-C) on the time course of light (450 pmol rn'2 5'1)-induced change in net Cl'
ﬂux from pea mesophyll tissues. Data points represent an average of 6-10 replicates.
Vertical bar represents LSD values at p<0.05. ‘

_

 

102

Table 3. Effects of K+ channel blocker on light-induced net ﬂuxes of H2 Ca“, K+

and Cl‘ from pea mesophyll tissues.

 

 

 

H+ Ca2+ K” CL
(nmol m'2 8")
Initial ﬂux response (inﬂux)
control +125 (10)§a +47 (8) +183 (10)a +91 (8) b
5.0 mM TEA +16 (8) b +42 (6) ns +120 (9) b +246 (7) a
2.0 mM CsCl +12 (5) b n.m. +142 (8) ab n.m.
Flux change during the ﬁrst min
control +4 (10) +21 (8) +41 (10) +15 (10) b
5.0 mM TEA +5 (8) +30 (6) ns +18 (9) +148 (7) a
2.0 mM CsCl +3 (5) ns n.m. +32 (8) ns n.m.
Flux change during the ﬁrst ﬁve min
control +11 (10) a +21 (10) +77 (10) ~34 (8) b
5.0 mM TEA +9 (8) ab +10 (6) ns +24 (9) +93 (7) a
2.0 mM CsCl +4 (5) b n.m. +53 (8) ns n.m.
Amplitude of PIFR‘
control ~18 (10) ~57 (8) ~189(10) a -l49 (8) a
5.0 mM TEA -20 (8) ~65 (6) ns ~64 (9) b ~39 (3) b
2.0 mM CsCl ~11 (5) ns n.m. ~95 (8) b n.m.

 

Different letters indicate signiﬁcant difference between treatments within columns at p < 0.05 according to

LSD test.
“ns” indicates no signiﬁcant difference.

§ The numbers in parentheses indicate the number of replicates.
1

~ or + indicates decrease or increase of net ion ﬂux and extracellular ion concentration, respectively.

‘ PIFR: Light-induced Post-Initial Flux Response, a result of either a decrease in net inﬂux or an increase

in net efflux, or both. This value is negative.

103

Both TEA and CsCl also signiﬁcantly reduced the light-induced initial response (36
and 52 %, respectively) (Table 3) and maximum increase (38 and 50 %, respectively, data
not shown) in not H inﬂux. The blocker TEA fluther increased the light-induced initial
response of net Cl' inﬂux, possibly because of high Cl’ concentrations in the bathing

solution. However, it did not affect the light responses of net Ca2+ ﬂux (Table 3).

Effects of Cl' Channel blocker

Two anion channel blockers, A~9~C (for the Slow-type channels) and DIDS (for the
Rapid-type channels), were used to determine whether anion channel activity was
correlated with the light-induced Cl' ﬂux change. Light elicited a rapid and short
response in net Cl‘ inﬂux and followed by a PIFR (Figure 6, Table 4, or see Chapter I).
The A-9-C treatment did not signiﬁcantly affect the light-induced initial response of net
Cl' inﬂux (Table 4). In contrast to A—9-C and the control, DIDS did not cause a net Cl'
efﬂux but a net inﬂux (48nmol m‘2 5") during the ﬁrst 5 min of illumination (Table 4). A
PIFR of Cl' occurred after 6.3 min in the presence of DIDS, whereas the control only
needed 2.3 min of light exposure. When the light was turned off, the net Cl' ﬂux changed
to a net efﬂux for the control whereas A-9-C and DIDS increased the net Cl‘ inﬂux
(Figure 6).

Both A-9-C and DIDS, when applied at the same concentration (0.1 mM), did not
signiﬁcantly inﬂuence the light-induced initial response of net inﬂux of IF, Ca” and K”
(K+ data for DIDS not measured experimentally). By comparison to the control, DIDS
signiﬁcantly reduced the light-induced response of H“ and Ca2+ inﬂuxes during the ﬁrst 5

min of illumination and extended the initial light response time of Ca2+ (Table 4).

104

Table 4. Effects of Cl' channel blocker on light-induced net ﬂuxes of H”, Ca“, K+
and CI’ and time response in pea mesophyll tissues.

 

 

H+ Ca2+ K” C1”
Initial ﬂux response (inﬂux) (nmol m2 8")
control +127 (8)§ +48 (6) +161 (8) +110 (8)
0.1 mM A-9-C +23 (11) +59 (7) +171 (8) ns +164 (7)
0.1 mM DIDS +18 (8) ns +45 (7) ns n.m. +201 (5) ns
Flux change during the ﬁrst min (nmol m'2 5")
control +8 (8) +22 (6) a +35 (8) a +64 (8)
0.1 mM A-9-C +7 (1 1) +21 (7) a ~43 (8) b +139 (7)
0.1 mM DIDS +4 (8) ns -7 (7) b n.m. +30 (7) ns
Flux change during the ﬁrst ﬁve min (nmol m'2 s")
control +13 (8) a +22 (6) a +68 (8) a ~30 (8) ab
0.1 mM A~9~C +12 (1 1) a +22 (7) b +25 (8) b ~99 (5) b
0.1 mM DIDS +6 (8) b +2 (7) b n.m. +48 (7) a
Initial light response time (min)
control 4.1 (10) 2.2 (6) b 4.3 (8) b 2.3 (8) b
0.1 mM A-9-C 3.2(11) 3.0 (7) b 8.0 (8) a 1.7 (7) b
0.1 mM DIDS 3.5 (8)ns 7.7 (7) a n.m. 6.3 (7) a

 

Different letters indicate signiﬁcant difference between treatments within columns at p < 0.05 according to

LSD test.
“ns” indicates no signiﬁcant difference.
“n.m.”. not experimentally measured.

§ The numbers in parentheses indicate the number of replicates.
I - or + indicates decrease or increase of net ion ﬂux and extracellular ion concentration, respectively.

105

Effects of Ca“ ionophore and external Ca“, K“ and Cl' concentrations

Treatment with the Ca2+~ionophore A23187 (5 uM) signiﬁcantly stimulated the light-
induced initial response in net Ca2+ inﬂux from 47 to 67 nmol m‘2 s'l (Figure 7).
Compared to the control, the light-induced initial response of not H’“, K+ and Cl' inﬂux
were not signiﬁcantly inﬂuenced by the 'Caztionophore (K+ and CT data not shown)
(Figure 7). In the same treatment, the light-induced PIFR of Cl' increased signiﬁcantly
from ~144 to -241 nmol m'2 8", whereas the PIFR of K+ was not signiﬁcantly affected
(Figure 7). Therefore, the Ca2+~ionophore and LaCl3 or EGTA produce different effects
on light-induced H“, Ca2+, Cl' and K+ ﬂux change in pea mesophyll tissues.

When the Ca2+ concentration in the external bathing solution was increased to 2.0 mM
(2.0 mM CaSO,, 1.0 mM KCI, pH 5.2), a signiﬁcant increase in net Ca“ inﬂux response
to light was observed (Figure 8). The light-induced PIFR of Cl' was also signiﬁcantly
enhanced by the higher Ca2+ concentration within the bathing solution (Figure 8).
Compared with the control (0.5 mM Ca2+), the light-induced initial response in net Hi, K+
and Cl' inﬂuxes were not signiﬁcantly affected (K+ and CT data not shown). However,
when the external Ca“ concentration was experimentally set at 0.1 mM, the PIFR of K+
and Cl' declined signiﬁcantly.

The light-induced initial response in net Kl inﬂux increased at higher external K+
concentration (5.0 mM) in the bathing solution and decreased at the lower concentration
(0.2 mM K”) (Table 5a). Thus, the initial light response of K+ ﬂux appears to be
concentration-dependent. However, changing the K+ concentration did not signiﬁcantly

affect the light-induced initial response nor the PIFR of Hi, Ca2+ and Cl' (Table 5a).

106

300

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a E
f: 150 - / _/ _
. - / / -
.5: 100 — % é _
g1 . 1150905) % / ‘
o 50 +- . ¢ ¢ / _

0 i % / / A ‘

 

 

 

 

 

 

 

 

 

WWW ..... mama" .... we new
Figure 7. Effect of 032+ ionophore A23187 on the light-induced ion ﬂux response of pea mesophyll
tissues. Changes in net K+ and CI' ﬂux indicate the amplitude of light (450 pmol m'2 s'1)-induced
Post-Initial Flux Response (PIFR), negative values. Data represents an average of 6-9 replicates.
Vertical bars represent LSD values at p<0.05.

 

 

 

 

 

     

 

 

 

 

 

 

 

 

 

 

300
r 1:) 0.1 men? .1
:21 0.5 mM of” (control)
._"‘ 250 L- - 2 mM ea “ _
'm
‘1‘ l .
E
'5 200 — _
E
5 ~ .
X
a 150 r- —l
C
‘5 L ‘
O
C
.s 100 ~ —
8, _ LSD(0.05) .
5 .
l. .
0 ra + ,
initial 11* hﬂux Initial Cruz" hﬂux Amplitude Amplitude of
response response PFIR for K PFIR for 0'

Figure 8. Effect of external Ca2+ concentration on the light-induced ion flux response of pea
mesophyll tissues. Changes in net K“ and or ﬂux indicate the amplitude of light (450 pmol

m'2 3'1)~induced Post-Initial Flux Response (PIFR), negative values. Data represents an average
of 6—9 replicates. Vertical bars represent LSD values at p<0.05.

107

Table 5. Effects of external Kr and Cl’ concentration on light-induced net ﬂuxes of
H”, Ca2+, K+ and Cl' from pea mesophyll tissues.

a: external K+ concentrations

 

 

 

 

 

 

 

Hi Ca2+ K+ Cl'
(nmol mas") '
Initial ﬂux response (inﬂux)
control(l.0 mM K”) +l21(7)§ +48 (8) +153 (6) b +91 (7)
0.2 mM K+ +19 (7) +43 (9) +49 (7) c +106 (7)
5.0mM K+ +25 (6) ns +38 (6) ns +255 (7)-a +153 (6) ns
Amplitude of PIFR‘
control(1.0 mM K‘) ~16 (7) ~51 (8) ~18] (6) a -143 (7)
0.2 mM K+ ~19 (7) -52 (9) ~82 (7) b ~119 (6)
5.0 mM K+ ~11 (6) ns ~51 (6) ns ~262 (7) ab ~111 (4) ns
b: external Cl concentrations
H+ Q24» K+ Cl-
(InnolnlL2 8")
Initial ﬂux response (inﬂux)
Control(1.0mM Cl') +27 (8) a +48 (8) a +176 (6) b +103 (8) b
0.2 mM Cl' +17 (5) a +28 (6) b +107 (8) b +101 (6) b
2.0 mM Cl’ +20 (7) a +43 (6) a n.m. +160 (5) b
10 mMKCl +8 (6) b +37 (7) ab +739 (8) a +312 (7) a
Amplitude of PIFR
Control(1.0mM Cl') ~24 (8) a -59 (8) -193 (6) b ~184 (8)
0.2 mM Cl' ~17 (5) a ~48 (5) ~155 (8) b ~112 (6)
2.0 mM Cl' -23 (7) a ~45 (6) n.m. ~126 (5)
10 mMKCI -8 (5) b ~51 (7) ns ~509 (8) a ~157 (6) ns

 

Different letters indicate signiﬁcant difference between treatments within columns at p < 0.05 according to

LSD test.
“ns” indicates no signiﬁcant difference.
“n.m.” not experimentally measured.

§ The numbers in parentheses indicate the number of replicates.

’

~ or + indicates decrease or increase of net ion ﬂux and extracellular ion concentration, respectively.

3 PIFR: Light-induced Post-Initial Flux Response, a result of either a decrease in net inﬂux or an increase

in net efflux, or both. This value is negative.

108

If the Cl' concentration in the bathing solution was lowered to 0.2 mM (did not alter
Ca2+ and K” concentration), the light-induced initial response in net Ca2+ inﬂux was
signiﬁcantly inhibited (Table 5b). Doubling the K” and Cl' concentrations in the external
bathing medium to 10 mM KCI dramatically reduced the light-induced initial response in
net H+ inﬂux and signiﬁcantly enhanced net K+ and Cl' inﬂux in response to light (Table

5b).

DISCUSSION

Ca2+ ﬂuxes and Ca2+ transporters

Increase in cytosolic free Ca2+ concentrations is considered to be necessary for cellular
signal transduction in response to external stimuli. Passive inﬂux of Ca2+, facilitated by
the opening of Ca2+-permeable channels in the plasma membrane, is one of the primary
means of increasing cytosolic free Ca2+. Numerous physiological, pharmacological and
Ca2+ ﬂux studies have provided indirect evidence that Ca2+ channels in the plasma
membrane of higher plant cells play a central role in the initiation of various signal
transduction sequences, including, plant-pathogen interactions (Bach et al., 1993), light-
induced leaf movement (Mayer et al., 1997), and honnone- and light-regulated stomatal
responses (Ward and Schroeder, 1997; Grabov and Blatt, 1998). However, direct
evidence for the light-induced Ca2+ channels in the plasma membrane of mesophyll cells
has not been obtained. Accurate measurement of Ca2+ inﬂux across the plasma
membrane is quite difﬁcult. In the current research, net Ca2+ ﬂux was measured by a
noninvasive, ion-selective, slowly vibrating microelectrode. A 1i ght-induced transient

increase in net Ca2+ inﬂux was identiﬁed and proposed as an initial depolarizing agent in

109

Chapter 1, suggesting that an increase in Ca2+ inﬂux not a decrease in Ca2+ efﬂux is one
major reason for the initial Ca2+ inﬂux response (see Chapter 1). Although H“~Ca2+
exchanges occurred in the cell wall of Chara australis cells (Ryan et al., 1992), the result
of net Ca2+ ﬂux mainly represents free Ca2+ movement within the apoplast of the tissues.
Reid and Smith (1992) did not ﬁnd signiﬁcant effects on Ca2+ inﬂux following changes in
external pH or following changes in internal pH by separating the cell wall from cell
contents of Chara corallina, indicating that exchanges between H+~Ca2+ are relatively
constant under a certain range of pH in the bathing solution. Therefore, it is reasonable to
believe that the light-induced net Ca2+ inﬂux is the result of Ca2+ uptake facilitated by
Ca2+~channels in the plasma membrane.

Further evidence for the role of putative Ca2+ channels is obtained by examining the
effects of a channel blocker and a Ca2+ chelator. Both LaCl3 and EGTA signiﬁcantly
suppressed the light-induced transient increase in net Ca2+ inﬂux (Table 2), demonstrating
the relationship between Ca2+ inﬂux and Ca2+ channel activity.

At least two major classes of Ca2+ channels reside in the plasma membrane (White,
1998). The ﬁrst class includes those that are relatively non-selective with respect to
cations and possess a high single-channel conductance; these are known as maxi-cation
channels (White, 1993; White, 1994). The second class is relatively more selective for
cations, and exhibits a smaller single-channel conductance. Since the addition of LaCl3
and EGTA also resulted in a signiﬁcant reduction of the light-induced initial response of
net K+ inﬂux (Table 2), the inﬂux of both Ca2+ and K+ is assumed to occur by diffusion
through non—selective cation channels. The same ﬁnding was reported in Samanea

saman protoplasts (Moran and Satter, 1990) and during light-induced swelling of

110

Phaseolus coccineus protoplasts (Mayer et al., 1997). The Ca2+ or K+ inﬂux across the
putative cation-channels was dependent upon the extemal concentration of Ca2+ or K+ but
not upon each other (Table 2, 5). However, an organic Ca2+ channel blocker, verapamil
(0.1 mM), did not affect light-induced Ca2+ inﬂux or other ion ﬂuxes (data not shown).
Reid and Smith (1992) also made similar observations in Chara. In animal cells, La“
can block at least two types (L~ and N-type) of voltage-activated Ca2+ channels, whereas
verapamil only blocks the L~type (Tsien et al., 1987). Apparently, not all types of cation-
channel are responsive to light in pea leaf tissues.

The effect of Ca2+ on the light-induced PIFR of Cl‘ provides more evidence for Ca2+
inﬂux as an initial response to light. Clearly, increased cytoplasmic Ca2+ has been shown
to trigger anion channels and to precede Cl' efﬂux (Schroeder, 1995). The C1' efﬂux
across the plasma membrane was activated by intracellular free Ca2+ in Chara cells
(Mimura and Shimmen, 1994; Johannes et al., 1998). A rapid light-induced PIFR of Cl'
after 2-3 min of illumination was detected and explained as a result of an increase in C1‘
efﬂux (See Chapter I ). This time allows for Ca” activation of anion channels and a
change in the directionality of the anion ﬂux. Three facts support the role of Ca” in
regulating Cl‘ efﬂux: (a) LaCl, and EGTA signiﬁcantly blocked the light-induced PIFR of
Cl’ (Table 2); (b) the addition of Cay-ionophore A23187 (Figure 7) or (0) increasing Ca2+
concentration in the bathing medium resulted in a signiﬁcant increase in the light-induced
PIF R of Cl‘ (Figure 8). Nevertheless, the light-induced Ca2+ inﬂux was not inﬂuenced by
C1” channel blockers (Table 4).

Both LaCl3 and EGTA dramatically inhibited the light-induced initial response in net

H” inﬂux (Table 2). F elle (1998) hypothesized that cation channels may contribute to the

111

regulation of the apoplastic pH in roots. He also observed that extracellular Ca2+ was
apparently less effective in inﬂuencing apoplastic pH. Using the Ca2+~ionophore A23187
as a Cay/H+ exchanger did not signiﬁcantly affect the light-induced Hi inﬂux (Figure 7).
This result is different from that of LaCl3 and EGTA treatments. A possible explanation
is that proton pumps are deactivated when cation channels are blocked. Kinoshita et al.
(1995) reported that the activation of IF-ATPase required elevated cytoplasmic Ca2+
concentration in guard cells. Under the light, perhaps the interaction of Ca2+ and H+ (and
other ions, such as K, Cl’) with Hi-ATPase in the plasma membrane stimulates its
phosphorylation/dephosphorylation, as occurs under stresses (Netting, 2000). No work
seems to have been done on the possible mechanisms for such a light-induced response.
The present result might constitute a linkage between Call-channels or Ca2+ messenger
and Hi ﬂux across the plasma membrane during light signal transduction. All
considerations taken together, the simplest interpretation is that light-induced transient
Ca2+ inﬂux facilitated by Ca2+ channels could initiate other ionic responses in pea
mesophyll.

It is interesting to note that external Ca2+ concentrations inﬂuenced ion ﬂux responses
to light. The ﬁrst effect of apoplastic Ca2+ is the light-induced initial response in net Ca2+
inﬂux which was signiﬁcantly increased by higher Ca2+ concentration (2.0 mM), whereas
the initial ﬂux responses in other ions were not signiﬁcantly inﬂuenced by changing
extracellular Ca2+ concentrations (Figure 8) (K+ and Cl' data not shown). However, the
light-induced PIFR of K and CT was signiﬁcantly inﬂuenced by apoplastic Ca2+
concentrations (Figure 8). These results suggest that apoplastic K+ also play an important

role in regulating ionic responses to light.

112

It is remarkable that light immediately stimulated a rapid PIFR of Ca2+ following a
transient Ca2+ inﬂux (see Chapter 1,). Perhaps the PIFR is associated with Ca” efﬂux and
reﬂects light-regulation of cytosolic Ca2+ homeostasis. When cytosolic Ca2+
concentration increases, active Ca2+~transport systems work to restore the resting
cytoplasmic Ca2+ to submicromolar levels (Trewavas, 1999). Vanadate is not completely
speciﬁc for the plasma membrane Hl-ATPase inhibition. The plasma membrane Ca2+-
ATPase also is inhibited by vanadate (Palmgren and Harper, 1999). Thus, it is not
surprising that vanadate and even DCCD signiﬁcantly reduced the light—induced PIF R of
Ca2+ (Table l) and enhanced the light-induced maximum increase in net Ca2+ inﬂux (data
not shown). On the other hand, Ca2+ channel blocker and EGTA also reduced the light-
induced PIFR of Ca2+ (Table 2). Therefore, the decrease in Ca2+ inﬂux is not a
contributor to this process. Ca2+ efﬂux across the plasma membrane mediated by Ca”-
ATPase in the plasma membrane would be expected to be a main pathway for the light-

induced PIF R of Ca2+.

Cl' ﬂuxes and Cl‘ transporters

Elzenga and Van Volkenburgh (1997) suggested that light-induced transient
depolarization across the plasma membrane of pea mesophyll cells is at least due in part
to an increased plasma membrane conductance to anions. They found that one of the
anion channels identiﬁed by the patch clamp technique is characterized with a single-
channel conductance of 32 picasiemens as a light-induced candidate. What is the role of
Cl' efﬂux and anion channels? Anion channels have higher selectivity for C1' than for

malate (Hedrich, 1994; Schroeder, 1995). In plant cells, anion channels regulate Cl'

113

efflux from cytoplasm into the extracellular space, driven by the anion-gradient across the
plasma membrane and negative membrane potentials. In two types of anion channels, the
S-type anion channels would produce a long-term and sustained depolarization, whereas
the R-type anion channels would be involved in short and transient depolarization. The
voltage-dependence and kinetics of R-type anion channels are also reminiscent of
voltage-dependent Ca2+ and K+ channels in excitable membranes (Bisson, 1984). The
data presented showed that the two anion channel inhibitors displayed different effects on
the light-induced change in net Cl' ﬂux. The S-type inhibitor, A-9-C, did not affect the
light-induced PIFR of Cl' (an increase in net Cl' efflux), which was eliminated by the R-
type blocker DIDS (Figure 8). A-9-C did not affect the light response in net inﬂux of
other ions (H1, Ca2+ and Kl). By comparison, DIDS signiﬁcantly reduced the inﬂux of H+
and Ca2+ inﬂux during the ﬁrst 5 min of illumination (Table 4). Thus, R-type, and not S-
type anion channels, appear to mediate Cl' efflux during the light response.

Most previous evidence supports the model that Cl’ efﬂux catalyzed by the opening of
anion channels is a component of light-induced depolarization (Spalding and Cosgrove,
1992; Trebacz et al., 1994; Elzenga and Van Volkenburgh, 1997). However, Cl' ﬂux was
not measured in their studies. The CI' ion can move inwardly or outwardly across the
plasma membrane. Indeed, light yielded an initial and short increase in net Cl' inﬂux; in
other words, Cl‘ inﬂux exceeds Cl' efﬂux during the initial light response (see Chapter I).
On the other hand, this result provides some evidence that‘ H+/Cl' symport might be a
pathway for the light-induced inﬂux of both Cl' and H1 as discussed in Chapter I.
Fusicoccin (FC) signiﬁcantly increased the light-induced initial response in net Cl' inﬂux

(Table l) which also was enhanced in acidic bathing medium (see Chapter 1). Thus, Cl'

114

efﬂux mediated by anion channels might be involved, but is probably not the initial
depolarizing component.

Calcium-dependent activation of anion channels seems to be one of its main pathways
in cellular signal transduction. Conversely, Cl' efﬂux in response to activation of anion
channels produces further depolarization, which, in turn, leads to rapid signal
ampliﬁcation as additional anion channels respond to increased depolarization (Ward and
Schroeder, 1997). Ward and Schroeder (1997) hypothesized that anion efflux induced by
cellular stimulation results in membrane depolarization which in turn triggers the
activation of voltage-dependent Ca2+~channels that mediate Ca2+ inﬂux. In this study, low
Cl' concentration (0.2 mM) in the bathing solution signiﬁcantly limited the light-induced
response in net Ca2+ inﬂux. At the same low concentration of exogenous Cl’, the light-
induced initial response in net Cl' inﬂux signiﬁcantly decreased (Table 5b), suggesting a
possible relationship between Ca2+ inﬂux and Cl' ﬂuxes or concentrations during the light
response. Elzenga et a1. (1995) showed that light-induced depolarization of pea
mesophyll was eliminated by high Cl‘ concentrations (about 10 mM, but they did not test
Cl' concentration below 1.0 mM). High external KCl concentrations, 10 mM, greatly
increased the light-induced response in net Cl‘ and K+ inﬂux but affected Hi inﬂux much
less (Table 5b). This provides an explanation for their results. However, the possibility
that high ion concentrations in the bathing medium may generate large variable ion ﬂux

changes cannot be excluded.

K+ ﬂuxes and K’r transporters

As shown previously, the ﬁrst K+ response to light is a rapid increase in net K” inﬂux

115

across the plasma membrane of pea mesophyll tissues (Figure 5). Uptake of K+ across
the plasma membrane is dominated by three possible pathways. One of these is K+ inﬂux
through a K+ inward rectiﬁer. When a K1 inward channel blocker (CsCl) was added to
the bathing solution, the light-induced initial response in net K+ inﬂux was reduced
slightly, but the maximum increase in net K+ inﬂux was inhibited later. Seemingly,
whether the K+ inwardly rectiﬁer is involved in the initial light response in net K“ inﬂux
is still unclear. Another candidate is the non-selective cation channels which are
permeable for both Ca2+ and K“. Both LaCl3 and EGTA resulted in signiﬁcant reductions
in the light-induced net K+ inﬂux response (Table 2), which were also dependent on
external K+ concentration (Table 5a). The ﬁnal possibility for K“ inﬂux is active K“~H+
symport. The proton motive force (pH gradient) generated by the H“ pump can
energetically drive K+ uptake through a carrier (Hedrich and Schroeder, 1989; Netting ,
2000). Two facts support the view that light increases K‘i-H+ symport across the plasma
membrane of pea mesophyll. First, changing apoplastic pH caused a signiﬁcant change
in the light—induced initial response in net K+ inﬂux (See Chapter I). Acidic external
bathing solution increased the K‘ inﬂux. Second, the plasma membrane Hl-ATPase
inhibitor, Na,VO,, decreased the light-induced response in net K+ inﬂux (Table l).
Roelfsema and Prins (1998) reported that light stimulated proton extrusion and resulted in
apoplastic acidiﬁcation enhancing the conductance of K+ inward rectifying channels.
Hedrich and Schroeder (1989) tried to compare the H+~K+ symporter with the Kl~inward
rectiﬁer. However, the ﬂux measurement of the present experiment could not distinguish
between these alternative pathways. To complicate things further, both K+ channel

blockers also signiﬁcantly reduced the light-induced initial response of not H* inﬂux

116

(Table 3), seemingly supporting the existence of a H+~K+ symporter. As discussed
previously, the light-induced K+ inﬂux is not a main depolarizing ionic event.
Considering light-induced transient apoplastic alkalinization and the CsCl effect'on the
initial response of net K+ inﬂux, the light-induced H+~K+ symport is proposed as an initial
pathway for K+ inﬂux.

Unlike Ca2+ and Cl', light only elicited a slow PIFR of K+ following the initial
response in net K+ inﬂux (see Chapter I). This PIFR may be due to a decrease of K+
inﬂux or an increase of K+ efﬂux mediated by K“ outward rectiﬁers. In pea mesophyll,
the K+ channel blocker TEA eliminated most the light-induced PIFR of K” (Table 3),
indicating that outwardly-directed K+ channels are a main pathway for K+ efﬂux and
contribute to the PIFR. However, the PIFR of K+ probably counteracts Cl’ efﬂux and
thus results in a membrane repolarization. Cay-activated K+ channels were found in
higher plants or green algae (Elzenga and Van Volkenburgh, 1993; Johannes et al., 1997;
Bauer etal., 1998). Exogenous LaCl3 and EGTA or low external Ca2+ concentration (0.1
mM) reduced the light-induced PIFR of K+ (Table 2, Figure 8). However, high external
Ca2+ concentration (2.0 mM) or addition of Ca2+~ionophore did not affect the PIP R of K
(Figure 7, 8). A possible reason is related to the decrease in net K+ inﬂux when inward
K+ rectiﬁers were deactivated by elevated cytosolic Ca” concentrations (Lemtiri-Chlieh
and MacRobbie, 1994; Blatting, 1999). In general, the Ki outward rectiﬁers are activated
by membrane depolarization and up-regulated by cytoplasmic pH increase, but are
virtually insensitive to cytosolic Ca2+ (Lemtiri-Chlieh and MacRobbie, 1994; Grabov and
Blatting, 1997). Keunecke and Hansen (2000) found a different pH-dependence of K+

channel activity between bundle sheath and mesophyll cells of maize leaves.

117

Alternatively, as in guard cells (Allan et al., 1994), Cay-independent pathways are
assumed to also exist during the light-induced PIFR of K in pea mesophyll. This process
may be associated with the regulation of H+ homeostasis and membrane potentials, which
also involve other ions. The regulation of K1 channel gating in guard cells may be
accomplished by G proteins and protein phosphorylation (Thiel and Wolf, 1997). K+
ﬂuxes in mesophyll do not have the clearly deﬁned specialized functions that are obvious
in guard cells (Assmann, 1996). The regulation of K” channel activation in mesophyll

cells still needs to be investigated.

The plasma membrane H+-ATPase is not directly involved in light-induced H+ inﬂux
and apoplastic alkalinization

Transient apoplastic alkalinization in mesophyll tissues has been demonstrated (Elzenga
et al., 1995; Shabala and Newman, 1999; or see Chapter I). Stahlberg and Van
Volkenburgh (1999) proposed that the plasma membrane Hl-ATPase does not directly
take part in the light-induced membrane depolarization and alkalinization of pea
mesophyll cells. However, previous results indicated that the plasma membrane H‘-
ATPase activity increased following illumination (Marré et al., 1989; Linnemeryer et al.,
1990; Remis et al., 1994). The resulting apoplastic acidiﬁcation is thought to be coupled
with H” efﬂux from the leaf cells. As proposed previously, CO2 uptake by mesophyll
cells and H+~K+ or Hl-Cl' symport are possible reasons for the alkalinization (see Chapter
I). In the present study, light induced an initial increase in extracellular pH (data not
shown) and in net H+ inﬂux both of which were inhibited by H+~ATPase inhibitors

(Figure 2). Why did the H+~ATPase inhibitor also reduce alkalinization? Possibly

118

because the inhibition of the H+ pump by vanadate or DCCD decreased H+ extrusion from
the cytoplasm thereby reducing the proton motive force, the H+ gradient across the
plasma membrane. The resulting decrease in the light-induced net K+ inﬂux could be
related to the blockage of H+-ATPase (Table 1). Considering the consistent change of H+
ﬂux and extracellular pH, it is assumed that the plasma membrane H+~ATPase may
contribute to the light-controlled apoplastic alkalinization. If light activates the plasma
membrane H+~ATPase and Hi extrusion, or if H+~ATPase is a primary channel for H1
transport, the net ﬂux rate should be shifted to efﬂux. In fact, however, a transient
increase in H+ inﬂux occurred after the onset of light stimulation, indicating that more H+
moves inwardly or H+ efﬂux was inhibited at least. Even though in the presence of FC,
which hyperpolarizes the plasma membrane by activating Hi extrusion and
simultaneously enhancing K+ inﬂux (Ullrich and Guem, 1990), light still elicited a rapid
increase in net H+ inﬂux (Figure 2, Table 1). Remarkably, H+ inﬂux is larger than its
efﬂux; in other words, at least Hl-ATPase does not function as a direct agent in the light-
induced initial response. Therefore, the present results support the conclusion of
Stahlberg and Van Volkenburgh (1999).

The ﬁmction of Hl-ATPase may involve feedback regulation of ion ﬂux.
Cytoplasmic pH homeostasis is attributed to the regulation of H+ pumps and other ionic
transporters, including K+ channels and anion channels such as in guard cells (Grabov and
Blatting, 1998). Stahlberg and Van Volkenburgh (1999) observed a long period of
apoplastic acidiﬁcation immediately following light-induced transient alkalinization of
pea mesophyll, whereas vanadate (0.5 mM) eliminated this acidiﬁcation but did not

inﬂuence the light-induced transient depolarization. Light induced a PIFR of Hi after a

119

rapid initial increase in net H+ inﬂux (Figure 2). An increase in H+ efﬂux or a decrease in
H“ inﬂux resulted in this process, which may contribute possibly to extracellular
acidiﬁcation. Treatment with F C signiﬁcantly increased the PIFR of H, which was
eliminated by vanadate or DCCD (Table 1), suggesting that the H+ pump could be
involved in this response by facilitating H+ efﬂux. Proton signalling has been proposed
as a second messenger in guard cells (Leckie et al., 1998). Alternatively, it is possible
that H+ ﬂux or pH, another candidates as signals, may also be involved in light responses
in pea mesophyll through a Ca2+~independent pathway. Dynamic cytoplasmic H+ changes
may link Caz‘ldependent pathways, or share some common signalling components in

cellular signal transduction. The role of the H* messenger remains to be investigated.

A model: ionic pathways for light response

Figure 9 presents a schematic model integrating the observed and hypothetical events in
light-induced ionic signal transduction in pea mesophyll. Upon activation of putative
photoreceptors, Ca2+ inﬂux through non-selective cation channels would play an initial
role in transmitting the light signal and depolarizing the membrane. K“ and Cl' uptake
coupled with H+ inﬂux contribute to apoplastic akalinization. Light-induced initiation of
Ca2+ channels drives several differential secondary signalling elements, e.g., intracellular
Ca2+ and pH, K”~ and anion-channels. A common feature for all ions measured is that
light induced PIFR of ion ﬂuxes from pea mesophyll. The processes are associated with
the activities of ionic transporters (e.g., Cay-ATPase, H+~ATPase, K+ channels and anion
Channels) in the plasma membrane or intracellular membrane systems, which contribute

to membrane repolarization.

120

However, this research does not exclude the possibility of Ca2+~independent
pathways. Proton signalling coupled with ion ﬂux changes in cellular signal transduction
challenges the hypothesis above. Identiﬁcation of some speciﬁc ion transporters, e.g.,
inward or outward K+ rectiﬁers, anion channels, Ca2+~ATPase, can allow discrimination
among speciﬁc pathways that are presumably involved in the rapid light response. Direct
measurement of dynamic changes of cytosolic ion concentration will provide more
valuable evidence. Further investigation will need to link the observed early events to
downstream steps, such as protein phosphorylation/dephosphorylation, Ca2+lcahnodulin

dependent—kinases and gene expression involved in photoreception.

121

apoplast

 

 

 

 

 

       

 

plasma membrane
ADP + Pi
H-t- « H+
cytoplasm \
H+~ATPase ATP
' ’ + '1'
repolarization K ——-.—-—- K /[pH]
.mm' 4.
anion channel ‘1'
(332+ Ca2+
v
Ca -- ’ase -
depolarization K+ K+

111+ H: [Ca2+] )

c1- ‘ Cl'
alkalinization V mm mm \

(332+ arm; C a”

 

 

 

 

Ca" channell j [intracellular stores]

,.
o
0'-
,.
a
a
'0
,0,"

'-

Light": — >C

photorecepto '

  
    
    
 

 

 

H+<

 

H’~ATPase

Figure 9. A hypothetical model for light-induced ion ﬂux response

in pea mesophyll cells

122

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127

Chapter III

Effects of light intensity and quality on ion ﬂux from pea

mesophyll tissues.

128

ABSTRACT

Mesophyll tissues from excised leaﬂet of pea (Pisum sativum L. cv. Argenteum) were
exposed to different light intensity treatments. Five ﬂuence rates (225, 450, 900, 1800
and 2800 pmol m"2 s") were used to test ﬂuence-response of ion ﬂux. Light-induced
changes in net Ca2+, H, K“ and Cl' ﬂux increased in a saturable manner with increasing
ﬂuence. However, high ﬂuence increased the temperature of the bathing solution, which
may have caues a heat effect on ion ﬂux. White light (WL) of 450 pmol m'2 s'1
stimulated signiﬁcant changes in net ﬂux of net Ca2+, Hi, K+ and Cl' from mesophyll
tissues. When leaf tissues were exposed to the blue-light (BL) and red-light (RL) of
similar intensity, the change in net Ca2+, IT, K+ and Cl‘ ﬂux exhibited a similar pattern to
WL. At an intensity of 225 nmol m'2 s", the RL-induced initial increase in net Kl inﬂux
was more rapid than under BL. However, there were no signiﬁcant differences in the
magnitude of change in net Ca2+, Hi, Ki and Cl' ﬂuxes between BL and RL. The BL~ and
RL-induced ﬂux responses of all ions were saturated at intensities higher than 16 pmol m‘
2 s‘1 (BL) and 75 pmol m'2 5’1 (RL), respectively. The result suggests that BL and RL play

a similar role in rapid ionic responses to light.

INTRODUCTION

Light provides plants with many different kinds of information, as a result of variations in
light ﬂuence, quantity, direction and duration. Plants sense light signals through a
complex sensory system consisting of different photoreceptors, which detect different
light qualities over a wide spectral range, but with overlapping action spectra

(Batschauer, 1998). Light energy is then used either directly or indirectly to optimize a

129

variety of physiological and morphological processes. Recently much attention has
focused on photosensory mechanisms in plants and several light signalling pathways have
been proposed (Chamovitz and Deng, 1996; Batschauer, 1998; Khurana et al., 1998).
The most important aspect of understanding the regulation of photosensory mechanisms
is to elucidate the function of photoreceptors. At least three classes of photosensory
pigments (photoreceptors) have been implicated in the perception of light signals in
plants, including phytochromes, blue/UV-A photoreceptors, and UV-B photoreceptors.
The multiple photoreceptor classes imply the presence of a highly responsive, intricate,
and yet ﬁnely tuned, cellular signal transduction machinery (Batschauer, 1998).
However, the chemical structure and function of receptors are not fully understood.

Early events in the transduction of light-mediated signals in plants are often
associated with transient changes of membrane potential. This bioelectrogensis reﬂects
changes in ion ﬂuxes across the plasma membrane. Light-induced rapid ion ﬂux changes
have been demonstrated in many plant species, such as Dunalliella acidophila (Remis et
al., 1994), Physcomitrella patens (Johannes et al., 1997), and Vicia fava (Shabala and
Newman, 1999). Both red and blue lights have been reported to elicit electrical signalling
or ionic ﬂux changes, especially for guard cells (Serrano and Zeiger, 1989; Assmann and
Shimazaki, 1999). Red light matching the absorption spectrum of chlorophyll, stimulates
an outward electrical current that may be canied by the plasma membrane Hi-ATPase
(Serrano et al., 1988). Blue light given in a single pulse stimulates stomatal opening
(Zeiger et al., 1987) and induces proton extrusion from guard cell protoplasts (Assmann
et al., 1985; Shimazaki et al., 1993). In addition, since an anion channel in Arabidopsis

hypocotyl tissue was shown to be activated by BL (Cho and Spalding, 1996), it is likely

130

that BL would affect similar anion channels in guard cells. Shacklock et al. (1992)
demonstrated that RL induces protoplast swelling and causes a transient Ca2+ increase
followed by a rapid reduction in cytoplasmic Ca2+ concentrations to below resting levels.
The BL-induced depolarization in etiolated cucumber hypocotyls involves the inhibition
of a proton-pumping ATPase and an inﬂux of Ca2+ (Spalding and Cosgrove, 1992).
However, the reports on pulvinar movements are complicated by the fact that
phytochromes, blue-light photoreceptors, and underlying circadian rhythms all play roles
in regulating turgor changes (Briggs and Liscum, 1997). Recently, Suh et al. (2000)
characterized BL activated K“ efﬂux channels in ﬂexor cells ﬁom Samanea saman. Lew
et a1. (1990) also had reported phytochrome-activated K+ channels in Mougeotis
protoplasts.

Clearly, light elicits transient electrical signalling which is mediated by ion ﬂuxes
across the plasma membranes of plant cells. However, the identity of the light quality
which plays a main role in regulating ion ﬂux remains to be investigated. In previous
reports, the light-induced rapid ﬂux changes of Ca2+, IT, K+ and Cl' had been
demonstrated in pea mesophyll. Some speciﬁc ion transporters were proposed to be
involved in the light~induced ionic ﬂux responses (See Chapter II). Based upon the
differential reports above, we hypothesize that either BL or RL, or both, may be involved
in the regulation of light—induced ion ﬂux responses. In addition, light intensity would be
expected to inﬂuence ion ﬂux changes in pea mesophyll cells. To test our hypotheses,
BL and RL, were used to evaluate the effects of light quality on ion ﬂuxes. Different

ﬂuence treatments were also imposed to determine ionic responses.

131

MATERIALS AND METHODS

Plant materials

Seeds of pea (Pisum sativum L. cv. Argenteum) were germinated for 4-5 d. Plants then
were transferred to a growth chamber with a 16 h photoperiod, day/night temperatures of
20/25 °C, 75% relative humidity, and a ﬂuence of 200 pmol m'2 s" at the top of the
canopy. The pea seedlings were cultured using modiﬁed Hoagland’s solution (Johnson et
al., 1957), gradually increasing the concentration from 0 to 1A to ‘/2 strength at 4-5 d

intervals.

Flux measurements

The ion ﬂuxes and extracellular ion concentrations at the surface of the leaf mesophyll
were measured using the MIFE system. Technical details of this system are similar to
that generally as described in Chapter I and by Shabala et al. (1997). From time course
data obtained by the MIFE system, the directionality and rates of ion ﬂux could be
estimated. Additional information on the MIFE is available at
http://www.phys.utas.edu.au/ physics/biophys. In brief, micropipettes with a tip diameter
of 2 - 3 urn were pulled ﬁ'om 1.5 mm-diameter borosilicate glass capillaries (catalog no.
MIB ISO-6; World Precision Instruments, Inc., Sarasota, FL) with a horizontal electrode
puller (PC-10, Narishige, Tokyo, Japan). After silanization, cooled microelectrodes were
back-ﬁlled with 500 mM CaCl2 for Ca“, 500 mM KCI for K” and Cl‘, and 15 mM NaCl
plus 40 mM KHZPO, (adjusted to pH 6.0 with NaOH) for H“. Electrode tips were then
front-ﬁlled with a liquid-membrane-type of the appropriate ion-selective sensor, Hi (no.

95291), Ca2+ (no. 21048), K” (no. 60031) and Cl' (no. 24902) cocktails (all from Fluka,

132

Milwaukee, WI), until a 50- to 80- um long column of ionophore was produced.
Microelectrodes were calibrated for each ion using a known set of standards before and
after use. The Ag/AgCl reference electrode was prepared using a plastic tube containing
1000 mM KCl in 2% agar.

After calibration, the ion-selective electrodes were slowly vibrated by a
micromanipulator which provided three-dimensional positioning. The microelectrode/
micromanipulator set-up was mounted on the stage of an inverted microscope (Leitz,
Wetzlar, Germany). A measuring chamber with leaf sample was placed on a holder that
was connected to a three-way hydraulic micromanipulator (WR 88, Narishige, Tokyo,
Japan) driven by a computer-controlled stepper motor (MO6l-CE08, Superior Electric,
Bristol, CT). The MIFE system allows measurement of three ions simultaneously. Three
microelectrodes can be vibrated at any angle in a two-dimensional plane by separately
controlling the amplitude of the slow vibration of the micromanipulator.

The MIFE system measured the voltage differences at the two extreme positions of
vibration, which provided the basis for estimation of both the rate and directionality of
net ion ﬂuxes. In this study the electrodes were moved in a 20-s square wave cycle
between 50 to 90 um above the leaf surface at a low ﬁequency (0.1 Hz). Ion
concentration of ions was calculated from the voltage potentials at each position using the
calculation curve for the speciﬁc microelectrode. The net ﬂux for each speciﬁc ion was
estimated ﬁom the measurements of the difference in the electrochemical potential
between these positions (Shabala et al., 1997). The ﬁrst 4 s of each half cycle (10 s) were
not used for concentration and ﬂux estimation. This was considered to be the minimum

delay time necessary to obtain a stable measurement following movement of the

133

microelectrodes. Instability was thought to be caused by solution turbulence and physical

vibrational noise following movement.

Light sources
Light sources were provided by a ﬁber-optic light projector (CUDA Products
Corporation, Jacksonville, FL) with a 250 W halogen bulb (GS Edmumd Scientiﬁc
Company, Barrington, NJ). A lens (no L~16-16, World Precision Instruments, Inc.,
Sarasota, FL) was fastened on the top of a light guide to focus the beam. The blue light
(BL) and red light (RL) treatments were achieved by a blue glass ﬁlter (5-60, thickness
4.5 mm, Kopp Glass Inc., Pittsburgh, PA) and a red glass ﬁlter (2-61, thickness 4.5 mm,
Kopp Glass Inc., Pittsburgh, PA), respectively, which were placed in front of the optics in
the lens. The spectra of white-, blue- and red-light sources were determined using Li-
1800 spectroradiometer (LI-COR, Lincoln, NE). The white light delivered a broad
spectra between 400-1000 nm (Figure 1A). However, most of light lies between 500-800
nm. The long wavelength (above 800 nm) ﬂuence was decreased by 45-47 % after
ﬁltering with distilled water (Figure 1). The blue source emitted light of wavelengths
between 375-525 nm with peak emission at around 450 nm (Figure l). The output of the
red source included light of wavelengths between 610-1000 nm. The peak of light
emission was between 650-740 nm (Figure 1). The blue— and red-light ﬂuence rates were
measured using a quantum sensor (model LI-189, LI-COR, Lincoln, NE).

In this study, White-light (WL) of 450 pmol m’2 s“ was used as light treatment. When
the WL was ﬁltered by either the blue- or red-glass, the BL of 32 pmol m'2 s'1 and the RL

of 225 pmol m'2 s'1 were generated, respectively.

134

 

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A

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Figure 1. Fluence of white, blue and red lights over a range

of wavelengths.

Wavelength (nm)

135

Experimental protocol

Young expanding leaﬂets were harvested and a leaf section with limited veinal tissue of
approximately 15 x 10 mm (LxW) was surgically cut from the lamina. A small strip of
epidermal tissue was peeled from the abaxial surface of the lamina of each section so as
to avoid incurring mechanical damage to the underlying veinal and mesophyll tissue
(Long and Widders, 1990). Isolated leaf segments were incubated in a buffered solution
(0.5 mM CaSO,, 1.0 mM KCI, 2.5 mM HEPES-HCl, and 10 mM sucrose at pH 5.2)
under 60 W rn‘2 illumination (Philips lamp). After 2.0-2.5 h, the cut segment was
mounted in a measuring chamber. The measuring chamber contained about 6-7 mL of
unbuffered solution or bathing solution (0.5 mM CaSO,, 1.0 mM KCI, pH 5.2), and the
segments then were exposed to 50 - 60 min of darlmess before light treatment.

All experiments were conducted in unbuffered solution at room temperature
(23:1°C). Flux measurement was continued for an average of 30 min from the onset of

illumination and then following the onset of darkness for 10-15 min.

Solution temperature

The temperatures of the solutions during illumination were measured using a
thermocouple thermometer (Model 600-2050, Barnant Company, Barrington, IL). To
increase the solution temperature, a warmer bathing solution was expelled into the
bathing solution (23i1°C) and then repeatedly sucked and expelled with a mL pipette for
5 - 6 times. The period of time during which warmer solution had been added, mixed and

equilibrated was ignored when analyzing ionic concentration and ﬂux data. This

136

represented about 3-4 min total. Change of temperature of the bathing solution could be
maintained within a range of 1-2 °C during 15-20 min measurement. All solutions were

unbuffered (0.5 mM CaSO,, 1.0 mM KCl, pH 5.2).

Data statistical analysis

Data analysis has been described in Chapter 1. Analysis of variance (ANOVA) of the

data was conducted using SAS 8 (SAS Institute Inc, Cary, NC).

RESULTS

Effects of light intensity on ion ﬂuxes

Since not ﬂux is a balance between inﬂux and efﬂux, a positive net inﬂux response is the
result of enhanced inﬂux or reduced efﬂux of the ion in question. In this study, ﬁve
ﬂuence rates of White-light (WL) (225, 450, 900, 1800 and 2800 umol m‘2 s") was used
to test the effects of light intensity. Under different light intensities, the net ﬂuxes of
Ca2+, H, K and Cl' around pea mesophyll exhibited a similar response trend, i.e. a light-
induced initial response in net ion inﬂux (more positive net ﬂux) followed by a Post-
Initial Flux Response (PIF R) (see Chapter I). The PIF R could theoretically involve either
independent changes or concurrent changes in both inﬂux and efﬂux. Therefore, either a
decrease in inﬂux or an increase in efﬂux or both can elicit a negative PIFR. In general,
the light-induced initial responses in net inﬂux of all ions were the smallest at the lowest
intensity (225 pmol m'2 s"). Further increase in light intensity did not produce a
signiﬁcant difference in the responses of net H+ and Cl’ inﬂux between intensities from

450 to 2800 umol m'zs" (Figure 2). Net Ca2+ and K+ ﬂuxes exhibited saturation kinetics

137

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in the initial inﬂux response at light intensities above 900 pmol m'2 s". The light-induced
PIFRs of W, Ca2+ and K+ were saturated above 450 pmol rn’2 s". In contrast, high light

intensities (1800 - 2800 pmol m’2 5") enhanced dramatically the amplitude of PIFR of Cl'

(Figure 2).

Effects of solution temperature on ion ﬂuxes

When the temperature of the bathing solution increased by 1.0-1.5 °C or 1.5-2.5 °C, the
net ﬂuxes of Ca2+, Hi, K1 and Cl‘ from pea mesophyll did not change signiﬁcantly in
darkness during 15-20 min of measurement (Figure 3). A 30 min illumination of 225 and
450 pmol m2 5'1 generated an increase in solution temperature of 0.6 °C and 1.3 °C,
respectively (Figure 4). This indicates that the heat effect of these two light intensities is
not consequential to ion ﬂux. However, when light intensity was further increased to
900, 1800 and 2800 pmol m'2 s", the temperature of the bathing solution was enhanced
by 2.6, 3.8 and 5.0 °C with 20 - 30 min, respectively (Figure 4). At 2800 umol m'2 s", in
particular, the increase of temperature was quite rapid. Red-light at intensities of 450,
1170 and 1490 pmol rn'2 s'l also heated up the bathing solution by 1.1, 2.1 and 3.2 °C
within 30 min, respectively (Figure 4). The highest ﬂuence of red-light (1490 pmol m'2 s’
I) produced a rapid increase in solution temperature. A large ﬂux change for Ca”, Hi, K+
and Cl' was observed when the temperature was increased by 3 - 4 or 4 - 5 °C in darkness
within 15-20 min (Figure 3). Therefore, high ﬂuence is expected to cause heat effects on

ion ﬂuxes during experiments of 30 min.

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solution. Data points represent an average of 3 replicates.

141

Effects of blue-light on ion ﬂuxes

Like WL, a BL exposure of 32 pmol m‘2 8" induced an initial response in net Hi, Ca2+, K+
and Cl' inﬂuxes (Figure 5). This was followed by Post-Initial Flux Responses (PIFRs) of
Hi, Ca2+, K+ and C1. The net ﬂuxes of all ions decreased to a net efﬂux (negative value)
or returned to the original value before the BL treatment when BL was turned off (Figure
5). Compared with the WL of 450 pmol m'2 s", the magnitude of BL-induced initial
response in net inﬂux of H+ and Ca2+ was signiﬁcantly smaller (Table 1). The BL-
induced changes in net H+ ﬂux during the ﬁrst min declined as compared to WL. In
particular, the BL-induced change in net K+ ﬂux was much smaller than WL-induced
during the ﬁrst 5 min (Figure 5). The BL-induced PIFRs for Ca”, K” and Cl’ decreased
signiﬁcantly, especially for K” and Cl' which were 58 % and 74 % lower than the WL-
induced PIFR, respectively. During the ﬁrst 5 min, the ﬂux change of Cl' was positive,
i.e., BL caused a signiﬁcant increase in net Cl' ﬂux. In contrast, WL caused a decrease in
net Cl' ﬂux (Figure 5). The BL-induced initial responses of net K+ and Cl‘ ﬂux were
approximately 3-4 min and 2-3 min slower, respectively, than the WL-induced initial
response (Table 1).

To examine the BL-ﬂuence-response relationships of ion ﬂux, ﬁve BL intensities
were used: 16, 32, 70, 160, and 225 pmol m'2 3". These BL treatments elicited a similar
ion ﬂux response as shown in Figure 5. The initial response of net ion inﬂux was the
smallest under the low BL intensity (16 pmol m'2 s" ) for all ions (Ca2+, Hi, K+ and Cl')
(Figure 6). Above 32 pmol m“2 s", the BL-induced initial response in net 11‘, K+ and Cl‘
inﬂux increased and exhibited light saturation kinetics. For Ca2+, higher BL intensities

(above 70 pmol m‘2 5") generated larger initial response in net inﬂux (Figure 6). With

142

 

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increasing intensity, the BL-induced PIFR of the four ions increased but was saturated
above 70 pmol m'2 s‘1 (for H+ and K) or above 160 pmol m‘2 s" (for Ca2+ and Cl') (Figure

6).

Effects of red-light on ion ﬂuxes

Red-light of 225 pmol m'2 3'1 produced similar net ﬂux responses for H, Ca2+, K+ and Cl‘
as observed following WL or BL exposure. A return to dark conditions caused a decrease
in net ion ﬂux (negative) similar to WL and BL (Figure 5). Compared with the WL of
450 nmol m'2 s", the initial net inﬂux responses of H", Ca2+, K” and Cl’ were not
signiﬁcantly inﬂuenced by RL of 225 nmol m’2 5’1 (Table 1). However, the RL-induced
PIFRs of K and Cl’ were signiﬁcantly lower than WL (Table 1). In most cases, there
were no signiﬁcant differences between RL- and BL—effects. The signiﬁcant difference
was that RL induced a rapid net K” inﬂux (75 and 38 nmol rn’2 s", respectively) which
was larger than BL (2 and 8 nmol m‘2 s", respectively) during the ﬁrst min and during the
ﬁrst 5 min after illumination (Figure 5). In addition, RL caused a decrease in net Cl' ﬂux
(~15 nmol m'2 5") whereas BL still increased net Cl’ ﬂux (+26 nmol m’2 8") during the
ﬁrst 5 min of illumination (Figure 5). The initial RL response time for C1’ (3.0 min) was
signiﬁcantly shorter than BL (5.5 min) (Table 1).

Five RL intensities, 75, 225, 450, 1170 and 1490 pmol m'2 s", were tested to evaluate
the RL ﬂuence-response on ion ﬂux. The highest two intensities caused an increase in
solution temperature of approximately 3-4 and 4-5 °C with in 20 - 30 min (Figure 4).
Therefore, these RL exposures would be expected to generate heat effects on ion ﬂux.

However, low ﬂuence (below 450 pmol m'2 8") did not cause signiﬁcant heating of the

145

Table 1. Effects of white-, blue- and red-light on net ﬂuxes of H+, Cu“, K" and Cl'
from pea mesophyll tissues.

 

Hi Ca2+ K+ Cl’
Initial ﬂux response (inﬂux) (nmol m'2 s")
White light +*22 (8)5a +44 (8)a +142 (10) +124 (8)
Blue-light +14(8) b +35 (8)b +118 (8) +93 (8)
Red-light +19(8) ab +41 (8) ab +145 (8)ns +80 (8) ns

Amplitude of PIFRI (nmol m’2 s")
White light -17 (8) -59 (8) a -l96 (10)a -161 (8) a
Blue-light -11 (8) ~41 (8) b -82 (8) b -39 (8) b
Red-light -19 (8) ns -45 (8) ab -111 (8) b -85 (8) c
Initial light response time (min)
White light 4.1 (8) 2.4 (8) 4.3 (10) b 2.8 (8) b
Blue-light 4.3 (8) 3.6 (8) 8.4 (8) a 5.5 (8) a

Red-light 3.0(8)ns 4.0(8)ns 2.5 (8) b 3.0(8)b

 

Different letters indicate signiﬁcant difference between treatments within columns at p < 0.05 according to
LSD test.
“ns” indicates no signiﬁcant difference.

White-light ﬂuence is 450umol m’2 5". Blue— and red-light ﬂuences are 32 and 225 pmol m'2 s",
respectively, as a result of ﬁltering the white light.

5 The numbers in parentheses indicate the number of replicates for each treatment.

' - or + indicates decrease or increase of net ion ﬂux and extracellular ion concentration, respectively.

3 PIFR: Light-induced Post-Initial Flux Response, a result of either a decrease in net inﬂux or an increase
in net efﬂux, or both. This value is negative.

146

solution. Like WL, all RL intensities exhibited a similar response in net ion ﬂuxes as
shown in Figure 5. There were no signiﬁcant differences of the initial response of net Hi,
Ca2+, and Cl' inﬂux among all RL intensities except at the lowest (75 pmol m‘2 s“)
(Figure 7). The RL-induced initial response of net K+ inﬂux was intensity-dependent
below 450 pmol m‘2 s‘1 but became saturated when RL intensity was further increased.
Higher RL ﬂuence rates (1170 and 1490 pmol m‘2 s") also enhanced the initial H+ inﬂux
response (Figure 7). This again was possibly due to heat effects. The RL-induced PIF R
by the four ions increased in magnitude but became saturated above 225 pmol 111'2 s" for
H”, Ca2+ and K”. However, the higher ﬂuence (1490 pmol m'2 5") caused a large increase

in the PIFR of Cl' which is also thought to be due to the heating effects (Figure 7).

Comparison between blue- and red-light effects on ion ﬂuxes

Comparison was conduced when all lights were a similar ﬂuence of 225 pmol m'2 s".
Blue-light produced a smaller change in net Ca2+ ﬂux than WL did during the ﬁrst min
(Table 2a). The PFIR of H+ induced by both BL and RL were signiﬁcantly larger than
the WL-induced PIFR. However, no signiﬁcant differences in the initial response of net
H” inﬂux were observed between the three lights. In the same manner, the WL, BL and
RL did also not produce a signiﬁcant difference in either the light-induced initial ﬂux

response or the light-induced PIFR of Cl' and K (Table 2a).

When both BL and RL intensities were reduced to 70-75 pmol m'2 s", no signiﬁcant differences of ionic
light response were observed between BL and RL. Both BL and RL

147

 

 

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148

Table 2. Effects of white-, blue- and red-light on ion ﬂux rate in pea mesophyll

tissues at similar light ﬂuences.

a: at 225 pmol m" s"

 

 

 

 

 

 

 

If Ca: K+ Cl‘
(nmol m“2 5")

Initial ﬂux response (inﬂux)

White light +ll4(5)§ +31 (5) +71 (3) +27 (3)

Blue-light +17 (5) +26 (3) +89 (4) +95 (4)

Red-light +21 (3) us +41 (4) ns +113 (4) ns +106 (4) ns
Flux change during the ﬁrst min

White light +4 (5) +24 (5) a +42 (3) +3 (3)

Blue-light +11 (5) -8 (3) b +5 (4) +12 (4)

Red-light +2 (5) ns +5 (4) ab +92 (4) ns +14 (5) ns
Amplitude of PIFRt

White light -6 (5) b -33 (5) -74 (3) -73 (3)

Blue-light -22 (3) a -45 (3) -124 (4) -61 (2)

Red-light -16 (5) a -43 (4) ns -1 l4 (4) ns -56 (3) ns
1): at 70-75 pmol m'2 s"

H Ca2+ K+ Cl'
(nmol m‘2 3")

Initial ﬂux response (inﬂux)

Blue-light +14 (4) +21(4) +115 (4) +83 (3)

Red-light +16 (4) ns +31(5) ns +84 (4) ns +42 (4) ns
Amplitude of PIFR

Blue-light -18 (4) -34 (4) -96 (4) -25 (2)

Red-light -11 (4) ns -31 (5) ns -63 (4) ns -43 (3) ns

 

Different letters indicate signiﬁcant difference between treatments within columns at p < 0.05 according to

LSD test.
“ns” indicates no signiﬁcant difference.

§ The numbers in parentheses indicate the number of replicates.
I - or + indicates decrease or increase of net ion ﬂux and extracellular ion concentration, respectively.
: PIFR: Light-induced Post-Initial Flux Response, a result of either a decrease in net inﬂux or an increase

in net efﬂux, or both. This value is negative.

149

produced similar initial ﬂux responses or amplitudes of PIFR of W, Ca2+, K+ and Cl

(Table 2b).

DISCUSSION
Effects of light intensity
Previous studies on light-induced ionic or electrical responses in plants have used an
intermediate ﬂuence within the range of 200 - 400 pmol In2 5". Only a few studies have
evaluated light responses to different light intensities. Elzenga et al. (1995) found that
light-induced depolarization in pea mesophyll tissues depended, in a saturable way, on
light intensities between 50-1000 pmol m'2 5". Lower intensities were observed to result
in less depolarization of the plasma membrane and a delay in the light response. The
light-induced polar pH reaction in Elodea canadensis was also found to be reduced at
lower light intensities (Elzenga and Prins, 1989). In the current study, the light responses
in the net ﬂuxes of Ca2+, H”, K+ and Cl' were limited at the lowest WL ﬂuence (225 pmol
rn’2 s" ) (Figure 2). Although higher ﬂuence (450 - 1800 pmol m‘2 5") produced a higher
magnitude of net inﬂux changes for all ions, that of net H” and Cl' inﬂux appeared to
saturate. Under 450 pmol m'2 s", the light-induced PIFR of Ca2+, H“ and K+ also were
saturated (Figure 2). Elzenga et al. (1995) also reported that light-induced depolarization
of pea mesophyll was saturated at approximately 400 pmol m'2 5".

However, the increase in solution temperature during light exposure constitutes a
potential problem in measurement of ion ﬂux at high ﬂuence. Both factors, light

intensity and solution temperature, should be considered when one evaluates the effect of

150

light on ion ﬂux. Most researchers ignore or do not consider the li ght-induced heat
effects on leaf tissues at lower radiance levels (Stahlberg and Van Volkenburgh, 1999,
Shabala and Newman, 1999). High ﬂuence was found to increase bathing solution
temperature by approximately 4 - 5 °C within 20 min (Figure 4). Therefore, temperature
effects caused by high ﬂuence on ion ﬂux must be considered and calculated. Bathing
solution temperature increase of 3 - 5 °C clearly increase net ﬂux kinetics of Ca“, H, K*
and Cl’ under darkness. By contrast, smaller increases in temperature (1.0 - 2.5 °C) did
not affect net ion ﬂux in a measurable manner (Figure 3). Therefore, high ﬂuence-
induced changes of ion ﬂuxes could be expected to result ﬁom two factors, light intensity
and heat. In other words, ion ﬂux changes induced by high intensities (1800 and 2800
pmol m‘2 3") should not be only explained on the basis of light ﬂuence, but also include
possible heat effects. Temperature directly affects the kinetics of ion transport across the
membranes in biological systems. For example, Lee and Satter (1988) found that
temperature had a large inﬂuence on H+ release ﬁ'om extensor cells of Samanea saman
pulvini. In this study, it is difﬁcult to differentiate between light- and temperature-effects
at high ﬂuence. The present work indicates that radiance of 450 of nmol m'2 s'1 is enough

to stimulate ionic responses to light.

Effects of light quality
Light (WL) induced a rapid increase in net H+ inﬂux from pea mesophyll tissues (see
Chapter 1). Both RL and BL also elicited a similar time-course change of net Hi (Figure

5). At the same ﬂuence, no signiﬁcant differences in the initial response in net H+ inﬂux

151

was observed among the three light treatments (Table 2). However, the BL- and RL-
induced PIFR for H were signiﬁcantly larger than that of WL- at 225 pmol m'2 5’1 (Table
2a). As discussed in Chapter H, the plasma membrane Hi-ATPase may contribute to the
light-induced PIFR of H+ causing apoplastic acidiﬁcation due to increased proton efﬂux
from mesophyll cells. Both lights stimulate proton efﬂux (Assmann et al., 1985; Serrano
et al., 1988). However, several contradictory opinions exist in the literature. Nishizaki
(1992) found that BL and RL elicited membrane depolarization and hyperpolarization,
respectively. He proposed that a pulse of BL inhibited the activity of the proton-ATPase
in pulvinar motor cells. Spalding and Cosgrove (1992), on the other hand, suggested that
the mechanism of BL-induced membrane depolarization of cucumber hypocotyls initially
involves inhibition of a HI-ATPase. This study provides indirect evidence for the role of
both BL and RL in stimulating proton efﬂux. Additionally, the ﬂuence-responses to both
BL and RL were similar for the initial response of net HI inﬂux (Figure 6, 7).

It is well established that light induces a transient increase in net Ca2+ inﬂux. Like Hi,
the effects of both BL and RL on net Ca2+ ﬂux were similar to that of WL (Figure 5).
Elevated concentrations of cytosolic Ca2+ were found in RL- (Shacklock et al., 1992;
Neuhaus et al., 1993; Ermolayeva et al., 1996) and BL-treated cells (Christie and Jenkins,
1996; Sharma et al., 1997). The role of Ca2+ as a cellular messenger, has been suggested
to play a role in both BL and RL signal transduction. However, evidence for a direct link
between Ca2+ transport and light quality is still lacking. The present results support the
hypothesis that both light qualities might initially increase net Ca2+ inﬂux. The most
likely pathway for Ca2+ inﬂux into cytoplasm would be through Ca2+ channels in the

plasma membrane (see Chapter II) resulting in a transient increase in cytosolic free Ca”

152

concentration.

Both BL and RL stimulated K+ uptake by guard cells (MacRobbie, 1988) and by bean
mesophyll (Blum et al., 1992). In pea mesophyll, RL induced a rapid and large increase
in net K+ inﬂux from pea mesophyll (Figure 5). In comparison, the BL-induced change
in net K+ inﬂux was slower and smaller during the ﬁrst min of illumination. However,
such result was not observed at low RL ﬂuence of 75 pmol rn‘2 5'1 (Table 2b). Similarly,
no difference between both BL and RL existed in the magnitude of the initial response in
net K+ inﬂux (Table 2). These differences are mainly reﬂected in the initial light
response time. Furthermore, the BL- and RL-induced PIFRs of K+ were also similar
(Table 2). As proposed in Chapter II, the light-induced PIFR of K+ ﬂux change may be
associated with a enhanced net KI efﬂux mediated by K+ channels. Both light qualities
were found to stimulate KI channel activity. For example, BL activated K” efﬂux
channels in ﬂexor cells of Samanea saman (Suh et al. 2000), whereas phytochrome
activated K+ channels in Mougeotis protoplasts (Lew et al., 1990). Subsequent research
demonstrated that one or two Ca2I-activated K+ channels were involved (Lew et al.,
1992). According to the present results in pea mesophyll, BL and RL should be
considered to play a role in the light-induced K+ ﬂux response in a similar manner.

Blue-light-activated anion channels have been identiﬁed in leaves of Arabidopsis
(Cho and Spalding, 1996; Lewis et al., 1997). These authors speculated that Cl‘ efﬂux,
mediated by anion channels, is responsible for BL-induced membrane depolarization.
However, the activity of anion channels does not explain fully the change of Cl' ﬂux

across the plasma membrane. Except efﬂux through anion channels, Cl' can move

153

 

inwardly into cytoplasm through a Hi/Cl‘ symport mechanism (Sanders et al., 1985). The
net ﬂux reﬂects a balance between inﬂux and efﬂux across the plasma membrane in pea
mesophyll. In the present study, both BL and RL stimulated similar changes in net Cl'
inﬂux and the PIFR of Cl' at the same ﬂuence (Table 2). The light-induced PIFR of Cl“
has been considered to be the result of Cl‘ efﬂux catalyzed by anion channels within the
plasma membrane (see Chapter 11).

Taken together the present data suggest that both BL and RL all induced ﬂux
responses of H’“, Ca2+, K+ and CI' in a similar manner. The same phenomenon was also
found in effects of BL and RL on the activity of ion transport across the plasma
membrane of guard cells (Assmann et al., 1985; Zeiger et al., 1987; Serranno et al., 1988;
Shimazaki et al., 1993). This indicates that BL + RL regions of the light spectrum can
elicit ionic ﬂux responses in pea mesophyll tissues. The similarity in ionic responses to
BL and RL are consistent with the conclusion in Chapter I that ion ﬂux responses in pea
mesophyll are photosynthesis dependent. This is supported by the ﬁnding that DCMU, a
photosynthestic inhibitor, also inhibited the light-induced initial responses in net ion ﬂux
(see Chapter I) and removed light-induced membrane depolarization (Stahlberg and Van
Volkenburgh, 1999) in pea mesophyll. The current study provides a starting point for

further investigations on potential involvement of photoreceptor.

154

 

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Assmann SM, Shmazaki K. 1999. The multisensory guard cell: Stomatal response to
blue light and abscisic acid. Plant Physiol 119:809-815.

Assmann SM, Simoncini L, Schroeder JI. 1985. Blue light activates electrogenic ion
pumping in guard cell protoplasts of Viciafava. Nature 318:285-287.

Batchauer A. 1998. Photoreceptors of higher plants. Planta 206:479-492.

Blum DE, Elzenga JTM, Linnemeyer PA, Van Volkenburgh E. 1992. Stimulation of
grth and ion uptake in bean leaves by red and blue light. Plant Physiol 100:1968-
1975.

Briggs WR, Liscum E. 1997. Blue light-activated signal transduction in higher plants.
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Chamovitz DA, Deng XW. 1996. Light signaling in plants. Crit Rev Plant Sci 15:455-
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Cho MH, Spalding EP. 1996. An action potential in Arabidopsis hypocotyls activated
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Elzenga JTM, Prins HBA. 1989. Light-induced polar pH changes in leaves of Elodea
canadensis. 1. effects of carbon concentration and light intensity. Plant Physiol 91 :62-67

Elzenga JTM, Prins HBA, Van Volkenburgh E. 1995. Light-induced membrane
potential changes of epidermal and mesophyll cells in growing leaves of Pisum sativum.
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Ermolayeva IE, Hohmeyer H, Johannes E, Sanders D. 1996. Calcium—depend membrane
depolarization activated by phytochrome in the moss Physcomitrella patens. Planta
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Johannes E, Ermolayeva E, Sanders D. 1997. Red-light-induced membrane potential
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Johnson CM, Stout PR, Broyer TC, Carton AB. 1957. Comparative chlorine
requirements of different plant species. Plant and Soil 8:337-353.

Lee Y, Satter RL. 1988. Effects of temperature on H+ uptake and release during

circadian rhythmic movements of excised Samanea motor organs. Plant Physiol 86:352-
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Lew RL, Serlin BS, Schauf CL, Stockton ME. 1990. Calcium activation of Mougeotia
potassium channels. Plant Physiol 92:831-836.

Lewis BD, Karlin-Neumann G, Davis RW and Spalding EP. 1997. Cay-activated anion
channels and membrane depolarization induced by blue light and cold in Arabidopsis
seedlings. Plant Physiol 114:1327-1334.

Long JM, Widders IE. 1990. Quantiﬁcation of apoplastic potassium content by elution
analysis of leaf lamina tissue from pea (Pisum sativum L. cv. Argenteum). Plant Physiol
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MacRobbie EAC. 1988. Control ion ﬂux in stomatal guard cells. Bot Acta 101:140-
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potential in pulvinar motor cells of Phaseolus vulgaria L. Plant Cell Physiol 33:1073-
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Remis D, Treffny B, Gimmler H. 1994. Light-induced H+ transport across the plasma
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mechanism of enhanced inﬂux after rapid external acidiﬁcation. Planta 163:411-418.

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Spalding EP, Cosgrove DJ. 1992. Mechanism of blue-light-induced plasma membrane
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function. Stanford University Press, Stanford, pp 209-227.

157

SUMMARY

Light signalling is becoming a main area of research in plant science. Plant scientists try
to elucidate photosensory mechanisms through genetic, biochemical and biophysical
approaches. Membrane potential changes are commonly observed responses to many
stimuli. However, the ionic basis for such stimulus-elcited membrane potential changes
remains to be determined. Ion transport mechanisms and kinetics are frequently not
understood. It is known that ion activity gradients and ﬂuxes exist across cellular
membranes, and this fact provides a potential target for studying electrical signalling.
Because electrical signals are short-lived, it is difﬁcult to use biochemical and molecular
techniques for investigation of such responses. In contrast, ion-microelectrode
technology can measure rapid ion kinetics in a time-course manner around plant cells
without tissue damage.

Microelectrode Ion Flux Estimation (MIFE) is a slowly vibrating ion-selective
microelectrode system developed by Shabala and Newman (Shabala et al., 1997; Shabala
and Newman, 1999). The MIFE has three advantages. First, it can measure the
simultaneous ﬂux of three ions, thus one can observe multiple ion movements across
mesophyll plasma membrane. Second, it provides a stable and anti-noise environment
during an experiment. In most cases, changes in extracellular ion concentration with are
consistent with changes in net ion ﬂux, the enabling the identiﬁcation and exclusion of

artifacts. Third, this system contains a soﬁware package to automatically record all

158

 

measurements and to estimate ﬂux. Therefore, the MIFE allows direct, dynamic and
continuous quantitative estimation of ion kinetics around cells.

To focus on mesophyll cells, a small strip of epidermal tissue was gently removed
from pea leaﬂets with little or no wounding. One cannot fully discount the possibility,
however, that peeling may elicit some type of long-term wound response which may pre-
dispose the tissue to responding to light in a certain manner and thus give rise to artifacts.
On the other hand, extensive research with peeled leaf lamina tissue indicates that ion
ﬂux kinetics is similar to intact tissue. The peeled mesophyll cells can easily be viewed
with an inverted microsc0pe. This conﬁguration ensures that measured ion ﬂuxes are
predominately from mesophyll cells.

Although Elzenga et al. (1995) observed a rapid light-induced membrane
depolarization (25 mV) in pea mesophyll cells, they did not provide further evidence for
the ionic basis of bioelectrogenesis. This dissertation demonstrates a signiﬁcant, short-
term, light-induced ion ﬂux response of pea mesophyll tissues. Rapid initial increases in
net Ca2+, H, K” and Cl' ﬂux from mesophyll cells were measured aﬁer onset of
illumination. The response of Ca2+ ﬂux was the most rapid (13 5), whereas HI, Cl‘ and K+
ﬂux responses started within approximately 29, 45, and 48 s, respectively, after
stimulation. Following the initial response, a light-induced Post-Initial Flux Response
(PIFR) occurred, which led to a decrease in net ﬂux. The PIFR time of Ca2+ and Cl' was
shorter (3 -4 min).

Ion transport across the plasma membrane can alter membrane potentials. Assuming
that Ca2+, HI, K+ and C1' are primary ions involved in light-induced changes in membrane

potential, this study showed that the time course of summed net charge ﬂux was similar

159

 

to that of the light-induced membrane potential reported previously (Elzenga et al., 1995;
Stahlberg and Van Volkenburgh, 1999). A light-induced initial increase in the net charge
ﬂux would be expected to depolarize the plasma membrane. Therefore, this dissertation
presents a effective and useﬁil method to estimate the involvement of ion ﬂuxes in
electrical signalling.

Calcium ﬁmctioning as a second messenger is thought to play a critical role in
cellular signal transduction in plant cells. An increase in free Ca2+ concentrations in the
cytoplasm occurs in most stimulus-responses. The present results support Ca2+ inﬂux as
an initial depolarizing agent in the light-induced membrane depolarization (Spalding et
al., 1992; Shabala and Newman, 1999). However, other researchers have not yet
provided experimental evidence for a Ca2+ inﬂux mechanism. This study showed that the
light-induced initial net Ca2+ inﬂux was signiﬁcantly inhibited by 42-44 % in the
presence of the Ca2+ channel blocker LaCl3 and the chelator EGTA. Apparently, the
light-induced initial response in net Ca2+ ﬂux is the result of an increase in inﬂux
mediated by Ca2+ channels, rather than a decrease in efﬂux. The increased Ca2+ inﬂux
would be expected to elevate cytoplasmic Ca2+ concentrations, thus initiating cellular
signal transduction. The effects of Ca2+ inﬂux on other ion ﬂux responses also provide
additional evidence for this hypothesis. Both LaCl3 and EGTA signiﬁcantly reduced the
light-induced initial H+ and K response and PIFR of C1‘. The light-induced PIFR of Cl'
were also signiﬁcantly enhanced by both Ca2+ ionophore A23187 and high exogenous
Ca2+ concentration. Therefore, this dissertation may be the ﬁrst to experimentally
demonstrate that Ca2+ inﬂux through Ca2+ channels represents an initial step in light-

induced membrane depolarization.

160

 

P.

1
~11...

A light-induced extracellular alkalinization (pH increase of 0.4 - 0.5) was observed in
the current study. The results were anticipated due to CO2 uptake and HI-mediated K+ or
Cl‘ uptake by mesophyll cells. Shabala and Newman (1999) also concluded that CO2
uptake of bean mesophyll cells was a reason of light-induced increase in apoplast pH.
However, the plasma membrane HI-ATPase inhibitors, Na3VO4 and DCCD, inhibited the
light-induced initial response in net H+ inﬂux by 50-60 % and eliminated the PIFR of H“.
The presence of Na,VO4 in the bathing solution also signiﬁcantly suppressed the light-
induced increase in net K+ inﬂux. In contrast, the ATPase activator, F usicoccin
signiﬁcantly increased the light-induced PIFR of H+ and the initial response in net Cl‘
inﬂux. In addition, external pH in the bathing solution affected ﬁtrther the light-induced
response in net KI and Cl' inﬂux. These results suggest that HI-ATPase in the plasma
membrane is not directly involved in the initial light response. The enzyme is involved
in mediating Hi/K+ and HI/Cl' symports, which contribute to light-induced initial
extracellular alkalinization.

Anion channels may also be involved in membrane potential changes. Elzenga and
Van Volkenburgh (1997) also reported increased activity of an anion channel in pea
mesophyll cells in response to light. However, they did not measure Cl' ﬂux, which was
a main reason for the light-induced PIFR of C1' in my study. Testing with anion channel
blockers indicated that rapid-type anion channels play a role in increasing Cl' efﬂux, but
anion channels are not involved in light-induced initial Cl‘ ﬂux response. This result is
different from that of Elzenga and Van Volkenburgh (1997).

Another contribution of this dissertation is the demonstration that the light-induced

PIFR is associated with ion transporters, including Ca2"-ATPase, HI-ATPase, K+ channels

161

and anion channels in the plasma membrane. Differential alteration in ion transporter
activity suggests that multiple ion transport pathways ﬁmction during short-term light
responses. To date, such ion ﬂux evidence has not been reported. Most research has just
focused on the effects of ion transporter inhibitors or activators on membrane potentials
or on ion concentration change in bulk solution surrounding plant tissues.

In this dissertation, I proposed a model for short-term light responses, based on
experimental evidence, and provided a possible explanation for bioelectrogenesis. Ion
ﬂux responses represent an important component of cellular signalling systems in plants
to external stimuli. This model also provides a potential approach for future research in
signal transduction of plants.

Both blue light and red light display a similar action in controlling stomotal
movement (Serranno and Zeiger, 1989; Assmann and Shimazaki et al., 1999). Similar
effects of both wavelengths on ion ﬂuxes in pea mesophyll cells were found in the
present study. The fact that DCMU treatment inhibited the light-induced H+ and Ca2+
ﬂux responses suggests that the ionic responses to light are photosynthesis-dependent.
The wide action spectra of the ion ﬂux response introduces a further question; namely
whether photoreceptors are ever involved in the light-induced ion ﬂux response?
Combining MIFE technology with speciﬁc mutants will enhance the possibility of
ascertaining the involvement of speciﬁc photoreceptors in the future research.

Although the MIFE system can estimate net ion ﬂux dynamics in the apoplast, the
technique does not directly measure intracellular ion concentration change. Some
attempts have been made to detect cytosolic ion concentrations and pH with improved

ion-selective microelectrode techniques, such as the double-barreled electrode and the

162

 

pH-electrode (Felle, 1993). In further studies, the MIFE system might be used to test
Ca2+ concentration and pH in the cytoplasm with improved microelectrodes.

The present study does not exclude the possibility of CaZI-independent mechanisms.
Hydrogen signalling, coupled with ion ﬂux changes may constitute an alternate
mechanism for cellular signal transduction. Further research is needed to test such a
hypothesis. A combination of both MIFE and patch-clamp techniques would provide
further discrimination among speciﬁc ion transporters, for instance, inward or outward K”
rectiﬁers and anion channels.

My data in this dissertation and studies by others (Shabala et al., 1997; Shabala and
Newman, 1999) indicate that, among the ion microelectrode techniques, the MIFE system
is a most powerﬁrl tool to measure ion ﬂux in plant cells. The ﬁirther improvement of the
MIF E system will open many new interesting avenues of research, including the
elucidation of signal transduction pathways in response to pathogen infection, salinity,
cold, wounding, etc. It would also be interesting to investigate localized ion ﬂux changes
in remote tissues of whole plants when stimuli have been shown to elicit systemic

response in plants, thus indicating the occurrence of long-distance signal systems.

163

 

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